Protein having glycoalkaloid biosynthetic enzyme activity and gene encoding the same

TECHNICAL FIELD

The present invention relates to a method for producing a glycoalkaloid compound characteristic of a solanaceous plant such as a potato, a glycoalkaloid biosynthetic enzyme, DNA encoding the glycoalkaloid biosynthetic enzyme, a method for breeding or selecting a novel solanaceous plant such as a potato using the DNA, and a solanaceous plant such as a potato that produces no glycoalkaloid.

BACKGROUND ART

Glycoalkaloids are members of plant-derived compounds and are said to be steroidal alkaloids. It has been reported that the glycoalkaloid structure has an N atom and is an isoprenoid having 27 carbon chains, and there are 422 compounds of glycoalkaloids from plants belonging to the genus Solanum (Non-Patent Literature 1: Chapter 7.8). In addition to solanaceous plants belonging to the genus Solanum, plants belonging to the family Liliaceae are known to contain glycoalkaloids. Important glycoalkaloids are chaconine and solanine from potatoes (Solanum tuberosum) belonging to the genus Solanum of the family Solanaceae and tomatine from tomatoes (Solanum lycopersicum).

The potato is the world's forth largest crop produced, following maize, rice, and wheat. It is well known that buds sprouting from the tubers and aerial parts of the potato contain chaconine and solanine, which are toxic substances. Chaconine and solanine cause toxic symptoms such as abdominal pain, dizziness, and mild disturbances of consciousness. Also, chaconine and solanine are likely to accumulate in tubers as a result of damage or exposure to sunlight. Therefore, there is the risk of accidental poisoning as a result of a failure in tuber management. Accidental glycoalkaloid poisoning sometimes occurs. In a recent case, accidental glycoalkaloid poisoning occurred in an elementary school in Nara city in Japan on Jul. 16, 2009 (reported by Asahi.com). The glycoalkaloid levels in potato tubers are controlled at 20 mg/100 g or lower by, for example, storing the tubers in dark places. Thus, in general, potato tubers are safe food products. However, in consideration of the risk of accidental poisoning described above, reduction of potato glycoalkaloid content is a key issue for those involved in breeding, production, storage, transportation, distribution, or purchasing in the potato-related industry. However, reduction of potato glycoalkaloid content has not been achieved thus far. This is because there is no wild-type potato line free from glycoalkaloids, the glycoalkaloid biosynthesis pathway has not been elucidated (Non-Patent Literature 1 (FIGS. 7.24 A and B) and Non-Patent Literature 2), and there has been little progress in identification of genes involved in the biosynthesis pathway.

It is known that glycoalkaloids have medicinal properties such as anticancer activity, liver-protecting effects, antispasmodic effects, immune-system-promoting effects, antifungal effects, antiprotozoal effects, and molluscicide activity, in addition to poisonous properties such as anticholinesterase activity and membrane disruption effects (Non-Patent Literature 1). It has been reported that esculeoside A, which is a glycoalkaloid metabolite, shows anti-arteriosclerotic effects in tomatoes (Non-Patent Literature 3). However, since the biosynthesis pathway has not been elucidated, there has been substantially no advance in research and development to suppress or efficiently produce metabolites.

In recent years, there have been some reports on genes involved in the glycosylation after transfer of a sugar to aglycone (Non-Patent Literature 4-6). Non-Patent Literature 4 reports that a UDP-galactosyltransferase gene is involved in the pathway of formation of .gamma.-solanine from solanidine (aglycone), and it also reports a strain in which the gene is suppressed. However, suppression of chaconine has been never achieved (Non-Patent Literature 4 (FIG. 2)). Non-Patent Literature 4 reports that a UDP-glucosyltransferase gene is involved in the pathway of formation of .gamma.-chaconine from solanidine, and it also reports a strain in which the gene is suppressed. However, suppression of chaconine and solanine has been substantially impossible (Non-Patent Literature 5 (FIG. 5)). Non-Patent Literature 6 reports a rhamnosyltransferase gene involved in the pathway of formation of .alpha.-chaconine from .beta.-chaconine and the pathway of formation of .alpha.-solanine from .beta.-solanine. In this case, .beta.-isomer or .gamma.-isomer increases while .alpha.-isomer decreases. Thus, it is understood that it has been very difficult to control total glycoalkaloid content, even though it has become possible to change the molecular species of glycoalkaloid by suppressing the glycosylation.

There is a report of an attempt to reduce glycoalkaloid through overexpression of genes involved in biosynthesis of plant sterols and plant hormones (Non-Patent Literature 7). However, in such case, it was merely possible to reduce the glycoalkaloid content up to almost half the initial amount (Non-Patent Literature 7 (FIG. 5)).

CITATION LIST

Non Patent Literature

Non-Patent Literature 1: Eich, Soloanaceae and Convolvulaceae: Secondary Metabolite (2008), Springer Non-Patent Literature 2: Ginzberg et al., Potato Research (2009) 52: 1-15 Non-Patent Literature 3: Fujiwara et al., Abstracts of the Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry 2008, 2B07, p. 22 Non-Patent Literature 4: McCue et al., Plant Sci. (2005) 168: 267-273 Non-Patent Literature 5: McCue et al., Phytochemistry (2006) 67: 1590-1597 Non-Patent Literature 6: McCue et al., Phytochemistry (1998) 68: 327-334 Non-Patent Literature 7: Arnqvist et al., Plant Physiol. (2003) 131: 1792-1799

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to provide a method for producing a glycoalkaloid compound characteristic of a solanaceous plant such as a potato, a glycoalkaloid biosynthetic enzyme, DNA encoding the glycoalkaloid biosynthetic enzyme, a method for breeding or selecting a novel solanaceous plant such as a potato using the DNA, and a solanaceous plant such as a potato that produces no glycoalkaloid.

Solution to Problem

The present inventors conducted intensive studies in order to achieve the above object. First, the present inventors focused on the process prior to aglycone formation. Then, the present inventors searched in silico for candidate genes involved in the biosynthesis pathway and suppressed expression of endogenous candidate genes by causing expression of parts of the candidate genes to induce RNAi. As a result, the present inventors succeeded in obtaining a potato having remarkably reduced glycoalkaloid content from the transformants and identifying the glycoalkaloid biosynthetic enzyme gene. In addition, the present inventors demonstrated that it is possible to obtain a solanaceous plant such as a potato lacking glycoalkaloids by selecting a plant in which the expression of the above gene is suppressed. Further, the present inventors demonstrated that it becomes possible to produce a novel glycoalkaloid compound by expression of the gene, and it also becomes possible to analyze polymorphisms by comparing the genomic sequence of the gene with the genomic sequences of different solanaceous plants such as potatoes, thereby making it possible to establish a newly bred solanaceous plant variety such as a potato variety. This has led to the completion of the present invention. Similarly, the present inventors succeeded in producing a tomato having reduced glycoalkaloid content by suppressing the endogenous gene in the above manner.

Specifically, the present invention encompasses the following inventions.

[1] A protein, which is the following (a) or (b):

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 18; or

(b) a protein having glycoalkaloid biosynthetic enzyme activity and comprising an amino acid sequence that has a deletion, substitution, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 1 or 18.

[2] A gene, which comprises DNA selected from among the following (c) to (f):

(c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or 19;

(d) DNA that hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or 19 and encodes a protein having glycoalkaloid biosynthetic enzyme activity;

(e) DNA that consists of a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2 or 19 and encodes a protein having glycoalkaloid biosynthetic enzyme activity; or

(f) DNA consisting of a degenerate isomer of the nucleotide sequence shown in SEQ ID NO: 2 or 19.

[3] A protein, which is the following (g) or (h):

(g) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 20; or

(h) a protein having glycoalkaloid biosynthetic enzyme activity and comprising an amino acid sequence that has a deletion, substitution, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 3 or 20.

[4] A gene, which comprises DNA selected from among the following (i) to (l):

(i) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 or 21;

(j) DNA that hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 or 21 and encodes a protein having glycoalkaloid biosynthetic enzyme activity;

(k) DNA that consists of a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 or 21 and encodes a protein having glycoalkaloid biosynthetic enzyme activity; or

(l) DNA consisting of a degenerate isomer of the nucleotide sequence shown in SEQ ID NO: 4 or 21.

[5] A recombinant vector, which comprises the gene according to [2] or [4].

[6] A transformant, to which the recombinant vector according to [5] is introduced.

[7] The transformant according to [6], which is a plant.

[8] A method for detecting the existence of a mutation and/or polymorphism of a gene encoding a glycoalkaloid biosynthetic enzyme in a plant, which comprises:

(i) a step of isolating a nucleic acid in the form of genomic DNA or RNA from a plant;

(ii) a step of synthesizing cDNA via reverse transcription when the nucleic acid in (i) is RNA;

(iii) a step of amplifying a gene fragment comprising the nucleotide sequence shown in SEQ ID NO: 2, 4, or 5 or the nucleotide sequence shown in SEQ ID NO: 19, 21, or 22 from DNA obtained in step (i) or (ii); and

(iv) a step of determining the existence of a mutation and/or polymorphism in the DNA.

[9] The method according to [8], wherein the plant is a plant belonging to the family Solanaceae.

[10] A method for selecting a plant having a mutation and/or polymorphism, which comprises detecting a mutation and/or polymorphism of a gene encoding a glycoalkaloid biosynthetic enzyme by the method according to [8] or [9].

[11] A plant having a mutation and/or a polymorphism in a gene encoding a glycoalkaloid biosynthetic enzyme, which is selected by the method according to [9].

[12] The plant according to [11], which is a plant belongs to the family Solanaceae.

[13] A method for selecting the plant according to [8] or [9], which comprises selecting a plant in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or the activity of a glycoalkaloid biosynthetic enzyme encoded by the gene is altered from that in an existing variety. [14] A plant selected by the method according to [13], in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or the activity of a glycoalkaloid biosynthetic enzyme encoded by the gene is altered from that in an existing variety. [15] The plant according to [14], which is a plant belonging to the family Solanaceae.

This description includes part or all of the contents as disclosed in the descriptions and/or drawings of Japanese Patent Application Nos. 2009-198889 and 2010-108445, which are priority documents of the present application.

Advantageous Effects of Invention

According to the present invention, it is possible to regulate expression of the activity of a protein having glycoalkaloid compound biosynthesis activity characteristic of a solanaceous plant such as a potato and that of a gene encoding the protein. Specifically, a method for producing a plant in which activity of such gene is regulated and a solanaceous plant such as a potato that produces no glycoalkaloid are provided. The present invention enables breeding of a solanaceous plant such as a potato having the feature of containing a glycoalkaloid compound. The use of the enzyme of the present invention allows the mass production of glycoalkaloid compounds that exhibit a variety of useful physiological activities at low prices.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows results of analysis of biosynthesis gene C homology between a potato (SEQ ID NO: 2; the top sequence in the pairwise alignment) and a tomato (SEQ ID NO: 4; the bottom sequence in the pairwise alignment) using the GENETYX DNA analysis software (Genetyx Corporation). The overall results indicate very high homology.

FIG. 1B shows results of analysis of biosynthesis gene C homology between a potato and a tomato using the GENETYX DNA analysis software (Genetyx Corporation) (continued from FIG. 1A).

FIG. 1C shows results of analysis of biosynthesis gene C homology between a potato and a tomato using the GENETYX DNA analysis software (Genetyx Corporation) (continued from FIG. 1B).

FIG. 2 shows a vector construct used for suppression of a candidate C gene. FIG. 2 shows the inner construct between the right border (RB) and the left border (LB) and restriction enzyme sites in T-DNA which is a gene fragment to be introduced.

FIG. 3 shows an MS spectrum for the protonated parent ion peak (M+H).sup.+ for .alpha.-solanine (A) and an MS spectrum for the protonated parent ion peak (M+H).sup.+ for .alpha.-chaconine (B).

FIG. 4 shows a calibration curve (LC-MS quantitative determination analysis system) for .alpha.-solanine (A), a calibration curve (LC-MS quantitative determination analysis system) for .alpha.-chaconine (B), and a calibration curve (LC-MS quantitative determination analysis system) for brassinolide (C).

FIG. 5 shows LC-MS chromatograms for reference products (.alpha.-solanine, .alpha.-chaconine, and brassinolide).

FIG. 6 shows LC-MS chromatograms for .alpha.-solanine, .alpha.-chaconine, and brassinolide in a stem-derived sample.

FIG. 7 is a chart showing the glycoalkaloid contents in in vitro stems of potato transformants. Each error bar indicates a standard deviation.

FIG. 8 shows RT-PCR results for mRNAs extracted from in vitro stems of potato transformants.

FIG. 9 is a chart showing the glycoalkaloid contents in tuber epidermis of potato transformants. Each error bar indicates a standard deviation.

FIG. 10 is a chart showing the glycoalkaloid contents in leaves of tomato transformants. Each error bar indicates a standard deviation.

FIG. 11A shows results of analysis of biosynthesis gene D homology between a potato (SEQ ID NO: 19; the top sequence in the pairwise alignment) and a tomato (SEQ ID NO: 21; the bottom sequence in the pairwise alignment) using the GENETYX DNA analysis software (Genetyx Corporation). The overall results indicate very high homology.

FIG. 11B shows results of analysis of biosynthesis gene D homology between a potato and a tomato using the GENETYX DNA analysis software (Genetyx Corporation) (continued from FIG. 11A).

FIG. 11C shows results of analysis of biosynthesis gene D homology between a potato and a tomato using the GENETYX DNA analysis software (Genetyx Corporation) (continued from FIG. 11B).

