Polynucleotides encoding plant prenyl proteases

SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs is incorporated herein by reference in its entirety: two copies of the Sequence Listing (COPY 1 and COPY 2) and a computer readable form copy of the Sequence Listing (CRF COPY), all on compact disc, each containing: file name: Final Sequence Listing-15342-00001; date recorded: Jul. 16, 2007; size: 316 KB.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding plant prenyl protease polypeptides, and fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides. Also included are transgenic plants expressing the novel polynucleotides. The present invention also includes transgenic plant cells, tissues and plants having novel phenotypes resulting from the expression of these polynucleotides in either the sense or antisense orientation. The invention further relates to methods of applying these novel plant polypeptides to the identification, prevention, and/or conferment of resistance, including resistance to various plant diseases and/or disorders, particularly drought resistance.

BACKGROUND OF THE INVENTION

Drought is one of the most limiting factors in plant growth and productivity. Crop and yield losses due to drought spells in crops such as soybeans, corn, rice and cotton represent a significant economic problem. Moreover, drought is also responsible for food shortages in many countries worldwide. Developing crops tolerant to drought is a strategy that has potential to alleviate some of these adverse situations.

Traditional plant breeding strategies to develop new lines of plants that exhibit tolerance to drought are relatively slow and require specific tolerant lines for crossing with the desired commercial lines. Limited germplasm resources for drought tolerance and incompatibility in crosses between distantly related plant species therefore represent significant problems encountered in conventional breeding. In contrast, plant genetic transformation and availability of useful genes subjected to specific expression patterns allow one to generate drought-tolerant plants using transgenic approaches.

Plants are exposed during their entire life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are extensive, the effects on plant development, growth and yield of most crop plants are profound.

The physiology of a drought-stressed plant is dramatically altered as compared with a plant grown under normal conditions. Most of the changes and their causes remain uncharacterized. Abscisic acid (ABA) plays a central role in mediating the processes between desiccation perception and cellular changes. ABA increases readily upon the onset of cell desiccation and exogenously applied ABA mimics many of the responses induced by water-stress. An increase in ABA causes the closure of stomata, thereby decreasing water loss through transpiration.

The identification of genes that transduce ABA into a cellular response opens the possibility of exploiting these regulators to enhance desiccation tolerance in crop species. In principle, these ABA signaling genes can be coupled with the appropriate controlling elements to allow optimal plant growth, development and productivity. Thus, not only would these genes allow the genetic tailoring of crops to withstand transitory environmental stresses, but they should also broaden the environments where traditional crops can be grown.

The regulation of protein phosphorylation by kinases and phosphatases is accepted as a universal mechanism of cellular control (Cohen 1992, Trends Biochem. Sci. 17: 408-413), and Ca.sup.2+ and calmodulin signals are frequently transduced via Ca.sup.2+ and calmodulin-dependent kinases and phosphatases (Roberts & Harmon 1992, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43: 375-414). Okadaic acid, a protein phosphatase inhibitor, has been found to affect both gibberellic (GA) and absisic acid (ABA) pathways (Kuo et al. 1996, Plant Cell. 8: 259-269). Although the molecular basis of GA and ABA signal transduction remains poorly understood, it seems well established that the two phytohormones are involved in overall regulatory processes in seed development (e.g. Ritchie & Gilroy 1998, Plant Physiol. 116: 765-776; Arenas-Huertero et al. 2000, Genes Dev. 14: 2085-2096). Likewise, the plant hormones ethylene (e.g. Zhou et al. 1998, Proc. Natl. Acad. Sci. USA 95: 10294-10299; Beaudoin et al. 2000, Plant Cell 2000: 1103-1115) and auxin (e.g. Colon-Carmona et al. 2000, Plant Physiol. 124:1728-1738) are involved in controlling plant development as well.

Protein farnesylation, the addition of a C-terminal, 15-carbon chain to proteins and subsequent processing, has been identified as being crucial for the mediating role of ABA in the desiccation-signal transduction chain. In short, protein farnesylation is required for ABA-induced stomata closure, thus for control of water loss.

Protein farnesylation is a three-step enzymatic reaction as shown in FIG. 1. Potentially, each of these steps could represent a target for genetic manipulation of the prenylation process to generate a desired phenotype such as stress tolerance.

The drought-tolerant phenotype of the era1 Arabidopsis mutant is due to a null mutation in the .beta.-subunit of the enzyme farnesyl transferase (FTase), the first enzyme in the protein farnesylation pathway. Farnesyl transferase is a heterodimeric enzyme that provides the specific addition of a farnesyl pyrophosphate moiety onto the substrate target sequence. The target sequence is defined as a sequence of four amino acids which are present at the carboxy terminus of the protein and is referred to as a CaaX motif in which the "C" is cysteine, "a" is any aliphatic amino acid and "X" is any amino acid. The .alpha. subunit is common with a second prenylation enzyme, geranylgeranyl transferase, that has a different .beta. subunit and adds a geranylgeranyl isoprenyl pyrophosphate moiety to the target sequence.

In plants, prenylation has been linked to cell cycle control, meristem development, and phytohormone signal transduction, however, few details of the role of prenylation, the substrate proteins or the extent to which the plant system will be analogous to the mammalian and yeast systems are known. The most characterized substrates for CaaX modification are the Ras and a-factor proteins of yeast. Although there are three steps to complete protein maturation, abolition or modification of any one step does not necessarily result in cessation of target biological activities. Ras function is attenuated if the -aaX tripeptide is not cleaved but not abolished and some proteins retain the -aaX tripeptide after farnesylation.

In Arabidopsis, more than 600 proteins contain a CaaX motif, suggesting a role for the post-translational modification by prenylation in numerous cellular processes. In Arabidopsis, it has been demonstrated that the loss-of-function of the .beta.-subunit of farnesyl transferase will result in an ABA-hypersensitive phenotype. Although it is still not clear why plants lacking the functional .beta.-subunit of farnesyl transferase become more sensitive to ABA, it clearly suggests that protein prenylation is involved in regulation of the homeostasis of ABA sensitivity. The balance of ABA cellular responses, whether more sensitive or less sensitive to ABA, is possibly regulated by the relative activities of prenylated proteins. The changes in Arabidopsis prenyl protease expression and gene activity may affect the activity of two pools of genes, one pool acting as positive regulators (pool A) and the second pool (pool B) as negative regulators, which require prenylation in order to function properly. Pool A may contain genes that can promote ABA sensitivity, and pool B genes that may reduce ABA sensitivity. The homeostasis of ABA sensitivity may therefore be governed by the ratio of activity of pool A to pool B. For example, in the case of up-regulation of Arabidopsis prenyl protease in Arabidopsis, the activity ratio of pool A over pool B may be increased due to differences in substrate affinity of pool A proteins toward Arabidopsis prenyl protease, thus the homeostasis of ABA sensitivity is changed, and the Arabidopsis prenyl protease over-expression plants are more sensitive to ABA.

There is a need in the art to identify new plant genes encoding these protein farnesylation enzymes as another opportunity to generate plants tolerant to environmental stress, such as drought.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel polynucleotides encoding prenyl protease polypeptides or active fragments thereof from Physcomitrella patens, Arabidopsis thaliana, rapeseed (Brassica napus), soybeans (Glycine max), and corn (Zea mays).

The present invention fulfills a need in the art, in part, by providing the isolated polynucleotide and polypeptide sequences of plant-derived PrPases from five species, namely, the PrPase sequences from moss (Physcomitrella patens; PpPrPase1), three PrPase sequences from Arabidopsis thaliana (AtPrPase1, AtPrPase2, and AtCPP), two PrPase from soybeans (Glycine max; GmPrPase2 and GmCPP), one PrPase from corn (Zea mays; ZmPrPase2), and one from rapeseed (Brassica napus; BnCPP). The invention also provides partial PrPase sequences from soybean (GmPrPase1) and corn (ZmPrPase1).

In a first embodiment, an isolated or recombinant polynucleotide is provided which encodes a plant prenyl protease having the amino acid sequence of SEQ ID NO: 4 or a homolog of SEQ ID NO: 4 having prenyl protease activity and having at least 70% identity at the amino acid level to SEQ ID NO: 4, with the proviso that the polynucleotide as set forth in SEQ ID NO: 27 and the polynucleotide as set forth in SEQ ID NO: 29 are excluded.

In a further embodiment, the invention provides an isolated or recombinant polynucleotide wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of a polynucleotide as set forth in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23; a polynucleotide encoding the polypeptide as set forth in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24; a polynucleotide encoding a polypeptide having at least 95% identity at the amino acid level to SEQ ID NO: 2, 4, 6, 15, 17, 22, or 24; a polynucleotide encoding a polypeptide having at least 99% identity at the amino acid level to SEQ ID NO: 8; a polynucleotide encoding a polypeptide having at least 96% identity at the amino acid level to SEQ ID NO: 12; a polynucleotide encoding a polypeptide having at least 85% identity at the amino acid level to SEQ ID NO: 19; and a polynucleotide complementary to any of these sequences.

The nucleic acid can be, for example, a genomic DNA fragment, a cDNA molecule, or is naturally occurring. In one aspect, the invention provides a nucleic acid that includes the sequence of SEQ ID NO: 84, 86, 88 or 90. The invention also provides a nucleic acid sequence that is complementary to the nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 18, for example, SEQ ID NO: 9, 13, or 20.

A further embodiment is an isolated polypeptide molecule that comprises the sequence of SEQ ID NO: 85, 87, 89, or 91.

Further described are transformation vectors useful to transform plants, such as Physcomitrella, Arabidopsis, rapeseed, soybeans, and corn plants. Such vectors comprise one or more of the nucleic acid molecules described herein. The invention provides for plants and host cells transformed with a PrPase nucleic acid or a vector comprising a PrPase nucleic acid. For example, a vector may comprise at least one of the nucleic acid sequences of SEQ ID NO: 41, 42, or 52-69.

In further embodiments, the invention provides methods of using the polynucleotides and polypeptides of the invention to create transgenic plants with one or more desirable traits, which include, but are not limited to, enhanced plant defense, increased tolerance to stress, drought tolerance, salt tolerance, ultraviolet (uv) tolerance, enhanced flower development, delayed senescence, increased ABA sensitivity, terpene synthesis, increased formation of seed storage compounds (like oil, sugars and proteins), increased yield, increased productivity and increased biomass compared to a wild type plant, and the identification, prevention, and/or conferment of resistance to various plant diseases and/or disorders by introducing into one or more cells of a plant a compound that alters (e.g., increases or decreases) PrPase expression or activity in the plant. In one aspect, the compound is a PrPase nucleic acid or polypeptide. In one embodiment, the nucleic acid is an inhibitor of farnesylation. Alternatively, the compound is a PrPase double stranded RNA-inhibition hair-pin nucleic acid or PrPase antisense nucleic acid.

The present invention further provides a general method for engineering drought-tolerant plants, said method being generally applicable to all plants.

Moreover, the present invention provides the first results suggesting that modulation of PrPase gene expression in a plant directly correlates with increased drought tolerance as compared to untransformed control plants. The present invention also describes methods of engineering drought-tolerant rapeseed, soybeans, and corn plants strains generated through modulation of PrPase expression.

In a preferred embodiment, the invention provides a method of producing a transgenic plant comprising an isolated or recombinant nucleic acid encoding a prenyl protease wherein the plant has increased tolerance to an environmental stress as compared to a wild type variety of the plant, comprising transforming a plant cell with an expression vector comprising a polynucleotide sequence encoding a prenyl protease and generating from the plant cell the transgenic plant with increased tolerance to stress, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 4 or a homolog of SEQ ID NO: 4 having prenyl protease activity and having at least 70% identity at the amino acid level to SEQ ID NO: 4.

In another preferred embodiment, the transgenic plant produced by the above method has increased tolerance to an environmental stress selected from the group consisting of drought, high temperature, and low temperature.

Further provided is the promoter of the Arabidopsis USP gene. This promoter is expressed most strongly during seed developmental stages, i.e. it is a seed-specific promoter.

Further provided is the promoter of the Arabidopsis FTase gene. This promoter is expressed most strongly in guard-cells, i.e. it is a guard-cell specific promoter. In another aspect of this invention, the promoter of an Arabidopsis PrPase is described. This promoter is guard-cell specific and can be used to engineer traits such as drought tolerance and regulation of gas exchange in the plant.

Moreover, the present invention provides novel polynucleotides encoding plant PrPase polypeptides, including a substantially purified PrPase polypeptide, and fragments and homologs thereof. Also provided are vectors, host cells, and recombinant methods for producing said polypeptides.

The invention further provides a method for producing a PrPase polypeptide by providing a cell containing a PrPase nucleic acid, e.g., a vector that includes a PrPase nucleic acid, and culturing the cell under conditions sufficient to express the PrPase polypeptide encoded by the nucleic acid. The expressed PrPase polypeptide can then be recovered from the cell. Preferably, the cell produces little or no endogenous PrPase polypeptide. The cell can be, e.g., a prokaryotic cell or eukaryotic cell.

Another aspect of this invention provides yeast expression vectors used to produce large quantities of the Arabidopsis PrPase in yeast.

The invention also provides methods for more particularly refining the function of the polynucleotides and/or polypeptides of the present invention.

The invention is also directed to plants transformed with the polynucleotides of the invention, to seed and progeny thereof, and to methods of producing these transgenic plants.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1--Schematic representation of the protein farnesylation pathway. This figure identifies the known enzymes involved in the protein farnesylation pathway, in addition to their functional relationship. For illustration purposes, a prospective target protein is represented by a dotted line; while the solid line represents the 15-carbon atom chain added to the C-terminus of the target protein at the "CaaX" site. "C" is Cysteine, "a" is any aliphatic amino acid, and "X" is any amino acid. The 15-carbon chain is added to a conserved Cysteine (C) by the enzyme farnesyl transferase (Ftase). The last three amino acid residues (aaX) are cleaved by the enzyme prenyl protease (PrPase). Lastly, the modified Cysteine is methylated by a methylase to create the final active product of the protein farnesylation pathway.

FIG. 2 is a schematic representation of the vector constructs, A) pBI121-AtCPP, B) pBI121-antisense-AtCPP, and C) pBI121-HP-AtCPP.

FIG. 3--Comparison of the Exon/Intron structure of the computer-predicted ORF of the Arabidopsis PrPase from genomic sequence of GenBank Accession No. AF007269 (SEQ ID NO: 30; labeled as Predicted Ara PrPase; predicted from Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information ("NCBI") website)) and experimentally demonstrated ORFs of the Arabidopsis PrPases (SEQ ID NO: 4 and 6; labeled as Ara PrPase1-2). The exons are numbered sequentially in both clones. Corresponding exons are placed below each other. The lines connecting the introns have no biological significance. Breaks were introduced in the sequences to make this figure.

FIG. 4A-N--Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, default parameters for ClustalW 1.82) of GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11), ZmPrPase2 (SEQ ID NO: 23), PpPrPase1 (SEQ ID NO: 1), and three other disclosed Arabidopsis PrPases sequences: AT4g01320 (SEQ ID NO: 27; GenBank Accession NO: AL161491), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25; GenBank Accession NO: AF353722). The consensus sequence (bottom line) is shown in SEQ ID NO: 84.

FIG. 5A-C--Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of GmCPP (SEQ ID NO: 19), GmPrPase2 (SEQ ID NO: 17), AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6), BnCPP (SEQ ID NO: 12), ZmPrPase2 (SEQ ID NO: 24), PpPrPase1 (SEQ ID NO: 2), three other disclosed Arabidopsis PrPases sequences: AT4g01320 (SEQ ID NO: 28; GenBank Accession NO: AL161491), AF007269 (SEQ ID NO: 30), and AFC1 (SEQ ID NO: 26; GenBank Accession NO: AF353722), and the sequence of the Saccharomyces cerevisiae PrPase (Swiss-Prot Accession No. P47154; SEQ ID NO: 31). The boxed area is the zinc metalloprotease motif (HEXXH). The consensus sequence (bottom line) is shown in SEQ ID NO: 85.

FIG. 6 is a comparison of nucleic acid sequence identities between the sequences of the invention (SEQ ID NOs: 1, 3, 5, 7, 11, 16, 18, 23), and three other disclosed Arabidopsis PrPases sequences (GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25)) as determined by EMBOSS Pairwise Alignment algorithms (Parameters: Blosum 62 matrix, 10.0 gap penalty, 0.1 extend penalty). FIG. 6A shows the comparison using the full-length sequences (including the 5'- and 3'-end non-coding sequences). FIG. 6B shows the comparison using the coding region of the sequences (excluding the 5'- and 3'-end non-coding sequences).

FIG. 7--Amino acid sequence comparison as determined by EMBOSS Pairwise Alignment algorithms (Parameters: Blosum 62 matrix, 10.0 gap penalty, 0.1 extend penalty) of the PrPase polypeptides of the present invention (SEQ ID NOS: 2, 4, 6, 8, 12, 17, 19, 24), other disclosed Arabidopsis PrPases sequences (GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26)), and sequence of the Saccharomyces cerevisiae PrPase (Swiss-Prot Accession No. P47154; SEQ ID NO: 31). The percent identity is shown with the percent similarity values in parenthesis.

FIG. 8 is an illustration showing the relative expression of AtCPP mRNA transcript (solid bars) and AtCPP protein levels (stippled bars) in several pBI121-AtCPP transgenic lines.

FIG. 9 is a histogram showing the percentage of lines which were categorized as ABA sensitive, moderately ABA sensitive or ABA insensitive. Seedlings were assessed on agar plates containing 1 .mu.M ABA and scored at 21 days growth. Thirty-six lines of the pBI121-AtCPP over-expression construct were assessed at 21 days by leaf and seedling development. Thirty-two lines of the 35S-HP-AtCPP down-regulation construct were assessed at 21 days for leaf and seedling development. Each line was assessed by plating approximately 100 seeds per plate and the seedlings scored and recorded as the percent insensitive seedlings per plate. Each line was then expressed as a percent of wild type (Wt). Lines were categorized as sensitive (less than 1% of Wt) solid bars, intermediate (1-50% of Wt) diagonally lined or insensitive (greater than 50% of Wt) stippled, based on their relationship to Wt and the percentage of each category plotted as a histogram.

FIG. 10 is a photograph showing the response of wild type and a pRD29A-HP-AtCPP transgenic line to various concentrations of ABA in two week old seedlings.

FIG. 11 is a histogram showing the analysis of transgenic plants containing the pBI121-AtCPP over-expression construct (SEQ ID NO: 41). Water loss is shown per gram of shoot dry weight after four days of water stress treatment. Lines that are marked with a star are those which were strongly ABA sensitive. Lines marked with a triangle are moderately ABA sensitive. Bars represent means of eight replicates. Lines marked with a filled dot above the bar represents lines which were significantly different from control at a p=0.05 value.

FIG. 12 is a histogram showing seed yield in grams of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal water conditions.

FIG. 13 is a bar chart showing growth and yield of transgenic Arabidopsis lines of pBI121-AtCPP grown under optimal watering conditions plus a biotic stress condition. Yields as a % of wild type, rosette leaf number, rosette leaf fresh weight and shoot dry weight are plotted.

FIG. 14 are photographs showing 12 day old seedling growth of transgenic Arabidopsis lines of pBI121-AtCPP (35S AtCPP) grown on agar plates. Changes to root growth visible (toluidin blue).

FIG. 15 is a bar chart showing growth of transgenic Arabidopsis lines of pRD29A-HP-AtCPP grown under optimal watering conditions. Rosette leaf number, rosette leaf dry weight and shoot dry weight are plotted.

FIG. 16A-K--Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the PrPase sequences of the invention from dicot plants: GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11); and three other disclosed dicot PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25). The consensus sequence (bottom line) is shown in SEQ ID NO: 86.

FIG. 17A-B--Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the PrPase sequences of the invention from dicot plants: GmCPP (SEQ ID NO: 19), GmPrPase2 (SEQ ID NO: 17), AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6), BnCPP (SEQ ID NO: 12); and three other disclosed dicot PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26). The consensus sequence (bottom line) is shown in SEQ ID NO: 87.

FIG. 18A-H--Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the Arabidopsis PrPase sequences of the invention: AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5); and three other disclosed Arabidopsis PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AF353722 (AFC1; SEQ ID NO: 25). The consensus sequence (bottom line) is shown as SEQ ID NO: 88.

FIG. 19A-B--Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the Arabidopsis PrPase sequences of the invention: AtCPP (SEQ ID NO: 8), AtPrPase1 (SEQ ID NO: 4), AtPrPase2 (SEQ ID NO: 6); and other disclosed Arabidopsis PrPases sequences: GenBank Accession NOs: AL161491 (AT4g01320; SEQ ID NO: 28), AF007269 (SEQ ID NO: 30), and AF353722 (AFC1; SEQ ID NO: 26). The consensus sequence (bottom line) is shown in SEQ ID NO: 89.

FIG. 20A-B--Nucleic acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 18) and GmPrPase2 (SEQ ID NO: 16). The consensus sequence (bottom line) is shown in SEQ ID NO: 90.

FIG. 21--Amino acid multiple sequence alignment (ClustalW algorithm, blosum scoring matrix, using default parameters) of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 19) and GmPrPase2 (SEQ ID NO: 17). The consensus sequence (bottom line) is shown in SEQ ID NO: 91.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein.

One aspect of this invention pertains to isolated nucleic acid molecules that encode PrPase polypeptides from Physcomitrella patens, Arabidopsis thaliana, soybeans, rapeseed, and corn. Moreover, this invention pertains to nucleic acid fragments originated from the clones mentioned above, as well as to other nucleic acid fragments from other organisms that can be isolated using the described nucleic acid fragments as probes in hybridization experiments.

As explained above, introduction of a PrPase into plants can improve tolerance to environmental stress. This invention also describes modulation of PrPase gene activity in a plant. The only condition necessary to realize either of these embodiments is the isolation of the corresponding PrPase genes from the target plants. The use of the described clones to isolate corresponding PrPase genes from other plants is something appreciated by someone skilled in the art.

Modulation of PrPase gene activity can be accomplished by reduction in PrPase, for example by: (a) antisense gene-expression repression, (b) targeted antibodies to PrPase, and (c) targeted, engineered promoter repression with for example zinc-finger derived transcription factors.

The present invention can make a significant contribution to the art by providing new strategies to engineer drought-tolerance in crop plants, especially the use of the previously unknown PrPase clones from plant origin. The polynucleotides and polypeptides of the present invention have uses which include conferring resistance to or modulating susceptibility to biotic and/or abiotic stresses such as heat, drought, and salt stress in plants.

In one embodiment, over-expression of a PrPase polypeptide of the present invention within a plant using a constitutive promoter (e.g., 35S, or other promoters disclosed herein), preferably not in the guard-cell, improves drought and salt tolerance in a plant.

In another embodiment, overexpression of a PrPase polypeptide of the present invention within a plant using a seed-specific promoter (e.g., unknown seed protein, USP, promoter) increases the amount of seed storage compounds.

Constitutive over-expression in plants of the farnesylation pathway, namely of a prenyl protease, may result in increased cell proliferation and increased plant growth. The polynucleotides and polypeptides of the present invention, including fragments thereof, have uses that include modulating plant growth, and potentially plant yield, preferably increasing plant growth.

The polynucleotides can also be used to express recombinant proteins for analysis, characterization and agronomic use, to express recombinant proteins to raise antibodies directed against polypeptides of the present invention, as markers for tissues in which the corresponding protein is expressed (e.g., preferentially, or non-preferentially), as hybridization markers on Southern gels, as genetic markers for breeding assistance, as RFLP markers, as markers for genotyping (varieties, etc), and the encoded protein, can, at the very least, be used as a molecular weight marker.

The polynucleotides of the present invention are also useful as chromosome markers or tags (when labeled) to identify chromosomes, to map related gene positions within a chromosome, or as a comparative reference to endogenous DNA sequences of mutant plants to identify allelic variants, and/or spontaneous or biotic mutations.

The polynucleotides of the present invention are also useful for genetic fingerprinting, for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns for particular genes, to differentiate intron and/or exon boundaries, to identify splice and/or allelic variants, and as diagnostic tools for identification of developmental stages, disease states, and/or nutrient levels.

The present invention encompasses polynucleotides that hybridize to the polynucleotides of the present invention under either stringent or non-stringent conditions and their uses as described herein. Such hybidization may be used to identify orthologs, homologs, allelic variants, variants, and/or mutants of the polynucleotides of the present invention. Additionally, the polynucleotides of the present invention may be used to clone orthologs, homologs, alleleic variants, variants, and/or mutants of the polynucleotides of the present by using oligonucleotides directed to polynucleotide sequences of the present invention, and performing PCR on plant cell or tissue samples.

The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol. Endocrinol., 9(10):1321-9, (1995); and Ann. NY Acad. Sci., 7; 766:279-81, (1995)).

Potential uses of polynucleotides and polypeptides of the present invention include nutrition (e.g., as an amino acid supplement), as a carbon source, as nitrogen source, as a carbohydrate source, modulating plant defense activity, modulating signal transduction, modulating metabolite transport (e.g., carbon, nitrogen fluxes, etc.), conferring abiotic stress tolerance and/or resistance (water, drought, cold, salt, etc.), conferring xenobiotic stress tolerance and/or resistance, and development control (for example, yield, flowering time, etc.).

The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of these or related sequences, as probes to "subtract-out" known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.

Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.

Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the molecules of the invention, non-coding regions of the molecules of the invention, regulatory sequences associated with the molecules of the invention, and secreted, mature, pro-, prepro- and other forms of the molecules of the invention.

In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution to create and identify novel variants with desired structural, functional, and/or physical characteristics.

The present invention in further preferred embodiments provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods and other procedures that could use sequence information from our clones to build a primer or a hybrid partner.

As used herein, the term "environmental stress" refers to any sub-optimal growing condition and includes, but is not limited to, sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be salinity, drought, or temperature, or combinations thereof, and in particular, can be high salinity, low water content or low temperature. It is also to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.

As also used herein, the term "nucleic acid" and "polynucleotide" refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The term "nucleic acid molecule" is intended to also include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. These terms also encompass untranslated sequences located at both the 3' and 5' ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR and in vitro or in vivo transcription.

An "isolated" nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (e.g., sequences encoding other polypeptides). Preferably, an "isolated" nucleic acid is free of some of the sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. In various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. In further embodiments, the isolated PrPase nucleic acid molecule can contain less than about 5 mb, 1 mb, 0.5 mb, 0.1 mb, 50 kb, 25 kb, 20 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection or other means of transformation. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

Specifically excluded from the definition of "isolated nucleic acids" are: naturally-occurring chromosomes (such as chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a specified nucleic acid makes up less than 5% of the number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including whole cell preparations that are mechanically sheared or enzymatically digested). Even further specifically excluded are the whole cell preparations found as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis wherein the nucleic acid of the invention has not further been separated from the heterologous nucleic acids in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).

One aspect of the invention pertains to isolated PrPase proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. In one embodiment, native PrPase proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, PrPase proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a PrPase protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PrPase protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of PrPase protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of PrPase protein having less than about 30% (by dry weight) of non-PrPase protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-PrPase protein, still more preferably less than about 10% of non-PrPase protein, and most preferably less than about 5% non-PrPase protein. When the PrPase protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of PrPase protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of PrPase protein having less than about 30% (by dry weight) of chemical precursors or non-PrPase chemicals, more preferably less than about 20% chemical precursors or non-PrPase chemicals, still more preferably less than about 10% chemical precursors or non-PrPase chemicals, and most preferably less than about 5% chemical precursors or non-PrPase chemicals.

Polynucleotides and Polypeptides of the Invention

The present invention provides novel polynucleotide and polypeptide sequences of plant-derived prenyl protease (PrPase) from five species namely, the nucleic acid PrPase sequences and the encoded polypeptides from the moss Physcomitrella patens (PpPrPase1, SEQ ID NOs: 1, 2), three from Arabidopsis thaliana (AtPrPase1, SEQ ID NOs: 3, 4; AtPrPase2, SEQ ID NOs: 5, 6; and AtCPP, SEQ ID NOs: 7, 8), two from soybeans Glycine max (GmPrPase2, SEQ ID NOs: 16, 17; and GmCPP, SEQ ID NOs: 18, 19), one from corn Zea mays (ZmPrPase2, SEQ ID NOs: 23, 24), and one from rapeseed Brassica napus (BnCPP, SEQ ID NOs: 11, 12). The invention also provides partial PrPase sequences from soybean (GmPrPase1, SEQ ID NOs: 14, 15) and corn (ZmPrPase1, SEQ ID NOs: 21, 22).

The sequences are collectively referred to as "PrPase nucleic acids", "PrPase polynucleotides" or "PrPase antisense nucleic acids" and the corresponding encoded polypeptide is referred to as a "PrPase polypeptide" or "PrPase protein". Unless indicated otherwise, "PrPase" is meant to refer to any of the novel sequences disclosed herein.

The prenyl proteases of the invention have recognizable homology at the nucleotide and amino acid level to both human and yeast prenyl proteases. Additionally, in a BLAST search of public sequence databases, it was found, for example, that the AtCPP nucleic acid sequence has 99.3% identity to an Arabidopsis thaliana CaaX processing zinc-metallo endoprotease (AFC1) mRNA (Genbank Accession No.: AF353722). FIG. 6A. The full amino acid sequence of the AtCPP protein of the invention was found to be 98.8% identical to Arabidopsis thaliana CaaX processing zinc-metallo endoprotease (AFC1) polypeptide (Genbank Accession No.: AAK39514). FIG. 7. A multiple alignment of the eight polypeptides of the invention (SEQ ID NO: 2, 4, 6, 8, 12, 17, 19, and 24), of yeast, and of three other published Arabidopsis PrPases is illustrated in FIG. 5 with the consensus sequence shown in SEQ ID NO: 85. Similarly, a multiple alignment of the polynucleotides of the invention (SEQ ID NO: 1, 3, 5, 7, 11, 16, 18, and 23) and of three other published Arabidopsis PrPase sequences is illustrated in FIG. 4 with the consensus sequence shown in SEQ ID NO: 84.

The percent identity between the various sequences of FIGS. 4 and 5 are tabulated in FIGS. 6 and 7. A multiple alignment of the PrPase sequences for dicot plants of the invention and three published PrPase are provided in FIGS. 16 and 17 with the consensus sequences shown in SEQ ID NOs: 86-87. A multiple alignment of the PrPase sequences for Arabidopsis are provided in FIGS. 18 and 19 with the consensus sequences shown in SEQ ID NOs: 88-89. A multiple alignment of the PrPase sequences of the invention for soybean plants are provided in FIGS. 20 and 21 with the consensus sequences shown in SEQ ID NOs: 90-91.

The previously described genomic sequence encoding the Arabidopsis PrPase (GenBank accession number AF007269, gene="A_IG002N01.21) was predicted by the computer program (Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information (NCBI) website)) to contain an ORF at positions 24979 to 28076. That predicted ORF does not reflect the real ORF for this gene as shown in FIG. 3.

Based on their structural and functional relatedness to known CaaX prenyl protease proteins, the PrPase proteins of the invention are novel members of the CaaX prenyl protease family of proteins. PrPase nucleic acids, and their encoded polypeptides, according to the invention are useful in a variety of applications and contexts. For example, the nucleic acids (i.e., sense or antisense PrPase nucleic acids) can be used to produce transgenic plants that have an increased resistance to biotic and abiotic stresses, e.g., chilling stress, salt stress, water stress (e.g., drought), wound healing, pathogen challenge, grazing pests, or herbicides. Additionally, the transgenic plants have an increased productivity during both optimal and suboptimal growth conditions, increased yield, increased biomass, or delayed senescence. Alternatively, the transgenic plants have an increased sensitivity to the phytohormone abscisic acid (ABA). By resistant is meant the plant grows under stress conditions (e.g., high salt, decreased water, low temperatures) or under conditions that normally inhibit, to some degree, the growth of an untransformed plant.

Although it is believed the encoded polypeptides may share at least some biological activities with prenyl proteases, a number of methods of determining the exact biological function of these clones are either known in the art or are described elsewhere herein. Briefly, the function of these clones may be determined, for example, by applying microarray methodology. The clones of the present invention may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been cold treated might indicate a function in modulating cold tolerance. Tissue deprived of water or stressed by other biotic or abiotic stresses (heat, drought, high light, high salt, etc.) should be used to extract RNA to prepare the probe. Moreover, different stages of seed development (early, middle, late) could be used to extract RNA to prepare the probe.

In addition, the function of the proteins may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of specific genes throughout the plant development cycle, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step (3 days germinated seedlings; 1 week old seedlings and their roots, shoots, and stems; roots, leaves and stems before the onset of flowering, flowers and their different parts; and/or developing embryos) is usually used to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of these polypeptides is encompassed by the present invention. Also encompassed by the present invention are PCR probes corresponding to the polynucleotide sequences provided or portions thereof.

The function of the proteins may also be assessed through complementation assays in yeast. For example, transforming yeast deficient in prenyl protease activity and assessing their ability to grow would provide convincing evidence that the clones of the invention have prenyl protease activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art.

Alternatively, the biological function of the encoded polypeptides may be determined by disrupting a homologue of the polypeptides in Synechosystis. Cyanobacteria (blue-green algae) is considered a precursor to the plant chloroplast. It possesses both photosynthetic systems and many other metabolic processes similar to those of plants. These processes are often targets for many commercial herbicides, and this organism has been widely used in the study of the mode of action of many classes of herbicides. Synechocystis is one of the best-studied cyanobacteria. In addition to most of the features common to cyanobacteria, it offers many other advantages. Synechocystis has a naturally occurring genetic transformation system, thus entailing vigorous and sophisticated genetic and molecular manipulation (e.g. targeted-gene disruption, gene replacement, etc.) applicable to some of the well-characterized systems (S. cerevisiae, E. coli). Most importantly, the availability of the complete genomic sequence information of the Synechocystis affords an avenue for the rapid identification and cloning of gene(s) of interest, and elucidation of gene function through genetic and molecular means.

Moreover, the biological function of polypeptides may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic plants. Expressing a particular gene in either sense or antisense orientation in a transgenic plant can lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the plant at all times using a strong ubiquitous promoter, or it can be expressed in one or more discrete parts of the plant using a well characterized tissue-specific promoter (i.e., a root promoter or a flower specific promoter or a seed-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of the transgenic plants of the invention, if no phenotype is apparent in normal growth conditions, observing the plants under stress conditions (deprivation of water, presence of high salt, or other biotic or abiotic stresses, such as cold, heat, drought, high light, etc.) may lead to understanding the function of the genes. Therefore, the application of antisense and/or sense methodology to the creation of transgenic plants to refine the biological function of the polypeptides is encompassed by the present invention.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to the sequences of the invention and may have been publicly available prior to conception of the present invention. Such related polynucleotides are specifically excluded from the scope of the present invention.

PpPrPase1

The polynucleotide sequence of the partial PrPase from Physcomitrella patens (Clone ID No: PpPrPase1) of the present invention is provided as SEQ ID NO: 1. The polynucleotide sequence contains a sequence of 1398 nucleotides. The deduced amino acid sequence of SEQ ID NO: 1 is provided as SEQ ID NO: 2 and contains 394 amino acids.

In further embodiments, deletion mutants of PpPrPase1 are encompassed by the present invention, the following N-terminal deletion mutants: L1-D394, K2-D394, L3-D394, S4-D394, N5-D394, L6-D394, P7-D394, A8-D394, P9-D394, L10-D394, K11-D394, G12-D394, I13-D394, V14-D394, S15-D394, Q16-D394, E17-D394, K18-D394, F19-D394, E20-D394, K21-D394, A22-D394, Q23-D394, A24-D394, Y25-D394, S26-D394, L27-D394, D28-D394, K29-D394, S30-D394, R31-D394, F32-D394, H33-D394, F34-D394, V35-D394, H36-D394, A37-D394, A38-D394, V39-D394, N40-D394, I41-D394, V42-D394, E43-D394, E44-D394, S45-D394, A46-D394, I47-D394, L48-D394, L49-D394, L50-D394, G51-D394, L52-D394, L53-D394, P54-D394, W55-D394, A56-D394, W57-D394, D58-D394, K59-D394, S60-D394, G61-D394, S62-D394, L63-D394, V64-D394, G65-D394, K66-D394, L67-D394, G68-D394, F69-D394, D70-D394, E71-D394, K72-D394, S73-D394, E74-D394, I75-D394, L76-D394, Q77-D394, T78-D394, L79-D394, S80-D394, F81-D394, L82-D394, A83-D394, V84-D394, T85-D394, T86-D394, L87-D394, W88-D394, S89-D394, Q90-D394, I91-D394, L92-D394, E93-D394, L94-D394, P95-D394, F96-D394, S97-D394, L98-D394, Y99-D394, S100-D394, T101-D394, F102-D394, V103-D394, I104-D394, E105-D394, A106-D394, R107-D394, H108-D394, G109-D394, F110-D394, N111-D394, K112-D394, Q113-D394, T114-D394, I115-D394, W116-D394, L117-D394, F118-D394, L119-D394, R120-D394, D121-D394, M122-D394, I123-D394, M124-D394, G125-D394, L126-D394, A127-D394, L128-D394, M129-D394, M130-D394, V131-D394, V132-D394, G133-D394, P134-D394, P135-D394, I136-D394, V137-D394, S138-D394, A139-D394, I140-D394, I141-D394, Y142-D394, I143-D394, V144-D394, Q145-D394, N146-D394, G147-D394, G148-D394, P149-D394, Y150-D394, L151-D394, A152-D394, L153-D394, Y154-D394, L155-D394, W156-D394, A157-D394, F158-D394, M159-D394, L160-D394, L161-D394, L162-D394, S163-D394, L164-D394, V165-D394, L166-D394, M167-D394, A168-D394, L169-D394, Y170-D394, P171-D394, V172-D394, L173-D394, I174-D394, A175-D394, P176-D394, L177-D394, F178-D394, N179-D394, T180-D394, F181-D394, T182-D394, P183-D394, L184-D394, P185-D394, E186-D394, G187-D394, Q188-D394, L189-D394, R190-D394, A191-D394, K192-D394, I193-D394, E194-D394, K195-D394, L196-D394, A197-D394, S198-D394, S199-D394, L200-D394, D201-D394, F202-D394, P203-D394, L204-D394, K205-D394, K206-D394, L207-D394, F208-D394, V209-D394, I210-D394, D211-D394, G212-D394, S213-D394, T214-D394, R215-D394, S216-D394, S217-D394, H218-D394, S219-D394, N220-D394, A221-D394, Y222-D394, M223-D394, Y224-D394, G225-D394, F226-D394, Y227-D394, N228-D394, S229-D394, K230-D394, R231-D394, I232-D394, V233-D394, L234-D394, Y235-D394, D236-D394, T237-D394, L238-D394, I239-D394, S240-D394, Q241-D394, C242-D394, K243-D394, N244-D394, E245-D394, E246-D394, E247-D394, V248-D394, V249-D394, A250-D394, V251-D394, I252-D394, A253-D394, H254-D394, E255-D394, L256-D394, G257-D394, H258-D394, W259-D394, K260-D394, L261-D394, S262-D394, H263-D394, T264-D394, M265-D394, Y266-D394, S267-D394, F268-D394, L269-D394, A270-D394, M271-D394, Q272-D394, V273-D394, L274-D394, T275-D394, L276-D394, L277-D394, Q278-D394, F279-D394, G280-D394, G281-D394, Y282-D394, T283-D394, L284-D394, V285-D394, R286-D394, N287-D394, S288-D394, S289-D394, G290-D394, L291-D394, F292-D394, L293-D394, S294-D394, F295-D394, G296-D394, F297-D394, S298-D394, T299-D394, Q300-D394, P301-D394, V302-D394, L303-D394, I304-D394, G305-D394, L306-D394, I307-D394, L308-D394, F309-D394, Q310-D394, H311-D394, T312-D394, I313-D394, M314-D394, P315-D394, F316-D394, H317-D394, H318-D394, L319-D394, V320-D394, S321-D394, F322-D394, A323-D394, L324-D394, N325-D394, L326-D394, L327-D394, S328-D394, R329-D394, A330-D394, F331-D394, E332-D394, F333-D394, Q334-D394, A335-D394, D336-D394, A337-D394, F338-D394, A339-D394, R340-D394, S341-D394, L342-D394, G343-D394, Y344-D394, R345-D394, E346-D394, P347-D394, L348-D394, R349-D394, A350-D394, G351-D394, L352-D394, I353-D394, K354-D394, L355-D394, Q356-D394, E357-D394, E358-D394, N359-D394, L360-D394, S361-D394, A362-D394, M363-D394, N364-D394, T365-D394, D366-D394, P367-D394, W368-D394, Y369-D394, S370-D394, A371-D394, Y372-D394, H373-D394, H374-D394, S375-D394, H376-D394, P377-D394, P378-D394, L379-D394, V380-D394, E381-D394, R382-D394, L383-D394, Q384-D394, A385-D394, L386-D394, D387-D394, E388-D394, of SEQ ID NO: 2. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

In further embodiments, deletion mutants of PpPrPase1 are encompassed by the present invention, including the following C-terminal deletion mutants: L1-D394, L1-T393, L1-K392, L1-K391, L1-S390, L1-T389, L1-E388, L1-D387, L1-L386, L1-A385, L1-Q384, L1-L383, L1-R382, L1-E381, L1-V380, L1-L379, L1-P378, L1-P377, L1-H376, L1-S375, L1-H374, L1-H373, L1-Y372, L1-A371, L1-S370, L1-Y369, L1-W368, L1-P367, L1-D366, L1-T365, L1-N364, L1-M363, L1-A362, L1-S361, L1-L360, L1-N359, L1-E358, L1-E357, L1-Q356, L1-L355, L1-K354, L1-I353, L1-L352, L1-G351, L1-A350, L1-R349, L1-L348, L1-P347, L1-E346, L1-R345, L1-Y344, L1-G343, L1-L342, L1-S341, L1-R340, L1-A339, L1-F338, L1-A337, L1-D336, L1-A335, L1-Q334, L1-F333, L1-E332, L1-F331, L1-A330, L1-R329, L1-S328, L1-L327, L1-L326, L1-N325, L1-L324, L1-A323, L1-F322, L1-S321, L1-V320, L1-L319, L1-H318, L1-H317, L1-F316, L1-P315, L1-M314, L1-I313, L1-T312, L1-H311, L1-Q310, L1-F309, L1-L308, L1-I307, L1-L306, L1-G305, L1-I304, L1-L303, L1-V302, L1-P301, L1-Q300, L1-T299, L1-S298, L1-F297, L1-G296, L1-F295, L1-S294, L1-L293, L1-F292, L1-L291, L1-G290, L1-S289, L1-S288, L1-N287, L1-R286, L1-V285, L1-L284, L1-T283, L1-Y282, L1-G281, L1-G280, L1-F279, L1-Q278, L1-L277, L1-L276, L1-T275, L1-L274, L1-V273, L1-Q272, L1-M271, L1-A270, L1-L269, L1-F268, L1-S267, L1-Y266, L1-M265, L1-T264, L1-H263, L1-S262, L1-L261, L1-K260, L1-W259, L1-H258, L1-G257, L1-L256, L1-E255, L1-H254, L1-A253, L1-I252, L1-V251, L1-A250, L1-V249, L1-V248, L1-E247, L1-E246, L1-E245, L1-N244, L1-K243, L1-C242, L1-Q241, L1-S240, L1-I239, L1-L238, L1-T237, L1-D236, L1-Y235, L1-L234, L1-V233, L1-I232, L1-R231, L1-K230, L1-S229, L1-N228, L1-Y227, L1-F226, L1-G225, L1-Y224, L1-M223, L1-Y222, L1-A221, L1-N220, L1-S219, L1-H218, L1-S217, L1-S216, L1-R215, L1-T214, L1-S213, L1-G212, L1-D211, L1-I210, L1-V209, L1-F208, L1-L207, L1-K206, L1-K205, L1-L204, L1-P203, L1-F202, L1-D201, L1-L200, L1-S199, L1-S198, L1-A197, L1-L196, L1-K195, L1-E194, L1-I193, L1-K192, L1-A191, L1-R190, L1-L189, L1-Q188, L1-G187, L1-E186, L1-P185, L1-L184, L1-P183, L1-T182, L1-F181, L1-T180, L1-N179, L1-F178, L1-L177, L1-P176, L1-A175, L1-I174, L1-L173, L1-V172, L1-P171, L1-Y170, L1-L169, L1-A168, L1-M167, L1-L166, L1-V165, L1-L164, L1-S163, L1-L162, L1-L161, L1-L160, L1-M159, L1-F158, L1-A157, L1-W156, L1-L155, L1-Y154, L1-L153, L1-A152, L1-L151, L1-Y150, L1-P149, L1-G148, L1-G147, L1-N146, L1-Q145, L1-V144, L1-I143, L1-Y142, L1-I141, L1-I140, L1-A139, L1-S138, L1-V137, L1-I136, L1-P135, L1-P134, L1-G133, L1-V132, L1-V131, L1-M130, L1-M129, L1-L128, L1-A127, L1-L126, L1-G125, L1-M124, L1-I123, L1-M122, L1-D121, L1-R120, L1-L119, L1-F118, L1-L117, L1-W116, L1-I115, L1-T114, L1-Q113, L1-K112, L1-N111, L1-F110, L1-G109, L1-H108, L1-R107, L1-A106, L1-E105, L1-I104, L1-V103, L1-F102, L1-T101, L1-S100, L1-Y99, L1-L98, L1-S97, L1-F96, L1-P95, L1-L94, L1-E93, L1-L92, L1-I91, L1-Q90, L1-S89, L1-W88, L1-L87, L1-T86, L1-T85, L1-V84, L1-A83, L1-L82, L1-F81, L1-S80, L1-L79, L1-T78, L1-Q77, L1-L76, L1-I75, L1-E74, L1-S73, L1-K72, L1-E71, L1-D70, L1-F69, L1-G68, L1-L67, L1-K66, L1-G65, L1-V64, L1-L63, L1-S62, L1-G61, L1-S60, L1-K59, L1-D58, L1-W57, L1-A56, L1-W55, L1-P54, L1-L53, L1-L52, L1-G51, L1-L50, L1-L49, L1-L48, L1-I47, L1-A46, L1-S45, L1-E44, L1-E43, L1-V42, L1-I41, L1-N40, L1-V39, L1-A38, L1-A37, L1-H36, L1-V35, L1-F34, L1-H33, L1-F32, L1-R31, L1-S30, L1-K29, L1-D28, L1-L27, L1-S26, L1-Y25, L1-A24, L1-Q23, L1-A22, L1-K21, L1-E20, L1-F19, L1-K18, L1-E17, L1-Q16, L1-S15, L1-V14, L1-I13, L1-G12, L1-K11, L1-L10, L1-P9, L1-A8, L1-P7, of SEQ ID NO: 2. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

AtPrPase1

The nucleotide sequence of the full-length PrPase (AtPrPase1) from Arabidopsis thaliana (Clone ID No: AtPrPase1) of the present invention is provided as SEQ ID NO: 3. The polynucleotide sequence contains a sequence of 1275 nucleotides. The deduced amino acid sequence of SEQ ID NO: 3 is provided as SEQ ID NO: 4 and contains 424 amino acids.

In a further embodiment, the invention also encompasses the promoter of the AtPrPase1 gene (SEQ ID NO: 10). The promoter has uses including, but not limited to, directing expression of a gene of interest in plant guard-cells. The gene of interest may be any gene endogenous to a plant, a non-plant derived gene (e.g., viral, mammalian, human, synthetic, molecularly evolved, bacterial, fungal, etc.), a reporter gene, a marker gene, a desired input trait, a desired output trait, a gene capable of conferring a specific phenotype in a plant, one or more genes of the present invention, antibody genes, antibody genes directed against the polypeptides of the present invention, antisense genes, in addition to other genes known in the art and/or disclosed herein.

