CN115894642B - Fruit control gene SlGT-2 and homologous gene and application thereof - Google Patents

Abstract

The invention discloses a regulating fruit type SlGT-2 protein and a homologous SlGTL1 protein thereof. The invention adopts CRISPR/Cas9 technology to edit the SlGT-2 gene and the SlGTL1 gene at fixed points, and knock out the tomato SlGT-2 gene and the SlGTL1 gene by causing frame shift mutation, so that the round multi-juice tomato material is converted into square few-juice tomato material. The invention has great application and popularization value.

Description

Fruit control gene SlGT-2 and homologous gene and application thereof

Technical Field

The invention relates to a fruit-type control gene SlGT-2 in the field of biotechnology and a homologous gene and application thereof.

Background

Tomato is one of the most widely consumed vegetables and is also an important fruit. The fruit type tomato is the most intuitive character of tomato fruit, not only affects the selection of consumers, but also affects the taste or nutrition quality of the fruit. The fruits of various crops are mostly round, oval, oblate and oblong, and few square fruits exist. Square is an extremely rare shape, has great attraction to consumers and has great demands on production, but square fruits are extremely difficult to cultivate, and a square fruit regulatory gene is not cloned at present.

Disclosure of Invention

The invention aims to solve the technical problems of regulating and controlling the shape of plant fruits and regulating and controlling the water content of pectin.

In order to solve the above technical problems, a first object of the present invention is to provide a protein which is SlGT-2 and/or its homologous protein SlGTL1;

the SlGT-2 is a protein of A1), A2) or A3) as follows:

a1 Amino acid sequence is protein of sequence 1 in a sequence table;

a2 Protein which is obtained by derivatizing the protein shown in A1) and is related to the shape of plant fruits and is obtained by substituting and/or deleting and/or adding more than one amino acid residue in any one of the amino acid sequences shown in the sequence 1 in the sequence table;

a3 Fusion proteins obtained by attaching protein tags to the N-terminal or/and C-terminal of A1) or A2);

the SlGTL1 is a protein of A4), A5) or A6) as follows:

a4 Amino acid sequence is protein of sequence 2 in the sequence table;

a5 Protein which is obtained by derivatizing the protein shown in A4) and is related to the shape of plant fruits and is obtained by substituting and/or deleting and/or adding more than one amino acid residue in any one of the amino acid sequences shown in the sequence 2 in the sequence table;

a6 Fusion proteins obtained by ligating protein tags at the N-terminus or/and the C-terminus of A5) or A6).

Wherein, the sequence 1 in the sequence table consists of 654 amino acid residues, and the sequence 2 in the sequence table consists of 651 amino acid residues.

The protein may be derived from tomato (Lycopersicon esculentum).

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

Among the proteins, A2) is the SlGT-2+T, and the amino acid sequence of the SlGT-2+T is the sequence 9 in the sequence table; a5 The protein is SlGTL1+AG, and the amino acid sequence of the SlGTL1+AG is sequence 10 in a sequence table.

The invention also provides a gene which is a slGT-2 gene encoding the slGT-2 or a slGTL1 gene encoding the slGTL1 or a slGT-2+T gene encoding the slGT-2+T or a slGTL1+AG gene encoding the slGTL1+AG;

the SlGT-2 gene is shown in the following B1) or B2):

b1 A coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 3 in the sequence table;

b2 The nucleotide of the coding strand is a DNA molecule of sequence 5 in the sequence table.

Wherein, the sequence 3 in the sequence table consists of 1965 nucleotides and codes the protein shown in the sequence 1 in the sequence table.

The SlGTL1 gene is a gene shown in the following B3) or B4):

b3 A coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 4 in the sequence table;

b4 The nucleotide of the coding strand is a DNA molecule of a sequence 6 in a sequence table.

Wherein, the sequence 4 in the sequence table consists of 1956 nucleotides, and codes the protein shown in the sequence 2 in the sequence table.

The nucleotide of the SLGT-2+T gene which is a coding chain is a DNA molecule of a sequence 11 in a sequence table;

the nucleotide of the coding chain of the SlGTL1+AG gene is the DNA molecule of the sequence 12 in the sequence table.