FIG. 12 shows a vector construct used for suppression of a candidate D gene. FIG. 12 shows the inner construct between the right border (RB) and the left border (LB) and restriction enzyme sites in T-DNA which is a gene fragment to be introduced.

FIG. 13 is a chart showing the glycoalkaloid contents in in vitro stems of potato transformants. Each error bar indicates a standard deviation.

FIG. 14 shows RT-PCR results for mRNAs extracted from in vitro stems of potato transformants.

FIG. 15 is a chart showing the glycoalkaloid contents in tuber epidermis of potato transformants. Each error bar indicates a standard deviation.

FIG. 16 is a chart showing the glycoalkaloid contents in leaves of tomato transformants. Each error bar indicates a standard deviation.

DESCRIPTION OF EMBODIMENTS

The present invention is described in detail below.

1. A Novel Glycoalkaloid Biosynthetic Enzyme

The protein/enzyme of the present invention is a glycoalkaloid biosynthetic enzyme contained in a solanaceous plant (Solanaceae) such as a potato. Plants such as potatoes belonging to the family Solanaceae include potatoes (Solanum tuberosum), tomatoes (Solanum lycopersicum), eggplants (Solanum melongena), and capsicums (Capsicum annum). In addition, the enzyme of the present invention is a membrane-bound cytochrome P450 monooxidase. Examples of glycoalkaloids obtained using the enzyme of the present invention include a glycoalkaloid synthesized by a solanaceous plant such as a potato. Examples thereof include glycoalkaloids such as chaconine and solanine contained in potatoes and glycoalkaloids such as tomatine contained in tomatoes.

Examples of a preferable steroid compound that can be used as a substrate for the glycoalkaloid biosynthetic enzyme of the present invention include cholesterols. Examples of cholesterols include cholesterol, sitosterol, campesterol, stigmasterol, and brassicasterol. The glycoalkaloid biosynthetic enzyme of the present invention is a hydroxylation enzyme that transfers a hydroxyl group to any of the above cholesterols.

The full-length amino acid sequence of the enzyme of the present invention is shown in SEQ ID NO: 1 or 3 (gene C) or SEQ ID NO: 18 or 20 (gene D). Further, the protein of the present invention includes a protein having glycoalkaloid biosynthetic enzyme activity and comprising an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 1 or 3 or the amino acid sequence shown in SEQ ID NO: 18 or 20. Here, an example of such a substantially identical amino acid sequence is an amino acid sequence that has a deletion, substitution, insertion, and/or addition of one or several amino acids (1 to 10 amino acids, preferably 1 to 7 amino acids, more preferably 1 to 5 amino acids, further preferably 1 to 3 amino acids, and even further preferably 1 amino acid or 2 amino acids) with respect to the above amino acid sequence. Alternatively, it is an amino acid sequence having at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more sequence identity to the above amino acid sequence when the sequence identity is calculated using, for example, BLAST (the Basic Local Alignment Search Tool at the National Center for Biological Information) (based on, for example, default (i.e., initial setting) parameters).

The glycoalkaloid biosynthetic enzyme of the present invention includes a natural glycoalkaloid biosynthetic enzyme isolated from a plant and a recombinant glycoalkaloid biosynthetic enzyme produced by a gene engineering technique.

2. A Gene Encoding a Glycoalkaloid Biosynthetic Enzyme

The gene of the present invention is a gene encoding a glycoalkaloid biosynthetic enzyme having activity of binding a hydroxyl group to a steroid compound, and it also encodes a protein having the above glycoalkaloid biosynthetic enzyme activity.

The DNA nucleotide sequence of the gene of the present invention is the nucleotide sequence shown in SEQ ID NO: 2 or 4 or the nucleotide sequence shown in SEQ ID NO: 19 or 21. Further, the DNA of the present invention includes: DNA that hybridizes under stringent conditions to DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 or 4 or the nucleotide sequence shown in SEQ ID NO: 19 or 21; DNA having at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2 or 4 or the nucleotide sequence shown in SEQ ID NO: 19 or 21 when the sequence identity is calculated using, for example, BLAST (the Basic Local Alignment Search Tool at the National Center for Biological Information) (based on, for example, default (i.e., initial setting) parameters); and DNA that encodes a protein comprising an amino acid sequence that has a deletion, substitution, insertion, and/or addition of one or several amino acids (1 to 10 amino acids, preferably 1 to 7 amino acids, preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids, and further preferably 1 amino acid or 2 amino acids) with respect to the amino acid sequence of a protein encoded by the above DNA and comprises a protein having glycoalkaloid biosynthetic enzyme activity. Here, the term "stringent conditions" refers to, for example, conditions comprising "1.times.SSC, 0.1% SDS, 37.degree. C." More stringent conditions comprise "0.5.times.SSC, 0.1% SDS, 42.degree. C." Further stringent conditions comprise "0.2.times.SSC, 0.1% SDS, 65.degree. C." In addition, the gene of the present invention includes DNA comprising a degenerate isomer of the nucleotide sequence shown in SEQ ID NO: 3 or 4.

3. Recombinant Vectors

The vector of the present invention is a recombinant vector into which DNA shown in SEQ ID NO: 2 or 4 or DNA shown in SEQ ID NO: 19 or 21 has been inserted. As such vector, a wide range of known vectors for yeasts, plant cells, insect cells, and the like can be used. Examples of known vectors for yeasts include pDR196, pYES-DEST 52, Yip5, Yrp17, and Yep24. Examples of known vectors for plant cells include the pGWB vector, pBiEl2-GUS, pIG121-Hm, pBI121, pBiHyg-HSE, pB119, pBI101, pGV3850, and pABH-Hm1. Examples of known vectors for insect cells include pBM030, pBM034, and pBK283. A vector used in the present invention incorporates components involved in gene expression or suppression such as a promoter, a terminator, and an enhancer. If necessary, the vector contains selection markers (e.g., a drug-resistant gene, an antibiotic-resistant gene, and a reporter gene). It is preferable for the components involved in gene expression or suppression to be incorporated into a recombinant vector in a manner such that they can independently function in accordance with their properties. A person skilled in the art can adequately carry out procedures of such incorporation.

4. Transformants

The transformant of the present invention is a transformant having the recombinant vector of the present invention. Such transformant can be obtained by introducing a recombinant vector into which a gene encoding an enzyme has been inserted into a host in a manner such that the gene of interest is expressed therein. A host appropriate for a vector can be used. Examples of hosts include yeasts, plant cells, insect cells (e.g., Sf9), and plant viruses. Preferable examples thereof include yeasts, plant cells, and plant viruses. A method for introducing a recombinant vector is not particularly limited as long as it is a method for introducing DNA into a microorganism. Examples of such method include a method using calcium ions (Cohen, S. N. et al.: Proc. Natl. Acad. Sci., USA, 69:2110 (1972)), an electroporation method, and a tri-parental mating method. In addition, an example of a method for producing a transformed plant is a method using, as a vector, a virus, a Ti plasmid or Ri plasmid of Agrobacterium, or the like. Examples of host plants include monocotyledonous plants such as rice, barley, and maize and dicotyledonous plants such as soybeans, rapeseeds, tomatoes, and potatoes. A transformed plant can be obtained through regeneration using plant cells transformed with the gene of the present invention. Regeneration of a plant from plant cells can be carried out by a known method.

5. A Method for Producing a Glycoalkaloid Biosynthetic Enzyme and a Method for Producing a Glycoalkaloid Compound

The glycoalkaloid biosynthetic enzyme of the present invention is a membrane-bound cytochrome P450 monooxidase, and it can be collected from a general plant (e.g., Collu et al., 2001, FEBS Lett. 508:215-220). In addition, the glycoalkaloid biosynthetic enzyme of the present invention can be produced via, for example, mass production using an expression system for microorganisms (e.g., yeast) or insect cells transformed with the gene of the present invention. An example of the use of insect cells has been reported by Morikawa et al. (2006, Plant Cell 18: 1008-1022).

The glycoalkaloid biosynthetic enzyme of the present invention can be expressed in the form of a highly active protein using such system. Therefore, a glycoalkaloid compound can be produced with the addition of the substrate of the glycoalkaloid biosynthetic enzyme to a transformed yeast or insect cell culture liquid. For example, a hydroxylated cholesterol can be efficiently mass-produced by administering, as a substrate, a cholesterol to a transformed yeast culture liquid. It has been reported that yeast has a pathway of biosynthesis of DMAPP in cytosol (mevalonate pathway), and a precursor or substrate can be produced by introducing a mevalonate pathway into Escherichia coli (Harada & Misawa, 2009 Aug. 12. Epub Appl Microbiol Biotechnol.). It becomes possible to simultaneously express a membrane-bound cytochrome P450 monooxidase and a different gene so as to produce a glycoalkaloid in the above manner. For example, a metabolite was obtained by causing the expression of a membrane-bound cytochrome P450 monooxidase using Escherichia coli (reported by Chang et al. (2007 Nat. Chem. Biol. 3:274-277)) and yeast (reported by Seki et al. (2008 PNAS 105:14204-14209)). The combined use of techniques used in the above cases allows production of a glycoalkaloid compound.

6. Selection of Gene Mutation, Polymorphism, and Gene Expression Mutation

According to the present invention, a method for detecting the existence of a glycoalkaloid biosynthetic enzyme gene mutation, a polymorphism such as a single nucleotide polymorphism (SNP), or a gene expression mutation in a plant is provided. A mutant may be obtained via radiation, chemical treatment, UV irradiation, or natural mutation.

The above method comprises: a step of isolating genomic DNA or RNA from a mutant plant, a plant of a different variety or breeding line and carrying out reverse transcription for cDNA synthesis if RNA is isolated; a step of amplifying a gene fragment containing a glycoalkaloid biosynthetic enzyme gene from DNA using a DNA amplification technique; and a step of determining the existence of a mutation in the DNA. A commercially available kit (e.g., DNeasy or RNeasy (QIAGEN)) can be used in a method for extracting DNA or RNA. Also, a commercially available kit (e.g., a SuperScript First-Strand System (Invitrogen)) can be used for cDNA synthesis. For example, techniques such as the so-called PCR and LAMP techniques can be used as methods for amplifying a gene fragment using a DNA amplification technique. Such techniques are included in a group of techniques involving the use of a polymerase so as to amplify (i.e., to increase the number of copies of) a specific DNA sequence in a continuous polymerase reaction. Such reaction can be employed instead of cloning. In such case, nucleic acid sequence information is the only requirement for the reaction. In order to carry out DNA amplification, primers complementary to a DNA sequence to be amplified are designed. Then, the primers are produced via automatic DNA synthesis. DNA amplification methods are known in the art, and thus a person skilled in the art can readily carry out such a method based on teachings and instructions described herein. Some PCR methods (and related techniques) are described in, for example, U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188, and "PCR Protocols: A guide to method and applications" edited by Innis et al.

In the step of determining the existence of a polymorphism or mutation in DNA, a detection method using homology between a mutant gene and a normal gene may be used. Examples of such method include the nucleotide sequence determination method (Applied Biosystems) and the TILLING method comprising detecting a mutant using an enzyme capable of cleaving one member of a mismatch pair (Till et al., 2003, Genome Res 13: 524-530). Detection can be carried out by comparing sequence data obtained by such method with the nucleotide sequence of a gene fragment shown in SEQ ID NO: 2, 4, or 5 or the nucleotide sequence of a gene fragment shown in SEQ ID NO: 19, 21, or 22.

In the step of determining a difference in the mRNA amount, the above cDNA is subjected to quantitative PCR which may be, for example, real-time PCR (with, for example, a LightCycler.RTM. (Roche Diagnostics)) with the use of primers produced based on the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or the nucleotide sequence shown in SEQ ID NO: 19 or 21. Then, a difference in the mRNA amount can be determined based on a comparison of the obtained result and the cDNA amount derived from the variety, "Sassy".

In a particularly preferable embodiment, the above defined method for determining the existence of a mutation of a glycoalkaloid biosynthetic enzyme gene is applied to a material obtained from a potato (Solanum tuberosum), which is a solanaceous plant (Solanaceae).

According to the above mutation and/or polymorphism determination method, a mutation or polymorphism of a gene encoding a glycoalkaloid biosynthetic enzyme can be identified at the nucleotide level. In addition, a plant in which a gene encoding a glycoalkaloid biosynthetic enzyme has a mutation and/or polymorphism can be selected. The present invention encompasses the thus obtained plant in which a gene encoding a glycoalkaloid biosynthetic enzyme has a mutation and/or polymorphism.

In addition, by determining a mutation or polymorphism or differences in the mRNA amount and by analysing a glycoalkaloid content described below, a plant in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or the activity of a glycoalkaloid biosynthetic enzyme has been altered can be selected.

Here, "alteration of the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or the activity of a glycoalkaloid biosynthetic enzyme" refers to a modification of the ability to express a gene or glycoalkaloid biosynthetic enzyme activity caused by mutation such as artificial mutation, and an alteration in the ability to express a gene or glycoalkaloid biosynthetic enzyme activity due to the existence of a polymorphism.