In further embodiments, deletion mutants of AtPrPase1 are encompassed by the present invention, including the following N-terminal deletion mutants: M1-D424, A2-D424, I3-D424, P4-D424, F5-D424, M6-D424, E7-D424, T8-D424, V9-D424, V10-D424, G11-D424, F12-D424, M13-D424, I14-D424, V15-D424, M16-D424, Y17-D424, I18-D424, F19-D424, E20-D424, T21-D424, Y22-D424, L23-D424, D24-D424, L25-D424, R26-D424, Q27-D424, L28-D424, T29-D424, A30-D424, L31-D424, K32-D424, L33-D424, P34-D424, T35-D424, L36-D424, P37-D424, K38-D424, T39-D424, L40-D424, V41-D424, G42-D424, V43-D424, I44-D424, S45-D424, Q46-D424, E47-D424, K48-D424, F49-D424, E50-D424, K51-D424, S52-D424, R53-D424, A54-D424, Y55-D424, S56-D424, L57-D424, D58-D424, K59-D424, S60-D424, Y61-D424, F62-D424, H63-D424, F64-D424, V65-D424, H66-D424, E67-D424, F68-D424, V69-D424, T70-D424, I71-D424, L72-D424, M73-D424, D74-D424, S75-D424, A76-D424, I77-D424, L78-D424, F79-D424, F80-D424, G81-D424, I82-D424, L83-D424, P84-D424, W85-D424, F86-D424, W87-D424, K88-D424, M89-D424, S90-D424, G91-D424, A92-D424, V93-D424, L94-D424, P95-D424, R96-D424, L97-D424, G98-D424, L99-D424, D100-D424, P101-D424, E102-D424, N103-D424, E104-D424, I105-D424, L106-D424, H107-D424, T108-D424, L109-D424, S110-D424, F111-D424, L112-D424, A113-D424, G114-D424, V115-D424, M116-D424, T117-D424, W118-D424, S119-D424, Q120-D424, I121-D424, T122-D424, D123-D424, L124-D424, P125-D424, F126-D424, S127-D424, L128-D424, Y129-D424, S130-D424, T131-D424, F132-D424, V133-D424, I134-D424, E135-D424, S136-D424, R137-D424, H138-D424, G139-D424, F140-D424, N141-D424, K142-D424, Q143-D424, T144-D424, I145-D424, W146-D424, M147-D424, F148-D424, I149-D424, R150-D424, D151-D424, M152-D424, I153-D424, K154-D424, G155-D424, T156-D424, F157-D424, L158-D424, S159-D424, V160-D424, I161-D424, L162-D424, G163-D424, P164-D424, P165-D424, I166-D424, V167-D424, A168-D424, A169-D424, I170-D424, I171-D424, F172-D424, I173-D424, V174-D424, Q175-D424, K176-D424, G177-D424, G178-D424, P179-D424, Y180-D424, L181-D424, A182-D424, I183-D424, Y184-D424, L185-D424, W186-D424, A187-D424, F188-D424, M189-D424, F190-D424, I191-D424, L192-D424, S193-D424, L194-D424, V195-D424, M196-D424, M197-D424, T198-D424, I199-D424, Y200-D424, P201-D424, V202-D424, L203-D424, I204-D424, A205-D424, P206-D424, L207-D424, F208-D424, N209-D424, K210-D424, F211-D424, T212-D424, P213-D424, L214-D424, P215-D424, D216-D424, G217-D424, D218-D424, L219-D424, R220-D424, E221-D424, K222-D424, I223-D424, E224-D424, K225-D424, L226-D424, A227-D424, S228-D424, S229-D424, L230-D424, K231-D424, F232-D424, P233-D424, L234-D424, K235-D424, K236-D424, L237-D424, F238-D424, V239-D424, V240-D424, D241-D424, G242-D424, S243-D424, T244-D424, R245-D424, S246-D424, S247-D424, H248-D424, S249-D424, N250-D424, A251-D424, Y252-D424, M253-D424, Y254-D424, G255-D424, F256-D424, F257-D424, K258-D424, N259-D424, K260-D424, R261-D424, I262-D424, V263-D424, L264-D424, Y265-D424, D266-D424, T267-D424, L268-D424, I269-D424, Q270-D424, Q271-D424, C272-D424, K273-D424, N274-D424, E275-D424, D276-D424, E277-D424, I278-D424, V279-D424, A280-D424, V281-D424, I282-D424, A283-D424, H284-D424, E285-D424, L286-D424, G287-D424, H288-D424, W289-D424, K290-D424, L291-D424, N292-D424, H293-D424, T294-D424, T295-D424, Y296-D424, S297-D424, F298-D424, I299-D424, A300-D424, V301-D424, Q302-D424, I303-D424, L304-D424, A305-D424, F306-D424, L307-D424, Q308-D424, F309-D424, G310-D424, G311-D424, Y312-D424, T313-D424, L314-D424, V315-D424, R316-D424, N317-D424, S318-D424, T319-D424, D320-D424, L321-D424, F322-D424, R323-D424, S324-D424, F325-D424, G326-D424, F327-D424, D328-D424, T329-D424, Q330-D424, P331-D424, V332-D424, L333-D424, I334-D424, G335-D424, L336-D424, I337-D424, I338-D424, F339-D424, Q340-D424, H341-D424, T342-D424, V343-D424, I344-D424, P345-D424, L346-D424, Q347-D424, H348-D424, P349-D424, V350-D424, S351-D424, F352-D424, G353-D424, L354-D424, N355-D424, L356-D424, V357-D424, S358-D424, R359-D424, A360-D424, F361-D424, E362-D424, F363-D424, Q364-D424, A365-D424, D366-D424, A367-D424, F368-D424, A369-D424, V370-D424, K371-D424, L372-D424, G373-D424, Y374-D424, A375-D424, K376-D424, D377-D424, L378-D424, R379-D424, P380-D424, T381-D424, L382-D424, V383-D424, K384-D424, L385-D424, Q386-D424, E387-D424, E388-D424, N389-D424, L390-D424, S391-D424, A392-D424, M393-D424, N394-D424, T395-D424, D396-D424, P397-D424, L398-D424, Y399-D424, S400-D424, A401-D424, Y402-D424, H403-D424, Y404-D424, S405-D424, H406-D424, P407-D424, P408-D424, L409-D424, V410-D424, E411-D424, R412-D424, L413-D424, R414-D424, A415-D424, I416-D424, D417-D424, G418-D424, of SEQ ID NO: 4. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

In further embodiments, deletion mutants of AtPrPase1 are encompassed by the present invention, including the following C-terminal deletion mutants: M1-D424, M1-T423, M1-K422, M1-K421, M1-D420, M1-E419, M1-G418, M1-D417, M1-I416, M1-A415, M1-R414, M1-L413, M1-R412, M1-E411, M1-V410, M1-L409, M1-P408, M1-P407, M1-H406, M1-S405, M1-Y404, M1-H403, M1-Y402, M1-A401, M1-S400, M1-Y399, M1-L398, M1-P397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-T381, M1-P380, M1-R379, M1-L378, M1-D377, M1-K376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-V370, M1-A369, M1-F368, M1-A367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-A360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-P349, M1-H348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-F322, M1-L321, M1-D320, M1-T319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-F306, M1-A305, M1-L304, M1-I303, M1-Q302, M1-V301, M1-A300, M1-I299, M1-F298, M1-S297, M1-Y296, M1-T295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-D276, M1-E275, M1-N274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-F232, M1-K231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-D218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-I199, M1-T198, M1-M197, M1-M196, M1-V195, M1-L194, M1-S193, M1-L192, M1-I191, M1-F190, M1-M189, M1-F188, M1-A187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-F172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-L162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-T156, M1-G155, M1-K154, M1-I153, M1-M152, M1-D151, M1-R150, M1-I149, M1-F148, M1-M147, M1-W146, M1-I145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-S136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-T117, M1-M116, M1-V115, M1-G114, M1-A113, M1-L112, M1-F111, M1-S110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-P100, M1-D100, M1-L99, M1-G98, M1-L97, M1-R96, M1-P95, M1-L94, M1-V93, M1-A92, M1-G91, M1-S90, M1-M89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-I82, M1-G81, M1-F80, M1-F79, M1-L78, M1-I77, M1-A76, M1-S75, M1-D74, M1-M73, M1-L72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-Y61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-V41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-T29, M1-L28, M1-Q27, M1-R26, M1-L25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-V15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-T8, M1-E7, of SEQ ID NO: 4. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

AtPrPase2

The nucleotide sequence of a full-length PrPase (AtPrPase2) from Arabidopsis thaliana (Clone ID No: AtPrPase2) of the present invention is provided as SEQ ID NO: 5. The polynucleotide sequence contains a sequence of 1275 nucleotides. SEQ ID NO: 3 and SEQ ID NO: 5 are full-length Arabidopsis AtPrPases with 8 nucleotide differences at nucleotide positions: 276, 504, 1046, 1062, 1068, 1141, 1182, and 1190, which are alternative embodiments of this sequence of the invention. The deduced amino acid sequence of SEQ ID NO: 5 is provided as SEQ ID NO: 6 and contains 424 amino acids. SEQ ID NO: 4 and SEQ ID NO: 6 are full-length Arabidopsis AtPrPases with 3 amino acid differences at amino acid positions: 349, 381, and 397.

In preferred embodiments, deletion mutants of AtPrPase2 are encompassed by the present invention, including the following N-terminal deletion mutants: M1-D424, A2-D424, I3-D424, P4-D424, F5-D424, M6-D424, E7-D424, T8-D424, V9-D424, V10-D424, G11-D424, F12-D424, M13-D424, I14-D424, V15-D424, M16-D424, Y17-D424, I18-D424, F19-D424, E20-D424, T21-D424, Y22-D424, L23-D424, D24-D424, L25-D424, R26-D424, Q27-D424, L28-D424, T29-D424, A30-D424, L31-D424, K32-D424, L33-D424, P34-D424, T35-D424, L36-D424, P37-D424, K38-D424, T39-D424, L40-D424, V41-D424, G42-D424, V43-D424, I44-D424, S45-D424, Q46-D424, E47-D424, K48-D424, F49-D424, E50-D424, K51-D424, S52-D424, R53-D424, A54-D424, Y55-D424, S56-D424, L57-D424, D58-D424, K59-D424, S60-D424, Y61-D424, F62-D424, H63-D424, F64-D424, V65-D424, H66-D424, E67-D424, F68-D424, V69-D424, T70-D424, I71-D424, L72-D424, M73-D424, D74-D424, S75-D424, A76-D424, I77-D424, L78-D424, F79-D424, F80-D424, G81-D424, I82-D424, L83-D424, P84-D424, W85-D424, F86-D424, W87-D424, K88-D424, M89-D424, S90-D424, G91-D424, A92-D424, V93-D424, L94-D424, P95-D424, R96-D424, L97-D424, G98-D424, L99-D424, D100-D424, P101-D424, E102-D424, N103-D424, E104-D424, I105-D424, L106-D424, H107-D424, T108-D424, L109-D424, S110-D424, F111-D424, L112-D424, A113-D424, G114-D424, V115-D424, M116-D424, T117-D424, W118-D424, S119-D424, Q120-D424, I121-D424, T122-D424, D123-D424, L124-D424, P125-D424, F126-D424, S127-D424, L128-D424, Y129-D424, S130-D424, T131-D424, F132-D424, V133-D424, I134-D424, E135-D424, S136-D424, R137-D424, H138-D424, G139-D424, F140-D424, N141-D424, K142-D424, Q143-D424, T144-D424, I145-D424, W146-D424, M147-D424, F148-D424, I149-D424, R150-D424, D151-D424, M152-D424, I153-D424, K154-D424, G155-D424, T156-D424, F157-D424, L158-D424, S159-D424, V160-D424, I161-D424, L162-D424, G163-D424, P164-D424, P165-D424, I166-D424, V167-D424, A168-D424, A169-D424, I170-D424, I171-D424, F172-D424, I173-D424, V174-D424, Q175-D424, K176-D424, G177-D424, G178-D424, P179-D424, Y180-D424, L181-D424, A182-D424, I183-D424, Y184-D424, L185-D424, W186-D424, A187-D424, F188-D424, M189-D424, F190-D424, I191-D424, L192-D424, S193-D424, L194-D424, V195-D424, M196-D424, M197-D424, T198-D424, I199-D424, Y200-D424, P201-D424, V202-D424, L203-D424, I204-D424, A205-D424, P206-D424, L207-D424, F208-D424, N209-D424, K210-D424, F211-D424, T212-D424, P213-D424, L214-D424, P215-D424, D216-D424, G217-D424, D218-D424, L219-D424, R220-D424, E221-D424, K222-D424, I223-D424, E224-D424, K225-D424, L226-D424, A227-D424, S228-D424, S229-D424, L230-D424, K231-D424, F232-D424, P233-D424, L234-D424, K235-D424, K236-D424, L237-D424, F238-D424, V239-D424, V240-D424, D241-D424, G242-D424, S243-D424, T244-D424, R245-D424, S246-D424, S247-D424, H248-D424, S249-D424, N250-D424, A251-D424, Y252-D424, M253-D424, Y254-D424, G255-D424, F256-D424, F257-D424, K258-D424, N259-D424, K260-D424, R261-D424, I262-D424, V263-D424, L264-D424, Y265-D424, D266-D424, T267-D424, L268-D424, I269-D424, Q270-D424, Q271-D424, C272-D424, K273-D424, N274-D424, E275-D424, D276-D424, E277-D424, I278-D424, V279-D424, A280-D424, V281-D424, I282-D424, A283-D424, H284-D424, E285-D424, L286-D424, G287-D424, H288-D424, W289-D424, K290-D424, L291-D424, N292-D424, H293-D424, T294-D424, T295-D424, Y296-D424, S297-D424, F298-D424, I299-D424, A300-D424, V301-D424, Q302-D424, I303-D424, L304-D424, A305-D424, F306-D424, L307-D424, Q308-D424, F309-D424, G310-D424, G311-D424, Y312-D424, T313-D424, L314-D424, V315-D424, R316-D424, N317-D424, S318-D424, T319-D424, D320-D424, L321-D424, F322-D424, R323-D424, S324-D424, F325-D424, G326-D424, F327-D424, D328-D424, T329-D424, Q330-D424, P331-D424, V332-D424, L333-D424, I334-D424, G335-D424, L336-D424, I337-D424, I338-D424, F339-D424, Q340-D424, H341-D424, T342-D424, V343-D424, I344-D424, P345-D424, L346-D424, Q347-D424, H348-D424, L349-D424, V350-D424, S351-D424, F352-D424, G353-D424, L354-D424, N355-D424, L356-D424, V357-D424, S358-D424, R359-D424, A360-D424, F361-D424, E362-D424, F363-D424, Q364-D424, A365-D424, D366-D424, A367-D424, F368-D424, A369-D424, V370-D424, K371-D424, L372-D424, G373-D424, Y374-D424, A375-D424, K376-D424, D377-D424, L378-D424, R379-D424, P380-D424, A381-D424, L382-D424, V383-D424, K384-D424, L385-D424, Q386-D424, E387-D424, E388-D424, N389-D424, L390-D424, S391-D424, A392-D424, M393-D424, N394-D424, T395-D424, D396-D424, L397-D424, L398-D424, Y399-D424, S400-D424, A401-D424, Y402-D424, H403-D424, Y404-D424, S405-D424, H406-D424, P407-D424, P408-D424, L409-D424, V410-D424, E411-D424, R412-D424, L413-D424, R414-D424, A415-D424, I416-D424, D417-D424, G418-D424, of SEQ ID NO: 6. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

In further embodiments, deletion mutants of AtPrPase2 are encompassed by the present invention, including the following C-terminal deletion mutants: M1-D424, M1-T423, M1-K422, M1-K421, M1-D420, M1-E419, M1-G418, M1-D417, M1-I416, M1-A415, M1-R414, M1-L413, M1-R412, M1-E411, M1-V410, M1-L409, M1-P408, M1-P407, M1-H406, M1-S405, M1-Y404, M1-H403, M1-Y402, M1-A401, M1-S400, M1-Y399, M1-L398, M1-L397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-A381, M1-P380, M1-R379, M1-L378, M1-D377, M1-K376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-V370, M1-A369, M1-F368, M1-A367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-A360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-L349, M1-H348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-F322, M1-L321, M1-D320, M1-T319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-F306, M1-A305, M1-L304, M1-I303, M1-Q302, M1-V301, M1-A300, M1-I299, M1-F298, M1-S297, M1-Y296, M1-T295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-D276, M1-E275, M1-N274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-F232, M1-K231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-D218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-I199, M1-T198, M1-M197, M1-M196, M1-V195, M1-L194, M1-S193, M1-L192, M1-I191, M1-F190, M1-M189, M1-F188, M1-A187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-F172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-L162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-T156, M1-G155, M1-K154, M1-I153, M1-M152, M1-D151, M1-R150, M1-I149, M1-F148, M1-M147, M1-W146, M1-I145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-S136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-T117, M1-M116, M1-V115, M1-G114, M1-A113, M1-L112, M1-F111, M1-S110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-P101, M1-D100, M1-L99, M1-G98, M1-L97, M1-R96, M1-P95, M1-L94, M1-V93, M1-A92, M1-G91, M1-S90, M1-M89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-I82, M1-G81, M1-F80, M1-F79, M1-L78, M1-I77, M1-A76, M1-S75, M1-D74, M1-M73, M1-L72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-Y61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-V41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-T29, M1-L28, M1-Q27, M1-R26, M1-L25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-V15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-T8, M1-E7, of SEQ ID NO: 6. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

GmPrPase

The nucleotide sequence of the full-length PrPase from soybeans (Glycine max) (Clone ID No: GmPrPase2) of the present invention is provided as SEQ ID NO: 16. The deduced amino acid sequence of SEQ ID NO: 16 is provided as SEQ ID NO: 17. A partial PrPase nucleotide sequence from soybean (Clone ID No: GmPrPase1) is provided as SEQ ID NO: 14 with the deduced amino acid sequence as SEQ ID NO: 15.

In further embodiments, deletion mutants of GmPrPase are encompassed by the present invention, including the following N-terminal deletion mutants: M1-C400, A2-C400, F3-C400, P4-C400, Y5-C400, M6-C400, E7-C400, A8-C400, V9-C400, V10-C400, G11-C400, F12-C400, M13-C400, I14-C400, L15-C400, M16-C400, Y17-C400, I18-C400, F19-C400, E20-C400, T21-C400, Y22-C400, L23-C400, D24-C400, V25-C400, R26-C400, Q27-C400, H28-C400, R29-C400, A30-C400, L31-C400, K32-C400, L33-C400, P34-C400, T35-C400, L36-C400, P37-C400, K38-C400, T39-C400, L40-C400, E41-C400, G42-C400, V43-C400, I44-C400, S45-C400, Q46-C400, E47-C400, K48-C400, F49-C400, E50-C400, K51-C400, S52-C400, R53-C400, A54-C400, Y55-C400, S56-C400, L57-C400, D58-C400, K59-C400, S60-C400, H61-C400, F62-C400, H63-C400, F64-C400, V65-C400, H66-C400, E67-C400, F68-C400, V69-C400, T70-C400, I71-C400, V72-C400, T73-C400, D74-C400, S75-C400, T76-C400, I77-C400, L78-C400, Y79-C400, F80-C400, G81-C400, V82-C400, L83-C400, P84-C400, W85-C400, F86-C400, W87-C400, K88-C400, K89-C400, S90-C400, G91-C400, D92-C400, F93-C400, M94-C400, T95-C400, I96-C400, A97-C400, G98-C400, F99-C400, N100-C400, A101-C400, E102-C400, N103-C400, E104-C400, I105-C400, L106-C400, H107-C400, T108-C400, L109-C400, A110-C400, F111-C400, L112-C400, A113-C400, G114-C400, L115-C400, M116-C400, I117-C400, W118-C400, S119-C400, Q120-C400, I121-C400, T122-C400, D123-C400, L124-C400, P125-C400, F126-C400, S127-C400, L128-C400, Y129-C400, S130-C400, T131-C400, F132-C400, V133-C400, I134-C400, E135-C400, A136-C400, R137-C400, H138-C400, G139-C400, F140-C400, N141-C400, K142-C400, Q143-C400, T144-C400, P145-C400, W146-C400, L147-C400, F148-C400, F149-C400, R150-C400, D151-C400, M152-C400, L153-C400, K154-C400, G155-C400, I156-C400, F157-C400, L158-C400, S159-C400, V160-C400, I161-C400, I162-C400, G163-C400, P164-C400, P165-C400, I166-C400, V167-C400, A168-C400, A169-C400, I170-C400, I171-C400, V172-C400, I173-C400, V174-C400, Q175-C400, K176-C400, G177-C400, G178-C400, P179-C400, Y180-C400, L181-C400, A182-C400, I183-C400, Y184-C400, L185-C400, W186-C400, V187-C400, F188-C400, T189-C400, F190-C400, G191-C400, L192-C400, S193-C400, I194-C400, V195-C400, M196-C400, M197-C400, T198-C400, L199-C400, Y200-C400, P201-C400, V202-C400, L203-C400, I204-C400, A205-C400, P206-C400, L207-C400, F208-C400, N209-C400, K210-C400, F211-C400, T212-C400, P213-C400, L214-C400, P215-C400, D216-C400, G217-C400, Q218-C400, L219-C400, R220-C400, E221-C400, K222-C400, I223-C400, E224-C400, K225-C400, L226-C400, A227-C400, S228-C400, S229-C400, L230-C400, N231-C400, Y232-C400, P233-C400, L234-C400, K235-C400, K236-C400, L237-C400, F238-C400, V239-C400, V240-C400, D241-C400, G242-C400, S243-C400, T244-C400, R245-C400, S246-C400, S247-C400, H248-C400, S249-C400, N250-C400, A251-C400, Y252-C400, M253-C400, Y254-C400, G255-C400, F256-C400, F257-C400, K258-C400, N259-C400, K260-C400, R261-C400, I262-C400, V263-C400, L264-C400, Y265-C400, D266-C400, T267-C400, L268-C400, I269-C400, Q270-C400, Q271-C400, C272-C400, K273-C400, D274-C400, D275-C400, E276-C400, E277-C400, I278-C400, V279-C400, A280-C400, V281-C400, I282-C400, A283-C400, H284-C400, E285-C400, L286-C400, G287-C400, H288-C400, W289-C400, K290-C400, L291-C400, N292-C400, H293-C400, T294-C400, V295-C400, Y296-C400, T297-C400, F298-C400, V299-C400, A300-C400, M301-C400, Q302-C400, I303-C400, L304-C400, T305-C400, L306-C400, L307-C400, Q308-C400, F309-C400, G310-C400, G311-C400, Y312-C400, T313-C400, L314-C400, V315-C400, R316-C400, N317-C400, S318-C400, A319-C400, D320-C400, L321-C400, Y322-C400, R323-C400, S324-C400, F325-C400, G326-C400, F327-C400, D328-C400, T329-C400, Q330-C400, P331-C400, V332-C400, L333-C400, I334-C400, G335-C400, L336-C400, I337-C400, I338-C400, F339-C400, Q340-C400, H341-C400, T342-C400, V343-C400, I344-C400, P345-C400, L346-C400, Q347-C400, Q348-C400, L349-C400, V350-C400, S351-C400, F352-C400, G353-C400, L354-C400, N355-C400, L356-C400, V357-C400, S358-C400, R359-C400, S360-C400, F361-C400, E362-C400, F363-C400, Q364-C400, A365-C400, D366-C400, G367-C400, F368-C400, A369-C400, K370-C400, K371-C400, L372-C400, G373-C400, Y374-C400, A375-C400, S376-C400, G377-C400, L378-C400, R379-C400, G380-C400, G381-C400, L382-C400, V383-C400, K384-C400, L385-C400, Q386-C400, E387-C400, E388-C400, N389-C400, L390-C400, S391-C400, A392-C400, M393-C400, N394-C400, of SEQ ID NO: 15 or 17. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

In further embodiments, deletion mutants of GmPrPase are encompassed by the present invention, including the following C-terminal deletion mutants: M1-C400, M1-S399, M1-C398, M1-P397, M1-D396, M1-T395, M1-N394, M1-M393, M1-A392, M1-S391, M1-L390, M1-N389, M1-E388, M1-E387, M1-Q386, M1-L385, M1-K384, M1-V383, M1-L382, M1-G381, M1-G380, M1-R379, M1-L378, M1-G377, M1-S376, M1-A375, M1-Y374, M1-G373, M1-L372, M1-K371, M1-K370, M1-A369, M1-F368, M1-G367, M1-D366, M1-A365, M1-Q364, M1-F363, M1-E362, M1-F361, M1-S360, M1-R359, M1-S358, M1-V357, M1-L356, M1-N355, M1-L354, M1-G353, M1-F352, M1-S351, M1-V350, M1-L349, M1-Q348, M1-Q347, M1-L346, M1-P345, M1-I344, M1-V343, M1-T342, M1-H341, M1-Q340, M1-F339, M1-I338, M1-I337, M1-L336, M1-G335, M1-I334, M1-L333, M1-V332, M1-P331, M1-Q330, M1-T329, M1-D328, M1-F327, M1-G326, M1-F325, M1-S324, M1-R323, M1-Y322, M1-L321, M1-D320, M1-A319, M1-S318, M1-N317, M1-R316, M1-V315, M1-L314, M1-T313, M1-Y312, M1-G311, M1-G310, M1-F309, M1-Q308, M1-L307, M1-L306, M1-T305, M1-L304, M1-I303, M1-Q302, M1-M301, M1-A300, M1-V299, M1-F298, M1-T297, M1-Y296, M1-V295, M1-T294, M1-H293, M1-N292, M1-L291, M1-K290, M1-W289, M1-H288, M1-G287, M1-L286, M1-E285, M1-H284, M1-A283, M1-I282, M1-V281, M1-A280, M1-V279, M1-I278, M1-E277, M1-E276, M1-D275, M1-D274, M1-K273, M1-C272, M1-Q271, M1-Q270, M1-I269, M1-L268, M1-T267, M1-D266, M1-Y265, M1-L264, M1-V263, M1-I262, M1-R261, M1-K260, M1-N259, M1-K258, M1-F257, M1-F256, M1-G255, M1-Y254, M1-M253, M1-Y252, M1-A251, M1-N250, M1-S249, M1-H248, M1-S247, M1-S246, M1-R245, M1-T244, M1-S243, M1-G242, M1-D241, M1-V240, M1-V239, M1-F238, M1-L237, M1-K236, M1-K235, M1-L234, M1-P233, M1-Y232, M1-N231, M1-L230, M1-S229, M1-S228, M1-A227, M1-L226, M1-K225, M1-E224, M1-I223, M1-K222, M1-E221, M1-R220, M1-L219, M1-Q218, M1-G217, M1-D216, M1-P215, M1-L214, M1-P213, M1-T212, M1-F211, M1-K210, M1-N209, M1-F208, M1-L207, M1-P206, M1-A205, M1-I204, M1-L203, M1-V202, M1-P201, M1-Y200, M1-L199, M1-T198, M1-M197, M1-M196, M1-V195, M1-I194, M1-S193, M1-L192, M1-G191, M1-F190, M1-T189, M1-F188, M1-V187, M1-W186, M1-L185, M1-Y184, M1-I183, M1-A182, M1-L181, M1-Y180, M1-P179, M1-G178, M1-G177, M1-K176, M1-Q175, M1-V174, M1-I173, M1-V172, M1-I171, M1-I170, M1-A169, M1-A168, M1-V167, M1-I166, M1-P165, M1-P164, M1-G163, M1-I162, M1-I161, M1-V160, M1-S159, M1-L158, M1-F157, M1-I156, M1-G155, M1-K154, M1-L153, M1-M152, M1-D151, M1-R150, M1-F149, M1-F148, M1-L147, M1-W146, M1-P145, M1-T144, M1-Q143, M1-K142, M1-N141, M1-F140, M1-G139, M1-H138, M1-R137, M1-A136, M1-E135, M1-I134, M1-V133, M1-F132, M1-T131, M1-S130, M1-Y129, M1-L128, M1-S127, M1-F126, M1-P125, M1-L124, M1-D123, M1-T122, M1-I121, M1-Q120, M1-S119, M1-W118, M1-I117, M1-M116, M1-L115, M1-G114, M1-A113, M1-L112, M1-F111, M1-A110, M1-L109, M1-T108, M1-H107, M1-L106, M1-I105, M1-E104, M1-N103, M1-E102, M1-A101, M1-N100, M1-F99, M1-G98, M1-A97, M1-I96, M1-T95, M1-M94, M1-F93, M1-D92, M1-G91, M1-S90, M1-K89, M1-K88, M1-W87, M1-F86, M1-W85, M1-P84, M1-L83, M1-V82, M1-G81, M1-F80, M1-Y79, M1-L78, M1-I77, M1-T76, M1-S75, M1-D74, M1-T73, M1-V72, M1-I71, M1-T70, M1-V69, M1-F68, M1-E67, M1-H66, M1-V65, M1-F64, M1-H63, M1-F62, M1-H61, M1-S60, M1-K59, M1-D58, M1-L57, M1-S56, M1-Y55, M1-A54, M1-R53, M1-S52, M1-K51, M1-E50, M1-F49, M1-K48, M1-E47, M1-Q46, M1-S45, M1-I44, M1-V43, M1-G42, M1-E41, M1-L40, M1-T39, M1-K38, M1-P37, M1-L36, M1-T35, M1-P34, M1-L33, M1-K32, M1-L31, M1-A30, M1-R29, M1-H28, M1-Q27, M1-R26, M1-V25, M1-D24, M1-L23, M1-Y22, M1-T21, M1-E20, M1-F19, M1-I18, M1-Y17, M1-M16, M1-L15, M1-I14, M1-M13, M1-F12, M1-G11, M1-V10, M1-V9, M1-A8, M1-E7, of SEQ ID NO: 15 or 17. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

ZmPrPase

The nucleotide sequence of the full-length PrPase from corn (Zea mays) (Clone ID No: ZmPrPase2) of the present invention is provided as SEQ ID NO: 23. The deduced amino acid sequence of SEQ ID NO: 23 is provided as SEQ ID NO: 24. A partial PrPase nucleotide sequence from corn (Clone ID No: ZmPrPase1) is provided as SEQ ID NO: 21 with the deduced amino acid sequence as SEQ ID NO: 22.

In further embodiments, deletion mutants of ZmPrPase are encompassed by the present invention, including the following N-terminal deletion mutants: T1-D329, R2-D329, L3-D329, S4-D329, A5-D329, E6-D329, N7-D329, E8-D329, I9-D329, I10-D329, H11-D329, T12-D329, L13-D329, A14-D329, F15-D329, L16-D329, A17-D329, G18-D329, S19-D329, M20-D329, V21-D329, W22-D329, S23-D329, Q24-D329, I25-D329, T26-D329, D27-D329, L28-D329, P29-D329, F30-D329, S31-D329, L32-D329, Y33-D329, S34-D329, T35-D329, F36-D329, V37-D329, I38-D329, E39-D329, A40-D329, R41-D329, H42-D329, G43-D329, F44-D329, N45-D329, K46-D329, Q47-D329, T48-D329, I49-D329, W50-D329, L51-D329, F52-D329, I53-D329, R54-D329, D55-D329, M56-D329, I57-D329, K58-D329, G59-D329, I60-D329, L61-D329, L62-D329, S63-D329, M64-D329, I65-D329, L66-D329, G67-D329, P68-D329, P69-D329, I70-D329, V71-D329, A72-D329, A73-D329, I74-D329, I75-D329, Y76-D329, I77-D329, V78-D329, Q79-D329, I80-D329, G81-D329, G82-D329, P83-D329, Y84-D329, L85-D329, A86-D329, I87-D329, Y88-D329, L89-D329, W90-D329, G91-D329, F92-D329, M93-D329, F94-D329, V95-D329, L96-D329, A97-D329, L98-D329, L99-D329, M100-D329, M101-D329, T102-D329, I103-D329, Y104-D329, P105-D329, I106-D329, V107-D329, I108-D329, A109-D329, P110-D329, L111-D329, F112-D329, N113-D329, K114-D329, F115-D329, T116-D329, P117-D329, L118-D329, P119-D329, E120-D329, G121-D329, V122-D329, L123-D329, R124-D329, E125-D329, K126-D329, I127-D329, E128-D329, K129-D329, L130-D329, A131-D329, A132-D329, S133-D329, L134-D329, K135-D329, F136-D329, P137-D329, L138-D329, K139-D329, K140-D329, L141-D329, F142-D329, V143-D329, V144-D329, D145-D329, G146-D329, S147-D329, T148-D329, R149-D329, S150-D329, S151-D329, H152-D329, S153-D329, N154-D329, A155-D329, Y156-D329, M157-D329, Y158-D329, G159-D329, F160-D329, F161-D329, K162-D329, N163-D329, K164-D329, R165-D329, I166-D329, V167-D329, L168-D329, Y169-D329, D170-D329, T171-D329, L172-D329, I173-D329, Q174-D329, Q175-D329, C176-D329, S177-D329, N178-D329, E179-D329, D180-D329, E181-D329, I182-D329, V183-D329, S184-D329, V185-D329, I186-D329, A187-D329, H188-D329, E189-D329, L190-D329, G191-D329, H192-D329, W193-D329, K194-D329, L195-D329, N196-D329, H197-D329, T198-D329, V199-D329, Y200-D329, S201-D329, F202-D329, V203-D329, A204-D329, V205-D329, Q206-D329, L207-D329, L208-D329, M209-D329, F210-D329, L211-D329, Q212-D329, F213-D329, G214-D329, G215-D329, Y216-D329, T217-D329, L218-D329, V219-D329, R220-D329, S221-D329, S222-D329, K223-D329, D224-D329, L225-D329, F226-D329, G227-D329, S228-D329, F229-D329, G230-D329, F231-D329, K232-D329, D233-D329, Q234-D329, P235-D329, V236-D329, I237-D329, I238-D329, G239-D329, L240-D329, I241-D329, I242-D329, F243-D329, P244-D329, H245-D329, T246-D329, I247-D329, I248-D329, P249-D329, I250-D329, Q251-D329, H252-D329, L253-D329, L254-D329, S255-D329, F256-D329, R257-D329, L258-D329, N259-D329, L260-D329, V261-D329, S262-D329, R263-D329, A264-D329, F265-D329, E266-D329, F267-D329, Q268-D329, A269-D329, D270-D329, A271-D329, F272-D329, A273-D329, K274-D329, N275-D329, L276-D329, G277-D329, Y278-D329, A279-D329, P280-D329, Q281-D329, L282-D329, R283-D329, A284-D329, A285-D329, L286-D329, V287-D329, K288-D329, L289-D329, Q290-D329, E291-D329, E292-D329, N293-D329, L294-D329, S295-D329, A296-D329, M297-D329, N298-D329, T299-D329, D300-D329, P301-D329, W302-D329, Y303-D329, S304-D329, A305-D329, Y306-D329, H307-D329, Y308-D329, S309-D329, H310-D329, P311-D329, P312-D329, L313-D329, V314-D329, E315-D329, R316-D329, L317-D329, Q318-D329, A319-D329, L320-D329, E321-D329, D322-D329, S323-D329, of SEQ ID NO: 22 or 24. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

In further embodiments, deletion mutants of ZmPrPase are encompassed by the present invention, including the following C-terminal deletion mutants: T1-D329, T1-E328, T1-K327, T1-K326, T1-D325, T1-D324, T1-S323, T1-D322, T1-E321, T1-L320, T1-A319, T1-Q318, T1-L317, T1-R316, T1-E315, T1-V314, T1-L313, T1-P312, T1-P311, T1-H310, T1-S309, T1-Y308, T1-H307, T1-Y306, T1-A305, T1-S304, T1-Y303, T1-W302, T1-P301, T1-D300, T1-T299, T1-N298, T1-M297, T1-A296, T1-S295, T1-L294, T1-N293, T1-E292, T1-E291, T1-Q290, T1-L289, T1-K288, T1-V287, T1-L286, T1-A285, T1-A284, T1-R283, T1-L282, T1-Q281, T1-P280, T1-A279, T1-Y278, T1-G277, T1-L276, T1-N275, T1-K274, T1-A273, T1-F272, T1-A271, T1-D270, T1-A269, T1-Q268, T1-F267, T1-E266, T1-F265, T1-A264, T1-R263, T1-S262, T1-V261, T1-L260, T1-N259, T1-L258, T1-R257, T1-F256, T1-S255, T1-L254, T1-L253, T1-H252, T1-Q251, T1-I250, T1-P249, T1-I248, T1-I247, T1-T246, T1-H245, T1-P244, T1-F243, T1-I242, T1-I241, T1-L240, T1-G239, T1-I238, T1-I237, T1-V236, T1-P235, T1-Q234, T1-D233, T1-K232, T1-F231, T1-G230, T1-F229, T1-S228, T1-G227, T1-F226, T1-L225, T1-D224, T1-K223, T1-S222, T1-S221, T1-R220, T1-V219, T1-L218, T1-T217, T1-Y216, T1-G215, T1-G214, T1-F213, T1-Q212, T1-L211, T1-F210, T1-M209, T1-L208, T1-L207, T1-Q206, T1-V205, T1-A204, T1-V203, T1-F202, T1-S201, T1-Y200, T1-V199, T1-T198, T1-H197, T1-N196, T1-L195, T1-K194, T1-W193, T1-H192, T1-G191, T1-L190, T1-E189, T1-H188, T1-A187, T1-I186, T1-V185, T1-S184, T1-V183, T1-I182, T1-E181, T1-D180, T1-E179, T1-N178, T1-S177, T1-C176, T1-Q175, T1-Q174, T1-I173, T1-L172, T1-T171, T1-D170, T1-Y169, T1-L168, T1-V167, T1-I166, T1-R165, T1-K164, T1-N163, T1-K162, T1-F161, T1-F160, T1-G159, T1-Y158, T1-M157, T1-Y156, T1-A155, T1-N154, T1-S153, T1-H152, T1-S151, T1-S150, T1-R149, T1-T148, T1-S147, T1-G146, T1-D145, T1-V144, T1-V143, T1-F142, T1-L141, T1-K140, T1-K139, T1-L138, T1-P137, T1-F136, T1-K135, T1-L134, T1-S133, T1-A132, T1-A131, T1-L130, T1-K129, T1-E128, T1-I127, T1-K126, T1-E125, T1-R124, T1-L123, T1-V122, T1-G121, T1-E120, T1-P119, T1-L118, T1-P117, T1-T116, T1-F115, T1-K114, T1-N113, T1-F112, T1-L111, T1-P110, T1-A109, T1-I108, T1-V107, T1-I106, T1-P105, T1-Y104, T1-I103, T1-T102, T1-M101, T1-M100, T1-L99, T1-L98, T1-A97, T1-L96, T1-V95, T1-F94, T1-M93, T1-F92, T1-G91, T1-W90, T1-L89, T1-Y88, T1-I87, T1-A86, T1-L85, T1-Y84, T1-P83, T1-G82, T1-G81, T1-I80, T1-Q79, T1-V78, T1-I77, T1-Y76, T1-I75, T1-I74, T1-A73, T1-A72, T1-V71, T1-I70, T1-P69, T1-P68, T1-G67, T1-L66, T1-I65, T1-M64, T1-S63, T1-L62, T1-L61, T1-I60, T1-G59, T1-K58, T1-I57, T1-M56, T1-D55, T1-R54, T1-I53, T1-F52, T1-L51, T1-W50, T1-I49, T1-T48, T1-Q47, T1-K46, T1-N45, T1-F44, T1-G43, T1-H42, T1-R41, T1-A40, T1-E39, T1-I38, T1-V37, T1-F36, T1-T35, T1-S34, T1-Y33, T1-L32, T1-S31, T1-F30, T1-P29, T1-L28, T1-D27, T1-T26, T1-I25, T1-Q24, T1-S23, T1-W22, T1-V21, T1-M20, T1-S19, T1-G18, T1-A17, T1-L16, T1-F15, T1-A14, T1-L13, T1-T12, T1-H11, T1-I10, T1-I9, T1-E8, T1-N7, of SEQ ID NO: 22 or 24. The invention includes the polynucleotide sequences encoding these mutant polypeptides.

TABLE-US-00001 TABLE 1 cDNA NT Total 5' NT AA Clone SEQ ID NT Seq. of Start 3' NT SEQ ID Total AA of ID Vector NO: X of Clone Codon of ORF of ORF NO: Y ORF PpPrPase1 pCR2.1 1 1398 33 1214 2 394 AtPrPase1 pCR2.1 3 1275 1 1272 4 424 AtPrPase2 pCR2.1 5 1275 1 1272 6 424 AtCPP 7 1275 1 1275 8 424 BnCPP 11 1275 1 1275 12 424 GmPrPase1 pCR2.1 14 1434 233 1432 15 400 GmPrPase2 16 1405 39 1313 17 424 GmCPP 18 1275 1 1275 19 424 ZmPrPase1 pCR2.1 21 1301 1 987 22 329 ZmPrPase2 23 1518 166 1443 24 424

Table 1 summarizes the information corresponding to each "cDNA Clone ID." The nucleotide sequences identified as NT SEQ ID NO: 1, 3, 5, 14, and 21 were assembled from partially homologous ("overlapping") sequences obtained from the corresponding "cDNA clone ID" identified in Table 1 and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified under "NT SEQ ID NO:X."

"Vector" in Table 1 refers to the type of vector contained in the cDNA Clone ID. pCR2.1 was obtained from Invitrogen, Inc.

"Total NT Seq. Of Clone" in Table 1 refers to the total number of nucleotides in the clone identified by "cDNA Clone ID." The nucleotide position of a sequence under NT SEQ ID NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon of ORF."

The translated amino acid sequence, beginning with the methionine, is identified in Table 1 as "AA SEQ ID NO:Y," although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The total number of amino acids within the open reading frame of a sequence under AA SEQ ID NO:Y is identified in Table 1 as "Total AA of ORF".

DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

For those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides the nucleotide sequence identified as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23 and the predicted translated amino acid sequence identified as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the cDNA, collecting the protein, and determining its sequence. Thus, minor errors in the nucleotide sequence can be recognized by persons skilled in the art.

The present invention also relates to the genes corresponding to SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, or SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologs of genes corresponding to SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, or SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, and 24, relying on the sequences disclosed herein. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers which correspond to the 5', 3', or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence to confer stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide can be substantially purified using techniques known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols known in the art.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.

The present invention encompasses polynucleotides which are complementary to the PrPase polynucleotides disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NOs: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NOs: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or a portion of these nucleotide sequence. A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NO:1, 4, 17, 76, 78, 80, 82, or 84 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, thereby forming a stable duplex. Exemplary complement nucleic acid sequences include the sequences of SEQ ID NO: 9, I3, and 20.

As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Hybridization

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Nucleic acid hybridization techniques are well known in the art. Examples of stringency conditions are shown in Table 2 below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.

TABLE-US-00002 TABLE 2 Hyridization Wash Stringency Polynucleotide Hybrid Length Temperature Temperature Condition Hybrid.dagger..dagger. (bp).dagger-dbl. and Buffer.dagger. and Buffer.dagger. A DNA:DNA > or equal to 50 65.degree. C.; 1xSSC -or- 65.degree. C.; 0.3xSSC 42.degree. C.; 1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67.degree. C.; 1xSSC 67.degree. C.; 0.3xSSC -or- 45.degree. C.; 1xSSC, 50% formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 50 70.degree. C.; 1xSSC 70.degree. C.; 0.3xSSC -or- 50.degree. C.; 1xSSC, 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 50 65.degree. C.; 4xSSC 65.degree. C.; 1xSSC -or- 45.degree. C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 50 67.degree. C.; 4xSSC 67.degree. C.; 1xSSC -or- 45.degree. C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 50 70.degree. C.; 4xSSC 67.degree. C.; 1xSSC -or- 40.degree. C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50 50.degree. C.; 4xSSC 50.degree. C.; 2xSSC -or- 40.degree. C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to 50 55.degree. C.; 4xSSC 55.degree. C.; 2xSSC -or- 42.degree. C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 50 60.degree. C.; 4xSSC 60.degree. C.; 2xSSC -or- 45.degree. C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC .dagger-dbl.The "hybrid length" in Table 2 is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucletotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc). .dagger.SSPE in Table 2 (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCl anmd 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr in Table 2. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10.degree. C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(.degree. C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(.degree. C.) = 81.5 + 16.6(log.sub.10[Na+]) + 0.41(% G + C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M). .dagger..dagger.The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a peptide nucleic acid ("PNA"), or a modified polynucleotide. Such modified polynucleotides are known in the art.

Additional examples of stringency conditions for polynucleotide hybridization are known to those skilled in the art and are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.

Preferably, such hybridizating polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art.

In another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500 or 750 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding PrPase proteins derived from species other than Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays, or Glycine max) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

Alternatively, the phrase "stringent hybridization conditions" can refer to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different depending upon circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60.degree. C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65.degree. C. This hybridization is followed by one or more washes in 0.2.times.SSC, 0.01% BSA at 50.degree. C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the nucleic acid sequences of the invention can correspond to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55.degree. C., followed by one or more washes in 1.times.SSC, 0.1% SDS at 37.degree. C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In yet another embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40.degree. C., followed by one or more washes in 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50.degree. C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981, Proc Natl Acad Sci USA 78: 6789-6792.

The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and "PCR Protocols, A Guide to Methods and Applications", Eds., Innis et al., Academic Press, New York, (1990).

The present invention includes mature forms of the polypeptide of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, and the polynucleotides which encode them. As used herein, a "mature" form of a polypeptide or protein described herein relates to the product of a naturally occurring polypeptide or precursor form or proprotein or any precursor or proprotein which is or can be processed to mature form. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, and 23, and/or the complementary strand thereto.

The present invention also encompasses variants of the polypeptide sequence, and/or fragments thereof, disclosed in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 due to the degeneracy of the genetic code. These nucleic acids thus encode the same PrPase protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, e.g., the polypeptide of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.

"Variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limiting examples, the polynucleotide sequence of the coding region of the sequence in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, the complementary strand of said coding region, a polynucleotide sequence encoding the polypeptide identified as SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. The invention also encompasses polynucleotide fragments of any of the polynucleotide sequences provided herein.

The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, and/or polypeptide fragments of any of the polypeptides provided herein.

Preferably, the present invention is directed to an isolated or recombinant polynucleotide wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of a polynucleotide as set forth in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23; or a polynucleotide encoding the polypeptide as set forth in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.

The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a nucleotide-by-nucleotide or residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison (e.g., gaps can be introduced in either of the sequences being compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) or amino acid residue occurs in both sequences is determined to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. As used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity."

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or amino acid sequence of the present invention can be determined conventionally using known computer programs. Homology or identity determination using computer programs is well known in the art. The skilled artisan will further appreciate that results may vary depending on the parameters and computer program used.

A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the EMBOSS Pairwise Alignment Algorithms program (publicly available online at EMBL-EBI (European Bioinformatics Institute)). This program uses the Needleman-Wunsch global alignment algorithm to find the optimum alignment (including gaps) of two sequences when considering their entire length (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Preferred parameters used in an EMBOSS Pairwise Alignment of DNA or protein sequences to calculate percent identity are: Matrix=BLOSUM62, Gap Open Penalty=10.0, Gap Extension Penalty=0.1.

The CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992), can also be used. In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA or protein sequences to calculate percent identity are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0, Scoring Method=Percent, Window Size=5 or the length of the subject sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the CLUSTALW program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score is what may be used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, by a nucleic acid having a nucleotide sequence at least 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence of one of the sequences of the invention, the ORF (open reading frame), or any fragment specified as described herein.

As another example, by a polypeptide having an amino acid sequence at least, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

In another embodiment, derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which is incorporated herein by reference in its entirety).

