Proteins imparting boron-tolerance and genes thereof

FIELD OF THE INVENTION

The present invention relates to a protein conferring a boric acid tolerance in Arabidopsis thaliana and a gene thereof, a recombinant vector containing the gene, a transformant introduced with the recombinant vector, and a screening method of a gene conferring a boric acid tolerance.

BACKGROUND OF THE INVENTION

Boron is one of the essential trace elements for higher plants (e.g., see nonpatent document 1). As boron also has toxicity, by over ingesting it, plant growth is inhibited and animal dies of acute intoxication. Boron exists in uncharged molecule state in soil solution. Therefore, boron eluviates with relative ease and boron deficiency is easily developed in agricultural crops. Lowering of yield point and quality in agriculture caused by boron deficiency is reported in 130 varieties in 80 or more countries worldwide including Japan (e.g., see nonpatent document 2). Boron is also known to have a restricted range of optimal concentration compared with other elements, and has little difference between the concentrations at which deficiency symptoms develop and excess symptoms develop. Therefore, the quantity adjustment of boron fertilizer application in agriculture is considered to be difficult. Especially, when boron is fertilized excessively, removal of the boron is difficult and crop production in the agricultural land would be affected. Further, as boron is contained in tap water, damages caused-by excessive boron often become a problem in drylands when irrigated agriculture is performed. In addition to agricultural lands over-fertilized with boron in this way, land areas with high concentration of boron are found worldwide. Countries having such areas have an important agenda for taking measures against damages caused by excessive boron in agricultural policy. Further, as boron is also present in agents for treating metal surface and bleaches, wastewater from factories using these agents and bleaches contains boron in appreciable quantities. Although lethal dose of boron for human is 15-20 mg, it is known that various disorders involving digestive organs and nervous systems are developed with less than the lethal dose of boron. At present, the amount of boron contained in wastewater from factories is becoming an issue.

Recently, a role of boron in plants has been elucidated. It was elucidated that boron bridges pectic polysaccharides in cell walls (e.g., see nonpatent document 3), and showed that the crossbridges are essential for plant growth (e.g., see nonpatent document 4). This is the first knowledge regarding the physiological function of boron at a molecular level in plants. On the other hand, many unclear points remains to be elucidated in the boron transportation mechanism in plants. It was thought for a long time that boron enters into cells by passive diffusion of lipid bilayer, and is transported in plant body by transpiration stream (e.g., see nonpatent document 5). In the meantime, it was known that nutrient conditions of boron, which are suited for growth, differ significantly among species and cultivars. Although absorption, translocation and difference of use efficiency were exemplified as possible causes, molecules of the contributing factors were unknown. In recent years, transportation via channels has been proposed (e.g., see nonpatent document 6), but the evidence was only in vitro experiments using an expression system or a membrane vesicle in Xenopus laevis oocytes, and it was not shown whether these channel molecules were involved in the boron transportation in actual individual plants. Further, the presence of active transport by a transporter was suggested from absorption experiments in roots of sunflower roots (e.g., see nonpatent document 7), however, the responsible transporter was not identified.

The present inventors isolated an efflux boron tolerance protein BOR1 from a plant model, Arabidopsis thaliana for the first time in animate nature (e.g., see patent document 1). It is thought that BOR1 is responsible for an active boron transportation to vessels under nutrient conditions of lower boron (e.g., see nonpatent document 8). Further, YNL275w of yeast, aside from BOR1 is known as tolerance being responsible for boron transportation (e.g., see nonpatent document 9).

Further, as described above, Boron (B) is an essential trace nutrient for plants (e.g., see nonpatent document 10) and animals (e.g., see nonpatent document 11), but toxic at high concentrations (e.g., see nonpatent documents 12 and 13) . Naturally occurring soils containing high concentration of B are distributed across the world and human activities such as fertilization with B, fossil combustion, and irrigation using B-containing water created an environment of high boron concentration (e.g., see nonpatent documents 12 and 13).

Symptoms of B toxicity in plants include chlorosis in leaf margin (e.g., see nonpatent document 13) and fruit disorder and/or bark necrosis (e.g., see nonpatent document 14). Excess B reduces the yield and quality of crops. B toxicity is a major obstruction of agricultural production worldwide. B is also toxic to animals and microorganisms at high concentration. The lethal dose of B is estimated to be about 140 mg/kg for adults and about 270 mg/kg for infants (.e.g., see nonpatent documents 15 and 16) . Long term-high B intake leads to poor appetite, nausea, weight loss, and decreased sexual activity for humans (e.g., see nonpatent document 17). At present, the acceptable safe intake of B for adults is suggested to be 13 mg per day (e.g., see nonpatent document 18). B has been contained in food preservatives for its sterilization effect on microorganisms (e.g., see nonpatent document 19) . In addition, B has been used as insecticides for many years, especially against cockroaches (e.g., see nonpatent document 20).

In the last several decades since B toxicity has been recognized, a number of studies were conducted to investigate toxic effects of B. Those were mostly physiological studies. For example, in soybean leaves, the activity of allantoate amidohydrolase is decreased by boric acid (e.g., see nonpatent document 21). The inhibitions of malate dehydrogenase and isocitrate dehydrogenase activities by B were observed in Chara corallina (e.g., see nonpatent document 22). A negative correlation between placental B levels and delta-aminolevulinic acid dehydratase activities involved in synthesis of porphobilinogen (an intermediate of porphyrin synthesis) in newborns has been also reported (e.g., see nonpatent document 23).

Solubilized borates are thought to play a major role in B toxicity. Boric acids in cells are partially converted into borates due to the higher internal pH. When boric acids with high concentration are supplied to cells, intracellular borate concentration rises to form borate complexes with a variety of cis-diol containing intracellular molecules. These cis-diols containing molecules include NAD.sup.+, ATP, S-Ado Met, RNA and several sugars (e.g., see nonpatent documents 24 and 25). Since these molecules are used as coenzymes and/or substrates for a number of enzymes, binding of borates is likely to induce loss of function or alteration of enzyme activities, inhibition of biochemical reactions, and finally metabolic disorders. Despite of the accumulation of biochemical and physiological analysis and speculation related to the toxic effect of B, molecular mechanism of B toxicity that leads to cell death has not been elucidated.

Patent document 1: Japanese Laid-Open Patent Application NO.2002-262872

Nonpatent document 1: Loomis, W. D.; Durst, R. W. (1992) Chemistry and biology of boron. Biofactors 3: 229-239

Nonpatent document 2: Shorrocks, V. M. (1997) The occurrence and correction of boron deficiency. Plant and Soil 193: 121-148

Nonpatent document 3: Matoh, T.; Ishigaki, K. I.; Ohno, K; Azuma, J. I. (1993) Isolation and characterization of a boron-polysaccharide complex from radish roots. Plant Cell Physiol. 34: 639-642

Nonpatent document 4: O'Neill, M. A.; Eberhard, S.; Albersheim, P.; Darvill, A. G. (2001) Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294: 846-849

Nonpatent document 5: Marschner, H. (1995) Mineral Nutritin of Higher Plants, 2nd ed. Academic Press, San Diego, Calif.

Nonpatent document 6: Dordas, C.; Chrispeels, M. J.; Brown, P. H. (2000) Permeability and channel-mediated transport of boric acid across membrane vesicles isolated from Squash roots. Plant Physiol. 124: 1349-1362

Nonpatent document 7: Dannel, F.; Heidrun, P; Romheld, V. (2000) Characterization of root boron pools, boron uptake and boron translocation in sunflower using the stable isotope 10B and 11B. Aust. J. Plant Physiol. 156: 756-761

Nonpatent document 8: Takano, J.; Noguchi, K.; Yasumori, M.; Kobayashi, M.; Gajdos, Z.; Miwa, K.; Hayashi, H.; Yoneyama, T.; Fujiwara, T. (2002) Arabidopsis boron transporter for xylem loading. Nature 420 (6913): 337-340

Nonpatent document 9: Zhao, R. M.; Reithmeier, R. A. F. (2001) Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae. American Journal of Physiology-Cell Physiology 281 (1): C33-C45

Nonpatent document 10: Warington, K. (1923) Ann. Bot. 37, 629-672

Nonpatent document 11: Park, M., Li, Q., Shcheynikov, N., Zeng, W., & Muallern, S. (2004) Mol. Cell 16, 331-341

Nonpatent document 12: Gupta, U. C., Jame, Y. W., Campbell, C. A., Leyshon, A. J., & Nicholaichuk, W. (1985) Can. J. Soil Sci.65, 381-409

Nonpatent document 13: Nable, R. O., Banuelos, G. S., & Paull, J. G. (1997) Plant Soil 193, 181-198

Nonpatent document 14: Brown, P. H., & Hu, H. (1996). Ann. Bot. 77, 497-505

Nonpatent document 15: Young, E. G., Smith, R. P., & MacIntosh, O. C. (1949) Can. Med. Assoc. J. 61, 447-450

Nonpatent document 16: Arena, J. M., & Drew, R. H. (1986) in Poisoning, (C. C. Thomas, Splingfield). pp. 131

Nonpatent document 17: Hunt, C. D. (1993) in Encyclopedia of Food Science, Food Technology and Nutrition, vol. 1, eds. Macrae, R., Robinson, R. K. & Sadler, M J. (Academic Press, London), pp 440-447

Nonpatent document 18: WHO/FAO/IAEA (1996) in Trace Elements in Human Nutrition and Health, (World Health Organization, Geneva), pp. 175-179

Nonpatent document 19: Nielsen, F. H. (1997) Plant Soil 193, 199-208

Nonpatent document 20: Cochran, D. G. (1995) Experientia 51, 561-563

Nonpatent document 21: Lukaszewski, K. M., Blevins, D. G., & Randall, D. D. (1992) Plant Physiol. 99, 1670-1676

Nonpatent document 22: Reid R. J., Hayes J. E., Post A., Stangoulis J. C. R., & Graham R. D. (2004) Plant Cell Environ. 27, 1405-1414

Nonpatent document 23: Huel, G., Yazbeck, C., Burnel, D., Missy, P., & Kloppmann. W. (2004) Toxicol. Sci. 80,304-309

Nonpatent document 24: Ralston, N. V. C., & Hunt, C. D. (2000) FASEB J. 14, A538

Nonpatent document 25: Ricardo, A., Carrigan, M. A., Olcott, A. N., & Benner, S. A. (2004) Science 303, 196

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

By introducing a gene that confers a boric acid tolerance to yeast into a plant, it has possibilities to generate plants having tolerance against excessive boron. It is thought that plant having boron tolerance can contribute to increase crop yields in places suffering from damages caused by excessive boron. Further, algae or bacteria wherein these genes have been introduced and boron tolerance has increased can be used to absorb boron contained in industrial water and to remove it, thus contributing to environmental cleanup. The present invention may provide a gene or protein conferring a boric acid tolerance to organisms, which has possibilities to generate plants having tolerance against excessive boron. Further, the present invention may provide a method for screening a gene conferring a boric acid tolerance effectively, by elucidating the toxicity mechanism of boric acid.

The present inventors devoted themselves to solve the above object and found 5 types of genes that can confer a boric acid tolerance to yeast, that is, AtPAB2, AtRBP47, AtRPS20B, AtMYB13 and AtMYB68, by expressing several genes of the higher plant Arabidopsis thaliana in yeast, which is an organism model of eukaryote. The present invention has been thus completed based on this knowledge. Further, the present inventors found that a key toxicity mechanism of boric acid exists in specific inhibition of splicing, and a gene related to enhancement of splicing efficiency also confers a boric acid tolerance, thus have completed the present invention.

That is, the present invention relates to (1) a DNA encoding a protein that may have an activity of conferring a boric acid tolerance and may consist of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (2) a DNA encoding a protein that may consist of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 and has an activity of conferring a boric acid tolerance; (3) a gene DNA conferring a boric acid tolerance, which may consist of the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 or a complementary sequence thereof; (4) a DNA encoding a protein that consists of a base sequence wherein one or a few bases may be deleted, substituted or added in the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29, and may have an activity of conferring a boric acid tolerance; (5) a DNA encoding a protein that may hybridize with the DNA according to "3" under stringent conditions and may have an activity of conferring a boric acid tolerance; (6) a protein that may have an activity of conferring a boric acid tolerance, which may consist of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (7) a protein consisting of an amino sequence wherein one or a few amino acids may be deleted, substituted or added in the amino sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; and may have an activity of conferring a boric acid tolerance; (8) a recombinant vector including the DNA according to any one of "1" to "5", which may express a protein conferring a boric acid tolerance; (9) a transformant wherein the recombinant vector according to "8" is introduced, which may express a protein conferring a boric acid tolerance; (10) the transformant according to "9" wherein the transformant may be yeast; (11) the transformant according to "9" wherein the transformant may be a plant; (12) a method for screening a gene conferring a boric acid tolerance, which may comprise the steps of transforming a YNL275w-disrupted yeast which is deficient in and not expressing YNL275w gene by using a gene library, culturing the obtained transformed YNL275w-disrupted yeast in medium containing boric acid, and measuring/evaluating an activity of conferring a boric acid tolerance of the transformed YNL275w-disrupted yeast; (13) a method for screening a gene conferring a boric acid tolerance wherein an enhancement level of splicing efficiency may be measured/evaluated by targeting a specific inhibition of splicing by boric acid; (14) the method for screening a gene conferring a boric acid tolerance according to "13", which may comprise the steps of expressing a test substance in yeast cells, culturing the expressed test substance in the presence of boric acid, and measuring/evaluating an improvement level of a specific inhibition of splicing by boric acid in an intron-containing gene in yeast, as an enhancement level of splicing efficiency; (15) the method for screening a gene conferring a boric acid tolerance according to "14" wherein the gene containing intron in yeast may be a gene RPL7B in Saccharomyces cerevisiae genome; (16) use of the DNA according to any one of "1" to "5" as a gene conferring a boric acid tolerance; (17) use of the DNA according to any one of "1" to "5" for producing a plant or yeast conferred a boric acid tolerance; (18) use of the protein according to "6" or "7" as a protein having an activity of conferring a boric acid tolerance; and (19) use of the protein according to "6" or "7" for producing a plant or yeast conferred a boric acid tolerance.

By introducing a gene that confers a boric acid tolerance of the present invention into a plant, it has possibilities to generate plants having tolerance against excessive boron. It is thought that plant having boron tolerance can contribute to increase crop yields in places suffering from damages caused by excessive boron. Algae or bacteria wherein these genes have been introduced and boron tolerance has increased can be used to absorb boron contained in industrial water and to remove it, thus contributing to environmental cleanup.

Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawing, in which:

FIG. 1 is a set of pictures showing the results of performance test of boric acid tolerance using yeast strain 1169. Yeast strain 1169 was transformed with pYES2 "2" and pYES2-BORI "7". Each yeast was streaked in SD solid medium containing 0 to 100 mM boric acid. The results after culturing at 26.5.degree. C. for 16 days are shown.

FIG. 2 is a set of pictures showing the growth results of yeast strain 1169 in excessive boric acid medium. Yeast strain 1169 was transformed with 46, 72, 84, 86 and 87. Each yeast was spotted in SD medium containing 80 mM boric acid after the liquid culture. The spots were diluted by 1/5 at a time from left to right. The results after culturing at 26.5.degree. C. for 9 days are shown.

FIG. 3 is a set of graphs showing the results of boric acid tolerance test of yeast strain 1169 in liquid medium. Yeast strain 1169 was transformed with 46, 72, 84, 86 and 87. Each yeast was subcultured to an OD.sub.600 of 0.1 in SD medium containing 80 mM boric acid after the liquid culture. The values of OD.sub.600were measured after culturing at 30.degree. C. for 4 days. The experiments of the test were performed in triplicate. The mean of the measurements and the standard deviation are shown using graph.

FIG. 4 is a set of pictures showing the growth results of yeast strain BY4741 in excessive boric acid medium. Yeast strain BY4741 was transformed with 46, 72, 84, 86 and 87. Each yeast was spotted on SD medium containing 100 mM boric acid after the liquid culture. The spots were diluted by 1/5 at a time from left to right. The results after culturing at 26.5.degree. C. for 10 days are shown.

FIG. 5 is a set of graphs showing the results of boric acid tolerance test of yeast strain BY4741 in liquid medium. Yeast strain BY4741 was transformed with 46, 72, 84, 86 and 87. Each yeast was subcultured to an OD.sub.600 of 0.1 in SD medium containing 80 mM boric acid after the liquid culture. The values of OD.sub.600 were measured after culturing at 30.degree. C. for 4 days. The experiments of the test were performed 3 times. The mean of the measurements and the standard deviation are shown using graphs.

FIG. 6 is a set of pictures and graphs showing the results of boric acid tolerance test for AtRBP47c'-related genes-transformed yeast cells. (A) Phylogenetic tree of AtRBP47c'-related family proteins. The dendrogram indicates relative evolutionary distance among the AtRBP47c'-related family proteins and was prepared by using NJ method. The bar indicates the genetic distance for 0.1 amino acid substitutions/site. (B) Boric acid tolerance in solid medium. Yeast cells were grown to an OD.sub.600 of 1.0, serially diluted, and then 10 .mu.l of the diluent was spotted in SD plate added with 0 or 80 mM boric acid. The growth was recorded after culturing for 10 days. Yeast cells transformed with an empty pFL61 vector were used as a control. (C) Boric acid tolerance in liquid medium. Yeast cells were grown to an OD.sub.600 of 1.0, and then diluted to an OD.sub.600 of 0.1 in SD medium added with 0 or 80 mM boric acid. The diluted yeast cells were cultured at 30.degree. C. and the values of OD.sub.600 in indicated time after the dilution were recorded. Vertical bars represent the standard deviation of the mean.+-.the mean of three replicate measurements.

FIG. 7 is a figure showing the effect of boric acid on slicing. (A) Schematic representations of splicing of RPL7B. Three types of mRNA can be generated from pre-mRNA of RPL7B by splicing. Arrowheads indicate the locations of primers used for RT-PCR. (B) The effect of boric acid on splicing of RPL7B. Yeast cells were grown to an OD.sub.600 of 1.0, and then boric acid was added to reach 80 mM at final concentrations. 24 hours later, the yeast cells were harvested and total RNA was isolated. cDNA was synthesized from the total RNA and was used a, a template for splicing analysis by PCR. In this analysis, yeast strain BY4741 (Wild Type) transformed with empty pFL61 vector or AtRBP47c'-expression vector (AtRBP47c') was used. (C) The effect of boric acid on splicing of RPL7A. Splicing of RPL7A was analyzed by RT-PCR in BY4741 transformed with pFL61.

FIG. 8 is a set of pictures and graphs showing the results of boric aid tolerance test for RPL7A- or RPL7B-disrupted yeast cells. (A) Boric acid tolerance in solid medium. Yeast cells were grown to an OD.sub.600 of 1.0, serially diluted, and then 10 .mu.l of the diluent was spotted in SD plate added with 0 or 80 mM boric acid. The growth was recorded after culturing for 7 days. (B) Boric acid tolerance in liquid medium. Yeast cells were grown to an OD.sub.600 of 1.0, and then diluted to an OD.sub.600 of 0.1 in SD medium added with 0 or 80 mM boric acid. The diluted yeast cells were cultured at 30.degree. C. for 21 hours (SD) and 60 hours (SD+80 mM boric acid) after the dilution, and then the values of OD.sub.600 were recorded. Vertical bars represent the standard deviation of the mean.+-.the mean of three replicate measurements. .DELTA.rpl7a and .DELTA.rpl7b represent RPL7A-disruption mutant (Y04443) and RPL7B-disruption mutant (Y01094), respectively. (C) The effect of over-expression of RPL7B on boric acid tolerance in RPL7A-disrupted yeast. Yeast cells were grown to an OD.sub.600 of 1.0, serially diluted, and then 10 .mu.l of the diluent was spotted in SD plate added with 0 or 80 mM boric acid. The growth was recorded after culturing for 5 days. Yeast cells transformed with an empty pDR195 vector were used as a control.

FIG. 9 is a set of pictures showing the effect of boric acid on splicing of genes containing noncanonical branchpoint sequences. Yeast were grown to an OD.sub.600 of 1.0, and then boric acid was added to reach 80 mM at final concentrations. 24 hours later, the yeast cells were harvested, and total RNA was isolated to use as a template for splicing analysis by PCR. In this analysis, yeast strain BY4741 (Wild Type) transformed with empty pFL61 vector or AtRBP47c'-expression vector (AtRBP47c') was used. White and black arrowheads indicate unspliced and spliced fragments, respectively.

FIG. 10 is a set of pictures showing the effects of salt on growth of AtRBP47c'-related genes-transformed yeast cells and on splicing of RPL7B. (A) Salt tolerance in solid medium. Yeast cells were grown to an OD.sub.600 of 1.0, serially diluted, and then 10 .mu.l of the diluent was spotted in SD plate containing 0, 1.75 or 2 M NaCl. The growth was recorded after culturing for 7 days. Yeast cells transformed with an empty pFL61 vector were used as a control. (B) The effect of salt on splicing of RPL7B. Yeast cells were grown to an OD.sub.600 of 1.0, and then NaCl or boric acid was added to reach 2 M or 80 mM at final concentrations, respectively. 24 hours later, the yeast cells were harvested and total RNA was isolated. cDNA was synthesized from the total RNA and was used as a template for splicing analysis by PCR.