"Modification of the glycoalkaloid biosynthetic enzyme activity in a plant caused by mutation" refers to modification of such activity in an existing variety of a plant species of interest. Such existing varieties include wild-type varieties. However, even if a wild-type variety is a naturally occurring variety, it is not included among the existing varieties if it is not an existing industrially applicable variety. The existing varieties include all varieties that have been confirmed to exist when a plant in which the glycoalkaloid biosynthetic enzyme activity has been modified has been obtained. A variety produced by artificial manipulation such as hybridization or gene manipulation is also included. In addition, in the case of modification of the activity, alteration in the activity does not necessarily take place compared to all existing varieties. If the activity in a specific existing variety is modified, the modified variety can be included among "plants having modified glycoalkaloid biosynthetic enzyme activity." Such "plants having modified glycoalkaloid biosynthetic enzyme activity" also include plants in which activity has been modified via spontaneous mutation without artificial manipulation. By the method of the present invention, a plant in which activity has been altered spontaneously can be selected and established as a new variety. In addition, in a case in which an existing variety is subjected to mutagenesis treatment so as to produce a plant having modified glycoalkaloid biosynthetic enzyme activity, the plant compared with the produced plant may be an existing variety identical to the variety subjected to mutagenesis treatment. Alternatively, it may be a different existing variety. Further, it is also possible to obtain a novel plant variety in which the mutation in the gene is fixed and the ability to express a glycoalkaloid biosynthetic enzyme gene or glycoalkaloid biosynthetic enzyme activity has been modified. Such plants can be obtained by crossing plants selected from nature or produced by mutagenesis treatment, and having a mutation or polymorphism in the gene encoding a glycoalkaloid biosynthetic enzyme.

If a plant is a potato (Solanum tuberosum), examples of existing varieties thereof include "Cynthia," "Sassy," "Cherie," "Irish Cobbler (i.e., Danshaku)," "May Queen," and "Sayaka (Norin registration number: Norin No. 36)." Here, "a plant in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or glycoalkaloid biosynthetic enzyme activity has been modified compared with an existing variety" refers to a plant in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme has been enhanced or reduced compared with an existing variety. It further refers to a plant in which glycoalkaloid biosynthetic enzyme activity has increased or decreased compared with an existing variety. The present invention also encompasses a plant in which the ability to express a gene encoding a glycoalkaloid biosynthetic enzyme or glycoalkaloid biosynthetic enzyme activity has been modified compared with an existing variety.

A plant in which the activity of biosynthetic enzyme of a glycoalkaloid that is a toxic substance has decreased is particularly preferable. In such plant, the amount of a glycoalkaloid biosynthetic enzyme synthesized is low or a glycoalkaloid biosynthetic enzyme cannot be synthesized. Also, the glycoalkaloid biosynthetic enzyme content is low or a glycoalkaloid biosynthetic enzyme is absent in the plant. Alternatively, the glycoalkaloid biosynthetic enzyme activity is low or nonexistent in the plant. Accordingly, the plant has low glycoalkaloid content or lacks glycoalkaloids. For instance, if the plant is a potato, a glycoalkaloid such as chaconine or solanine is not synthesized, and thus the amount of a synthesized or existing glycoalkaloid such as chaconine or solanine in the potato tubers is low. In addition, if the plant is a tomato, a glycoalkaloid such as tomatine is not synthesized, and thus the amount of a synthesized or existing glycoalkaloid such as tomatine in tomato fruits is low.

If the plant in which the glycoalkaloid biosynthetic enzyme activity is low or nonexistent is a potato, a glycoalkaloid such as chaconine or solanine is not synthesized in tubers, or the amount of a glycoalkaloid such as chaconine or solanine synthesized in tubers is lower than that in an existing variety described above. Also, in such a case, the content of a glycoalkaloid such as chaconine or solanine present in tubers may be low.

7. Glycoalkaloid Analysis and Purification

Known glycoalkaloid content analysis methods and glycoalkaloid purification methods using liquid chromatography have been disclosed by, for example, Matsuda et al. (Phytochem. Anal. 15:121-124, 2004) and Kozukue et al. (J. Agric. Food Chem. 52: 2079-2083, 2004). However, in any case, pretreatment of samples is labor-consuming, sufficient measurable limits cannot be achieved, or the use of a strong acid imposes a burden upon a column or apparatus. This has been problematic. Therefore, according to the present invention, the method using liquid chromatography with an alkali-resistant reversed-phase chromatography column capable of efficiently purifying GAs (glycoalkaloids) and performing high-precision analysis (disclosed in JP Patent Application No. 2009-170317) can be used. An example in which this method is applied to a potato (Solanum tuberosum L.) is described in Example 5.

Any column can be used as a column in the above method as long as it is an excellent alkali-resistant column. An example of an excellent alkali-resistant column that can be used is an ethylene-crosslinked column. Preferably, a column with the brand name of Xbridge (trademark) (Waters) is used. Particularly preferably, the Waters XBridge.TM. Shield RP18 (Waters) and the Waters XBridge.TM. C18 are used. According to the method of the present invention, the XBridge.TM. Shield RP18 column is advantageous in that the time required for treatment of a single sample is short. Meanwhile, the Waters XBridge.TM. C18 column is advantageous in that it has good durability.

An alkaline buffer can be used as a mobile phase for liquid chromatography. Preferably, a volatile alkaline buffer is used. When a sample purified by liquid chromatography is subjected to mass spectrometry, it is convenient to use a volatile alkaline buffer as a mobile phase so as to prevent the alkaline buffer from remaining in the sample. Examples of a volatile alkaline buffer that can be used include triethylamine and ammonium hydrogen carbonate. However, ammonium hydrogen carbonate having excellent buffering effects is preferably used.

The concentration of ammonium hydrogen carbonate used as a mobile phase is 5 to 20 mM, preferably 5 to 15 mM, and more preferably 10 mM. The pH of ammonium hydrogen carbonate can be adjusted to preferably pH 9.0 to 11.0 and more preferably the pH 10.0. If the pH of a mobile phase is adjusted to 10.0, the buffering performance of ammonium hydrogen carbonate can be further improved.

GAs may be eluted into a mobile phase using an alkaline buffer and an organic solvent with an isocratic method or a gradient method. However, it is preferable to carry out elution with an isocratic method, which is convenient in terms of operation.

Examples of an organic solvent that can be used for a mobile phase include, but are not limited to, methanol, ethanol, tetrahydrofuran (THF), and acetonitrile (MeCN). Preferably, MeCN is used.

In an isocratic method, an alkaline buffer and an organic solvent, which are preferably an ammonium hydrogen carbonate solution and MeCN, are adequately used at a ratio of 30:70 to 70:30 and preferably 40:60 to 60:40 depending on the type of the GA of interest. For instance, if the GA of interest is .alpha.-solanine or .alpha.-chaconine, an alkaline buffer and an organic solvent, which are preferably an ammonium hydrogen carbonate solution and MeCN, are used at a ratio of 40:60. If the GA of interest is .alpha.-tomatine, an alkaline buffer and an organic solvent, which are preferably an ammonium hydrogen carbonate solution and MeCN, are used at a ratio of 60:40.

Liquid chromatography can be carried out using a commercially available HPLC apparatus. Column equilibration and flow rate can be adequately determined depending on the column size or sample volume.

Fractions obtained by liquid chromatography can be analyzed using mass spectrometry, a UV or multi-wavelength detector, or the like described below.

Preferably, a plant-derived sample is pretreated as described below prior to liquid chromatography for crude purification.

A plant-derived sample contains GAs and various polymers as foreign substances (e.g., starch, proteins, and cellulose). Thus, it is necessary to remove polymers contained as foreign substances in a sample and subject GAs to crude purification and washing in order to achieve efficient purification and high-precision analysis of GAs.

As a method for removing polymers as foreign substances, a general method used by a person skilled in the art such as an alcohol precipitation method can be used. Ethanol or methanol can be used as alcohol. However, methanol is preferable. In such case, acid is added to alcohol so as to extract GAs in salt form with good efficiency. Examples of acids that can be used include, but are not limited to, acetic acid, hydrochloric acid, and formic acid. Preferably, formic acid is added. The amount of acid added to alcohol is adequately determined so that the GA of interest is not damaged. If formic acid is used, it is added to alcohol so as to result in a concentration of 0.1% to 2% (v/v) and preferably 0.1% (v/v). If an acid other than formic acid is used, it can be added until the concentration thereof reaches the level equivalent to the above normality of added formic acid.

In the case of a conventional sample preparation method (see Matsuda et al., Phytochem. Anal. 15: 121-124, 2004), lengthy and complex pretreatment is required. This comprises steps of homogenizing a sample for many hours, carrying out centrifugation a plurality of times to remove polymers contained in the sample in large amounts as foreign substances such as starch, and filtering the resultant. Meanwhile, according to the preparation method of the present invention, it is possible to rapidly remove polymers as foreign substances such as starch via alcohol precipitation from plant pieces obtained by rapid disruption as described above. Therefore, sample preparation can be carried out in a short time in a convenient manner.

After alcohol precipitation, the supernatant containing GAs is diluted with acid such as 0.1% to 2% (v/v) formic acid or acetic acid and preferably 0.1% (v/v) formic acid. The dilution is subjected to liquid chromatography under the above conditions.

Fractions purified via liquid chromatography can be further subjected to mass spectrometry. In such case, mass spectrometry may be carried out by LC-MS, which is a technique that combines liquid chromatography with mass spectrometry.

Mass spectrometry can be carried out by sector field mass spectrometry, double-focusing sector field mass spectrometry, quadrupole mass spectrometry, quadrupole ion trap mass spectrometry, time-of-flight mass spectrometry, ion-cyclotron mass spectrometry (Fourier transform mass spectrometry), or the like.

Examples of a method for ionizing a sample for mass spectrometry that can be used include an EI (electron ionization) method, a CI (chemical ionization) method, a DEI (desorption electron ionization) method, a DCI (desorption chemical ionization) method, an FAB (fast atom bombardment) method, an FRIT-FAB (FRIT-fast atom bombardment) method, an ESI (electrospray ionization) method, and an MALDI (matrix-assisted laser desorption ionization) method.

Mass spectrometry conditions are specifically described in the Examples. However, a person skilled in the art can adequately determine conditions depending on the type of GA used as an analyte.

It is possible to analyze reference analytes of GAs using LC-MS to create a calibration curve according to a general method used by a person skilled in the art. .beta.-D-glucosamine pentaacetate can be used as an internal reference substance for a potato-derived sample, particularly in an .alpha.-solanine or .alpha.-chaconine analysis system. However, it is preferable to use brassinolide having a steroid skeleton similar to that of .alpha.-solanine or .alpha.-chaconine. Meanwhile, a water-soluble amine is preferably used for a tomato-derived sample, particularly in an .alpha.-tomatine analysis system. Examples of a water-soluble amine that can be used as an internal reference substance include serine methyl ester and alanine methyl ester. However, alanine methyl ester is particularly preferable because sufficient retention in a column is achieved. Therefore, the reliability of quantitative analysis can be significantly improved using brassinolide for a potato-derived sample and alanine methyl ester for a tomato-derived sample.

According to the method of the present invention, a column with a size widely used for HPLC can be used. The conditions used herein can be directly applied to analysis using a UV or multi-wavelength detector.

EXAMPLES

The present invention is hereafter described in greater detail with reference to the following examples, although the present invention is not limited thereto.

(Example 1) Acquisition of the Full-Length Sequence of Candidate Glycoalkaloid Biosynthetic Gene C

mRNA was extracted from sprouts of a potato (Solanum tuberosum) variety, "Sassy" using RNeasy (QIAGEN). Total cDNA synthesis was carried out using a SuperScript First-Strand System (Invitrogen). It is said that aglycone of a glycoalkaloid is formed with cholesterol, but this has not been proved (Non-Patent Literature 1). However, assuming that the aglycone is formed with a cholesterol-related compound, there must be some steps of hydroxylation. In this case, at least three types of enzymes (i.e., cytochrome P450 monooxygenase, dioxygenase, and/or NADPH-flavin reductase) are probably involved in the steps of hydroxylation. Of these, cytochrome P450 monooxygenase was designated herein as a target. As a gene expressed in a potato, the TC135549 gene, for which many EST clones have been isolated from sprouts, was selected based on the information disclosed in Release 11.0 of the DFCI Potato Gene Index (compbio.dfci.harvard.edu/tgi/plant.html).

PCR was performed based on the above sequence using primers (U841: GCTTGCTCTGTTCTTGTACATCTC (SEQ ID NO: 6); and U842: TGAAAAGCAGAATTAGCAGCA (SEQ ID NO: 7)) (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 3 minutes; and 72.degree. C. for 10 minutes). The amplification product was subjected to cloning using a TOPOTA cloning kit for sequencing (Invitrogen). Further, the nucleotide sequence was determined using an ABI310 (Applied Biosystems). The sequence comprising the ORF region is shown in SEQ ID NO: 2 and the amino acid sequence of an enzyme encoded by the cDNA sequence is shown in SEQ ID NO: 1. The homologous gene of tomato used herein corresponds to TC192845 in the DFCI Tomato Gene Index as in the above case. The sequence comprising the ORF region is shown in SEQ ID NO: 4 and the amino acid sequence of an enzyme encoded by the cDNA sequence is shown in SEQ ID NO: 3. As a result of a comparison of the nucleotide sequences of these genes, homology therebetween was found to be 95% (FIGS. 1A-1C). In addition, the homologous gene of tomato was identical to the sequence reported by Bartoszewski et al. (Acta Physiol. Plant. 22, 269-271 (2000)). Bartoszewski et al. reported that a sequence homologous (75%) to the sequence of CYP72A2 found in tobacco was identified, that wound-induced expression of the gene takes place, that the gene exhibits circadian rhythm, and that the gene is frequently expressed in young tissues. However, no results or discussions about the functions of the gene were reported. Therefore, nothing about the involvement of the gene in glycoalkaloid biosynthesis was reported.