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a PrPase polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Exemplary homologous nucleic acid sequences include the nucleic acid sequences of SEQ ID NO: 84, 86, 88, and 90. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions in sequences of the invention, as well as a polypeptide having PrPase activity, e.g. substrate binding.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter substantially the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

In addition to the Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays, or Glycine max PrPase nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of PrPase may exist within a population (e.g., of the plant). Such genetic polymorphism in the PrPase gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a PrPase protein, preferably a plant PrPase protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the PrPase gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in PrPase that are the result of natural allelic variation and that do not alter the functional activity of PrPase are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding PrPase proteins from other species, and thus that have a nucleotide sequence that differs from the sequences of the invention are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the PrPase polynucleotides of the invention can be isolated based on their homology to the Arabidopsis thaliana, Physcomitrella patens, Brassica napus, Zea mays, or Glycine max PrPase nucleic acids disclosed herein using the cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

In addition to naturally-occurring allelic variants of the PrPase sequence that may exist, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention, thereby leading to changes in the amino acid sequence of the encoded PrPase protein, without significantly altering the function of the PrPase protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of PrPase without substantially altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the PrPase proteins of the present invention are predicted to be less amenable to alteration.

In general, a PrPase-like variant that preserves PrPase-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as explained above.

Mutations can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Following mutagenesis of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. For example, a mutant PrPase protein can be assayed for (1) the ability to form protein:protein interactions with other PrPase proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant PrPase protein and a PrPase receptor; (3) the ability of a mutant PrPase protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind PrPase protein; or (5) the ability to specifically bind an anti-PrPase protein antibody.

Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants created through the application of molecular evolution ("DNA Shuffling") methodology to the polynucleotide disclosed as SEQ ID NO:X, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:Y. Such DNA Shuffling technology is known in the art (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)).

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.

In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence which is a portion of that shown in SEQ ID NO:X (wherein X is any polynucleotide of the invention) or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y (wherein Y is any polypeptide of the invention). The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 or more contiguous bases from the nucleotide sequence shown in SEQ ID NO:X. In this context "about" includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context "about" includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y. Protein (polypeptide) fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Alternatively, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, e.g., a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of PrPase. Fragments provided herein can also be defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention, which can include mature forms, as measured in a particular biological assay, with or without dose dependency. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention. A nucleic acid fragment encoding a "biologically active portion of PrPase" can be prepared by isolating a portion of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 that encodes a polypeptide having a PrPase biological activity, expressing the encoded portion of PrPase protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of PrPase. In another embodiment, a nucleic acid fragment encoding a biologically active portion of PrPase includes one or more regions.

In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan.

Biologically active portions of a PrPase protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the PrPase protein, e.g., the amino acid sequence shown in SEQ ID NO: 8 that include fewer amino acids than the full length PrPase proteins, and exhibit at least one activity of a PrPase protein, e.g. substrate binding. Typically, biologically active portions comprise a domain or motif with at least one activity of the PrPase protein. A biologically active portion of a PrPase protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.

A biologically active portion of a PrPase protein of the present invention may contain at least one of the above-identified domains conserved between the PrPase proteins. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native PrPase protein.

A biologically active portion or a PrPase protein can be the N-terminal domain of the PrPase polypeptide. Alternatively, a biologically active portion or a PrPase protein can be the C-terminal domain of the PrPase polypeptide. Preferably, the biologically active portion comprises at least 75 amino acids of the C-terminal domain. More preferably, the biologically active portion comprises at least 25 amino acids of the C-terminal domain. Most preferably, the biologically active portion comprises at least 10 amino acids of the C-terminal.

In an embodiment, the PrPase protein has an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24. In other embodiments, the PrPase protein is substantially homologous to SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24 and retains the functional activity of the protein of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. Accordingly, in another embodiment, the PrPase protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24 and retains the functional activity of the PrPase proteins of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24.

Exemplary homologous PrPase polypeptides include for example the polypeptide sequences of SEQ ID NO: 85, 87, 89, and 91.

Fusion Proteins

The invention also provides PrPase chimeric or fusion proteins, i.e. a PrPase polypeptide (all or portion) fused-in-frame to be linked to a non-PrPase polypeptide. The non-PrPase polypeptide can be fused to the N-terminus or C-terminus of the PrPase polypeptide. A PrPase chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. Any polypeptide of the present invention can be used to generate fusion proteins. Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. The polypeptides of the present invention can also be fused to marker sequences (also referred to as "tags").

Polypeptides of the present invention, including fragments and/or variants thereof, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann N Y Acad. Sci. 1999; 886:233-5), HC toxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248), BT endotoxin, or pseudomonas endotoxin. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.

Polypeptide Libraries

In a further embodiment, libraries of fragments of the PrPase protein coding sequence can be used to generate a variegated population of PrPase fragments for screening and subsequent selection of variants of a PrPase protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PrPase coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the PrPase protein.

Variants of the PrPase protein that function as either PrPase agonists (mimetics) or as PrPase antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the PrPase protein for PrPase protein agonist or antagonist activity. In one embodiment, a variegated library of PrPase variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of PrPase variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PrPase sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of PrPase sequences therein. There are a variety of methods which can be used to produce libraries of potential PrPase variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PrPase sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 11:477.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PrPase proteins and can be amenable to high throughput analysis.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The vectors of the invention can be recombinant expression vectors which comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

The polynucleotide insert of the present invention should be operatively linked to an appropriate promoter, such as the 35S promoter, the 34S promoter, CMV promoter, phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters, promoters of retroviral LTRs, constitutive promoters, and ABA inducible promoters to name a few. In addition, it may be desirable, or required, in some instances to direct expression of the nucleotide sequence only in certain host cells by having tissue-specific or cell type-specific promoters operably linked to a polynucleotide of the present invention. Examples of suitable plant-expressible promoters selectively expressed in particular tissues or cell types are well known in the art and include, but are not limited to, guard cell specific promoters, seed specific promoters (e.g., WO 89/03887), organ-primordia specific promoters (An et al., Plant Cell, 8:15-30, (1996)), stem-specific promoters (Keller et al., EMBO J., 7:3625-3633, (1988)), leaf specific promoters (Hudspeth et al., Plant. Mol. Biol., 12:579-589, (1989)), mesophyl-specific promoters (such as the light inducible Rubisco promoters), root-specific promoters (Keller et al., Genes Devel., 3:1639-1646, (1989)), tuber-specific promoters (Keil et al., EMBO J., 8:1323-1330, (1989)), vascular tissue specific promoters (Peleman et al., Gene, 84:359-369, (1989)), meristem specific promoters (such as the promoter of the SHOOTMERISTEMLESS (STM) gene, Long, et al., Nature, 379:66-69, (1996)), primodia specific promoter (such as the Antirrhinum CycD3a gene promoter, Doonan et al., in "Plant Cell Division" (Francis, Duditz, and Inze, Eds), Portland Press, London, (1998)), another specific promoters (WO 89/10396, WO 92/13956, and WO 92/13957), stigma-specific promoters (WO 91/02068), degiscence-zone specific promoters (WO 97/13865), seed-specific promoters (WO 89/03887), etc. Organ-specific promoters are also well known. For example, the patatin class I promoter is transcriptionally activated only in the potato tuber and can be used to target gene expression in the tuber (Bevan, M., 1986, Nucleic Acids Research 14:4625-4636). Another potato-specific promoter is the granule-bound starch synthase (GBSS) promoter (Visser, R. G. R, et al., 1991, Plant Molecular Biology 17:691-699). Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, P., 1986, Trans. R. Soc. London B314:343).

Further examples of suitable promoters include promoters from genes such as rice actin (McElroy, et al., Plant Cell, 163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol. Biol., 12: 619-632 (1992); and Christensen, et al., Plant Mol. Biol., 18: 675-689 (1992)); pEMU (Last, et al., Theor. Appl. Genet., 81: 581-588 (1991)); MAS (Velten, et al., EMBO J., 3: 2723-2730 (1984)); maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231: 276-285 (1992); and Atanassvoa, et al., Plant Journal, 2(3): 291-300 (1992)), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the rubisco promoter, the GRP1-8 promoter, ALS promoter, (WO 96/30530), a synthetic promoter, such as, Rsyn7, SCP and UCP promoters, ribulose-1,3-diphosphate carboxylase, fruit-specific promoters, heat shock promoters. Additional promoters that may be operably linked to a polynucleotide of the present invention may be found in McElroy and Brettel, Tibtech, Vol. 12, February, 1994. Moreover, a number of promoters are currently being used for transformation of dicotyledonous plants. These promoters come from a variety of different sources. One group of commonly used promoters were isolated from Agrobacterium tumefaciens, where they function to drive the expression of opine synthase genes carried on the T-DNA segment that is integrated into the plant genome during infection. These promoters include the octopine synthase (ocs) promoter (L. Comai et al., 1985; C. Waldron et al., 1985), the mannopine synthase (mas) promoter (L. Comai et al., 1985; K. E. McBride and K. R. Summerfelt, 1990) and the nopaline synthase (nos) promoter (M. W. Bevan et al., 1983; L. Herrera-Estrella et al., 1983, R. T. Fraley et al., 1983, M. De Block et al., 1984; R. Hain et al., 1985). These promoters are active in a wide variety of plant tissue.

In addition, the promoters disclosed in the following publications may also be operably linked to a polynucleotide of the present invention: U.S. Pat. Nos. 5,623,067; 5,683,439; 5,712,112; 5,723,751; 5,723,754; 5,723,757; 5,744,334; 5,750,385; 5,750,399; 5,767,363; 5,783,393; 5,789,214; 5,792,922; 5,792,933; 5,801,027; 5,804,694; 5,814,618; 5,824,857; 5,824,863; 5,824,865; 5,824,866; 5,824,872; and 5,929,302; and International Publication Nos. WO 96/30530, WO 97/49727, WO 98/00533, WO 98/03655, WO 98/07846, WO 98/08961, WO 98/08962, WO 98/10734, WO 98/16634, WO 98/22593, WO 98/38295, and WO 98/44097; and European Patent Application No. EP 0 846 770.

Several viral promoters are also used to, drive heterologous gene expression in dicots (J. C. Kridl and R. M. Goodman, 1986) and may be operably linked to a polynucleotide of the present invention. The Cauliflower Mosaic Virus 35S promoter is one of the promoters used most often for dicot transformation because it confers high levels of gene expression in almost all tissues (J. Odell et al., 1985; D. W. Ow et al., 1986; D. M. Shah et al., 1986). Modifications of this promoter are also used, including a configuration with two tandem 35S promoters (R. Kay et al., 1987) and the mas-35S promoter (L. Comai et al., 1990), which consists of the mannopine synthase promoter in tandem with the 35S promoter. Both of these promoters drive even higher levels of gene expression than a single copy of the 35S promoter. Other viral promoters that have been used include the Cauliflower Mosaic Virus 19S promoter (J. Paszkowski et al., 1984; E. Balazs et al.; Mogen, et al., 1990, Plant Cell, 2: 1261-1272) and the 34S promoter from the figwort mosaic virus (M. Sanger et al., 1990).

Alternatively, the polynucleotide insert of the present invention could be operatively linked to any of a number of inducible promoters known in the art, which include, but are not limited to: tetracycline inducible promoters, small-molecule inducible promoters, light inducible promoters, chemical compounds (e.g., safeners, herbicides, glucocorticoids, etc.), abiotic stress inducible promoters (e.g., wounding, heavy metals, cold-sensitive promoters, heat-sensitive promoters, salt sensitive promoters, drought sensitive promoters, hypoxia inducible (such as those disclosed in EP 1012317), etc.), biotic stress promoters (e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms, and mycoplasma-like organisms, etc.). Examples of plant-expressible inducible promoters suitable for the invention are: nematode inducible promoters (such as those disclosed in WO 92/21757 and/or EP1007709), fungus inducible promoters (WO 93/19188, WO 96/28561), chemically inducible Arabidopsis PR-1 promoter (WO 98/03536), the inducible promoters disclosed in WO 98/45445, the inducible promoters disclosed in U.S. Pat. No. 5,804,693, the tomato soft fruit inducible promoter disclosed in U.S. Pat. No. 5,821,398, promoters inducible after application of glucocorticoids such as dexamethasone, or promoters repressed or activated after application of tetracyclin (Gatz et al., PNAS USA, 85:1394-1397, (1988)). Other suitable inducible promoters will be known to the skilled artisan.

Exemplary expression vector constructs include for example the constructs of SEQ ID NO: 41, 42, 52, 53, 55, 56, 57, 58, 60, 61, 63, 64, 66, 67, and 69. Additional exemplary expression vector constructs include constructs comprising PrPase anti-sense nucleic acid such as SEQ ID NO: 51, 54, 59, 62, 65, and 68.

In addition, the polynucleotide insert of the present invention could be operatively linked to "artificial" or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in plants. Further, the trans-acting transcription factors of such "artificial" promoters could also be "artificial" or chimeric in design themselves and could act as activators or repressors to said "artificial" promoter. For example, a chimeric promoter of the invention could comprise one or more, upstream activating sequences from the Octopine Synthase gene (OCS), matrix attachment regions (MAR), etc.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors or plant transformation vectors, binary or otherwise, which serve equivalent functions.

The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

The expression constructs may additionally comprise 5' leader sequences in the expression constructs. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, 0., Fuerst, T. R., and Moss, B. (1989) PNAS USA, 86:6126-6130); polyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) (Virology, 154:9-20); and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., and Sarnow, P. (1991) Nature, 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., (1987) Nature, 325:622-625); tobacco mosaic virus leader (TW), (Gallie, D. R. et al. (1989) Molecular Biology of RNA, pages 237-256); and maize chlorotic mottle virus leader (MCNW) (Lommel, S. A. et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology., 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

Additional regulatory elements that may be connected to a PrPase encoding nucleic acid sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell. Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements PrPase gene are known, and include, but are not limited to, 3' termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan, et al., Nucl. Acids Res., 12: 369-385 (1983)); the potato proteinase inhibitor II (PINII) gene (Keil, et al., Nucl. Acids Res., 14: 5641-5650 (1986) and hereby incorporated by reference); and An, et al., Plant Cell, 1: 115-122 (1989)); and the CaMV 19S gene (Mogen, et al., Plant Cell, 2: 1261-1272 (1990)).

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include, but are not limited to, dihydrofolate reductase, G418 or neomycin resistance, kanamycin resistance, hygromycin resistance, bialaphos resistance, sulfonoamide resistance, stretomycin resistance, spectinomycin resistance, chlorosulfuron resistance, glyphosphate resistance, and methotrexate resistance, for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Markers may also include resistance to bleomycin and gentamicin. For Example, after transforming plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic/marker. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis, and Salmonella typhimurium cells (or any bacterial strain capable of expressing heterologous polypeptides); fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris (ATCC Accession No. 201178), Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; plant cells, and specifically plant cells and/or tissues derived from any of the plants listed in Table 3. Plant host cells include, for example, plant cells that could function as suitable hosts for the expression of a polynucleotide of the invention include epidermal cells, mesophyll and other ground tissues, and vascular tissues in leaves, stems, floral organs, and roots. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

The polynucleotides and polypeptides of the present invention can be targeted to the chloroplast or amyloplast for expression. In this manner, the expression construct will additionally contain a polynucleotide sequence encoding a transit peptide operably linked to a polynucleotide of the present invention to direct the polynucleotide of the present invention to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

The expression construct may also comprise any other necessary regulators such as nuclear localization signals (Kalderon et al. (1984) Cell 39:499-509; and Lassner et al. (1991) Plant Molecular Biology 17:229-234); plant translational consensus sequences (Joshi, C. P. (1987) Nucleic Acids Research 15:6643 6653), introns (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81-93) and the like, operably linked to a polynucleotide of the present invention.

Plant signal sequences, including, but not limited to, signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell (Dratewka-Kos, et al., J. Biol. Chem., 264: 4896-4900 (1989)) and the Nicotiana plumbaginifolia extension gene (DeLoose, et al., Gene, 99: 95-100 (1991)), or signal peptides which target proteins to the vacuole like the sweet potato sporamin gene (Matsuka, et al., Proc. Nat'l Acad. Sci. (USA), 88: 834 (1991)) and the barley lectin gene (Wilkins, et al., Plant Cell, 2: 301-313 (1990)), or signals which cause proteins to be secreted such as that of PRIb (Lind, et al., Plant Mol. Biol., 18: 47-53 (1992)), or those which target proteins to the plastids such as that of rapeseed enoyl-ACP reductase (Verwaert, et al., Plant Mol. Biol., 26: 189-202 (1994)) are useful in the invention.

It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., PrPase proteins, mutant forms of PrPase proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of PrPase proteins in prokaryotic or eukaryotic cells. For example, PrPase proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells, plant cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

The polynucleotide sequences encoding the proteins or polypeptides of the present invention may be particularly useful in the genetic manipulation of plants. In this manner, the polynucleotides of the invention are provided in expression cassettes for expression in the plant of interest. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant preferred codons for improved expression specific to a particular species. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Depending upon the species in which the DNA sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research. In this manner, the polynucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

Additionally, it may be desirable to selectively express a polypeptide of the present invention in a specific target cell or tissue of a plant by synthesizing the encoding polynucleotide sequence to contain codons optimized for high translational efficiency within the particular target cell or tissue. Such methods are known in the art and are specifically provided in PCT International Publication No. WO 00/42190 (which is hereby incorporated herein by reference).

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures.

For in situ production of the antisense mRNA of GST, those regions of the glutathione S-transferase ("GST") gene which are transcribed into GST mRNA, including the untranslated regions thereof, are inserted into the expression vector under control of the promoter system in a reverse orientation. The resulting transcribed mRNA is then complementary to that normally produced by the plant.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.).

In another embodiment, the PrPase expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, PrPase can be expressed in insect cells using baculovirus expression vectors.

In a preferred embodiment, a nucleic acid of the invention is expressed in plants cells using a plant expression vector. Examples of plant expression vectors systems include tumor inducing (Ti) plasmid or portion thereof found in Agrobacterium, cauliflower mosaic virus (CAMV) DNA and vectors such as pBI121. Preferred expression vectors in plant systems include, but are not limited to, Bin 19 (ATCC Deposit No: 37327), GA437 (ATCC Deposit No: 37350), pAK1003 (ATCC Deposit No: 37425), pAS2022 (ATCC Deposit No: 37426), pAS2023 (ATCC Deposit No: 37427), pAP2034 (ATCC Deposit No: 37428), pC22 (ATCC Deposit No: 37493), pHS24 (ATCC Deposit No: 37841), pHS85 (ATCC Deposit No: 37842), pPM1 (ATCC Deposit No: 40172), pGV3111SE (ATCC Deposit No: 53213), pCGN978 (ATCC Deposit No: 67064), pFL61 (ATCC Deposit No: 77215), pGPTV-KAN (ATCC Deposit No: 77388), pGPTV-HPT (ATCC Deposit No: 77389), pGPTV-DHFR (ATCC Deposit No: 77390), pGPTV-BAR (ATCC Deposit No: 77391), pGPTV-BLEO (ATCC Deposit No: 77392), and/or pPE1000 (ATCC Deposit No: 87573). The skilled artisan would appreciate that any of the above vectors could easily be modified to either include or delete specific elements as may be required for operability. Other suitable vectors will be readily apparent to the skilled artisan.

For expression in plants, the recombinant expression cassette will contain in addition to the PrPase nucleic acids, a plant promoter region, a transcription initiation site (if the coding sequence to transcribed lacks one), and a transcription termination/polyadenylation sequence. The termination/polyadenylation region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.

Introduction of the construct into the host cell can be effected by biolistic transformation (Klein et al., Nature, 327:70-73 (1987)), PEG-mediated transfection (Paskowski, et al., EMBO J., 3:2717, (1984)), calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation (Fromm, et al., PNAS, USA, 82:5824 (1985)), transduction, infection, Agrobacterium tumefaciens-directed infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods.

Alternatively, a polypeptide or protein may also be expressed in a form which will facilitate purification. Polypeptides of the present invention, can also be recovered from: products purified from natural sources, including tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, and higher plant cells.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, I497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8.degree.-20.degree. C., vs. 4.degree.-16.degree. C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

In addition, polypeptides, analogs, derivatives, and/or fragments of the invention can be chemically synthesized.

Each of the polynucleotides identified herein can be used in numerous ways as reagents, including for chromosome identification and mapping, as a diagnostic or prognostic marker, for control of gene expression through triple helix formation or antisense DNA or RNA, for producing a new trait in the host cell, for identifying organisms from minute biological samples, as polymorphic markers, for identifying the source of a particular tissue.

In preferred embodiments, the polynucleotides are used for modulating, inhibiting, increasing, decreasing, or introducing traits in a plant including one or more of drought tolerance, UV tolerance, flower development, terpene synthesis, abiotic stressed tolerance, heat stress tolerance, cold stress tolerance, nutritional stress tolerance, xenobiotic stress tolerance, protein storage capability, oil storage capability, amino acid content, amino acid composition, carbohydrate storage capability, oil content, oil composition, carbohydrate content, carbohydrate composition, fiber content, fiber composition, metabolite content, metaboliter composition, vitamin content, and/or vitamin composition. The polynucleotides of the invention, are useful in modulating plant yield, plant development, plant differentiation, root growth, root morphology, plant color, plant aroma, plant flavor, palatability of plant tissue, plant organoleptic properties, may be useful in phytoremediation, and/or plant defense. Moreover, the polypeptides of the invention may also be useful in modulating the plants ability to serve as a plant neutriceutical, pharmaceutical, or phytoceutical. Alternatively, polypeptides of the invention may also be useful in modulating the plants ability to produce plant neutriceuticals, pharmaceuticals, or phytoceuticals of either endogenous or exogenous origin (e.g., from another plant species, a human, a mammal, an animal, or other organism). In these contexts, the term "plant" may be applied to mean any plant cell, plant tissue, plant fluid, or plant feature, and includes plant infection structures, which may include, but are not limited to an appressorium, a gall, a canker, and/or nodules. In these contexts, the term "modulate" may be applied to mean the qualitative or quantitative increase, decrease, introduction of, inhibition of, complete loss of, or over-expression of a specific trait or characteristic.

Each of the polypeptides identified herein can be used in numerous ways appreciated by those skilled in the art, including assaying protein levels in a biological sample using antibody-based techniques and in vivo imaging.

The PrPase proteins can be used to screen compounds that modulate the PrPase protein activity or expression. In addition, anti-PrPase antibodies can be made and used to detect and isolate PrPase proteins and modulate PrPase activity.

Another aspect of the present invention relates to methods for the introduction of nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an organism, preferably a plant, to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention operatively linked to a promoter and other genetic elements necessary for the expression of the polypeptide by the organism or a target tissue. Such transgenic and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

The polynucleotide constructs can be delivered by any method that delivers materials to the cells of an organism, such as, biolistic injection into the plant tissues (apical meristem, root, flower, stem, and the like). The polynucleotide constructs may be delivered in an acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs of the invention may integrate into the host genome and may replicate. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include the 35S, 34S, and actin promoters, in addition to any other promoter known in the art and/or described elsewhere herein. The promoter also may be the native promoter for the polynucleotides of the invention.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct injection, direct needle injection at the delivery site, topical administration, and so-called "gene guns". These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA that comprises a sequence encoding polypeptides of the invention. Retroviral integration has been detected to occur in plants based upon the identification of a pararetrovirus sequences within the tobacco genome. Since such integration was determined to occur at very limited integration sites, such a pararetrovirus may represent a desirable genetic transformation vehicle for the polynucleotides of the present invention (Jakowitsch, J., et al., PNAS 96(23):13241-6 (1999).

The present invention also encompasses the application of retrotransposons to the genetic transformation of plants. The retrotransposons would preferably represent retrotransposons with known plant host range and would comprise polynucleotides encoding polypeptides of the present invention. Many retrotransposons are known in the art, some of which are described by Bennetzen J L, Trends Microbiol., 4(9):347-53 (1996) which is hereby incorporated herein by reference.

Polynucleotide constructs are made using standard techniques known in the art.

Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transformed. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Plant Transformation

The invention includes protoplast, plants cells, plant tissue and plants (e.g., monocots and dicots) transformed with a PrPase nucleic acid, a vector containing a PrPase nucleic acid or an expression vector containing a PrPase nucleic acid (i.e., sense or antisense). As used herein, "plant" is meant to include not only a whole plant but also a portion thereof (i.e., cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds and the progeny derived therefrom).

The plant can be any plant type including, for example, species from the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Gossypium, Picea, Caco, and Populus.

Preferred plants, may include the following non-limiting examples, including barley, oats, rye, sorghum, pea, sunflower, tobacco, cotton, petunia, tomato, broccoli, lettuce, apple, plum, orange, and lemon, and more preferrably rice, maize, conola, wheat, sugarbeet, sugarcane, and soybean, in addition to other plants known in the art and referenced more particularly elsewhere herein (e.g., Table 3).

Non-limiting examples of suitable recipient plants for introducing polynucleotides of the invention, polynucleotides encoding the polypeptides of the invention, and/or fragments, and variants therein, are listed in Table 3 below:

TABLE-US-00003 TABLE 3 RECIPIENT PLANTS COMMON NAME FAMILY LATIN NAME Maize Gramineae Zea mays Maize, Dent Gramineae Zea mays dentiformis Maize, Flint Gramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microsperma Maize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea mays amyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, Waxy Gramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum spelta Wheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticum turgidum Wheat, Large Spelt Pooideae Triticum spelta Wheat, Polish Pooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidum Wheat, Singlegrained Pooideae Triticum monococcum Wheat, Small Spelt Pooideae Triticum monococcum Wheat, Soft Pooideae Triticum aestivum Rice Gramineae Oryza sativa Rice, American Wild Gramineae Zizania aquatica Rice, Australian Gramineae Oryza australiensis Rice, Indian Gramineae Zizania aquatica Rice, Red Gramineae Oryza glaberrima Rice, Tuscarora Gramineae Zizania aquatica Rice, West African Gramineae Oryza glaberrima Barley Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeum irregulare Intermediate, also Irregular Barley, Ancestral Pooideae Hordeum spontaneum Tworow Barley. Beardless Pooideae Hordeum trifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley, fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed Pooideae Hordeum vulgare hexastichon Barley, Tworowed Pooideae Hordeum distichon Cotton, Abroma Dicotyledoneae Abroma augusta Cotton, American Malvaceae Gossypium hirsutum Upland Cotton, Asiatic Tree, also Malvaceae Gossypium arboreum Indian Tree Cotton, Brazilian, also, Malvaceae Gossypium barbadense Kidney, and, brasiliense Pernambuco Cotton, Levant Malvaceae Gossypium herbaceum Cotton, Long Silk, also Malvaceae Gossypium barbadense Long Staple, Sea Island Cotton, Mexican, also Malvaceae Gossypium hirsutum Short Staple Soybean, Soya Leguminosae Glycine max Sugar beet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plant Arenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, Cherry Solanaceae Lycopersicon esculentum cerasiforme Tomato, Common Solanaceae Lycopersicon esculentum commune Tomato, Currant Solanaceae Lycopersicon pimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato, Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersicon esculentum pyriforme Tomato, Tree Solanaceae Cyphomandra betacea Potato Solanaceae Solanum tuberosum Potato, Spanish, Sweet Convolvulaceae Ipomoea batatas potato Rye, Common Pooideae Secale cereale Rye, Mountain Pooideae Secale montanum Pepper, Bell Solanaceae Capsicum annuum grossum Pepper, Bird, also Solanaceae Capsicum annuum Cayenne, Guinea minimum Pepper, Bonnet Solanaceae Capsicum sinense Pepper, Bullnose, also Solanaceae Capsicum annuum grossum Sweet Pepper, Cherry Solanaceae Capsicum annuum cerasiforme Pepper, Cluster, also Red Solanaceae Capsicum annuum Cluster fasciculatum Pepper, Cone Solanaceae Capsicum annuum conoides Pepper, Goat, also Spur Solanaceae Capsicum frutescens Pepper, Long Solanaceae Capsicum frutescens longum Pepper, Oranamental Solanaceae Capsicum annuum Red, also Wrinkled abbreviatum Pepper, Tabasco Red Solanaceae Capsicum annuum conoides Lettuce, Garden Compositae Lactuca sativa Lettuce, Asparagus, also Compositae Lactuca sativa asparagina Celery Lettuce, Blue Compositae Lactuca perennis Lettuce, Blue, also Compositae Lactuca pulchella Chicory Lettuce, Cabbage, also Compositae Lactuca sativa capitata Head Lettuce, Cos, also Compositae Lactuca sativa longifolia Longleaf, Romaine Lettuce, Crinkle, also Compositae Lactuca sativa crispa Curled, Cutting, Leaf Celery Umbelliferae Apium graveolens dulce Celery, Blanching, also Umbelliferae Apium graveolens dulce Garden Celery, Root, also Umbelliferae Apium graveolens Turniprooted rapaceum Eggplant, Garden Solanaceae Solanum melongena Sorghum Sorghum All crop species Alfalfa Leguminosae Medicago sativum Carrot Umbelliferae Daucus carota sativa Bean, Climbing Leguminosae Phaseolus vulgaris vulgaris Bean, Sprouts Leguminosae Phaseolus aureus Bean, Brazilian Broad Leguminosae Canavalia ensiformis Bean, Broad Leguminosae Vicia faba Bean, Common, also Leguminosae Phaseolus vulgaris French, White, Kidney Bean, Egyptian Leguminosae Dolichos lablab Bean, Long, also Leguminosae Vigna sesquipedalis Yardlong Bean, Winged Leguminosae Psophocarpus tetragonolobus Oat, also Common, Side, Avena Avena sativa Tree Oat, Black, also Bristle, Avena Avena strigosa Lopsided Oat, Bristle Avena Pea, also Garden, Green, Leguminosae Pisum, sativum sativum Shelling Pea, Blackeyed Leguminosae Vigna sinensis Pea, Edible Podded Leguminosae Pisum sativum axiphium Pea, Grey Leguminosae Pisum sativum speciosum Pea, Winged Leguminosae Tetragonolobus purpureus Pea, Wrinkled Leguminosae Pisum sativum medullare Sunflower Compositae Helianthus annuus Squash, Autumn, Winter Dicotyledoneae Cucurbita maxima Squash, Bush, also Dicotyledoneae Cucurbita pepo melopepo Summer Squash, Turban Dicotyledoneae Cucurbita maxima turbaniformis Cucumber Dicotyledoneae Cucumis sativus Cucumber, African, also Momordica charantia Bitter Cucumber, Squirting, Ecballium elaterium also Wild Cucumber, Wild Cucumis anguria Poplar, California Woody-Plant Populus trichocarpa Poplar, European Black Populus nigra Poplar, Gray Populus canescens Poplar, Lombardy Populus italica Poplar, Silverleaf, also Populus alba White Poplar, Western Balsam Populus trichocarpa Tobacco Solanaceae Nicotiana Arabidopsis thaliana Cruciferae Arabidopsis thaliana Turfgrass Lolium Turfgrass Agrostis Other families of turfgrass Clover Leguminosae

Numerous methods for introducing foreign genes into plants are known and can be used to insert a gene into a plant host, including biological and physical plant transformation protocols. See, for example, Miki et al., (1993) "Procedure for Introducing Foreign DNA into Plants", In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88 and Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, polyethylene glycol (PEG) transformation, microorganism-mediated gene transfer such as Agrobacterium (Horsch, et al., Science, 227: 1229-31 (1985)), electroporation, protoplast transformation, micro-injection, flower dipping and biolistic bombardment.

Also included in the invention are methods of producing a transgenic plant. The method includes introducing into one or more plant cells a compound that alters PrPase expression or activity in the plant to generate a transgenic plant cell and regenerating a transgenic plant from the transgenic cell. In some aspects the compound increases alters PrPase expression or activity. Alternatively, the compound decrease alters PrPase expression or activity. The compound can be, e.g., (i) a PrPase polypeptide; (ii) a nucleic acid encoding a PrPase polypeptide; (iii) a nucleic acid that increases expression of a nucleic acid that encodes a PrPase polypeptide; (iv) a nucleic acid that decreases the expression of a nucleic acid that encodes a PrPase polypeptide; (v) a PrPase antisense nucleic acid and derivatives, fragments, analogs and homologs thereof. A nucleic acid that increases expression of a nucleic acid that encodes a PrPase polypeptide includes, e.g., promoters, enhancers. The nucleic acid can be either endogenous or exogenous. Preferably, the "compound" is a nucleic acid encoding a PrPase polypeptide of the invention. For example, the compound comprises the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or a fragment thereof. Alternatively, the compound is a PrPase antisense nucleic acid. For example, the compound comprises the nucleic acid sequence of SEQ ID NO: 9, 13, or 20.

In various aspects the transgenic plant has an altered phenotype as compared to a wild type plant (i.e., untransformed). By altered phenotype is meant that the plant has a one or more characteristic that is different from the wild type plant. For example, the transgenic plant has an increased resistance to stress. An "increased" stress means that the transgenic plant can grow under stress conditions (e.g., high salt, decreased water, low temperatures, high temperatures) or under conditions that normally inhibit the growth of an untransformed plant. Stresses include, for example, chilling stress, heat stress, heat shock, salt stress, water stress (i.e., drought), nutritional stress, disease, grazing pests, wound healing, pathogens such as for example fungi, bacteria, nematodes, viruses or parasitic weed and herbicides. Alternatively, the transformed plant has an increased (i.e., enhanced) ABA sensitivity. The enhanced ABA sensitivity is at the seedling growth stage. Alternatively, the enhanced ABA sensitivity is at the mature plant stage. Additional altered phenotypes include for example, enhanced vegetative growth (e.g., increased leaf number, thickness and overall biomass), delayed reproductive growth (e.g., flowering later); enhanced seedling vigor (e.g., increased root biomass and length), enhanced lateral root formation and therefore soil penetration more extensive vascular system resulting in an enhanced transport system. In preferred embodiments, the growth and/or yield of transgenic plants is improved relative to a wild-type under a stress condition.

The polynucleotides of the invention are introduced into plant cells using, for example, Agrobacterium-mediated transformation, microprojectile-mediated transformation, sonication of target cells, liposome or spheroplast fusion, direct uptake into protoplasts by CaCl.sub.2 precipitation, electroporation of protoplasts and whole cells and tissues, wounding by particle bombardment followed by use of Agrobacterium for DNA delivery, intact meristem transformation, split meristem method.

Once a single transformed plant has been obtained, conventional plant breeding methods can be used to transfer the gene and associated regulatory sequences via crossing and backcrossing.

Methodologies to determine plant growth or response to stress include for example, height measurements, weight measurements, leaf area, ability to flower, water use, transpiration rates and yield.

Infectious Agents and Pests

A polynucleotide or polypeptide of the present invention can be used to detect, prevent, and/or confer resistance to infectious agents and/or pests which may, for example, inhibit the plants ability to control cellular division, differentiation, and development; absorption of water and minerals from the soil and the translocation of these substances throughout the plant; photosynthesis and translocation of the photosynthetic products to areas of use or storage; metabolism of synthesized compounds; reproduction; and storage of plant food-stuffs for overwintering or reproduction. For example, infection of the root (e.g., root rot), interferes with absorption of water and nutrients from the soil; infection of the xylem vessels (e.g., vascular wilts, cankers, etc.) interferes with translocation of water and minerals to the crown of the plant; infection of the foliage (e.g., leaf spots, blights, mosaics, etc.) interferes with photosynthesis; infection of the cortex (e.g., cortical canker, viral and mycoplasmal infections of phloem, etc.) interferes with the downward translocation of photosynthetic products; flower infections (e.g., bacterial and fungal blights, viral, mycoplasmal, and fungal infections of flowers, etc.) interfere with reproduction; and infections of fruit (e.g., fruit rot, etc.) interfere with reproduction or storage of reserve food stuffs for the new plant.

Infectious agents and pests include, but are not limited to, viruses, bacteria, fungi, parasitic agents (i.e. parasitic weeds), herbaceous species, nematodes, and insects.

Defense Mechanisms and Plant Hormones

A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to increase a plants defense mechanisms against either environmental or pathogenic stresses (e.g., viral, fungal, mycoplasma, bacterial, nematode, herbicidal, insecticidal, acid rain, drought, chemical, etc.). Such defense mechanisms may be a combination of structural characteristics (i.e., to serve as a physical barrier to inhibit a pathogen, for example, from entering or spreading throughout the plant), and biochemical reactions either on the scale of the whole plant or of individual cells (e.g., producing substances that are either toxic to the pathogen, or create an environment that is non-permissive for pathogen survival, etc.).

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention can be used to modulate the hormone levels within a plant (including any of its cells, tissues, and/or organs, etc.). Examples of hormones that may be modulated by the present invention, either directly or indirectly, generally include, but are not limited to, auxins, indoleacetic acid, gibberellins, cytokinins, ethylene, abscisic acid, polyamines, jasmonates, salicylic acid, and brassinolides (see, for example, Davies, P. J., in "Plant Hormones: Physiology, Biochemistry, and Molecular Biology", Kluwer Academic Publishers, Boston, 1995; which is hereby incorporated by reference in its entirety herein).

Regeneration

The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988, Plant Molecular Biology, Methods in Enzymology, Vol. 118, Academic Press, Orlando, Fla.). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing a foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983). In particular, U.S. Pat. No. 5,349,124 (specification incorporated herein by reference) details the creation of genetically transformed lettuce cells and plants resulting therefrom which express hybrid crystal proteins conferring insecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, or pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

A preferred transgenic plant is an independent segregant and can transmit the PrPase gene and its activity to its progeny. A more preferred transgenic plant is homozygous for the gene, and transmits that gene to all of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for increased expression of the PrPase transgene.

A polynucleotide or polypeptide of the present invention can be used to modulate a plants nutritional status.

A polynucleotide or polypeptide of the present invention may increase the plants ability, either directly or indirectly, to initiate and/or maintain biotic associations with other organisms. Such associations may be symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature.

In another embodiment, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase the plants ability to synthesize and/or release a pheromone.

A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity.

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Antisense, Ribozymes, PNA Moieties

In further embodiments, the invention encompasses antagonists which correspond to the polynucleotide sequences of the invention, and in particular, the complementary strand of the PrPase polynucleotides.

Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire PrPase coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a PrPase protein of SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24, or antisense nucleic acids complementary to a PrPase nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 11, 14, 16, 18, 21, or 23 are additionally provided. Exemplary PrPase anti-sense nucleic acid include the nucleic acid sequences of SEQ ID NO: 9, 13, and 20.

The antisense nucleic acids may be transiently generated within the organism (e.g., sequence contained within an inducible or constitutively expressed vector introduced into the cells of an organism), stably generated within the organism (e.g., sequence contained within an inducible or constitutively expressed vector introduced into the cells of an organism using transgenic methods, including viral integration, etc.) or may be exogenously administered. For a nucleic acid to serve an antisense role, it is only necessary that it has sequence homology to the sense RNA product of the gene of interest. A number of methods of administering antisense nucleic acids, their compositions, and designs are known in the art and encompassed by the invention (see for example, Agrawal S, et al., Mol Med. Today. 2000 February; 6(2):72-81; Yacyshyn B R, et al, Can J Gastroenterol. 1999 November; 13(9):745-51; Mrsny R J., J Drug Target. 1999; 7(1):1-10; Toulme J J, et al, Nucleic Acids Symp Ser. 1997; (36):39-41), Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988); and Cooper S R, et al., Pharmacol Ther. 1999 May-June; 82(2-3):427-35). Likewise, a number of methods have been developed regarding the application of triple helix antisense technology to modulating gene expression (see, for example, Gowers D M, et al, Nucleic Acids Res. 1999 Apr. 1; 27(7):1569-77; and Chan P P, et al., J Mol. Med. 1997 April; 75(4):267-82).

Antisense technology has wide-ranging applications in plants. For example, antisense RNA has been shown to effectively downregulate a variety of plant genes as described by Shimada, et al., Theor. Appl. Genet., 86:665-672, (1993); Kull, et al., J. Genet. Breed., 49:67-76, (1995)., Slabas and Elborough, WO 97/07222; Knutzon et al., Proc. Natl. Acad. Sci. USA, 89:2624-2628, (1992), and Baulcombe D C., Plant Mol. Biol. 1996 October; 32(1-2):79-88).

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Antisense oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5'- or 3'-non-translated, non-coding regions of a polynucleotide sequence of the invention could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.

Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of Physcomitrella patens, Arabidopsis thaliana, Brassica napus, Zea mays, or Glycine max PrPase corresponds to SEQ ID NO: 2, 4, 6, 8, 12, 15, 17, 19, 22, or 24). The antisense nucleic acid molecule can be complementary to the entire coding region of PrPase mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of PrPase mRNA. Whether designed to hybridize to the 5'-, 3'- or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. In another specific aspect, an antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

The antisense oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810, published Dec. 15, I988) or blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).

The oligonucleotide can also be a peptido-nucleic acid ("PNA") based on a linked N-(2-aminoethyl)glycine backbone to which normal DNA bases have been attached (Egholm et al., 1993, Nature 365:566-67). This PNA obeys specific Watson-Crick base pairing, but with greater free energy of binding and correspondingly higher melting temperatures. Suitable oligomers may be constructed entirely from PNAs or from mixed PNA and DNA and/or RNA oligomers. In fact, PNA:DNA chimeras have increased solubility characteristics, as compared to DNA:DNA or DNA:RNA chimeras of the same sequence. Most notably, PNAs have the unique ability to displace one strand of a DNA double-helix thus making them highly suitable in antisense applications (Uhlmann E., Biol. Chem. 1998 August-September; 379(8-9):1045-52).

In various embodiments, the nucleic acids of PrPase can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" can also refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.

PNAs of PrPase can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of PrPase can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).

In another embodiment, PNAs of PrPase can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of PrPase can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-11124.

In a further embodiment, the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In another embodiment, the anti-sense oligonucleotide of the invention may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

In another embodiment, the anti-sense oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the anti-sense oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The anti-sense oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligos may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.

An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.

Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PrPase protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

In a specific embodiment, the oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225; Hasselhoff, et al., Nature 342:76-79 (1988)). Ribozymes have been used to downregulate gene expression, and more recently in the downregulation of plant proteins (seem e.g., PCT International Publication WO 97/10328).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave PrPase mRNA transcripts to thereby inhibit translation of PrPase mRNA. A ribozyme having specificity for a PrPase-encoding nucleic acid can be designed based upon the nucleotide sequence of a PrPase DNA disclosed herein (i.e., SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 18). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PrPase-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, PrPase mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, PrPase gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the PrPase (e.g., the PrPase promoter and/or enhancers) to form triple helical structures that prevent transcription of the PrPase gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an .alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .beta.-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). In another embodiment, the oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in applications.

Double Stranded RNA Inhibition (RNAi) by Hairpin Nucleic Acids

Another aspect of the invention pertains to the use of post transcriptional gene silencing (PTGS) to repress gene expression, including use of the sequences disclosed herein to produce small interfering RNAs. Double stranded RNA can initiate the sequence specific repression of gene expression in plants and animals. Double stranded RNA is processed to short duplex oligomers of 21-23 nucleotides in length. These small interfering RNAs suppress the expression of endogenous and heterologous genes in a sequence specific manner (Fire et al. Nature 391:806-811, Carthew, Curr. Opin. in Cell Biol., 13:244-248, Elbashir et al., Nature 411:494-498). An RNAi suppressing construct can be designed in a number of ways, for example, transcription of a inverted repeat which can form a long hair pin molecule, inverted repeats separated by a spacer sequence that could be an unrelated sequence such as GUS or an intron sequence. Transcription of sense and antisense strands by opposing promoters or cotranscription of sense and antisense genes.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXAMPLES

Example 1

Growth of Physcomitrella patens Cultures

For this study, plants of the species Physcomitrella patens (Hedw.) B.S.G. from the collection of the genetic studies section of the University of Hamburg were used. They originated from the strain 16/14 collected by H.L.K. Whitehouse in Gransden Wood, Huntingdonshire (England), which was subcultured from a spore by Engel (1968, Am J Bot 55, 438-446). Proliferation of the plants was carried out by means of spores and by means of regeneration of the gametophytes. The protonema developed from the haploid spore as a chloroplast-rich chloronema and chloroplast-low caulonema, on which buds formed after approximately 12 days. These grew to give gametophores bearing antheridia and archegonia. After fertilization, the diploid sporophyte with a short seta and the spore capsule resulted, in which the meiospores mature.

Culturing was carried out in a climatic chamber at an air temperature of 25.degree. C. and light intensity of 55 micromol s.sup.-1 m.sup.-2 (white light; Philips TL 65W/25 fluorescent tube) and a light/dark change of 16/8 hours. The moss was either modified in liquid culture using Knop medium according to Reski and Abel (1985, Planta 165, 354-358) or cultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke, England).

The protonemas used for RNA and DNA isolation were cultured in aerated liquid cultures. The protonemas were comminuted every 9 days and transferred to fresh culture medium.

Example 2

Isolation of Total RNA and Poly-(A)+ RNA and cDNA Library Construction from Physcomitrella patens

For the investigation of transcripts, both total RNA and poly-(A)+ RNA were isolated. The total RNA was obtained from wild-type 9 d old protonemata following the GTC-method (Reski et al. 1994, Mol. Gen. Genet., 244:352-359).

The Poly(A)+ RNA was isolated using Dyna Beads.sup.R (Dynal, Oslo, Norway) following the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70 degree C.

RNA Preparation from Arabidiopsis Seeds--"Hot" Extraction:

1. Buffers, Enzymes and Solutions

2M KCl Proteinase K Phenol (for RNA) Chloroform:Isoamylalcohol (Phenol:choloroform 1:1; pH adjusted for RNA) 4 M LiCl, DEPC-treated DEPC-treated water 3M NaOAc, pH 5, DEPC-treated Isopropanol 70% ethanol (made up with DEPC-treated water) Resuspension buffer: 0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC-treated water as this solution can not be DEPC-treated Extraction Buffer: 0.2M Na Borate 30 mM EDTA 30 mM EGTA 1% SDS*(250 .mu.l of 10% SDS-solution for 2.5 ml buffer) 1% Deoxycholate (25 mg for 2.5 ml buffer) 2% PVPP (unsoluable--50 mg for 2.5 ml buffer) 2% PVP 40K (50 mg for 2.5 ml buffer) 10 mM DTT* 100 mM-Mercaptoethanol*(fresh, handle under fume hood--use 35 .mu.l of 14.3M solution for 5 ml buffer) 2. Extraction

Heat extraction buffer up to 80.degree. C. Grind tissue in liquid nitrogen-cooled mortar, transfer tissue powder to 1.5 ml tube. Tissue should kept frozen until buffer is added so transfer the sample with precooled spatula and keep the tube in liquid nitrogen all time. Add 350 .mu.l preheated extraction buffer (here for 100 mg tissue. Buffer volume can be as much as 500 .mu.l for bigger samples) to tube, vortex and heat tube to 80.degree. C. for .about.1 min. Keep then on ice. Vortex sample, grind additionally with electric mortar.

3. Digestion

Add Proteinase K (0.15 mg/100 mg tissue), vortex and keep at 37.degree. C. for one hour.

4. First Purification

Add 27 .mu.l 2M KCl. Chill on ice for 10 min. Centrifuge at 12.000 rpm for 10 minutes at room temperature. Transfer supernatant to fresh, RNAase-free tube and do one phenol extraction, followed by a choloroform:isoamylalcohol extraction. Add 1 vol. isopropanol to supernatant and chill on ice for 10 min. Pellet RNA by centrifugation (7000 rpm for 10 min at RT). Resolve pellet in 1 ml 4M LiCl by 10 to 15 min vortexing. Pellet RNA by 5 min centrifugation.