DETAILED DESCRIPTION

As for a gene DNA of the present invention, it is not especially limited as long as it is a gene conferring a boric acid tolerance consisting of the following: (A) a DNA encoding a protein that has an activity of conferring a boric acid tolerance and consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; (B) a DNA encoding a protein that consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 and has an activity of conferring a boric acid tolerance; (C) a gene DNA conferring a boric acid tolerance, which consists of the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 or a complementary sequence thereof; (D) a DNA encoding a protein that consists of a base sequence wherein one or a few bases are deleted, substituted or added in the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29, and has an activity of conferring a boric acid tolerance; or (E) a DNA encoding a protein that hybridizes with a DNA conferring a boric acid tolerance which consists of the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 under stringent conditions and has an activity of conferring a boric acid tolerance.

Further, as for a protein of the present invention, it is not especially limited as long as the protein is the following: (A) a protein having an activity of conferring a boric acid tolerance, which consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; or (B) a protein Insisting of an amino sequence wherein one or a few amino acids are deleted, substituted or added in the amino sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; and having an activity of conferring a boric acid tolerance. Here, the term "a gene conferring a boric acid tolerance" relates to a gene that can confer a boric acid tolerance to a living organism, and the term "a protein conferring a boric acid tolerance" relates to a protein that can confer a boric acid tolerance to a living organism.

The above-mentioned phrase "a protein which has an activity of conferring a boric acid tolerance" relates to a protein having an activity that can confer tolerance against boric acid in a living organism such as yeast and plant, and the yeast and plant highly-expressing the protein can be grown even in the presence of boric acid in high concentration.

AtPAB2 gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 1, AtPAB2 as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 2, AtRBP47c' gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 3, AtRBP47c' as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 4, AtRPS20B gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 5, AtRPS20B as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 6, AtMYB13 gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 7, AtMYB13 as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 8, AtMYB68 gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 9, AtMYB68 as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 10, AtRBP45a gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 11, AtRBP45a as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 12, AtRBP45b gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 13, AtRBP45b as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 14, AtRBP45c gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 15, AtRBP45c as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 16, AtRBP45d gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 17, AtRBP45d as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 18, AtRBP47a gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 19, AtRBP47a as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 20, AtRBP47b gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 21, AtRBP47b as a-protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 22, AtRBP47c gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 23, AtRBP47c as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 24, AtUBP1a gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 25, AtUBP1a as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 26, AtUBP1b gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 27, AtUBP1b as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 28, AtUBP1c gene as a gene conferring a boric acid tolerance consisting of the base sequence shown by SEQ ID NO: 29, AtUBP1c as a protein conferring a boric acid tolerance consisting of the amino acid sequence shown by SEQ ID NO: 30, can be exemplified respectively.

The above-mentioned phrase "an amino sequence wherein one or a few amino acids are deleted, substituted or added" relates to an amino sequence wherein, for example, any number of 1 to 20, preferably 1 to 15, more preferably 1 to 10, furthermore preferably 1-5 amino acids are deleted, substituted or added. Further, the above-mentioned phrase "a base sequence wherein one or a few bases are deleted, substituted or added" relates to a base sequence wherein, for example, any number of 1 to 20, preferably 1 to 15, more preferably 1 to 10, furthermore preferably 1 to 5 bases are deleted, substituted or added.

For example, a DNA, which consists a base sequence wherein one or a few bases are deleted, substituted or added (mutated DNA), can be produced by any methods such as chemical synthesis, genetic engineering method and mutagenesis, which are known to those skilled in the art. Specifically, a mutated DNA can be obtained by introducing a mutation into a DNA that consists of the base sequence shown by SEQ ID NO: 1, 3, 5, 7 or 9, with the use of methods such as a method of allowing to contact and react an agent to be a mutagen, a method of irradiating ultraviolet and a genetic engineering method. Site-specific mutagenesis which one of the genetic engineering methods is a useful method that can introduce a specific mutant into a specific site, and can be performed according to methods described previously such as Molecular Cloning, A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter, abbreviated as "Molecular Cloning 2nd Ed."); Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997). By expressing this mutated DNA with the use of a suitable expression system, a protein encoded by an amino sequence wherein one or a few amino acids are deleted, substituted or added can be obtained.

The above-mentioned phrase "a base sequence which hybridizes under stringent conditions" relates to a base sequence obtained by using methods such as colony hybridization, plaque hybridization, and Southern blotting, with the use of nucleic acids such as DNA and RNA as a probe. Specifically, DNA that can be identified by hybridizing by using a filter-immobilized DNA derived from a colony or a plaque, or a fragment thereof, at 65.degree. C. in the presence of 0.7-1.0 M NaCl; by washing the filter under the condition of 65.degree. C. with the use of SSC solution of approximately 0.1-2.0-fold concentration (one-fold concentration of SSC solution is composed of 150 MM NaCl and 15 mM sodium citrate); can be exemplified. Hybridization can be performed according to the method described in Molecular Cloning 2nd Ed. and the like.

For example, as a DNA that can hybridize under stringent conditions, a DNA having above a certain level of homology with a base sequence of DNA used as a probe can be exemplified, and a DNA having, for example, 60% or more, preferably 70% or more, more preferably 80% or more, furthermore preferably 90% or more, especially preferably 95% or more, most preferably 98% or more of homology, can be exemplified.

Methods for obtaining and preparing genes of the present invention are not especially limited; and it can be prepared by isolating the desired genes through preparing a suitable probe or primer based on the base sequence information shown by SEQ ID NO: 1, 3, 5, 7 or 9, or the amino sequence information shown by SEQ ID NO: 2, 4, 6, 8 or 10 disclosed in the present specification, and screening a cDNA library wherein the presence of the genes are expected with the use of the above probe or primer; or by chemical synthesis according to ordinary methods.

Specifically, a gene of the present invention can be obtained by preparing a cDNA library from Arabidopsis thaliana from where the gene of the present invention was isolated, according to ordinary methods; and selecting the desired clone with the use of a specific and appropriate probe for the gene of the present invention. As the origin of the above cDNA, a variety of cells and tissues derived from the above plant can be exemplified; and further, isolation of all RNA from these cells or tissues, purification and isolation of mRNA, obtaining cDNA and the cloning thereof, and the like, can all be performed according to ordinary methods. As for a method for screening genes of the present invention from a cDNA library, for example, methods which are generally used by those skilled in the art such as methods described in Molecular Cloning 2nd Ed., and the like, can be exemplified.

Furthermore, a mutated gene or homologous gene of the present invention which consists of the base sequence shown by any one of the above (B) to (F) can be isolated, with the use of a DNA fragment having, the base sequence shown by SEQ ID NO: 1, 3, 5, 7 or 9, or part thereof, by screening a homolog of the DNA under appropriate conditions from other organisms and the like. Furthermore, it can be prepared by the above-mentioned methods for preparing the mutated DNA.

Methods for obtaining and preparing proteins of the present invention are not especially limited, and any one of the following proteins can be used: a natural occurring protein, a chemical synthetic protein, or a recombinant protein prepared by transgenesis. When obtaining a natural occurring protein, a protein of the present invention can be obtained from the cells or tissues expressing the protein, by combining appropriately the methods of isolation/purification of protein. When preparing a protein by chemical synthesis, for example, a protein of the present invention can be synthesized according to chemical synthesis such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). Further, a protein of the present invention can be also synthesized with the use of various types of peptide synthesizer being marketed. When preparing a protein by transgenesis, a protein of the present invention can be prepared by introducing a DNA that consists of a base sequence encoding the protein into a preferable expression system. Among the above methods, preparation by transgenesis which manipulation is relatively easy and by which a large amount of preparation can be available, is preferable.

For example, when preparing a protein of the present invention by transgenesis, known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, and preferably high-performance liquid chromatography are used for collecting and purifying the protein from cell culture. Particularly, as for a column to use for affinity chromatography, for example, by using a column bound with antibodies such as monoclonal antibodies against a protein of the present invention; when a normal peptide tag is added to the above protein of the present invention, by using a column bound with certain materials that have an affinity for the peptide tag, purified products of these proteins can be obtained. Further, when a protein of the present invention is expressed on a cell membrane, purified preparations can be obtained by performing the above purification treatment after allowing to act a cell membrane catabolic enzyme.

In addition, a protein consisting of an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30, or a protein consists of the amino acid sequence having 60% or more of homology with the amino acid shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 can be prepared or obtained conveniently by those skilled in the art according to the base sequence information shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 which shows one of the examples of the base sequences encoding the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 respectively. For example, a homolog of a DNA having the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29, or part thereof can be isolated from organisms other than Arabidopsis thaliana by screening under appropriate conditions with the use of the DNA as a probe. A protein encoded by the homolog DNA can be prepared by integrating into an expression vector to express in an appropriate host after cloning a full length of the homolog DNA.

As for a recombinant vector of the present invention, it is not especially limited as long as it is a recombinant vector that contains the above gene of the present invention and can express a protein conferring a boric acid tolerance, and a recombinant vector of the present invention can be constructed by integrating the gene of the present invention appropriately into an expression vector. As for an expression vector, a vector that can self-replicate in host cells or can be integrated in chromosomes of host cells, is preferable; moreover, vectors which contain regulatory sequences such as promoter, enhancer and terminator at a position where a gene of the present invention can be expressed, can be used preferably. As for an expression vector, an expression vector for yeast, an expression vector for plant cells, an expression vector for bacteria, an expression vector for animal cells and the like can be used; however, a recombinant vector using an expression vector for yeast or expression vector for plant cells is preferable.

As for an expression vector for yeast, pYES2 (Invitrogen), YEp13 (ATCC37115), YEp24 (ATCC37051), Ycp50 (ATCC37419), pHS19 and pHS15 can be exemplified. As for a promoter for yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, GAL1 promoter, GAL10 promoter, heat shock protein promoter, MF.alpha.1 promoter and CUP1 promoter can be specifically exemplified.

As for an expression vector for plant cells, plasmids such as Ti plasmid (Tumor inducing plasmid), pSPORT1, pT7Blue-T vector, pIG121-Hm [Plant Cell Report, 15, 809-814(1995)], pBI121 [EMBO J. 6, 3901-3907(1987)], or plant viral vectors such as tobacco mosaic virus, cauliflower mosaic virus and geminivirus can be exemplified. As for a promoter for plant cells, cauliflower mosaic virus 35S promoter [Mol.Gen.Genet (1990) 220, 389-392] and ribulose bisphosphate carboxylase small subunit promoter can be exemplified, and as for a terminator, nopaline synthase gene terminator can be exemplified.

Further, as for a transformant of the present invention, it is not especially limited as long as it is a transformant wherein the above recombinant vector of the present invention is introduced and which expresses a protein conferring a boric acid tolerance. Transgenic yeasts, transgenic plants (cells, tissues, individuals), transgenic bacteria, transgenic animals (cells, tissues, individuals), can be exemplified, while transgenic yeasts and transgenic plants (cells, tissues, individuals) are preferable.

As for a host yeast to use for producing a transgenic yeast, Saccharomyces cerevisiae, Schizosaccharomyces prombe, Kluyveromyces lactis, Trichosporon pullulans and Schwanniomyces alluvius can be exemplified. As for a method for introducing a recombinant vector to host yeast, for example, electroporation, spheroplast method and lithium acetate method can be exemplified.

As for a host plant (cell, tissue, individual) to use for producing a transgenic plant (cell, tissue, individual), species is not especially limited, and it can be appropriately selected from plants such as flowers and ornamental plants, fruit plants, vegetables, root crops, cereals, foliage plants and trees including fruit trees, for example, plants belonging to Solanaceae, Poaceae, Brassicaceae, Asteraceae, Pedaliaceae, Oleaceae, Myrtaceae, Rosaceae, Leguminosae, Palmae or rubiaceae, and cultured cells and tissues thereof (seed, callus and the like) To produce a transgenic plant, a method for introducing a gene DNA of the present invention into genomic DNA within plant cells, by introducing the above recombinant vector into plant cells with the use of the recombinant vector of the present invention containing a gene of the present invention can be used. Transformation of a plant can be performed by appropriately using known methods such as leaf disk cocultivation method, electroporation, Agrobacterium method and particle gun method, according to species of the plant. Other methods for producing transgenic plant, including a method by directly incorporating a recombinant vector of the present invention into a receptor cell can be also used, by physically or chemically enhancing the permeability of plant cells.

As for a method for screening a gene conferring a boric acid tolerance of the present invention is not especially limited as long as it is a method for measuring/evaluating an activity of conferring a boric acid tolerance of the transformed YNL275w-disrupted yeast by transforming a YNL275w-disrupted yeast which is deficient in and not expressing YNL275w gene with the use of a gene library such as a variety of plants or yeasts, and by culturing the obtained transformed YNL275w-disrupted yeast in medium containing boric acid. As for a measurement/evaluation of an activity of conferring a boric acid tolerance, a measurement/evaluation of a level of growth/proliferation of transgenic yeast in culture medium containing boric acid can be exemplified. Further, as for a YNL275w-disrupted strain, Saccharomyces cerevisiae strain 1169 (Winzeler, E. A.; Shoemaker, D. D.; Astromoff, A.; Liang, H.; Anderson, K.; Andre, B.; Bangham, R.; Benito, R.; Boeke, J. D.; Bussey, H., Chu, A. M.; Connelly, C.; Davis, K.; Dietrich, F.; Dow, S. W.; El Bakkoury, M.; Foury, F.; Friend, S. H.; Gentalen, E.; Giaever, G.; Hegemann, J. H.; Jones, T.; Laub, M.; Liao, H.; Liebundguth, N.; Lockhart, D. J.; Lucau-Danila, A.; Lussier, M.; M'Rabet, N.; Menard, P.; Mittmann, M.; Pai, C.; Rebischung, C.; Revuelta, J. L.; Riles, L.; Roberts, C. J.; Ross-MacDonald, P.; Scherens, B.; Snyder, M.; Sookhai-Mahadeo, S.; Storms, R. K.; Veronneau, S.; Voet, M.; Volckaert, G.; Ward, T. R.; Wysocki, R.; Yen, G. S.; Yu, K. X.; Zimmermann, K.; Philippsen, P.; Johnston, M.; Davis, R. W. (1999) Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285: 901-906) can be preferably exemplified. As for yeast to use for screening, it is not limited to YNL275w-disrupted strains, and wild types can be used.

Further, as for a screening method of a gene conferring a boric acid tolerance of the present invention, a method for measuring/evaluating an enhancement level of splicing efficiency can be exemplified, for example, a method for measuring/evaluating an improvement level of a specific inhibition of splicing by boric acid in an intron-containing gene in yeast by expressing a test substance in yeast cells and culturing the expressed test substance in the presence of boric acid, as an enhancement level of splicing efficiency, can be exemplified. As for an intron-containing gene in yeast, specifically RPL7B gene (SEQ ID NO: 33) which is a gene encoding large subunit protein of essential ribosome in Saccharomyces cerevisiae genome, can be exemplified. The improvement level of specific inhibition of splicing by boric acid can be measured, for example, by RT-PCR, and at that time, AtRBP47c' gene, which is a gene conferring a boric acid tolerance is preferably used as a positive control.

In the present invention, use of (a method for) using the above DNA of the present invention as a gene conferring a boric acid tolerance, use of (a method for) using the above DNA of the present invention for producing plants or yeast conferred a boric acid tolerance, use of (a method for) using the above protein of the present invention as a protein having an activity of conferring a boric acid tolerance, and use of (a method for) using the above protein of the present invention for producing plants or yeast conferred a boric acid tolerance are included. Therefore, using the above gene conferring a boric acid tolerance and the above protein having an activity of conferring a boric acid tolerance (protein conferring a boric acid tolerance) for producing plants or yeast conferred a boric acid tolerance are included in the embodiments of the present invention.

The invention will now be further described by way of the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

EXAMPLES

Example 1

1.1. Test Yeasts and Plasmids

As for yeasts, Saccharomyces cerevisiae strain 1169 (purchased from Research Genetics) and Saccharomyces cerevisiae strain BY4741 (purchased from Research Genetics) are used. Genotypes for strain 1169 are MATa, his3.DELTA.1, leu2.DELTA.0, met15.DELTA.0, ura3.DELTA.0, YNL275w, kanMX4; and MATa, his3.DELTA.1, leu2.DELTA.0, met15.DELTA.0, ura3.DELTA.0 for strain BY4741 respectively. As for plasmids, pYES2 (purchased from Invitrogen Genetics) and pFLM61 (provided from Dr. Nicolaus von Wiren in Hohenheim University, Germany; Minet M., Dufour M. -E., and Lacroute F. (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J. 2, 417-422) were used. pFL61 was used to produce an Arabidopsis thaliana expression library. Boric acid tolerance test on yeast-strain 1169

The performance of boric acid tolerance in the used yeast strain 1169 was evaluated. A single colony of yeast strain 1169 which was transformed with pYES2 and pYES2-BOR1 (to which inserted CDS of BOR1, a boron tolerance gene of Arabidopsis thaliana downstream of GAL1 promoter of pYES2 vector) was picked by a platinum loop, and shaking cultured to an OD.sub.600 of around 1.0 in SD liquid medium. The culture solution was respectively streaked in SD solid medium containing 0, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM boric acid, and cultured at 26.5.degree. C. for 16 days. It was then evaluated whether or not the yeast can form colony in each medium.

1.2. Screening of Genes Conferring Boric Acid Tolerance

Yeast strain 1169 was transformed with lithium acetate method with the use of Arabidopsis thaliana expression library (provided from Dr. Nicolaus von Wiren in Hohenheim University, Germany; Schaaf G., Catoni E., Fits M., Schwacke R., Schneider A., von Wiren N., and Frommer W.B. (2002) A putative role for the vacuoler calcium/manganese proton antiporter AtCAX2 in heavy metal detoxification. Plant Biol. 4; 612-618). The transgenic yeast was streaked in SD medium added 80 mM boric acid (6.7 g/l yeast nitrogen base without amino acids, 5 g/l ammonium sulfate, 20 g/l glucose, 2 g/l histidine, 2 g/l methionine, 3 g/l leucine, 20 g/l agar, pH 5.5) and cultured at 26.5.degree. C. After 10 to 14 days, plasmids were collected from the yeast that formed a colony. The collected plasmids were introduced into yeast again and the repeatability of the performance of boric acid tolerance was identified.

1.3. Boric Acid Tolerance Tests

Spot assays and tests in liquid culture were performed. Spot assays were performed by the following procedures. Each of the yeast was shaking cultured to an OD.sub.600 of 0.5-1.0 at 30.degree. C. in SD liquid medium. Each yeast culture was diluted until the values of OD.sub.600 are equal in SD medium. 1/5, 1/25, 1/125 or 1/625 diluted diluent which values of OD.sub.600 are equal was prepared for each yeast culture medium. Each diluent was spotted with 5 .mu.l at a time by pipetman (Gilson) in SD solid medium with boric acid, and in SD solid medium without boric acid as a control. It was also spotted from left to right to lower the concentration for the same. The plate spotted yeast was cultured at 30.degree. C. for around 10 days and growth states of the yeast were observed.

Test in liquid culture was performed as follows. Each yeast was shaking cultured to an OD.sub.600 of around 1.0 at 30.degree. C. in SD medium. Each culture medium was subcultured in SD solid medium with boric acid, and in SD solid medium without boric acid as a control, to an OD.sub.600 of 0.1, and shaking cultured at 30.degree. C., then the values of OD.sub.600 were measured every 24 hours.

1.4. Sequences of Genes Conferring Boric Acid Tolerance

Analysis of the base sequences of 6 cDNA clones obtained by screening was performed as follows. The base sequences were analyzed by performing sequence reaction using fluorescent dye-terminator terminator, with the use of ABI 310 genetic analyzer. A gene encoding the base sequences was identified by BLAST search of TAIR (see the website for The Arabidopsis Information Resource) from the obtained base sequences.

1.5. Screening Results of Genes Conferring Boric Acid Tolerance

First, the performance of boric acid tolerance in yeast strain 1169 used in the present experiments was evaluated. The yeast 1169 was transformed with pYES2 and pYES2-BOR1. pYES2 and pYES2-BOR1 were used for the transformation, because these vectors retain URA3 that is the same one as vector pFL61, which is used in the Arabidopsis thaliana expression library that is used for the following screening, as a selection marker. Further, in SD medium, as the expression of BOR1 gene of pYES-BOR1 is not induced, the same level of boric acid tolerance as in transformant of pYES2 should be induced. The yeasts transformed with pYES2 and pYES2-BOR1 were named "2" and "7", respectively. "2" and "7" were shaking cultured in SD liquid medium, and streaked in SD solid medium containing 0, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM boric acid. As a result, it was revealed that the transformant by either vector could not also be grown in SD medium containing 80 mM or more of boric acid (FIG. 1).