(Example 2) Isolation of the Genomic Gene of Candidate Glycoalkaloid Biosynthetic Gene C

Genomic DNA was extracted from "Sassy" using RNeasy (QIAGEN). PCR was performed using the primers used in Example 1 for determination of the nucleotide sequence of the full-length genomic DNA (SEQ ID NO: 5). The DNA was found to contain four introns.

(Example 3) Vector Construction for Production of a Transformant Having the Suppressed Candidate Glycoalkaloid Biosynthetic Gene C

The above gene was suppressed through transformation by a method comprising inducing expression of a gene fragment of a reverse complementary strand structured to be driven by a powerful promoter (which is generally referred to as an RNAi method for plants) (Chuang and Meyerowitz, Proc Natl Acad Sci, USA, 97, 4985-90 (2000); Wesley et al., Plant J., 27, 581-90 (2001)). The full-length cDNA obtained in Example 1 was subjected to PCR using primers (U724: GAGCTCTAGAGAAGCAAAGAAAACACC (SEQ ID NO: 8); and U725: GGATCCATATGCTAACCAATTCCTCCCATC (SEQ ID NO: 9)) (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds; and 72.degree. C. for 10 minutes). Thus, a gene fragment was obtained. A pKT226 vector for plant transformation was prepared using a pKT11 binary vector (JP Patent Publication (Kokai) No. 2001-161373 A) as a reference vector by ligating a cauliflower mosaic virus 35S RNA promoter, the gene fragment (in the forward direction), the 7th intron of the Arabidopsis thaliana phytoene desaturase gene (AT4g14210), the gene fragment (in the reverse direction), and a cauliflower mosaic virus 35S RNA terminator in such order (FIG. 2).

(Example 4) Production of a Transformed Potato Plant

The vector prepared in Example 3 was introduced into the Agrobacterium tumefaciens GV3110 strain by the electroporation method (Gelvin and Schilperoor, Plant Molecular Biology Manual, C2, 1-32 (1994), Kluwer Academic Publishers). The Agrobacterium tumefaciens GV3110 strain comprising the vector was subjected to shake culture in a YEB liquid medium supplemented with 50 ppm Kanamycin (5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, and 2 mM magnesium sulfate (pH 7.2)) at 28.degree. C. for 12 hours. The culture liquid (1.5 ml) was centrifuged at 10,000 rpm for 3 minutes for harvest, followed by washing with an LB medium (1 ml) for removal of Kanamycin. Further, centrifugation was performed at 10,000 rpm for 3 minutes for harvest. The resultant was resuspended in an MS medium containing 3% sucrose (1.5 ml) (Murashige & Skoog, Physiol. Plant., 15, 473-497 (1962)). Thus, a bacterial suspension for infection was obtained.

Transformation of a potato was carried out according to a conventional method (Monma (1990), Plant Biotechnology 7: 57-63). Microtubers obtained from a potato variety, "Sassy" (Kirin Agribio Company, Limited.) were sliced to thicknesses of 2 to 3 mm, and thus materials for Agrobacterium infection were prepared. The slices were immersed in the above Agrobacterium cell suspension and then placed on sterilized filter paper for removal of excessive Agrobacterium cells. The slices were placed on an MS medium (supplemented with Zeatin (1 ppm), IAA (0.1 ppm), acetosyringone (100 .mu.M), and agar (0.8%)) in a petri dish. Culture was carried out under conditions comprising illumination for 16 hours (photon flux density: 32 .mu.E/m.sup.2s)/non-illumination for 8 hours at 25.degree. C. for 3 days. Next, culture was carried out in a medium supplemented with carbenicillin (250 ppm) instead of acetosyringone for 1 week. Then, the culture product was further transferred onto a medium supplemented with Kanamycin (50 ppm), followed by subculture at 2-week intervals. During subculture, adventitious bud formation and then shoot formation took place. The grown shoots were placed on an MS medium containing carbenicillin (250 ppm) and Kanamycin (100 ppm) and lacking plant growth regulators. Each individual having a Kanamycin-resistant gene as a foreign gene was detected from among Kanamycin-resistant plants grown from the rooting shoots by PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and 72.degree. C. for 10 minutes), thereby confirming that the redifferentiated plant was a transformed plant. Here, the following primers were used as primers capable of specifically amplifying the Kanamycin-resistant gene sequence: TAAAGCACGAGGAAGCGGT (SEQ ID NO: 10); and GCACAACAGACAATCGGCT (SEQ ID NO: 11). Accordingly, 28 transformed potato plant lines transfected with the pKT226 vector were obtained.

(Example 5) Analysis of the Glycoalkaloid Content and the Expression of Candidate Gene C in Transformed Plants

In vitro stems of the 28 lines obtained in Example 4 were allowed to grow for one month after subculture. Two to four stems were collected to adjust the weight to approximately 100 mg. The glycoalkaloid content was determined by the method comprising liquid chromatography using an alkali-resistant reversed-phase chromatography column (which has been disclosed in Japanese Patent Application No. 2009-170317) described below.

Analysis of GAs (.alpha.-Solanine and .alpha.-Chaconine) Contained in a Potato

1. Sample Preparation

In vitro stems of the 28 individuals obtained in Example 4 were allowed to grow for one month after subculture. Two to four stems were collected to adjust the weight to approximately 100 mg. 80% MeOH aq. (990 .mu.L) containing 0.1% formic acid and brassinolide (Brassino Co., Ltd.) (10 .mu.g/10 .mu.L) used as an internal reference were added thereto, followed by disruption using a mixer mill ( 1/25 sec, 5 min, 4.degree. C.). The obtained disruptant was centrifuged (10,000 rpm, 5 min), followed by alcohol precipitation. Then, a portion of the supernatant (25 .mu.L) was collected and adjusted to 500 .mu.L with a 0.1% formic acid solution. The thus obtained sample was subjected to LC-MS under the conditions described below. LCMS-2010EV (Shimadzu Corporation) was used as an LC-MS apparatus.

2. LC-MS Conditions

(i) LC Conditions

An ethylene-crosslinked column (XBridge.TM. Shield RP18-5 (.PHI.2.1.times.150 mm, Waters)) having excellent alkali resistance was employed for the LC system. The following mobile phases were used at a ratio of A:B=40:60 with the above sample solvent under isocratic conditions: mobile phase A: 10 mM ammonium hydrogen carbonate solution (pH 10); and mobile phase B: MeCN. Other conditions applied herein are described below.

Flow rate: 0.2 mL/min

Column oven: 40.degree. C.

(ii) MS Conditions

First, the MS spectrum for each component was confirmed via scan mode (see FIG. 3). As a result, a detection method was used via SIM mode (m/z: 481 (brassinolide), 869 (.alpha.-solanine), and 853 (.alpha.-chaconine)).

Other MS conditions used herein are described below.

MS detection: Positive ion mode

Ionization method: ESI

Event time: 1 sec

Detector voltage: 1.5 kV

Analysis time: 8 min

3. Creation of Calibration Curves Using Reference Products (.alpha.-Solanine, .alpha.-Chaconine, and Brassinolide)

.alpha.-solanine (Wako Pure Chemical Industries, Ltd.) (2 mg) and .alpha.-chaconine (Sigma-Aldrich) (2 mg) were separately dissolved in a 0.1% (v/v) formic acid solution (1 mL) (so as to obtain a 2 .mu.g/.mu.L solution for each product). Equivalent volumes of the two different solutions were mixed so as to prepare a solution containing .alpha.-solanine and .alpha.-chaconine at a concentration of 1 .mu.g/.mu.L (=1000 ng/.mu.L). The solution was diluted 10 times with a 0.1% (v/v) formic acid solution in a stepwise manner, followed by LC-MS. Thus, calibration curves were created. In addition, measurable limits of the both substances were obtained.

Brassinolide (Brassino Co., Ltd.) (1 mg) was dissolved in an MeOH solution (1 mL) (1 .mu.g/.mu.g). The resulting solution was diluted 10 times with 50% (v/v) aqueous MeOH in a stepwise manner, followed by LC-MS. Thus, a calibration curve was created.

FIG. 4 shows calibration curves created for .alpha.-solanine, .alpha.-chaconine, and brassinolide. Good linearity with a confidence coefficient of 0.99 or more was confirmed within the range of 0.05 to 50 ng for .alpha.-solanine and .alpha.-chaconine as shown in FIG. 4. For the both substances, when the content exceeded 100 ng, signal saturation was observed, resulting in loss of linearity. In addition, the measurable limit was 0.02 ng (2 .mu.L per injection) for both substances.

Meanwhile, good linearity was confirmed within the range of 2 to 200 ng for brassinolide (see FIG. 4). When the content was not less than 500 ng, this caused signal saturation as in the above case.

FIG. 5 shows typical chromatograms of the reference products (.alpha.-solanine, .alpha.-chaconine, and brassinolide).

4. LC-MS Analysis of GAs in a Potato Using Brassinolide as an Internal Reference

Each sample prepared in 1 above (10 .mu.L or 20 .mu.L) was injected into an LC-MS system under the above conditions.

The recovery rate of brassinolide used as an internal reference was found to be 50% to 110%. Correction was carried out based on the quantitative value of brassinolide, followed by quantitative determination of the amounts of .alpha.-solanine and .alpha.-chaconine in each sample based on the above calibration curves. The amounts of .alpha.-solanine and .alpha.-chaconine per 100 mg (FW) of each sample were calculated.

FIG. 6 shows typical chromatograms for the analysis.

The amounts of accumulated glycoalkaloids were found to be low with good reproducibility in 4 lines (#20, #35, #45, and #67) selected from among 28 lines. Therefore, as described above, in vitro stems of the 4 lines were disrupted in liquid nitrogen. A half portion of each disruptant was used for determination of the glycoalkaloid content. The other half portion thereof was subjected to mRNA extraction using RNeasy (QIAGEN). Total cDNA synthesis was carried out using a SuperScript First-Strand System (Invitrogen). The amounts of accumulated glycoalkaloids in individual of these lines were found to be remarkably lower than those in a non-transformant (2 individuals) (FIG. 7). Further, as a result of RT-PCR using primers (U724: GAGCTCTAGAGAAGCAAAGAAAACACC (SEQ ID NO: 12); and U840: GGGCATGAACATAGGAAGGA (SEQ ID NO: 13)), it was found that mRNA was expressed at a remarkably low level or was impossible to observe in any of the individuals (FIG. 8). These results revealed that suppression of expression of the candidate gene C caused significant reduction of glycoalkaloid accumulation, and they also revealed that the candidate gene C was a gene encoding a glycoalkaloid biosynthetic enzyme. The in vitro plants of the 4 lines were allowed to proliferate with the non-transformant. Three individuals of each line were habituated in commercially available culture soil for vegetables and cultivated in a biohazard greenhouse according to a general method, followed by harvest of tubers. Each of the individuals of the 4 lines (#20, #35, #45, and #67) was found to be comparable in growth to the non-transformant. Although line #35 was found to have a low average weight of 1 tuber, it was possible to harvest tubers having average weights comparable to the average weight of the non-transformant (table 1).

TABLE-US-00001 TABLE 1 Potato transformant tuber yield Total Number Average weight of Standard weight of of 1 Standard Line number tubers deviation 1 tuber (g) stock (g) deviation Non- 9.7 5.5 13.4 129.3 60.8 transformant #20 8.7 3.8 15.2 131.7 6.4 #35 12.0 5.0 8.5 102.2 40.9 #45 12.3 3.5 12.1 149.2 4.9 #67 11.3 1.2 11.1 125.5 5.2

Further, the epidermis of the center portion of each of three harvested tubers of each line was peeled to result in thicknesses of approximately 1 mm. Then, the glycoalkaloid content was analyzed in the above manner. As a result, surprisingly, it was confirmed that the glycoalkaloid content in tubers was extremely low, having a value that was lower than that determined in the same manner for "Sayaka," which is a variety known to have low glycoalkaloid content (FIG. 9)

(Example 6) Production of Transformed Tomato Plants

Tomato transformation was carried out according to a conventional method (Sun et al. (2006) Plant Cell Physiol. 47: 426-431). The Agrobacterium tumefaciens AGL0 strain comprising a pKT226 vector prepared in Example 3 was cultured so as to obtain a bacterial suspension for infection. Sections (thickness: 5 mm or less) of cotyledons of a plant of a tomato (Solanum lycopersicum) experimental line called "Micro-Tom" obtained via sterile seeding were immersed in the above Agrobacterium suspension for infection for 10 minutes and then placed on sterilized filter paper for removal of excessive Agrobacterium cells. The leaf sections were placed on a coculture MS medium (supplemented with zeatin (1.5 mg/l), acetosyringone (40 .mu.M), and Gelrite.RTM. (0.3%)) (Murashige & Skoog, Physiol. Plant., 15, 473-497 (1962)) in a petri dish. The petri dish was subjected to culture in a dark place at 25.degree. C. for 3 days. The sections were subjected to subculture at 2-week intervals using selective MS medium 1 (supplemented with zeatin (1.5 mg/l), Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)) under conditions comprising illumination for 16 hours (photon flux density: 32 .mu.E/m.sup.2s)/non-illumination for 8 hours at 25.degree. C. During subculture, adventitious bud formation and then shoot formation took place. In order to allow the shoots to further grow, the shoots were transplanted to selective MS medium 2 (supplemented with zeatin (1.0 mg/l), Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)). The grown shoots were rooted in a selective 1/2 concentration MS medium (supplemented with Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)). Each individual having a Kanamycin-resistant gene as a foreign gene was detected from among Kanamycin-resistant plants grown from the rooting shoots by PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and 72.degree. C. for 10 minutes), thereby confirming that the redifferentiated plant was a transformed plant. Here, the following primers were used as primers capable of specifically amplifying the Kanamycin-resistant gene sequence: TAAAGCACGAGGAAGCGGT (SEQ ID NO: 14); and GCACAACAGACAATCGGCT (SEQ ID NO: 15). Accordingly, 32 transformed tomato plant lines transfected with the pKT226 vector were obtained. The obtained 28 individuals were habituated in a greenhouse and cultivated for approximately 2 months. Three newly developed leaves were weighed to approximately 100 mg for each line. The glycoalkaloid content was determined as in the case of potato by the method comprising liquid chromatography using an alkali-resistant reversed-phase chromatography column described in Example 5. Note that, regarding analysis conditions, the following mobile phases were used at a ratio of A:B=60:40 with the above sample solvent under isocratic conditions: mobile phase A: 10 mM ammonium hydrogen carbonate solution (pH 10); and mobile phase B: MeCN. As a result, 7 out of 32 lines were found to have remarkably low tomatine content of 50 .mu.g or less per 100 mg of fresh weight (FW) (FIGS. 11A-11C).