5. Second Purification

Resuspend pellet in 500 .mu.l Resuspension buffer. Add 500 .mu.l phenol and vortex. Add 250 .mu.l chloroform:isoamylalcohol and vortex. Spin for 5 min. and transfer supernatant to fresh tube. Repeat choloroform:isoamylalcohol extraction until interface is clear. Transfer supernatant to fresh tube and add 1/10 vol 3M NaOAc, pH 5 and 600 .mu.l isopropanol. Keep at -20 for 20 min or longer. Pellet RNA by 10 min centrifugation. Wash pellet once with 70% ethanol. Remove all remaining alcohol before resolving pellet with 15 to 20 .mu.l DEPC-water. Determine quantity and quality by measuring the absorbance of a 1:200 dilution at 260 and 280 nm. 40 .mu.g RNA/ml=1OD260

For cDNA library construction first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12 degree C. (2 h), 16 degree C. (1 h) and 22 degree C. (1 h). The reaction was stopped by incubation at 65 degree C. (10 min) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37 degree C. (30 min). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12 degree C., overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37 degree C., 30 min). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 basepairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.

Example 3

Sequencing and Function Annotation of Physcomitrella patens ESTs

cDNA libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). Random Sequencing was carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands. Plasmid DNA was prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing ampicillin (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) on a Qiagene DNA preparation robot (Qiagen, Hilden) according to the manufacturers protocols. Sequencing primers with the following nucleotide sequences were used:

TABLE-US-00004 Qiagen1: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 32) Qiagen2: 5'-CTAAAGGGAACAAAAGCTG-3' (SEQ ID NO: 33) Qiagen3: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 34)

Sequences were processed and annotated using the software package EST-MAX commercially provided by Bio-Max (Munich, Germany). The program incorporates practically all bioinformatics methods important for functional and structural characterization of protein sequences.

The most important algorithms incorporated in EST-MAX are:

FASTA: Very sensitive sequence database searches with estimates of statistical significance; Pearson W. R. (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98.

BLAST: Very sensitive sequence database searches with estimates of statistical significance. Altschul S. F., Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignment search tool. Journal of Molecular Biology 215:403-10.

PREDATOR: High-accuracy secondary structure prediction from single and multiple sequences. Frishman, D. and Argos, P. (1997) 75% accuracy in protein secondary structure prediction. Proteins, 27:329-335.

CLUSTALW: Multiple sequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680.

TMAP: Transmembrane region prediction from multiply aligned sequences. Persson, B. and Argos, P. (1994) Prediction of transmembrane segments in proteins utilising multiple sequence alignments. J. Mol. Biol. 237:182-192.

ALOM2: Transmembrane region prediction from single sequences. Klein, P., Kanehisa, M., and DeLisi, C. Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221-226 (1984). Version 2 by Dr. K. Nakai.

PROSEARCH: Detection of PROSITE protein sequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M., Smith J. E. (1992) ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13, 919-921.

BLIMPS: Similarity searches against a database of ungapped blocks. J. C. Wallace and Henikoff S., (1992)

PATMAT: A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford.

Example 4

Identification of Arabidopsis, Soybean and Corn ORFs Corresponding to PrPase

The Physcomitrella patens, PpPrPase (SEQ ID NO: 1), was identified in EST-MAX through BLAST analysis. The top hit of the BLAST is an Arabidopsis unknown ORF. The second and the third hits are human and yeast caax prenyl protease. Further analysis of the unknown Arabidopsis ORF revealed that it is a predicted ORF from computer analysis, using the program Genefinder (P. Green and L. Hillier, National Center for Biotechnology Information). The ORF is located on the complementary strand of the BAC clone AF007269 (GenBank accession number, gene="A_IG002N01.21) from 24979 to 28076.

Using this computer predicted Arabidopsis AtPrPase cDNA as a query, BLAST search in various maize and soybean databases has identified one corn ZmPrPase EST (SEQ ID NO: 21) and one soybean GmPrPase EST (SEQ ID NO: 14).

Example 5

Cloning of Arabidopsis cDNAs Encoding for PrPase

Total RNA Isolation from Arabidopsis thaliana

The total RNA was obtained from wild-type 14 day old Arabidopsis thaliana following the Van Slogteren (1983 Plant Mol. Biol. 2: 321-333) method with slight modifications. Tissue (200 mg) was frozen with liquid nitrogen and ground to a fine powder with a mortar and pestle. The powder was placed in a microfuge tube and the RNA was extracted with 500 ul of extraction buffer (phenyl: 0.1 M LiCl, 100 mM Tris-HCl [pH8.0], 10 mM EDTA, 1% SDS (w/v) [1:1]) preheated to 90.degree. C. The mixture was heated further for 1 min at 90.degree. C. and then vortexed for 5 mins. Proteins were extracted by adding 250 ul of chloroform: isoamyl:alcohol (24:1) and the mixture was vortexed for 5 mins and centrifuged for 15 mins at 13,000 rpm in an Eppendorf centrifuge 5414 at 4.degree. C. The aqueous layer was removed and the protein extraction was repeated twice more. One volume of 4 mM LiCl was added and the RNA was allowed to precipitate overnight at 4.degree. C. To collect the RNA, the mixture was centrifuged for 15 min at 4.degree. C. at 13,000 rpm in an Eppendorf centrifuge 5414. The pellet was resuspended in 250 ul sterile, deionized water. To precipitate the RNA, 0.1 vols of 3M sodium acetate (pH5.2) and 2 vols 100% ethanol were added. An aliquote was taken and centrifuged for 20 mins at 4.degree. C. at 13,000 rpm in an Eppendorf centrifuge 5414. The pellet was washed with 70% ethanol to remove salts from the pellet and dried using a speed vac. The pellet was resuspended in 25 ul DEPC H.sub.2O and analyzed for integrity via electrophoresis. The RNA was stored at -70.degree. C.

RT-PCR and Cloning of Arabidopsis AtPrPase

The synthetic oligonucleotide primers (MWG-Biotech) were designed based on the BAC clone sequence (GenBank accession number AF007269, gene="A_IG002N01.21", complement 24979 . . . 28076).

TABLE-US-00005 APP forward: (SEQ ID NO: 35) 5' CCGTTAACAGCCATGGCGATTCCTTTCATGGAA 3' APP reverse: (SEQ ID NO: 36) 5' GTCCCGGGACTTAATCTGTCTTCTTGTCTT 3'

The primers designed contained a HpaI site in the 5' region and an XmaI site in the 3' region for cloning purpose.

The synthesis of the first strand cDNA was achieved using AMV Reverse Transcriptase (Roche, Mannheim, Germany). The resulting single-stranded cDNA was amplified via Polymerase Chain Reaction (PCR) utilizing the two gene specific primers. The conditions for the reaction were standard conditions with Expand High Fidelity PCR system (Roche). The parameters for the reaction were: five minutes at 94.degree. C. followed by five cycles of 40 seconds at 94.degree. C., 40 seconds at 50.degree. C. and 1.5 minutes at 72.degree. C. This was followed by thirty cycles of 40 seconds at 94.degree. C., 40 seconds at 65.degree. C. and 1.5 minutes at 72.degree. C. The fragment generated under these RT-PCR conditions was 1.3 kilobase long.

The fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacture's instructions. Recombinant vectors were transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 .mu.g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 0.8 mg IPTG (isopropylthio-.beta.-D-galactoside) grown overnight at 37.degree. C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 .mu.g/ml ampicillin and grown overnight at 37.degree. C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions.

The RT-PCR cloned Arabidopsis AtPrPase1 and AtPrPase2 were sequenced to obtain their complete cDNA sequences (SEQ ID NO: 3 and SEQ ID NO: 5).

Example 6

In Vivo Complementation of the SM3614 Yeast (PrPase) Mutant

The fragment containing the Arabidopsis AtPrPase1 cDNA was excised from the recombinant PCR2.1 TOPO vector by digestion with EcoRI (Roche) according to manufacture's instructions. The subsequent fragment was excised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen) according to manufacture's instructions and ligated into the yeast expression vector pYES2 (Invitrogen), also cleaved with EcoRI and dephosphorylated prior to ligation.

The recombinant expression pYES2 vector containing the Arabidopsis AtPrPase1 cDNA in the sense orientation under the yeast GAL1 promoter was transformed into the yeast mutant SM3614 (MATa rcel .DELTA.::TRP1 step24D::LEU2) (Tam et al. 1998) following Invitrogen's protocol. The transformed cells were selected for on Complete Supplement Mixture (CSM) minus Uracil 0.8% agar (Bio 101, Inc.) grown at 30.degree. C. for two days. The transformed colonies were selected to make master plates containing patches of the transformed SM3614 on CSM plates minus Uracil supplemented with 2% galactose for induction of the expression of the Arabidopsis AtPrPase1. The plates were grown at 30.degree. C. for two days. The master plates were replica plated onto a lawn of wild type yeast SM 1068 (MAT.alpha. lysl) (Tam et al. 1998, The Journal of Cell Biology, 142, 635-649) on SD plates supplemented with 2% galactose under various mating conditions and incubated at 30.degree. C. for two days.

Example 7

Cloning of Soybean and Corn cDNAs Encoding for PrPase

Construction of cDNA Libraries of Soybean and Corn

To isolate the clones encoding PrPase from soybean and corn the cDNA libraries were created with SMART RACE cDNA Amplification kit (Clontech Laboratories) following manufacture's instructions. Total RNA created as described in Example 5 was used as the template. Three-week old corn leaves and stems and soybean leaves were used for total RNA preparation respectively.

Cloning of Soybean and Corn cDNAs Encoding for PrPase

The EST sequences for ZmPrPase and GmPrPase identified from the database search as described in Example 4 were used to design oligos for RACE. The extended partial sequences for ZmPrPase and GmPrPase were obtained by performing Rapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR) using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNA amplification kit (Clontech Laboratories) using a Biometra T3 Thermocycler following the manufacture's instructions. The gene specific synthetic oligonucleotide primer (MWG-Biotech) used were:

For ZmPrPase:

TABLE-US-00006 (SEQ ID NO: 37) 5' RACE oligo: 5' AGCAGCCACGATTGGTGGCCCCAAT 3' (SEQ ID NO: 38) 3' RACE oligo: 5' GGGCCACCAATCGTGGCTGCTATCA 3'

For GmPrPase:

TABLE-US-00007 (SEQ ID NO: 39) 5' RACE oligo: 5' CGCAGCCAGTCCTCATTGGGCTCATC 3' (SEQ ID NO: 40) 3' RACE oligo: 5' CGGATAGTTGAGGGAGGAAGCAAG 3'

The sequences obtained from the RACE reactions were compiled to give the nucleotide sequences for the partial GmPrPase (SEQ ID NO: 14) and the partial ZmPrPase (SEQ ID NO: 21).

Having the partial sequences for soybean and corn, full-length sequences were obtained. The nucleotide sequence of the full-length PrPase from soybean (Glycine max) (Clone ID No: GmPrPase2) is provided as SEQ ID NO: 16 with the deduced amino acid sequence as SEQ ID NO: 17. The nucleotide sequence of the full-length PrPase from corn (Zea mays) (Clone ID No: ZmPrPase2) is provided as SEQ ID NO: 23 with the deduced amino acid sequence as SEQ ID NO: 24.

Example 8

Engineering Drought-Tolerant Arabidopsis Plants

Binary Vector Construction: pGMSG and pGMGG

The pLMNC53 vector (Mankin, 2000, PHD thesis) was digested with HindIII (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) according to manufacture's instructions. This fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The purified fragment was then digested with EcoRI (Roche) according to manufacture's instructions. This fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The resulting 1.4 kilobase fragment, the gentamycin cassette, included the nos promoter (Becker et al., 1992 Plant Molecular Biology 20: 1195-7), aacCI gene (Hajdukiewicz et al., 1994 Plant Molecular Biology 25: 989-94), and the g7 terminator (Becker et al., 1992).

The vector pBlueScript was digested with EcoRI and SmaI (Roche) according to manufacture's instructions. The resulting fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. The digested pBlueScript vector and the gentamycin cassette fragments were ligated with T4 DNA Ligase (Roche) according to manufacture's instructions, joining the two respective EcoRI sites and joining the blunt-ended HindIII site with the SmaI site.

The recombinant vector (pGMBS) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 0.8 mg IPTG (isopropylthio-.beta.-D-galactoside), grown overnight at 37.degree. C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 .mu.g/ml ampicillin and grown overnight at 37.degree. C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).

Both the pGMBS vector and plbxSuperGUS vector were digested with XbaI and KpnI (Roche) according to manufacture's instructions, excising the gentamycin cassette from pGMBS and producing the backbone from the plbxSuperGUS vector. The resulting fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacture's instructions.

The resulting recombinant vector (pGMSG) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 .mu.g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 0.8 mg IPTG (isopropylthio-.beta.-D-galactoside), grown overnight at 37.degree. C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 .mu.g/ml ampicillin and grown overnight at 37.degree. C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).

Another example for a plant binary vector is the pGMGG vector, where both the pBinK vector containing the guard cell-specific promoter KST1 (Bernd Muller-Rober, 1999) and pGMSG vector were digested with XbaI and SmaI according to manufacture's instructions, excising KST1 from pBinK and producing the backbone from the pGMSG vector. The resulting fragments were extracted from agarose gel with QIAquick Gel Extraction Kit (Qiagen) according to manufacture's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacture's instructions.

The resulting recombinant vector (pGMGG) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 .mu.g/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 0.8 mg IPTG (isopropylthio-.beta.-D-galactoside), grown overnight at 37.degree. C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 .mu.g/ml ampicillin and grown overnight at 37.degree. C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacture's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al. 1989).

Subcloning of Arabidopsis AtPrPases into the Binary Vectors

The fragment containing the Arabidopsis PrPase cDNA was excised from the recombinant PCR2.1 TOPO vector by digestion with HpaI and XmaI (Roche) according to manufacture's instructions. The subsequent fragment was excised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen) according to manufacture's instructions and ligated into the binary vectors pGMSG, cleaved with XmaI and Ecl136II and dephosphorylated prior to ligation.

The clones AtPrPase1 (SEQ ID NO: 3) and AtPrPase2 (SEQ ID NO: 5) were cloned into pGMSG vectors in sense orientation. The resulting recombinant pGMSG vectors contained the Arabidopsis prenyl protease under the control of the constitutive super promoter.

Agrobacterium Transformation

The recombinant vectors were transformed into Agrobacterium tumefaciens C58C1 and PMP90 according to standard conditions (Hoefgen and Willmitzer, 1990).

Plant Transformation

Arabidopsis thaliana ecotypes C24 and Col-2 were grown and transformed according to standard conditions (Bechtold 1993, Acad. Sci. Paris. 316:1194-1199, Bent et al. 1994, Science 265:1856-1860).

Screening of Transformed Plants

Seeds were sterilized according to standard protocols (Xiong et al. 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were plated on 1/2 MS 0.6% agar supplemented with 1-3% sucrose and 50-150 .mu.g/ml gentamycin. Seeds on plates were vernalized for two days at 4.degree. C. The seeds were germinated in a climatic chamber at an air temperature of 22.degree. C. and light intensity of 55 micromols.sup.-1 m.sup.-2 (white light; Philips TL 65W/25 fluorescent tube) and 24 hour light. Transformed seedlings were selected after 7-14 and transferred to 1/2 MS 0.6% agar plates supplemented with 1% sucrose and allowed to recover for 1-5 days.

Drought Tolerance Screening

The transgenic plants were screened for their improved drought tolerance. Seedlings were transferred to dry, sterile filter paper and allowed to desiccate for 4 hours. Seedlings were then removed and placed on 1/2 MS 0.6% agar plates and scored after two days.

Salt Tolerance Screening

The transgenic plants are screened for the improved salt tolerance. Seedlings are transferred to 1/2 MS liquid supplemented with 600 mM NaCl and allowed to incubate for 2-4 hours. Seedlings are then removed and placed on 1/2 MS 0.6% agar plates and scored for surviving seedlings after two days.

Example 9

Engineering Drought-Tolerant Soybean Plants

The GmPrPase clones (SEQ ID NO: 14 and 16) are cloned into pGMSG vectors in sense orientation. These constructs are used to transform soybean as described below.

Seeds of soybean are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at RT, and resuspended in MS (Murashige and Skoog, 1962) medium supplemented with 100 .mu.M acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at RT before use. The axis of soybean zygotic seed embryos at approximately 15% moisture content are imbibed for 2 hours at RT with the pre-induced Agrobacterium suspension culture. The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at RT. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to kill the agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25.degree. C., under 150 .mu.mol m.sup.-2 sec.sup.-1 and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favor the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25.degree. C., under 150 .mu.mol m.sup.-2 sec.sup.-1 light intensity and 12 hours photoperiod for about 80 days.

The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.

Example 10

Engineering Drought-Tolerant Rapeseed Plants with the AtPrPase Clones

The AtPrPase constructs of Example 8 are used to transform rapeseed as described below.

The method of plant transformation described in Example 8 is also applicable to Brassica and other crops. Seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes, at RT with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. Then the seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period the seeds lose approx. 85% of its water content. The seeds are then stored at room temperature in a sealed Petri dish until further use. DNA constructs and embryo imbibitions are as described in Example 8. Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.

The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.

Example 11

Engineering Drought-Tolerant Corn Plants

The ZmPrPase clones (SEQ ID NO: 21 and 23) are cloned into pGMSG vectors in sense orientation. These constructs are used to transform corn.

The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes. The experimental protocol is the same as described in Example 8 but using maize seeds as the source of embryos.

The transgenic plants are screened for their improved drought tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers drought tolerance.

Example 12

Guard-Cell Specific Expression of the AtPrPase1 Promoter

The promoter region of AtPrPase (SEQ ID NO: 10) is cloned into pGMSG in place of the superpromoter, driving the reporter gene GUS (Jefferson et al., 1987). The resulting construct is transformed into Arabidopsis plants as described in Example 8.

The transgenic plants are screened for their guard-cell specific staining to demonstrate that the transgene expression confers guard-cell specific promoter activity.

Example 13

Over-Expression of PrPase in Plants Leads to Increased Stress Tolerance and Plant Growth

The AtPrPase clones (SEQ ID NO: 3 and 5) used to transform Arabidopsis, as described in Example 8 are used to transform soybean, rapeseed, and corn as described in Examples 9, 10, and 11, respectively.

The transgenic plants are screened for their improved stress tolerance according to the screening method described in Example 8 to demonstrate that transgene expression confers stress tolerance.

The transgenic plants are further screened for their growth rate to demonstrate that transgene expression confers increased growth rates.

Specifically, transgenic Arabidopsis plants transformed with AtPrPase1 (SEQ ID NO: 3 encoding SEQ ID NO: 4) were assessed for stress tolerance and plant growth. The transgenic Arabidopsis plants were grown for three weeks in a growth chamber at a temperature of 22.degree. C., 55% relative humidity and light intensity of 40 micromols.sup.-1 m.sup.-2 (white light; Philips TL 65W/25 fluorescent tube) and 16 hours light and 8 hours dark day length cycle. Soil moisture was maintained throughout this time at approximately 50% of the maximum water-holding capacity of soil. After 3 weeks, the entire above-group plant biomass was collected, dried at 65.degree. C. for 2 days and weighed.

Three independent experiments were conducted with data shown in Table 4. In each experiment, 10 transgenic events were selected, 10-12 transgenic plants were assayed for each event (including the control event). Different control plants were used for comparison in each experiment. The dry weight was calculated by averaging plants of the same transformation event (given in grams in Table 4). The analysis was run using a Least Square Mean (LSMEAN).

The first experiment used Arabidopsis ecotype C24 as control. The vector control plants were generated by transforming into the wild type C24 plants the T-DNA of a binary vector containing between the left and right borders a selectable marker cassette (Promoter-selectable marker-Terminator) and a cassette with no gene of interest (promoter-terminator).

The second experiment used both MTXC24 and BPSC24 as controls, where MTXC24 and BPSC24 were single seed descent variants of the Arabidopsis C24 ecotype.

The third experiment used Arabidopsis ecotype Col-0 as control.

In all three experiments, AtPrPase1 (SEQ ID NO: 3 encoding SEQ ID NO: 4) transgenic plants in each of the corresponding control background, under limited water condition, accumulated more dry weight than the corresponding control plants as shown in Table 4.

TABLE-US-00008 TABLE 4 Control Control Dry Weight AtPrPase1 Dry Weight Experiment used LSMean Std. Error LSMean Std. Error 1 SC24 0.098269 0.0045776 0.141823 0.0043416 2 MTX C24 0.0547 0.0035 0.1047 0.0028 BPS C24 0.0581 0.0035 3 Col-0 0.189 0.009 0.213 0.003

Example 14

Transformation of Dicotyledons

The polynucleotides of the present invention, including the polynucleotides encoding the polypeptides of the present invention, may be used to transform dicotyledons in an effort to confer specific traits into the plant. Such polynucleotides may be either the full-length polynucleotide, fragments, the complementary strand, or variants thereof, and may be either by themselves or operably fused to heterologous polynucleotides.

Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques which do not require Agrobacterium. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. The many crop species which are routinely transformable by Agrobacterium include tobacco, tomato, sunflower, cotton, oilseed rape, potato, soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432 (tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No. 4,795,855 (poplar)). Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.

Other methods for the transformation of dicotyledons are known in the art. Thus, this example should be not construed as limiting the scope of the invention to only those examples illustrated above or elsewhere herein.

Example 15

Transformation of Monocotyledons

The polynucleotides of the present invention, including the polynucleotides encoding the polypeptides of the present invention, may be used to transform monocotyledons in an effort to confer specific traits into the plant. Such polynucleotides may be either the full-length polynucleotide, fragments, the complementary strand, or variants thereof, and may be either by themselves or operably fused to heterologous polynucleotides as described in more detail elsewhere herein.

Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complex vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent Applications EP 0 292 435 (to Ciba-Geigy), EP 0 392 225 (to Ciba-Geigy) and WO 93/07278 (to Ciba-Geigy) describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Fromm et al., Biotechnology 8: 833-839 (1990) have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al., Biotechnology 11: 194-200 (1993) describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

Patent Application EP 0 332 581 (to Ciba-Geigy) describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation was been described by Vasil et al., Biotechnology 10: 667-674 (1992) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al., Biotechnology 11: 1553-1558 (1993) and Weeks et al., Plant Physiol. 102: 1077-1084 (1993) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashige & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont Biolistics, helium device using a burst pressure of about 1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile containers known as "GA7s" which contained half-strength MS, 2% sucrose, and the same concentration of selection agent.

An additional method of transforming monocots is found in International Publication No. WO 00/12734, and describes the application of the Ac-Ds transposon system to the insertion of transgenes into plants.

Other methods for the transformation of monocotyledons are known in the art. Thus, this example should be not construed as limiting the scope of the invention to only those examples illustrated above or elsewhere herein.

Example 16

Method of Transforming Plants Using Vacuum Infiltration

Transformation of plants may serve as a vital tool in assessing the biological function of a particular polynucleotide or polypeptide. For example, a plant may be transformed with a vector capable of downregulating a particular gene via anti-sense regulation (i.e., the vector may express a transcript of the gene of interest in the anti-sense direction), or the vector may simply be capable of overexpressing a particular polypeptide, for example. By observing the resulting phenotypes of the transformant, one may derive protein function using techniques known in the art and described elsewhere herein.

The following method of transforming plant material may be applicable to any plant species, though is particularly suited for use in Arabidopsis.

Arabidopsis is grown at 20.degree. C., 8 hr light, 18.degree. C. 16 dark until needed for transformation and is fertilized once a week from below. Plants are thinned to .about.1 per square inch and are used immediately upon bolting. Short days allow stronger vegetative plant growth and increase seed yield.

Plants are transferred to 20.degree. C., 16 hr light, 18.degree. C. 8 hr dark. The plants should bolt quickly, and they are ready to infiltrate when the primary inflorescences are 10-15 cm tall and the secondary inflorescences are appearing at the rosette.

In the meantime, constructs are transformed into Agrobacterium tumefaciens strain EHA105 (Hood et al., Transgenic Research 2:208-218 (1993)) (see Direct Agrobacterium Transformation:Freeze-Thaw Method below). When the plants are ready to transform, 1 ml of an overnight culture is used to innoculate a 500 ml culture of YEB medium (2 L flask) containing the appropriate antibiotic for the construct and 50 ug/mL rifampicin (C58 Agro and or 25 mg/mL gentamycin (pMP90)). Cultures are grown for 2 days at 28.degree. C., .about.275 rmp. YEB media is described below.

When OD.sub.600 is greater than 2.0, the culture is centrifuged for 30 min, at 3500 rpm and resuspended in 0.5-1.0 ml of infiltration medium described below.

The resuspended culture is placed in a container with a large bell jar, and pots containing plants to be infiltrated are inverted into the infiltration medium so that the entire plant is covered (including the rosette, but not too much soil). Any large air bubbles under the plants are removed. A vacuum (.about.700 Hg) is drawn, the suction is closed, and the plants are allowed to sit under vacuum for 5 min. The vacuum pressure is quickly released and the pots are briefly drained.

Infiltrated plants are grown as before at 20.degree. C., 16 hr light, and 18.degree. C. 8 hr dark. Plants are staked as the bolts grow. When the plants are finished flowering, the T.sub.0 seeds are harvested.

Seeds are sterilized and screened for transformants on the selective medium described below. Dark green (resistant) plants are transferred to secondary selection plate a week after germination, then to soil after 6-10 days. New transplants are kept covered for several days.

TABLE-US-00009 Plant media: Vacuum Infiltration Selection For 1 L Medium Medium MS Salts 2.2 g 4.3 g B5Vitamins, 1000X 1.0 mL 1.0 mL Sucrose 50 g 10 g MES, 200 mg/mL pH 5.7 with KOH 2.5 mL 2.5 mL Benzylamonipurine (BAP, 1 mg/mL) 44 .mu.L -- Silweet L-77 200 .mu.L -- Phytagar -- 8 g Pursuit (1 mM) 100 .mu.L

TABLE-US-00010 Bacterial Media YEP Yeast extract 1.0 g Beef Extract 5.0 g Peptone 5.0 g Sucrose 5.0 g MgSO.sub.4 0.5 g

The skilled artisan would appreciate that the above transformation method could be modified to apply to other species of plants. Such modification may include the addition of new steps, the deletion of any of the steps described, and/or substitution of reagents.

Direct Agrobacterium Transformation:Freeze-Thaw Method.

An Agrobacterium strain containing the appropriate helper Ti plasmid is grown in 5 mL of YEP medium overnight at 28.degree. C. 2 ml of the overnight culture is added to 50 ml YEP medium in a 259 mL flask and shaken vigorously (250 rpm) at 28.degree. C. until the culture grows to an OD.sub.600 of 0.5 to 1.0. The culture is chilled on ice. The cell suspension is centrifuged at 3000 g for 5 min at 4.degree. C.

The supernatant solution is discarded. The cells are resuspend in 1 mL of 20 mM CaCl.sub.2 solution (ice-cold). 0.1 mL aliquots are dispensed into prechilled Eppendorf tubes. About 1 ug of plasmid DNA is added to the cells.

The cells are frozen in liquid nitrogen. The cells are thawed by incubating the test tube in a 37.degree. C. water bath for 5 min. 1 mL of YEP medium is added to the tube and the tube incubated at 28.degree. C. for 2-4 hr with gentle rocking. This period allows the bacteria to express the antibiotic resistance genes. The tubes are centrifuged for 30 s in an Eppendorf centrifuge. The supernatant solution is discarded and the cells resuspended in 0.1 mL YEP medium

The cells are spread onto a YEP agar plate containing 3-5 ug/mL tetracycline and 10-25 ug/mL kanamycin. The plate is incubated at 28.degree. C. Transformed colonies should appear in 2-3 days.

The skilled artisan would appreciate that the above transformation method could be modified. Such modification may include the addition of new steps, the deletion of any of the steps described, and/or substitution of reagents.

Example 17

Functional Determination of Proteins Using Metabolite Profiling

The present invention encompasses the application of metabolite profiling to the identification of gene function for the polypeptides of the present invention. In one example, transgenic plants could be produced which are either incapable of expressing a protein of the present invention, or that have decreased expression levels of a protein of the present invention. Such transgenic plants could be produced by creating knockout constructs to inactivate or delete the endogenous gene, for example, using methods known in the art. Alternatively, the transgenic plants could be produced by inserting into the plant a construct that expresses antagonists of a protein of the present invention (e.g., antisense oligonucleotides, antisense genes, antibodies, etc.). Other examples of methods of producing transgenic plants, including specific strategies, are known in the art, some of which are described elsewhere herein.

Once a protein of the present invention is inactivated, or its expression inhibited, the resulting metabolite profile of the plant can be ascertained, and the function of the protein assigned. Some of the anticipated metabolic profiles of inhibiting or inactivating the expression of a protein of the present invention in a plant may resemble known nutritional deficiencies, pathogenic diseases, biotic stresses, or abiotic stresses, for example, many of which are disclosed elsewhere herein. In addition, the metabolic profile of a transgenic plant of the present invention may be useful in identifying the specific pathways the polypeptide of the present invention is a member of, in addition, to identifying the potential downstream and/or upstream effectors or affectors, respectively. In addition, it may be possible to identify the mode of action of a polypeptide of the present invention.

A number of methods are known in the art for identifying the metabolic profile of a plant. A non-limiting example is provided by Sauter, H., et al., in "Metabolic Profiling of Plants: A New Diagnostic Technique", Synthesis and Chemistry of Agrochemicals II, Baker, D. R., Fenyes, J. G., and Moberg, W. K., eds, ACS Symposium Series, 433, Chapter 24, pp. 288-299, (1991). Briefly, transgenic plants of the invention, or plants in which the expression of a polypeptide of the present invention is inhibited or inactivated, are grown in growth chambers. The shoots are harvested and immediately deep frozen until further treatment. The frozen plant samples are weighed and a threefold amount (W:W) of ethanol is added. The mixture is then macerated in a mixer and the resulting suspension is left for 2 hours for extraction. The next steps are filtration, evaporation, and silylation with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). Internal standard alkanes are also added, thus allowing for the calculation of retention coefficients, as well as, quantification. The crude mixture is then subjected to gas chromotography on a methyl silicon gum fused silica capillary column (30 m DB-1. Injection temperature 230.degree. C. Oven temperature 100.degree.-320.degree. C., 4.degree. C./min; 15 min 320.degree. C.). Retention coefficients are then calculated relative to internal standards (n-C10H22=1000, n-C28H58=2800).

The above protocol can be applied to numerous test plants, in addition, to controls. The data from the resulting profiles are then grouped together (i.e., one group for the test plants, another group for the controls) to arrive at an average profile for each group. In the latter step, the corresponding peaks (i.e., those peaks with equal retention coefficients) are grouped together and the peak heights are subjected to statistical analysis.

The differences in metabolic profiles between the test and control plants are determined by calculating the "difference profile" between the two groups. The difference profile is calculated by dividing the peal heights. This difference profile provides a semiquantitative estimate of the change in magnitude of one metabolic with respect to the other.

Once the above is completed, the peaks are then associated with particular metabolites (i.e., the metabolite identify of each peak is determined). By comparing the metabolite profile of proteins known to modulate specific pathways in a plant, to those of the present invention, clues for and/or identification of the function of a polypeptide of the present invention may be determined. Other methods are known in the art, and any one or more steps, may be equally substituted with such methods.

Example 18

RT-PCR Amplification and Cloning of CaaX Prenyl Proteases

Total RNA was isolated from leaf tissue of Arabidopsis thaliana, Brassica napus and Glycine max, using the Qiagen RNeasy kit and used as template to amplify the CaaX prenyl proteases (CPP) genes by RT-PCR. Reaction conditions were as follows; 1.times. reaction buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl.sub.2, 50 mM KCl), dNTP's at 200 .mu.M, 1 pM AtCPP BamFW and AtCPP SmaRV primers, 2.5 U. Pfu DNA polymerase, and template plus water to a final volume of 100 .mu.L. Reactions were run at 1 minute 94.degree. C., 1 minute 60.degree. C., 1 minute 72.degree. C., for 30 cycles. Primers used to PCR amplify Arabidopsis and Brassica sequences were those identified by SEQ ID NO: 43 and SEQ ID NO: 44. Primers used to PCR amplify the Glycine sequence were those identified by SEQ ID NO: 70 and SEQ ID NO: 71. PCR products were separated from the RT-PCR reaction mixture using the Qiagen PCR column spin kit and ligated into the prepared cloning vector, pBluescript KS+. The vector had been prepared by digestion with EcoRV and treated with Taq polymerase in the presence of dTTP to produce a 3' overhand suitable for ligation with the PCR products. The ligation products were transformed into E. coli DH5.alpha. cells, positive colonies selected and the resulting inserts sequenced. The above methodology is applicable to obtain homologous sequences and may require alternative primers.

TABLE-US-00011 AtCPP BamFW: (SEQ ID NO: 43) 5'-AAAGGATCCATGGCGATTCCTTTCATGG-3' AtCPP SmaRV: (SEQ ID NO: 44) 5'-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3' GmCPP SmaFW: (SEQ ID NO: 70) 5'-AAACCCGGGATGGCGTTTCCCTACATGGAAGCC-3' GmCPP SacRV: (SEQ ID NO: 71) 5'-AAAGAGCTCTTAGTCTTCCTTCTTATCCGGTTCG-3'

Example 19

Vector Construction

Construction of the pBI121-AtCPP construct (SEQ ID NO: 41) was prepared as follows. The pBI121 vector was digested with BamHI and SmaI. The AtCPP, 1.4 kb DNA fragment from RT-PCR (SEQ ID NO: 7) was digested with BamHI and SmaI and ligated into the pBI121 vector. The GUS sequence was then removed by digestion with SmaI and EcolCRI and the vector ligated after purification of the vector from the GUS insert to produce the pBI121-AtCPP vector (FIG. 2A). This construct was used to further generate constructs expressing the CPP gene from Brassica and Glycine. To produce the pBI121-BnCPP construct (SEQ ID NO: 63) primer pairs identified by SEQ ID NO: 43 and SEQ ID NO: 44 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-GmCPP construct (SEQ ID NO: 57) primer pairs identified by SEQ ID NO: 70 and SEQ ID NO: 71 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.

Construction of the pBI121-antisense-AtCPP construct (SEQ ID NO: 51). The antisense fragment was produced using PCR amplification with SEQ ID NO: 7 as template and primers identified as SEQ ID NO: 48 and SEQ ID NO: 49, listed in Table 5. This fragment was digested with BamHI and SmaI and used to replace the sense fragment of the pBI121-AtCPP construct (SEQ ID NO: 41), to yield SEQ ID NO: 51 (FIG. 2B). This construct, SEQ ID NO: 51, was used to further generate constructs expressing the antisense CPP gene from Brassica and Glycine. To produce the pBI121-antisense-BnCPP construct (SEQ ID NO: 65) primer pairs identified by SEQ ID NO: 72 and SEQ ID NO: 73 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector. To produce the pBI121-antisense-GmCPP construct (SEQ ID NO: 59) primer pairs identified by SEQ ID NO: 74 and SEQ ID NO: 75 are used to PCR amplify the appropriate fragment which is ligated into the prepared parent vector.

Construction of the pBI121-HP-AtCPP construct (SEQ ID NO: 42). The cloning strategy involved truncating the GUS gene of pBI121 and flanking the GUS sequence with a AtCPP fragment in the antisense orientation upstream of the GUS and in the sense orientation on the downstream side of GUS. The pBI121 vector was digested with SmaI and SacI, the GUS sequence and the vector fragments were purified from one another. The isolated GUS fragment was digested using EcoRV and the 1079 bp. blunt ended EcoRV/SacI fragment isolated. This was ligated back into the digested parent vector at the SmaI/SacI sites. This intermediate vector was used in the subsequent production of the hair-pin vectors. The AtCPP fragment to be used as the gene specific hair-pin sequence was isolated by PCR. Primers identified as SEQ ID NO: 45 and SEQ ID NO: 46, listed in Table 5, were used to generate a 596 bp fragment. Cloning of the sense orientation fragment was achieved by digesting the PCR AtCPP fragment with SacI and ligation into the SacI site at the 3' end of GUS. To insert the same fragment upstream of GUS, the BamHI site was opened and the ends blunted with Klenow. The PCR amplified AtCPP fragment was digested with EcolCRI, which is an isoschizomer of SacI but leaves blunt ends, and ligated into the blunted BamHI site of the vector to yield the final construct (FIG. 2C). The intermediate construct used to produce SEQ ID NO: 42 above contained only the truncated GUS gene and no CPP sequences this intermediate vector was used to further generate constructs expressing hair-pin CPP gene constructs from Brassica and Glycine. To produce the pBI121-HP-BnCPP construct (SEQ ID NO: 64) primer pairs identified by SEQ ID NO: 74 and SEQ ID NO: 75 are used to PCR amplify the sense fragment and primer pairs identified by SEQ ID NO: 76 and SEQ ID NO: 77 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above. To produce the pBI121-HP-GmCPP construct (SEQ ID NO: 58) primer pairs identified by SEQ ID NO: 78 and SEQ ID NO: 79 are used to PCR amplify the sense fragment and primer pairs identified by SEQ ID NO: 80 and SEQ ID NO: 81 are used to PCR amplify the antisense fragment. These fragments are cloned into the prepared intermediate vector described above.

The above vector constructs were modified to place the genes under the control of alternative promoters, such as, but not limited to, the RD29A or MuA. This was accomplished by excising the 35S promoter sequence and replacing it with an appropriate promoter sequence. In this way SEQ ID NOs: 55 and 56 were generated and SEQ ID NOs: 54, 57-69 can be constructed.

TABLE-US-00012 TABLE 5 AtCPP-HP-SacFW 5'-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3' (SEQ ID NO: 45) AtCPP-HP-SacRV 5'-ATTGAGCTCCCAATGTCCAAGCTCGTGTGCAATA-3' (SEQ ID NO: 46) AtCPP-anti-SmaFW 5'-AAACCCGGGATGGCGATTCCTTTCATGG-3' (SEQ ID NO: 48) AtCPP-anti-BamRV 5'-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCCA-3' (SEQ ID NO: 49) BnCPP-anti-SmaFW 5'-AAACCCGGGATGGCGATTCCTTTCATGG-3' (SEQ ID NO: 72) BnCPP-anti-BamRV 5'-AAAGGATCCTTAATCTGTCTTCTTGTCTTCTCC-3' (SEQ ID NO: 73) BnCPP-HP-Sac-FW 5'-AAAGAGCTCTTCTACCAATGGTGGGACTCG-3' (SEQ ID NO: 74) BnCPP-HP-Sac-RV 5'-AAAGAGCTCCCAGTGTCCCAGCTCGTGTG-3' (SEQ ID NO: 75) BnCPP-HP-BamFW 5'-AAAGGATCCTTCTACCAATGGTGGGACTCG-3' (SEQ ID NO: 76) BnCPP-HP-XbaRV 5'-AAATCTAGACCAGTGTCCCAGCTCGTGTG-3' (SEQ ID NO: 77) GmCPP-HP-Sac-FW 5'-GATGAGCTCACAAGATCAAGTCACAGCAATGCCT-3' (SEQ ID NO: 78) GmCPP-HP-Sac-RV 5'-AAAGAGCTCCCGGTTCGTCCAGCGCGGCC-3' (SEQ ID NO: 79) GmCPP-HP-BamFW 5'-GATGGATCCACAAGATCAAGTCACAGCAATGCCT-3' (SEQ ID NO: 80) GmCPP-HP-XbaRV 5'-CCTTCTAGACCGGTTCGTCCAGCGCGGCC-3' (SEQ ID NO: 81)

Example 20

Sequence Analysis

Arabidopsis thaliana PrPase (AtCPP)

A disclosed nucleic acid of 1275 nucleotides referred to as AtCPP is shown in SEQ ID NO: 7.

A disclosed PrPase polypeptide encoded by SEQ ID NO: 7 has 424 amino acid residues and is presented as SEQ ID NO: 8.

The present invention also includes a nucleic acid sequence complementary to the Arabidopsis thaliana CaaX prenyl protease of SEQ ID NO: 7. The disclosed complementary sequence is shown as SEQ ID NO: 9.

Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5' and 3' non-translated regions. The percent identities of the Arabidopsis thaliana nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7.

Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.

Brassica napus PrPase (BnCPP)

A disclosed nucleic acid of 1275 nucleotides referred to as BnCPP is shown in SEQ ID NO: 11. A disclosed PrPase polypeptide encoded by SEQ ID NO: 11 has 424 amino acid residues and is presented in SEQ ID NO: 12.

The present invention also includes a nucleic acid sequence complementary to the Brassica napus CaaX prenyl protease of SEQ ID NO: 11. The disclosed complementary sequence is shown as SEQ ID NO: 13.

Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5' and 3' non-translated regions. The percent identities of the Brassica napus nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7.

Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.

Glycine max PrPase (GmCPP)

A disclosed nucleic acid of 1275 nucleotides referred to as GmCPP is shown in SEQ ID NO: 18.

A disclosed PrPase polypeptide encoded by SEQ ID NO: 18 has 424 amino acid residues and is presented in SEQ ID NO: 19.

The present invention also includes a nucleic acid sequence complementary to the Glycine max CaaX prenyl protease of SEQ ID NO: 18. The disclosed complementary sequence is shown as SEQ ID NO: 20.

Due to the nature of the cloning strategy the sequence presented is not full length but is missing the 5' and 3' non-translated regions. The percent identities of the Glycine max nucleotide sequence and its encoded amino acid sequence to that of other PrPase sequences as determined by EMBOSS Pairwise Alignment analysis are shown in FIGS. 6 and 7.

Using the sequences disclosed herein as hybridization probes, one is able to screen and isolate full length sequences from cDNA or genomic libraries or use the rapid amplification of cDNA ends (RACE) technology or other such PCR techniques.

The PrPase nucleic acids and amino acids disclosed above have homology to other disclosed PrPase sequences (GenBank ID NOs: AL161491 (AT4g01320), AF007269 and AF353722; WO 02/16625 A2). The nucleic acid and amino acid homology between these and other sequences is shown in the EMBOSS Pairwise Alignment analysis shown in FIGS. 4 and 5.

Example 21

Plant Transformation

Arabidopsis transgenic plants were made by the method of dipping flowering plants into an Agrobacterium culture, based on the method of Andrew Bent in, Clough S J and Bent A F, 1998. Floral dipping: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Wild type plants were grown under standard conditions until the plant has both developing flowers and open flowers. The plant was inverted for 2 minutes into a solution of Agrobacterium culture carrying the appropriate gene construct. Plants were then left horizontal in a tray and kept covered for two days to maintain humidity and then righted and bagged to continue growth and seed development. Mature seed was bulk harvested.

Transformed T1 plants were selected by germination and growth on MS plates containing 50 .mu.g/ml kanamycin. Green, kanamycin resistant (Kan.sup.R) seedlings were identified after 2 weeks growth and transplanted to soil. Plants were bagged to ensure self fertilization and the T2 seed of each plant harvested separately. During growth of T1 plants leaf samples were harvested, DNA extracted and Southern blot and PCR analysis performed.

T2 seeds were analysed for Kan.sup.R segregation. From those lines that showed a 3:1 resistant phenotype, surviving T2 plants were grown, bagged during seed set, and T3 seed harvested from each line. T3 seed was again used for Kan.sup.R segregation analysis and those lines showing 100% Kan.sup.R phenotype were selected as homozygous lines. Further molecular and physiological analysis was done using T3 seedlings.

Transgenic Brassica napus, Glycine max and Zea mays plants were produced using Agrobacterium mediated transformation of cotyledon petiole tissue. Seeds were sterilized as follows. Seeds were wetted with 95% ethanol for a short period of time such as 15 seconds. Approximately 30 ml of sterilizing solution I was added (70% Javex, 100 .mu.l Tween20) and left for approximately 15 minutes. Solution I was removed and replaced with 30 ml of solution II (0.25% mecuric chloride, 100 .mu.l Tween20) and incubated for about 10 minutes. Seeds were rinsed with at least 500 ml double distilled sterile water and stored in a sterile dish. Seeds were germinated on plates of 1/2 MS medium, pH 5.8, supplemented with 1% sucrose and 0.7% agar. Fully expanded cotyledons were harvested and placed on Medium I (Murashige minimal organics (MMO), 3% sucrose, 4.5 mg/L benzyl adenine (BA), 0.7% phytoagar, pH5.8). An Agrobacterium culture containing the nucleic acid construct of interest was grown for 2 days in AB Minimal media. The cotyledon explants were dipped such that only the cut portion of the petiole is contacted by the Agrobacterium solution. The explants were then embedded in Medium I and maintained for 5 days at 24.degree. C., with 16, 8 hr light dark cycles.

Explants were transferred to Medium II (Medium 1,300 mg/L timentin) for a further 7 days and then to Medium III (Medium II, 20 mg/L kanamycin). Any root or shoot tissue which had developed at this time was dissected away. Transfer explants to fresh plates of Medium III after 14-21 days. When regenerated shoot tissue developed the regenerated tissue was transferred to Medium IV (MMO, 3% sucrose, 1.0% phytoagar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin). Once healthy shoot tissue developed shoot tissue dissected from any callus tissue was dipped in 10.times.IBA and transferred to Medium V (Murashige and Skooge (MS), 3% sucrose, 0.2 mg/L indole butyric acid (IBA), 0.7% agar, 300 mg/L timentin, 20 mg/L 20 mg/L kanamycin) for rooting. Healthy plantlets were transferred to soil. The above method, with or without modifications, is suitable for the transformation of numerous plant species including Glycine max, Zea mays and cotton.

Transgenic Glycine max, Zea mays and cotton can be produced using Agrobacterium-based methods which are known to one of skill in the art. Alternatively one can use a particle or non-particle biolistic bombardment transformation method. An example of non-particle biolistic transformation is given in U.S. Patent Application 20010026941. This method has been used to produce transgenic Glycine max and Zea mays plants. Viable plants are propagated and homozygous lines are generated. Plants are tested for the presence of drought tolerance, physiological and biochemical phenotypes as described elsewhere.

The following table identifies the constructs and the species which they have been transformed.

TABLE-US-00013 TABLE 6 Transformation List SEQ ID NO: Construct Species Transformed 41 pBII121-AtCPP A. thaliana, B. napus 42 pBII121-HP-AtCPP A. thaliana 52 pRD29A-AtCPP A. thaliana, B. napus 53 pRD29A-HP-AtCPP A. thaliana 55 MuA-AtCPP Glycine max, Zea mays

Non-limiting examples of vector constructs suitable for plant transformation are given in SEQ ID NOs: 41, 42, 51-69. The right and left border repeats correspond to the first 24 nucleotide positions and the last 25 nucleotide positions of each construct.

SEQ ID NO: 41 is the nucleic acid sequence of pBI121-AtCPP. The 35S promoter is at positions 2515-3318 and the AtCPP sense sequence is at positions 3334-4608 of SEQ ID NO: 41.

SEQ ID NO: 42 is the nucleic acid sequence of pBI121-HP-AtCPP. The 35S promoter is at positions 2515-3318, the AtCPP anti-sense sequence at positions 3336-3925, the truncated GUS fragment at positions 3936-4958; and the AtCPP sense sequence at positions 4959-5548 of SEQ ID NO: 42.

SEQ ID NO: 51 is the nucleic acid sequence of pBI121-antisense-AtCPP. The 35S promoter is at positions 2515-3318 and the AtCPP anti-sense sequence at positions 3334-4608 of SEQ ID NO: 51.

SEQ ID NO: 52 is the nucleic acid sequence of RD29A-AtCPP. The RD29A promoter is at positions 2515-2865 and the AtCPP sense sequence at positions 3458-4732 of SEQ ID NO: 52.

SEQ ID NO: 53 is the nucleic acid sequence of RD29A-HP-AtCPP. The RD29A promoter is at positions 2515-3442, the AtCPP anti-sense sequence at positions 3460-4069, the truncated GUS fragment at positions 4060-5082, and the A. thaliana CaaX prenyl protease sense fragment at positions 5083-5675 of SEQ ID NO: 53.

SEQ ID NO: 54 is the nucleic acid sequence of RD29A-antisense-AtCPP. The RD29A promoter is at positions 2515-3442 and the AtCPP anti-sense sequence is at positions 3458-4732 of SEQ ID NO: 54.