To isolate ones inferring a boric acid tolerance, Arabidopsis thaliana genes that can grow the yeast in SD medium containing 80 mM boric acid by expressing the genes in yeast strain 1169 were searched in the present experiment. Therefore, around 1.2 million yeasts transformed in Arabidopsis thaliana expression library were streaked in SD medium containing 80 mM boric acid. As a result, 6 transgenic yeasts: 46, 66, 72, 84, 86 and 87 that induce tolerance against 80 mM boric acid were obtained. The performances of boric acid tolerance in transgenic yeasts: 46, 72, 84, 86 and 87 by spot assays are shown in FIG. 2 (Since 66 encodes the same gene as 46 does, it is shown in the following, only the result of 46 is shown) . Yeast strain 1169 can hardly form colony in SD medium containing 80 mM boric acid, as it is shown in the upper half of FIG. 2. On the other hand, any of these transgenic yeasts could form more colonies compared to 1169 strain. Next, test in liquid culture was performed. In the liquid culture, 46, 72, 86 and 87 showed around 3-fold growth potential compared to strain 1169 in boric acid medium, as shown in FIG. 3. However, 84 had variable growth rates and no significant difference was observed compared to strain 1169 in boric acid tolerance. Further, these genes could confer a boric acid tolerance when they were introduced into yeast strain BY4741 as well as when they were introduced into 1169 strains. The results from spot assays are shown in FIG. 4, and the results from liquid culture are shown in FIG. 5. When they were introduced into strains BY4741, in all of the transformed yeasts, significant differences were also observed in boric acid tolerance in the liquid culture (FIG. 5).

1.6. Sequences of Genes Conferring Boric Acid Tolerance

6 base sequences of the cDNA clones obtained from screening were determined, and genes encoding them were identified by BLAST searches. As a result, it was revealed that 46 and 66, 72, 84, 86, and 87 matched AtPAB2, AtMYB68, AtMYB13, AtRPS20B, and AtRBP47, respectively. The respective sequences of the genes are shown in the following sequence listing. AtPAB2, AtMYB13 and AtMYB68, AtRPS20, and AtRBP47 are genes encoding polyA-binding protein, Myb-like transcription factor, ribosomal protein, and RNA-binding protein, respectively.

Example 2

2.1. Yeast Strains and Screening

Saccharomyces cerevisiae strain BY4741 (MATa his3D1 leu2D0 met15D0 ura3D0), Y01169 (MATa his3D1 leu2D0 met15D0 ura3D0 YNL275W::kanMX4), Y04443 (MATa his3D1 leu2D0 met15D0 ura3D0 YGL076C::kanMX4), and Y01094 (MATa his3D1 leu2D0 met15D0 ura3D0 YPL198W::kanMX4), were used in this study. Strains: Y01169, Y04443, and Y01094 were constructed from BY4741 by insertional mutagenesis (Winzeler et al., 1999) and obtained from EUROSCARF.

Yeast competent cells were transformed with an Arabidopsis thaliana cDNA library cloned in the expression plasmid pFL61(Minet et al., 1992) by using the lithium acetate method (Gietz and Schiestl, 1995). The strain Y01169 was used as a host because it lacks YNL275W (hereinafter, referred to as BOR1), an efflux B transporter, and sensitive to boric acid compared with the corresponding wild type strain (data not shown). Transformants were screened on SD solid medium (Sherman, 1991) containing 80 mM boric acid at 26.5.degree. C. SD medium contained 2% glucose, 0.67% yeast nitrogen base without amino acids, 0.05% ammonium sulfate, and the amino acids (20 mg/L His, 30 mg/L Leu, and 20 mg/L Met), which are required for the growth of the mutant, and the pH was adjusted to 5.5 with Tris. Agar (2% w/v) was added for making the solid medium. Colony formation of the nontransformed Y01169 (.DELTA.bor1) cells was completely suppressed by addition of 80 mM boric acid. Among the transformed cells, those that formed colonies on media containing 80 mM boric acid after two-week incubation at 26.5.degree. C. were selected and their tolerance were confirmed by testing their growth in the presence of 80 mM boric acid. To confirm that the phenotype was conferred by the plasmids, plasmids were isolated from the positive isolates and re-transformed into the yeast strain Y01169. Tolerant isolates were subjected to fluoro-orotic acid-induced plasmid loss (Boeke, J. D., LaCroute, F., & Fink, G. R. (1984) Mol. Gen. Genet. 197, 345-346) to select only those clones showing plasmid-dependent boric acid tolerance.

2.2. Construction of Plasmids

ORF sequences of AtRBP47c''-related genes and RPL7B (see SEQ ID NO: 34) were amplified by PCR using the primer sets listed in Table 1. The amplified products were sub-cloned into pGEM-T easy vector (Promega). These plasmids were treated with NotI, and the resultant ORF fragments of AtRBP45a, AtRBP47b, AtRBP47c, AtRBP47c' and AtUBP1 were cloned into the NotI site of the pFL61 expression vector (Minet et al., 1992), and the ORF fragments of RPL7B were cloned into the NotI site of the pDR195 expression vector (Rentsch et al., 1995). pFL61 And pDR195 carry PGK and PMA1 promoters for expression, respectively.

TABLE-US-00001 TABLE 1 (SEQ ID NOS 35-48, respectively, in order of appearance) Gene Primer sequences AtRBP45a 5'-AAAAAGCAGGCTTAATGCAGCAACCACCGTCAAACGC C-3' 5'-AGAAAGCTGGGTTTCACTGACGTTGCTGCTGATAGT T-3' AtRBP47a 5'-AAAAAGCAGGCTTAATGCAGACACCAAACAACAACGG T-3' 5'-AGAAAGCTGGGTTTCAAGAAGCTCCCGGGACTGCAG C-3' AtRBP47b 5'-AAAAAGCAGGCTTAATGCAGACAACCAACGGCTCAGA T-3' 5'-AGAAAGCTGGGTTTCAATTCTCCCCATGATAGTTGT T-3' AtRBP47c 5'-AAAAAGCAGGCTTAATGGCAGACGTCAAGATTCAATC C-3' 5'-AGAAAGCTGGGTTTCAGCTAACTTGTTGCTGATGAC C-3' AtRBP47c' 5'-AAAAAGCAGGCTTAATGGCAGACGTCAAGGTTCAATC C-3' 5'-AGAAAGCTGGGTTTCAGCTAACTTGTTGCTGATGAC C-3' AtUBP1a 5'-AAAAAGCAGGCTTAATGCAGAATCAAAGGCTTATTAA G-3' 5'-AGAAAGCTGGGTTTTACTGATAGTACATGAGCTGCT G-3' RPL7B 5'-AAAAAGCAGGCTTAATGTCCACTGAAAAAATCTT-3' 5'-AGAAAGCTGGGTTTTAGTTCATAGCCTTAACCA-3'

2.3. Boric Acid Tolerance Assays

For boric acid tolerance assay of AtRBP47c''-related family genes, the expression plasmids were introduced into the Saccharomyces cerevisiae strain BY4741. As controls, empty vectors without insert were also introduced into BY4741. The transformants were grown to stationary phase in the SD liquid medium, and then cell densities of the cultures were adjusted to OD.sub.600=1.0. These cell density-adjusted cultures were diluted to 1/5, 1/25, 1/125, and 1/625 with the SD liquid medium and 10 .mu.L of diluted cultures were dropped on the SD solid medium with or without 80 mM boric acid and incubated at 30.degree. C. for 7 days.

For analysis in liquid culture, the transformants were grown to stationary phase in the SD liquid medium, and then diluted in the SD liquid medium with or without 80 mM boric acid to adjust the value of OD.sub.600 to 0.1 for the performance test of high concentration boric acid tolerance.

For analysis of boric acid tolerance of .DELTA.rpl7a (Y04443) and .DELTA.rpl7b (Y01094) mutants, SD medium containing 2% glucose, 0.67% yeast nitrogen base without amino acids, and 0.05% ammonium sulfate was used, adjusting to pH 5.5 with Tris, and the required amino acids (20 mg/L His, 30 mg/L Leu, 20 mg/L Met, and 20 mg/L Ura) were added. The mutants were obtained from EUROSCARF. To further examine the role of RPL7B in boric acid tolerance, RPL7B was over-expressed in the yeast strain Y04443. Boric acid tolerance assays were carried out as described above.

2.4 Detection of Unspliced Transcripts by RT-PCR

Yeast cells were grown to exponential phase (OD.sub.600=0.5-1.0) in SD liquid medium, and then boric acid was added to be 80 mM at final concentration. After 24h incubation at 30.degree. C., one-ml of samples were taken, and the cells were collected by centrifugation, frozen in liquid nitrogen, and stored at -80.degree. C. until use.

Total RNA was extracted from the yeast cells by using an RNeasy Mini Kit (Qiagen), and 1 .mu.g of total RNA was reverse-transcribed by using MuLV reverse-transcriptase (Applied Biosystems) and oligo (dT).sub.16 primer. One-fifteenth of the RT products was subjected to PCR with the following cycle: 40-50 times at 94.degree. C. for 30 sec, 45.degree. C. for 30 sec, and 72.degree. C. for 1 min. PCR was carried out with a Smart Cycler (Cepheid) using a DNA polymerase, Ex taq (Takara) . Primer sets used in this analysis are listed in Table 2, which is published as expanded information on the PNAS web site. Amplified transcripts were separated on 2% agarose gel and detected after staining with Etd bromide.

TABLE-US-00002 TABLE 2 (SEQ ID NOS 49-134, respectively, in order of appearance) Gene Primer sequences SNR17A 5'-AATCTGTGTCGACGTACTTC-3' (Forward) 5'-AGAAGTACATAGGATGGGTC-3' (Reverse) SNR17B 5'-AAAAATTGTCGACGTACTTC-3' (Forward) 5'-AAAGGAAGTTATCACAATTG-3' (Reverse) YBR230C 5'-CCAGCATCTATGTCTGCAAC-3' (Forward) 5'-CGTATCTGGAGTAGTATTTC-3' (Reverse) VMA10 5'-GCAAGGTATACAAAGCAGAA-3' (Forward) 5'-TCATCCTTTTTCTTCTCTGC-3' (Reverse) SEC27 5'-GACACGATGAAGTTGGATAT-3' (Forward) 5'-TGACTGTCAAATCATCACTG-3' (Reverse) YNL050C 5'-CAGTATAAAAATGTCTGAAT-3' (Forward) 5'-TGGTTGATTATTTCTTCTTC-3' (Reverse) RPL7B 5'-ATCAACGTCATAATGTCCAC-3' (Forward) 5'-TACCAGAGTTGATTCTTGTC-3' (Reverse) MUD1 5'-ACCTAAAGAAACCATGTCAG-3' (Forward) 5'-TATCAAGGTTGTACGTTTCG-3' (Reverse) SNC1 5'-ATGTACAGTCTAAGTCAAGG-3' (Forward) 5'-GACTAAAGTGAACAGCAATG-3' (Reverse) POP8 5'-GAGAATGGCAATATTTCAAG-3' (Forward) 5'-TGTTCTTCTTCTTCCATTAC-3' (Reverse) ARP2 5'-TGGACCCACATAATCCAATT-3' (Forward) 5'-TTTCGAACATTACCTCACAC-3' (Reverse) CNB1 5'-GTGGATGGTCTTTTAGAAGA-3' (Forward) 5'-AACTCCTCGAAACTTAAACG-3' (Reverse) RPS22B 5'-TATTGAGACCTTCTTCCAAG-3' (Forward) 5'-AAGATTTTACCGGAAACGTG-3' (Reverse) YML025C 5'-GACGATAAAAAGAAATTTGGTG-3' (Forward) 5'-CTCAAAGCGTTGTTGAAAG-3' (Reverse) TUB3 5'-GAGAGAGGTCATTAGTATTA-3' (Forward) 5'-TTTTCTAATAACAGGGAACC-3' (Reverse) STO1 5'-GTTTAATAGAAAAAGAAGAGGAG-3' (Forward) 5'-TAGTTCATCAACTAAAAACATGG-3' (Reverse) RPS16A 5'-AGCTGTCCCAAGTGTTCAA-3' (Forward) 5'-ACCCTTACCACCGAATTTC-3' (Reverse) SAR1 5'-GTTGGGATATTTTTGGTTGG-3' (Forward) 5'-AAAGGAACGTCCTTCAATTC-3' (Reverse) PM140 5'-AACAAGCTGTTCAGGTTAGA-3' (Forward) 5'-GGTTTGTGATTATCATCAGG-3' (Reverse) RPL7A 5'-AATTAAAGATCACAATGGCCG-3' (Forward) 5'-CTTGGTAACTTTGACGAATG-3' (Reverse) YBL091C-A 5'-CAGAAAAGCTGGTGTTCAAG-3' (Forward) 5'-TGATTCTGCATCGTGGTTTC-3' (Reverse) RPL19A 5'-TTGATTAAGAACTCCAAAGC-3' (Forward) 5'-TCTTCTCAAGACACGTAATC-3' (Reverse) PCH2 5'-AGATGAGGTTGAAGCAATAG-3' (Forward) 5'-CAAGGGCAATTTCCTTATTG-3' (Reverse) RPS9B 5'-TAAGACTAAGCAACAATGCC-3' (Forward) 5'-AAACCCAACTTGTAGACTTG-3' (Reverse) YBR230C 5'-GCATCTCATAATATGTCTGC-3' (Forward) 5'-TTGTTGCTAAGACTGTAGAG-3' (Reverse) YDR381C-A 5'-CAAATCCATTTCAAAATATAGG-3' (Forward) 5'-CTCCTCCTATCTAAAAAACC-3' (Reverse) YRA1 5'-AAGAAGAGTTGGTAAGCAAG-3' (Forward) 5'-CACCGTTTTTGAATGTGATG-3' (Reverse) UBC8 5'-AGCGTAATACGAAAGATGAG-3' (Forward) 5'-AGCTTCGTTATTCAAGGGAT-3' (Reverse) MND1 5'-GTATCATAAACATTCAACAATG-3' (Forward) 5'-CGGATCTGTTGTTTATTCTC-3' (Reverse) MER3 5'-AAACAAAGTTTGATCGCCTG-3' (Forward) 5'-TCGTGCTCAAACATTTCTTC-3' (Reverse) ERV1 5'-AAAATGACGGATAATCCACC-3' (Forward) 5'-TTCAAAGTCTTTAGCACACC-3' (Reverse) SRB2 5'-CAATCCATCATGGGAAAATC-3' (Forward) 5'-CTTGGACGACAAAATAGTGT-3' (Reverse) MOB1 5'-AGGACTTCAATTTCCATGTC-3' (Forward) 5'-AGTGTCATCTCCACAATTTG-3' (Reverse) RPS21A 5'-GAAAACGATAAGGGCCAATT-3' (Forward) 5'-CGTTCTTTAACAAACCATCG-3' (Reverse) NYV1 5'-TACCAAATGAAACGCTTTAATG-3' (Forward) 5'-TCTTCATGGAAAGAGTCTAG-3' (Reverse) YLR211C 5'-ATGGAATGAGTACTTTAGCG-3' (Forward) 5'-CTTCATTTCCGAGTTTTTGG-3' (Reverse) TAD3 5'-AATAGAAAATCGGCTTCTGC-3' (Forward) 5'-TATTTGATCATTGGGGTTGC-3' (Reverse) ERV41 5'-GATTGAAGACATTTGATGCG-3' (Forward) 5'-TCGCCACTAACTCTATTTAC-3' (Reverse) SPO1 5'-ACCATTTCAGGTACAATGTC-3' (Forward) 5'-CTTCGGAAATATCGAATTCC-3' (Reverse) YOL048C 5'-CTGAAACGATACCAACAATG-3' (Forward) 5'-TTTGTGGTTTAGGCAATACC-3' (Reverse) RPS9A 5'-ATACAAAAGTATACAACATGCC-3' (Forward) 5'-TTTCCAAGAAATCTTCGACC-3' (Reverse) CIN2 5'-CTTTACTGCGAAGATAAAGG-3' (Forward) 5'-GCCACTATAATCTGTTGTTG-3' (Reverse) YRP098C 5'-TCAAAACTACGGCTCATTTG-3' (Forward) 5'-TGAACAAAAGACTCAATCCG-3' (Reverse)

2.4. Salt Tolerance Assays

Salt tolerance assay were carried out as in the above-described. boric acid tolerance assays, except that SD media containing 1.75 M or 2 M NaCl were used.

2.5. Accession Numbers

The GenBank accession numbers for the sequences described in Example 2 are as follows: Arabidopsis thaliana sequences AtRBP45a, MN124872; AtRBP45b, MN101037; AtRBP45c, MN118834; AtRBP45d, MN121940; AtRBP47a, MN103848; AtRBP47b, MN112800; AtRBP47c, MN103642; AtRBP47c', MN103643; AtUBP1a, MN104285; AtUBP1b, MN101598; AtUBP1c, MN112266; and Saccharomyces cerevisiae sequences RPL7A, X62627; RPL7B, Z73554.

2.6. Result of Isolation of Arabidopsis thaliana cDNA Clones that Confer High Boric Acid Tolerance to Yeast

Saccharomyces cerevisiae strain Y01169 was transformed with an Arabidopsis thaliana cDNA expression library (Minet, M., Dufour, M. -E., & Lacroute, F. (1992) Plant J. 2, 417-422) and the transformants were selected on dishes containing 80 mM of boric acid. Boric acid at this concentration completely suppressed the formation of colonies of Y01169 cells even after two-week incubation at 26.5.degree. C. In this screening, several colonies of yeast which showed enhanced boric acid tolerances were isolated. It was shown that one of the cDNA clones encodes an RNA binding protein, AtRBP47c'.

2.7. Expression of AtRBP47c'-Related Genes from Arabidopsis thaliana Confers Boric Acid Tolerance to Yeast

AtRBP47c' has three RNA recognition motifs (RRM). In Arabidopsis thaliana genome, there are eleven genes encoding a protein which has three RRMs and 100 or more of sequence identity scores to AtRBP47c' in BLASTP program. The phylogenetic tree of these AtRBP47c'-related family proteins is shown in FIG. 6A.

To investigate whether or not the expression of these Arabidopsis thaliana genes confers a boric acid tolerance to yeast, ORF sequences corresponding to 6 genes AtRBP45a, AtRBP47a, AtRBP47b, AtRBP47c, AtRBP47c', and AtUBP1a) were cloned into pFL61 expression vector. The plasmids were introduced into the yeast strain BY4741 and boric acid tolerances of these transformants were investigated. As shown in FIG. 6B, all of the 6 constructs conferred the ability to the yeast strains to grow on 80 mM boric acid-containing SD solid medium to varying extents. To compare the level of boric acid tolerances among those transformants, their growth rates in the presence of boric acid were analyzed in liquid culture. All transformants showed faster growth rate than the control. In the graph, the AtRBP47c'-expressing line showed the fastest growth rate (FIG. 6C).

2.8. Boric Acid Treatment Inhibits Splicing of RPL7B, but not RPL7A, in Yeast

The present inventors found that the over-expression of AtRBP47c'-related genes conferred a boric acid tolerance. Although roles of these genes in A. thaliana are still unknown, similar genes in other plant species were characterized. Nicotiana plumbaginifolia RBP45 (Simpson, C. G., Jennings, S. N., Clark, G. P., Thow, G., & Brown, J. W. S. (2004) Plant J. 37, 82-91) and UBP1 (Lambermon, M. H., Simpson, G. G., Wieczorek Kirk, D. A., Hemmings-Mieszczak, M., Klahre, U., & Filipowicz, W. (2000) EMBO J. 19, 1638-1649) were shown to enhance splicing efficiency. This led the present inventors to investigate the effect of boric acid on splicing of randomly selected 20 intron-containing genes in Saccharomyces cerevisiae by RT-PCR. Among the 6317 nuclear genes in the Saccharomyces cerevisiae genome, only 231 genes contain introns (see the website for the Munich Information Center for Protein Sequences. Among the 20 genes investigated, the increase by boric acid treatment in the accumulation of unspliced fragments compared to that of spliced fragments was observed in RPL7B, a gene encoding an essential ribosomal large subunit protein. This suggests that the splicing of RPL7B was inhibited in boric acid-treated yeast (FIG. 7B).

The RPL7B contains two introns. The size of unspliced fragments indicated that these fragments were derived from splicing of either one of the first and second introns (see FIG. 7A). To determine which intron is more susceptible to boric acid, the unspliced fragments were cloned and DNA sequences of the eight clones were determined. Six and two clones contained the first intron and the second intron, respectively. This suggests that inhibition occurs both at the first and the second introns and the first intron is more susceptible to high boric acid than the second one. The results also indicate that one of the two introns were correctly spliced, i.e., those unspliced fragments did not derive from genome DNA contamination but from the reverse transcription reaction of RNA.

Moreover, the inhibition of splicing of RPL7B by boric acid was not observed in yeast expressing AtRBP47c' (FIG. 7B) . This result suggest that AtRBP47c' elevate splicing efficiency of RPL7B in the presence of high boric acid. It is possible that enhancement of splicing efficiency may be the cause of boric acid tolerance in yeast.