The invention also provides a method for regulating and controlling the shape of the plant fruits, which comprises the following steps: inhibiting the expression of the SlGT-2 gene and the SlGTL1 gene in the receptor round fruit plant to obtain the target plant with square fruits.

In the above method, the inhibition of expression of the SlGT-2 gene of claim 2 and the SlGTL1 gene of claim 2 in the recipient circular fruit plant is achieved by gene editing of the SlGT-2 gene and the SlGTL1 gene in the plant. The gene editing is achieved by means of a CRISPR/Cas9 system.

In the above method, the CRISPR/Cas9 system comprises a plasmid expressing Cas9 and sgrnas, wherein the sgrnas may be the sgrnas 1 and 2 for the target sequence 1 and 2, the target sequence 1 may be the 40 th to 59 th positions of the sequence 5 in the sequence table, and the target sequence 2 may be the 116 th to 136 th positions of the sequence 6 in the sequence table.

In the method, the nucleotide sequence of the sgRNA1 is shown as a sequence 7 in a sequence table, and the nucleotide sequence of the sgRNA2 is shown as a sequence 8 in the sequence table.

In the above method, the target plant is a plant satisfying the following conditions: a plant having a mutation in both the target sequence 1 and the target sequence 2 region.

In the above method, the expression of the SlGT-2 gene and the SlGTL1 gene in the recipient circular fruit plant is expressed as the following X1 and X2:

x1 mutates the SlGT-2 gene shown in a sequence 5 in a sequence table into a SlGT-2+T gene, wherein the coding sequence of the SlGT-2+T gene is shown in a sequence 11;

and X2 mutates the SlGTL1 gene shown in a sequence 6 in the sequence table into a SlGTL1+AG gene, wherein the coding sequence of the SlGTL1+AG gene is shown in a sequence 12.

In the method, the amino acid sequence of the mutant SlGT-2 protein in the target plant is shown as a sequence 9, and the coding sequence is shown as a sequence 11; the amino acid sequence of the mutated SlGTL1 protein is shown in sequence 10, and the coding sequence is shown in sequence 12.

In the above method, the target plant may be a plant in which homozygous mutation is made in both the SlGT-2 gene and the SlGTL1 gene.

The plant described above may be any one of F1) -F4):

f1 Tubular flower plants;

f2 Solanaceae plant;

f3 Plants of the genus Lycopersicon;

f4 Tomato (tomato).

In order to solve the technical problems, the invention also provides a reagent for regulating and controlling plant fruit type, and the active ingredients of the reagent are substances for inhibiting the expression of the coding SLGT-2 gene and the SLGTL1 gene, reducing the abundance of the SLGT-2 protein and the SLGTL1 protein and/or knocking out the SLGT-2 gene and the SLGTL1 gene.

In the above reagent, the substance contains the following F1), F2) or F3):

f1 sgRNA, siRNA, shRNA, miRNA or antisense RNA targeting the gene;

f2 A DNA molecule that produces sgrnas targeting the gene, a DNA molecule that produces sirnas targeting the gene, a DNA molecule that produces shrnas targeting the gene, a DNA molecule that produces mirnas targeting the gene, or a DNA molecule that produces antisense RNAs targeting the gene;

f3 Producing an expression vector for sgrnas targeting the gene, producing an expression vector for sirnas targeting the gene, producing an expression vector for shrnas targeting the gene, producing an expression vector for mirnas targeting the gene, or producing an expression vector for antisense RNAs targeting the gene.

The active ingredients of the agents described above may also contain other biological or/and non-biological ingredients, and the other active ingredients of the agents described above may be determined by one skilled in the art based on the fruit type of the plant.

The invention also provides application of the protein, the gene or the reagent in regulating plant fruit shape and/or pectin water content.

The invention also provides application of the method in regulating and controlling the water content of the pectin in plants.

The inventor of the invention utilizes the laboratory to separate and clone a fruit-related protein SlGT-2 and homologous protein SlGTL1 thereof from tomatoes, adopts CRISPR/Cas9 technology to edit the SlGT-2 gene and the SlGTL1 gene at fixed points, and can convert round juicy tomato materials into square juicy tomato materials by knocking out the SlGT-2 gene and the SlGTL1 gene in tomatoes through causing frame shift mutation. The invention has great application and popularization value.