SEQ ID NO: 55 is the nucleic acid sequence of MuA-AtCPP. The MuA promoter is at positions 2502-2863 and the A. thaliana CaaX prenyl protease sense sequence at positions 2864-4138 of SEQ ID NO: 55.

SEQ ID NO: 56 is the nucleic acid sequence of MuA-GmCPP. The MuA promoter is at positions 2502-2865 and the G. max CaaX prenyl protease sense sequence at positions 2866-4140 of SEQ ID NO: 56.

SEQ ID NO: 57 is the nucleic acid sequence of pBI121-GmCPP. The 35S promoter is at positions 2515-3318 and the G. max CaaX prenyl protease sense sequence at positions 3339-4613 of SEQ ID NO: 57.

SEQ ID NO: 58 is the nucleic acid sequence of pBI121-HP-GmCPP. The 35S promoter is at positions 2515-3318, the antisense prenyl protease fragment of G. max at positions 3328-3856, the G. max sense prenyl protease fragment is at positions 4888-5416, and the truncated GUS fragment at positions 3865-4827 of SEQ ID NO: 58.

SEQ ID NO: 59 is the nucleic acid sequence of pBI121-antisense-GmCPP. The 35S promoter is at positions 2515-3318 is the GmCPP anti-sense sequence at positions 3339-4613 of SEQ ID NO: 59.

SEQ ID NO: 60 is the nucleic acid sequence of pRD29A-GmCPP. The RD29A promoter is at positions 2515-3442 and the GmCPP sense sequence at positions 3463-4737 of SEQ ID NO: 60.

SEQ ID NO: 61 is the nucleic acid sequence of pRD29A-HP-GmCPP. The RD29A promoter is at positions 2515-3442, the GmCPP antisense sequence at positions 3452-3980, and the GmCPP sense sequence at positions 5012-5540 of SEQ ID NO: 61.

SEQ ID NO: 62 is the nucleic acid sequence of pRD29A-antisense-GmCPP. The RD29A promoter is at positions 2515-3442 and the GmCPP antisense sequence at positions 3463-4737 of SEQ ID NO: 62.

SEQ ID NO: 63 is the nucleic acid sequence of pBI121-BnCPP. The 35S promoter is at positions 2515-3318 and the BnCPP antisense sequence at positions 3334-4608 of SEQ ID NO: 63.

SEQ ID NO: 64 is the nucleic acid sequence of pBI121-HP-BnCPP. The 35S promoter is at positions 2515-3318, the BnCPP antisense sequence at positions 3328-3917, the BnCPP sense fragment at positions 4949-5538, and the truncated GUS fragment at positions 3926-4888 of SEQ ID NO: 64.

SEQ ID NO: 65 is the nucleic acid sequence of pBI121-antisense-BnCPP. The 35S promoter is at positions 2515-3318 and the BnCPP antisense sequence at positions 3334-4608 of SEQ ID NO: 65.

SEQ ID NO: 66 is the nucleic acid sequence of pRD29A-BnCPP. The RD29A promoter is at positions 2515-3420 and the BnCPP sense sequence at positions 3458-4732 of SEQ ID NO: 66.

SEQ ID NO: 67 is the nucleic acid sequence of pRD29A-HP-BnCPP. The RD29A promoter is at positions 2515-3442, the BnCPP antisense sequence at positions 3452-4041, the BnCPP sense fragment at positions 5073-5640, and the truncated GUS fragment at positions 4050-5072 of SEQ ID NO: 67.

SEQ ID NO: 68 is the nucleic acid sequence of pRD29A-antisense-BnCPP. The RD29A promoter is at positions 2515-3420 and the BnCPP antisense sequence at positions 3458-4732 of SEQ ID NO: 68.

SEQ ID NO: 69 is the nucleic acid sequence of MuA-BnCPP. The MuA promoter is at positions 2502-2820 and the BnCPP sense sequence at positions 2863-4138 of SEQ ID NO: 69.

Example 22

Southern Analysis

Genomic Southern blot analysis of transgenic Arabidopsis was performed using standard techniques known to one skilled in the art. Typically, 10 .mu.g of DNA was electrophoresed in a 0.8% agarose gel and transferred to an appropriate membrane such as Hybond N+ (Amersham Pharmacia Biotech). Pre-hybridization and hybridization conditions were as suggested by the membrane manufacturer, typically at 65.degree. C. The final stringency wash was typically at 1.times.SSC and 0.1% SDS at 65.degree. C. The NPTII coding region was typically used as the radiolabeled probe in Southern blot analysis.

Thirty-seven Arabidopsis lines were selected as homozygous pBI121-AtCPP over-expression lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pBI121-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.

Thirty-three Arabidopsis lines were selected as homozygous pBI121-HP-AtCPP hair-pin down-regulation lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pBI121-HP-AtCPP hair-pin construct. All lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.

Arabidopsis lines were selected as homozygous pRD29A-AtCPP over-expression lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pRD29A-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.

Arabidopsis lines were selected as homozygous pRD29A-HP-AtCPP lines for further examination. The Southern blot depicted a representative blot confirming the presence of the pRD29A-HP-AtCPP transgene. Lines were confirmed to be transgenic by PCR analysis using transgene specific primers in the PCR assays.

Example 23

PCR Analysis of Transgenic Plants

PCR was used as a method to confirm the presence of the transgene in all transgenic lines and every construct. Typical PCR mixtures contained: 1.times. reaction buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl.sub.2, 50 mM KCl), dNTP's at 200 .mu.M, 1 pM forward and reverse primer, 2.5 U. Taq DNA polymerase, and template plus water to a final volume of 50 .mu.L. Reactions were run at 1 minute 94.degree. C., 1 minute 60.degree. C., 1 minute 72.degree. C., for 30 cycles. Primers used in the analysis of pBI121-AtCPP and pBI121-HP-AtCPP transgenic plants were as shown in Table 7. Primers used in the analysis of pRD29A-AtCPP were RD29AP1 (SEQ ID NO: 82) and SEQ ID NO: 44. Primers used in the analysis of pRD29A-HP-AtCPP transgenic plants were those identified as RD29AP1 (SEQ ID NO: 82), SEQ ID NO: 44 and SEQ ID NO: 45, Nosterm-RV (SEQ ID NO: 83).

TABLE-US-00014 TABLE 7 pBI121-AtCPP BamFW: 5'-GCCGACAGTGGTCCCAAAGATGG-3' (SEQ ID NO: 47) p35S-AtCPP SmaRV: 5'-AAACCCGGGTTAATCTGTCTTCTTGTCTTCTCCA-3' (SEQ ID NO: 44) p35S-HP-AtCPP BamFW: 5'-CTGGAGCTCTTTTACCGAGGTTGGGCCTTGATCC-3' (SEQ ID NO: 45) p35S-HP-AtCPP SmaRV: 5'-GCAAGACCGGCAACAGGA-3' (SEQ ID NO: 50) pRD29AP1: 5'-TTTAAGCTTGGAGCCATAGATGCAATTCAA-3' (SEQ ID NO: 82) Nosterm-RV: 5'-GCAAGACCGGCAACAGGA-3' (SEQ ID NO: 83)

Example 24

Northern Analysis of Transgenic Plants

Total RNA was isolated from developing leaf tissue of 27 35S-AtCPP Arabidopsis lines (T3 plants). Approximately 10 .mu.g of total RNA was loaded into each lane. The Northern blot was first probed with P.sup.32 labeled, single-stranded antisense transcript of AtCPP which detects sense transcript, then stripped and re-probed with cDNA of .beta.-tubulin that was used as a reference. The hybridizing bands of AtCPP and .beta.-tubulin were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA), and the ratio of the two hybridizing bands for each sample was obtained. The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Twenty-one out of twenty-seven lines showed higher expression of AtCPP transcript as compared to the wild type. Values ranged from 104% to 282% of wild type. The results of five lines (35, 84, 76, 136, and 156) of the 21 over-expressing lines is shown in FIG. 8.

Example 25

Production of Polyclonal Antibodies Against AtCPP

Anti-AtCPP antibodies were generated using AtCPP fusion protein over-expressed in E. coli. The over-expression vector, pMAL-p2, contains 1175 bp malE gene that is located upstream of AtCPP and encodes a 43 KDa maltose-binding protein (MBP). The 1275 bp BamHI/SmaI DNA fragment of AtCPP was inserted into pMAL-p2 at BamHI and SalI sites. The SalI site was converted into blunt end using Klenow fragment. The resulting fusion protein MBP-AtCPP was then over-expressed in DH5.alpha., and purified by one-step affinity for MBP as described by the manufacturer (New England Biolab). The soluble fraction of the crude bacterial extract containing the MBP-AtCPP fusion protein was loaded to a amylose column (1.5 cm.times.10.0 cm), and the proteins were eluted with 10 mM maltose in column buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 200 mM NaCl). Fractions containing purified MBP-AtCPP fusion protein were pooled, and concentrated with a Centriprep-30 concentrator (Amicon). All purification steps were carried out at 4.degree. C. To generate an antibody, the purified fusion protein was further separated by SDS-PAGE and the Coomassie stained band corresponding to the fusion protein was excised. The identity of the fusion protein was confirmed by Western analysis using anti-MBP antibodies (purchased from New England Biolab). The protein was eluted from the gel slice by electroelution and then emulsified in Ribi adjuvant (Ribi Immunochem) to a final volume of 1 ml. MBP-AtCPP protein was injected into a 3 kg New Zealand rabbit on day 1 and booster injections were given on day 21 and day 35 with 175 .mu.g of the protein each time. High-titer antisera were obtained one week after the final injection.

Example 26

Western Blot Analysis of 35S-AtCPP Transgenic Lines Using Anti-AtCPP Antibodies

Western analysis was performed to examine expression level of AtCPP in the transgenic lines compared with that of wild type plants. Anti-Bip antibody, an ER lumenal protein (Stressgen, Victoria, BC, Canada) was used as a reference. Total proteins were extracted from developing leaf tissue of five ABA.sup.S lines and a wild type control. The antigenic protein bands of AtCPP and Bip were scanned and quantified using the UN-Scan-It programme (Silk Scientific, Utah, USA) and the ratio of the two protein bands for each sample was obtained. The ratio of the wild type plants was set to 100%, and was compared with those of the transgenic lines. Data is presented in FIG. 8 indicating that the AtCPP protein level was increased in the transgenic lines compared to the wild type plants.

Example 27

ABA Sensitivity of Transgenic Seedlings

Approximately 100 seeds were assessed per line per 9 cm plate. Seeds were plated on minimal medium (1/2 MS) supplemented with no ABA or 1.0 .mu.M ABA. Plates were chilled for 3 days at 4.degree. C. in the dark, and incubated for up to 21 days at 22.degree. C. with 24 hour continuous light. Plates were assessed for germination, cotyledon expansion, true leaf development and seedling vigor. Seedlings were assessed for ABA sensitivity over 21 days of growth at which time sensitive seedlings were arrested at the cotyledon stage, lacked true leaves, and showed inhibition of root growth. Wild type control Columbia plants had two to three pairs of true leaves and a well developed root system. Lines were categorized as ABA sensitive (ABA.sup.S) if less than 1% of plants looked like control, moderately ABA sensitive (ABA.sup.MS) if more than 1% but less than 50% of looked like control, or ABA insensitive (ABA.sup.Wt) if greater than 50% looked like control.

For example, if a plate had 20 healthy seedlings and the control plate had 60 healthy seedlings, the line would be 33% of control and categorized as moderately ABA sensitive.

All four vector constructs (pBI121-AtCPP, pBI121Hp-AtCPP, pRD29AHp-AtCPP, pRD29A-ATCPP) have resulted in transgenic lines of Arabidopsis which have increased sensitivity to ABA which is indicative of stress tolerance. The data for all 4 constructs is shown in FIG. 9. Of the lines transformed with the pBI121-AtCPP construct to over-express the AtCPP gene, 58% (21 out of 36) were classified as sensitive and an added 30% (11 out of 36) were classified as moderately sensitive. These lines were tested again in T4 and T5 generations and their ABA sensitivity was still present indicating that ABA sensitivity is an inheritable trait. Of the lines transformed with the pBI121-HP-AtCPP construct to down-regulate the AtCPP gene by double stranded RNA-inhibition, 15% (7 out of 45) were classified as sensitive and 31% (14 out of 45) were classified as moderately sensitive. To illustrate the increased sensitivity of transgenic lines to ABA, FIG. 10 shows the results of germination and seedling development over a range of ABA concentrations. Wild type and pRD29A-HP-AtCPP are compared. Of the lines transformed with pRD29AHp-AtCPP 70% (12 out of 17) showed high sensitivity and 24% (4 out of 17) showed moderate sensitivity to ABA. Of the lines transformed with pRD29A-AtCPP 29% (5 out of 17) showed high sensitivity and 12% (2 out of 17) moderate sensitivity to ABA. Clearly all 4 transgene constructs are altering ABA sensitivity and ABA signal transduction.

Example 28

Drought Experiments

Arabidopsis plants were grown five plants per 4'' or 3'' pot, in a replicated water-stress experiment. All pots were filled with equal amounts of homogeneous premixed and wetted soil. Plants were grown under 16 hour daylight (150-200 .mu.mol/m.sup.2/s) at 22.degree. C. and 70% relative humidity. On the day that the first flower opened drought treatment was initiated. First soil water content in each pot was equalized on a weight basis and any further watering of plants was stopped. Daily measurements of soil water content were taken by recording total pot weight. At the end of the drought treatment (6 to 9 days for experiments in 4'' pots and 4-5 days for experiments in 3'' pots) plants were harvested and shoot dry weights determined. Differences in plant growth were factored into the analysis by expressing water loss on a per gram shoot dry weight basis.

28a) pBI121-AtCPP, Drought Stress Screen:

Analysis of pBI121-AtCPP transgenic lines during water-stress treatment experiments of up to an eight day period, shows a strong trend towards increased soil water content and reduced water loss per gram of shoot biomass. After three days of water-stress treatment most lines had increased soil water content relative to the wild type control with four out of twenty-four lines, 146, 149, 156 and 97, showing a statistically significant difference. The amount of water lost per gram of shoot biomass was lower for all lines except one (95), and thirteen of these lines were significantly different from the wild type Columbia control (FIG. 11). All of the lines showing a statistically significant lower water loss per gram shoot biomass also showed an increased ABA sensitivity. There is also a strong trend, for all but one line (95), which is ABA.sup.Wt, towards greater shoot biomass at the end of the drought stress treatment. Seven of those lines 136, 146, 23, 46, 76, 84 and 9, were statistically significant from control at a p=0.05 value.

28b) pBI121-AtCPP, Water Loss Per Gram Shoot Biomass During Water Stress Treatment:

Lines 35, 76, 95 and a wild type control were grown and placed under a water-stress treatment as above. Plants were harvested at 2 days, 4 days and 6 days of drought treatment. The ABA.sup.S lines, 35 and 76, showed a statistically significant reduction in water-loss relative to shoot dry weight at all three time points (Table 8). Additionally, the two ABA.sup.S transgenic lines had increased shoot biomass, due to increased leaf biomass, and maintained higher soil water contents during drought treatment.

TABLE-US-00015 TABLE 8 Water loss (g) per Shoot dry weight (g) after 2, 4 and 6 days of drought-stress treatment. Values in bold indicate statistically significant differences from Columbia. 2 days 4 days 6 days Line Mean Std. Error Mean Std. Error Mean Std. Error 35 212.5 3.5 308.0 9.9 297.7 11.2 76 227.2 5.8 321.2 8.5 293.8 5.0 95 287.0 5.1 377.3 14.8 348.5 25.5 Columbia 265.3 11.8 408.2 7.7 345.9 6.7 Wild type

28c) pBI121-AtCPP, Drought Stress and Shoot Recovery:

Water-stress tolerance and determination of post drought-treatment recovery ability was assessed using 20 of the 24 pBI121-AtCPP transgenic lines. Drought treatment was imposed for 6 days after which the plants were watered and allowed to grow for 6 days. Recovered shoot fresh biomass was then determined. Soil water content of these plants was measured daily during the drought treatment and the results confirm previously seen trends. All ABA sensitive (ABA.sup.S) lines that showed a statistically significantly reduction of water loss on a per gram dry weight basis in experiment 34a, continued to show a significant greater soil water content than control plants in this experiment (Table 9). Additionally, Table 9 shows that the recovered shoot fresh biomass after 6 days of drought treatment was significantly greater in all the ABAs lines than Columbia.

TABLE-US-00016 TABLE 9 Soil water content on day 3 of drought treatment and recovered shoot fresh weight after 6 days of drought treatment (values in bold were significantly different from Columbia at p = 0.05) soil water content day 3 recovered shoot ABA status Mean (% biomass Line ABA initial) Std Error Mean (g) Std Error 136 ABA.sup.S 46.6 1.9 4.5 0.16 14 ABA.sup.S 50.25 0.7 4.1 0.12 146 ABA.sup.S 45.9 2.5 4.0 0.11 147 ABA.sup.S 45.1 1.7 4.0 0.15 149 ABA.sup.S 45.3 1.8 3.8 0.17 156 ABA.sup.S 47.1 1.9 4.0 0.134 23 ABA.sup.S 49 1.4 4.0 0.17 33 ABA.sup.S 46.9 1.6 4.3 0.14 35 ABA.sup.S 41.7 1.7 4.0 0.11 46 ABA.sup.S 44.8 1.7 3.8 0.09 63 ABA.sup.S 46.3 1.4 4.0 0.19 76 ABA.sup.S 47.8 1.0 3.9 0.17 79 ABA.sup.S 45.4 1.1 4.1 0.09 84 ABA.sup.S 46.8 1.9 4.1 0.16 85 ABA.sup.S 45.3 1.9 4.0 0.12 9 ABA.sup.S 45.2 2.1 3.9 0.12 93 ABA.sup.wt 43.5 1.2 2.8 0.07 94 ABA.sup.S 46.9 1.5 3.9 0.13 97 ABA.sup.S 53 1.2 3.8 0.16 95 ABA.sup.Wt 41.9 1.2 2.7 0.06 Columbia ABA.sup.Wt 41.3 1.0 2.7 0.04

28d) pBI121-AtCPP, Seed Yield After Drought Stress Treatment:

Seed yield after drought stress during flowering was examined using ten pBI121-AtCPP transgenic lines, eight of which were ABA.sup.S. Plants were grown one per 4'' pot and were exposed to 9 days of drought treatment as described above. A second group of plants was grown and maintained under well watered conditions as the optimal group. After 9 days of drought treatment plants were re-watered and allowed to continue growth and seed set to maturity. After drought-treatment conditions all eight ABA.sup.S lines had increased yields relative to controls, which ranged from 109% to 126% of the Columbia (Table 10). Drought-treatment resulted in a reduction of yield in all lines, including controls, relative to plants grown under optimal conditions. Expression of the seed yields obtained from drought-treated group relative to the same line under optimal conditions shows that the transgenics preserve a larger percentage of optimal seed yield than do wild type lines.

TABLE-US-00017 TABLE 10 Seed Yield following 9 days drought-treatment Seed Yield ABA status (g per plant) % Line ABA Mean (g) Std Error Columbia % Optimal 156 ABA.sup.S 0.735 0.044 126.2 83.7 63 ABA.sup.S 0.675 0.061 116.0 71.0 146 ABA.sup.S 0.666 0.053 114.4 72.9 94 ABA.sup.S 0.644 0.052 110.6 68.8 84 ABA.sup.S 0.642 0.049 110.4 61.8 76 ABA.sup.S 0.631 0.055 108.5 66.6 136 ABA.sup.S 0.630 0.051 108.3 74.1 35 ABA.sup.S 0.614 0.054 105.6 74.2 93 ABA.sup.Wt 0.567 0.041 97.5 60.0 95 ABA.sup.Wt 0.388 0.088 66.7 43.4 Columbia ABA.sup.Wt 0.582 0.060 100 53.8

28e) pBI121-AtCPP Seed Yield and Growth Under Optimal Water Conditions:

The lines evaluated above and a number of additional lines were examined in a growth and yield experiment under optimal, well-watered conditions. Results indicated that the ABA.sup.S lines were shorter at the stage of first open flower, had more rosette leaves, however, by maturity there were no differences in plant height of transgenics and Columbia. Moreover, the ABA.sup.S transgenics showed similar or higher seed yields ranging from 95% to 121% of the wild type control (FIG. 12).

28f) pRD29A-HP-AtCPP Screen for Drought Tolerant Phenotype:

Analysis of 17 transgenic lines identified 7 candidate drought tolerant lines (12, 22, 23, 47, 82, 83, 90) on the basis of higher soil water content and lower water loss per g of shoot dry weight (Table 11). All 7 drought tolerant candidate lines showed strong ABA sensitivity and lines that did not show drought tolerance did not show ABA sensitivity.

TABLE-US-00018 TABLE 11 Soil water content after 3 days of drought treatment and water lost per g shoot dry weight. Values in bold are statistically different from those of Columbia wild type (p = 0.05) soil water content water lost in day 2 2 days/g shootDW ABA status Mean Mean Line ABA (% initial) Std Error (g/g) Std Error 10 ABA.sup.S 33.4 1.6 199.1 4.5 11 ABA.sup.S 34.6 3.3 173.1 1.6 12 ABA.sup.S 36.2 2.0 179.5 5.0 126 ABA.sup.MS 32.5 2.6 199.1 4.1 127 ABA.sup.MS 33.5 2.0 195.6 10.6 14 ABA.sup.S 32.7 1.2 203 4.9 17 ABA.sup.S 29.9 1.8 200.7 7.3 22 ABA.sup.S 39.3 2.1 170.0 3.0 23 ABA.sup.S 35.7 1.4 174.9 2.6 42 ABA.sup.MS 28 0.7 185.4 5.8 47 ABA.sup.S 35.9 2.2 181.2 7.7 7 ABA.sup.Wt 35 1.3 201.8 5.1 82 ABA.sup.S 36.7 2.2 178.3 4.0 83 ABA.sup.S 40 1.4 180.7 6.9 9 ABA.sup.S 31.4 1.4 173.8 8.7 90 ABA.sup.S 38.2 1.3 177.6 6.2 93 ABA.sup.Wt 30.7 1.8 175.3 4.6 Columbia ABA.sup.Wt 32.1 1.2 196.9 6.2

Example 29

Growth Analysis

The growth analysis of most promising constructs has been set up at 3 stages. Eight plants per line were grown in 3'' pots with one plant per pot at 22 C, 16 hr light (150-200 .mu.mol/m.sup.2/s) and 70% RH. Plants were harvested at vegetative growth stage (2 week old seedlings), bolting growth stage (at first open flower) and mid-flowering growth stage (5 to 7 days from first open flower). Also, in some growth experiments additional group of plants was grown in 4'' pots (one per pot and 10 plants per line) to maturity for seed yield determinations.

29a) pBI121-AtCPP Growth Under Optimal and Biotic Stress Conditions

The growth and productivity of pBI121-AtCPP transgenic Arabidopsis lines was examined at several stages of development under optimal growth conditions. Although optimal growth conditions were maintained, plants were assessed to be under a degree of stress that was later determined to be a result of the soil properties. Soil analysis found a fungal contaminant that was believed to be responsible for the biotic stress. This stress could be negated by sterilization of the soil prior to use. Eight ABA.sup.S lines, two with normal ABA sensitivity (ABA.sup.Wt) and a wild type Columbia control were analyzed.

FIG. 13 presents the results of various growth (from mid-flowering stage) and yield parameters and each trait is expressed as a percentage of the Columbia control. The results strongly support an enhanced growth phenotype. This enhanced growth phenotype is present at all growth stages. At the vegetative stage, all ABA.sup.S transgenic plants showed an increase in leaf number relative to that of the wild type with four of the eight lines showing a statistically significant difference. The two ABA.sup.Wt lines showed the same or fewer leaves relative to wild type.

At the bolting stage ABA.sup.S transgenics showed an increase in leaf number but plants were shorter at this stage (first open flower) than controls. The shoot fresh weight of transgenics was significantly increased relative to that of controls, ranging from 80% to 342% of the wild type. The ABA.sup.S transgenics displayed a delay in flowering from one to three days. The ABA.sup.Wt transgenics did not show delayed flowering, increased shoot fresh weight or increased height.

At the flowering stage of development the enhanced growth phenotype is maintained (greater leaf number and fresh weight), however, there were no observable differences in plant height indicating that transgenics bolt shorter but reach same final plant height.

Of particular significance is the observation, that under these conditions (biotic stress due to presence of fungi in the soil) yields of the ABA.sup.S transgenics were significantly higher, ranging from 120% to 229% of the wild type control. The ABA.sup.Wt lines showed similar or slightly reduced yields relative to the Columbia control. This finding indicates that ABA.sup.S transgenic lines are affected less by the biotic stress. This observation has been confirmed, where 5 of the drought tolerant lines were grown in contaminated soil to maturity. The seed yields of transgenic lines, even though greatly reduced relative to optimal conditions, were 2.5 to 4.5 fold higher than those of Columbia wild type (Table 12).

TABLE-US-00019 TABLE 12 Seed yield of pBI121-AtCPP lines grown in contaminated soil. Values in bold indicate statistical differences at p = 0.05 ABA Seed Yield per plant Line sensitivity (g) % of Columbia 156 ABA.sup.S 0.33 .+-. 0.04 316% 23 ABA.sup.S 0.35 .+-. 0.05 336% 76 ABA.sup.S 0.31 .+-. 0.04 296% 84 ABA.sup.S 0.25 .+-. 0.33 237% 9 ABA.sup.S 0.48 .+-. 0.05 455% Columbia ABA.sup.Wt 0.11 .+-. 0.03

29b) pBI121-AtCPP Early Seedling Growth:

Four ABA.sup.S and one ABA.sup.Wt line plus Columbia were examined for early seedling growth on agar plates. Twenty seeds were plated in a line on agar plates containing 50% MS with 1% sucrose and vitamins and 6 plates per line were used. Plates were placed on slants, which allowed roots to grow downwards. Root length was measured on 7-day old seedlings and shoot and root biomass determined on 11-day old seedlings. Two of the ABA.sup.S transgenic lines had significantly longer roots and all 4 ABA.sup.S lines had shoot dry weights 114% to 123% of controls and root dry weights of 116% to 151% of controls. As a result, the shoot biomass to rootbiomass ratios were slightly reduced in transgenics. These results indicate that enhanced growth of these transgenics is evident in the early growth stage, shortly after germination, and the root growth is more enhanced relative to shoot growth. In a different experiment seedlings were pulled out of agar and roots were stained with toluidine blue to show their structure. FIG. 14 shows that transgenic lines had more extensive lateral root system, which would account for greater root biomass.

29c) pRD29A-HP-AtCPP Optimal Growth Characteristics

An optimal growth study has been conducted with 10 lines as described before. Vegetative growth data showed that two of the lines (12 and 9) had significantly more leaves and seven of the lines (12, 22, 23, 47, 82, 9) had significantly greater shoot biomass. Bolting data showed that eight of the lines (12, 22, 23, 47, 82, 9, 90, 93) were significantly delayed in flowering by one to two days, and seven of the lines were significantly shorter than Columbia at first open flower. All of the lines except 42 and 7 had significantly greater number of rosette leaves and shoot FW and this trend is maintained into the mid-flowering harvest (FIG. 15). The plant height, however, by mid-flowering harvest was not significantly different between the transgenic lines and control. All the lines that showed this enhanced growth also showed drought tolerance and ABA sensitivity.

Example 30

Ultrastructure pBI121-AtCPP

Two of the drought tolerant and ABA.sup.S lines (35 and 76) plus Wt Columbia were used to examine stem and root cross-sections for any differences in ultrastructure. Free hand sections of mature stems (plants flowering for 10 days) were obtained from above the first node, stained with toluidine blue and preserved with glycerol. The stems of transgenic plants appeared to have more dense cellular structure and contain one or two more vascular bundles than those of Columbia Wt indicating more enhanced water and nutrient transport system.

Leaf disks were taken and fresh weights determined. Transgenic leaf disks were significantly heavier, 20-24% greater than corresponding wild type controls. This increase is believed to be as a result of a thicker leaf.

Example 31

Cold Stress Experiment pBI121-AtCPP

Four drought tolerant, ABA.sup.S lines (156, 23, 35, 76) and one ABA.sup.Wt (95) line plus wild type Columbia were included in a cold stress study. Plants were grown in 3'' pots one per pot) with 10 replicate pots per line at 22 C for 10 days (7 days on agar plates and 4 in soil). The cold stress group was moved into 7.degree. C. for 5 days while the optimal group was left at 22 C. After 5 days in the cold both cold stress group and the optimal group were harvested for shoot biomass determination. ABA.sup.S and drought tolerant lines had significantly greater shoot biomass than Columbia in both optimal (25 to 39% greater shoot fresh weight) and cold stress groups (18 to 44% greater shoot DW) (Table 13). Results of an eight-day cold stress showed that differences between the transgenic lines and Columbia were even more pronounced (53 to 61% greater shoot fresh weight). This result indicates greater plant vigor and better ability of transgenics to cope with cold stress.

TABLE-US-00020 TABLE 13 Shoot fresh weight of optimal and cold stressed (5 C. for 5 d) pBI121-AtCPP. Values in bold indicate statistical difference at p = 0.05 Optimal shoot FW Cold stress shoot FW ABA % of % of Line sensitivity mg Columbia mg Columbia 156 ABA.sup.S 95.4 .+-. 3.7 137% 23.1 0.7 118% 23 ABA.sup.S 96.3 .+-. 3.9 139% 28.3 1.5 144% 35 ABA.sup.S 87.0 .+-. 1.7 125% 25.3 1.4 130% 76 ABA.sup.S 94.7 .+-. 2.2 136% 27.3 1.5 140% 95 ABAWt 67 .+-. 2.4 96% 21.4 1.0 109% Columbia ABAWt 69 .+-. 1.9 19.6 1.1

Example 32

Drought Stress Under High Temperature pBI121-AtCPP

A drought stress experiment was conducted as described above except that day temperature of 32.degree. C. (16 hr) and night temperature of 22.degree. C. (8 hr) was maintained. These temperatures were achieved daily over a 2 hr ramping period. Four ABA.sup.S and one ABA.sup.Wt line plus Columbia were included. Plants were monitored daily for water loss and soil water content and after 5 days of drought treatment half of the plants were harvested and the other half was re- and watered and allowed to recover for four days. Shoots were harvested and shoot fresh weight determined. The results (Table 14) of this experiment showed that previously identified drought tolerant lines maintained their drought tolerant phenotype at high temperature and were able to recover well from the drought stress at high temperature

TABLE-US-00021 TABLE 14 Soil water content on day 2 and water lost in 2 days/final shoot dry weight plus recovery shoot FW after 5 days of drought stress at 32 C. day and 22 C. night temperatures. Values in bold indicate significant differences from the Columbia control. ABA soil water water lost in recovered shoot line sensitivity content day 2 2 d/shoot DW FW (g) 136 ABA.sup.S 50.4 .+-. 1.1 485.7 .+-. 18.5 1.30 .+-. 0.04 146 ABA.sup.S 52.1 .+-. 1.0 504.5 .+-. 7.9 1.15 .+-. 0.04 35 ABA.sup.S 52.2 .+-. 0.8 502.8 .+-. 15.8 1.19 .+-. 0.02 76 ABA.sup.S 52.1 .+-. 0.6 435.6 .+-. 10.5 1.11 .+-. 0.03 95 ABAWt 50.0 .+-. 0.9 518.2 .+-. 13.0 0.86 .+-. 0.03 Columbia ABAWt 48.6 .+-. 0.6 559.7 .+-. 19.0 0.84 .+-. 0.03

Example 33

Heat Stress and Seed Yield pBI121-AtCPP

Two ABA.sup.S lines and one ABA.sup.Wt line plus Columbia were examined for the effect of heat stress during flowering on the final seed yield. Plants were grown in 4 inch pots (one/pot) as described above and 9 days from first open flower the temperature was ramped from 22 C to 43 C over 2 hours and plants were kept at 43 C for 2 hr. Temperature was then ramped back to 22 C over 2 hours and plants were grown under optimal conditions until maturity. The seed yields from this experiment are shown in Table 15. One of the drought-tolerant lines (35) had significantly greater yield than Columbia.

TABLE-US-00022 TABLE 15 Seed yield of pBI121-AtCPP lines after two hour 43 C. heat stress 9 days from first open flower. Values in bold are statistically significant from Columbia. ABA seed yield seed yield line sensitivity (g/plant) (% of col.) 35 ABA.sup.S 0.55 .+-. 0.05 347% 76 ABA.sup.S 0.24 .+-. 0.03 148% 95 ABAWt 0.11 .+-. 0.02 69% Columbia ABAWt 0.16 .+-. 0.03

The effect of heat shock on lines of pBI121-AtCPP at the early flowering stage was assessed. Three ABA.sup.S lines (76, 136, 97) a ABA.sup.Wt line (95) and a Columbia wild type control were seeded in 128 cell flats, one flat per line. At the early flowering stage flats were exposed to a temperature of 46.8.degree. C. for 50 minutes and then returned to normal growth conditions. Lack of continued growth from main meristems was defined as main meristem death and scored for each line. Data is shown in Table 16.

TABLE-US-00023 TABLE 16 Meristem death due to heat shock Line Wt 95 76 136 97 % Death 91 97 79 59 18

Example 34

Stomata Density Determinations pBI121AtCPP

Two ABA.sup.S lines (76 and 35) plus Columbia were examined for stomata density on the upper and lower leaf surface. Nail polish imprints of the upper and lower epidermis were obtained from a fully expanded leaf #5. These imprints were analyzed under the microscope and the number of stomata per 8.7.times.10.sup.-8 m.sup.2 were counted. There were no significant differences found between transgenics and Columbia in the stomata of the upper or lower epidermis (Table 17). The increases seen in drought tolerance and reduced water loss is not attributable to a reduced number of leaf stomata.

TABLE-US-00024 TABLE 17 Stomata numbers per 8.7 .times. 10.sup.-8 m.sup.2 of abaxial and adaxial epidermis of fully expanded leaf #5 in pBI121AtCPP. stomata stomata ABA on upper on lower line sensitivity epidermis epidermis 35 ABA.sup.S 68 .+-. 5 103 .+-. 7 76 ABA.sup.S 58 .+-. 6 120 .+-. 16 Columbia ABAWt 57 .+-. 6 116 .+-. 11

Example 35

PrPase Consensus Sequences

Also included in the invention are the PrPase consensus sequences. The consensus sequences were generated by alignment of the PrPase polypeptide and nucleic acid sequences.

The consensus sequence for the nucleic acid sequence alignment of GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11), ZmPrPase2 (SEQ ID NO: 23), PpPrPase1 (SEQ ID NO: 1), and three other disclosed Arabidopsis PrPases sequences (AT4g01320 (SEQ ID NO: 27); AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 4 is provided in SEQ ID NO: 84. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 5 is provided in SEQ ID NO: 85.

The consensus sequence for the nucleic acid alignment of the PrPase sequences of the invention from dicot plants (GmCPP (SEQ ID NO: 18), GmPrPase2 (SEQ ID NO: 16), AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5), BnCPP (SEQ ID NO: 11)) and three other disclosed dicot PrPases sequences (AT4g01320 (SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 16 is provided in SEQ ID NO: 86. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 17 is provided in SEQ ID NO: 87.

The consensus sequence for the nucleic acid alignment of the Arabidopsis PrPase sequences of the invention (AtCPP (SEQ ID NO: 7), AtPrPase1 (SEQ ID NO: 3), AtPrPase2 (SEQ ID NO: 5)) and three other disclosed Arabidopsis PrPases sequences (AT4g01320 (SEQ ID NO: 27), AF007269 (SEQ ID NO: 29), and AFC1 (SEQ ID NO: 25)) as shown in FIG. 18 is provided in SEQ ID NO: 88. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 19 is provided in SEQ ID NO: 89.

The consensus sequence for the nucleic acid alignment of the soybean PrPase sequences of the invention: GmCPP (SEQ ID NO: 18) and GmPrPase2 (SEQ ID NO: 16) as shown in FIG. 20 is provided in SEQ ID NO: 90. The consensus sequence for the corresponding amino acid sequence alignment shown in FIG. 21 is provided in SEQ ID NO: 91.

The "X" in the consensus sequence represents any amino acid and the "N" represents any nucleotide. Preferably "X" is a conservative amino acid substitution and the "N" a conservative nucleotide substitution. More preferably, "X" is the amino acid and "N" the nucleotide most prevalent at a given position.

SEQUENCE LISTINGS

1

9111398DNAPhyscomitrella patens 1ggatcccccg ggctgcagga attcggcacg agctcaagct gtccaatctg ccagcgcctc 60tcaagggaat agttagtcaa gagaaatttg agaaagcgca ggcgtacagc ttagacaaga 120gccgattcca ttttgtgcac gcggctgtga atatcgtgga ggaatcggca attcttctgc 180tggggttgtt gccgtgggcg tgggataaga gtggatcgtt agtagggaag ctagggtttg 240atgagaagag cgaaattttg cagacgcttt cttttcttgc ggtgaccacg ttgtggtcgc 300agatacttga gcttccattc tcgctctact ccacgtttgt catcgaggcc cgccatggct 360tcaacaagca aaccatatgg ttgtttttac gggatatgat catggggctg gctctcatga 420tggtggttgg cccacccata gtgtcggcaa ttatctatat tgtgcagaac ggtgggccat 480atcttgccct ctatctgtgg gcctttatgt tgctgttatc cctcgtgttg atggccctat 540atcccgttct catcgcgcct cttttcaaca cattcacacc cttgccagaa gggcagcttc 600gtgccaagat cgagaagctg gcatcctcct tggacttccc attgaagaaa ttgtttgtaa 660ttgacggttc tactcggtca agccatagca acgcctacat gtatggattt tacaacagca 720agcgcatcgt tctgtacgac actctaatat cgcaatgtaa gaatgaggaa gaagtagtgg 780cagttatagc tcatgagctt ggccattgga agctgagcca cactatgtac tcgttcctgg 840ccatgcaggt gcttacactg ttgcaattcg gaggctatac gcttgttcgg aactctagtg 900gcctgttttt gagcttcggt ttctccacac agccagtgct tatcgggctg atcctattcc 960agcacactat tatgcccttc catcatcttg taagctttgc tctcaacctg ttgagccgag 1020ccttcgaatt tcaggcggat gcgttcgccc gctcattagg gtacagagag ccattgagag 1080ctggcctgat caagctgcag gaggagaatc tgtctgccat gaacacggat ccgtggtatt 1140cagcgtatca tcattcacac cccccgcttg ttgagcgatt gcaagctctt gatgaaacgt 1200ccaagaaaac ggattagaac ttaccccctt cggaccgtag ttgagatttg taggaatata 1260gcttcttcag gagaaagaaa caaaatgagc tatgtcctag cacatccact gtagaattca 1320ctgatgaatg acgaatagta catgaacact cattctttaa aaaaaaaaaa aaaaaactcg 1380agggggggcc cggtaccc 13982394PRTPhyscomitrella patens 2Leu Lys Leu Ser Asn Leu Pro Ala Pro Leu Lys Gly Ile Val Ser Gln 1 5 10 15Glu Lys Phe Glu Lys Ala Gln Ala Tyr Ser Leu Asp Lys Ser Arg Phe 20 25 30His Phe Val His Ala Ala Val Asn Ile Val Glu Glu Ser Ala Ile Leu 35 40 45Leu Leu Gly Leu Leu Pro Trp Ala Trp Asp Lys Ser Gly Ser Leu Val 50 55 60Gly Lys Leu Gly Phe Asp Glu Lys Ser Glu Ile Leu Gln Thr Leu Ser 65 70 75 80Phe Leu Ala Val Thr Thr Leu Trp Ser Gln Ile Leu Glu Leu Pro Phe 85 90 95Ser Leu Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys 100 105 110Gln Thr Ile Trp Leu Phe Leu Arg Asp Met Ile Met Gly Leu Ala Leu 115 120 125Met Met Val Val Gly Pro Pro Ile Val Ser Ala Ile Ile Tyr Ile Val 130 135 140Gln Asn Gly Gly Pro Tyr Leu Ala Leu Tyr Leu Trp Ala Phe Met Leu145 150 155 160Leu Leu Ser Leu Val Leu Met Ala Leu Tyr Pro Val Leu Ile Ala Pro 165 170 175Leu Phe Asn Thr Phe Thr Pro Leu Pro Glu Gly Gln Leu Arg Ala Lys 180 185 190Ile Glu Lys Leu Ala Ser Ser Leu Asp Phe Pro Leu Lys Lys Leu Phe 195 200 205Val Ile Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr 210 215 220Gly Phe Tyr Asn Ser Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Ser225 230 235 240Gln Cys Lys Asn Glu Glu Glu Val Val Ala Val Ile Ala His Glu Leu 245 250 255Gly His Trp Lys Leu Ser His Thr Met Tyr Ser Phe Leu Ala Met Gln 260 265 270Val Leu Thr Leu Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser 275 280 285Ser Gly Leu Phe Leu Ser Phe Gly Phe Ser Thr Gln Pro Val Leu Ile 290 295 300Gly Leu Ile Leu Phe Gln His Thr Ile Met Pro Phe His His Leu Val305 310 315 320Ser Phe Ala Leu Asn Leu Leu Ser Arg Ala Phe Glu Phe Gln Ala Asp 325 330 335Ala Phe Ala Arg Ser Leu Gly Tyr Arg Glu Pro Leu Arg Ala Gly Leu 340 345 350Ile Lys Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp 355 360 365Tyr Ser Ala Tyr His His Ser His Pro Pro Leu Val Glu Arg Leu Gln 370 375 380Ala Leu Asp Glu Thr Ser Lys Lys Thr Asp385 39031275DNAArabidopsis thaliana 3atggcgattc ctttcatgga aaccgtcgtg ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacagtct tgacaaaagc 180tattttcact ttgttcatga gtttgtaact atacttatgg actctgcaat tttgttcttt 240gggatcttgc cttggttttg gaagatgtct ggagctgttt taccgaggtt gggccttgat 300ccagagaatg aaatactgca tactctttca ttcttggctg gtgttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaacatt cctctctgtc 480atactaggcc cacccattgt tgccgcgata attttcatag tccagaaagg aggtccttat 540cttgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600ccggtcttga tagcaccgct cttcaacaag ttcactcctc ttccagatgg agacctccgg 660gagaagattg agaaacttgc ttcttctcta aagtttcctt tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagcaat gcttacatgt atggtttctt taagaacaaa 780aggattgttc tttatgatac gttgattcag cagtgcaaga atgaggatga aattgtggcg 840gttattgcac acgagcttgg acattggaaa ctgaatcaca ctacatactc gttcattgca 900gttcaaatcc ttgccttctt acaatttgga ggatacactc ttgtcagaaa ctccactgat 960ctcttcagga gtttcggatt tgatacacag cctgttctca ttggtttgat catatttcag 1020cacactgtaa taccactgca acatccagta agctttggcc tcaaccttgt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgctgtg aagcttggct atgcaaaaga tcttcgtcct 1140actctagtga aactacagga agagaactta tcagcaatga atactgatcc attgtactca 1200gcttatcact actcacatcc tcctcttgtt gaaaggcttc gagccattga tggagaagac 1260aagaagacag attaa 12754424PRTArabidopsis thaliana 4Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser Tyr Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Leu Met Asp Ser Ala Ile Leu Phe Phe 65 70 75 80Gly Ile Leu Pro Trp Phe Trp Lys Met Ser Gly Ala Val Leu Pro Arg 85 90 95Leu Gly Leu Asp Pro Glu Asn Glu Ile Leu His Thr Leu Ser Phe Leu 100 105 110Ala Gly Val Met Thr Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val145 150 155 160Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 180 185 190Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln Ile Leu 290 295 300Ala Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Thr Asp305 310 315 320Leu Phe Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln His Pro Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Val Lys Leu Gly Tyr Ala Lys Asp Leu Arg Pro Thr Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Leu Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Arg Ala Ile 405 410 415Asp Gly Glu Asp Lys Lys Thr Asp 42051275DNAArabidopsis thaliana 5atggcgattc ctttcatgga aaccgtcgtg ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacagtct tgacaaaagc 180tattttcact ttgttcatga gtttgtaact atacttatgg actctgcaat tttgttcttt 240gggatcttgc cttggttttg gaagatgtct ggagcagttt taccgaggtt gggccttgat 300ccagagaatg aaatactgca tactctttca ttcttggctg gtgttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaacatt cctctctgtc 480atactaggcc cacccattgt tgctgcgata attttcatag tccagaaagg aggtccttat 540cttgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600ccggtcttga tagcaccgct cttcaacaag ttcactcctc ttccagatgg agacctccgg 660gagaagattg agaaacttgc ttcttctcta aagtttcctt tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagcaat gcttacatgt atggtttctt taagaacaaa 780aggattgttc tttatgatac gttgattcag cagtgcaaga atgaggatga aattgtggcg 840gttattgcac acgagcttgg acattggaaa ctgaatcaca ctacatactc gttcattgca 900gttcaaatcc ttgccttctt acaatttgga ggatacactc ttgtcagaaa ctccactgat 960ctcttcagga gtttcggatt tgatacacag cctgttctca ttggtttgat catatttcag 1020cacactgtaa taccactgca acatctagta agctttggcc tgaacctcgt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgctgtg aagcttggct atgcaaaaga tcttcgtcct 1140gctctagtga aactacagga agagaactta tcagcaatga acactgatct attgtactca 1200gcttatcact actcacatcc tcctcttgtt gaaaggcttc gagccattga tggagaagac 1260aagaagacag attaa 12756424PRTArabidopsis thaliana 6Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser Tyr Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Leu Met Asp Ser Ala Ile Leu Phe Phe 65 70 75 80Gly Ile Leu Pro Trp Phe Trp Lys Met Ser Gly Ala Val Leu Pro Arg 85 90 95Leu Gly Leu Asp Pro Glu Asn Glu Ile Leu His Thr Leu Ser Phe Leu 100 105 110Ala Gly Val Met Thr Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val145 150 155 160Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 180 185 190Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln Ile Leu 290 295 300Ala Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Thr Asp305 310 315 320Leu Phe Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln His Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Val Lys Leu Gly Tyr Ala Lys Asp Leu Arg Pro Ala Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Leu Leu Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Arg Ala Ile 405 410 415Asp Gly Glu Asp Lys Lys Thr Asp 42071275DNAArabidopsis thaliana 7atggcgattc ctttcatgga aaccgtcgtg ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacagtct tgacaaaagc 180tattttcact ttgttcatga gtttgtaact atacttatgg actctgcaat tttgttcttt 240gggatcttgc cttggttttg gaagatgtct ggagctgttt taccgaggtt gggccttgat 300ccggagaatg aaatactgca tactctttca ttcttggctg gtgttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaacatt cctctctgtc 480atactaggcc cacccattgt tgctgcgata attttcatag tccagaaagg aggtccttat 540cttgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600ccggtcttga tagcaccgct cttcaacaaa ttcactcctc ttccagatgg agacctccgg 660gagaagattg agaaacttgc ttcttcccta aagtttcctt tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagcaat gcttacatgt atggtttctt taagaacaaa 780aggattgttc tttatgatac gttgattcag cagtgcaaga atgaggatga aattgtggcg 840gttattgcac acgagcttgg acattggaaa ctgaatcaca ctacatactc gttcattgca 900gttcaaatcc ttgccttctt acaatttgga ggatacactc ttctcagaaa ctccactgat 960ctcttcagga gtttcggatt tgatacacag cctgttctca ttggtttgat catatttcag 1020cacactgtaa taccactgca acatctagta agctttggcc tgaacctcgt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgctgtg aagcttgact atgcaaaaga tcttcgtcct 1140gctctagtga aactacagga agagaactta tcaacaatga acactgatcc attgtactca 1200gcttatcact actcacatcc tcctcttgtt gaaaggcttc gagccactga tggagaagac 1260aagaagacag attaa 12758424PRTArabidopsis thaliana 8Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser Tyr Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Leu Met Asp Ser Ala Ile Leu Phe Phe 65 70 75 80Gly Ile Leu Pro Trp Phe Trp Lys Met Ser Gly Ala Val Leu Pro Arg 85 90 95Leu Gly Leu Asp Pro Glu Asn Glu Ile Leu His Thr Leu Ser Phe Leu 100 105 110Ala Gly Val Met Thr Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val145 150 155 160Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 180 185 190Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln Ile Leu 290 295 300Ala Phe Leu Gln Phe Gly Gly Tyr Thr Leu Leu Arg Asn Ser Thr