RPL7B has a paralog, RPL7A (SEQ ID NO: 32), in the yeast genome. RPL7A gene (SEQ ID NO: 31) also has two introns as in RPL7B gene. The effect of boric acid on the splicing of RPL7A was examined. The splicing inhibition by boric acid was not observed unlike in the case of RPL7B (FIG. 7C).

2.9. Disruption of RPL7A in Yeast Reduces Boric Acid Tolerance

RPL7A and RPL7B Double Disruption Mutant is Lethal (see the website for the Saccharomyces Genome Database), indicating that RPL7 proteins are essential for yeast growth. Considering the differential sensitivity of boric acid to splicing between the two genes, it is possible that the boric acid tolerances of RPL7A- and RPL7B-disruption mutants differ. The .DELTA.rpl7b (Y01094) showed a similar level of boric acid tolerance to the wild type Saccharomyces cerevisiae, whereas a boric acid tolerance of the .DELTA.rpl7a (Y04443) was lower than the wild type (FIG. 8A). The difference in a boric acid tolerance was also evident in liquid culture (FIG. 8B). These results suggest that the inhibition of RPL7B splicing by boric acid is caused by reduction in a boric acid tolerance of .DELTA.rpl7a.

2.10. Expression of Intronless RPL7B in RPL7A-Disrupted Yeast Increases Boric Acid Tolerance

If the reduction in a boric acid tolerance of .DELTA.rpl7a is due to the reduction in the level of RPL7 protein by inhibition of RPL7B splicing, expression of intronless RPL7B cDNA should increase the tolerance of .DELTA.rpl7a.

It was examined whether the expression of intronless RPL7B in .DELTA.rpl7a increases boric acid tolerance. ORF sequence of RPL7B was cloned into pDR195 expression vector. The plasmid was then introduced into the .DELTA.rpl7a and a boric acid tolerance in the transformant was investigated. As shown in FIG. 8C, the expression of intronless RPL7B increased boric acid tolerance in .DELTA.rpl7a. This result indicates that the inhibition of RPL7B splicing is the cause of growth cessation by highly concentrated boric acid in .DELTA.rpl7a.

2.11. Analysis of Splicing Inhibition in Genes Containing Noncanonical Branchpoint Sequences by Boric Acid Treatment

RPL7B has a noncanonical branchpoint sequence in its first intron (see Table 3). 28 genes containing such noncanonical branchpoint sequences among 231 nuclear intron-containing genes were found. Among the 28 genes, increase in the level of unspliced fragments by boric acid treatment compared to that of spliced fragments was observed in nine genes (FIG. 9). These genes are ERV1, ERV41, NYV1, RPS9A, RPS9B, SRB2, YOL048C, YPR098C, and YRA1.

TABLE-US-00003 TABLE 3 ##STR00001##

Table 3 shows three consensus sequences, 5' splice site, branchpoint, and 3' splice site, that were recognized in yeast. A transition point from A to G in branchpoint of the first intron is represented by white letter in black background. Y represents pyrimidine ribonucleotides (C or U).

The effects of over-expression of AtRBP47c' on the splicing inhibition of those genes by boric acid was analyzed. As shown in FIG. 9, the level of splicing inhibition of NYV1 and SRB2 was impaired in yeast expressing AtRBP47c'. NYV1 and SRB2 encode v-SNARE protein and RNA polymerase II holoenzyme protein, respectively. These results strongly suggest that the mechanism of conferring a boric acid tolerance to yeast by over-expression of AtRBP47c' is the enhancement of splicing efficiency.

2.12. Effects of Salt Treatment are Different from those of Boric Acid Treatment

Over-expression of splicing factor genes confers salt tolerance to yeast and/or plants (Forment, J., Naranjo, M. A., Roldan, M., Serrano, R., & Vicente, O. (2002) Plant J. 30, 511-519, 2002; Serrano, R., Gaxiola, R., Rios, G., Forment, J., Vicente, O., & Ros, R. (2003) Monatsh. Chem. 134, 1445-1464). It was examined whether AtRBP47c'-related genes also confer salt tolerance to yeast. All six AtRBP47c'-related genes tested in this study did not increase the salt tolerance in yeast (FIG. 10A). Furthermore, inhibition of splicing of RPL7B was not observed in cells exposed to high salt (FIG. 10B) . These results suggest that AtRBP47c'-related genes do not function in salt tolerance and that inhibition of RPL7B splicing is likely to be unique to boric acid treatment.

2.13. Discussion

AtRBP47c' was isolated from Arabidopsis thaliana as a gene that confers a boric acid tolerance to yeast cells by yeast complementation. In yeast genome, there are seven genes encoding a protein-which has three RRMs and 100 or more of sequence identity scores to AtRBP47c' in BLASTP program. Among these genes, the most similar gene to AtRBP47c' is NAM8. Although NAM8 was originally isolated as a suppressor of mitochondrial splicing deficiencies (Ekwall, K., Kermorgant, M., Dujardin, G., Groudinsky, O., & Slonimski, P. P. (1992) Mol. Gene. Genet.233, 136-144), subsequent analysis showed that NAM8 interacts with U1snRNA and that NAM8 is indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' slice sites (Gottschalk, A, Tang, J., Puig, O., Salgado, J., Neubauer, G., Colot, H. V., Mann, M., Seraphin, B., Rosbash, M., Luhrmann, R., & Fabrizio, P. (1998) RNA 4, 374-393.; Puig, O., Gottschalk, A., Fabrizio, P., & Seraphin, B. (1999) Gene. Dev. 13, 569-580). From these observations, it was hypothesized that AtRBP47c' might play a similar role with NAM8 in a boric acid tolerance. However, over-expression of NAM8 did not confer a boric acid tolerance to yeast and NAM8-disrupted mutants were tolerant to boric acid as well as wild type, indicating that AAtRBP47c' has possibilities to be involved in an another step of splicing processes and/or other reaction(s) in boric acid tolerance.

In this study, it was found that boric acid could inhibit splicing of RPL7B among randomly selected 20 genes in yeast (FIG. 7B) . By analysis of the DNA sequence in the first intron of this gene, it became clear that the first intron has a transition in the consensus sequence of the branchpoint. As shown in Table 1, the second A in the branchpoint consensus sequence is converted to G in the first intron of RPL7B. The binding of branchpoint bridging protein (BBP) to the branchpoint is a critical step in splicing progression (Abovich and Rosbash, 1997). Affinity between BBP and branchpoint sequence is known to be an important factor for splicing efficiency (Champion-Arnaud, et al., 1995). It has been reported that especially, this type of transition from A to G in second nucleotide of branchpoint sequence showed an approximately 10% decrease in the affinity with BBP (Berglund, J. A., Chua, K., Abovich, N., Reed, R., & Rosbash, M. (1997) Cell 89, 781-787). Therefore, it is likely that RPL7B is one of the genes with low splicing efficiency.

It is reported that the second step of splicing is inhibited by boric acid treatment in HeLa cell in vitro splicing system (Shomron, N., & Ast, G. (2003) FEBS Lett. 552, 219-224). The second step of splicing is a process in which the treated 3' end of an exon is ligated to 5' end of the next exon. Considering that boric acid binds to cis-diol in ribose (Ralston, N. V. C., & Hunt, C. D. (2000) FASEB J. 14, A538; Nicholas et al., 2001; Ricardo, A., Carrigan, M. A., Olcott, A. N., & Benner, S. A. (2004) Science 303, 196), it is likely that the ligation reaction in second step of splicing is inhibited by the binding of boric acid to the 3' end of an exon. The above Shomron and Ast (2003) has been reported that inhibition of splicing by boric acid at the second step is a general phenomenon, as five different mRNA precursors exhibited a similar pattern of inhibition. In that case, inhibition of the splicing in yeast should occur similarly with all introns. However, among the 20 genes tested in the initial step of this study, the only gene in which inhibition was observed was RPL7B (FIG. 7B).

A possible explanation of this specific inhibition is as follows. The inhibition of splicing in the second step by boric acid takes place with all intron-containing genes in yeast. At this step, intron-including splicing intermediates, which should be rapidly degraded when the splicing progresses normally, accumulate. The accumulation of intermediates inhibits normal turnover. In such a situation, genes having introns with low splicing efficiency are likely to be more susceptible to the inhibition of splicing by boric acid. As one of such genes, a gene containing a noncanonical branchpoint sequence such as RPL7B can be exemplified. This speculation was verified by analysis of the inhibition of splicing by high boric acid on other genes having the same feature (FIG. 9). In the analysis, it was found that high boric acid treatment inhibits splicing of nine genes containing noncanonical branchpoint sequences except for RPL7B. This result clearly indicates that one of the toxic mechanisms of boric acid is inhibition of splicing of genes having introns with low splicing efficiency. Moreover, it was found that the splicing inhibitions of two genes among those nine genes were impaired by over-expression of AtRBP47c' (FIG. 9). This result suggests that a boric acid tolerance by over-expression of AtRBP47c' may be achieved by the enhancement of splicing efficiency of part of genes among many genes of which splicing is inhibited during high boric acid treatment. Hence, splicing inhibition of a limited number of genes might be a cause of growth inhibition.

The AtRBP47c'-related proteins have three RRMs. RNA binding activity of RBP45, RBP47, and UBP1 of N. plumbaginifolia has been confirmed. All of these proteins tend to bind with U-rich sequence (Lambermon, M. H., Simpson, G. G., Wieczorek Kirk, D. A., Hemmings-Mieszczak, M., Klahre, U., & Filipowicz, W. (2000) EMBO J. 19, 1638-1649: Lorkovic, Z. J., Wieczorek Kirk, D. A., Klahre, U., Hemmings-Mieszczak, M., & Filipowicz, W. (2000). RNA 6, 1610-1624). Moreover, deletion analysis of RBP45 in N. plumbaginifolia indicated that at least two RRMs are required for interaction with RNA (Lorkovic et al., 2000). Although an RRM was thought to be involved in RNA binding, it was shown that an RRM of a certain protein participates in interaction with other proteins (Kielkopf, C. L., Lucke, S., & Green, M. R. (2004) Gene Dev. 18, 1513-1526). Especially, yeast U2AF.sup.65, a splicing factor containing three RRMs, is reported that the third RRM is bound to BBP (Rain, J. C., Rafi, Z., Rhani, Z., Legrain, P., & Kramer, A. (1998) RNA 4, 551-565). These results suggest that AtRBP47c' may also interact with BBP. Furthermore, analysis of RBP7B first intron and SRB2 intron sequences revealed that there are U-rich sequences at the 3' side of the branchpoint. Taking the results together, it was hypothesized that AtRBP47c' stabilizes the interaction of BBP with branchpoint and the U-rich sequence of the branchpoint by binding with BBP, and as a result, the efficiency of splicing is increased.

It is reported that splicing is also inhibited by salt stress. Furthermore, over-production of several splicing factors such as SR protein have been also reported to increase salt tolerance in yeast and plants (Forment, J., Naranjo, M. A., Roldan, M., Serrano, R., & Vicente, O. (2002) Plant J. 30,511-519, 2002; Serrano, R., Gaxiola, R., Rios, G., Forment, J., Vicente, O., & Ros, R. (2003) Monatsh. Chem. 134, 1445-1464). In the present study, however, over-expression of AtRBP47c'-related genes did not confer salt tolerance to yeast (FIG. 10A), and inhibition of splicing of RPL7B was not detected after salt treatment (FIG. 10B). These results suggest that the mechanism of splicing inhibition is different between salt treatment and boric acid treatment.

Example 2 is the first report showing that the key of the toxic mechanisms of boric acid is the specific inhibition of splicing and that genes involved in enhancement of splicing efficiency lead to the boric acid tolerance. However, the toxic mechanisms other than the inhibition of splicing should exist, since-toxic effect of boric acid is observed in the prokaryotes in which splicing are not performed.

The invention is further described by the following numbered paragraphs:

1. A DNA encoding a protein that has an activity of conferring a boric acid tolerance and consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30.

2. A DNA encoding a protein that consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 and has an activity of conferring a boric acid tolerance.

3. A gene DNA conferring a boric acid tolerance, which consists of the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 or a complementary sequence thereof.

4. A DNA encoding a protein that consists of a base sequence wherein one or a few bases are deleted, substituted or added in the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29, and has an activity of conferring a boric acid tolerance.

5. A DNA encoding a protein that hybridizes with the DNA according to paragraph 3 under stringent conditions and has an activity of conferring a boric acid tolerance.

6. A protein having an activity of conferring a boric acid tolerance, which consists of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30.

7. A protein consisting of an amino sequence wherein one or a few amino acids are deleted, substituted or added in the amino sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30; and having an activity of conferring a boric acid tolerance.

8. A recombinant vector including the DNA according to any one of paragraphs 1 to 5, which can express a protein conferring a boric acid tolerance.

9. A transformant wherein the recombinant vector according to paragraph 8 is introduced, which can express a protein conferring a boric acid tolerance.

10. The transformant according to paragraph 9 wherein the transformant is yeast.

11. The transformant according to paragraph 9 wherein the transformant is a plant.

12. A method for screening a gene conferring a boric acid tolerance, comprising the steps of transforming a YNL275w-disrupted yeast which is deficient in and not expressing YNL275w gene by using a gene library, culturing the obtained transformed YNL275w-disrupted yeast in a medium containing boric acid, and measuring/evaluating an activity of conferring a boric acid tolerance of the transformed YNL275w-disrupted yeast.

13. A method for screening a gene conferring a boric acid tolerance wherein an enhancement level of splicing efficiency is measured/evaluated by targeting a specific inhibition of splicing by boric acid.

14. The method for screening a gene conferring a boric acid tolerance according to paragraph 13, comprising the steps of expressing a test substance in yeast cells, culturing the expressed test substance in the presence of boric acid, and measuring/evaluating an improvement level of a specific inhibition of splicing by boric acid in an intron-containing gene in yeast, as an enhancement level of splicing efficiency.

15. The method for screening a gene conferring a boric acid tolerance according to paragraph 14 wherein the gene containing intron in yeast is a gene RPL7B in Saccharomyces cerevisiae genome.

16. Use of the DNA according to any one of paragraphs 1 to 5 as a gene conferring a boric acid tolerance.

17. Use of the DNA according to any one of paragraphs 1 to 5 for producing a plant or yeast conferred a boric acid tolerance.

18. Use of the protein according to paragraph 6 or 7 as a protein having an activity of conferring a boric acid tolerance.

19. Use of the protein according to paragraph 6 or 7 for producing a plant or yeast conferred a boric acid tolerance.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