(Example 7) Screening of Plants with Mutation of Candidate Glycoalkaloid Biosynthetic Gene C

Leaves were collected from 10 individuals of an in vitro plant obtained by subjecting a potato variety ("Sassy") to mutation treatment involving particle beam irradiation (an NIRS-HIMAC irradiation apparatus; a 0.1 Gy to 3 Gy argon ion beam (500 MeV/nucleon), a 0.2 Gy to 3 Gy neon ion beam (400 Mev/nucleon), or a 0.5 Gy to 5 Gy carbon ion beam (290 MeV/nucleon)) (provided by Dr. Okamura (chief researcher), Kirin Agribio Company, Limited.). Then, genomic DNA was obtained using DNeasy. The structural gene of the genomic DNA was subjected to PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 5 minutes; and 72.degree. C. for 10 minutes) using primers (U841: GCTTGCTCTGTTCTTGTACATCTC (SEQ ID NO: 16); and U842: TGAAAAGCAGAATTAGCAGCA (SEQ ID NO: 17)). Thus, the gene region was obtained. In addition, cloning was carried out using a TOPOTA cloning kit for sequencing. Further, the nucleotide sequence was determined using ABI310. As a result, it was found that a line having a mutated gene was not included among 10 stocks provided herein. However, it is possible to obtain a plant having a mutated gene by repeatedly carrying out the above procedures using a plant subjected to sufficient mutation treatment.

(Example 8) Acquisition of the Full-Length Sequence of Candidate Glycoalkaloid Biosynthetic Gene D

mRNA was extracted from sprouts of a potato (Solanum tuberosum) variety, "Sassy" using RNeasy (QIAGEN). Total cDNA synthesis was carried out using a SuperScript First-Strand System (Invitrogen). It is said that aglycone of a glycoalkaloid is formed with cholesterol, but this has not been proved (Non-Patent Literature 1). However, assuming that the aglycone is formed with a cholesterol-related compound, there must be some steps of hydroxylation. In this case, at least three types of enzymes (i.e., cytochrome P450 monooxygenase, dioxygenase, and NADPH-flavin reductase) are probably involved in the steps of hydroxylation. Of these, cytochrome P450 monooxygenase was designated herein as a target. As a gene expressed in a potato, the TC141445 gene, for which many EST clones have been isolated from sprouts, was selected based on the information disclosed in Release 11.0 of the DFCI Potato Gene Index (compbio.dfci.harvard.edu/tgi/plant.html).

PCR was performed based on the above sequence using primers (U883: AGCAATCAAACATGGGTATTG (SEQ ID NO: 23); and U876: TGATGTGAACTTGAGATTGGTG (SEQ ID NO: 24)) (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 3 minutes; and 72.degree. C. for 10 minutes). The amplification product was subjected to cloning using a TOPOTA cloning kit for sequencing (Invitrogen). Further, the nucleotide sequence was determined using an ABI310 (Applied Biosystems). The sequence comprising the ORF region is shown in SEQ ID NO: 19 and the amino acid sequence of an enzyme encoded by the cDNA sequence is shown in SEQ ID NO: 18. The homologous gene of tomato used herein corresponds to SGN-U567668 in the Lycopersicon Combined (Tomato) Unigenes in the sol genomics network (solgenomics.net/). The sequence comprising the ORF region is shown in SEQ ID NO: 21 and the amino acid sequence of an enzyme encoded by the cDNA sequence is shown in SEQ ID NO: 20. As a result of a comparison of the nucleotide sequences of these genes, homology therebetween was found to be 95% (FIGS. 11A and 11B). The genomic sequence of the homologous gene of tomato was identical to the sequence reported as SL1.00sc04687 with the genomic structure in the family Solanaceae genomic network (solgenomics.net/index.pl) which has been reported to comprise 4 introns. However, nothing about its functions has been reported on the website.

(Example 9) Isolation of the Genomic Gene of Candidate Glycoalkaloid Biosynthetic Gene D

Genomic DNA was extracted from "Sassy" using RNeasy (QIAGEN). PCR was performed using the primers used in Example 1 for determination of the nucleotide sequence of the full-length genomic DNA (SEQ ID NO: 22). The DNA was found to contain four introns.

(Example 10) Vector Construction for Production of a Transformant Having the Suppressed Candidate Glycoalkaloid Biosynthetic Gene D

The above gene was suppressed through transformation by a method comprising inducing expression of a gene fragment of a reverse complementary strand structured to be driven by a powerful promoter (which is generally referred to as an RNAi method for plants) (Chuang and Meyerowitz, Proc Natl Acad Sci, USA, 97, 4985-90 (2000); Wesley et al., Plant J., 27, 581-90 (2001)). The full-length cDNA obtained in Example 1 was subjected to PCR using primers (U726: GAGCTCTAGAGGTTAAGAGTTTGTGCCAACG (SEQ ID NO: 25); and U727: GGATCCATATGGCTTTCTCTTGCCAATCTG (SEQ ID NO: 26)) (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds; and 72.degree. C. for 10 minutes). Thus, a gene fragment was obtained. A pKT227 vector for plant transformation was prepared using a pKT11 binary vector (JP Patent Publication (Kokai) No. 2001-161373 A) as a reference vector by ligating a cauliflower mosaic virus 35S RNA promoter, the gene fragment (in the forward direction), the 3rd intron of the Arabidopsis thaliana phytoene desaturase gene (AT4g14210), the gene fragment (in the reverse direction), and a nopaline synthetase gene terminator in such order (FIG. 12).

(Example 11) Production of a Transformed Potato Plant

The vector prepared in Example 10 was introduced into the Agrobacterium tumefaciens GV3110 strain by the electroporation method (Gelvin and Schilperoor, Plant Molecular Biology Manual, C2, 1-32 (1994), Kluwer Academic Publishers). The Agrobacterium tumefaciens GV3110 strain comprising the vector was subjected to shake culture in a YEB liquid medium supplemented with 50 ppm Kanamycin (5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, and 2 mM magnesium sulfate (pH7.2)) at 28.degree. C. for 12 hours. The culture liquid (1.5 ml) was centrifuged at 10,000 rpm for 3 minutes for harvest, followed by washing with an LB medium (1 ml) for removal of Kanamycin. Further, centrifugation was performed at 10,000 rpm for 3 minutes for harvest. The resultant was resuspended in an MS medium containing 3% sucrose (1.5 ml) (Murashige & Skoog, Physiol. Plant., 15, 473-497 (1962)). Thus, a bacterial suspension for infection was obtained.

Transformation of a potato was carried out according to a conventional method (Monma (1990), Plant Biotechnology 7: 57-63). Microtubers obtained from a potato variety, "Sassy" (Kirin Agribio Company, Limited.) were sliced to thicknesses of 2 to 3 mm, and thus materials for Agrobacterium infection were prepared. The slices were immersed in the above Agrobacterium cell suspension and then placed on sterilized filter paper for removal of excessive Agrobacterium cells. The slices were placed on an MS medium (supplemented with Zeatin (1 ppm), IAA (0.1 ppm), acetosyringone (100 .mu.M), and agar (0.8%)) in a petri dish. Culture was carried out under conditions comprising illumination for 16 hours (photon flux density: 32 .mu.E/m.sup.2s)/non-illumination for 8 hours at 25.degree. C. for 3 days. Next, culture was carried out in a medium supplemented with carbenicillin (250 ppm) instead of acetosyringone for 1 week. Then, the culture product was further transferred onto a medium supplemented with Kanamycin (50 ppm), followed by subculture at 2-week intervals. During subculture, adventitious bud formation and then shoot formation took place. The grown shoots were placed on an MS medium containing carbenicillin (250 ppm) and Kanamycin (100 ppm) and lacking plant growth regulators. Each individual having a Kanamycin-resistant gene as a foreign gene was detected from among Kanamycin-resistant plants grown from the rooting shoots by PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and 72.degree. C. for 10 minutes), thereby confirming that the redifferentiated plant was a transformed plant. Here, the following primers were used as primers capable of specifically amplifying the Kanamycin-resistant gene sequence: TAAAGCACGAGGAAGCGGT (SEQ ID NO: 10); and GCACAACAGACAATCGGCT (SEQ ID NO: 11). Accordingly, 31 transformed potato plant lines transfected with the pKT227 vector were obtained.

(Example 12) Analysis of the Glycoalkaloid Content and the Expression of Candidate Gene D in Transformed Plants

Thirty one lines obtained in Example 11 were subjected to glycoalkaloid content measurement in the manner used in Example 5.

The amounts of accumulated glycoalkaloids were found to be low with good reproducibility in 4 lines (#9, #28, #45, and #59) selected from among 31 lines. Therefore, as described above, in vitro stems of the 4 lines were disrupted in liquid nitrogen. A half portion of each disruptant was used for determination of the glycoalkaloid content. The other half portion thereof was subjected to mRNA extraction using RNeasy (QIAGEN). Total cDNA synthesis was carried out using a SuperScript First-Strand System (Invitrogen). The amounts of accumulated glycoalkaloids in individuals of these lines were found to be remarkably lower than those in a non-transformant (2 individuals) (FIG. 13). Further, as a result of RT-PCR using primers (U871: TCGGGTGAGTTCAGAAAACC (SEQ ID NO: 27); and U727: GGATCCATATGGCTTTCTCTTGCCAATCTG (SEQ ID NO: 28)), it was found that mRNA was expressed at a remarkably low level or was impossible to observe in any of the individuals (FIG. 14). These results revealed that suppression of expression of the candidate gene D caused significant reduction of glycoalkaloid accumulation, and they also revealed that the candidate gene D was a gene encoding a glycoalkaloid biosynthetic enzyme. The in vitro plants of the 4 lines were allowed to proliferate with the non-transformant. Three individuals of each line were habituated in commercially available culture soil for vegetables (in a single planter) and cultivated in a biohazard greenhouse according to a general method, followed by harvest of tubers. Each of the individuals of the 4 lines (#9, #28, #45, and #59) was found to be comparable in growth to each non-transformant (of the two transformants in two planters). There were no differences in terms of the number of tubers among the transformants. Meanwhile, almost all of the transformants were found to be inferior to the non-transformants in terms of the average weight of a single tuber and the total weight of a single stock. However, it was possible to harvest tubers comparable to those of the non-transformants in terms of the conditions other than the weight (table 2).

TABLE-US-00002 TABLE 2 Potato transformant tuber yield Average number Average weight Total weight of Line number of tubers of 1 tuber (g) 1 stock Non-transformant 9.3 20.6 192.2 Non-transformant 25.0 6.9 172.1 #9 8.3 5.9 49.0 #28 14.3 3.9 56.4 #45 11.7 7.9 91.9 #59 9.7 11.6 111.8

Further, the epidermis of the center portion of each of three harvested tubers of each line was peeled to result in thicknesses of approximately 1 mm. Then, the glycoalkaloid content was analyzed in the above manner. As a result, surprisingly, it was confirmed that the glycoalkaloid content in tubers was extremely low, having a value that was lower than that determined in the same manner for "Sayaka," which is a variety known to have low glycoalkaloid content (FIG. 15)

(Example 13) Production of Transformed Tomato Plants

Tomato transformation was carried out according to a conventional method (Sun et al. (2006) Plant Cell Physiol. 47: 426-431). The Agrobacterium tumefaciens AGL0 strain comprising a pKT227 vector prepared in Example 10 was cultured so as to obtain a bacterial suspension for infection. Sections (thickness: 5 mm or less) of cotyledons of a plant of a tomato (Solanum lycopersicum) experimental line called "Micro-Tom" obtained via sterile seeding were immersed in the above Agrobacterium suspension for infection for 10 minutes and then placed on sterilized filter paper for removal of excessive Agrobacterium cells. The leaf sections were placed on a coculture MS medium (supplemented with zeatin (1.5 mg/l), acetosyringone (40 .mu.M), and Gelrite.RTM. (0.3%)) (Murashige & Skoog, Physiol. Plant., 15, 473-497 (1962)) in a petri dish. The petri dish was subjected to culture in a dark place at 25.degree. C. for 3 days. The sections were subjected to subculture at 2-week intervals using selective MS medium 1 (supplemented with zeatin (1.5 mg/l), Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)) under conditions comprising illumination for 16 hours (photon flux density: 32 .mu.E/m.sup.2s)/non-illumination for 8 hours at 25.degree. C. During subculture, adventitious bud formation and then shoot formation took place. In order to allow the shoots to further grow, the shoots were transplanted to selective MS medium 2 (supplemented with zeatin (1.0 mg/l), Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)). The grown shoots were rooted in a selective 1/2 concentration MS medium (supplemented with Kanamycin (100 mg/l), Augmentin (375 mg/l), and Gelrite.RTM. (0.3%)). Each individual having a Kanamycin-resistant gene as a foreign gene was detected from among Kanamycin-resistant plants grown from the rooting shoots by PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1 minute; and 72.degree. C. for 10 minutes), thereby confirming that the redifferentiated plant was a transformed plant. Here, the following primers were used as primers capable of specifically amplifying the Kanamycin-resistant gene sequence: TAAAGCACGAGGAAGCGGT (SEQ ID NO: 14); and GCACAACAGACAATCGGCT (SEQ ID NO: 15). Accordingly, 21 transformed tomato plant lines transfected with the pKT227 vector were obtained. The obtained 21 lines were habituated in a greenhouse and cultivated for approximately 2 months. Three newly developed leaves were weighed to approximately 100 mg for each line. The glycoalkaloid content was determined as in the case of potato by the method comprising liquid chromatography using an alkali-resistant reversed-phase chromatography column described in Example 5. Note that, regarding analysis conditions, the following mobile phases were used at a ratio of A:B=60:40 with the above sample solvent under isocratic conditions: mobile phase A: 10 mM ammonium hydrogen carbonate solution (pH 10); and mobile phase B: MeCN. As a result, 6 out of 21 lines were found to have remarkably low tomatine content of 50 .mu.g or less per 100 mg of fresh weight (FIG. 16).