Asp305 310 315 320Leu Phe Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln His Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Val Lys Leu Asp Tyr Ala Lys Asp Leu Arg Pro Ala Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Thr Met Asn Thr Asp Pro Leu Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Arg Ala Thr 405 410 415Asp Gly Glu Asp Lys Lys Thr Asp 42091275DNAArtificial SequenceDescription of Artificial Sequence sequence complementary to seq id no 7 9ttaatctgtc ttcttgtctt ctccatcagt ggctcgaagc ctttcaacaa gaggaggatg 60tgagtagtga taagctgagt acaatggatc agtgttcatt gttgataagt tctcttcctg 120tagtttcact agagcaggac gaagatcttt tgcatagtca agcttcacag caaaagcatc 180agcctgaaac tcaaacgctc gactaacgag gttcaggcca aagcttacta gatgttgcag 240tggtattaca gtgtgctgaa atatgatcaa accaatgaga acaggctgtg tatcaaatcc 300gaaactcctg aagagatcag tggagtttct gagaagagtg tatcctccaa attgtaagaa 360ggcaaggatt tgaactgcaa tgaacgagta tgtagtgtga ttcagtttcc aatgtccaag 420ctcgtgtgca ataaccgcca caatttcatc ctcattcttg cactgctgaa tcaacgtatc 480ataaagaaca atccttttgt tcttaaagaa accatacatg taagcattgc tatggcttga 540ccttgtagat ccatcgacaa caaacagctt cttcaaagga aactttaggg aagaagcaag 600tttctcaatc ttctcccgga ggtctccatc tggaagagga gtgaatttgt tgaagagcgg 660tgctatcaag accgggtata tagtcatcat cactagagac aggataaaca tgaatgccca 720cagatagatg gcaagataag gacctccttt ctggactatg aaaattatcg cagcaacaat 780gggtgggcct agtatgacag agaggaatgt tcctttgatc atgtccctaa tgaacatcca 840tattgtttgt ttgttgaacc catgccgaga ctcgatcacg aaagttgagt acaaagaaaa 900tggcaaatca gtgatctgtg accatgtcat aacaccagcc aagaatgaaa gagtatgcag 960tatttcattc tccggatcaa ggcccaacct cggtaaaaca gctccagaca tcttccaaaa 1020ccaaggcaag atcccaaaga acaaaattgc agagtccata agtatagtta caaactcatg 1080aacaaagtga aaatagcttt tgtcaagact gtatgctcgt gatttctcaa acttctcttg 1140gctaattaca ccaaccaagg ttttcgggag agttggaagc ttgagagcag tgagttgcct 1200cagatccaaa tacgtctcaa aaatgtacat cactatcata aaacccacga cggtttccat 1260gaaaggaatc gccat 1275102047DNAArabidopsis thaliana 10ttgatgatga aaaagagaag atcgaaacca agtcttacaa agaagagcga tttctctcaa 60aggggaaggt aaaattgaca aatccacgcg ctagcttttc acgttcactc acgcgctaca 120ttttgtataa tccacaaaac tttcaataaa ttacagaaaa accttgataa atttttacca 180taacaacaag atccctgata ttattttcaa attgactcat aaagcattac aaaaggagat 240ggtttttctg aaacatgaaa tggttggtta cagaagacga tacatacaat aggcagctat 300gttcatcatc tctttccttt tcctttagca tcaaagtgat gagactttag tttcttcttc 360cgcactatcg cgcctgtgct gccaccacct ccttccctga aaggcattcc cattagagcc 420aatagtttct gtccttcttg atcgctttta gccgttgtgc tgatgcatac atccattcct 480ctcgtttttc caacggcatc aaacctagag tgtaataaac caaccaacca acatagagtt 540tagttgctcg gttgtaaaca aagagtggga ctaaagagaa accatgcttg atcttgttga 600accagcggat aacaagttag tagttttgat tttgagggta gatacaatag aactaacctg 660atttcaggga atacaccttg gtctttcaca ccaatactgt agtttccgtt cccatcaaag 720ctactgggac tcacaccttg gaaatctcga gttctcggaa gggctaagtt gataagacga 780tccaagaagg agtacattac ctgtcacaac agtcaaaatc acaaaatgtt aatacataaa 840taactcaaaa ggaaagtttg attagtcaag agaaatatag ctcacatctc ctctgagagt 900gacagcaatc ccaagaggtt gatcttccct gatcttgaaa gtagcaatgg aagctctagc 960tcgtgtctta ataggtttct gccctgtgat aagcgcgata tccttcatcg cagcctccaa 1020acccttgtcg ttctgcgccg catctccaat accacaattc actacaatct tctgtacctt 1080tggaacctgg tagagaagat gtccaagcag atatacaaca atgatgagat tgacaaattc 1140acattcacat ttgtaaatgg cacatcccaa cagcacgatt ttatcaacac aacaatctta 1200aatctaggct tcttcactac attttctaga agaaatcaat tcaaacttat cattaaaagc 1260aaattaggtt taacattcgc tcaaaaaatt cgaattcact gagaattaga cttcaatcaa 1320tcgcaacaga acaaaaacta ggtttgagct cagaggggaa ggatttgggg ttacctggtg 1380aatattaacg tacttgaact cttctttgag cgcagggata atcctctcga ggtaagcggt 1440tttgaggcgt tgagttttct cggcttcaga tttctcgacc agtacagttc cagacgccga 1500gactttcacc acgtttctga gcggcggaga gagcattcgt gcggaggatg gagccgctaa 1560tggtgagaaa cgtccgtgaa acgaagaagc ggaagactgc agaagcgaag gagacgccat 1620tgtcgaagct ccaagtggat aaagtgtgaa gtgagagctc tcggcgttcg ttgttgatta 1680aacccaatgg caccttcgta atttgttgac agtttgagga taaggagttt tccgtttagc 1740ccctttaaaa cataatattt caactaaggc cccaatattt gataatatac tattatactt 1800agagatttta gataaaatat aggggttgtt tcatatgggc ctaaatctca gcccgtttac 1860tatttgggct tctaaggtat aacccgtacc cgtgtttttg ttgttttaca tatccacacc 1920gacctgagaa gagtcaaaaa cgaaaaacct ctcttttgtc gttcctctgc tttcttcgat 1980ttgcttctgc tttcttcgat ttgcttctgc tttcatcgcg gttcaggtca ctcttttctc 2040agccatg 2047111275DNABrassica napus 11atggcgattc ctttcatgga aaccgtcgtt ggttttatga tagtgatgta cgtttttgag 60acgtatttgg atctgaggca acatactgct ctcaagcttc ccactctccc aaagactttg 120gttggagtca ttagccaaga gaagtttgag aaatctcgag cttacagtct tgacaaaagc 180cattttcact ttgttcatga gtttgttact atacttatgg actctgcgat tctgttcttt 240gggatcttgc cttggttttg gaagatatct ggcggctttc taccaatggt gggactcgat 300ccagagaatg aaatcctgca cactctttca ttcttggctg gtcttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaatact cctctctgtc 480atacctgccc ctcctatcgt tgccgcaatt attgttatag ttcagaaagg aggtccttac 540ctcgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600cctgttttga ttgcacctct tttcaacaag ttcactcctc ttcctgatgg agacctccgg 660gagaagattg agaaacttgc ttcttctcta aagtttcctc tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagtaat gcttacatgt atggtttctt caagaacaaa 780aggattgttc tttatgacac attgattcag cagtgccaga atgagaatga aattgtggcg 840gttattgcac acgagctggg acactggaag ctgaatcaca ctacatactc gttcattgct 900gttcaaatcc ttgccttctt gcaatttgga ggatacactc ttgtcagaaa ctccactgat 960ctcttcagga gttttggttt tgatacacaa ccagttctca ttggtttgat catatttcag 1020cacactgtaa taccacttca acacctagta agctttgacc tcaaccttgt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgcagtg aatcttggtt atgcaaagga tctacgtcct 1140gccctagtga agctacagga agagaactta tcagcgatga acacagaccc attgtactca 1200gcttatcact actcacaccc tcctcttgta gagaggcttc gagccattga tggagaagac 1260aagaagacag attaa 127512424PRTBrassica napus 12Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Val Phe Glu Thr Tyr Leu Asp Leu Arg Gln His Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser His Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Leu Met Asp Ser Ala Ile Leu Phe Phe 65 70 75 80Gly Ile Leu Pro Trp Phe Trp Lys Ile Ser Gly Gly Phe Leu Pro Met 85 90 95Val Gly Leu Asp Pro Glu Asn Glu Ile Leu His Thr Leu Ser Phe Leu 100 105 110Ala Gly Leu Met Thr Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Ile Leu Leu Ser Val145 150 155 160Ile Pro Ala Pro Pro Ile Val Ala Ala Ile Ile Val Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 180 185 190Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Gln Asn Glu Asn Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln Ile Leu 290 295 300Ala Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Thr Asp305 310 315 320Leu Phe Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln His Leu Val Ser Phe 340 345 350Asp Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Val Asn Leu Gly Tyr Ala Lys Asp Leu Arg Pro Ala Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Leu Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Arg Ala Ile 405 410 415Asp Gly Glu Asp Lys Lys Thr Asp 420131275DNAArtificial SequenceDescription of Artificial Sequence complementary sequence to Seq id no 11 13ttaatctgtc ttcttgtctt ctccatcaat ggctcgaagc ctctctacaa gaggagggtg 60tgagtagtga taagctgagt acaatgggtc tgtgttcatc gctgataagt tctcttcctg 120tagcttcact agggcaggac gtagatcctt tgcataacca agattcactg caaaagcatc 180agcctgaaac tcaaacgctc gactaacaag gttgaggtca aagcttacta ggtgttgaag 240tggtattaca gtgtgctgaa atatgatcaa accaatgaga actggttgtg tatcaaaacc 300aaaactcctg aagagatcag tggagtttct gacaagagtg tatcctccaa attgcaagaa 360ggcaaggatt tgaacagcaa tgaacgagta tgtagtgtga ttcagcttcc agtgtcccag 420ctcgtgtgca ataaccgcca caatttcatt ctcattctgg cactgctgaa tcaatgtgtc 480ataaagaaca atccttttgt tcttgaagaa accatacatg taagcattac tatggcttga 540ccttgtagat ccatcgacaa caaacagctt cttcagagga aactttagag aagaagcaag 600tttctcaatc ttctcccgga ggtctccatc aggaagagga gtgaacttgt tgaaaagagg 660tgcaatcaaa acagggtata tagtcatcat cactagagac aggataaaca tgaatgccca 720cagatagatg gcgaggtaag gacctccttt ctgaactata acaataattg cggcaacgat 780aggaggggca ggtatgacag agaggagtat tcctttgatc atgtccctaa tgaacatcca 840tattgtttgt ttgttgaacc catgccgaga ctcgatcacg aaagttgagt acaaagaaaa 900tggcaaatca gtgatctgtg accatgtcat aagaccagcc aagaatgaaa gagtgtgcag 960gatttcattc tctggatcga gtcccaccat tggtagaaag ccgccagata tcttccaaaa 1020ccaaggcaag atcccaaaga acagaatcgc agagtccata agtatagtaa caaactcatg 1080aacaaagtga aaatggcttt tgtcaagact gtaagctcga gatttctcaa acttctcttg 1140gctaatgact ccaaccaaag tctttgggag agtgggaagc ttgagagcag tatgttgcct 1200cagatccaaa tacgtctcaa aaacgtacat cactatcata aaaccaacga cggtttccat 1260gaaaggaatc gccat 1275141434DNAGlycine max 14ctaatacgac tcactatagg gcaagcagtg gtaacaacgc agagtacgcg gggggagacg 60catggttctg aactaattgt tataaataat acctaaaatt ttgagttgtc ctaaacattg 120gggtttaaac aaatccaatc tctcaatata aaacccaatg atctcaccct cactccgttt 180ctgatttctc actcttcgtt tctcgttcgg ttcatcagcg tgtgtctcag ccatggcgtt 240tccctacatg gaagccgttg tcggatttat gatattaatg tacatttttg aaacttactt 300ggatgtgcga caacataggg ccctcaaact tcctactctt ccaaagactt tagaaggtgt 360tatcagccaa gagaaatttg agaaatctag agcctatagt cttgataaaa gccacttcca 420ttttgttcac gagtttgtga caatagtgac agactctaca attttgtact ttggggtatt 480gccctggttt tggaagaaat caggagattt tatgacaata gctggtttca atgctgagaa 540tgaaatactg catacccttg ccttcttagc agggctgatg atttggtcac agataacaga 600tttgcccttt tctctgtact caacttttgt gattgaggcc cgtcatggtt ttaataagca 660aacaccatgg ttattcttta gggacatgct taaaggaatt ttcctttccg taataattgg 720tccacctatt gtggctgcaa tcattgtaat agtacagaaa ggaggtccat acttggccat 780ctatctttgg gtttttacgt ttggtctttc tattgtgatg atgacccttt atccagtact 840aatagctcca ctcttcaata agttcactcc acttccagat ggtcaactca gggagaaaat 900cgagaaactt gcttcctccc tcaactatcc gttaaagaaa ctatttgttg tcgatggatc 960cacaagatca agtcacagca atgcctatat gtatggattc ttcaagaaca agaggattgt 1020cctttatgac acattaattc aacagtgcaa agacgatgag gaaattgttg ctgttattgc 1080ccatgagttg ggacactgga agctcaacca tactgtgtac acatttgttg ctatgcagat 1140tcttacactt ctacaatttg gaggatatac actagtgcga aattcagctg atctgtatcg 1200aagctttggg tttgatacgc agccagtcct cattgggctc atcatatttc agcatactgt 1260aatcccactt cagcaattgg tcagctttgg tctgaaccta gtcagccgat catttgaatt 1320tcaggctgat ggctttgcca agaagcttgg atatgcatct ggattacgcg gtggtcttgt 1380gaaactacag gaggagaatc tgtcagctat gaatacagat ccttgctcgt gccg 143415400PRTGlycine max 15Met Ala Phe Pro Tyr Met Glu Ala Val Val Gly Phe Met Ile Leu Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Val Arg Gln His Arg Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Glu Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser His Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Val Thr Asp Ser Thr Ile Leu Tyr Phe 65 70 75 80Gly Val Leu Pro Trp Phe Trp Lys Lys Ser Gly Asp Phe Met Thr Ile 85 90 95Ala Gly Phe Asn Ala Glu Asn Glu Ile Leu His Thr Leu Ala Phe Leu 100 105 110Ala Gly Leu Met Ile Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 130 135 140Pro Trp Leu Phe Phe Arg Asp Met Leu Lys Gly Ile Phe Leu Ser Val145 150 155 160Ile Ile Gly Pro Pro Ile Val Ala Ala Ile Ile Val Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Val Phe Thr Phe Gly Leu 180 185 190Ser Ile Val Met Met Thr Leu Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Gln Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Asn Tyr Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asp Asp Glu Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Val Tyr Thr Phe Val Ala Met Gln Ile Leu 290 295 300Thr Leu Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Ala Asp305 310 315 320Leu Tyr Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln Gln Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ser Phe Glu Phe Gln Ala Asp Gly Phe 355 360 365Ala Lys Lys Leu Gly Tyr Ala Ser Gly Leu Arg Gly Gly Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Cys Ser Cys385 390 395 400161405DNAGlycine max 16gcgagctctc gttcggttca tcagcgtgtg tctcagccat ggcgtttccc tacatggaag 60ccgttgtcgg atttatgata ttaatgtaca tttttgaaac ttacttggat gtgcgacaac 120atagggccct caaacttcct actcttccaa agactttaga aggtgttatc agccaagaga 180aatttgagaa atctagagcc tatagtcttg ataaaagcca cttccatttt gttcacgagt 240ttgtgacaat agtgacagac tctacaattt tgtactttgg ggtattgccc tggttttgga 300agaaatcagg agattttatg acaatagctg gtttcaatgc tgagaatgaa atactgcata 360cccttgcctt cttagcaggg ctgatgattt ggtcacagat aacagatttg cccttttctc 420tgtactcaac ttttgtgatt gaggcccgtc atggttttaa taagcaaaca ccatggttat 480tctttaggga catgcttaaa ggaattttcc tttctgtaat aattggtcca cctattgtgg 540ctgcaatcat tgtaatagta cagaaaggag gtccatactt ggccatctat ctttgggttt 600ttacgtttgg tctttctatt gtgatgatga ccctttatcc agtactaata gctccactct 660tcaataagtt cactccactt ccagatggtc aactcaggga gaaaatcgag aaacttgctt 720cctccctcaa ctatccgtta aagaaactat ttgttgtcga tggatccaca agatcaagtc 780acagcaatgc ctatatgtat ggattcttca agaacaagag gattgtcctt tatgacacat 840taattcaaca gtgcaaagac gatgaggaaa ttgttgctgt tattgcccat gagttgggac 900actggaagct caaccatact gtgtacacat ttgttgctat gcagattctt acacttctac 960aatttggagg atatacacta gtgcgaaatt cagctgatct gtatcgaagc tttgggtttg 1020atacgcagcc agtcctcatt gggctcatca tatttcagca tactgtaatc ccacttcagc 1080aattggtcag ctttggtctg aacctagtca gccgatcatt tgaatttcag gctgatggct 1140ttgccaagaa gcttggatat gcatctggat tacgcggtgg tcttgtgaaa ctacaggagg 1200agaatctgtc agctatgaat acagatcctt ggtactctgc ttatcactat tctcatcctc 1260cccttgttga aagattggct gtgctggacg aaccggataa gaaggaagac taagcaagta 1320acttaaagat gaagagctgc aaaaattggc tataccctaa cttgctatga tttagtgctg 1380caatagctgt aatatctccc gggat 140517424PRTGlycine max 17Met Ala Phe Pro Tyr Met Glu Ala Val Val Gly Phe Met Ile Leu Met 1 5

10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Val Arg Gln His Arg Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Glu Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser His Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Val Thr Asp Ser Thr Ile Leu Tyr Phe 65 70 75 80Gly Val Leu Pro Trp Phe Trp Lys Lys Ser Gly Asp Phe Met Thr Ile 85 90 95Ala Gly Phe Asn Ala Glu Asn Glu Ile Leu His Thr Leu Ala Phe Leu 100 105 110Ala Gly Leu Met Ile Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 130 135 140Pro Trp Leu Phe Phe Arg Asp Met Leu Lys Gly Ile Phe Leu Ser Val145 150 155 160Ile Ile Gly Pro Pro Ile Val Ala Ala Ile Ile Val Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Val Phe Thr Phe Gly Leu 180 185 190Ser Ile Val Met Met Thr Leu Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Gln Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Asn Tyr Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asp Asp Glu Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Val Tyr Thr Phe Val Ala Met Gln Ile Leu 290 295 300Thr Leu Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Ala Asp305 310 315 320Leu Tyr Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln Gln Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ser Phe Glu Phe Gln Ala Asp Gly Phe 355 360 365Ala Lys Lys Leu Gly Tyr Ala Ser Gly Leu Arg Gly Gly Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Ala Val Leu 405 410 415Asp Glu Pro Asp Lys Lys Glu Asp 420181275DNAGlycine max 18atggcgtttc cctacatgga agccgttgtc ggatttatga tattaatgta catttttgaa 60acttacttgg atgtgcgaca acatagggcc ctcaaacttc ctactcttcc aaagacttta 120gagggtgtta tcagccaaga gaaatttgag aaatctagag cctatagtct tgataaaagc 180cacttccatt ttgttcacga gtttgtgaca atagtgacag actctacaat tttgtacttt 240ggggtattgc cctggttttg gaagaaatca ggagatttta tgacaatagc tggtttcaat 300gctgagaatg aaatactgca tacccttgcc ttcttagcag ggctgatgat ttggtcacag 360ataacagatt tgcccttttc tctgtactca acttttgtga ttgaggcccg tcatggtttt 420aataagcaaa caccatggtt attctttagg gacatgctta aaggaatttt cctttctgta 480ataattggtc cacctattgt ggctgcaatc attgtaatag tacagaaagg aggtccatac 540ttggccatct atctttgggt ttttacgttt ggtctttcta ttgtgatgat gaccctttat 600ccagtactaa tagctccact cttcaataag ttcactccac ttccagatgg tcaactcagg 660gagaaaatcg agaaacttgc ttcctccctc aactatccgt taaagaaact atttgttgtc 720gatggatcca caagatcaag tcacagcaat gcctatatgt atggattctt caagaacaag 780aggattgtcc cttatgacac attaattcaa cagtgcaaag acgatgagga aattgttgct 840gttattgccc atgagttggg acactggaag ctcaaccata ctgtgtacac atttgttgct 900atgcagattc ttacacttct acaatttgga ggatatacac tagtgcgaaa ttcagctgat 960ctgtatcgaa gctttgggtt tgatacgcag ccagtcctca ttgggctcat catatttcag 1020catactgtaa tcccacttca gcaattggtc agctttggtc tgaacctagt cagccgatca 1080tttgaatttc aggctgatgg ctttgccaag aagcttggat atgcatctgg attacgcggt 1140ggtcttgtga aactacagga ggagaatctg tcagctatga atacagatcc ttggtactct 1200gcttatcact attctcatcc tccccttgtt gaaagattgg ccgcgctgga cgaaccggat 1260aagaaggaag actaa 127519424PRTGlycine max 19Met Ala Phe Pro Tyr Met Glu Ala Val Val Gly Phe Met Ile Leu Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Val Arg Gln His Arg Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Glu Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser His Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Val Thr Asp Ser Thr Ile Leu Tyr Phe 65 70 75 80Gly Val Leu Pro Trp Phe Trp Lys Lys Ser Gly Asp Phe Met Thr Ile 85 90 95Ala Gly Phe Asn Ala Glu Asn Glu Ile Leu His Thr Leu Ala Phe Leu 100 105 110Ala Gly Leu Met Ile Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 130 135 140Pro Trp Leu Phe Phe Arg Asp Met Leu Lys Gly Ile Phe Leu Ser Val145 150 155 160Ile Ile Gly Pro Pro Ile Val Ala Ala Ile Ile Val Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Val Phe Thr Phe Gly Leu 180 185 190Ser Ile Val Met Met Thr Leu Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Gln Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Asn Tyr Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Pro Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asp Asp Glu Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Val Tyr Thr Phe Val Ala Met Gln Ile Leu 290 295 300Thr Leu Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Ala Asp305 310 315 320Leu Tyr Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln Gln Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ser Phe Glu Phe Gln Ala Asp Gly Phe 355 360 365Ala Lys Lys Leu Gly Tyr Ala Ser Gly Leu Arg Gly Gly Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Ala Ala Leu 405 410 415Asp Glu Pro Asp Lys Lys Glu Asp 420201275DNAArtificial SequenceDescription of Artificial Sequence complement to seq id no 18 20ttagtcttcc ttcttatccg gttcgtccag cgcggccaat ctttcaacaa ggggaggatg 60agaatagtga taagcagagt accaaggatc tgtattcata gctgacagat tctcctcctg 120tagtttcaca agaccaccgc gtaatccaga tgcatatcca agcttcttgg caaagccatc 180agcctgaaat tcaaatgatc ggctgactag gttcagacca aagctgacca attgctgaag 240tgggattaca gtatgctgaa atatgatgag cccaatgagg actggctgcg tatcaaaccc 300aaagcttcga tacagatcag ctgaatttcg cactagtgta tatcctccaa attgtagaag 360tgtaagaatc tgcatagcaa caaatgtgta cacagtatgg ttgagcttcc agtgtcccaa 420ctcatgggca ataacagcaa caatttcctc atcgtctttg cactgttgaa ttaatgtgtc 480ataagggaca atcctcttgt tcttgaagaa tccatacata taggcattgc tgtgacttga 540tcttgtggat ccatcgacaa caaatagttt ctttaacgga tagttgaggg aggaagcaag 600tttctcgatt ttctccctga gttgaccatc tggaagtgga gtgaacttat tgaagagtgg 660agctattagt actggataaa gggtcatcat cacaatagaa agaccaaacg taaaaaccca 720aagatagatg gccaagtatg gacctccttt ctgtactatt acaatgattg cagccacaat 780aggtggacca attattacag aaaggaaaat tcctttaagc atgtccctaa agaataacca 840tggtgtttgc ttattaaaac catgacgggc ctcaatcaca aaagttgagt acagagaaaa 900gggcaaatct gttatctgtg accaaatcat cagccctgct aagaaggcaa gggtatgcag 960tatttcattc tcagcattga aaccagctat tgtcataaaa tctcctgatt tcttccaaaa 1020ccagggcaat accccaaagt acaaaattgt agagtctgtc actattgtca caaactcgtg 1080aacaaaatgg aagtggcttt tatcaagact ataggctcta gatttctcaa atttctcttg 1140gctgataaca ccctctaaag tctttggaag agtaggaagt ttgagggccc tatgttgtcg 1200cacatccaag taagtttcaa aaatgtacat taatatcata aatccgacaa cggcttccat 1260gtagggaaac gccat 1275211301DNAZea mays 21acgaggctga gtgctgagaa tgagataata cacacccttg ctttcttagc tggttccatg 60gtttggtcgc agattacaga cttgccgttc tctctctatt caacttttgt tatagaggct 120cgacatggtt ttaacaagca aactatatgg ctcttcatta gggatatgat caaaggaatt 180ttactatcca tgatattggg gccaccaatc gtggctgcta tcatctacat agtacagatt 240ggaggacctt acctggctat atatctctgg ggttttatgt ttgtattagc tctactgatg 300atgacaatat accccattgt gatagctcct ctgttcaaca agttcactcc tcttcctgaa 360ggagtcctca gggaaaaaat agagaagctg gcagcttccc tcaagtttcc tttgaaaaag 420cttttcgtgg tagatgggtc taccagatca agccacagta atgcctacat gtatggtttt 480ttcaagaaca agcgcatagt actctatgac acattgattc agcagtgtag caatgaggat 540gagatagttt ctgttatagc acatgaactt ggacactgga aactcaatca tactgtctat 600tcctttgtag ctgtccagct gcttatgttt cttcaatttg gaggatatac tctagtaagg 660agctccaaag atctatttgg aagttttggc ttcaaggacc agccagtaat aattggattg 720atcattttcc cgcacaccat aatacccatc caacaccttc tgagctttcg cctgaacctt 780gtcagcagag catttgaatt tcaggctgat gcctttgcca agaaccttgg atatgcccct 840cagctccgag cagcccttgt taaactacag gaggagaact tgtctgcgat gaacaccgat 900ccttggtatt cggcatatca ctactcccac ccaccactcg tcgagaggct gcaagctttg 960gaagattcag acgacaaaaa agaagattag tcgatccttg tatgaggttt acatatggat 1020ttttccctgc cacatgcaca ccgattcagt gcttggatgg tgagggtttt gacataggag 1080tgttgtcaaa gctttagagt gcatctttcg gtcaggtgca acagcctttc ggtcattgag 1140acatataagc gaattagcta ttaaaaaaaa cagaactgtt gcatcaaaaa aaaaaaaaaa 1200aaagaaacaa aaaaaaaaaa aaaaaaaaaa aagaaaaaaa aaaaaaaaaa aaaagtgctc 1260tgcgttgtta ccactgcttg ccctatagtg atcgtatcag a 130122329PRTZea mays 22Thr Arg Leu Ser Ala Glu Asn Glu Ile Ile His Thr Leu Ala Phe Leu 1 5 10 15Ala Gly Ser Met Val Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 20 25 30Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 35 40 45Ile Trp Leu Phe Ile Arg Asp Met Ile Lys Gly Ile Leu Leu Ser Met 50 55 60Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Tyr Ile Val Gln Ile 65 70 75 80Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Gly Phe Met Phe Val Leu 85 90 95Ala Leu Leu Met Met Thr Ile Tyr Pro Ile Val Ile Ala Pro Leu Phe 100 105 110Asn Lys Phe Thr Pro Leu Pro Glu Gly Val Leu Arg Glu Lys Ile Glu 115 120 125Lys Leu Ala Ala Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val 130 135 140Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe145 150 155 160Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 165 170 175Ser Asn Glu Asp Glu Ile Val Ser Val Ile Ala His Glu Leu Gly His 180 185 190Trp Lys Leu Asn His Thr Val Tyr Ser Phe Val Ala Val Gln Leu Leu 195 200 205Met Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Ser Ser Lys Asp 210 215 220Leu Phe Gly Ser Phe Gly Phe Lys Asp Gln Pro Val Ile Ile Gly Leu225 230 235 240Ile Ile Phe Pro His Thr Ile Ile Pro Ile Gln His Leu Leu Ser Phe 245 250 255Arg Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 260 265 270Ala Lys Asn Leu Gly Tyr Ala Pro Gln Leu Arg Ala Ala Leu Val Lys 275 280 285Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp Tyr Ser 290 295 300Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Gln Ala Leu305 310 315 320Glu Asp Ser Asp Asp Lys Lys Glu Asp 325231518DNAZea mays 23aaatccgagc tccggaatcg agcaaagcac ccgagcctgc gccgcgtcaa gccgtcaaag 60ctcccccgct tcataccatt ccctcccccg atccctcgcc tcagccctca gctccgcccc 120tcggtttccg aagcgccacc gccgacgtgg cctccctcgg ctccaatggc gttgccctac 180ctggaggccg tgctttgctt tatgattttc atgtacatat ttgagacata tcttgacatc 240cgtcagcata gagccctcaa gctgccaact ttgccaaaac ccctgctggg agtaattagt 300gacgaaaagt ttgaacgctc tagagcttat agcctcgaca aaagctattt ccattttgtt 360catgaggctg tgactatttt aatggatact acaatactat actatagagt tcttccctgg 420ttttggaaga aatctggaga gttagttacc agtgttgggc tgagtgctga gaatgagata 480atacacaccc ttgctttctt agctggttcc atggtttggt cgcagattac agacttgccg 540ttctctctct attcaacttt tgttatagag gctcgacatg gttttaacaa gcaaactata 600tggctcttca ttagggatat gatcaaagga attttactat ccatgatatt ggggccacca 660atcgtggctg ctatcatcta catagtacag attggaggac cttacctggc tatatatctc 720tggggtttta tgtttgtatt agctctactg atgatgacaa tataccccat tgtgatagct 780cctctgttca acaagttcac tcctcttcct gaaggagtcc tcagggaaaa aatagagaag 840ctggcagctt ccctcaagtt tcctttgaaa aagcttttcg tggtagatgg gtctaccaga 900tcaagccaca gtaatgccta catgtatggt tttttcaaga acaagcgcat agtactctat 960gacacattga ttcagcagtg tagcaatgag gatgagatag tttctgttat agcacatgaa 1020cttggacact ggaaactcaa tcatactgtc tattcctttg tagctgtcca gctgcttatg 1080tttcttcaat ttggaggata tactctagta aggagctcca aagatctatt tggaagtttt 1140ggcttcaagg accagccagt aataattgga ttgatcattt tccagcacac cataataccc 1200atccaacacc ttctgagctt ttgcctgaac cttgtcagca gagcatttga atttcaggct 1260gatgcctttg ccaagaacct tggatatgcc cctcagctcc gagcagccct tgttaaacta 1320caggaggaga acttgtctgc gatgaacacc gatccttggt attcggcata tcactactcc 1380cacccaccac tcgtcgagag gctgcaagct cttgaagatt cagacagcaa aaaagaagat 1440tagtcgatcc ttgtatgagg tttacatatg gatttttccc tgccacatgc acaccgattc 1500agtgcttgga tggtgagg 151824425PRTZea mays 24Met Ala Leu Pro Tyr Leu Glu Ala Val Leu Cys Phe Met Ile Phe Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Ile Arg Gln His Arg Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Pro Leu Leu Gly Val Ile Ser Asp Glu Lys 35 40 45Phe Glu Arg Ser Arg Ala Tyr Ser Leu Asp Lys Ser Tyr Phe His Phe 50 55 60Val His Glu Ala Val Thr Ile Leu Met Asp Thr Thr Ile Leu Tyr Tyr 65 70 75 80Arg Val Leu Pro Trp Phe Trp Lys Lys Ser Gly Glu Leu Val Thr Ser 85 90 95Val Gly Leu Ser Ala Glu Asn Glu Ile Ile His Thr Leu Ala Phe Leu 100 105 110Ala Gly Ser Met Val Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Leu Phe Ile Arg Asp Met Ile Lys Gly Ile Leu Leu Ser Met145 150 155 160Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Tyr Ile Val Gln Ile 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Gly Phe Met Phe Val Leu 180 185 190Ala Leu Leu Met Met Thr Ile Tyr Pro Ile Val Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Glu Gly Val Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ala Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Ser Asn Glu Asp Glu Ile Val Ser Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Val Tyr Ser Phe Val Ala Val Gln Leu Leu 290 295 300Met Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Ser Ser Lys Asp305 310 315 320Leu Phe Gly Ser Phe Gly Phe Lys Asp Gln Pro Val Ile Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Ile Ile Pro Ile Gln His Leu Leu Ser Phe 340 345 350Cys Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Lys Asn Leu Gly Tyr Ala Pro Gln

Leu Arg Ala Ala Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Gln Ala Leu 405 410 415Glu Asp Ser Asp Ser Lys Lys Glu Asp 420 425251275DNAArabidopsis thaliana 25atggcgattc ctttcatgga aaccgtcgtg ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacagtct tgacaaaagc 180tattttcact ttgttcatga gtttgtaact atacttatgg actctgcaat tttgttcttt 240gggatcttgc cttggttttg gaagatgtct ggagctgttt taccgaggtt gggccttgat 300ccagagaatg aaatactgca tactctttca ttcttggctg gtgttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaacatt cctctctgtc 480atactaggcc cacccattgt tgctgcgata attttcatag tccagaaagg aggtccttat 540cttgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600ccggtcttga tagcaccgct cttcaacaag ttcactcctc ttccagatgg agacctccgg 660gagaagattg agaaacttgc ttcttctcta aagtttcctt tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagcaat gcttacatgt atggtttctt taagaacaaa 780aggattgttc tttatgatac gttgattcag cagtgcaaga atgaggatga aattgtggcg 840gttattgcac acgagcttgg acattggaaa ctgaatcaca ctacatactc gttcattgca 900gttcaaatcc ttgccttctt acaatttgga ggatacactc ttgtcagaaa ctccactgat 960ctcttcagga gtttcggatt tgatacacag cctgttctca ttggtttgat catatttcag 1020cacactgtaa taccactgca acatctagta agctttggcc tgaacctcgt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgccgtg aagcttggct atgcaaaaga tcttcgtcct 1140gctctagtga aactacagga agagaactta tcagcaatga acactgatcc attgcactca 1200gcttatcact actcacatcc tcctcttgtt gaaaggcttc gagccattga tggagaagac 1260aagaagacag attaa 127526424PRTArabidopsis thaliana 26Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser Tyr Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Leu Met Asp Ser Ala Ile Leu Phe Phe65 70 75 80Gly Ile Leu Pro Trp Phe Trp Lys Met Ser Gly Ala Val Leu Pro Arg 85 90 95Leu Gly Leu Asp Pro Glu Asn Glu Ile Leu His Thr Leu Ser Phe Leu 100 105 110Ala Gly Val Met Thr Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 130 135 140Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val145 150 155 160Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 180 185 190Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln Ile Leu 290 295 300Ala Phe Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Thr Asp305 310 315 320Leu Phe Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln His Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe 355 360 365Ala Val Lys Leu Gly Tyr Ala Lys Asp Leu Arg Pro Ala Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Leu His Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Arg Ala Ile 405 410 415Asp Gly Glu Asp Lys Lys Thr Asp 420271380DNAArabidopsis thaliana 27atggcgattc ctttcatgga aaccgtcgtg ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacaggga tatcatcact 180gagaacttta atatatgcag ctattttcac tttgttcatg agtttgtaac tatacttatg 240gactctgcaa ttttgttctt tgggatcttg ccttggtttt ggaagatgtc tggagctgtt 300ttaccgaggt tgggccttga tccagagaat gaaatactgc atactctttc attcttggct 360ggtgttatga catggtcaca gatcactgat ttgccatttt ctttgtactc aactttcgtg 420atcgagtctc ggcatgggtt caacaaacaa acaatatgga tgttcattag ggacatgatc 480aaaggaacat tcctctctgt catactaggc ccacccattg ttgctgcgat aattttcata 540gtccagaaag gaggtcctta tcttgccatc tatctgtggg cattcatgtt tatcctgtct 600ctagtgatga tgactatata cccggtcttg atagcaccgc tcttcaacaa gttcactcct 660cttccagatg gagacctccg ggagaagatt gagaaacttg cttcttctct aaagtttcct 720ttgaagaagc tgtttgttgt cgatggatct acaaggtcaa gccatagcaa tgcttacatg 780tatggtttct ttaagaacaa aaggattgtt ctttatgata cgttgattca gcagtgcaag 840aatgaggatg aaattgtggc ggttattgca cacgagcttg gacattggaa actgaatcac 900actacatact cgttcattgc agttcaaatc cttgccttct tacaatttgg aggatacact 960cttgtcagaa actccactga tctcttcagg agtttcggat ttgatacaca gcctgttctc 1020attggtttga tcatatttca gcacactgta ataccactgc aacatctagt aagctttggc 1080ctgaacctcg ttagtcgagc gtttgagttt caggctgatg cttttgctgt gaagcttggc 1140tatgcaaaag atcttcgtcc tgctctagtg aaactacagg tcagagaaga taacaacaga 1200acacaaactg ttacctcaat ttgtgtcaca cacttaaatg gattttttgt tgggattttg 1260caggaagaga acttatcagc aatgaacact gatccattgt actcagctta tcactactca 1320catcctcctc ttgttgaaag gcttcgagcc attgatggag aagacaagaa gacagattaa 138028459PRTArabidopsis thaliana 28Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Val Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Arg Asp Ile Ile Thr Glu Asn Phe Asn 50 55 60Ile Cys Ser Tyr Phe His Phe Val His Glu Phe Val Thr Ile Leu Met 65 70 75 80Asp Ser Ala Ile Leu Phe Phe Gly Ile Leu Pro Trp Phe Trp Lys Met 85 90 95Ser Gly Ala Val Leu Pro Arg Leu Gly Leu Asp Pro Glu Asn Glu Ile 100 105 110Leu His Thr Leu Ser Phe Leu Ala Gly Val Met Thr Trp Ser Gln Ile 115 120 125Thr Asp Leu Pro Phe Ser Leu Tyr Ser Thr Phe Val Ile Glu Ser Arg 130 135 140His Gly Phe Asn Lys Gln Thr Ile Trp Met Phe Ile Arg Asp Met Ile145 150 155 160Lys Gly Thr Phe Leu Ser Val Ile Leu Gly Pro Pro Ile Val Ala Ala 165 170 175Ile Ile Phe Ile Val Gln Lys Gly Gly Pro Tyr Leu Ala Ile Tyr Leu 180 185 190Trp Ala Phe Met Phe Ile Leu Ser Leu Val Met Met Thr Ile Tyr Pro 195 200 205Val Leu Ile Ala Pro Leu Phe Asn Lys Phe Thr Pro Leu Pro Asp Gly 210 215 220Asp Leu Arg Glu Lys Ile Glu Lys Leu Ala Ser Ser Leu Lys Phe Pro225 230 235 240Leu Lys Lys Leu Phe Val Val Asp Gly Ser Thr Arg Ser Ser His Ser 245 250 255Asn Ala Tyr Met Tyr Gly Phe Phe Lys Asn Lys Arg Ile Val Leu Tyr 260 265 270Asp Thr Leu Ile Gln Gln Cys Lys Asn Glu Asp Glu Ile Val Ala Val 275 280 285Ile Ala His Glu Leu Gly His Trp Lys Leu Asn His Thr Thr Tyr Ser 290 295 300Phe Ile Ala Val Gln Ile Leu Ala Phe Leu Gln Phe Gly Gly Tyr Thr305 310 315 320Leu Val Arg Asn Ser Thr Asp Leu Phe Arg Ser Phe Gly Phe Asp Thr 325 330 335Gln Pro Val Leu Ile Gly Leu Ile Ile Phe Gln His Thr Val Ile Pro 340 345 350Leu Gln His Leu Val Ser Phe Gly Leu Asn Leu Val Ser Arg Ala Phe 355 360 365Glu Phe Gln Ala Asp Ala Phe Ala Val Lys Leu Gly Tyr Ala Lys Asp 370 375 380Leu Arg Pro Ala Leu Val Lys Leu Gln Val Arg Glu Asp Asn Asn Arg385 390 395 400Thr Gln Thr Val Thr Ser Ile Cys Val Thr His Leu Asn Gly Phe Phe 405 410 415Val Gly Ile Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro 420 425 430Leu Tyr Ser Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu 435 440 445Arg Ala Ile Asp Gly Glu Asp Lys Lys Thr Asp 450 455293098DNAArabidopsis thaliana 29atggcgattc ctttcatgga aaccgtcgtg ggtaagcttc aaaacctttt tctgagacat 60tttactatcc tgtttcactc atcgtatttc gtttttgttt gggttttgct ttctgtgttg 120tgtgtgttga gattccatga ctcgtttgtt tcatatacca tcgtctctgc ttctcgtttc 180taaattttgt tcttttctaa tagtgcgtac cttgatctga ggttttatta ctcctactag 240tttcttgtct tactcgtgcg tttgatttga tttgagctta tgtgatttca tcatctcttc 300ctcggtttta gaatgtacgg agcttctctg ttaaccaaaa tctaggattt gggaagaaaa 360gtcggagtct tttttttcct cattcccgat tggaaattga gaatcttgaa atttttcttt 420gttcaagtca tacagcttga ggttttgggt tttcttgtca gggtattatt atgttcgtga 480ctgcaactag agttttctgg agttttttga aatgggtttt gtgttgtgga accgtatgtg 540aatgttgcat caaaactctt tcagtgctcc aatgtttcca tcagtagtca gcacaagaga 600tctttttata tctggttgat caaaaaagta gatgatgtta ttgaattttc agtgatggag 660tatctgttgt tgtggcattt agagtagatt cgtatttcat cttctgtttt attctttttc 720ttacaggttt tatgatagtg atgtacattt ttgagacgta tttggatctg aggcaactca 780ctgctctcaa gcttccaact ctcccgaaaa ccttggttgg tgtaattagc caagagaagt 840ttgagaaatc acgagcatac agtcttgaca aaaggtttcg tcttgatcat atttatatca 900ttttagtttt ttataattgc caggggatat catcactgag aactttaata tatgcagcta 960ttttcacttt gttcatgagt ttgtaactat acttatggac tctgcaattt tgttctttgg 1020gatcttgcct tggttttgga aggtacatat ctggtttcgg tatacagtat ctcattttga 1080atatagagtt gttacattac aattgtaaag ttttcatttt taccttagat gtctggagct 1140gttttaccga ggttgggcct tgatccagag aatgaaatac tgcatactct ttcattcttg 1200gctggtgtta tgacatggtc acaggtgttc caaataaacc ccttcatata gtcctatacg 1260tttagcatca aaatatctat tttcttaaga taataatatt tcttttatat tctgatgcag 1320atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 1380aacaaagtat gtcgtatttc caacactacc ttgtgactta cgttttttta tcagagatgt 1440ggattaaatt tgcttctaaa ttctgttgac agcaaacaat atggatgttc attagggaca 1500tgatcaaagg aacattcctc tctgtcatac taggcccacc cattgttgct gcgataattt 1560tcatagtcca ggtttgatga ttctggattc atcttatttc tgagtttttc acatggatga 1620ctattctcca ttgagtgtga gcttcaaagt ttttagtttt cgtgttaaaa atttaaaatt 1680tgcttctctg agcatgaagt ttctatcttt ttccagaaag gaggtcctta tcttgccatc 1740tatctgtggg cattcatgtt tatcctgtct ctagtgatga tgactatata cccggtcttg 1800atagcaccgc tcttcaacaa gttcactcct gtgtgtattt ctgtcatggc cattttacaa 1860ttcactgctt gtttgcatat gttgttacca gacaatataa tctcccgctt ttttatggct 1920atagcttcca gatggagacc tccgggagaa gattgagaaa cttgcttctt ctctaaagtt 1980tcctttgaag aagctgtttg ttgtcgatgg atctacaagg tcaagccata gcaatgtgag 2040aagcttgaga tctcttccta cctactttac tctagtttac cattagaagc ttacgtatct 2100tgttacatca tacaggctta catgtatggt ttctttaaga acaaaaggat tgttctttat 2160gatacgttga ttcagcaggt actgtgactc ttgatgcttc aaacgagcta tactcacatt 2220tctgtttctg gttctgaaac ataacataat cttctattgt gcagtgcaag aatgaggatg 2280aaattgtggc ggttattgca cacgagcttg gacattggaa actgaatcac actacatact 2340cgttcattgc agttcaagtg aggctcaacc gacagttcaa aaacttactc acatctacat 2400ttcacttaag aaatcatgtc ttatgaccct ctctcaatgt tttgcttgca gatccttgcc 2460ttcttacaat ttggaggata cactcttgtc agaaactcca ctgatctctt caggagtttc 2520ggatttgata cacagcctgt tctcattggt ttgatcatat ttcaggtttg ttatttttgc 2580cttttgacac taatctaatg aatcaaggat ggattaagaa aaaaaaactc taaacctttg 2640gttatatctc ctgtctgatt atcacagcac actgtaatac cactgcaaca tctagtaagc 2700tttggcctga acctcgttag tcgagcgttt gagtttcagg taccatctta caatccctca 2760agatccaacc atagtttctt tattgcaatg gcagcctcat ctactaatct gagttaacgt 2820tccttttgca ggctgatgct tttgctgtga agcttggcta tgcaaaagat cttcgtcctg 2880ctctagtgaa actacaggtc agagaagata acaacagaac acaaactgtt acctcaattt 2940gtgtcacaca cttaaatgga ttttttgttg ggattttgca ggaagagaac ttatcagcaa 3000tgaacactga tccattgtac tcagcttatc actactcaca tcctcctctt gttgaaaggc 3060ttcgagccat tgatggagaa gacaagaaga cagattaa 309830316PRTArabidopsis thaliana 30Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Ile Thr Asp Leu Pro Phe Ser Leu 35 40 45Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 50 55 60Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val 65 70 75 80Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 85 90 95Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 100 105 110Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 115 120 125Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 130 135 140Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val145 150 155 160Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 165 170 175Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 180 185 190Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 195 200 205Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln His Thr 210 215 220Val Ile Pro Leu Gln His Leu Val Ser Phe Gly Leu Asn Leu Val Ser225 230 235 240Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe Ala Val Lys Leu Gly Tyr 245 250 255Ala Lys Asp Leu Arg Pro Ala Leu Val Lys Leu Gln Val Arg Glu Asp 260 265 270Asn Asn Arg Thr Gln Thr Glu Glu Asn Leu Ser Ala Met Asn Thr Asp 275 280 285Pro Leu Tyr Ser Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg 290 295 300Leu Arg Ala Ile Asp Gly Glu Asp Lys Lys Thr Asp305 310 31531453PRTSaccharomyces cerevisiae 31Met Phe Asp Leu Lys Thr Ile Leu Asp His Pro Asn Ile Pro Trp Lys 1 5 10 15Leu Ile Ile Ser Gly Phe Ser Ile Ala Gln Phe Ser Phe Glu Ser Tyr 20 25 30Leu Thr Tyr Arg Gln Tyr Gln Lys Leu Ser Glu Thr Lys Leu Pro Pro 35 40 45Val Leu Glu Asp Glu Ile Asp Asp Glu Thr Phe His Lys Ser Arg Asn 50 55 60Tyr Ser Arg Ala Lys Ala Lys Phe Ser Ile Phe Gly Asp Val Tyr Asn 65 70 75 80Leu Ala Gln Lys Leu Val Phe Ile Lys Tyr Asp Leu Phe Pro Lys Ile 85 90 95Trp His Met Ala Val Ser Leu Leu Asn Ala Val Leu Pro Val Arg Phe 100 105 110His Met Val Ser Thr Val Ala Gln Ser Leu Cys Phe Leu Gly Leu Leu 115 120 125Ser Ser Leu Ser Thr Leu Val Asp Leu Pro Leu Ser Tyr Tyr Ser His 130 135 140Phe Val Leu Glu Glu Lys Phe Gly Phe Asn Lys Leu Thr Val Gln Leu145 150 155 160Trp Ile Thr Asp Met Ile Lys Ser Leu Thr Leu Ala Tyr Ala Ile Gly 165 170 175Gly Pro Ile Leu Tyr Leu Phe Leu Lys Ile Phe Asp Lys Phe Pro Thr 180 185 190Asp Phe Leu Trp Tyr Ile Met Val Phe Leu Phe Val Val Gln Ile Leu 195 200 205Ala Met Thr Ile Ile Pro Val Phe Ile Met Pro Met Phe Asn Lys Phe 210 215 220Thr Pro Leu Glu Asp Gly Glu Leu Lys Lys Ser Ile Glu Ser Leu Ala225 230 235 240Asp Arg Val Gly