SEQUENCE LISTINGS

1

13411332DNAArabidopsis thaliana 1atgttgctca atgacaagca agtgtatgtg ggtcctttcc tgaggagaca agaaagagac 60tccactgcta acaaaacgaa attcaccaat gtgtatgtga agaatctcgc ggaaagtact 120accgatgatg acttgaagaa tgcttttggc gagtatggaa agataacaag tgctgtcgtg 180atgaaagatg gagaagggaa gtccaagggc tttgggtttg tcaactttga aaatgctgat 240gatgctgcta gggctgtgga gtctctcaat gggcacaaat ttgatgataa ggagtggtat 300gttggtagag cccagaagaa gtcagagagg gaaacagaat taagggtccg ttatgaacag 360aatttgaagg aagctgcaga caagtttcaa agttcaaact tgtatgttaa gaatttggat 420cctagcattt cagatgagaa acttaaagag atcttttctc cttttggtac cgttacatct 480agcaaggtga tgcgggatcc taatggaaca agcaaaggct caggttttgt tgctttcgca 540actcccgaag aagcaactga agctatgtca cagttgagcg gtaaaatgat cgaaagcaag 600ccactctatg tggctattgc acagcggaag gaagacagaa gggtcagact acaggctcag 660ttttcccaag tgaggccagt tgcaatgcag ccgtctgttg gtccccgcat gccagtgtat 720cccccgggtg gtcctggtat tggacaacaa atgttctatg gtcaggcccc tcctgccatg 780attcctcccc agcctgggta tggataccaa cagcagcttg ttcctggaat gagacctggt 840gggggtcctg tacccagttt cttcatgcct atggttcagc cacagcagca gcgtcctgga 900ggaggaagac gtcctggggg aatccaacac tcccagcagc aaaatcccat gatgcagcaa 960cagatgcatc caaggggtcg gatgttccgg tatccccaag ggcgtggtgg tagtggtgat 1020gtgcctccat atgatatggg caacaacatg ccattgacta ttggagcttt ggcttcaaat 1080ctgtctaatg ctactccaga gcaacagagg acgatgctgg gtgaggtgct gtacccgttg 1140gtggagcagg ttgaggcaga gtctgcagcc aaagtgactg ggatgctttt ggagatggac 1200cagactgaag tgctccatct gttggagtca ccagaagctc tcaaggccaa agttgcagag 1260gctatggatg ttctcaggag tgtcgctgct ggtggtgcaa ccgagcagct cgcttccttg 1320aacctctctt aa 13322443PRTArabidopsis thaliana 2Met Leu Leu Asn Asp Lys Gln Val Tyr Val Gly Pro Phe Leu Arg Arg1 5 10 15Gln Glu Arg Asp Ser Thr Ala Asn Lys Thr Lys Phe Thr Asn Val Tyr 20 25 30Val Lys Asn Leu Ala Glu Ser Thr Thr Asp Asp Asp Leu Lys Asn Ala 35 40 45Phe Gly Glu Tyr Gly Lys Ile Thr Ser Ala Val Val Met Lys Asp Gly 50 55 60Glu Gly Lys Ser Lys Gly Phe Gly Phe Val Asn Phe Glu Asn Ala Asp65 70 75 80Asp Ala Ala Arg Ala Val Glu Ser Leu Asn Gly His Lys Phe Asp Asp 85 90 95Lys Glu Trp Tyr Val Gly Arg Ala Gln Lys Lys Ser Glu Arg Glu Thr 100 105 110Glu Leu Arg Val Arg Tyr Glu Gln Asn Leu Lys Glu Ala Ala Asp Lys 115 120 125Phe Gln Ser Ser Asn Leu Tyr Val Lys Asn Leu Asp Pro Ser Ile Ser 130 135 140Asp Glu Lys Leu Lys Glu Ile Phe Ser Pro Phe Gly Thr Val Thr Ser145 150 155 160Ser Lys Val Met Arg Asp Pro Asn Gly Thr Ser Lys Gly Ser Gly Phe 165 170 175Val Ala Phe Ala Thr Pro Glu Glu Ala Thr Glu Ala Met Ser Gln Leu 180 185 190Ser Gly Lys Met Ile Glu Ser Lys Pro Leu Tyr Val Ala Ile Ala Gln 195 200 205Arg Lys Glu Asp Arg Arg Val Arg Leu Gln Ala Gln Phe Ser Gln Val 210 215 220Arg Pro Val Ala Met Gln Pro Ser Val Gly Pro Arg Met Pro Val Tyr225 230 235 240Pro Pro Gly Gly Pro Gly Ile Gly Gln Gln Met Phe Tyr Gly Gln Ala 245 250 255Pro Pro Ala Met Ile Pro Pro Gln Pro Gly Tyr Gly Tyr Gln Gln Gln 260 265 270Leu Val Pro Gly Met Arg Pro Gly Gly Gly Pro Val Pro Ser Phe Phe 275 280 285Met Pro Met Val Gln Pro Gln Gln Gln Arg Pro Gly Gly Gly Arg Arg 290 295 300Pro Gly Gly Ile Gln His Ser Gln Gln Gln Asn Pro Met Met Gln Gln305 310 315 320Gln Met His Pro Arg Gly Arg Met Phe Arg Tyr Pro Gln Gly Arg Gly 325 330 335Gly Ser Gly Asp Val Pro Pro Tyr Asp Met Gly Asn Asn Met Pro Leu 340 345 350Thr Ile Gly Ala Leu Ala Ser Asn Leu Ser Asn Ala Thr Pro Glu Gln 355 360 365Gln Arg Thr Met Leu Gly Glu Val Leu Tyr Pro Leu Val Glu Gln Val 370 375 380Glu Ala Glu Ser Ala Ala Lys Val Thr Gly Met Leu Leu Glu Met Asp385 390 395 400Gln Thr Glu Val Leu His Leu Leu Glu Ser Pro Glu Ala Leu Lys Ala 405 410 415Lys Val Ala Glu Ala Met Asp Val Leu Arg Ser Val Ala Ala Gly Gly 420 425 430Ala Thr Glu Gln Leu Ala Ser Leu Asn Leu Ser 435 44031305DNAArabidopsis thaliana 3atggcagacg tcaaggttca atccgaatcc gaatcctcgg attctcatcc cttggtcgac 60tatcaatcac ttccacctta tcctccgccg catccaccgg ttgaagtaga ggagaatcaa 120ccaaaaacat ctccgactcc gccgccgcca cactggatgc gttatccacc ggtgttaatg 180cctcagatga tgtacgcgcc gccgccaccg atgccgttct caccttatca tcaatatccg 240aatcaccacc actttcacca tcaatctcgt ggtaataagc atcaaaacgc ttttaatggt 300gagaataaaa ctatttgggt tggtgatttg caaaactgga tggatgaggc ttatcttaat 360tctgctttta cttccgccga agagagagag attgtttcgc tgaaggtgat tcgtaataag 420cacaatggtt catcggaagg atatggattt gtggagtttg agtcccatga tgtagctgat 480aaggttttgc aggagtttaa cggggcgcct atgccaaata ctgaccaacc ttttcgtttg 540aactgggcta gttttagcac cggtgagaag cggttagaga acaatggacc tgatctctct 600atatttgttg gggatttggc gccagatgtt tcggatgctt tgttgcacga gaccttctct 660gagaagtatc cgtcggttaa agctgccaaa gttgtccttg atgctaatac tggtagatca 720aaggggtatg ggtttgtgag gtttggagat gagaatgaaa ggaccaaagc aatgactgag 780atgaatggtg ttaaatgctc tagtagagct atgcgtatcg gtcctgctac cccaaggaaa 840actaatggtt atcaacaaca aggtggatac atgccgagtg gtgcctttac gcgttctgaa 900ggggacacaa tcaacacaac aatatttgtt ggagggcttg actctagtgt cactgatgaa 960gacttaaagc aacctttctc tgaattcggg gaaatagtgt ctgtcaagat tcctgttggt 1020aaaggatgcg gatttgttca gtttgttaac agaccaaatg cagaggaggc tttggaaaaa 1080ctcaatggga ctgtaattgg caaacaaaca gtccggcttt cttggggccg taatccagcc 1140aataagcagc ctagagataa gtatggaaac caatgggttg atccgtacta tggaggacag 1200ttttacaatg ggtatggata catggtacct caacctgacc cgagaatgta tcctgctgca 1260ccttactatc caatgtacgg tggtcatcag caacaagtta gctga 13054434PRTArabidopsis thaliana 4Met Ala Asp Val Lys Val Gln Ser Glu Ser Glu Ser Ser Asp Ser His1 5 10 15Pro Leu Val Asp Tyr Gln Ser Leu Pro Pro Tyr Pro Pro Pro His Pro 20 25 30Pro Val Glu Val Glu Glu Asn Gln Pro Lys Thr Ser Pro Thr Pro Pro 35 40 45Pro Pro His Trp Met Arg Tyr Pro Pro Val Leu Met Pro Gln Met Met 50 55 60Tyr Ala Pro Pro Pro Pro Met Pro Phe Ser Pro Tyr His Gln Tyr Pro65 70 75 80Asn His His His Phe His His Gln Ser Arg Gly Asn Lys His Gln Asn 85 90 95Ala Phe Asn Gly Glu Asn Lys Thr Ile Trp Val Gly Asp Leu Gln Asn 100 105 110Trp Met Asp Glu Ala Tyr Leu Asn Ser Ala Phe Thr Ser Ala Glu Glu 115 120 125Arg Glu Ile Val Ser Leu Lys Val Ile Arg Asn Lys His Asn Gly Ser 130 135 140Ser Glu Gly Tyr Gly Phe Val Glu Phe Glu Ser His Asp Val Ala Asp145 150 155 160Lys Val Leu Gln Glu Phe Asn Gly Ala Pro Met Pro Asn Thr Asp Gln 165 170 175Pro Phe Arg Leu Asn Trp Ala Ser Phe Ser Thr Gly Glu Lys Arg Leu 180 185 190Glu Asn Asn Gly Pro Asp Leu Ser Ile Phe Val Gly Asp Leu Ala Pro 195 200 205Asp Val Ser Asp Ala Leu Leu His Glu Thr Phe Ser Glu Lys Tyr Pro 210 215 220Ser Val Lys Ala Ala Lys Val Val Leu Asp Ala Asn Thr Gly Arg Ser225 230 235 240Lys Gly Tyr Gly Phe Val Arg Phe Gly Asp Glu Asn Glu Arg Thr Lys 245 250 255Ala Met Thr Glu Met Asn Gly Val Lys Cys Ser Ser Arg Ala Met Arg 260 265 270Ile Gly Pro Ala Thr Pro Arg Lys Thr Asn Gly Tyr Gln Gln Gln Gly 275 280 285Gly Tyr Met Pro Ser Gly Ala Phe Thr Arg Ser Glu Gly Asp Thr Ile 290 295 300Asn Thr Thr Ile Phe Val Gly Gly Leu Asp Ser Ser Val Thr Asp Glu305 310 315 320Asp Leu Lys Gln Pro Phe Ser Glu Phe Gly Glu Ile Val Ser Val Lys 325 330 335Ile Pro Val Gly Lys Gly Cys Gly Phe Val Gln Phe Val Asn Arg Pro 340 345 350Asn Ala Glu Glu Ala Leu Glu Lys Leu Asn Gly Thr Val Ile Gly Lys 355 360 365Gln Thr Val Arg Leu Ser Trp Gly Arg Asn Pro Ala Asn Lys Gln Pro 370 375 380Arg Asp Lys Tyr Gly Asn Gln Trp Val Asp Pro Tyr Tyr Gly Gly Gln385 390 395 400Phe Tyr Asn Gly Tyr Gly Tyr Met Val Pro Gln Pro Asp Pro Arg Met 405 410 415Tyr Pro Ala Ala Pro Tyr Tyr Pro Met Tyr Gly Gly His Gln Gln Gln 420 425 430Val Ser5369DNAArabidopsis thaliana 5atggcgtatg aaccgatgaa gcccacgaaa gctggtttgg aggctcctct ggagcagatt 60cataagatca ggatcactct ctcttcaaaa aatgtgaaga acttggaaaa agtgtgcact 120gatttggtcc gtggagctaa ggataagaga cttagagtta agggaccagt gagaatgccc 180actaaggttc ttaagatcac taccagaaag gcaccttgtg gtgaaggtac caatacttgg 240gacaggtttg agctcagggt tcacaagcgt gtcatcgatc tcttcagctc ccctgacgtt 300gttaagcaaa tcacgtctat caccattgag cccggtgttg aggtcgaggt cactattgct 360gactcttag 3696122PRTArabidopsis thaliana 6Met Ala Tyr Glu Pro Met Lys Pro Thr Lys Ala Gly Leu Glu Ala Pro1 5 10 15Leu Glu Gln Ile His Lys Ile Arg Ile Thr Leu Ser Ser Lys Asn Val 20 25 30Lys Asn Leu Glu Lys Val Cys Thr Asp Leu Val Arg Gly Ala Lys Asp 35 40 45Lys Arg Leu Arg Val Lys Gly Pro Val Arg Met Pro Thr Lys Val Leu 50 55 60Lys Ile Thr Thr Arg Lys Ala Pro Cys Gly Glu Gly Thr Asn Thr Trp65 70 75 80Asp Arg Phe Glu Leu Arg Val His Lys Arg Val Ile Asp Leu Phe Ser 85 90 95Ser Pro Asp Val Val Lys Gln Ile Thr Ser Ile Thr Ile Glu Pro Gly 100 105 110Val Glu Val Glu Val Thr Ile Ala Asp Ser 115 1207741DNAArabidopsis thaliana 7atggggagaa gaccatgctg tgagaagata ggattgaaga aagggccatg gagtgctgaa 60gaagatcgaa tcttgatcaa ttatattagt ctccatggcc atcccaattg gagagctctc 120cctaaactag ccgggctact tcggtgcgga aaaagttgca ggcttcgttg gattaattat 180ttgagaccag acatcaaacg tggcaatttc actcctcatg aagaagatac tatcatcagc 240ttacatcaac tcttaggcaa cagatggtct gcgatagctg caaaattgcc tggacgaaca 300gacaacgaaa ttaaaaatgt ttggcacact catttaaaga aaagactcca ccacagtcaa 360gatcaaaaca acaaggaaga tttcgtctct actacagctg cggagatgcc aacctctccg 420caacaacaat ctagtagtag tgccgacatt tcagcaatta caacattggg aaacaacaat 480gacatctcca atagcaacaa agactccgcg acgtcatccg aagatgttct tgcaattata 540gatgagagct tttggtcaga agtggtattg atggactgtg acatttcagg aaatgagaag 600aatgagaaaa agatagagaa ttgggagggc tcactagata gaaacgataa gggatataac 660catgacatgg agttttggtt tgaccatctc actagtagta gttgtataat tggagaaatg 720tccgacattt ctgagttttg a 7418246PRTArabidopsis thaliana 8Met Gly Arg Arg Pro Cys Cys Glu Lys Ile Gly Leu Lys Lys Gly Pro1 5 10 15Trp Ser Ala Glu Glu Asp Arg Ile Leu Ile Asn Tyr Ile Ser Leu His 20 25 30Gly His Pro Asn Trp Arg Ala Leu Pro Lys Leu Ala Gly Leu Leu Arg 35 40 45Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp 50 55 60Ile Lys Arg Gly Asn Phe Thr Pro His Glu Glu Asp Thr Ile Ile Ser65 70 75 80Leu His Gln Leu Leu Gly Asn Arg Trp Ser Ala Ile Ala Ala Lys Leu 85 90 95Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Val Trp His Thr His Leu 100 105 110Lys Lys Arg Leu His His Ser Gln Asp Gln Asn Asn Lys Glu Asp Phe 115 120 125Val Ser Thr Thr Ala Ala Glu Met Pro Thr Ser Pro Gln Gln Gln Ser 130 135 140Ser Ser Ser Ala Asp Ile Ser Ala Ile Thr Thr Leu Gly Asn Asn Asn145 150 155 160Asp Ile Ser Asn Ser Asn Lys Asp Ser Ala Thr Ser Ser Glu Asp Val 165 170 175Leu Ala Ile Ile Asp Glu Ser Phe Trp Ser Glu Val Val Leu Met Asp 180 185 190Cys Asp Ile Ser Gly Asn Glu Lys Asn Glu Lys Lys Ile Glu Asn Trp 195 200 205Glu Gly Ser Leu Asp Arg Asn Asp Lys Gly Tyr Asn His Asp Met Glu 210 215 220Phe Trp Phe Asp His Leu Thr Ser Ser Ser Cys Ile Ile Gly Glu Met225 230 235 240Ser Asp Ile Ser Glu Phe 24591125DNAArabidopsis thaliana 9atgggaagag caccgtgttg tgataaggcc aacgtgaaga aagggccttg gtctcctgag 60gaagacgcca aactcaaaga ttacatcgag aatagtggca caggaggcaa ctggattgct 120ttgcctcaga aaattggttt aaggagatgt gggaagagtt gcaggctaag gtggctcaac 180tatttgagac caaacatcaa acatggtggc ttctccgagg aagaagacaa catcatttgt 240aacctctatg ttactattgg tagcaggtgg tctataattg ctgcacaatt gccgggaaga 300accgacaacg atatcaaaaa ctattggaac acgaggctga agaagaagct tctgaacaaa 360caaaggaaag agttccaaga agcgcgaatg aagcaagaga tggtgatgat gaaaaggcaa 420caacaaggac aaggacaagg tcaaagtaat ggtagtacgg atctttatct taacaacatg 480tttggatcat caccatggcc attactacca caacttcctc ctccacatca tcaaatacct 540cttggaatga tggaaccaac aagctgtaac tactaccaaa cgacaccgtc ttgtaaccta 600gaacaaaagc cattgatcac actcaagaac atggtcaaga ttgaagaaga acaggaaagg 660acaaaccctg atcatcatca tcaagattct gtcacaaacc cttttgattt ctctttctct 720cagcttttgt tagatcccaa ttactatctg ggatcaggag ggggaggaga aggagatttt 780gctatcatga gcagcagcac aaactcacca ttaccaaaca caagtagtga tcaacatcca 840agtcaacagc aagagattct tcaatggttt gggagcagta actttcagac agaagcaatc 900aacgatatgt tcataaacaa caacaacaac atagtgaatc ttgagaccat cgagaacaca 960aaagtctatg gagacgcctc agtagccgga gccgctgtcc gagcagcttt gggcggaggg 1020acaacgagta catcggcgga tcaaagtaca ataagttggg aggatataac ttctctagtt 1080aattccgaag atgcaagtta cttcaatgcg ccaaatcatg tgtaa 112510374PRTArabidopsis thaliana 10Met Gly Arg Ala Pro Cys Cys Asp Lys Ala Asn Val Lys Lys Gly Pro1 5 10 15Trp Ser Pro Glu Glu Asp Ala Lys Leu Lys Asp Tyr Ile Glu Asn Ser 20 25 30Gly Thr Gly Gly Asn Trp Ile Ala Leu Pro Gln Lys Ile Gly Leu Arg 35 40 45Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Leu Asn Tyr Leu Arg Pro 50 55 60Asn Ile Lys His Gly Gly Phe Ser Glu Glu Glu Asp Asn Ile Ile Cys65 70 75 80Asn Leu Tyr Val Thr Ile Gly Ser Arg Trp Ser Ile Ile Ala Ala Gln 85 90 95Leu Pro Gly Arg Thr Asp Asn Asp Ile Lys Asn Tyr Trp Asn Thr Arg 100 105 110Leu Lys Lys Lys Leu Leu Asn Lys Gln Arg Lys Glu Phe Gln Glu Ala 115 120 125Arg Met Lys Gln Glu Met Val Met Met Lys Arg Gln Gln Gln Gly Gln 130 135 140Gly Gln Gly Gln Ser Asn Gly Ser Thr Asp Leu Tyr Leu Asn Asn Met145 150 155 160Phe Gly Ser Ser Pro Trp Pro Leu Leu Pro Gln Leu Pro Pro Pro His 165 170 175His Gln Ile Pro Leu Gly Met Met Glu Pro Thr Ser Cys Asn Tyr Tyr 180 185 190Gln Thr Thr Pro Ser Cys Asn Leu Glu Gln Lys Pro Leu Ile Thr Leu 195 200 205Lys Asn Met Val Lys Ile Glu Glu Glu Gln Glu Arg Thr Asn Pro Asp 210 215 220His His His Gln Asp Ser Val Thr Asn Pro Phe Asp Phe Ser Phe Ser225 230 235 240Gln Leu Leu Leu Asp Pro Asn Tyr Tyr Leu Gly Ser Gly Gly Gly Gly 245 250 255Glu Gly Asp Phe Ala Ile Met Ser Ser Ser Thr Asn Ser Pro Leu Pro 260 265 270Asn Thr Ser Ser Asp Gln His Pro Ser Gln Gln Gln Glu Ile Leu Gln 275 280 285Trp Phe Gly Ser Ser Asn Phe Gln Thr Glu Ala Ile Asn Asp Met Phe 290 295 300Ile Asn Asn Asn Asn Asn Ile Val Asn Leu Glu Thr Ile Glu Asn Thr305 310 315 320Lys Val Tyr Gly Asp Ala Ser Val Ala Gly Ala Ala Val Arg Ala Ala 325 330 335Leu Gly Gly Gly Thr Thr Ser Thr Ser Ala Asp Gln Ser Thr Ile Ser 340 345 350Trp Glu Asp Ile Thr Ser Leu Val Asn Ser Glu Asp Ala Ser