(Example 14) Screening of Plants with Mutation of Candidate Glycoalkaloid Biosynthetic Gene D

Leaves were collected from 10 individuals of an in vitro plant obtained by subjecting a potato variety ("Sassy") to mutation treatment involving particle beam irradiation (an NIRS-HIMAC irradiation apparatus; a 0.1 Gy to 3 Gy argon ion beam (500 MeV/nucleon), a 0.2 Gy to 3 Gy neon ion beam (400 Mev/nucleon), or a 0.5 Gy to 5 Gy carbon ion beam (290 MeV/nucleon)) (provided by Dr. Okamura (chief researcher), Kirin Holdings Company, Limited.). Then, genomic DNA was obtained using DNeasy. The structural gene of the genomic DNA was subjected to PCR (PCR conditions: 95.degree. C. for 5 minutes; 30 cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 5 minutes; and 72.degree. C. for 10 minutes) using primers (U883: AGCAATCAAACATGGGTATTG (SEQ ID NO: 27); and U876: TGATGTGAACTTGAGATTGGTG (SEQ ID NO: 28)). Thus, the gene region was obtained. In addition, cloning was carried out using a TOPOTA cloning kit for sequencing. Further, the nucleotide sequence was determined using ABI310. As a result, it was found that a line having a mutated gene was not included among 10 stocks provided herein. However, it is possible to obtain a plant having a mutated gene by repeatedly carrying out the above procedures using a plant subjected to sufficient mutation treatment.

All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The glycoalkaloid biosynthetic enzyme and the organism production/detection method using the gene of the present invention are useful for the development of production of glycoalkaloid compounds using organisms such as plants and selection of solanaceous plant varieties such as potatoes.