Phe Pro Leu Asp Lys Ile Phe Val Ile Asp Gly Ser 245 250 255Lys Arg Ser Ser His Ser Asn Ala Tyr Phe Thr Gly Leu Pro Phe Thr 260 265 270Ser Lys Arg Ile Val Leu Phe Asp Thr Leu Val Asn Ser Asn Ser Thr 275 280 285Asp Glu Ile Thr Ala Val Leu Ala His Glu Ile Gly His Trp Gln Lys 290 295 300Asn His Ile Val Asn Met Val Ile Phe Ser Gln Leu His Thr Phe Leu305 310 315 320Ile Phe Ser Leu Phe Thr Ser Ile Tyr Arg Asn Thr Ser Phe Tyr Asn 325 330 335Thr Phe Gly Phe Phe Leu Glu Lys Ser Thr Gly Ser Phe Val Asp Pro 340 345 350Val Ile Thr Lys Glu Phe Pro Ile Ile Ile Gly Phe Met Leu Phe Asn 355 360 365Asp Leu Leu Thr Pro Leu Glu Cys Ala Met Gln Phe Val Met Ser Leu 370 375 380Ile Ser Arg Thr His Glu Tyr Gln Ala Asp Ala Tyr Ala Lys Lys Leu385 390 395 400Gly Tyr Lys Gln Asn Leu Cys Arg Ala Leu Ile Asp Leu Gln Ile Lys 405 410 415Asn Leu Ser Thr Met Asn Val Asp Pro Leu Tyr Ser Ser Tyr His Tyr 420 425 430Ser His Pro Thr Leu Ala Glu Arg Leu Thr Ala Leu Asp Tyr Val Ser 435 440 445Glu Lys Lys Lys Asn 4503218DNAArtificial SequenceDescription of Artificial Sequence Primer 32caggaaacag ctatgacc 183319DNAArtificial SequenceDescription of Artificial Sequence Primer 33ctaaagggaa caaaagctg 193418DNAArtificial SequenceDescription of Artificial Sequence Primer 34tgtaaaacga cggccagt 183533DNAArtificial SequenceDescription of Artificial Sequence Primer 35ccgttaacag ccatggcgat tcctttcatg gaa 333630DNAArtificial SequenceDescription of Artificial Sequence Primer 36gtcccgggac ttaatctgtc ttcttgtctt 303725DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37agcagccacg attggtggcc ccaat 253825DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38gggccaccaa tcgtggctgc tatca 253926DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39cgcagccagt cctcattggg ctcatc 264024DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40cggatagttg agggaggaag caag 24415544DNAArtificial SequenceDescription of Artificial Sequence pBI121-AtCPP vector sequence 41gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccatggcga ttcctttcat ggaaaccgtc 3360gtgggtttta tgatagtgat gtacattttt gagacgtatt tggatctgag gcaactcact 3420gctctcaagc ttccaactct cccgaaaacc ttggttggtg taattagcca agagaagttt 3480gagaaatcac gagcatacag tcttgacaaa agctattttc actttgttca tgagtttgta 3540actatactta tggactctgc aattttgttc tttgggatct tgccttggtt ttggaagatg 3600tctggagctg ttttaccgag gttgggcctt gatccggaga atgaaatact gcatactctt 3660tcattcttgg ctggtgttat gacatggtca cagatcactg atttgccatt ttctttgtac 3720tcaactttcg tgatcgagtc tcggcatggg ttcaacaaac aaacaatatg gatgttcatt 3780agggacatga tcaaaggaac attcctctct gtcatactag gcccacccat tgttgctgcg 3840ataattttca tagtccagaa aggaggtcct tatcttgcca tctatctgtg ggcattcatg 3900tttatcctgt ctctagtgat gatgactata tacccggtct tgatagcacc gctcttcaac 3960aaattcactc ctcttccaga tggagacctc cgggagaaga ttgagaaact tgcttcttcc 4020ctaaagtttc ctttgaagaa gctgtttgtt gtcgatggat ctacaaggtc aagccatagc 4080aatgcttaca tgtatggttt ctttaagaac aaaaggattg ttctttatga tacgttgatt 4140cagcagtgca agaatgagga tgaaattgtg gcggttattg cacacgagct tggacattgg 4200aaactgaatc acactacata ctcgttcatt gcagttcaaa tccttgcctt cttacaattt 4260ggaggataca ctcttctcag aaactccact gatctcttca ggagtttcgg atttgataca 4320cagcctgttc tcattggttt gatcatattt cagcacactg taataccact gcaacatcta 4380gtaagctttg gcctgaacct cgttagtcga gcgtttgagt ttcaggctga tgcttttgct 4440gtgaagcttg actatgcaaa agatcttcgt cctgctctag tgaaactaca ggaagagaac 4500ttatcaacaa tgaacactga tccattgtac tcagcttatc actactcaca tcctcctctt 4560gttgaaaggc ttcgagccac tgatggagaa gacaagaaga cagattaacc cctcgaattt 4620ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 4680tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 4740atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 4800atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 4860atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 4920accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 4980atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgccc 5040gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5100ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 5160aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 5220cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 5280ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggaacca 5340ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 5400ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 5460ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg 5520tttacaccac aatatatcct gcca 5544426484DNAArtificial SequenceDescription of Artificial Sequence pBI121-HP-AtCPP vector sequence 42gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tcctcccaat gtccaagctc gtgtgcaata 3360accgccacaa tttcatcctc attcttgcac tgctgaatca acgtatcata aagaacaatc 3420cttttgttct taaagaaacc atacatgtaa gcattgctat ggcttgacct tgtagatcca 3480tcgacaacaa acagcttctt caaaggaaac tttagggaag aagcaagttt ctcaatcttc 3540tcccggaggt ctccatctgg aagaggagtg aatttgttga agagcggtgc tatcaagacc 3600gggtatatag tcatcatcac tagagacagg ataaacatga atgcccacag atagatggca 3660agataaggac ctcctttctg gactatgaaa attatcgcag caacaatggg tgggcctagt 3720atgacagaga ggaatgttcc tttgatcatg tccctaatga acatccatat tgtttgtttg 3780ttgaacccat gccgagactc gatcacgaaa gttgagtaca aagaaaatgg caaatcagtg 3840atctgtgacc atgtcataac accagccaag aatgaaagag tatgcagtat ttcattctcc 3900ggatcaaggc ccaacctcgg taaaagagga tccccatcta cccgcttcgc gtcggcatcc 3960ggtcagtggc agtgaagggc gaacagttcc tgattaacca caaaccgttc tactttactg 4020gctttggtcg tcatgaagat gcggacttgc gtggcaaagg attcgataac gtgctgatgg 4080tgcacgacca cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc 4140cttacgctga agagatgctc gactgggcag atgaacatgg catcgtggtg attgatgaaa 4200ctgctgctgt cggcttttcg ctctctttag gcattggttt cgaagcgggc aacaagccga 4260aagaactgta cagcgaagag gcagtcaacg gggaaactca gcaagcgcac ttacaggcga 4320ttaaagagct gatagcgcgt gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca 4380acgaaccgga tacccgtccg caaggtgcac gggaatattt cgcgccactg gcggaagcaa 4440cgcgtaaact cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc tgcgacgctc 4500acaccgatac catcagcgat ctctttgatg tgctgtgcct gaaccgttat tacggatggt 4560atgtccaaag cggcgatttg gaaacggcag agaaggtact ggaaaaagaa cttctggcct 4620ggcaggagaa actgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata 4680tgtatcaccg cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta tggaatttcg 4740ccgattttgc gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa gggatcttca 4800ctcgcgaccg caaaccgaag tcggcggctt ttctgctgca aaaacgctgg actggcatga 4860acttcggtga aaaaccgcag cagggaggca aacaatgaat caacaactct cctggcgcac 4920catcgtcggc tacagcctcg ggaattgcta ccgagctctt ttaccgaggt tgggccttga 4980tccggagaat gaaatactgc atactctttc attcttggct ggtgttatga catggtcaca 5040gatcactgat ttgccatttt ctttgtactc aactttcgtg atcgagtctc ggcatgggtt 5100caacaaacaa acaatatgga tgttcattag ggacatgatc aaaggaacat tcctctctgt 5160catactaggc ccacccattg ttgctgcgat aattttcata gtccagaaag gaggtcctta 5220tcttgccatc tatctgtggg cattcatgtt tatcctgtct ctagtgatga tgactatata 5280cccggtcttg atagcaccgc tcttcaacaa attcactcct cttccagatg gagacctccg 5340ggagaagatt gagaaacttg cttcttccct aaagtttcct ttgaagaagc tgtttgttgt 5400cgatggatct acaaggtcaa gccatagcaa tgcttacatg tatggtttct ttaagaacaa 5460aaggattgtt ctttatgata cgttgattca gcagtgcaag aatgaggatg aaattgtggc 5520ggttattgca cacgagcttg gacattggga gctcgaattt ccccgatcgt tcaaacattt 5580ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat 5640ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga 5700gatgggtttt tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa 5760tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcggg 5820aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 5880aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 5940gatcgccctt cccaacagtt gcgcagcctg aatggcgccc gctcctttcg ctttcttccc 6000ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 6060agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg 6120ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 6180gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta 6240ttcttttgat ttataaggga ttttgccgat ttcggaacca ccatcaaaca ggattttcgc 6300ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag 6360ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccccagt acattaaaaa 6420cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatcct 6480gcca 64844328DNAArtificial SequenceDescription of Artificial Sequence PCR primer 43aaaggatcca tggcgattcc tttcatgg 284434DNAArtificial SequenceDescription of Artificial Sequence PCR primer 44aaacccgggt taatctgtct tcttgtcttc tcca 344534DNAArtificial SequenceDescription of Artificial Sequence PCR primer

45ctggagctct tttaccgagg ttgggccttg atcc 344634DNAArtificial SequenceDescription of Artificial Sequence PCR primer 46attgagctcc caatgtccaa gctcgtgtgc aata 344723DNAArtificial SequenceDescription of Artificial Sequence PCR primer 47gccgacagtg gtcccaaaga tgg 234828DNAArtificial SequenceDescription of Artificial Sequence PCR primer 48aaacccggga tggcgattcc tttcatgg 284934DNAArtificial SequenceDescription of Artificial Sequence PCR primer 49aaaggatcct taatctgtct tcttgtcttc tcca 345018DNAArtificial SequenceDescription of Artificial Sequence PCR primer 50gcaagaccgg caacagga 18515544DNAArtificial SequenceDescription of Artificial Sequence pBI121-antisense-AtCPP vector sequence 51gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccttaatct gtcttcttgt cttctccatc 3360agtggctcga agcctttcaa caagaggagg atgtgagtag tgataagctg agtacaatgg 3420atcagtgttc attgttgata agttctcttc ctgtagtttc actagagcag gacgaagatc 3480ttttgcatag tcaagcttca cagcaaaagc atcagcctga aactcaaacg ctcgactaac 3540gaggttcagg ccaaagctta ctagatgttg cagtggtatt acagtgtgct gaaatatgat 3600caaaccaatg agaacaggct gtgtatcaaa tccgaaactc ctgaagagat cagtggagtt 3660tctgagaaga gtgtatcctc caaattgtaa gaaggcaagg atttgaactg caatgaacga 3720gtatgtagtg tgattcagtt tccaatgtcc aagctcgtgt gcaataaccg ccacaatttc 3780atcctcattc ttgcactgct gaatcaacgt atcataaaga acaatccttt tgttcttaaa 3840gaaaccatac atgtaagcat tgctatggct tgaccttgta gatccatcga caacaaacag 3900cttcttcaaa ggaaacttta gggaagaagc aagtttctca atcttctccc ggaggtctcc 3960atctggaaga ggagtgaatt tgttgaagag cggtgctatc aagaccgggt atatagtcat 4020catcactaga gacaggataa acatgaatgc ccacagatag atggcaagat aaggacctcc 4080tttctggact atgaaaatta tcgcagcaac aatgggtggg cctagtatga cagagaggaa 4140tgttcctttg atcatgtccc taatgaacat ccatattgtt tgtttgttga acccatgccg 4200agactcgatc acgaaagttg agtacaaaga aaatggcaaa tcagtgatct gtgaccatgt 4260cataacacca gccaagaatg aaagagtatg cagtatttca ttctccggat caaggcccaa 4320cctcggtaaa acagctccag acatcttcca aaaccaaggc aagatcccaa agaacaaaat 4380tgcagagtcc ataagtatag ttacaaactc atgaacaaag tgaaaatagc ttttgtcaag 4440actgtatgct cgtgatttct caaacttctc ttggctaatt acaccaacca aggttttcgg 4500gagagttgga agcttgagag cagtgagttg cctcagatcc aaatacgtct caaaaatgta 4560catcactatc ataaaaccca cgacggtttc catgaaagga atcgccatcc cctcgaattt 4620ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 4680tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 4740atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 4800atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 4860atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 4920accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 4980atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgccc 5040gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5100ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 5160aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 5220cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 5280ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggaacca 5340ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 5400ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 5460ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg 5520tttacaccac aatatatcct gcca 5544525668DNAArtificial SequenceDescription of Artificial Sequence vector sequence 52gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatccatg gcgattcctt tcatggaaac 3480cgtcgtgggt tttatgatag tgatgtacat ttttgagacg tatttggatc tgaggcaact 3540cactgctctc aagcttccaa ctctcccgaa aaccttggtt ggtgtaatta gccaagagaa 3600gtttgagaaa tcacgagcat acagtcttga caaaagctat tttcactttg ttcatgagtt 3660tgtaactata cttatggact ctgcaatttt gttctttggg atcttgcctt ggttttggaa 3720gatgtctgga gctgttttac cgaggttggg ccttgatccg gagaatgaaa tactgcatac 3780tctttcattc ttggctggtg ttatgacatg gtcacagatc actgatttgc cattttcttt 3840gtactcaact ttcgtgatcg agtctcggca tgggttcaac aaacaaacaa tatggatgtt 3900cattagggac atgatcaaag gaacattcct ctctgtcata ctaggcccac ccattgttgc 3960tgcgataatt ttcatagtcc agaaaggagg tccttatctt gccatctatc tgtgggcatt 4020catgtttatc ctgtctctag tgatgatgac tatatacccg gtcttgatag caccgctctt 4080caacaaattc actcctcttc cagatggaga cctccgggag aagattgaga aacttgcttc 4140ttccctaaag tttcctttga agaagctgtt tgttgtcgat ggatctacaa ggtcaagcca 4200tagcaatgct tacatgtatg gtttctttaa gaacaaaagg attgttcttt atgatacgtt 4260gattcagcag tgcaagaatg aggatgaaat tgtggcggtt attgcacacg agcttggaca 4320ttggaaactg aatcacacta catactcgtt cattgcagtt caaatccttg ccttcttaca 4380atttggagga tacactcttc tcagaaactc cactgatctc ttcaggagtt tcggatttga 4440tacacagcct gttctcattg gtttgatcat atttcagcac actgtaatac cactgcaaca 4500tctagtaagc tttggcctga acctcgttag tcgagcgttt gagtttcagg ctgatgcttt 4560tgctgtgaag cttgactatg caaaagatct tcgtcctgct ctagtgaaac tacaggaaga 4620gaacttatca acaatgaaca ctgatccatt gtactcagct tatcactact cacatcctcc 4680tcttgttgaa aggcttcgag ccactgatgg agaagacaag aagacagatt aacccctcga 4740atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 4800gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 4860tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 4920tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 4980tgtcatctat gttactagat cgggaattca ctggccgtcg ttttacaacg tcgtgactgg 5040gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 5100cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 5160gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 5220agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 5280caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 5340tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 5400aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 5460accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5520ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 5580aaaaccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 5640tttgtttaca ccacaatata tcctgcca 5668536608DNAArtificial SequenceDescription of Artificial Sequence pRD29A-HP-AtCPP vector sequence 53gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt

aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatcctcc caatgtccaa gctcgtgtgc 3480aataaccgcc acaatttcat cctcattctt gcactgctga atcaacgtat cataaagaac 3540aatccttttg ttcttaaaga aaccatacat gtaagcattg ctatggcttg accttgtaga 3600tccatcgaca acaaacagct tcttcaaagg aaactttagg gaagaagcaa gtttctcaat 3660cttctcccgg aggtctccat ctggaagagg agtgaatttg ttgaagagcg gtgctatcaa 3720gaccgggtat atagtcatca tcactagaga caggataaac atgaatgccc acagatagat 3780ggcaagataa ggacctcctt tctggactat gaaaattatc gcagcaacaa tgggtgggcc 3840tagtatgaca gagaggaatg ttcctttgat catgtcccta atgaacatcc atattgtttg 3900tttgttgaac ccatgccgag actcgatcac gaaagttgag tacaaagaaa atggcaaatc 3960agtgatctgt gaccatgtca taacaccagc caagaatgaa agagtatgca gtatttcatt 4020ctccggatca aggcccaacc tcggtaaaag aggatcccca tctacccgct tcgcgtcggc 4080atccggtcag tggcagtgaa gggcgaacag ttcctgatta accacaaacc gttctacttt 4140actggctttg gtcgtcatga agatgcggac ttgcgtggca aaggattcga taacgtgctg 4200atggtgcacg accacgcatt aatggactgg attggggcca actcctaccg tacctcgcat 4260tacccttacg ctgaagagat gctcgactgg gcagatgaac atggcatcgt ggtgattgat 4320gaaactgctg ctgtcggctt ttcgctctct ttaggcattg gtttcgaagc gggcaacaag 4380ccgaaagaac tgtacagcga agaggcagtc aacggggaaa ctcagcaagc gcacttacag 4440gcgattaaag agctgatagc gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt 4500gccaacgaac cggatacccg tccgcaaggt gcacgggaat atttcgcgcc actggcggaa 4560gcaacgcgta aactcgaccc gacgcgtccg atcacctgcg tcaatgtaat gttctgcgac 4620gctcacaccg ataccatcag cgatctcttt gatgtgctgt gcctgaaccg ttattacgga 4680tggtatgtcc aaagcggcga tttggaaacg gcagagaagg tactggaaaa agaacttctg 4740gcctggcagg agaaactgta caccgacatg tggagtgaag agtatcagtg tgcatggctg 4800gatatgtatc accgcgtctt tgatcgcgtc agcgccgtcg tcggtgaaca ggtatggaat 4860ttcgccgatt ttgcgacctc gcaaggcata ttgcgcgttg gcggtaacaa gaaagggatc 4920ttcactcgcg accgcaaacc gaagtcggcg gcttttctgc tgcaaaaacg ctggactggc 4980atgaacttcg gtgaaaaacc gcagcaggga ggcaaacaat gaatcaacaa ctctcctggc 5040gcaccatcgt cggctacagc ctcgggaatt gctaccgagc tcttttaccg aggttgggcc 5100ttgatccgga gaatgaaata ctgcatactc tttcattctt ggctggtgtt atgacatggt 5160cacagatcac tgatttgcca ttttctttgt actcaacttt cgtgatcgag tctcggcatg 5220ggttcaacaa acaaacaata tggatgttca ttagggacat gatcaaagga acattcctct 5280ctgtcatact aggcccaccc attgttgctg cgataatttt catagtccag aaaggaggtc 5340cttatcttgc catctatctg tgggcattca tgtttatcct gtctctagtg atgatgacta 5400tatacccggt cttgatagca ccgctcttca acaaattcac tcctcttcca gatggagacc 5460tccgggagaa gattgagaaa cttgcttctt ccctaaagtt tcctttgaag aagctgtttg 5520ttgtcgatgg atctacaagg tcaagccata gcaatgctta catgtatggt ttctttaaga 5580acaaaaggat tgttctttat gatacgttga ttcagcagtg caagaatgag gatgaaattg 5640tggcggttat tgcacacgag cttggacatt gggagctcga atttccccga tcgttcaaac 5700atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 5760taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 5820atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 5880aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 5940cgggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 6000acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 6060caccgatcgc ccttcccaac agttgcgcag cctgaatggc gcccgctcct ttcgctttct 6120tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 6180ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg 6240atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 6300ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 6360gctattcttt tgatttataa gggattttgc cgatttcgga accaccatca aacaggattt 6420tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt 6480gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc cagtacatta 6540aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata 6600tcctgcca 6608545668DNAArtificial SequenceDescription of Artificial Sequence pRD29A-antisense-AtCPP vector sequence 54gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatcctta atctgtcttc ttgtcttctc 3480catcagtggc tcgaagcctt tcaacaagag gaggatgtga gtagtgataa gctgagtaca 3540atggatcagt gttcattgtt gataagttct cttcctgtag tttcactaga gcaggacgaa 3600gatcttttgc atagtcaagc ttcacagcaa aagcatcagc ctgaaactca aacgctcgac 3660taacgaggtt caggccaaag cttactagat gttgcagtgg tattacagtg tgctgaaata 3720tgatcaaacc aatgagaaca ggctgtgtat caaatccgaa actcctgaag agatcagtgg 3780agtttctgag aagagtgtat cctccaaatt gtaagaaggc aaggatttga actgcaatga 3840acgagtatgt agtgtgattc agtttccaat gtccaagctc gtgtgcaata accgccacaa 3900tttcatcctc attcttgcac tgctgaatca acgtatcata aagaacaatc cttttgttct 3960taaagaaacc atacatgtaa gcattgctat ggcttgacct tgtagatcca tcgacaacaa 4020acagcttctt caaaggaaac tttagggaag aagcaagttt ctcaatcttc tcccggaggt 4080ctccatctgg aagaggagtg aatttgttga agagcggtgc tatcaagacc gggtatatag 4140tcatcatcac tagagacagg ataaacatga atgcccacag atagatggca agataaggac 4200ctcctttctg gactatgaaa attatcgcag caacaatggg tgggcctagt atgacagaga 4260ggaatgttcc tttgatcatg tccctaatga acatccatat tgtttgtttg ttgaacccat 4320gccgagactc gatcacgaaa gttgagtaca aagaaaatgg caaatcagtg atctgtgacc 4380atgtcataac accagccaag aatgaaagag tatgcagtat ttcattctcc ggatcaaggc 4440ccaacctcgg taaaacagct ccagacatct tccaaaacca aggcaagatc ccaaagaaca 4500aaattgcaga gtccataagt atagttacaa actcatgaac aaagtgaaaa tagcttttgt 4560caagactgta tgctcgtgat ttctcaaact tctcttggct aattacacca accaaggttt 4620tcgggagagt tggaagcttg agagcagtga gttgcctcag atccaaatac gtctcaaaaa 4680tgtacatcac tatcataaaa cccacgacgg tttccatgaa aggaatcgcc atcccctcga 4740atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 4800gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 4860tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 4920tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 4980tgtcatctat gttactagat cgggaattca ctggccgtcg ttttacaacg tcgtgactgg 5040gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 5100cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 5160gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 5220agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 5280caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 5340tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 5400aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 5460accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5520ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 5580aaaaccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 5640tttgtttaca ccacaatata tcctgcca 5668555074DNAArtificial SequenceDescription of Artificial Sequence MuA-AtCPP vector sequence 55gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc tgggaaattt ttcgccagtt 2520ctaaatatcc ggaaacctct tgggatgcca ttgcccatct atctgtaatt tattgacgaa 2580atagacgaaa aggaaggtgg ctcctataaa gcacatcatt gcgataacag aaaggccatt 2640gttgaagata cctctgctga cattggtccc caagtggaag caccacccca tgaggagcac 2700cgtggagtaa gaagacgttc gagccacgtc gaaaaagcaa gtgtgttgat gtagtatctc 2760cattgacgta agggatgacg cacaatccaa ctatccatcg caagaccatt gctctatata 2820agaaagttaa tatcatttcg agtggccacg ctgaggggga tccatggcga ttcctttcat 2880ggaaaccgtc gtgggtttta tgatagtgat gtacattttt gagacgtatt tggatctgag 2940gcaactcact gctctcaagc ttccaactct cccgaaaacc ttggttggtg taattagcca 3000agagaagttt gagaaatcac gagcatacag tcttgacaaa agctattttc actttgttca 3060tgagtttgta actatactta tggactctgc aattttgttc tttgggatct tgccttggtt 3120ttggaagatg tctggagctg ttttaccgag gttgggcctt gatccggaga atgaaatact 3180gcatactctt tcattcttgg ctggtgttat gacatggtca cagatcactg atttgccatt 3240ttctttgtac tcaactttcg tgatcgagtc tcggcatggg ttcaacaaac aaacaatatg 3300gatgttcatt agggacatga tcaaaggaac attcctctct gtcatactag gcccacccat 3360tgttgctgcg ataattttca tagtccagaa aggaggtcct tatcttgcca tctatctgtg 3420ggcattcatg tttatcctgt ctctagtgat gatgactata tacccggtct tgatagcacc 3480gctcttcaac aaattcactc ctcttccaga tggagacctc cgggagaaga ttgagaaact 3540tgcttcttcc ctaaagtttc ctttgaagaa gctgtttgtt gtcgatggat ctacaaggtc 3600aagccatagc aatgcttaca tgtatggttt ctttaagaac aaaaggattg ttctttatga 3660tacgttgatt cagcagtgca agaatgagga tgaaattgtg gcggttattg cacacgagct 3720tggacattgg aaactgaatc acactacata ctcgttcatt gcagttcaaa tccttgcctt 3780cttacaattt ggaggataca ctcttctcag aaactccact gatctcttca ggagtttcgg 3840atttgataca cagcctgttc tcattggttt gatcatattt cagcacactg taataccact 3900gcaacatcta gtaagctttg gcctgaacct cgttagtcga gcgtttgagt ttcaggctga 3960tgcttttgct gtgaagcttg actatgcaaa agatcttcgt cctgctctag tgaaactaca 4020ggaagagaac ttatcaacaa tgaacactga tccattgtac tcagcttatc actactcaca 4080tcctcctctt gttgaaaggc ttcgagccac tgatggagaa gacaagaaga cagattaacc 4140cctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 4200ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 4260ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat 4320tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 4380gcgcggtgtc atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt 4440gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 4500agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 4560aatggcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 4620ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 4680cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 4740ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 4800tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 4860ttcggaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg 4920ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg 4980aaaagaaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 5040cgtcaatttg tttacaccac aatatatcct gcca 5074565076DNAArtificial SequenceDescription of Artificial Sequence MuA-GmCPP vector sequence 56gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc

180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc tgggaaattt ttcgccagtt 2520ctaaatatcc ggaaacctct tgggatgcca ttgcccatct atctgtaatt tattgacgaa 2580atagacgaaa aggaaggtgg ctcctataaa gcacatcatt gcgataacag aaaggccatt 2640gttgaagata cctctgctga cattggtccc caagtggaag caccacccca tgaggagcac 2700cgtggagtaa gaagacgttc gagccacgtc gaaaaagcaa gtgtgttgat gtagtatctc 2760cattgacgta agggatgacg cacaatccaa ctatccatcg caagaccatt gctctatata 2820agaaagttaa tatcatttcg agtggccacg ctgaggggga tcgggatggc gtttccctac 2880atggaagccg ttgtcggatt tatgatatta atgtacattt ttgaaactta cttggatgtg 2940cgacaacata gggccctcaa acttcctact cttccaaaga ctttagaggg tgttatcagc 3000caagagaaat ttgagaaatc tagagcctat agtcttgata aaagccactt ccattttgtt 3060cacgagtttg tgacaatagt gacagactct acaattttgt actttggggt attgccctgg 3120ttttggaaga aatcaggaga ttttatgaca atagctggtt tcaatgctga gaatgaaata 3180ctgcataccc ttgccttctt agcagggctg atgatttggt cacagataac agatttgccc 3240ttttctctgt actcaacttt tgtgattgag gcccgtcatg gttttaataa gcaaacacca 3300tggttattct ttagggacat gcttaaagga attttccttt ctgtaataat tggtccacct 3360attgtggctg caatcattgt aatagtacag aaaggaggtc catacttggc catctatctt 3420tgggttttta cgtttggtct ttctattgtg atgatgaccc tttatccagt actaatagct 3480ccactcttca ataagttcac tccacttcca gatggtcaac tcagggagaa aatcgagaaa 3540cttgcttcct ccctcaacta tccgttaaag aaactatttg ttgtcgatgg atccacaaga 3600tcaagtcaca gcaatgccta tatgtatgga ttcttcaaga acaagaggat tgtcccttat 3660gacacattaa ttcaacagtg caaagacgat gaggaaattg ttgctgttat tgcccatgag 3720ttgggacact ggaagctcaa ccatactgtg tacacatttg ttgctatgca gattcttaca 3780cttctacaat ttggaggata tacactagtg cgaaattcag ctgatctgta tcgaagcttt 3840gggtttgata cgcagccagt cctcattggg ctcatcatat ttcagcatac tgtaatccca 3900cttcagcaat tggtcagctt tggtctgaac ctagtcagcc gatcatttga atttcaggct 3960gatggctttg ccaagaagct tggatatgca tctggattac gcggtggtct tgtgaaacta 4020caggaggaga atctgtcagc tatgaataca gatccttggt actctgctta tcactattct 4080catcctcccc ttgttgaaag attggccgcg ctggacgaac cggataagaa ggaagactaa 4140gagctcgaat ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc 4200tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat 4260aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca 4320attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc 4380gcgcgcggtg tcatctatgt tactagatcg ggaattcact ggccgtcgtt ttacaacgtc 4440gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 4500ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 4560tgaatggcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 4620ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 4680ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 4740acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 4800actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 4860atttcggaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct 4920tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg 4980tgaaaagaaa aaccacccca gtacattaaa aacgtccgca atgtgttatt aagttgtcta 5040agcgtcaatt tgtttacacc acaatatatc ctgcca 5076575549DNAArtificial SequenceDescription of Artificial Sequence pBI121-GmCPP vector sequence 57gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccccgggat ggcgtttccc tacatggaag 3360ccgttgtcgg atttatgata ttaatgtaca tttttgaaac ttacttggat gtgcgacaac 3420atagggccct caaacttcct actcttccaa agactttaga gggtgttatc agccaagaga 3480aatttgagaa atctagagcc tatagtcttg ataaaagcca cttccatttt gttcacgagt 3540ttgtgacaat agtgacagac tctacaattt tgtactttgg ggtattgccc tggttttgga 3600agaaatcagg agattttatg acaatagctg gtttcaatgc tgagaatgaa atactgcata 3660cccttgcctt cttagcaggg ctgatgattt ggtcacagat aacagatttg cccttttctc 3720tgtactcaac ttttgtgatt gaggcccgtc atggttttaa taagcaaaca ccatggttat 3780tctttaggga catgcttaaa ggaattttcc tttctgtaat aattggtcca cctattgtgg 3840ctgcaatcat tgtaatagta cagaaaggag gtccatactt ggccatctat ctttgggttt 3900ttacgtttgg tctttctatt gtgatgatga ccctttatcc agtactaata gctccactct 3960tcaataagtt cactccactt ccagatggtc aactcaggga gaaaatcgag aaacttgctt 4020cctccctcaa ctatccgtta aagaaactat ttgttgtcga tggatccaca agatcaagtc 4080acagcaatgc ctatatgtat ggattcttca agaacaagag gattgtccct tatgacacat 4140taattcaaca gtgcaaagac gatgaggaaa ttgttgctgt tattgcccat gagttgggac 4200actggaagct caaccatact gtgtacacat ttgttgctat gcagattctt acacttctac 4260aatttggagg atatacacta gtgcgaaatt cagctgatct gtatcgaagc tttgggtttg 4320atacgcagcc agtcctcatt gggctcatca tatttcagca tactgtaatc ccacttcagc 4380aattggtcag ctttggtctg aacctagtca gccgatcatt tgaatttcag gctgatggct 4440ttgccaagaa gcttggatat gcatctggat tacgcggtgg tcttgtgaaa ctacaggagg 4500agaatctgtc agctatgaat acagatcctt ggtactctgc ttatcactat tctcatcctc 4560cccttgttga aagattggcc gcgctggacg aaccggataa gaaggaagac taagagctcg 4620aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc 4680ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac 4740atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac 4800atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg 4860gtgtcatcta tgttactaga tcgggaattc actggccgtc gttttacaac gtcgtgactg 4920ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 4980gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 5040cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5100aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5160ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5220ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 5280caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5340aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 5400actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 5460aaaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt attaagttgt ctaagcgtca 5520atttgtttac accacaatat atcctgcca 5549586352DNAArtificial SequenceDescription of Artificial Sequence pBI121-HP-GmCPP vector sequence 58gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagaccg gttcgtccag cgcggccaat ctttcaacaa 3360ggggaggatg agaatagtga taagcagagt accaaggatc tgtattcata gctgacagat 3420tctcctcctg tagtttcaca agaccaccgc gtaatccaga tgcatatcca agcttcttgg 3480caaagccatc agcctgaaat tcaaatgatc ggctgactag gttcagacca aagctgacca 3540attgctgaag tgggattaca gtatgctgaa atatgatgag cccaatgagg actggctgcg 3600tatcaaaccc aaagcttcga tacagatcag ctgaatttcg cactagtgta tatcctccaa 3660attgtagaag tgtaagaatc tgcatagcaa caaatgtgta cacagtatgg ttgagcttcc 3720agtgtcccaa ctcatgggca ataacagcaa caatttcctc atcgtctttg cactgttgaa 3780ttaatgtgtc ataagggaca atcctcttgt tcttgaagaa tccatacata taggcattgc 3840tgtgacttga tcttgtggat ccccatctac ccgcttcgcg tcggcatccg gtcagtggca 3900gtgaagggcg aacagttcct gattaaccac aaaccgttct actttactgg ctttggtcgt 3960catgaagatg cggacttgcg tggcaaagga ttcgataacg tgctgatggt gcacgaccac 4020gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc ttacgctgaa 4080gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac tgctgctgtc 4140ggcttttcgc tctctttagg cattggtttc gaagcgggca acaagccgaa agaactgtac 4200agcgaagagg cagtcaacgg ggaaactcag caagcgcact tacaggcgat taaagagctg 4260atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa cgaaccggat 4320acccgtccgc aaggtgcacg ggaatatttc gcgccactgg cggaagcaac

gcgtaaactc 4380gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca caccgatacc 4440atcagcgatc tctttgatgt gctgtgcctg aaccgttatt acggatggta tgtccaaagc 4500ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac ttctggcctg gcaggagaaa 4560ctgtacaccg acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc 4620gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc cgattttgcg 4680acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac tcgcgaccgc 4740aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga ctggcatgaa cttcggtgaa 4800aaaccgcagc agggaggcaa acaatgaatc aacaactctc ctggcgcacc atcgtcggct 4860acagcctcgg gaattgctac cgagctcaca agatcaagtc acagcaatgc ctatatgtat 4920ggattcttca agaacaagag gattgtccct tatgacacat taattcaaca gtgcaaagac 4980gatgaggaaa ttgttgctgt tattgcccat gagttgggac actggaagct caaccatact 5040gtgtacacat ttgttgctat gcagattctt acacttctac aatttggagg atatacacta 5100gtgcgaaatt cagctgatct gtatcgaagc tttgggtttg atacgcagcc agtcctcatt 5160gggctcatca tatttcagca tactgtaatc ccacttcagc aattggtcag ctttggtctg 5220aacctagtca gccgatcatt tgaatttcag gctgatggct ttgccaagaa gcttggatat 5280gcatctggat tacgcggtgg tcttgtgaaa ctacaggagg agaatctgtc agctatgaat 5340acagatcctt ggtactctgc ttatcactat tctcatcctc cccttgttga aagattggcc 5400gcgctggacg aaccgggagc tcgaatttcc ccgatcgttc aaacatttgg caataaagtt 5460tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 5520acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 5580tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 5640actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttcactggcc 5700gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 5760gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 5820caacagttgc gcagcctgaa tggcgcccgc tcctttcgct ttcttccctt cctttctcgc 5880cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 5940tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg 6000gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 6060tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt 6120ataagggatt ttgccgattt cggaaccacc atcaaacagg attttcgcct gctggggcaa 6180accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg 6240ttgcccgtct cactggtgaa aagaaaaacc accccagtac attaaaaacg tccgcaatgt 6300gttattaagt tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc ca 6352595549DNAArtificial SequenceDescription of Artificial Sequence pBI121-antisense-GmCPP vector sequence 59gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccccgggtt agtcttcctt cttatccggt 3360tcgtccagcg cggccaatct ttcaacaagg ggaggatgag aatagtgata agcagagtac 3420caaggatctg tattcatagc tgacagattc tcctcctgta gtttcacaag accaccgcgt 3480aatccagatg catatccaag cttcttggca aagccatcag cctgaaattc aaatgatcgg 3540ctgactaggt tcagaccaaa gctgaccaat tgctgaagtg ggattacagt atgctgaaat 3600atgatgagcc caatgaggac tggctgcgta tcaaacccaa agcttcgata cagatcagct 3660gaatttcgca ctagtgtata tcctccaaat tgtagaagtg taagaatctg catagcaaca 3720aatgtgtaca cagtatggtt gagcttccag tgtcccaact catgggcaat aacagcaaca 3780atttcctcat cgtctttgca ctgttgaatt aatgtgtcat aagggacaat cctcttgttc 3840ttgaagaatc catacatata ggcattgctg tgacttgatc ttgtggatcc atcgacaaca 3900aatagtttct ttaacggata gttgagggag gaagcaagtt tctcgatttt ctccctgagt 3960tgaccatctg gaagtggagt gaacttattg aagagtggag ctattagtac tggataaagg 4020gtcatcatca caatagaaag accaaacgta aaaacccaaa gatagatggc caagtatgga 4080cctcctttct gtactattac aatgattgca gccacaatag gtggaccaat tattacagaa 4140aggaaaattc ctttaagcat gtccctaaag aataaccatg gtgtttgctt attaaaacca 4200tgacgggcct caatcacaaa agttgagtac agagaaaagg gcaaatctgt tatctgtgac 4260caaatcatca gccctgctaa gaaggcaagg gtatgcagta tttcattctc agcattgaaa 4320ccagctattg tcataaaatc tcctgatttc ttccaaaacc agggcaatac cccaaagtac 4380aaaattgtag agtctgtcac tattgtcaca aactcgtgaa caaaatggaa gtggctttta 4440tcaagactat aggctctaga tttctcaaat ttctcttggc tgataacacc ctctaaagtc 4500tttggaagag taggaagttt gagggcccta tgttgtcgca catccaagta agtttcaaaa 4560atgtacatta atatcataaa tccgacaacg gcttccatgt agggaaacgc catgagctcg 4620aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc 4680ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac 4740atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac 4800atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg 4860gtgtcatcta tgttactaga tcgggaattc actggccgtc gttttacaac gtcgtgactg 4920ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 4980gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 5040cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5100aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5160ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5220ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 5280caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5340aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 5400actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 5460aaaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt attaagttgt ctaagcgtca 5520atttgtttac accacaatat atcctgcca 5549605673DNAArtificial SequenceDescription of Artificial Sequence pRD29A-GmCPP vector sequence 60gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatccccg ggatggcgtt tccctacatg 3480gaagccgttg tcggatttat gatattaatg tacatttttg aaacttactt ggatgtgcga 3540caacataggg ccctcaaact tcctactctt ccaaagactt tagagggtgt tatcagccaa 3600gagaaatttg agaaatctag agcctatagt cttgataaaa gccacttcca ttttgttcac 3660gagtttgtga caatagtgac agactctaca attttgtact ttggggtatt gccctggttt 3720tggaagaaat caggagattt tatgacaata gctggtttca atgctgagaa tgaaatactg 3780catacccttg ccttcttagc agggctgatg atttggtcac agataacaga tttgcccttt 3840tctctgtact caacttttgt gattgaggcc cgtcatggtt ttaataagca aacaccatgg 3900ttattcttta gggacatgct taaaggaatt ttcctttctg taataattgg tccacctatt 3960gtggctgcaa tcattgtaat agtacagaaa ggaggtccat acttggccat ctatctttgg 4020gtttttacgt ttggtctttc tattgtgatg atgacccttt atccagtact aatagctcca 4080ctcttcaata agttcactcc acttccagat ggtcaactca gggagaaaat cgagaaactt 4140gcttcctccc tcaactatcc gttaaagaaa ctatttgttg tcgatggatc cacaagatca 4200agtcacagca atgcctatat gtatggattc ttcaagaaca agaggattgt cccttatgac 4260acattaattc aacagtgcaa agacgatgag gaaattgttg ctgttattgc ccatgagttg 4320ggacactgga agctcaacca tactgtgtac acatttgttg ctatgcagat tcttacactt 4380ctacaatttg gaggatatac actagtgcga aattcagctg atctgtatcg aagctttggg 4440tttgatacgc agccagtcct cattgggctc atcatatttc agcatactgt aatcccactt 4500cagcaattgg tcagctttgg tctgaaccta gtcagccgat catttgaatt tcaggctgat 4560ggctttgcca agaagcttgg atatgcatct ggattacgcg gtggtcttgt gaaactacag 4620gaggagaatc tgtcagctat gaatacagat ccttggtact ctgcttatca ctattctcat 4680cctccccttg ttgaaagatt ggccgcgctg gacgaaccgg ataagaagga agactaagag 4740ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 4800tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 4860taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 4920atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 4980cgcggtgtca tctatgttac tagatcggga attcactggc cgtcgtttta caacgtcgtg 5040actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 5100gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5160atggcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 5220cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 5280gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 5340gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 5400ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 5460tcggaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 5520tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga 5580aaagaaaaac caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc 5640gtcaatttgt ttacaccaca atatatcctg cca 5673616476DNAArtificial SequenceDescription of Artificial Sequence pRD29A-HP-GmCPP vector sequence 61gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc

aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag accggttcgt ccagcgcggc caatctttca 3480acaaggggag gatgagaata gtgataagca gagtaccaag gatctgtatt catagctgac 3540agattctcct cctgtagttt cacaagacca ccgcgtaatc cagatgcata tccaagcttc 3600ttggcaaagc catcagcctg aaattcaaat gatcggctga ctaggttcag accaaagctg 3660accaattgct gaagtgggat tacagtatgc tgaaatatga tgagcccaat gaggactggc 3720tgcgtatcaa acccaaagct tcgatacaga tcagctgaat ttcgcactag tgtatatcct 3780ccaaattgta gaagtgtaag aatctgcata gcaacaaatg tgtacacagt atggttgagc 3840ttccagtgtc ccaactcatg ggcaataaca gcaacaattt cctcatcgtc tttgcactgt 3900tgaattaatg tgtcataagg gacaatcctc ttgttcttga agaatccata catataggca 3960ttgctgtgac ttgatcttgt ggatccccat ctacccgctt cgcgtcggca tccggtcagt 4020ggcagtgaag ggcgaacagt tcctgattaa ccacaaaccg ttctacttta ctggctttgg 4080tcgtcatgaa gatgcggact tgcgtggcaa aggattcgat aacgtgctga tggtgcacga 4140ccacgcatta atggactgga ttggggccaa ctcctaccgt acctcgcatt acccttacgc 4200tgaagagatg ctcgactggg cagatgaaca tggcatcgtg gtgattgatg aaactgctgc 4260tgtcggcttt tcgctctctt taggcattgg tttcgaagcg ggcaacaagc cgaaagaact 4320gtacagcgaa gaggcagtca acggggaaac tcagcaagcg cacttacagg cgattaaaga 4380gctgatagcg cgtgacaaaa accacccaag cgtggtgatg tggagtattg ccaacgaacc 4440ggatacccgt ccgcaaggtg cacgggaata tttcgcgcca ctggcggaag caacgcgtaa 4500actcgacccg acgcgtccga tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga 4560taccatcagc gatctctttg atgtgctgtg cctgaaccgt tattacggat ggtatgtcca 4620aagcggcgat ttggaaacgg cagagaaggt actggaaaaa gaacttctgg cctggcagga 4680gaaactgtac accgacatgt ggagtgaaga gtatcagtgt gcatggctgg atatgtatca 4740ccgcgtcttt gatcgcgtca gcgccgtcgt cggtgaacag gtatggaatt tcgccgattt 4800tgcgacctcg caaggcatat tgcgcgttgg cggtaacaag aaagggatct tcactcgcga 4860ccgcaaaccg aagtcggcgg cttttctgct gcaaaaacgc tggactggca tgaacttcgg 4920tgaaaaaccg cagcagggag gcaaacaatg aatcaacaac tctcctggcg caccatcgtc 4980ggctacagcc tcgggaattg ctaccgagct cacaagatca agtcacagca atgcctatat 5040gtatggattc ttcaagaaca agaggattgt cccttatgac acattaattc aacagtgcaa 5100agacgatgag gaaattgttg ctgttattgc ccatgagttg ggacactgga agctcaacca 5160tactgtgtac acatttgttg ctatgcagat tcttacactt ctacaatttg gaggatatac 5220actagtgcga aattcagctg atctgtatcg aagctttggg tttgatacgc agccagtcct 5280cattgggctc atcatatttc agcatactgt aatcccactt cagcaattgg tcagctttgg 5340tctgaaccta gtcagccgat catttgaatt tcaggctgat ggctttgcca agaagcttgg 5400atatgcatct ggattacgcg gtggtcttgt gaaactacag gaggagaatc tgtcagctat 5460gaatacagat ccttggtact ctgcttatca ctattctcat cctccccttg ttgaaagatt 5520ggccgcgctg gacgaaccgg gagctcgaat ttccccgatc gttcaaacat ttggcaataa 5580agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 5640aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 5700tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 5760gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggaattcact 5820ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 5880tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 5940ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc ccttcctttc 6000tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 6060gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta 6120gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 6180atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg 6240atttataagg gattttgccg atttcggaac caccatcaaa caggattttc gcctgctggg 6300gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca 6360gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa aacgtccgca 6420atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgcca 6476625673DNAArtificial SequenceDescription of Artificial Sequence pRD29A-antisense-GmCPP vector sequence 62gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatccccg ggttagtctt ccttcttatc 3480cggttcgtcc agcgcggcca atctttcaac aaggggagga tgagaatagt gataagcaga 3540gtaccaagga tctgtattca tagctgacag attctcctcc tgtagtttca caagaccacc 3600gcgtaatcca gatgcatatc caagcttctt ggcaaagcca tcagcctgaa attcaaatga 3660tcggctgact aggttcagac caaagctgac caattgctga agtgggatta cagtatgctg 3720aaatatgatg agcccaatga ggactggctg cgtatcaaac ccaaagcttc gatacagatc 3780agctgaattt cgcactagtg tatatcctcc aaattgtaga agtgtaagaa tctgcatagc 3840aacaaatgtg tacacagtat ggttgagctt ccagtgtccc aactcatggg caataacagc 3900aacaatttcc tcatcgtctt tgcactgttg aattaatgtg tcataaggga caatcctctt 3960gttcttgaag aatccataca tataggcatt gctgtgactt gatcttgtgg atccatcgac 4020aacaaatagt ttctttaacg gatagttgag ggaggaagca agtttctcga ttttctccct 4080gagttgacca tctggaagtg gagtgaactt attgaagagt ggagctatta gtactggata 4140aagggtcatc atcacaatag aaagaccaaa cgtaaaaacc caaagataga tggccaagta 4200tggacctcct ttctgtacta ttacaatgat tgcagccaca ataggtggac caattattac 4260agaaaggaaa attcctttaa gcatgtccct aaagaataac catggtgttt gcttattaaa 4320accatgacgg gcctcaatca caaaagttga gtacagagaa aagggcaaat ctgttatctg 4380tgaccaaatc atcagccctg ctaagaaggc aagggtatgc agtatttcat tctcagcatt 4440gaaaccagct attgtcataa aatctcctga tttcttccaa aaccagggca ataccccaaa 4500gtacaaaatt gtagagtctg tcactattgt cacaaactcg tgaacaaaat ggaagtggct 4560tttatcaaga ctataggctc tagatttctc aaatttctct tggctgataa caccctctaa 4620agtctttgga agagtaggaa gtttgagggc cctatgttgt cgcacatcca agtaagtttc 4680aaaaatgtac attaatatca taaatccgac aacggcttcc atgtagggaa acgccatgag 4740ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 4800tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 4860taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 4920atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 4980cgcggtgtca tctatgttac tagatcggga attcactggc cgtcgtttta caacgtcgtg 5040actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 5100gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5160atggcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 5220cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 5280gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 5340gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 5400ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 5460tcggaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 5520tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga 5580aaagaaaaac caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc 5640gtcaatttgt ttacaccaca atatatcctg cca 5673635544DNAArtificial SequenceDescription of Artificial Sequence pBI121-BnCPP vector sequence 63gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccatggcga ttcctttcat ggaaaccgtc 3360gttggtttta tgatagtgat gtacgttttt gagacgtatt tggatctgag gcaacatact 3420gctctcaagc ttcccactct cccaaagact ttggttggag tcattagcca agagaagttt 3480gagaaatctc gagcttacag tcttgacaaa agccattttc actttgttca tgagtttgtt 3540actatactta tggactctgc gattctgttc tttgggatct tgccttggtt ttggaagata 3600tctggcggct ttctaccaat ggtgggactc gatccagaga atgaaatcct gcacactctt 3660tcattcttgg ctggtcttat gacatggtca cagatcactg atttgccatt ttctttgtac 3720tcaactttcg tgatcgagtc tcggcatggg ttcaacaaac aaacaatatg gatgttcatt 3780agggacatga tcaaaggaat actcctctct gtcatacctg cccctcctat cgttgccgca 3840attattgtta tagttcagaa aggaggtcct tacctcgcca tctatctgtg ggcattcatg 3900tttatcctgt ctctagtgat gatgactata taccctgttt tgattgcacc tcttttcaac 3960aagttcactc ctcttcctga tggagacctc cgggagaaga ttgagaaact tgcttcttct 4020ctaaagtttc ctctgaagaa gctgtttgtt gtcgatggat ctacaaggtc aagccatagt 4080aatgcttaca tgtatggttt cttcaagaac aaaaggattg ttctttatga cacattgatt 4140cagcagtgcc agaatgagaa tgaaattgtg