Tyr Phe 355 360 365Asn Ala Pro Asn His Val 370111164DNAArabidopsis thaliana 11atgcagcaac caccgtcaaa cgccgccgga gctggacaga taccatcagg acaacagcat 60ttgtggatga tgatgcaaca gcagcagcag cagcagcaga tgcagttgtc tgcggcgcca 120ctaggtcaac atcagtacgg tattggatct cagaatccag gatccgctag cgatgttaag 180tcgttgtgga tcggagactt gcagcaatgg atggacgaga actacatcat gagcgtcttt 240gctcagtctg gcgaggctac atcagctaaa gtcattcgta ataagctgac gggacaatct 300gaaggttatg gattcattga gttcgtcagc cactctgtag cagagcgggt tttgcagact 360tacaatggtg ctcccatgcc gagcactgaa cagacgttta ggctcaactg ggctcaggct 420ggggctggag agaaacgatt ccagactgaa gggcctgacc ataccatttt cgtaggtgac 480ttggcacctg aggtgactga ctatatgctc tcggacacat tcaagaatgt gtatgggtct 540gtcaaagggg ctaaagttgt gcttgacagg accactggaa ggtccaaggg gtatgggttt 600gttaggtttg cggatgaaaa tgagcagatg cgtgccatga ctgaaatgaa tggtcaatac 660tgctcgacaa ggcctatgcg tattggtccg gctgccaata agaatgctct tccgatgcaa 720ccagctatgt atcaaaacac tcaaggagca aatgctggag ataatgatcc taataacaca 780acaatttttg ttggaggtct ggatgctaat gttacagacg atgaattaaa gtcaattttt 840ggtcaatttg gtgaacttct tcatgtgaaa atacctccag gaaaacgttg tggattcgtt 900caatatgcca acaaggcgtc tgcagagcat gcactttcgg tgctgaatgg aacacaatta 960ggtggacaaa gcatccgtct ttcgtgggga cgtagtccaa acaagcagtc tgatcaagcg 1020caatggaacg gtggtggata ctatggatac cctccacagc cacagggcgg ctatggttat 1080gcagctcaac caccaactca agaccctaat gcgtactatg gtggttacac tggctatggc 1140aactatcagc agcaacgtca gtga 116412387PRTArabidopsis thaliana 12Met Gln Gln Pro Pro Ser Asn Ala Ala Gly Ala Gly Gln Ile Pro Ser1 5 10 15Gly Gln Gln His Leu Trp Met Met Met Gln Gln Gln Gln Gln Gln Gln 20 25 30Gln Met Gln Leu Ser Ala Ala Pro Leu Gly Gln His Gln Tyr Gly Ile 35 40 45Gly Ser Gln Asn Pro Gly Ser Ala Ser Asp Val Lys Ser Leu Trp Ile 50 55 60Gly Asp Leu Gln Gln Trp Met Asp Glu Asn Tyr Ile Met Ser Val Phe65 70 75 80Ala Gln Ser Gly Glu Ala Thr Ser Ala Lys Val Ile Arg Asn Lys Leu 85 90 95Thr Gly Gln Ser Glu Gly Tyr Gly Phe Ile Glu Phe Val Ser His Ser 100 105 110Val Ala Glu Arg Val Leu Gln Thr Tyr Asn Gly Ala Pro Met Pro Ser 115 120 125Thr Glu Gln Thr Phe Arg Leu Asn Trp Ala Gln Ala Gly Ala Gly Glu 130 135 140Lys Arg Phe Gln Thr Glu Gly Pro Asp His Thr Ile Phe Val Gly Asp145 150 155 160Leu Ala Pro Glu Val Thr Asp Tyr Met Leu Ser Asp Thr Phe Lys Asn 165 170 175Val Tyr Gly Ser Val Lys Gly Ala Lys Val Val Leu Asp Arg Thr Thr 180 185 190Gly Arg Ser Lys Gly Tyr Gly Phe Val Arg Phe Ala Asp Glu Asn Glu 195 200 205Gln Met Arg Ala Met Thr Glu Met Asn Gly Gln Tyr Cys Ser Thr Arg 210 215 220Pro Met Arg Ile Gly Pro Ala Ala Asn Lys Asn Ala Leu Pro Met Gln225 230 235 240Pro Ala Met Tyr Gln Asn Thr Gln Gly Ala Asn Ala Gly Asp Asn Asp 245 250 255Pro Asn Asn Thr Thr Ile Phe Val Gly Gly Leu Asp Ala Asn Val Thr 260 265 270Asp Asp Glu Leu Lys Ser Ile Phe Gly Gln Phe Gly Glu Leu Leu His 275 280 285Val Lys Ile Pro Pro Gly Lys Arg Cys Gly Phe Val Gln Tyr Ala Asn 290 295 300Lys Ala Ser Ala Glu His Ala Leu Ser Val Leu Asn Gly Thr Gln Leu305 310 315 320Gly Gly Gln Ser Ile Arg Leu Ser Trp Gly Arg Ser Pro Asn Lys Gln 325 330 335Ser Asp Gln Ala Gln Trp Asn Gly Gly Gly Tyr Tyr Gly Tyr Pro Pro 340 345 350Gln Pro Gln Gly Gly Tyr Gly Tyr Ala Ala Gln Pro Pro Thr Gln Asp 355 360 365Pro Asn Ala Tyr Tyr Gly Gly Tyr Thr Gly Tyr Gly Asn Tyr Gln Gln 370 375 380Gln Arg Gln385131218DNAArabidopsis thaliana 13atgatgcagc agccaccacc cggaggtatc cttccacatc acgctcctcc tccttctgcg 60caacaacagt acggttacca acaaccttac gggattgctg gagctgctcc accaccacca 120cagatgtgga atcctcaagc ggcggcgccg ccatcagttc agcctacgac cgctgacgag 180atccggactc tttggatcgg ggacttacag tattggatgg atgagaattt cctctacggt 240tgctttgctc ataccggaga gatggtttct gctaaagtga ttcgtaacaa gcaaaccggt 300caagttgaag gatacggttt cattgaattc gcatctcatg ctgctgctga aagagttcta 360caaacattca acaacgctcc tatcccgagc tttcctgatc agctctttag actgaactgg 420gcatcattga gttcaggaga taaacgagac gattcaccgg actacacgat atttgtcggt 480gatctggctg ctgatgttac ggattatatc ttacttgaga cgttcagagc ctcttatccg 540tcagtgaagg gtgcaaaggt tgttattgac agagtcactg gacgtacaaa aggatatggg 600tttgttaggt tttctgatga aagtgaacag atccgtgcta tgacggagat gaatggcgtt 660ccttgttcta ctagacctat gagaattggt cccgctgcta gcaagaaagg tgtaactggt 720caaagagatt cataccagag ctctgctgca ggggtaacaa ctgataatga tccaaataac 780acaactgttt ttgttggtgg attagatgca tctgtcacgg atgatcatct gaagaatgtc 840tttagccaat atggtgagat tgtgcatgtg aaaatacccg ctggaaagcg ctgtggattc 900gttcagtttt ccgagaagag ctgtgcagag gaagctctta gaatgctgaa tggagtgcaa 960ttaggcggaa caaccgtcag gctctcatgg ggccgaagtc cttcgaacaa acagtcgggg 1020gatccgagcc agttttacta cggtgggtat ggacaaggac aggagcagta tgggtacacg 1080atgcctcaag accctaatgc atattacgga ggctactctg gtggaggata cagcggtggt 1140taccagcaga caccacaggc aggacagcaa ccaccacaac agccaccaca gcagcaacaa 1200gtcgggttta gctactaa 121814405PRTArabidopsis thaliana 14Met Met Gln Gln Pro Pro Pro Gly Gly Ile Leu Pro His His Ala Pro1 5 10 15Pro Pro Ser Ala Gln Gln Gln Tyr Gly Tyr Gln Gln Pro Tyr Gly Ile 20 25 30Ala Gly Ala Ala Pro Pro Pro Pro Gln Met Trp Asn Pro Gln Ala Ala 35 40 45Ala Pro Pro Ser Val Gln Pro Thr Thr Ala Asp Glu Ile Arg Thr Leu 50 55 60Trp Ile Gly Asp Leu Gln Tyr Trp Met Asp Glu Asn Phe Leu Tyr Gly65 70 75 80Cys Phe Ala His Thr Gly Glu Met Val Ser Ala Lys Val Ile Arg Asn 85 90 95Lys Gln Thr Gly Gln Val Glu Gly Tyr Gly Phe Ile Glu Phe Ala Ser 100 105 110His Ala Ala Ala Glu Arg Val Leu Gln Thr Phe Asn Asn Ala Pro Ile 115 120 125Pro Ser Phe Pro Asp Gln Leu Phe Arg Leu Asn Trp Ala Ser Leu Ser 130 135 140Ser Gly Asp Lys Arg Asp Asp Ser Pro Asp Tyr Thr Ile Phe Val Gly145 150 155 160Asp Leu Ala Ala Asp Val Thr Asp Tyr Ile Leu Leu Glu Thr Phe Arg 165 170 175Ala Ser Tyr Pro Ser Val Lys Gly Ala Lys Val Val Ile Asp Arg Val 180 185 190Thr Gly Arg Thr Lys Gly Tyr Gly Phe Val Arg Phe Ser Asp Glu Ser 195 200 205Glu Gln Ile Arg Ala Met Thr Glu Met Asn Gly Val Pro Cys Ser Thr 210 215 220Arg Pro Met Arg Ile Gly Pro Ala Ala Ser Lys Lys Gly Val Thr Gly225 230 235 240Gln Arg Asp Ser Tyr Gln Ser Ser Ala Ala Gly Val Thr Thr Asp Asn 245 250 255Asp Pro Asn Asn Thr Thr Val Phe Val Gly Gly Leu Asp Ala Ser Val 260 265 270Thr Asp Asp His Leu Lys Asn Val Phe Ser Gln Tyr Gly Glu Ile Val 275 280 285His Val Lys Ile Pro Ala Gly Lys Arg Cys Gly Phe Val Gln Phe Ser 290 295 300Glu Lys Ser Cys Ala Glu Glu Ala Leu Arg Met Leu Asn Gly Val Gln305 310 315 320Leu Gly Gly Thr Thr Val Arg Leu Ser Trp Gly Arg Ser Pro Ser Asn 325 330 335Lys Gln Ser Gly Asp Pro Ser Gln Phe Tyr Tyr Gly Gly Tyr Gly Gln 340 345 350Gly Gln Glu Gln Tyr Gly Tyr Thr Met Pro Gln Asp Pro Asn Ala Tyr 355 360 365Tyr Gly Gly Tyr Ser Gly Gly Gly Tyr Ser Gly Gly Tyr Gln Gln Thr 370 375 380Pro Gln Ala Gly Gln Gln Pro Pro Gln Gln Pro Pro Gln Gln Gln Gln385 390 395 400Val Gly Phe Ser Tyr 405151248DNAArabidopsis thaliana 15atgatgcagc agccacctcc agcttccaac ggtgctgcaa cagggccagg gcagattcct 60tccgaccaac aagcttacct ccagcagcag cagtcgtgga tgatgcagca ccagcagcaa 120caacaaggtc agccgcctgc aggatggaat cagcagtctg caccgtcttc tggtcaacca 180cagcagcagc agtatggtgg tggtggatct cagaatccag gatcagctgg tgagatccgg 240tccctgtgga tcggtgactt gcagccatgg atggatgaga actatctcat gaacgtcttt 300ggtcttactg gcgaggctac agcagctaaa gttattcgca ataaacagaa cggatattca 360gaaggttatg gctttattga gtttgtgaac catgctacag ctgagaggaa tttacagact 420tacaatggtg ctccgatgcc gagcagtgag caggccttca ggttgaactg ggctcagctt 480ggagctggag agagacgcca ggctgaaggg cctgagcaca cagtttttgt tggagacttg 540gcacctgatg ttaccgacca catgcttact gaaacgttta aagctgtgta ttcctctgtc 600aagggagcta aagttgtgaa tgataggact actggacggt ccaagggtta tggatttgtc 660aggtttgcgg atgaaagtga gcagattcgt gccatgactg aaatgaatgg tcaatactgc 720tcatcaaggc ctatgcgtac tggtcctgct gccaacaaga agcctcttac aatgcaacca 780gcttcatatc agaacactca aggaaattca ggagaaagtg atccaactaa cacaacaatt 840tttgttggag ctgtggatca aagtgtaaca gaagatgatt tgaagtcagt ttttggtcaa 900tttggtgaac tagttcatgt gaaaataccc gcaggaaaac gttgcggatt tgttcaatac 960gccaataggg catgtgctga gcaagcactt tctgtgttga acggaacaca acttggggga 1020caaagcattc gtctttcatg gggtcgcagt ccttccaaca aacagactca acctgatcaa 1080gcccagtatg gtggtggtgg aggatactat gggtatcctc ctcaaggata tgaagcatac 1140ggatatgcac ctcctcctca ggaccctaac gcctactacg gtggttatgc tgggggcggc 1200tatggaaact accagcagcc tggtggatac cagcagcaac agcagtga 124816415PRTArabidopsis thaliana 16Met Met Gln Gln Pro Pro Pro Ala Ser Asn Gly Ala Ala Thr Gly Pro1 5 10 15Gly Gln Ile Pro Ser Asp Gln Gln Ala Tyr Leu Gln Gln Gln Gln Ser 20 25 30Trp Met Met Gln His Gln Gln Gln Gln Gln Gly Gln Pro Pro Ala Gly 35 40 45Trp Asn Gln Gln Ser Ala Pro Ser Ser Gly Gln Pro Gln Gln Gln Gln 50 55 60Tyr Gly Gly Gly Gly Ser Gln Asn Pro Gly Ser Ala Gly Glu Ile Arg65 70 75 80Ser Leu Trp Ile Gly Asp Leu Gln Pro Trp Met Asp Glu Asn Tyr Leu 85 90 95Met Asn Val Phe Gly Leu Thr Gly Glu Ala Thr Ala Ala Lys Val Ile 100 105 110Arg Asn Lys Gln Asn Gly Tyr Ser Glu Gly Tyr Gly Phe Ile Glu Phe 115 120 125Val Asn His Ala Thr Ala Glu Arg Asn Leu Gln Thr Tyr Asn Gly Ala 130 135 140Pro Met Pro Ser Ser Glu Gln Ala Phe Arg Leu Asn Trp Ala Gln Leu145 150 155 160Gly Ala Gly Glu Arg Arg Gln Ala Glu Gly Pro Glu His Thr Val Phe 165 170 175Val Gly Asp Leu Ala Pro Asp Val Thr Asp His Met Leu Thr Glu Thr 180 185 190Phe Lys Ala Val Tyr Ser Ser Val Lys Gly Ala Lys Val Val Asn Asp 195 200 205Arg Thr Thr Gly Arg Ser Lys Gly Tyr Gly Phe Val Arg Phe Ala Asp 210 215 220Glu Ser Glu Gln Ile Arg Ala Met Thr Glu Met Asn Gly Gln Tyr Cys225 230 235 240Ser Ser Arg Pro Met Arg Thr Gly Pro Ala Ala Asn Lys Lys Pro Leu 245 250 255Thr Met Gln Pro Ala Ser Tyr Gln Asn Thr Gln Gly Asn Ser Gly Glu 260 265 270Ser Asp Pro Thr Asn Thr Thr Ile Phe Val Gly Ala Val Asp Gln Ser 275 280 285Val Thr Glu Asp Asp Leu Lys Ser Val Phe Gly Gln Phe Gly Glu Leu 290 295 300Val His Val Lys Ile Pro Ala Gly Lys Arg Cys Gly Phe Val Gln Tyr305 310 315 320Ala Asn Arg Ala Cys Ala Glu Gln Ala Leu Ser Val Leu Asn Gly Thr 325 330 335Gln Leu Gly Gly Gln Ser Ile Arg Leu Ser Trp Gly Arg Ser Pro Ser 340 345 350Asn Lys Gln Thr Gln Pro Asp Gln Ala Gln Tyr Gly Gly Gly Gly Gly 355 360 365Tyr Tyr Gly Tyr Pro Pro Gln Gly Tyr Glu Ala Tyr Gly Tyr Ala Pro 370 375 380Pro Pro Gln Asp Pro Asn Ala Tyr Tyr Gly Gly Tyr Ala Gly Gly Gly385 390 395 400Tyr Gly Asn Tyr Gln Gln Pro Gly Gly Tyr Gln Gln Gln Gln Gln 405 410 415171278DNAArabidopsis thaliana 17atggcgatga tgcatcctcc gcagccgccg caaggctcct atcaccatcc tcagacgctc 60gaagaagttc gaactctttg gattggtgat ttgcagtact gggtcgacga aaattacctc 120acttcctgct tctcccaaac cggcgagctc gtttctgtca aggtaatacg taacaagatc 180acgggacagc cagaggggta tggttttata gagtttatat ctcatgcagc agcagagaga 240actctgcaga cgtacaatgg gacacagatg cctggaactg agttaacttt tcggttaaat 300tgggcttctt ttggttcagg acagaaagtt gatgctggac ctgatcattc tatctttgtt 360ggagatttag cacctgatgt tacagattat cttcttcaag agacattccg tgttcattat 420tcttctgtta gaggtgccaa ggttgttact gatccaagta ctggacgatc aaagggttat 480ggatttgtaa aatttgcaga ggaaagtgaa aggaatcggg ctatggctga aatgaatggt 540ttgtattgct caacaaggcc tatgcgtatt agcgcagcaa cacctaaaaa aaacgtcggt 600gtgcagcaac aatatgtcac caaagctgtt tacccagtta cagtcccatc tgcagttgct 660gcaccagtcc aagcatacgt tgctccacct gaaagtgatg tcacctgtac aacgatttca 720gttgccaatt tggaccaaaa tgttacagag gaagagctga agaaagcatt ctcccaatta 780ggagaggtta tttatgtcaa aatacctgca acaaagggat atggttatgt tcaattcaaa 840accaggcctt ctgcagaaga agctgttcaa agaatgcagg gacaagtgat tggtcaacaa 900gcagttcgca tctcttggag taaaaatcca ggacaggatg gttgggttac acaagcagat 960ccgaatcagt ggaatgggta ttatggttat gggcaaggct atgatgcata tgcttatggg 1020gcaactcaag atccatccgt gtacgcatat ggtggatatg gctatcccca gtatccgcaa 1080cagggagagg gtacacaaga catttcgaac tctgcggcgg gtggagtagc aggtgcagag 1140caagagttgt atgatcctct ggccactcct gatgtagaca agttaaatgc tgcttacctt 1200tcggttcatg caagtgccat attaggaagg ccaatgtggc agcggacctc atcgctcaca 1260tcacaattgg gcaaatga 127818425PRTArabidopsis thaliana 18Met Ala Met Met His Pro Pro Gln Pro Pro Gln Gly Ser Tyr His His1 5 10 15Pro Gln Thr Leu Glu Glu Val Arg Thr Leu Trp Ile Gly Asp Leu Gln 20 25 30Tyr Trp Val Asp Glu Asn Tyr Leu Thr Ser Cys Phe Ser Gln Thr Gly 35 40 45Glu Leu Val Ser Val Lys Val Ile Arg Asn Lys Ile Thr Gly Gln Pro 50 55 60Glu Gly Tyr Gly Phe Ile Glu Phe Ile Ser His Ala Ala Ala Glu Arg65 70 75 80Thr Leu Gln Thr Tyr Asn Gly Thr Gln Met Pro Gly Thr Glu Leu Thr 85 90 95Phe Arg Leu Asn Trp Ala Ser Phe Gly Ser Gly Gln Lys Val Asp Ala 100 105 110Gly Pro Asp His Ser Ile Phe Val Gly Asp Leu Ala Pro Asp Val Thr 115 120 125Asp Tyr Leu Leu Gln Glu Thr Phe Arg Val His Tyr Ser Ser Val Arg 130 135 140Gly Ala Lys Val Val Thr Asp Pro Ser Thr Gly Arg Ser Lys Gly Tyr145 150 155 160Gly Phe Val Lys Phe Ala Glu Glu Ser Glu Arg Asn Arg Ala Met Ala 165 170 175Glu Met Asn Gly Leu Tyr Cys Ser Thr Arg Pro Met Arg Ile Ser Ala 180 185 190Ala Thr Pro Lys Lys Asn Val Gly Val Gln Gln Gln Tyr Val Thr Lys 195 200 205Ala Val Tyr Pro Val Thr Val Pro Ser Ala Val Ala Ala Pro Val Gln 210 215 220Ala Tyr Val Ala Pro Pro Glu Ser Asp Val Thr Cys Thr Thr Ile Ser225 230 235 240Val Ala Asn Leu Asp Gln Asn Val Thr Glu Glu Glu Leu Lys Lys Ala 245 250 255Phe Ser Gln Leu Gly Glu Val Ile Tyr Val Lys Ile Pro Ala Thr Lys 260 265 270Gly Tyr Gly Tyr Val Gln Phe Lys Thr Arg Pro Ser Ala Glu Glu Ala 275 280 285Val Gln Arg Met Gln Gly Gln Val Ile Gly Gln Gln Ala Val Arg Ile 290 295 300Ser Trp Ser Lys Asn Pro Gly Gln Asp Gly Trp Val Thr Gln Ala Asp305 310 315 320Pro Asn Gln Trp Asn Gly Tyr Tyr Gly Tyr Gly Gln Gly Tyr Asp Ala 325 330 335Tyr Ala Tyr Gly Ala Thr Gln Asp Pro Ser Val Tyr Ala Tyr Gly Gly 340 345 350Tyr Gly Tyr Pro Gln Tyr Pro Gln Gln Gly Glu Gly Thr Gln Asp Ile 355 360 365Ser Asn Ser Ala Ala Gly Gly Val Ala Gly Ala Glu Gln Glu Leu Tyr 370 375 380Asp Pro Leu Ala Thr Pro Asp Val Asp Lys Leu