FREE TEXT OF SEQUENCE LISTINGS

Primers: SEQ ID NOS: 6 to 17 and 23 to 28

SEQUENCE LISTINGS

1

281498PRTSolanum tuberosum 1Met Ala Ile Ala Thr Val Ile Gly Ala Thr Ile Gly Ile Leu Ile Ala 1 5 10 15 Ile Phe Cys Val Lys Ser Phe Tyr Thr Leu Trp Trp Trp Pro Lys Met 20 25 30 Ile Glu Lys Lys Leu Lys Lys Glu Gly Ile His Gly Leu Pro Tyr Gln 35 40 45 Phe Leu Phe Gly Asn Leu Lys Glu Met Thr Arg Met Ser Arg Glu Ala 50 55 60 Lys Lys Thr Pro Leu Val Asn His Asp Ile Val Pro Trp Val Asn Pro 65 70 75 80 Phe Ile Leu His Leu Ser Lys Thr Tyr Glu Arg Leu Phe Val Met Trp 85 90 95 Ala Gly Pro Thr Pro Arg Ile Val Val Ser Asp Pro Lys Leu Ile Lys 100 105 110 Glu Val Val Asn Arg His Asn Glu Phe Gln Lys Pro Gln Ala Asn Ala 115 120 125 Phe Ile Asp Met Phe Val Thr Gly Leu Ala Ser Tyr Asn Gly Gln Lys 130 135 140 Trp Asp His His Arg Lys Ile Leu Asn Pro Ala Phe His Ile Glu Lys 145 150 155 160 Ile Lys Arg Leu Tyr Pro Ala Phe Cys Glu Cys Cys Asp Glu Met Ile 165 170 175 Asn Arg Trp Glu Glu Leu Val Ser Lys Ser Gly Ser Cys Glu Leu Asp 180 185 190 Val Ala Asp Glu Phe Leu Asn Val Gly Gly Asp Val Ile Ser Arg Ala 195 200 205 Ala Phe Gly Ser Asn Ile Glu Glu Gly Arg Thr Ile Phe Ile Leu Gln 210 215 220 Lys Glu Gln Cys Asp Leu Ile Leu Ala Ser Pro Phe Thr Leu Phe Phe 225 230 235 240 Pro Leu Leu Arg Phe Phe Pro Thr Ala Ser Asn Arg Arg Ala Arg Tyr 245 250 255 Ile Tyr Lys Lys Val Leu Ser Leu Ile Asn Gly Ile Ile Glu Lys Lys 260 265 270 Lys Asp Thr Met Arg Arg Gly Val Ser Gln Ser Asp Asp Ile Leu Gly 275 280 285 Leu Leu Leu Lys Gly Gly Leu Ser Thr Thr Glu Ile Ile Glu Glu Cys 290 295 300 Lys Glu Phe Tyr Leu Ala Gly Gln Asp Thr Thr Thr Ala Leu Leu Ser 305 310 315 320 Trp Thr Leu Val Ala Leu Ser Met His Pro Glu Trp Gln Asp Lys Ala 325 330 335 Arg Asn Glu Val Phe Gln Val Leu Gly Lys Asn Lys Pro Lys Phe Glu 340 345 350 Asp Leu Asn Gln Leu Lys Ile Met Asn Met Ile Phe Gln Glu Val Leu 355 360 365 Arg Leu Tyr Pro Ala Leu Thr Leu Met Arg Ser Thr Val Lys Asn Thr 370 375 380 Lys Leu Gly Asp Met Thr Ile Pro Ala Gly Val Gln Ile Phe Val Pro 385 390 395 400 Ile Tyr Ile Ala His Arg Asp Pro Gln Val Trp Gly Asp Asp Ala Leu 405 410 415 Ile Phe Asn Pro Asn Arg Phe Ser Glu Gly Val Ser Lys Ala Ala Lys 420 425 430 Glu Pro Leu Tyr Phe Pro Phe Gly Trp Gly Pro Arg Met Cys Ile Gly 435 440 445 Asn Asn Phe Gly Met Ala Glu Ala Lys Leu Val Leu Ser Gln Ile Leu 450 455 460 Gln Arg Phe Trp Phe Lys Leu Ser Pro Ser Tyr Val His Ala Pro Gln 465 470 475 480 Ala Ile Leu Val Met Lys Pro Gln Tyr Gly Ala Gln Ile Ile Leu Asn 485 490 495 Lys Leu 21494DNASolanum tuberosum 2atggcaattg caacagtaat tggtgcaaca attggtattt tgatagcgat tttttgtgta 60aaatcgtttt acacattatg gtggtggcca aagatgatcg aaaagaagct gaagaaggaa 120ggtattcatg ggctgcccta ccaatttctg tttggaaatc tgaaagagat gacgagaatg 180tctagagaag caaagaaaac accgttagta aatcatgata tcgttccttg ggttaatcct 240tttattcttc atctttctaa aacttacgag agattatttg tgatgtgggc tggaccaaca 300cctcggattg tagtatcaga tccaaagcta attaaagaag tggtgaacag acataatgaa 360tttcagaagc ctcaagccaa tgcgttcatt gacatgtttg ttactggact tgctagttac 420aatggtcaaa aatgggacca ccatagaaag atactaaacc ctgcttttca tatagagaag 480attaagaggt tgtacccagc attttgtgag tgttgtgatg aaatgataaa tagatgggag 540gaattggtta gcaaaagtgg aagttgtgag ttggatgtgg cagatgaatt cctaaatgta 600ggtggagatg ttatatctag agctgctttt ggtagcaata ttgaagaagg aaggactatt 660ttcatacttc agaaagagca gtgcgatctt attttggctt ctccatttac tcttttcttt 720cccttactaa gattctttcc aacagcatca aacagaagag caagatacat ctacaagaaa 780gtgttatcat tgattaacgg aataatagag aagaaaaaag acactatgcg aagaggagtc 840tcacaaagtg atgatatttt agggttactc ttaaaaggag gactatcaac cactgaaata 900attgaagaat gtaaggaatt ctatcttgca ggacaagata caaccacagc tttgctctct 960tggacattgg ttgccttgag tatgcaccct gagtggcaag acaaagctag aaatgaagtc 1020tttcaagtcc ttggaaaaaa caaaccaaag tttgaggact tgaatcaatt aaaaataatg 1080aacatgatct tccaagaggt gttgagatta tatccagcac tcacccttat gcgaagcacc 1140gtaaagaaca ctaaattggg agatatgaca attcctgcag gagtacaaat atttgtgcct 1200atatatatag cacatcgcga tccccaagta tggggagacg atgcattgat attcaatcca 1260aataggttct cagaaggggt atccaaagct gcaaaagagc ccttgtattt cccctttggt 1320tggggtcctc gaatgtgcat tggtaataac tttggcatgg cagaagccaa gctcgtttta 1380tctcaaattc tgcagcgttt ttggtttaag ctctctcctt cctatgttca tgcccctcag 1440gcaatactcg ttatgaagcc tcagtatggt gctcagataa tcctcaacaa gctt 14943498PRTSolanum lycopersicum 3Met Ala Ile Val Thr Val Ile Gly Ala Thr Ile Gly Ile Leu Ile Ala 1 5 10 15 Leu Phe Phe Val Lys Ser Phe Tyr Thr Leu Trp Trp Trp Pro Lys Met 20 25 30 Ile Glu Lys Lys Leu Lys Lys Glu Gly Ile His Gly Gln Pro Tyr Gln 35 40 45 Phe Leu Phe Gly Asn Leu Lys Glu Met Thr Arg Met Ser Arg Glu Ala 50 55 60 Lys Lys Lys Pro Leu Val Asn His Asp Ile Val Pro Trp Val Asn Pro 65 70 75 80 Phe Ile Leu His Leu Ser Lys Thr Tyr Glu Arg Leu Phe Val Met Trp 85 90 95 Ala Gly Pro Thr Pro Arg Ile Thr Val Thr Asp Pro Lys Leu Ile Lys 100 105 110 Glu Val Val Asn Arg His Asn Glu Phe Gln Lys Pro Gln Ala Asn Ala 115 120 125 Phe Ile Asp Met Phe Val Thr Gly Leu Ala Ser Tyr Asn Gly Gln Lys 130 135 140 Trp Asp His His Arg Lys Ile Leu Asn Pro Ala Phe His Ile Glu Lys 145 150 155 160 Ile Lys Arg Leu Tyr Pro Ala Phe Cys Glu Cys Cys Asp Glu Met Ile 165 170 175 Asn Arg Trp Glu Asp Leu Val Ser Lys Thr Gly Ser Cys Glu Leu Asp 180 185 190 Val Ala Asp Glu Phe Leu Asn Val Gly Gly Asp Val Ile Ser Arg Ala 195 200 205 Ala Phe Gly Ser Asn Ile Glu Glu Gly Arg Thr Ile Phe Ile Leu Gln 210 215 220 Lys Glu Gln Cys Asp Leu Ile Leu Ala Ser Pro Phe Thr Leu Phe Phe 225 230 235 240 Pro Leu Leu Arg Phe Phe Pro Thr Glu Ser Asn Arg Arg Ala Arg Tyr 245 250 255 Ile Tyr Lys Lys Val Leu Ser Leu Ile Lys Gly Ile Ile Glu Lys Lys 260 265 270 Glu Asp Ala Met Arg Arg Gly Val Ser Glu Ser Asp Asp Ile Leu Gly 275 280 285 Leu Leu Leu Lys Gly Gly Leu Ser Thr Thr Glu Ile Ile Glu Glu Cys 290 295 300 Lys Glu Phe Tyr Leu Ala Gly Gln Asp Thr Thr Thr Ala Leu Leu Ser 305 310 315 320 Trp Thr Leu Val Ala Leu Ser Met His Pro Glu Trp Gln Asp Lys Ala 325 330 335 Arg Asn Glu Val Phe Gln Val Leu Gly Lys Asn Lys Pro Lys Phe Glu 340 345 350 Asp Leu Asn Gln Leu Lys Ile Met Asn Met Ile Phe Gln Glu Val Leu 355 360 365 Arg Leu Tyr Pro Ala Leu Thr Leu Met Arg Ser Thr Ser Lys Asp Thr 370 375 380 Lys Leu Gly Glu Met Thr Ile Pro Ala Gly Val Gln Ile Phe Val Pro 385 390 395 400 Ile Tyr Ile Ala His Arg Asp Pro Gln Val Trp Gly Asp Asp Ala Leu 405 410 415 Ile Phe Asn Pro Asn Arg Phe Ser Glu Gly Val Ser Lys Ala Ala Lys 420 425 430 Glu Pro Leu Tyr Phe Pro Phe Gly Trp Gly Pro Arg Met Cys Ile Gly 435 440 445 Asn Asn Phe Gly Met Ala Glu Ala Lys Leu Val Leu Ser Gln Ile Leu 450 455 460 Gln Arg Phe Trp Phe Lys Leu Ser Pro Ser Tyr Val His Ala Pro Gln 465 470 475 480 Ala Ile Leu Val Met Lys Pro Gln Tyr Gly Ala Gln Ile Ile Leu Asn 485 490 495 Lys Leu 41494DNASolanum lycopersicum 4atggcaattg ttacagtaat tggtgcaacg attggcattt tgatagccct attttttgta 60aaatcgtttt atacattatg gtggtggcca aagatgatcg aaaagaagct gaagaaggaa 120ggtattcatg gtcagccgta ccaatttctg tttggaaatc tgaaagagat gacgagaatg 180tctagagaag caaagaaaaa accattagta aatcacgata ttgttccttg ggtgaatcct 240tttattcttc atctttctaa aacttacgag agattatttg tgatgtgggc tggaccaacc 300cctcggatta cagtaacaga tccaaagcta ataaaagaag tggtgaacag acataatgaa 360tttcaaaagc ctcaagccaa tgccttcatt gatatgtttg ttactggact tgctagttac 420aatggtcaaa aatgggatca ccatagaaag atactaaacc cggcttttca tatagagaag 480attaagaggt tgtacccagc attttgcgag tgttgtgatg aaatgataaa tagatgggag 540gacttggtta gcaaaactgg aagttgtgaa ttggatgtag cagatgaatt tctaaatgta 600ggtggagatg ttatatcgag agctgctttt ggtagcaata ttgaagaagg aaggactatt 660ttcatacttc agaaagagca gtgcgatctt attttggctt ctccatttac tctcttcttt 720cccttactaa gattctttcc aacagaatca aacagaagag caagatacat ctacaaaaaa 780gtgttatcat tgatcaaagg aatcatagag aagaaagaag acgctatgcg aagaggagtc 840tcagaaagtg atgatatatt aggattactc ttaaaaggag gactgtcaac cactgaaata 900attgaagaat gtaaggaatt ctatcttgca ggacaagata caaccacagc tttgctctcc 960tggacattgg tagccttgag tatgcatcct gagtggcaag acaaagctag aaatgaagta 1020tttcaagtac ttggaaaaaa caaaccaaag tttgaggact tgaatcaatt aaaaataatg 1080aacatgatct tccaagaggt gttgagatta tacccagcac tcacccttat gcgaagcacc 1140tcaaaggaca ctaaattggg agaaatgaca attcctgcag gagtacaaat ttttgtgcct 1200atatacatag cacatcgcga cccccaagta tggggagacg atgcactgat tttcaatcca 1260aataggttct cagaaggggt atccaaagct gcaaaagagc cattgtattt ccccttcggt 1320tggggtcctc gaatgtgcat tggtaataac tttggaatgg cagaagcaaa gctcgtttta 1380tctcaaattc tgcagaggtt ttggttcaag ctctctcctt cctatgttca tgcccctcag 1440gcaatactcg ttatgaagcc tcagtatggt gctcagataa tcctcaacaa gctc 149451974DNASolanum tuberosum 5gcttgctctg ttcttgtaca tctcaacctc actattcatt catcagttga tagaaaatat 60ctaggctatg gcaattgcaa cagtaattgg tgcaacaatt ggtattttga tagcgatttt 120ttgtgtaaaa tcgttttaca cattatggtg gtggccaaag atgatcgaaa agaagctgaa 180gaaggaaggt attcatgggc tgccctacca atttctgttt ggaaatctga aagagatgac 240gagaatgtct agagaagcaa agaaaacacc gttagtaaat catgatatcg ttccttgggt 300taatcctttt attcttcatc tttctaaaac ttacggtaag ccagccatgt atatttcttt 360aaaacacatt ccatatatat tttgttaaaa ctttacctaa ttaggtatgg tgcagagaga 420ttatttgtga tgtgggctgg accaacacct cggattgtag tatcagatcc aaagctaatt 480aaagaagtgg tgaacagaca taatgaattt cagaagcctc aagccaatgc gttcattgac 540atgtttgtta ctggacttgc tagttacaat ggtcaaaaat gggaccacca tagaaagata 600ctaaaccctg cttttcatat agagaagatt aaggtattga actagttatt cctctcttct 660ttttggatga ccattcattt tcctattttt gtgtgactgc acagaggttg tacccagcat 720tttgtgagtg ttgtgatgaa atgataaata gatgggagga attggttagc aaaagtggaa 780gttgtgagtt ggatgtggca gatgaattcc taaatgtagg tggagatgtt atatctagag 840ctgcttttgg tagcaatatt gaagaaggaa ggactatttt catacttcag aaagagcagt 900gcgatcttat tttggcttct ccatttactc ttttctttcc cttactaagg tgagtattta 960tctaatttct tcaaaaatat atatatgtat tgttataact aacatacatt ttaactacgc 1020agattctttc caacagcatc aaacagaaga gcaagataca tctacaagaa agtgttatca 1080ttgattaacg gaataataga gaagaaaaaa gacactatgc gaagaggagt ctcacaaagt 1140gatgatattt tagggttact cttaaaagga ggactatcaa ccactgaaat aattgaagaa 1200tgtaaggaat tctatcttgc aggacaagat acaaccacag ctttgctctc ttggacattg 1260gttgccttga gtatgcaccc tgagtggcaa gacaaagcta gaaatgaagt ctttcaagtc 1320cttggaaaaa acaaaccaaa gtttgaggac ttgaatcaat taaaaatagt aagctctctc 1380tttagtcttt atgatgatac agagtcctaa ttttactact gagtactact ataaacatgt 1440cattaatatt tgtgtattaa tactagatga acatgatctt ccaagaggtg ttgagattat 1500atccagcact cacccttatg cgaagcaccg taaagaacac taaattggga gatatgacaa 1560ttcctgcagg agtacaaata tttgtgccta tatatatagc acatcgcgat ccccaagtat 1620ggggagacga tgcattgata ttcaatccaa ataggttctc agaaggggta tccaaagctg 1680caaaagagcc cttgtatttc ccctttggtt ggggtcctcg aatgtgcatt ggtaataact 1740ttggcatggc agaagccaag ctcgttttat ctcaaattct gcagcgtttt tggtttaagc 1800tctctccttc ctatgttcat gcccctcagg caatactcgt tatgaagcct cagtatggtg 1860ctcagataat cctcaacaag ctttgacctt tcgggctggt agttaattat aagtggtact 1920agttctttca ataaatcagg ttgagttgta ttctgctgct aattctgctt ttca 1974624DNAArtificial SequenceSynthetic oligonucleotide primer 6gcttgctctg ttcttgtaca tctc 24721DNAArtificial SequenceSynthetic oligonucleotide primer 7tgaaaagcag aattagcagc a 21827DNAArtificial SequenceSynthetic oligonucleotide primer 8gagctctaga gaagcaaaga aaacacc 27930DNAArtificial SequenceSynthetic oligonucleotide primer 9ggatccatat gctaaccaat tcctcccatc 301019DNAArtificial SequenceSynthetic oligonucleotide primer 10taaagcacga ggaagcggt 191119DNAArtificial SequenceSynthetic oligonucleotide primer 11gcacaacaga caatcggct 191227DNAArtificial SequenceSynthetic oligonucleotide primer 12gagctctaga gaagcaaaga aaacacc 271320DNAArtificial SequenceSynthetic oligonucleotide primer 13gggcatgaac ataggaagga 201419DNAArtificial SequenceSynthetic oligonucleotide primer 14taaagcacga ggaagcggt 191519DNAArtificial SequenceSynthetic oligonucleotide primer 15gcacaacaga caatcggct 191624DNAArtificial SequenceSynthetic oligonucleotide primer 16gcttgctctg ttcttgtaca tctc 241721DNAArtificial SequenceSynthetic oligonucleotide primer 17tgaaaagcag aattagcagc a 2118517PRTSolanum tuberosum 18Met Gly Ile Ala Val Phe Ile Ala Leu Ala Val Cys Leu Pro Phe Ser 1 5 10 15 Phe Trp Cys Leu Lys Leu Leu Tyr Phe Val Trp Trp Arg Pro Lys Thr 20 25 30 Val Glu Asn Glu Leu Arg Gln Gln Gly Ile Tyr Gly Arg Pro Tyr Arg 35 40 45 Phe Leu Phe Gly Asn Leu Lys Glu Met Ile Glu Met Asn Lys Ile Ala 50 55 60 Lys Ser Lys Pro Met Pro Leu His His Asp Phe Thr Pro Arg Leu Asn 65 70 75 80 Pro Leu Phe Tyr Glu Leu Ala Thr Thr Tyr Lys Lys Leu Tyr Leu Phe 85 90 95 Trp Leu Gly Pro Ile Pro Arg Leu Thr Ile Leu Asp Pro Lys Leu Ile 100 105 110 Lys Glu Val Leu Ser Asn Lys Ser Gly Glu Phe Arg Lys Pro Asn Ile 115 120 125 Ser Ala Phe Leu Lys Leu Phe Val Thr Gly Leu Gly Thr Tyr Asp Gly 130 135 140 Glu Lys Trp Ala Lys His Arg Lys Ile Leu Asn Pro Ala Phe His Met 145 150 155 160 Glu Lys Leu Lys Val Met Leu Gly Leu Phe Val Asn Cys Thr Asp Asp 165 170 175 Met Ile Ser Arg Trp Asp Lys Leu Thr Gly Ser Thr Gly Gly Ser Cys 180 185 190 Glu Val Asp Ile Ser Gln Glu Phe His Asn Leu Thr Gly Asp Met Leu 195 200 205 Ser Lys Ala Ala Phe Gly Ser Asn Phe Glu Glu Gly Lys Leu Val Phe 210 215 220 Ser Leu Leu Arg Glu Gln Cys Glu Leu Ile Phe Thr Ala Lys Leu Ala 225 230 235 240 Ile Asn Val Phe Pro Trp Leu Arg Phe Val Pro Thr Lys Thr Asn Arg 245 250 255 Arg Arg Leu Tyr Ile Tyr Asn Thr Val Arg Ser Ser Leu Lys Ala Ile 260 265 270 Ile Glu Lys Arg Glu Lys Glu Val Gln Ser Gly Lys Ser His Asn Glu 275 280 285 Asp Leu Leu Gly Leu Leu Met Lys Ser Asn Gln Glu Glu Gln Gln Gly 290 295 300 Asn Lys Asn Ser Asn Lys Gly Met Ser Thr Glu