gcggttattg cacacgagct gggacactgg 4200aagctgaatc acactacata ctcgttcatt gctgttcaaa tccttgcctt cttgcaattt 4260ggaggataca ctcttgtcag aaactccact gatctcttca ggagttttgg ttttgataca 4320caaccagttc tcattggttt gatcatattt cagcacactg taataccact tcaacaccta 4380gtaagctttg acctcaacct tgttagtcga gcgtttgagt ttcaggctga tgcttttgca 4440gtgaatcttg gttatgcaaa ggatctacgt cctgccctag tgaagctaca ggaagagaac 4500ttatcagcga tgaacacaga cccattgtac tcagcttatc actactcaca ccctcctctt 4560gtagagaggc ttcgagccat tgatggagaa gacaagaaga cagattaacc cctcgaattt 4620ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 4680tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 4740atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 4800atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 4860atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 4920accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 4980atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgccc 5040gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5100ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 5160aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 5220cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 5280ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggaacca 5340ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 5400ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 5460ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg 5520tttacaccac aatatatcct gcca 5544646474DNAArtificial SequenceDescription of Artificial Sequence pBI121-HP-BnCPP vector sequence 64gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagacca gtgtcccagc tcgtgtgcaa taaccgccac 3360aatttcattc tcattctggc actgctgaat caatgtgtca taaagaacaa tccttttgtt 3420cttgaagaaa ccatacatgt aagcattact atggcttgac cttgtagatc catcgacaac 3480aaacagcttc ttcagaggaa actttagaga agaagcaagt ttctcaatct tctcccggag 3540gtctccatca ggaagaggag tgaacttgtt gaaaagaggt gcaatcaaaa cagggtatat 3600agtcatcatc actagagaca ggataaacat gaatgcccac agatagatgg cgaggtaagg 3660acctcctttc tgaactataa caataattgc ggcaacgata ggaggggcag gtatgacaga 3720gaggagtatt cctttgatca tgtccctaat gaacatccat attgtttgtt tgttgaaccc 3780atgccgagac tcgatcacga aagttgagta caaagaaaat ggcaaatcag tgatctgtga 3840ccatgtcata agaccagcca agaatgaaag agtgtgcagg atttcattct ctggatcgag 3900tcccaccatt ggtagaagga tccccatcta cccgcttcgc gtcggcatcc ggtcagtggc 3960agtgaagggc gaacagttcc tgattaacca caaaccgttc tactttactg gctttggtcg 4020tcatgaagat gcggacttgc gtggcaaagg attcgataac gtgctgatgg tgcacgacca 4080cgcattaatg gactggattg gggccaactc ctaccgtacc tcgcattacc cttacgctga 4140agagatgctc gactgggcag atgaacatgg catcgtggtg attgatgaaa ctgctgctgt 4200cggcttttcg ctctctttag gcattggttt cgaagcgggc aacaagccga aagaactgta 4260cagcgaagag gcagtcaacg gggaaactca gcaagcgcac ttacaggcga ttaaagagct 4320gatagcgcgt gacaaaaacc acccaagcgt ggtgatgtgg agtattgcca acgaaccgga 4380tacccgtccg caaggtgcac gggaatattt cgcgccactg gcggaagcaa cgcgtaaact 4440cgacccgacg cgtccgatca cctgcgtcaa tgtaatgttc tgcgacgctc acaccgatac 4500catcagcgat ctctttgatg tgctgtgcct gaaccgttat tacggatggt atgtccaaag 4560cggcgatttg gaaacggcag agaaggtact ggaaaaagaa cttctggcct ggcaggagaa 4620actgtacacc gacatgtgga gtgaagagta tcagtgtgca tggctggata tgtatcaccg 4680cgtctttgat cgcgtcagcg ccgtcgtcgg tgaacaggta tggaatttcg ccgattttgc 4740gacctcgcaa ggcatattgc gcgttggcgg taacaagaaa gggatcttca ctcgcgaccg 4800caaaccgaag tcggcggctt ttctgctgca aaaacgctgg actggcatga acttcggtga 4860aaaaccgcag cagggaggca aacaatgaat caacaactct cctggcgcac catcgtcggc 4920tacagcctcg ggaattgcta ccgagctctt ctaccaatgg tgggactcga tccagagaat 4980gaaatcctgc acactctttc attcttggct ggtcttatga catggtcaca gatcactgat 5040ttgccatttt ctttgtactc aactttcgtg atcgagtctc ggcatgggtt caacaaacaa 5100acaatatgga tgttcattag ggacatgatc aaaggaatac tcctctctgt catacctgcc 5160cctcctatcg ttgccgcaat tattgttata gttcagaaag gaggtcctta cctcgccatc 5220tatctgtggg cattcatgtt tatcctgtct ctagtgatga tgactatata ccctgttttg 5280attgcacctc ttttcaacaa gttcactcct cttcctgatg gagacctccg ggagaagatt 5340gagaaacttg cttcttctct aaagtttcct ctgaagaagc tgtttgttgt cgatggatct 5400acaaggtcaa gccatagtaa tgcttacatg tatggtttct tcaagaacaa aaggattgtt 5460ctttatgaca cattgattca gcagtgccag aatgagaatg aaattgtggc ggttattgca 5520cacgagctgg gacactggga gctcgaattt ccccgatcgt tcaaacattt ggcaataaag 5580tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 5640ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 5700tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 5760aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcggg aattcactgg 5820ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 5880cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 5940cccaacagtt gcgcagcctg aatggcgccc gctcctttcg ctttcttccc ttcctttctc 6000gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 6060tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 6120gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 6180agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat 6240ttataaggga ttttgccgat ttcggaacca ccatcaaaca ggattttcgc ctgctggggc 6300aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc 6360tgttgcccgt ctcactggtg aaaagaaaaa ccaccccagt acattaaaaa cgtccgcaat 6420gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatcct gcca 6474655544DNAArtificial SequenceDescription of Artificial Sequence pBI121-antisense-BnCPP vector sequence 65gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagcccaca 2520gatggttaga gaggcttacg cagcaggtct catcaagacg atctacccga gcaataatct 2580ccaggaaatc aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa 2640ctgcatcaag aacacagaga aagatatatt tctcaagatc agaagtacta ttccagtatg 2700gacgattcaa ggcttgcttc acaaaccaag gcaagtaata gagattggag tctctaaaaa 2760ggtagttccc actgaatcaa aggccatgga gtcaaagatt caaatagagg acctaacaga 2820actcgccgta aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa 2880gaaaatcttc gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga 2940tacagtctca gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa 3000cctcctcgga ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga 3060aggtggctcc tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc 3120tgccgacagt ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga 3180cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 3240tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 3300tttggagaga acacggggga ctctagagga tccttaatct gtcttcttgt cttctccatc 3360aatggctcga agcctctcta caagaggagg gtgtgagtag tgataagctg agtacaatgg 3420gtctgtgttc atcgctgata agttctcttc ctgtagcttc actagggcag gacgtagatc 3480ctttgcataa ccaagattca ctgcaaaagc atcagcctga aactcaaacg ctcgactaac 3540aaggttgagg tcaaagctta ctaggtgttg aagtggtatt acagtgtgct gaaatatgat 3600caaaccaatg agaactggtt gtgtatcaaa accaaaactc ctgaagagat cagtggagtt 3660tctgacaaga gtgtatcctc caaattgcaa gaaggcaagg atttgaacag caatgaacga 3720gtatgtagtg tgattcagct tccagtgtcc cagctcgtgt gcaataaccg ccacaatttc 3780attctcattc tggcactgct gaatcaatgt gtcataaaga acaatccttt tgttcttgaa 3840gaaaccatac atgtaagcat tactatggct tgaccttgta gatccatcga caacaaacag 3900cttcttcaga ggaaacttta gagaagaagc aagtttctca atcttctccc ggaggtctcc 3960atcaggaaga ggagtgaact tgttgaaaag aggtgcaatc aaaacagggt atatagtcat 4020catcactaga gacaggataa acatgaatgc ccacagatag atggcgaggt aaggacctcc 4080tttctgaact ataacaataa ttgcggcaac gataggaggg gcaggtatga cagagaggag 4140tattcctttg atcatgtccc taatgaacat ccatattgtt tgtttgttga acccatgccg 4200agactcgatc acgaaagttg agtacaaaga aaatggcaaa tcagtgatct gtgaccatgt 4260cataagacca gccaagaatg aaagagtgtg caggatttca ttctctggat cgagtcccac 4320cattggtaga aagccgccag atatcttcca aaaccaaggc aagatcccaa agaacagaat 4380cgcagagtcc ataagtatag taacaaactc atgaacaaag tgaaaatggc ttttgtcaag 4440actgtaagct cgagatttct caaacttctc ttggctaatg actccaacca aagtctttgg 4500gagagtggga agcttgagag cagtatgttg cctcagatcc aaatacgtct caaaaacgta 4560catcactatc ataaaaccaa cgacggtttc catgaaagga atcgccatcc cctcgaattt 4620ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 4680tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 4740atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 4800atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 4860atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 4920accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 4980atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgccc 5040gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5100ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 5160aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 5220cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 5280ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggaacca 5340ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 5400ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 5460ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg 5520tttacaccac aatatatcct gcca 5544665668DNAArtificial SequenceDescription of Artificial Sequence pRD29A-BnCPP vector sequence 66gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc

gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatccatg gcgattcctt tcatggaaac 3480cgtcgttggt tttatgatag tgatgtacgt ttttgagacg tatttggatc tgaggcaaca 3540tactgctctc aagcttccca ctctcccaaa gactttggtt ggagtcatta gccaagagaa 3600gtttgagaaa tctcgagctt acagtcttga caaaagccat tttcactttg ttcatgagtt 3660tgttactata cttatggact ctgcgattct gttctttggg atcttgcctt ggttttggaa 3720gatatctggc ggctttctac caatggtggg actcgatcca gagaatgaaa tcctgcacac 3780tctttcattc ttggctggtc ttatgacatg gtcacagatc actgatttgc cattttcttt 3840gtactcaact ttcgtgatcg agtctcggca tgggttcaac aaacaaacaa tatggatgtt 3900cattagggac atgatcaaag gaatactcct ctctgtcata cctgcccctc ctatcgttgc 3960cgcaattatt gttatagttc agaaaggagg tccttacctc gccatctatc tgtgggcatt 4020catgtttatc ctgtctctag tgatgatgac tatataccct gttttgattg cacctctttt 4080caacaagttc actcctcttc ctgatggaga cctccgggag aagattgaga aacttgcttc 4140ttctctaaag tttcctctga agaagctgtt tgttgtcgat ggatctacaa ggtcaagcca 4200tagtaatgct tacatgtatg gtttcttcaa gaacaaaagg attgttcttt atgacacatt 4260gattcagcag tgccagaatg agaatgaaat tgtggcggtt attgcacacg agctgggaca 4320ctggaagctg aatcacacta catactcgtt cattgctgtt caaatccttg ccttcttgca 4380atttggagga tacactcttg tcagaaactc cactgatctc ttcaggagtt ttggttttga 4440tacacaacca gttctcattg gtttgatcat atttcagcac actgtaatac cacttcaaca 4500cctagtaagc tttgacctca accttgttag tcgagcgttt gagtttcagg ctgatgcttt 4560tgcagtgaat cttggttatg caaaggatct acgtcctgcc ctagtgaagc tacaggaaga 4620gaacttatca gcgatgaaca cagacccatt gtactcagct tatcactact cacaccctcc 4680tcttgtagag aggcttcgag ccattgatgg agaagacaag aagacagatt aacccctcga 4740atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 4800gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 4860tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 4920tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 4980tgtcatctat gttactagat cgggaattca ctggccgtcg ttttacaacg tcgtgactgg 5040gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 5100cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 5160gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 5220agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 5280caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 5340tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 5400aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 5460accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5520ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 5580aaaaccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 5640tttgtttaca ccacaatata tcctgcca 5668676598DNAArtificial SequenceDescription of Artificial Sequence pRD29A-HP-BnCPP vector sequence 67gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag accagtgtcc cagctcgtgt gcaataaccg 3480ccacaatttc attctcattc tggcactgct gaatcaatgt gtcataaaga acaatccttt 3540tgttcttgaa gaaaccatac atgtaagcat tactatggct tgaccttgta gatccatcga 3600caacaaacag cttcttcaga ggaaacttta gagaagaagc aagtttctca atcttctccc 3660ggaggtctcc atcaggaaga ggagtgaact tgttgaaaag aggtgcaatc aaaacagggt 3720atatagtcat catcactaga gacaggataa acatgaatgc ccacagatag atggcgaggt 3780aaggacctcc tttctgaact ataacaataa ttgcggcaac gataggaggg gcaggtatga 3840cagagaggag tattcctttg atcatgtccc taatgaacat ccatattgtt tgtttgttga 3900acccatgccg agactcgatc acgaaagttg agtacaaaga aaatggcaaa tcagtgatct 3960gtgaccatgt cataagacca gccaagaatg aaagagtgtg caggatttca ttctctggat 4020cgagtcccac cattggtaga aggatcccca tctacccgct tcgcgtcggc atccggtcag 4080tggcagtgaa gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg 4140gtcgtcatga agatgcggac ttgcgtggca aaggattcga taacgtgctg atggtgcacg 4200accacgcatt aatggactgg attggggcca actcctaccg tacctcgcat tacccttacg 4260ctgaagagat gctcgactgg gcagatgaac atggcatcgt ggtgattgat gaaactgctg 4320ctgtcggctt ttcgctctct ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac 4380tgtacagcga agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag 4440agctgatagc gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt gccaacgaac 4500cggatacccg tccgcaaggt gcacgggaat atttcgcgcc actggcggaa gcaacgcgta 4560aactcgaccc gacgcgtccg atcacctgcg tcaatgtaat gttctgcgac gctcacaccg 4620ataccatcag cgatctcttt gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc 4680aaagcggcga tttggaaacg gcagagaagg tactggaaaa agaacttctg gcctggcagg 4740agaaactgta caccgacatg tggagtgaag agtatcagtg tgcatggctg gatatgtatc 4800accgcgtctt tgatcgcgtc agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt 4860ttgcgacctc gcaaggcata ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg 4920accgcaaacc gaagtcggcg gcttttctgc tgcaaaaacg ctggactggc atgaacttcg 4980gtgaaaaacc gcagcaggga ggcaaacaat gaatcaacaa ctctcctggc gcaccatcgt 5040cggctacagc ctcgggaatt gctaccgagc tcttctacca atggtgggac tcgatccaga 5100gaatgaaatc ctgcacactc tttcattctt ggctggtctt atgacatggt cacagatcac 5160tgatttgcca ttttctttgt actcaacttt cgtgatcgag tctcggcatg ggttcaacaa 5220acaaacaata tggatgttca ttagggacat gatcaaagga atactcctct ctgtcatacc 5280tgcccctcct atcgttgccg caattattgt tatagttcag aaaggaggtc cttacctcgc 5340catctatctg tgggcattca tgtttatcct gtctctagtg atgatgacta tataccctgt 5400tttgattgca cctcttttca acaagttcac tcctcttcct gatggagacc tccgggagaa 5460gattgagaaa cttgcttctt ctctaaagtt tcctctgaag aagctgtttg ttgtcgatgg 5520atctacaagg tcaagccata gtaatgctta catgtatggt ttcttcaaga acaaaaggat 5580tgttctttat gacacattga ttcagcagtg ccagaatgag aatgaaattg tggcggttat 5640tgcacacgag ctgggacact gggagctcga atttccccga tcgttcaaac atttggcaat 5700aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 5760tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 5820tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 5880gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattca 5940ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 6000cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 6060ccttcccaac agttgcgcag cctgaatggc gcccgctcct ttcgctttct tcccttcctt 6120tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 6180ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 6240tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 6300taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt 6360tgatttataa gggattttgc cgatttcgga accaccatca aacaggattt tcgcctgctg 6420gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat 6480cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc cagtacatta aaaacgtccg 6540caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgcca 6598685668DNAArtificial SequenceDescription of Artificial Sequence pRD29A-antisense-BnCPP vector sequence 68gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcagggagcc 2520atagatgcaa ttcaatcaaa ctgaaatttc tgcaagaatc tcaaacacgg agatctcaaa 2580gtttgaaaga aaatttattt cttcgactca aaacaaactt acgaaattta ggtagaactt 2640atatacatta tattgtaatt ttttgtaaca aaatgttttt attattatta tagaatttta 2700ctggttaaat taaaaatgaa tagaaaaggt gaattaagag gagagaggag gtaaacattt 2760tcttctattt tttcatattt tcaggataaa ttattgtaaa agtttacaag atttccattt 2820gactagtgta aatgaggaat attctctagt aagatcatta tttcatctac ttcttttatc 2880ttctaccagt agaggaataa acaatattta gctcctttgt aaatacaaat taattttcct 2940tcttgacatc attcaatttt aattttacgt ataaaataaa agatcatacc tattagaacg 3000attaaggaga aatacaattc gaatgagaag gatgtgccgt ttgttataat aaacagccac 3060acgacgtaaa cgtaaaatga ccacatgatg ggccaataga catggaccga ctactaataa 3120tagtaagtta cattttagga tggaataaat atcataccga catcagtttt gaaagaaaag 3180ggaaaaaaag aaaaaataaa taaaagatat actaccgaca tgagttccaa aaagcaaaaa 3240aaaagatcaa gccgacacag acacgcgtag agagcaaaat gactttgacg tcacaccacg 3300aaaacagacg cttcatacgt gtccctttat ctctctcagt ctctctataa acttagtgag 3360accctcctct gttttactca caaatatgca aactagaaaa caatcatcag gaataaaggg 3420tttgattact tctattggaa aggactctag aggatcctta atctgtcttc ttgtcttctc 3480catcaatggc tcgaagcctc tctacaagag gagggtgtga gtagtgataa gctgagtaca 3540atgggtctgt gttcatcgct gataagttct cttcctgtag cttcactagg gcaggacgta 3600gatcctttgc ataaccaaga ttcactgcaa aagcatcagc ctgaaactca aacgctcgac 3660taacaaggtt gaggtcaaag cttactaggt gttgaagtgg tattacagtg tgctgaaata 3720tgatcaaacc aatgagaact ggttgtgtat caaaaccaaa actcctgaag agatcagtgg 3780agtttctgac aagagtgtat cctccaaatt gcaagaaggc aaggatttga acagcaatga 3840acgagtatgt

agtgtgattc agcttccagt gtcccagctc gtgtgcaata accgccacaa 3900tttcattctc attctggcac tgctgaatca atgtgtcata aagaacaatc cttttgttct 3960tgaagaaacc atacatgtaa gcattactat ggcttgacct tgtagatcca tcgacaacaa 4020acagcttctt cagaggaaac tttagagaag aagcaagttt ctcaatcttc tcccggaggt 4080ctccatcagg aagaggagtg aacttgttga aaagaggtgc aatcaaaaca gggtatatag 4140tcatcatcac tagagacagg ataaacatga atgcccacag atagatggcg aggtaaggac 4200ctcctttctg aactataaca ataattgcgg caacgatagg aggggcaggt atgacagaga 4260ggagtattcc tttgatcatg tccctaatga acatccatat tgtttgtttg ttgaacccat 4320gccgagactc gatcacgaaa gttgagtaca aagaaaatgg caaatcagtg atctgtgacc 4380atgtcataag accagccaag aatgaaagag tgtgcaggat ttcattctct ggatcgagtc 4440ccaccattgg tagaaagccg ccagatatct tccaaaacca aggcaagatc ccaaagaaca 4500gaatcgcaga gtccataagt atagtaacaa actcatgaac aaagtgaaaa tggcttttgt 4560caagactgta agctcgagat ttctcaaact tctcttggct aatgactcca accaaagtct 4620ttgggagagt gggaagcttg agagcagtat gttgcctcag atccaaatac gtctcaaaaa 4680cgtacatcac tatcataaaa ccaacgacgg tttccatgaa aggaatcgcc atcccctcga 4740atttccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 4800gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 4860tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 4920tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 4980tgtcatctat gttactagat cgggaattca ctggccgtcg ttttacaacg tcgtgactgg 5040gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 5100cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 5160gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 5220agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 5280caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 5340tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 5400aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 5460accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 5520ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 5580aaaaccaccc cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa 5640tttgtttaca ccacaatata tcctgcca 5668695074DNAArtificial SequenceDescription of Artificial Sequence MuA-BnCPP vector sequence 69gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60aatctgatca tgagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 120acaagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actcagccgc 180gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 240agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 300tccataaatt cccctcggta tccaattaga gtctcatatt cactctcaat ccaaataatc 360tgcaccggat ctggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 420gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 480gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 540ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg 600acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 660ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 720gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 780ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 840gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 900aggctcaagg cgcgcatgcc cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc 960ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1020ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1080ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1140cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa 1200tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct 1260atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg 1320gggatctcat gctggagttc ttcgcccacg ggatctctgc ggaacaggcg gtcgaaggtg 1380ccgatatcat tacgacagca acggccgaca agcacaacgc cacgatcctg agcgacaata 1440tgatcgggcc cggcgtccac atcaacggcg tcggcggcga ctgcccaggc aagaccgaga 1500tgcaccgcga tatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 1560tgatccccga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 1620gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 1680tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 1740tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 1800tgtcatctat gttactagat cgggcctcct gtcaatgctg gcggcggctc tggtggtggt 1860tctggtggcg gctctgaggg tggtggctct gagggtggcg gttctgaggg tggcggctct 1920gagggaggcg gttccggtgg tggctctggt tccggtgatt ttgattatga aaagatggca 1980aacgctaata agggggctat gaccgaaaat gccgatgaaa acgcgctaca gtctgacgct 2040aaaggcaaac ttgattctgt cgctactgat tacggtgctg ctatcgatgg tttcattggt 2100gacgtttccg gccttgctaa tggtaatggt gctactggtg attttgctgg ctctaattcc 2160caaatggctc aagtcggtga cggtgataat tcacctttaa tgaataattt ccgtcaatat 2220ttaccttccc tccctcaatc ggttgaatgt cgcccttttg tctttggccc aatacgcaaa 2280ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 2340tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 2400caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 2460tttcacacag gaaacagcta tgaccatgat tacgccaagc tgggaaattt ttcgccagtt 2520ctaaatatcc ggaaacctct tgggatgcca ttgcccatct atctgtaatt tattgacgaa 2580atagacgaaa aggaaggtgg ctcctataaa gcacatcatt gcgataacag aaaggccatt 2640gttgaagata cctctgctga cattggtccc caagtggaag caccacccca tgaggagcac 2700cgtggagtaa gaagacgttc gagccacgtc gaaaaagcaa gtgtgttgat gtagtatctc 2760cattgacgta agggatgacg cacaatccaa ctatccatcg caagaccatt gctctatata 2820agaaagttaa tatcatttcg agtggccacg ctgaggggga tccatggcga ttcctttcat 2880ggaaaccgtc gttggtttta tgatagtgat gtacgttttt gagacgtatt tggatctgag 2940gcaacatact gctctcaagc ttcccactct cccaaagact ttggttggag tcattagcca 3000agagaagttt gagaaatctc gagcttacag tcttgacaaa agccattttc actttgttca 3060tgagtttgtt actatactta tggactctgc gattctgttc tttgggatct tgccttggtt 3120ttggaagata tctggcggct ttctaccaat ggtgggactc gatccagaga atgaaatcct 3180gcacactctt tcattcttgg ctggtcttat gacatggtca cagatcactg atttgccatt 3240ttctttgtac tcaactttcg tgatcgagtc tcggcatggg ttcaacaaac aaacaatatg 3300gatgttcatt agggacatga tcaaaggaat actcctctct gtcatacctg cccctcctat 3360cgttgccgca attattgtta tagttcagaa aggaggtcct tacctcgcca tctatctgtg 3420ggcattcatg tttatcctgt ctctagtgat gatgactata taccctgttt tgattgcacc 3480tcttttcaac aagttcactc ctcttcctga tggagacctc cgggagaaga ttgagaaact 3540tgcttcttct ctaaagtttc ctctgaagaa gctgtttgtt gtcgatggat ctacaaggtc 3600aagccatagt aatgcttaca tgtatggttt cttcaagaac aaaaggattg ttctttatga 3660cacattgatt cagcagtgcc agaatgagaa tgaaattgtg gcggttattg cacacgagct 3720gggacactgg aagctgaatc acactacata ctcgttcatt gctgttcaaa tccttgcctt 3780cttgcaattt ggaggataca ctcttgtcag aaactccact gatctcttca ggagttttgg 3840ttttgataca caaccagttc tcattggttt gatcatattt cagcacactg taataccact 3900tcaacaccta gtaagctttg acctcaacct tgttagtcga gcgtttgagt ttcaggctga 3960tgcttttgca gtgaatcttg gttatgcaaa ggatctacgt cctgccctag tgaagctaca 4020ggaagagaac ttatcagcga tgaacacaga cccattgtac tcagcttatc actactcaca 4080ccctcctctt gtagagaggc ttcgagccat tgatggagaa gacaagaaga cagattaacc 4140cctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 4200ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 4260ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat 4320tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 4380gcgcggtgtc atctatgtta ctagatcggg aattcactgg ccgtcgtttt acaacgtcgt 4440gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 4500agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 4560aatggcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 4620ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 4680cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 4740ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 4800tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 4860ttcggaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg 4920ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg 4980aaaagaaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 5040cgtcaatttg tttacaccac aatatatcct gcca 50747033DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 70aaacccggga tggcgtttcc ctacatggaa gcc 337134DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 71aaagagctct tagtcttcct tcttatccgg ttcg 347228DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 72aaacccggga tggcgattcc tttcatgg 287333DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 73aaaggatcct taatctgtct tcttgtcttc tcc 337430DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 74aaagagctct tctaccaatg gtgggactcg 307529DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 75aaagagctcc cagtgtccca gctcgtgtg 297630DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 76aaaggatcct tctaccaatg gtgggactcg 307729DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 77aaatctagac cagtgtccca gctcgtgtg 297834DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 78gatgagctca caagatcaag tcacagcaat gcct 347929DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 79aaagagctcc cggttcgtcc agcgcggcc 298034DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 80gatggatcca caagatcaag tcacagcaat gcct 348129DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 81ccttctagac cggttcgtcc agcgcggcc 298230DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 82tttaagcttg gagccataga tgcaattcaa 308318DNAArtificial SequenceDescription of Artificial Sequence PCR Primer sequence 83gcaagaccgg caacagga 18841217DNAArtificial sequenceDescription of Artificial Sequence Consensus sequence 84nnntnnnnng nnntnnnnna nnnnnnnncn tcaanctnnc nantntnccn nnnncnntnn 60nnggnntnnt nagnnangan aantttgann nnncnnnngc ntanagnntn nanannnnna 120gcnnnttnca ntttgtncan gngnntgtna nnatnntnnn nganncnncn atnntnnnnn 180nnngnntnnt nccntggnnn tggnanannn nnggnnnnnt nntnnnnann nnnggnntnn 240ntnnnnagan nganatnntn canacncttn cnttnntngc ngnnnnnang nnntggtcnc 300agatnnnnga nntnccnttn tcnntntant cnacnttngt natngagncn cgncatggnt 360tnaanaanca aacnnnatgg ntnttnntnn ggganatgnt nannggnnnn nnnctnnnnn 420tnntnnnngn nccnccnatn gtnncngcna tnatnnnnat ngtncagann ggnggnccnt 480anntngcnnt ntatctntgg gnnttnangt tnnnnntnnc nntnntgntg atgncnntnt 540anccnntnnt natngcnccn ctnttcaana nnttcacncc nntnccngan ggnnnnctnn 600gngnnaanat ngagaanctn gcnncntcnn tnnantnncc nntnaanaan ntnttngtnn 660tnganggntc nacnngntca agncanagna angcntanat gtatggnttn tnnaananca 720anngnatngt ncnntangan acnntnatnn nncantgnnn nnangannan ganntngtnn 780cngttatngc ncanganntn ggncantgga anctnannca nactnnntan ncnttnntng 840cnntncannt ncttnnnntn ntncaattng gaggntanac nctnntnngn anntcnnnng 900nnctntnnnn nagnttnggn ttnnnnnnnc anccngtnnt natnggnntn atcntnttnc 960agcanacnnt natnccnntn cancannnnn tnagctttnn nctnaacctn ntnagnngan 1020cnttngantt tcaggcngat gnnttngcnn nnnnnntngn ntannnnnnn nnnntnngnn 1080nnnnnctnnt naanctncag gangagaann tntcnncnat gaanacngan cnntngnant 1140cngcntatca nnantcncan ccnccnctng tnganngnnt nnnngnnnnn gangnnnnnn 1200nnannaannn ngannan 121785268PRTArtificial sequenceDescription of Artificial Sequence Consensus sequence 85Leu Xaa Xaa Xaa Xaa Leu Pro Xaa Xaa Leu Xaa Xaa Xaa Leu Pro Xaa 1 5 10 15Ser Xaa Tyr Ser Xaa Phe Val Xaa Glu Xaa Xaa Xaa Gly Phe Asn Lys 20 25 30Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Asp Met Xaa Xaa Xaa Xaa Xaa Leu 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Pro Ile Xaa Xaa Xaa Xaa Xaa Xaa Ile Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Phe Xaa Xaa 65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Met Xaa Xaa Xaa Pro Xaa Xaa Ile Xaa Pro 85 90 95Xaa Phe Asn Xaa Phe Thr Pro Leu Xaa Xaa Gly Xaa Leu Xaa Xaa Xaa 100 105 110Ile Glu Xaa Leu Ala Xaa Xaa Xaa Xaa Xaa Pro Leu Xaa Lys Xaa Phe 115 120 125Val Xaa Asp Gly Ser Xaa Arg Ser Ser His Ser Asn Ala Tyr Xaa Xaa 130 135 140Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa Xaa Xaa Xaa Glu Xaa Xaa Xaa Val Xaa Ala His Glu Xaa 165 170 175Gly His Trp Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Leu 195 200 205Xaa Ser Arg Xaa Xaa Glu Xaa Gln Ala Asp Xaa Xaa Ala Xaa Xaa Leu 210 215 220Xaa Tyr Xaa Xaa Xaa Leu Xaa Xaa Asn Leu Ser Xaa Met Asn Xaa Asp225 230 235 240Xaa Xaa Xaa Ser Xaa Tyr His Xaa Ser His Pro Xaa Leu Xaa Glu Arg 245 250 255Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Xaa 260 265861275DNAArtificial SequenceDescription of Artificial Sequence Consensus sequence 86atnncnnntn cntnnatnnn nnnnntnnnn ggntttatga tantnatgta cntttttgan 60acntanttgg atntgngnca acnnanngcn ctcaancttc cnactctncc naanacnttn 120gnnggngtna tnagccaaga gaantttgag aaatcnngag cntanagnnt tnananaagc 180nanttncant ttgttcanga gtttgtnacn atantnanng actctncnat tntgtncttt 240gggntnttgc cntggttttg gaaganntcn ggngnnnttn tnncnannnn nggnntnnat 300ncngagaatg aaatnctgca nacncttncn ttcttngcng gnntnatgan ntggtcacag 360atnacngatt tgccnttttc tntgtactca actttngtga tngagncncg ncatggnttn 420aanaancaaa cannatggnt nttcnttagg gacatgntna aaggaannnt cctntctgtn 480atannngnnc cnccnatngt ngcngcnatn attntnatag tncagaaagg aggtccntan 540ntngccatct atctntgggn nttnangttt nnnctntctn tngtgatgat gacnntntan 600ccngtnntna tngcnccnct nttcaanaan ttcactccnc ttccngatgg nnanctcngg 660gagaanatng agaaacttgc ttcntcnctn aantntccnn tnaagaanct ntttgttgtc 720gatggatcna caagntcaag ncanagnaat gcntanatgt atggnttctt naagaacaan 780aggattgtnc nttatganac nttnattcan cagtgcnann angannanga aattgtngcn 840gttattgcnc angagntngg acantggaan ctnaancana ctnnntacnc nttnnttgcn 900ntncanatnc ttncnntnnt ncaatttgga ggatanacnc tnntnngaaa ntcnnctgat 960ctntnnngna gnttnggntt tgatacncan ccngtnctca ttggnntnat catatttcag 1020canactgtaa tnccactnca ncannnngtn agctttgnnc tnaacctngt nagncgancn 1080tttgantttc aggctgatgn ntttgcnnng aancttgnnt atgcannngn nntncgnnnt 1140nnnctngtga anctacagga ngagaanntn tcancnatga anacngancn ntngnactcn 1200gcttatcact antcncancc tccncttgtn ganagnntnn nngnnnnnga ngnannngan 1260aagaagnnag antaa 127587298PRTArtificial SequenceDescription of Artificial Sequence Consensus sequence 87Met Ala Xaa Pro Xaa Met Glu Xaa Val Val Gly Phe Met Ile Xaa Met 1 5 10 15Tyr Xaa Phe Glu Thr Tyr Leu Asp Xaa Arg Gln Xaa Xaa Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Xaa Thr Asp Leu Pro Phe Ser Leu 35 40 45Tyr Ser Thr Phe Val Ile Glu Xaa Arg His Gly Phe Asn Lys Gln Thr 50 55 60Xaa Trp Xaa Phe Xaa Arg Asp Met Xaa Lys Gly Xaa Xaa Leu Ser Val 65 70 75 80Ile Xaa Xaa Pro Pro Ile Val Ala Ala Ile Ile Xaa Ile Val Gln Lys 85 90 95Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Xaa Phe Xaa Phe Xaa Leu 100 105 110Ser Xaa Val Met Met Thr Xaa Tyr Pro Val Leu Ile Ala Pro Leu Phe 115 120 125Asn Lys Phe Thr Pro Leu Pro Asp Gly Xaa Leu Arg Glu Lys Ile Glu 130 135 140Lys Leu Ala Ser Ser Leu Xaa Xaa Pro Leu Lys Lys Leu Phe Val Val145 150 155 160Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 165 170 175Phe Lys Asn Lys Arg Ile Val Xaa Tyr Asp Thr Leu Ile Gln Gln Cys 180 185 190Xaa Xaa Xaa Xaa Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 195 200 205Trp Lys Leu Asn His Thr Xaa Tyr Xaa Phe Xaa Ala Xaa Gln His Thr 210 215 220Val Ile Pro Leu Gln Xaa Xaa Val Ser Phe Xaa Leu Asn Leu Val Ser225 230 235 240Arg Xaa Phe Glu Phe Gln Ala Asp Xaa Phe Ala Xaa Xaa Leu Xaa Tyr 245 250 255Ala Xaa Xaa Leu Arg Glu Asn

Leu Ser Xaa Met Asn Thr Asp Xaa Xaa 260 265 270Xaa Ser Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Xaa 275 280 285Xaa Xaa Asp Xaa Xaa Asp Lys Lys Xaa Asp 290 295881275DNAArtificial sequenceDescription of Artificial Sequence Consensus sequence 88atnncnnntn ctttnatnnn nnnnntnnnn ggttttatga tagtgatgta catttttgag 60acgtatttgg atctgaggca actcactgct ctcaagcttc caactctccc gaaaaccttg 120gttggtgtaa ttagccaaga gaagtttgag aaatcacgag catacagnnt tnananaagc 180tattttcact ttgttcatga gtttgtaact atacttatgg actctgcaat tttgttcttt 240gggatcttgc cttggttttg gaagatgtct ggagcngttt taccgaggtt gggccttgat 300ccngagaatg aaatactgca tactctttca ttcttggctg gtgttatgac atggtcacag 360atcactgatt tgccattttc tttgtactca actttcgtga tcgagtctcg gcatgggttc 420aacaaacaaa caatatggat gttcattagg gacatgatca aaggaacatt cctctctgtc 480atactaggcc cacccattgt tgcngcgata attttcatag tccagaaagg aggtccttat 540cttgccatct atctgtgggc attcatgttt atcctgtctc tagtgatgat gactatatac 600ccggtcttga tagcaccgct cttcaacaan ttcactcctc ttccagatgg agacctccgg 660gagaagattg agaaacttgc ttcttcncta aagtttcctt tgaagaagct gtttgttgtc 720gatggatcta caaggtcaag ccatagcaat gcttacatgt atggtttctt taagaacaaa 780aggattgttc tttatgatac gttgattcag cagtgcaaga atgaggatga aattgtggcg 840gttattgcac acgagcttgg acattggaaa ctgaatcaca ctacatactc gttcattgca 900gttcaaatcc ttgccttctt acaatttgga ggatacactc ttntcagaaa ctccactgat 960ctcttcagga gtttcggatt tgatacacag cctgttctca ttggtttgat catatttcag 1020cacactgtaa taccactgca acatcnagta agctttggcc tnaacctngt tagtcgagcg 1080tttgagtttc aggctgatgc ttttgcngtg aagcttgnct atgcaaaaga tcttcgtcct 1140nctctagtga aactacagga agagaactta tcancaatga anactgatcn attgnactca 1200gcttatcact actcacatcc tcctcttgtt gaaaggcttc gagccantga tggagaagac 1260aagaagacag attaa 127589306PRTArtificial sequenceDescription of Artificial Sequence Consensus sequence 89Met Ala Ile Pro Phe Met Glu Thr Val Val Gly Phe Met Ile Val Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Leu Arg Gln Leu Thr Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Xaa Thr Asp Leu Pro Phe Ser Leu 35 40 45Tyr Ser Thr Phe Val Ile Glu Ser Arg His Gly Phe Asn Lys Gln Thr 50 55 60Ile Trp Met Phe Ile Arg Asp Met Ile Lys Gly Thr Phe Leu Ser Val 65 70 75 80Ile Leu Gly Pro Pro Ile Val Ala Ala Ile Ile Phe Ile Val Gln Lys 85 90 95Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Ala Phe Met Phe Ile Leu 100 105 110Ser Leu Val Met Met Thr Ile Tyr Pro Val Leu Ile Ala Pro Leu Phe 115 120 125Asn Lys Phe Thr Pro Leu Pro Asp Gly Asp Leu Arg Glu Lys Ile Glu 130 135 140Lys Leu Ala Ser Ser Leu Lys Phe Pro Leu Lys Lys Leu Phe Val Val145 150 155 160Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 165 170 175Phe Lys Asn Lys Arg Ile Val Leu Tyr Asp Thr Leu Ile Gln Gln Cys 180 185 190Lys Asn Glu Asp Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 195 200 205Trp Lys Leu Asn His Thr Thr Tyr Ser Phe Ile Ala Val Gln His Thr 210 215 220Val Ile Pro Leu Gln His Xaa Val Ser Phe Gly Leu Asn Leu Val Ser225 230 235 240Arg Ala Phe Glu Phe Gln Ala Asp Ala Phe Ala Val Lys Leu Xaa Tyr 245 250 255Ala Lys Asp Leu Arg Pro Xaa Leu Val Lys Leu Gln Xaa Glu Asn Leu 260 265 270Ser Xaa Met Asn Thr Asp Xaa Leu Xaa Ser Ala Tyr His Tyr Ser His 275 280 285Pro Pro Leu Val Glu Arg Leu Arg Ala Xaa Asp Gly Glu Asp Lys Lys 290 295 300Thr Asp305901275DNAArtificial sequenceDescription of Artificial Sequence Consensus sequence 90atggcgtttc cctacatgga agccgttgtc ggatttatga tattaatgta catttttgaa 60acttacttgg atgtgcgaca acatagggcc ctcaaacttc ctactcttcc aaagacttta 120ganggtgtta tcagccaaga gaaatttgag aaatctagag cctatagtct tgataaaagc 180cacttccatt ttgttcacga gtttgtgaca atagtgacag actctacaat tttgtacttt 240ggggtattgc cctggttttg gaagaaatca ggagatttta tgacaatagc tggtttcaat 300gctgagaatg aaatactgca tacccttgcc ttcttagcag ggctgatgat ttggtcacag 360ataacagatt tgcccttttc tctgtactca acttttgtga ttgaggcccg tcatggtttt 420aataagcaaa caccatggtt attctttagg gacatgctta aaggaatttt cctttctgta 480ataattggtc cacctattgt ggctgcaatc attgtaatag tacagaaagg aggtccatac 540ttggccatct atctttgggt ttttacgttt ggtctttcta ttgtgatgat gaccctttat 600ccagtactaa tagctccact cttcaataag ttcactccac ttccagatgg tcaactcagg 660gagaaaatcg agaaacttgc ttcctccctc aactatccgt taaagaaact atttgttgtc 720gatggatcca caagatcaag tcacagcaat gcctatatgt atggattctt caagaacaag 780aggattgtcc nttatgacac attaattcaa cagtgcaaag acgatgagga aattgttgct 840gttattgccc atgagttggg acactggaag ctcaaccata ctgtgtacac atttgttgct 900atgcagattc ttacacttct acaatttgga ggatatacac tagtgcgaaa ttcagctgat 960ctgtatcgaa gctttgggtt tgatacgcag ccagtcctca ttgggctcat catatttcag 1020catactgtaa tcccacttca gcaattggtc agctttggtc tgaacctagt cagccgatca 1080tttgaatttc aggctgatgg ctttgccaag aagcttggat atgcatctgg attacgcggt 1140ggtcttgtga aactacagga ggagaatctg tcagctatga atacagatcc ttggtactct 1200gcttatcact attctcatcc tccccttgtt gaaagattgg cngngctgga cgaaccggat 1260aagaaggaag actaa 127591424PRTArtificial sequenceDescription of Artificial Sequence Consensus sequence 91Met Ala Phe Pro Tyr Met Glu Ala Val Val Gly Phe Met Ile Leu Met 1 5 10 15Tyr Ile Phe Glu Thr Tyr Leu Asp Val Arg Gln His Arg Ala Leu Lys 20 25 30Leu Pro Thr Leu Pro Lys Thr Leu Glu Gly Val Ile Ser Gln Glu Lys 35 40 45Phe Glu Lys Ser Arg Ala Tyr Ser Leu Asp Lys Ser His Phe His Phe 50 55 60Val His Glu Phe Val Thr Ile Val Thr Asp Ser Thr Ile Leu Tyr Phe 65 70 75 80Gly Val Leu Pro Trp Phe Trp Lys Lys Ser Gly Asp Phe Met Thr Ile 85 90 95Ala Gly Phe Asn Ala Glu Asn Glu Ile Leu His Thr Leu Ala Phe Leu 100 105 110Ala Gly Leu Met Ile Trp Ser Gln Ile Thr Asp Leu Pro Phe Ser Leu 115 120 125Tyr Ser Thr Phe Val Ile Glu Ala Arg His Gly Phe Asn Lys Gln Thr 130 135 140Pro Trp Leu Phe Phe Arg Asp Met Leu Lys Gly Ile Phe Leu Ser Val145 150 155 160Ile Ile Gly Pro Pro Ile Val Ala Ala Ile Ile Val Ile Val Gln Lys 165 170 175Gly Gly Pro Tyr Leu Ala Ile Tyr Leu Trp Val Phe Thr Phe Gly Leu 180 185 190Ser Ile Val Met Met Thr Leu Tyr Pro Val Leu Ile Ala Pro Leu Phe 195 200 205Asn Lys Phe Thr Pro Leu Pro Asp Gly Gln Leu Arg Glu Lys Ile Glu 210 215 220Lys Leu Ala Ser Ser Leu Asn Tyr Pro Leu Lys Lys Leu Phe Val Val225 230 235 240Asp Gly Ser Thr Arg Ser Ser His Ser Asn Ala Tyr Met Tyr Gly Phe 245 250 255Phe Lys Asn Lys Arg Ile Val Xaa Tyr Asp Thr Leu Ile Gln Gln Cys 260 265 270Lys Asp Asp Glu Glu Ile Val Ala Val Ile Ala His Glu Leu Gly His 275 280 285Trp Lys Leu Asn His Thr Val Tyr Thr Phe Val Ala Met Gln Ile Leu 290 295 300Thr Leu Leu Gln Phe Gly Gly Tyr Thr Leu Val Arg Asn Ser Ala Asp305 310 315 320Leu Tyr Arg Ser Phe Gly Phe Asp Thr Gln Pro Val Leu Ile Gly Leu 325 330 335Ile Ile Phe Gln His Thr Val Ile Pro Leu Gln Gln Leu Val Ser Phe 340 345 350Gly Leu Asn Leu Val Ser Arg Ser Phe Glu Phe Gln Ala Asp Gly Phe 355 360 365Ala Lys Lys Leu Gly Tyr Ala Ser Gly Leu Arg Gly Gly Leu Val Lys 370 375 380Leu Gln Glu Glu Asn Leu Ser Ala Met Asn Thr Asp Pro Trp Tyr Ser385 390 395 400Ala Tyr His Tyr Ser His Pro Pro Leu Val Glu Arg Leu Ala Xaa Leu 405 410 415Asp Glu Pro Asp Lys Lys Glu Asp 420