Asn Ala Ala Tyr Leu385 390 395 400Ser Val His Ala Ser Ala Ile Leu Gly Arg Pro Met Trp Gln Arg Thr 405 410 415Ser Ser Leu Thr Ser Gln Leu Gly Lys 420 425191338DNAArabidopsis thaliana 19atgcagacac caaacaacaa cggttcaaca gattcagtgt taccaccaac atcagccgga 60acaacaccac caccaccgtt gcagcaatca acaccaccac cgcagcagca acaacaacaa 120cagtggcaac aacaacaaca atggatggct gcgatgcagc aataccctgc agctgctatg 180gctatgatgc aacaacaaca gatgatgatg tatcctcacc ctcaatacgc tccttacaat 240caagctgctt atcaacagca tcctcagttt caatacgctg cttatcaaca gcagcagcag 300caacatcacc agagtcagca gcagccacgc ggtggatctg gtggtgatga tgtcaagact 360ctttgggttg gtgatcttct tcattggatg gatgagactt atctccatac ctgtttctct 420cacaccaatg aggtttcttc tgtgaaagtt atacgcaaca agcaaacttg tcaatctgaa 480ggatatgggt ttgttgagtt tctttcacgt tcagcagctg aggaagctct tcagagcttt 540agcggtgtta caatgccgaa cgcggaacag cctttccgtt taaactgggc atctttcagt 600actggtgaga aaagagcatc agagaatggt cctgacctat ccatatttgt tggagatttg 660gctccagatg tgagtgatgc tgtcttgctt gagacttttg ctggtagata tccatctgtc 720aaaggtgcta aagttgtgat tgattccaac actgggcgtt ccaaaggtta cgggtttgtt 780aggtttggtg atgagaatga gcgatcaaga gctatgacag aaatgaatgg tgctttctgt 840tcaagcaggc aaatgcgtgt tggtatcgca accccgaaaa gggctgctgc ttacggccaa 900caaaatggtt cacaagctct tacacttgct ggtggacatg gagggaatgg ttcaatgtct 960gatggagaat caaataactc aacaatattt gttggcggtc ttgatgctga tgttactgaa 1020gaagacctca tgcaaccttt ttccgatttt ggggaggttg tttcagtgaa gatcccagta 1080gggaaaggat gtggctttgt ccaatttgct aacaggcaaa gtgctgagga agccatcggg 1140aacttgaacg ggacagtcat tgggaagaac actgtccgcc tttcatgggg aagaagcccc 1200aacaaacagt ggagaagtga ctctggcaac caatggaatg gaggatattc aagaggtcaa 1260ggatacaaca atggatatgc caatcaggac tcaaacatgt acgctactgc agcggctgca 1320gtcccgggag cttcttga 133820445PRTArabidopsis thaliana 20Met Gln Thr Pro Asn Asn Asn Gly Ser Thr Asp Ser Val Leu Pro Pro1 5 10 15Thr Ser Ala Gly Thr Thr Pro Pro Pro Pro Leu Gln Gln Ser Thr Pro 20 25 30Pro Pro Gln Gln Gln Gln Gln Gln Gln Trp Gln Gln Gln Gln Gln Trp 35 40 45Met Ala Ala Met Gln Gln Tyr Pro Ala Ala Ala Met Ala Met Met Gln 50 55 60Gln Gln Gln Met Met Met Tyr Pro His Pro Gln Tyr Ala Pro Tyr Asn65 70 75 80Gln Ala Ala Tyr Gln Gln His Pro Gln Phe Gln Tyr Ala Ala Tyr Gln 85 90 95Gln Gln Gln Gln Gln His His Gln Ser Gln Gln Gln Pro Arg Gly Gly 100 105 110Ser Gly Gly Asp Asp Val Lys Thr Leu Trp Val Gly Asp Leu Leu His 115 120 125Trp Met Asp Glu Thr Tyr Leu His Thr Cys Phe Ser His Thr Asn Glu 130 135 140Val Ser Ser Val Lys Val Ile Arg Asn Lys Gln Thr Cys Gln Ser Glu145 150 155 160Gly Tyr Gly Phe Val Glu Phe Leu Ser Arg Ser Ala Ala Glu Glu Ala 165 170 175Leu Gln Ser Phe Ser Gly Val Thr Met Pro Asn Ala Glu Gln Pro Phe 180 185 190Arg Leu Asn Trp Ala Ser Phe Ser Thr Gly Glu Lys Arg Ala Ser Glu 195 200 205Asn Gly Pro Asp Leu Ser Ile Phe Val Gly Asp Leu Ala Pro Asp Val 210 215 220Ser Asp Ala Val Leu Leu Glu Thr Phe Ala Gly Arg Tyr Pro Ser Val225 230 235 240Lys Gly Ala Lys Val Val Ile Asp Ser Asn Thr Gly Arg Ser Lys Gly 245 250 255Tyr Gly Phe Val Arg Phe Gly Asp Glu Asn Glu Arg Ser Arg Ala Met 260 265 270Thr Glu Met Asn Gly Ala Phe Cys Ser Ser Arg Gln Met Arg Val Gly 275 280 285Ile Ala Thr Pro Lys Arg Ala Ala Ala Tyr Gly Gln Gln Asn Gly Ser 290 295 300Gln Ala Leu Thr Leu Ala Gly Gly His Gly Gly Asn Gly Ser Met Ser305 310 315 320Asp Gly Glu Ser Asn Asn Ser Thr Ile Phe Val Gly Gly Leu Asp Ala 325 330 335Asp Val Thr Glu Glu Asp Leu Met Gln Pro Phe Ser Asp Phe Gly Glu 340 345 350Val Val Ser Val Lys Ile Pro Val Gly Lys Gly Cys Gly Phe Val Gln 355 360 365Phe Ala Asn Arg Gln Ser Ala Glu Glu Ala Ile Gly Asn Leu Asn Gly 370 375 380Thr Val Ile Gly Lys Asn Thr Val Arg Leu Ser Trp Gly Arg Ser Pro385 390 395 400Asn Lys Gln Trp Arg Ser Asp Ser Gly Asn Gln Trp Asn Gly Gly Tyr 405 410 415Ser Arg Gly Gln Gly Tyr Asn Asn Gly Tyr Ala Asn Gln Asp Ser Asn 420 425 430Met Tyr Ala Thr Ala Ala Ala Ala Val Pro Gly Ala Ser 435 440 445211308DNAArabidopsis thaliana 21atgcagacaa ccaacggctc agattcgacg ttggcaactt ccggagccac accgccgaat 60caacaaaccc ctcctccacc tcagcagtgg cagcagcagc aacagcaaca gcaacagtgg 120atggctgcca tgcaatatcc accagcggcg gcgatgatga tgatgcagca gcaacagatg 180ctgatgtatc ctcatcaata tgttccgtat aatcaaggtc cttatcagca gcatcatcct 240cagcttcacc aatacgggtc ttatcaacag caccagcacc agcaacacaa ggctattgac 300cgtggatctg gagatgatgt caagactctt tgggttggtg atcttcttca ttggatggat 360gagacttatc tccattcttg cttttctcac accggcgagg tttcttctgt gaaagttata 420cgtaacaagc tcacttctca atcagaaggg tatgggtttg ttgagtttct ttcacgtgct 480gcagctgaag aagttcttca gaactatagt ggttcagtga tgccaaactc ggaccaaccc 540ttccgtataa actgggcatc ttttagtact ggtgaaaaaa gagcagtgga aaatggtcca 600gacctatctg tttttgtggg agacttgtct ccagatgtca ctgacgtttt attgcatgag 660accttttctg atagatatcc ttctgtcaaa agcgccaaag ttgtgattga ttccaacacc 720ggccggtcca aaggttatgg ttttgtgagg ttcggtgatg aaaatgagag gtcaagggct 780ttgacagaaa tgaatggagc ttactgttcg aacaggcaaa tgcgtgtagg tattgcaact 840cccaaaagag cgattgctaa tcagcaacaa cattcttcac aagctgtgat tctggctggt 900ggacatggat caaatggttc catgggttat ggctcgcagt ctgatggcga atcaactaac 960gcaacaatat ttgttggcgg cattgaccct gatgttattg atgaagacct cagacaacct 1020ttttcccagt ttggagaggt tgtttcagtg aagatcccag tagggaaagg atgtggattt 1080gtccaatttg ctgacaggaa gagtgctgaa gatgctatcg agagtttgaa cgggacagtc 1140atcggcaaga acactgtcag actctcctgg ggacgaagcc caaacaagca gtggagagga 1200gactcagggc agcagtggaa tggaggatac tcacgaggac atggttacaa caatggagga 1260ggatatgcta accaccacga ctccaacaac tatcatgggg agaattga 130822435PRTArabidopsis thaliana 22Met Gln Thr Thr Asn Gly Ser Asp Ser Thr Leu Ala Thr Ser Gly Ala1 5 10 15Thr Pro Pro Asn Gln Gln Thr Pro Pro Pro Pro Gln Gln Trp Gln Gln 20 25 30Gln Gln Gln Gln Gln Gln Gln Trp Met Ala Ala Met Gln Tyr Pro Pro 35 40 45Ala Ala Ala Met Met Met Met Gln Gln Gln Gln Met Leu Met Tyr Pro 50 55 60His Gln Tyr Val Pro Tyr Asn Gln Gly Pro Tyr Gln Gln His His Pro65 70 75 80Gln Leu His Gln Tyr Gly Ser Tyr Gln Gln His Gln His Gln Gln His 85 90 95Lys Ala Ile Asp Arg Gly Ser Gly Asp Asp Val Lys Thr Leu Trp Val 100 105 110Gly Asp Leu Leu His Trp Met Asp Glu Thr Tyr Leu His Ser Cys Phe 115 120 125Ser His Thr Gly Glu Val Ser Ser Val Lys Val Ile Arg Asn Lys Leu 130 135 140Thr Ser Gln Ser Glu Gly Tyr Gly Phe Val Glu Phe Leu Ser Arg Ala145 150 155 160Ala Ala Glu Glu Val Leu Gln Asn Tyr Ser Gly Ser Val Met Pro Asn 165 170 175Ser Asp Gln Pro Phe Arg Ile Asn Trp Ala Ser Phe Ser Thr Gly Glu 180 185 190Lys Arg Ala Val Glu Asn Gly Pro Asp Leu Ser Val Phe Val Gly Asp 195 200 205Leu Ser Pro Asp Val Thr Asp Val Leu Leu His Glu Thr Phe Ser Asp 210 215 220Arg Tyr Pro Ser Val Lys Ser Ala Lys Val Val Ile Asp Ser Asn Thr225 230 235 240Gly Arg Ser Lys Gly Tyr Gly Phe Val Arg Phe Gly Asp Glu Asn Glu 245 250 255Arg Ser Arg Ala Leu Thr Glu Met Asn Gly Ala Tyr Cys Ser Asn Arg 260 265 270Gln Met Arg Val Gly Ile Ala Thr Pro Lys Arg Ala Ile Ala Asn Gln 275 280 285Gln Gln His Ser Ser Gln Ala Val Ile Leu Ala Gly Gly His Gly Ser 290 295 300Asn Gly Ser Met Gly Tyr Gly Ser Gln Ser Asp Gly Glu Ser Thr Asn305 310 315 320Ala Thr Ile Phe Val Gly Gly Ile Asp Pro Asp Val Ile Asp Glu Asp 325 330 335Leu Arg Gln Pro Phe Ser Gln Phe Gly Glu Val Val Ser Val Lys Ile 340 345 350Pro Val Gly Lys Gly Cys Gly Phe Val Gln Phe Ala Asp Arg Lys Ser 355 360 365Ala Glu Asp Ala Ile Glu Ser Leu Asn Gly Thr Val Ile Gly Lys Asn 370 375 380Thr Val Arg Leu Ser Trp Gly Arg Ser Pro Asn Lys Gln Trp Arg Gly385 390 395 400Asp Ser Gly Gln Gln Trp Asn Gly Gly Tyr Ser Arg Gly His Gly Tyr 405 410 415Asn Asn Gly Gly Gly Tyr Ala Asn His His Asp Ser Asn Asn Tyr His 420 425 430Gly Glu Asn 435231299DNAArabidopsis thaliana 23atggcagacg tcaagattca atccgaatcc gaatcctcgg attctcatcc agtggtcgac 60aatcaaccac ctcctccgcc tccgccgccg caacagccgg cgaaagaaga ggagaatcaa 120ccaaaaacat ctccgactcc gccgccacac tggatgcggt atccaccaac ggtgataatc 180cctcatcaga tgatgtacgc gccgccgccg ttcccacctt atcatcagta tccgaatcac 240caccaccttc accatcaatc tcgtggtaat aagcatcaaa acgcttttaa tggtgagaat 300aaaaccatat gggttggtga tttgcatcac tggatggatg aggcttatct taattcttct 360tttgcttccg gcgacgagag agagattgtt tcggtgaagg tgattcgtaa taagaacaat 420ggtttatcag aaggatatgg atttgtggag tttgagtccc atgatgtagc tgataaggtt 480ttgcgggagt ttaacgggac gactatgcca aatactgacc aaccttttcg tttgaactgg 540gctagtttta gcaccggtga gaagcggtta gagaacaatg gacctgatct ctctattttc 600gtgggggatt tgtcaccaga tgtttcggat aatttgttgc acgagacctt ctctgagaag 660tatccgtcgg ttaaagctgc gaaagttgtc cttgatgcta atactggtag gtcaaagggg 720tatgggtttg tgaggtttgg tgatgagaat gaaaggacca aagcaatgac tgagatgaat 780ggtgttaaat gttctagtag agctatgcgc atcggtcctg ctaccccgag gaagactaat 840ggttatcaac aacaaggtgg atacatgccg aatggtacct tgacgcgtcc tgaaggggac 900ataatgaaca caacaatatt tgttggaggg cttgactcta gtgtcactga tgaagactta 960aagcaacctt tcaatgaatt cggggaaata gtctctgtca agattcctgt tggtaaagga 1020tgcggatttg ttcagtttgt taacagacca aatgcagagg aggctttgga gaaactaaat 1080gggactgtaa ttggaaaaca aacagttcgg ctttcttggg gacgtaatcc cgccaataag 1140cagcctagag ataagtatgg aaaccaatgg gttgatccgt actatggagg acagttttac 1200aatgggtatg gatacatggt acctcaacct gacccgagaa tgtatcccgc tgcaccttac 1260tatccaatgt acggtggtca tcagcaacaa gttagctga 129924432PRTArabidopsis thaliana 24Met Ala Asp Val Lys Ile Gln Ser Glu Ser Glu Ser Ser Asp Ser His1 5 10 15Pro Val Val Asp Asn Gln Pro Pro Pro Pro Pro Pro Pro Pro Gln Gln 20 25 30Pro Ala Lys Glu Glu Glu Asn Gln Pro Lys Thr Ser Pro Thr Pro Pro 35 40 45Pro His Trp Met Arg Tyr Pro Pro Thr Val Ile Ile Pro His Gln Met 50 55 60Met Tyr Ala Pro Pro Pro Phe Pro Pro Tyr His Gln Tyr Pro Asn His65 70 75 80His His Leu His His Gln Ser Arg Gly Asn Lys His Gln Asn Ala Phe 85 90 95Asn Gly Glu Asn Lys Thr Ile Trp Val Gly Asp Leu His His Trp Met 100 105 110Asp Glu Ala Tyr Leu Asn Ser Ser Phe Ala Ser Gly Asp Glu Arg Glu 115 120 125Ile Val Ser Val Lys Val Ile Arg Asn Lys Asn Asn Gly Leu Ser Glu 130 135 140Gly Tyr Gly Phe Val Glu Phe Glu Ser His Asp Val Ala Asp Lys Val145 150 155 160Leu Arg Glu Phe Asn Gly Thr Thr Met Pro Asn Thr Asp Gln Pro Phe 165 170 175Arg Leu Asn Trp Ala Ser Phe Ser Thr Gly Glu Lys Arg Leu Glu Asn 180 185 190Asn Gly Pro Asp Leu Ser Ile Phe Val Gly Asp Leu Ser Pro Asp Val 195 200 205Ser Asp Asn Leu Leu His Glu Thr Phe Ser Glu Lys Tyr Pro Ser Val 210 215 220Lys Ala Ala Lys Val Val Leu Asp Ala Asn Thr Gly Arg Ser Lys Gly225 230 235 240Tyr Gly Phe Val Arg Phe Gly Asp Glu Asn Glu Arg Thr Lys Ala Met 245 250 255Thr Glu Met Asn Gly Val Lys Cys Ser Ser Arg Ala Met Arg Ile Gly 260 265 270Pro Ala Thr Pro Arg Lys Thr Asn Gly Tyr Gln Gln Gln Gly Gly Tyr 275 280 285Met Pro Asn Gly Thr Leu Thr Arg Pro Glu Gly Asp Ile Met Asn Thr 290 295 300Thr Ile Phe Val Gly Gly Leu Asp Ser Ser Val Thr Asp Glu Asp Leu305 310 315 320Lys Gln Pro Phe Asn Glu Phe Gly Glu Ile Val Ser Val Lys Ile Pro 325 330 335Val Gly Lys Gly Cys Gly Phe Val Gln Phe Val Asn Arg Pro Asn Ala 340 345 350Glu Glu Ala Leu Glu Lys Leu Asn Gly Thr Val Ile Gly Lys Gln Thr 355 360 365Val Arg Leu Ser Trp Gly Arg Asn Pro Ala Asn Lys Gln Pro Arg Asp 370 375 380Lys Tyr Gly Asn Gln Trp Val Asp Pro Tyr Tyr Gly Gly Gln Phe Tyr385 390 395 400Asn Gly Tyr Gly Tyr Met Val Pro Gln Pro Asp Pro Arg Met Tyr Pro 405 410 415Ala Ala Pro Tyr Tyr Pro Met Tyr Gly Gly His Gln Gln Gln Val Ser 420 425 430251281DNAArabidopsis thaliana 25atgcagaatc aaaggcttat taagcagcaa caacaacaac aacaacagca acatcaacaa 60gctatgattc aacaagctat gatgcaacaa catccttctc tttatcatcc tggtgttatg 120gctcctcctc agatggagcc tttaccaagt ggaaaccttc ctcctggttt tgatccaact 180acttgccgta gtgtgtatgc tggaaacatt catacgcagg tcacagagat tcttcttcaa 240gagatttttg caagtactgg tcctattgaa agctgtaaac tcatcagaaa ggataagtca 300tcatatggat ttgttcacta ctttgatcga agatgtgcta gtatggctat aatgactctt 360aacggaaggc atatatttgg acagcctatg aaagttaatt gggcgtatgc aactggtcaa 420agggaagata catcaagtca tttcaacatt tttgttggag atcttagtcc agaggttact 480gatgcagcat tgtttgatag cttttctgct tttaacagct gctcggacgc aagagtaatg 540tgggaccaga aaactggacg ctcaagaggc tttggttttg tttccttccg taatcagcag 600gatgctcaaa ctgccataaa tgagatgaat ggtaaatggg taagtagcag acagatcaga 660tgcaactggg cgacaaaagg tgctactttt ggcgaggaca aacatagctc tgatggaaaa 720agtgttgtag aacttactaa cggatcttca gaggatggta gagagctgtc aaatgaagat 780gcccctgaaa acaatcctca atttacaact gtctatgtag gaaatctctc tccagaagta 840actcagcttg atctacaccg tctattctat acccttggtg ctggagtgat cgaagaggtc 900cgtgtccagc gagacaaagg gtttggtttt gtgagatata acactcatga cgaggctgct 960cttgctattc agatgggcaa cgctcagcct ttcctcttta gcagacagat aaggtgttcc 1020tggggaaaca aaccaactcc atcaggcaca gcctcaaacc cacttccccc accagccccg 1080gcatcagtcc cttctctgtc tgcaatggac ctcttagcct acgagaggca actggctcta 1140gccaagatgc atcctcaggc tcaacattct ctgaggcaag caggtcttgg agtcaatgtt 1200gctggaggaa ctgcagctat gtatgatggt ggctatcaga atgtagctgc ggcccatcag 1260cagctcatgt actatcagta a 128126426PRTArabidopsis thaliana 26Met Gln Asn Gln Arg Leu Ile Lys Gln Gln Gln Gln Gln Gln Gln Gln1 5 10 15Gln His Gln Gln Ala Met Ile Gln Gln Ala Met Met Gln Gln His Pro 20 25 30Ser Leu Tyr His Pro Gly Val Met Ala Pro Pro Gln Met Glu Pro Leu 35 40 45Pro Ser Gly Asn Leu Pro Pro Gly Phe Asp Pro Thr Thr Cys Arg Ser 50 55 60Val Tyr Ala Gly Asn Ile His Thr Gln Val Thr Glu Ile Leu Leu Gln65 70 75 80Glu Ile Phe Ala Ser Thr Gly Pro Ile Glu Ser Cys Lys Leu Ile Arg 85 90 95Lys Asp Lys Ser Ser Tyr Gly Phe Val His Tyr Phe Asp Arg Arg Cys 100 105 110Ala Ser Met Ala Ile Met Thr Leu Asn Gly Arg His Ile Phe Gly Gln 115 120 125Pro Met Lys Val Asn Trp Ala Tyr Ala Thr Gly Gln Arg Glu Asp Thr 130 135 140Ser Ser His Phe Asn Ile Phe Val Gly Asp Leu Ser Pro Glu Val Thr145 150 155 160Asp Ala Ala Leu Phe Asp Ser Phe Ser Ala Phe Asn Ser Cys Ser Asp 165 170 175Ala Arg Val Met Trp Asp Gln Lys Thr Gly Arg Ser Arg Gly Phe Gly 180 185 190Phe Val Ser Phe Arg Asn Gln Gln Asp Ala Gln Thr Ala Ile Asn Glu 195 200 205Met Asn Gly Lys Trp Val Ser