Asp Met Ile Glu Glu 305 310 315 320 Cys Asn Ser Phe Tyr Phe Ala Gly Gln Glu Thr Thr Ala Thr Leu Leu 325 330 335 Thr Trp Thr Ala Ile Val Leu Thr Met His Pro Asp Trp Gln Glu Lys 340 345 350 Ala Arg Lys Glu Val Leu Glu Val Ile Gly Lys Asp Glu Pro Lys Phe 355 360 365 Asp Gln Leu Asn His Leu Lys Ile Val Thr Met Ile Leu His Glu Val 370 375 380 Leu Arg Leu Tyr Pro Ser Gly Ser Leu Val Arg Glu Thr Asn Lys Lys 385 390 395 400 Thr Lys Leu Gly Glu Tyr Thr Ile Pro Ala Gly Ala Gln Leu Leu Val 405 410 415 Pro Leu Gln Thr Ile His Arg Asp Thr Glu Ala Trp Gly Glu Asp Ala 420 425 430 Leu Ile Phe Asn Pro Glu Arg Phe Ser Glu Gly Val Ser Lys Ala Ser 435 440 445 Lys Asp Leu Met Tyr Phe Pro Phe Gly Trp Gly Ser Arg Ile Cys Leu 450 455 460 Gly Met Asn Val Ser Met Ile Gln Gly Lys Leu Val Leu Ala Lys Ile 465 470 475 480 Leu Gln Asn Tyr Ser Phe Glu Leu Ser Pro Ser Tyr Ala His Gly Pro 485 490 495 Thr Met Pro Ala Leu Val Leu Gln Pro Gln Tyr Gly Ala Pro Met Ile 500 505 510 Leu Arg Lys Leu Ser 515 191551DNASolanum tuberosum 19atgggtattg cagttttcat tgctttggcc gtatgtttgc ctttcagttt ttggtgccta 60aaattgctct actttgtatg gtggcgtccc aaaacagtag aaaatgaact gcggcagcaa 120ggaatatatg ggcgtcccta tagatttcta tttggaaatc taaaggagat gatagagatg 180aataaaatag ccaagtctaa acccatgcca ttgcaccacg atttcacacc tcgacttaat 240ccattgttct atgaactggc caccacttac aagaaacttt acttgttttg gctaggaccg 300atacctcgat taaccatttt ggatcccaag ttaattaagg aagtactgtc aaacaaatcg 360ggtgagttca gaaaaccaaa catcagcgct ttcctgaagc tatttgtaac ggggctgggg 420acttacgatg gtgaaaaatg ggcgaaacac agaaaaattc ttaatccggc tttccacatg 480gaaaaattga aggtgatgtt gggattattt gttaactgta ccgatgacat gataagcaga 540tgggacaagc taactggttc aacgggtggt tcttgtgaag tagatatttc tcaagaattt 600cataatttaa ctggagatat gctatcgaaa gcagccttcg gtagcaattt tgaagaaggg 660aagttggtat tttcacttct gagagagcaa tgtgaactaa ttttcactgc aaagcttgct 720attaatgtct tcccatggtt aaggtttgtg ccaacgaaaa ctaataggag aagattgtac 780atctataaca cagttcgtag ttcgctaaaa gcaataattg agaaacgaga gaaagaggta 840caatctggaa aatcacacaa tgaagatctg ttgggtttgc taatgaaatc taatcaggaa 900gaacagcaag ggaataagaa ctcgaacaaa ggaatgagta cagaggatat gatagaagag 960tgcaactctt tctattttgc tggtcaagag actactgcaa ctttgttaac atggactgca 1020attgtcttga ctatgcatcc agattggcaa gagaaagcca ggaaagaagt tcttgaagtc 1080attggaaaag atgagcctaa gtttgatcaa ctcaaccatc taaagattgt aactatgatc 1140ttgcacgagg ttctgaggtt atatccatca ggttctcttg ttagagaaac aaacaaaaag 1200acaaagcttg gagagtatac aatcccagca ggtgcgcaac ttttagttcc tctacaaaca 1260atccatcgcg atacagaggc atggggagaa gatgctctaa ttttcaatcc agaaaggttt 1320tcagaagggg tatcaaaagc atcaaaggac ctgatgtact ttccgtttgg ttggggttct 1380cggatatgcc ttggaatgaa tgtttccatg attcaaggga agcttgtttt ggctaaaatc 1440ttacagaact actcctttga gctttccccc tcctatgctc atggtccaac catgccagct 1500cttgttctac aaccacaata tggtgctcct atgatccttc gaaagctatc a 155120516PRTSolanum lycopersicum 20Met Ala Ile Ala Ile Phe Ile Ala Leu Ala Ile Phe Phe Pro Phe Thr 1 5 10 15 Phe Trp Cys Leu Lys Leu Leu Tyr Phe Val Trp Trp Arg Pro Lys Thr 20 25 30 Val Glu Asn Glu Leu Arg His Gln Gly Ile Tyr Gly Arg Pro Tyr Arg 35 40 45 Phe Leu Phe Gly Asn Leu Lys Glu Met Ile Glu Met Asn Lys Ile Ala 50 55 60 Lys Ser Lys Pro Met Pro Leu His His Asp Phe Thr Pro Arg Leu Asn 65 70 75 80 Pro Leu Phe Tyr Glu Leu Ala Thr Thr Tyr Lys Lys Leu Tyr Leu Phe 85 90 95 Trp Leu Gly Pro Ile Pro Arg Leu Thr Ile Leu Asp Pro Lys Leu Ile 100 105 110 Lys Glu Val Leu Ser Asn Lys Ser Gly Glu Phe Arg Lys Pro Lys Ile 115 120 125 Ser Ala Phe Leu Lys Leu Phe Val Thr Gly Leu Gly Thr Tyr Asp Gly 130 135 140 Glu Lys Trp Ala Lys His Arg Lys Ile Leu Asn Pro Ala Phe His Met 145 150 155 160 Glu Lys Leu Lys Val Met Leu Gly Leu Phe Val Glu Cys Thr Asp Asp 165 170 175 Met Ile Ser Arg Trp Asp Lys Leu Thr Gly Ser Thr Gly Ser Cys Glu 180 185 190 Leu Asp Ile Ser Gln Glu Phe His Asn Leu Thr Gly Asp Met Leu Ser 195 200 205 Lys Ala Ala Phe Gly Ser Asn Phe Glu Glu Gly Lys Leu Val Phe Ser 210 215 220 Leu Leu Arg Glu Gln Cys Glu Leu Ile Phe Thr Ala Lys Leu Ala Ile 225 230 235 240 Asn Val Phe Pro Trp Leu Arg Phe Val Pro Thr Lys Thr Asn Arg Arg 245 250 255 Arg Leu Tyr Ile Tyr Asn Thr Val Arg Ser Ser Leu Lys Ser Ile Ile 260 265 270 Glu Lys Arg Glu Lys Glu Val Gln Ser Gly Lys Ser His Asn Glu Asp 275 280 285 Leu Leu Gly Leu Leu Met Lys Ser Asn Gln Glu Glu Gln Gln Gly Asn 290 295 300 Lys Asn Ser Asn Lys Gly Met Ser Thr Glu Asp Met Ile Glu Glu Cys 305 310 315 320 Asn Ser Phe Tyr Phe Ala Gly Gln Glu Thr Thr Ala Thr Leu Leu Thr 325 330 335 Trp Thr Ala Ile Val Leu Thr Met His Pro Asp Trp Gln Glu Lys Ala 340 345 350 Arg Lys Glu Val Leu Gln Val Ile Gly Lys Asp Glu Pro Lys Phe Asp 355 360 365 Gln Leu Asn His Leu Lys Ile Val Thr Met Ile Leu His Glu Val Leu 370 375 380 Arg Leu Tyr Pro Ser Gly Ser Leu Val Arg Glu Thr Asn Lys Lys Thr 385 390 395 400 Lys Leu Gly Gly Tyr Thr Ile Pro Ala Gly Ala Gln Leu Leu Val Pro 405 410 415 Leu Gln Thr Ile His Arg Asp Thr Glu Ala Trp Gly Glu Asp Ala Leu 420 425 430 Ile Phe Asn Pro Glu Arg Phe Ser Glu Gly Val Ser Lys Ala Ser Lys 435 440 445 Asp Leu Met Tyr Phe Pro Phe Gly Trp Gly Ser Arg Ile Cys Leu Gly 450 455 460 Met Asn Val Ser Met Ile Gln Gly Lys Leu Val Leu Ala Lys Ile Leu 465 470 475 480 Gln Asn Tyr Ser Phe Glu Leu Ser Pro Ser Tyr Ala His Gly Pro Thr 485 490 495 Met Pro Ala Leu Val Leu Gln Pro Gln Tyr Gly Ala Pro Met Ile Val 500 505 510 Arg Lys Leu Glu 515 211548DNASolanum lycopersicum 21atggctattg caattttcat agctttggcc atattttttc ctttcacttt ttggtgccta 60aaattgctct actttgtatg gtggcgtccg aaaacagtag aaaatgaact gcgtcatcaa 120ggaatctatg ggcgtcccta tagatttcta tttggaaatc taaaagagat gatagagatg 180aacaaaatag ccaaatctaa acccatgcca ttgcaccacg atttcacacc tcgacttaat 240ccattgttct atgaactcgc taccacttac aagaaacttt acttgttttg gctaggaccc 300atccctcgat taaccatttt ggatcccaag ttaataaagg aagtactgtc aaacaaatct 360ggtgagttca gaaaaccaaa aatcagtgct tttctgaagc tatttgtaac agggctaggg 420acttacgatg gggaaaaatg ggccaaacat cgaaaaattc ttaatccggc attccacatg 480gaaaaattga aggtgatgct gggattattt gttgaatgca cggatgacat gataagcaga 540tgggataagc taacgggttc aacgggttct tgtgaattgg atatttctca agaatttcat 600aatttaactg gagatatgct atcgaaagca gctttcggta gcaattttga agaagggaaa 660ttggtatttt cacttctgag agagcaatgt gaactaattt tcactgcaaa gcttgctatt 720aatgtcttcc catggttaag gtttgtgcca acgaaaacta ataggagaag attgtacatc 780tataacacag ttcgtagttc gttaaaatca atcattgaga aacgagagaa agaggtacaa 840tctggaaaat cacacaacga agatctattg ggtttgttga tgaaatctaa tcaggaagaa 900cagcaaggga ataagaactc gaataaagga atgagtacag aggatatgat agaagagtgt 960aactctttct actttgctgg tcaagagact actgcaactt tgttaacatg gactgcaatt 1020gtgttgacta tgcatccaga ttggcaagag aaagctagga aagaagttct tcaagtcatt 1080ggaaaagatg aacctaagtt tgatcaactc aaccacctaa agattgtaac aatgattttg 1140cacgaggttc tgaggctcta tccatcaggt tctctcgtta gagaaacaaa caaaaaaaca 1200aagctcggag ggtatacaat cccagcaggt gcgcaacttt tagtgcctct acaaacaatt 1260catcgggata cagaggcatg gggtgaagat gcattaattt tcaatccaga aaggttttca 1320gaaggggtat caaaagcatc aaaggacctg atgtacttcc catttggttg gggttctcgg 1380atatgccttg gaatgaatgt ttcgatgatt caagggaagc ttgttttggc taaaatctta 1440cagaactact catttgagct ttccccatcc tatgctcatg gtccaacaat gccagctctt 1500gttctacaac cacaatatgg tgctcctatg attgttcgaa agctagaa 1548222102DNASolanum tuberosum 22agcaatcaaa catgggtatt gcagttttca ttgctttggc cgtatgtttg cctttcagtt 60tttggtgcct aaaattgctc tactttgtat ggtggcgtcc caaaacagta gaaaatgaac 120tgcggcagca aggaatatat gggcgtccct atagatttct atttggaaat ctaaaggaga 180tgatagagat gaataaaata gccaagtcta aacccatgcc attgcaccac gatttcacac 240ctcgacttaa tccattgttc tatgaactgg ccaccactta cagtaattat tctttctaat 300ttccaatggc cttgttacag taacattttt aatttgaaca ctgattgtgg agtggttatt 360attgcagaga aactttactt gttttggcta ggaccgatac ctcgattaac cattttggat 420cccaagttaa ttaaggaagt actgtcaaac aaatcgggtg agttcagaaa accaaacatc 480agcgctttcc tgaagctatt tgtaacgggg ctggggactt acgatggtga aaaatgggcg 540aaacacagaa aaattcttaa tccggctttc cacatggaaa aattgaaggt atttactagt 600attagttttc tggtatacaa tttccttaag tgatttatta ttataattgt tggtgtttgc 660acaggtgatg ttgggattat ttgttaactg taccgatgac atgataagca gatgggacaa 720gctaactggt tcaacgggtg gttcttgtga agtagatatt tctcaagaat ttcataattt 780aactggagat atgctatcga aagcagcctt cggtagcaat tttgaagaag ggaagttggt 840attttcactt ctgagagagc aatgtgaact aattttcact gcaaagcttg ctattaatgt 900cttcccatgg ttaaggtact tgtagtaatt aactaatgcc ttcgttttcc ttgcattgga 960tcttaacaat tgatgatgta tgtaataggt ttgtgccaac gaaaactaat aggagaagat 1020tgtacatcta taacacagtt cgtagttcgc taaaagcaat aattgagaaa cgagagaaag 1080aggtacaatc tggaaaatca cacaatgaag atctgttggg tttgctaatg aaatctaatc 1140aggaagaaca gcaagggaat aagaactcga acaaaggaat gagtacagag gatatgatag 1200aagagtgcaa ctctttctat tttgctggtc aagagactac tgcaactttg ttaacatgga 1260ctgcaattgt cttgactatg catccagatt ggcaagagaa agccaggaaa gaagttcttg 1320aagtcattgg aaaagatgag cctaagtttg atcaactcaa ccatctaaag attgtaagaa 1380gatactatcg ttttcatttc ccattcatac tttggaattt ttacatttga gtaaaccgtg 1440tttaattata acaagtagaa tcacgaccca ccccataaac ttcaaattct agatctagga 1500gtactagtga tttgaattct ggattcatct attaattaat aattgtgcta tatatgtagg 1560taactatgat cttgcacgag gttctgaggt tatatccatc aggttctctt gttagagaaa 1620caaacaaaaa gacaaagctt ggagagtatc caatcccagc aggtgcgcaa cttttagttc 1680ctctacaaac aatccatcgc gatacagagg catggggaga agatgctcta attttcaatc 1740cagaaaggtt ttcagaaggg gtatcaaaag catcaaagga cctgatgtac tttccgtttg 1800gttggggttc tcggatatgc cttggaatga atgtttccat gattcaaggg aagcttgttt 1860tggctaaaat cttacagaac tactcctttg agctttcccc ctcctatgct catggtccaa 1920ccatgccagc tcttgttcta caaccacaat atggtgctcc tatgatcctt cgaaagctat 1980catgatattg gtcaccctaa catgaatcaa taaatcactt ctctgtttcc tattgttgtg 2040aacttttctt cattctatcc acaaacagga ttgctgttta caccaatctc aagttcacat 2100ca 21022321DNAArtificial SequenceSynthetic oligonucleotide primer 23agcaatcaaa catgggtatt g 212422DNAArtificial SequenceSynthetic oligonucleotide primer 24tgatgtgaac ttgagattgg tg 222531DNAArtificial SequenceSynthetic oligonucleotide primer 25gagctctaga ggttaagagt ttgtgccaac g 312630DNAArtificial SequenceSynthetic oligonucleotide primer 26ggatccatat ggctttctct tgccaatctg 302720DNAArtificial SequenceSynthetic oligonucleotide primer 27tcgggtgagt tcagaaaacc 202830DNAArtificial SequenceSynthetic oligonucleotide primer 28ggatccatat ggctttctct tgccaatctg 30