Ser Arg Gln Ile Arg Cys Asn Trp Ala 210 215 220Thr Lys Gly Ala Thr Phe Gly Glu Asp Lys His Ser Ser Asp Gly Lys225 230 235 240Ser Val Val Glu Leu Thr Asn Gly Ser Ser Glu Asp Gly Arg Glu Leu 245 250 255Ser Asn Glu Asp Ala Pro Glu Asn Asn Pro Gln Phe Thr Thr Val Tyr 260 265 270Val Gly Asn Leu Ser Pro Glu Val Thr Gln Leu Asp Leu His Arg Leu 275 280 285Phe Tyr Thr Leu Gly Ala Gly Val Ile Glu Glu Val Arg Val Gln Arg 290 295 300Asp Lys Gly Phe Gly Phe Val Arg Tyr Asn Thr His Asp Glu Ala Ala305 310 315 320Leu Ala Ile Gln Met Gly Asn Ala Gln Pro Phe Leu Phe Ser Arg Gln 325 330 335Ile Arg Cys Ser Trp Gly Asn Lys Pro Thr Pro Ser Gly Thr Ala Ser 340 345 350Asn Pro Leu Pro Pro Pro Ala Pro Ala Ser Val Pro Ser Leu Ser Ala 355 360 365Met Asp Leu Leu Ala Tyr Glu Arg Gln Leu Ala Leu Ala Lys Met His 370 375 380Pro Gln Ala Gln His Ser Leu Arg Gln Ala Gly Leu Gly Val Asn Val385 390 395 400Ala Gly Gly Thr Ala Ala Met Tyr Asp Gly Gly Tyr Gln Asn Val Ala 405 410 415Ala Ala His Gln Gln Leu Met Tyr Tyr Gln 420 425271260DNAArabidopsis thaliana 27atgcagaggt tgaagcagca gcagcagcag caacaagtta tgatgcagca agctcttatg 60cagcaacagt ctctctacca tcctggtctc cttgccccgc cacagataga accaatccca 120agtggaaatc tcccccctgg ttttgatcca agtacttgcc gcagtgtgta cgttggaaac 180atccatattc aggtgacgga acctctgctt caagaggttt ttgctggcac tggtcctgta 240gaaagctgta aactaattag gaaagaaaag tcttcttatg ggtttgtgca ctactttgat 300cgaagatcgg ctggtcttgc aatcctttct ctcaatggaa ggcatttgtt tgggcaacct 360atcaaggtta actgggctta tgcgagtggc cagagggagg atacatcaag tcacttcaat 420atatttgttg gggatttgag tccggaggtt actgatgcaa tgctgtttac ttgcttctct 480gtctacccga cttgctcgga tgcaagagtt atgtgggatc agaaaactgg gcgttcaaga 540ggatttggat ttgtttcctt ccgtaaccaa caggatgccc agactgcaat agatgagata 600actgggaaat ggcttggttc caggcagata cgttgcaact gggcgacaaa gggagccact 660tctggtgagg acaaacagag ctctgattcc aaaagcgtcg tggaacttac cagtggtgtc 720tcggaggatg gtaaagatac tactaatggt gaagctcctg agaacaatgc tcagtacaca 780actgtttacg tcggtaatct tgctccagag gtgtcccagg ttgatcttca ccgccacttc 840cattcccttg gtgctggggt catagaggaa gtccgtgttc aaagagacaa aggtttcgga 900tttgtgagat actctactca tgtagaggca gccctcgcta ttcagatggg aaacacacat 960tcctacctta gtggcaggca aatgaagtgt tcttggggaa gcaagccaac tccagcagga 1020acagcttcaa acccgcttcc tccaccagct cctgcaccaa tcccgggatt ctcagcgagt 1080gatctcttgg cttacgagag gcaactagcg atgagcaaga tggcaggaat gaatccgatg 1140atgcatcacc cgcagggaca acatgctttt aaacaagctg caatgggagc cactggttca 1200aaccaggcaa tatatgacgg tggttaccag aacgcgcagc agctcatgta ctaccagtaa 126028419PRTSaccharomyces cerevisiae 28Met Gln Arg Leu Lys Gln Gln Gln Gln Gln Gln Gln Val Met Met Gln1 5 10 15Gln Ala Leu Met Gln Gln Gln Ser Leu Tyr His Pro Gly Leu Leu Ala 20 25 30Pro Pro Gln Ile Glu Pro Ile Pro Ser Gly Asn Leu Pro Pro Gly Phe 35 40 45Asp Pro Ser Thr Cys Arg Ser Val Tyr Val Gly Asn Ile His Ile Gln 50 55 60Val Thr Glu Pro Leu Leu Gln Glu Val Phe Ala Gly Thr Gly Pro Val65 70 75 80Glu Ser Cys Lys Leu Ile Arg Lys Glu Lys Ser Ser Tyr Gly Phe Val 85 90 95His Tyr Phe Asp Arg Arg Ser Ala Gly Leu Ala Ile Leu Ser Leu Asn 100 105 110Gly Arg His Leu Phe Gly Gln Pro Ile Lys Val Asn Trp Ala Tyr Ala 115 120 125Ser Gly Gln Arg Glu Asp Thr Ser Ser His Phe Asn Ile Phe Val Gly 130 135 140Asp Leu Ser Pro Glu Val Thr Asp Ala Met Leu Phe Thr Cys Phe Ser145 150 155 160Val Tyr Pro Thr Cys Ser Asp Ala Arg Val Met Trp Asp Gln Lys Thr 165 170 175Gly Arg Ser Arg Gly Phe Gly Phe Val Ser Phe Arg Asn Gln Gln Asp 180 185 190Ala Gln Thr Ala Ile Asp Glu Ile Thr Gly Lys Trp Leu Gly Ser Arg 195 200 205Gln Ile Arg Cys Asn Trp Ala Thr Lys Gly Ala Thr Ser Gly Glu Asp 210 215 220Lys Gln Ser Ser Asp Ser Lys Ser Val Val Glu Leu Thr Ser Gly Val225 230 235 240Ser Glu Asp Gly Lys Asp Thr Thr Asn Gly Glu Ala Pro Glu Asn Asn 245 250 255Ala Gln Tyr Thr Thr Val Tyr Val Gly Asn Leu Ala Pro Glu Val Ser 260 265 270Gln Val Asp Leu His Arg His Phe His Ser Leu Gly Ala Gly Val Ile 275 280 285Glu Glu Val Arg Val Gln Arg Asp Lys Gly Phe Gly Phe Val Arg Tyr 290 295 300Ser Thr His Val Glu Ala Ala Leu Ala Ile Gln Met Gly Asn Thr His305 310 315 320Ser Tyr Leu Ser Gly Arg Gln Met Lys Cys Ser Trp Gly Ser Lys Pro 325 330 335Thr Pro Ala Gly Thr Ala Ser Asn Pro Leu Pro Pro Pro Ala Pro Ala 340 345 350Pro Ile Pro Gly Phe Ser Ala Ser Asp Leu Leu Ala Tyr Glu Arg Gln 355 360 365Leu Ala Met Ser Lys Met Ala Gly Met Asn Pro Met Met His His Pro 370 375 380Gln Gly Gln His Ala Phe Lys Gln Ala Ala Met Gly Ala Thr Gly Ser385 390 395 400Asn Gln Ala Ile Tyr Asp Gly Gly Tyr Gln Asn Ala Gln Gln Leu Met 405 410 415Tyr Tyr Gln291284DNAArabidopsis thaliana 29atgcagaatc cgagactgaa gcaacatcag cagcaacaac aacaacaagc tatgatgcag 60caacaagctc tgatgcagca acactctctt taccatcctg gtgttttggc tcctcctcag 120ttagagcctg ttccaagtgg aaaccttcct cctggttttg atcccagtac ttgccgtagc 180gtgtatgttg gaaacatcca tacacaggtc acagagcctt tgcttcaaga gatttttaca 240agcactggcc ctgttgaaag cagtaaactc atcagaaagg ataagtcatc atatggattt 300gttcactact ttgatcgaag atccgctgct ctggctatac tgtctctgaa cggaaggcat 360ctgtttggac agcctatcaa agtcaattgg gcgtatgcca ctggtcagag ggaagataca 420tcaagtcatt tcaacatttt tgttggagat ctcagtccag aggtcactga tgcaacatta 480tatcaaagct tttctgtctt ttccagttgt tcggatgcga gagttatgtg ggaccaaaaa 540actgggcgct cgagaggctt tgggtttgtt tccttccgca atcaacagga tgctcaaact 600gccattaatg agatgaatgg taagtggtta agtagcagac aaatcagatg caactgggcc 660acgaagggcg ctacttctgg tgatgataag ctcagttctg atggaaaaag tgttgtggaa 720cttacaactg gctcatcaga ggatggtaaa gagacattaa atgaggaaac acctgaaaat 780aattctcagt ttaccactgt ttatgtggga aaccttgctc cagaggtaac tcagcttgat 840ctacaccgtt acttccatgc tcttggcgct ggagttattg aggaggtccg tgtccaacga 900gacaaaggct ttggtttcgt gagatataac actcatcccg aagctgctct tgctattcag 960atgggtaaca ctcagcctta cctctttaac agacagataa agtgctcatg gggaaacaag 1020ccaactccac caggtacagc ctcaaaccca cttcccccac ctgccccagc tccagttcct 1080ggtctatctg cagctgatct cctaaactat gagaggcaat tggcacttag caagatggca 1140agtgtgaatg cgttaatgca tcaacagggt caacaccctc taaggcaggc tcatggaata 1200aatgccgctg gagcaactgc agccatgtat gatggtggct ttcagaatgt agccgccgca 1260cagcaactca tgtactatca gtaa 128430427PRTArabidopsis thaliana 30Met Gln Asn Pro Arg Leu Lys Gln His Gln Gln Gln Gln Gln Gln Gln1 5 10 15Ala Met Met Gln Gln Gln Ala Leu Met Gln Gln His Ser Leu Tyr His 20 25 30Pro Gly Val Leu Ala Pro Pro Gln Leu Glu Pro Val Pro Ser Gly Asn 35 40 45Leu Pro Pro Gly Phe Asp Pro Ser Thr Cys Arg Ser Val Tyr Val Gly 50 55 60Asn Ile His Thr Gln Val Thr Glu Pro Leu Leu Gln Glu Ile Phe Thr65 70 75 80Ser Thr Gly Pro Val Glu Ser Ser Lys Leu Ile Arg Lys Asp Lys Ser 85 90 95Ser Tyr Gly Phe Val His Tyr Phe Asp Arg Arg Ser Ala Ala Leu Ala 100 105 110Ile Leu Ser Leu Asn Gly Arg His Leu Phe Gly Gln Pro Ile Lys Val 115 120 125Asn Trp Ala Tyr Ala Thr Gly Gln Arg Glu Asp Thr Ser Ser His Phe 130 135 140Asn Ile Phe Val Gly Asp Leu Ser Pro Glu Val Thr Asp Ala Thr Leu145 150 155 160Tyr Gln Ser Phe Ser Val Phe Ser Ser Cys Ser Asp Ala Arg Val Met 165 170 175Trp Asp Gln Lys Thr Gly Arg Ser Arg Gly Phe Gly Phe Val Ser Phe 180 185 190Arg Asn Gln Gln Asp Ala Gln Thr Ala Ile Asn Glu Met Asn Gly Lys 195 200 205Trp Leu Ser Ser Arg Gln Ile Arg Cys Asn Trp Ala Thr Lys Gly Ala 210 215 220Thr Ser Gly Asp Asp Lys Leu Ser Ser Asp Gly Lys Ser Val Val Glu225 230 235 240Leu Thr Thr Gly Ser Ser Glu Asp Gly Lys Glu Thr Leu Asn Glu Glu 245 250 255Thr Pro Glu Asn Asn Ser Gln Phe Thr Thr Val Tyr Val Gly Asn Leu 260 265 270Ala Pro Glu Val Thr Gln Leu Asp Leu His Arg Tyr Phe His Ala Leu 275 280 285Gly Ala Gly Val Ile Glu Glu Val Arg Val Gln Arg Asp Lys Gly Phe 290 295 300Gly Phe Val Arg Tyr Asn Thr His Pro Glu Ala Ala Leu Ala Ile Gln305 310 315 320Met Gly Asn Thr Gln Pro Tyr Leu Phe Asn Arg Gln Ile Lys Cys Ser 325 330 335Trp Gly Asn Lys Pro Thr Pro Pro Gly Thr Ala Ser Asn Pro Leu Pro 340 345 350Pro Pro Ala Pro Ala Pro Val Pro Gly Leu Ser Ala Ala Asp Leu Leu 355 360 365Asn Tyr Glu Arg Gln Leu Ala Leu Ser Lys Met Ala Ser Val Asn Ala 370 375 380Leu Met His Gln Gln Gly Gln His Pro Leu Arg Gln Ala His Gly Ile385 390 395 400Asn Ala Ala Gly Ala Thr Ala Ala Met Tyr Asp Gly Gly Phe Gln Asn 405 410 415Val Ala Ala Ala Gln Gln Leu Met Tyr Tyr Gln 420 42531735DNASaccharomyces cerevisiae 31atggccgctg aaaaaatctt gaccccagaa tctcagttga agaagtctaa ggctcaacaa 60aagactgctg aacaagtcgc tgctgaaaga gctgctcgta aggctgctaa caaggaaaag 120agagccatta ttttggaaag aaacgccgct taccaaaagg aatacgaaac tgctgaaaga 180aacatcattc aagctaagcg tgatgccaag gctgctggtt cctactacgt cgaagctcaa 240cacaagttgg tcttcgttgt cagaatcaag ggtattaaca agatcccacc taagccaaga 300aaggttctac aattgctaag attgacaaga atcaactctg gtacattcgt caaagttacc 360aaggctactt tggaactatt gaagttgatt gaaccatacg ttgcttacgg ttacccatcg 420tactctacta ttagacaatt ggtctacaag agaggtttcg gtaagatcaa caagcaaaga 480gttccattgt ccgacaatgc tatcatcgaa gccaacttgg gtaagtatgg tatcttgtcc 540attgacgatt tgattcacga aatcatcact gttggtccac acttcaagca agctaacaac 600tttttgtggc cattcaagtt gtccaaccca tctggtggtt ggggtgtccc aagaaagttc 660aagcacttta tccaaggtgg ttctttcggt aaccgtgaag aattcatcaa caaattggtt 720aagtccatga actaa 73532244PRTSaccharomyces cerevisiae 32Met Ala Ala Glu Lys Ile Leu Thr Pro Glu Ser Gln Leu Lys Lys Ser1 5 10 15Lys Ala Gln Gln Lys Thr Ala Glu Gln Val Ala Ala Glu Arg Ala Ala 20 25 30Arg Lys Ala Ala Asn Lys Glu Lys Arg Ala Ile Ile Leu Glu Arg Asn 35 40 45Ala Ala Tyr Gln Lys Glu Tyr Glu Thr Ala Glu Arg Asn Ile Ile Gln 50 55 60Ala Lys Arg Asp Ala Lys Ala Ala Gly Ser Tyr Tyr Val Glu Ala Gln65 70 75 80His Lys Leu Val Phe Val Val Arg Ile Lys Gly Ile Asn Lys Ile Pro 85 90 95Pro Lys Pro Arg Lys Val Leu Gln Leu Leu Arg Leu Thr Arg Ile Asn 100 105 110Ser Gly Thr Phe Val Lys Val Thr Lys Ala Thr Leu Glu Leu Leu Lys 115 120 125Leu Ile Glu Pro Tyr Val Ala Tyr Gly Tyr Pro Ser Tyr Ser Thr Ile 130 135 140Arg Gln Leu Val Tyr Lys Arg Gly Phe Gly Lys Ile Asn Lys Gln Arg145 150 155 160Val Pro Leu Ser Asp Asn Ala Ile Ile Glu Ala Asn Leu Gly Lys Tyr 165 170 175Gly Ile Leu Ser Ile Asp Asp Leu Ile His Glu Ile Ile Thr Val Gly 180 185 190Pro His Phe Lys Gln Ala Asn Asn Phe Leu Trp Pro Phe Lys Leu Ser 195 200 205Asn Pro Ser Gly Gly Trp Gly Val Pro Arg Lys Phe Lys His Phe Ile 210 215 220Gln Gly Gly Ser Phe Gly Asn Arg Glu Glu Phe Ile Asn Lys Leu Val225 230 235 240Lys Ser Met Asn33735DNASaccharomyces cerevisiae 33atgtccactg aaaaaatctt gactcctgaa tctcaattga agaagactaa agctcaacaa 60aagactgcag aacaaattgc tgcagagaga gctgcccgta aagccgctaa caaggaaaaa 120agagctatta ttttggaaag aaacgccgct taccaaaagg aatacgaaac tgctgaaaga 180aacatcattc aagctaagcg tgatgccaag gctgctggtt cctactacgt cgaagctcaa 240cacaagttgg tcttcgttgt cagaatcaag ggtattaaca agattccacc taagccaaga 300aaggttctac aattgctaag attgacaaga atcaactctg gtacattcgt caaagttacc 360aaggctactt tggaactatt gaagttgatt gaaccatacg ttgcttacgg ttacccatcc 420tactctacta ttagacaatt ggtctacaag agaggtttcg gtaagatcaa caagcaaaga 480gttccattgt ccgacaatgc tatcatcgaa gccaacttgg gtaagtatgg tatcttgtcc 540attgacgatt tgattcacga aatcatcact gttggtccac acttcaagca agctaacaac 600tttttgtggc cattcaagtt gtccaaccca tctggtggtt ggggtgtccc aagaaagttc 660aagcatttca tccaaggtgg ttctttcggt aaccgtgaag aattcatcaa taaattggtt 720aaggctatga actaa 73534244PRTSaccharomyces cerevisiae 34Met Ser Thr Glu Lys Ile Leu Thr Pro Glu Ser Gln Leu Lys Lys Thr1 5 10 15Lys Ala Gln Gln Lys Thr Ala Glu Gln Ile Ala Ala Glu Arg Ala Ala 20 25 30Arg Lys Ala Ala Asn Lys Glu Lys Arg Ala Ile Ile Leu Glu Arg Asn 35 40 45Ala Ala Tyr Gln Lys Glu Tyr Glu Thr Ala Glu Arg Asn Ile Ile Gln 50 55 60Ala Lys Arg Asp Ala Lys Ala Ala Gly Ser Tyr Tyr Val Glu Ala Gln65 70 75 80His Lys Leu Val Phe Val Val Arg Ile Lys Gly Ile Asn Lys Ile Pro 85 90 95Pro Lys Pro Arg Lys Val Leu Gln Leu Leu Arg Leu Thr Arg Ile Asn 100 105 110Ser Gly Thr Phe Val Lys Val Thr Lys Ala Thr Leu Glu Leu Leu Lys 115 120 125Leu Ile Glu Pro Tyr Val Ala Tyr Gly Tyr Pro Ser Tyr Ser Thr Ile 130 135 140Arg Gln Leu Val Tyr Lys Arg Gly Phe Gly Lys Ile Asn Lys Gln Arg145 150 155 160Val Pro Leu Ser Asp Asn Ala Ile Ile Glu Ala Asn Leu Gly Lys Tyr 165 170 175Gly Ile Leu Ser Ile Asp Asp Leu Ile His Glu Ile Ile Thr Val Gly 180 185 190Pro His Phe Lys Gln Ala Asn Asn Phe Leu Trp Pro Phe Lys Leu Ser 195 200 205Asn Pro Ser Gly Gly Trp Gly Val Pro Arg Lys Phe Lys His Phe Ile 210 215 220Gln Gly Gly Ser Phe Gly Asn Arg Glu Glu Phe Ile Asn Lys Leu Val225 230 235 240Lys Ala Met Asn3538DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35aaaaagcagg cttaatgcag caaccaccgt caaacgcc 383637DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36agaaagctgg gtttcactga cgttgctgct gatagtt 373738DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37aaaaagcagg cttaatgcag acaccaaaca acaacggt 383837DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38agaaagctgg gtttcaagaa gctcccggga ctgcagc 373938DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39aaaaagcagg cttaatgcag acaaccaacg gctcagat 384037DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40agaaagctgg gtttcaattc tccccatgat agttgtt 374138DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41aaaaagcagg cttaatggca gacgtcaaga ttcaatcc 384237DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42agaaagctgg gtttcagcta acttgttgct gatgacc 374338DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43aaaaagcagg cttaatggca gacgtcaagg ttcaatcc 384437DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer

44agaaagctgg gtttcagcta acttgttgct gatgacc 374538DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45aaaaagcagg cttaatgcag aatcaaaggc ttattaag 384637DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46agaaagctgg gttttactga tagtacatga gctgctg 374734DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47aaaaagcagg cttaatgtcc actgaaaaaa tctt 344833DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48agaaagctgg gttttagttc atagccttaa cca 334920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49aatctgtgtc gacgtacttc 205020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50agaagtacat aggatgggtc 205120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51aaaaattgtc gacgtacttc 205220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52aaaggaagtt atcacaattg 205320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53ccagcatcta tgtctgcaac 205420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54cgtatctgga gtagtatttc 205520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55gcaaggtata caaagcagaa 205620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56tcatcctttt tcttctctgc 205720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 57gacacgatga agttggatat 205820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 58tgactgtcaa atcatcactg 205920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59cagtataaaa atgtctgaat 206020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60tggttgatta tttcttcttc 206120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61atcaacgtca taatgtccac 206220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 62taccagagtt gattcttgtc 206320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 63acctaaagaa accatgtcag 206420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 64tatcaaggtt gtacgtttcg 206520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 65atgtacagtc taagtcaagg 206620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 66gactaaagtg aacagcaatg 206720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 67gagaatggca atatttcaag 206820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 68tgttcttctt cttccattac 206920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 69tggacccaca taatccaatt 207020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70tttcgaacat tacctcacac 207120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71gtggatggtc ttttagaaga 207220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 72aactcctcga aacttaaacg 207320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 73tattgagacc ttcttccaag 207420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 74aagattttac cggaaacgtg 207522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 75gacgataaaa agaaatttgg tg 227619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 76ctcaaagcgt tgttgaaag 197720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 77gagagaggtc attagtatta 207820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 78ttttctaata acagggaacc 207923DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 79gtttaataga aaaagaagag gag 238023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 80tagttcatca actaaaaaca tgg 238119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 81agctgtccca agtgttcaa 198219DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 82acccttacca ccgaatttc 198320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 83gttgggatat ttttggttgg 208420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 84aaaggaacgt ccttcaattc 208520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 85aacaagctgt tcaggttaga 208620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 86ggtttgtgat tatcatcagg 208721DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 87aattaaagat cacaatggcc g 218820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 88cttggtaact ttgacgaatg 208920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 89cagaaaagct ggtgttcaag 209020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 90tgattctgca tcgtggtttc 209120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 91ttgattaaga actccaaagc 209220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 92tcttctcaag acacgtaatc 209320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 93agatgaggtt gaagcaatag 209420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 94caagggcaat ttccttattg 209520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 95taagactaag caacaatgcc 209620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 96aaacccaact tgtagacttg 209720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 97gcatctcata atatgtctgc 209820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 98ttgttgctaa gactgtagag 209922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 99caaatccatt tcaaaatata gg 2210020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 100ctcctcctat ctaaaaaacc 2010120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 101aagaagagtt ggtaagcaag 2010220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 102caccgttttt gaatgtgatg 2010320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 103agcgtaatac gaaagatgag 2010420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 104agcttcgtta ttcaagggat 2010522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 105gtatcataaa cattcaacaa tg 2210620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 106cggatctgtt gtttattctc 2010720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 107aaacaaagtt tgatcgcctc 2010820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 108tcgtgctcaa acatttcttc 2010920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 109aaaatgacgg ataatccacc 2011020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 110ttcaaagtct ttagcacacc 2011120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 111caatccatca tgggaaaatc 2011220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 112cttggacgac aaaatagtgt 2011320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 113aggacttcaa tttccatgtc 2011420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 114agtgtcatct ccacaatttg 2011520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 115gaaaacgata agggccaatt 2011620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 116cgttctttaa caaaccatcg 2011722DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 117taccaaatga aacgctttaa tg 2211820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 118tcttcatgga aagagtctag 2011920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 119atggaatgag tactttagcg 2012020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 120cttcatttcc gagtttttgg 2012120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 121aatagaaaat cggcttctgc 2012220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 122tatttgatca ttggggttgc 2012320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 123gattgaagac atttgatgcg 2012420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 124tcgccactaa ctctatttac 2012520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 125accatttcag gtacaatgtc 2012620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 126cttcggaaat atcgaattcc 2012720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 127ctgaaacgat accaacaatg 2012820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 128tttgtggttt aggcaatacc 2012922DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 129atacaaaagt atacaacatg cc 2213020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 130tttccaagaa atcttcgacc 2013120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 131ctttactgcg aagataaagg 2013220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 132gccactataa tctgttgttg 2013320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 133tcaaaactac ggctcatttg 2013420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 134tgaacaaaag actcaatccg 20