Drawings

FIG. 1 is a photograph showing the outline of the AC fruits of the 1-SlGT-2-SlGTL1 gene-edited plant and the control wild tomato variety in example 1 of the present invention.

FIG. 2 is a photograph of a longitudinal cut of the AC fruit of the 1-SlGT-2-SlGTL1 gene-edited plant and control wild tomato variety of example 1 of the present invention.

FIG. 3 shows the measured positions of the width of the top 5% of the fruit in the longitudinal direction and the width of the middle position in the longitudinal direction in the square calculation formula of the present invention. Wherein, the first horizontal line from top to bottom indicates the position of 5% of the longitudinal top end of the fruit, and the second horizontal line from top to bottom indicates the longitudinal middle position of the fruit.

FIG. 4 is a graph showing the statistical results of fruit sizes of the 1-SlGT-2-SlGTL1 gene editing plants and the control wild tomato variety AC in example 1 of the present invention. Data shown are mean ± standard deviation, repeat number 15, significance differences for each group were analyzed with t-test, representing significance analysis results P <0.01.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Tomato variety AC (Ailsa Craig) in the examples below is the American tomato genetic resource center product (TGRC, http:// TGRC. Ucdavis. Edu /), accession number LA2838A.

CRISPR/Cas9 vector pTX041 in the examples described below is described in non-patent literature "Deng et al Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system. Journal of Genetics and Genomics 45 (2018) 51-54", available to the public from national academy of sciences genetic and developmental biology research to repeat the experiments of the present application, and is not useful for other applications.

The preparation method of the seed growth medium in the following examples is (1L as an example): 2.2g of MS salt and 10g of sucrose are dissolved in water to a volume of 1L, the pH is adjusted to between 5.8 and 6.0 by using 1mol/L KOH, 8g of agar is added, and the mixture is autoclaved.

The preparation method of the preculture medium in the following examples is (1L as an example): 4.4g of MS salt, 1.0mg of Zeatin (Zeatin) and 30g of sucrose are dissolved in water to a volume of 1L, the pH is adjusted to between 5.8 and 6.0 by using 1mol/L KOH, 8g of agar is added, and the mixture is autoclaved.

The preparation method of the liquid MS culture medium in the following examples is (1L as an example): dissolving 4.4g of MS salt and 30g of sucrose in water to a volume of 1L, adjusting the pH to between 5.8 and 6.0 by using 1mol/L KOH, and sterilizing under high pressure.

The preparation method of the screening differentiation medium in the following examples is (1L as an example): dissolving 4.4g of MS salt, 2.0mg of zeatin, 50mg of kanamycin, 100mg of inositol, 0.5mg of folic acid and 30g of sucrose in water to reach a volume of 1L, adjusting the pH to between 5.8 and 6.0 by using 1mol/L KOH, adding 8g of agar, and sterilizing under high pressure.

The rooting medium was prepared by the following method in the examples (1L as an example): 4.4g of MS salt, 50mg of kanamycin, 0.5mg of folic acid, 0.5mg of indolebutyric acid and 30g of sucrose are dissolved in water to be fixed to 1L, the pH is adjusted to between 5.8 and 6.0 by using 1mol/L KOH, 8g of agar is added, and the mixture is autoclaved.

Example 1 tomato with square few juice fruits was obtained by editing tomato SlGT-2 gene and homologous gene SlGTL1 thereof using CRISPR/Cas9 method

The inventors have found for the first time the SlGT-2 protein and its homologous protein SlGTL1 in tomato, which controls the formation of square fruits. The amino acid sequence of the SLGT-2 protein is sequence 1 in a sequence table, the gene for encoding the SLGT-2 protein is the SLGT-2 gene, the nucleotide sequence from the start codon to the stop codon of the SLGT-2 gene in genome DNA is sequence 5 in the sequence table (wherein the 1 st to 284 th positions and the 1037 th to 2717 th positions are exons), and the open reading frame in cDNA is sequence 3 in the sequence table. The amino acid sequence of the SLGTL1 protein is sequence 2 in a sequence table, the gene for encoding the SLGTL1 protein is the SLGTL1 gene, the nucleotide sequence from the start codon to the stop codon of the SLGTL1 gene in the genome DNA is sequence 6 in the sequence table (wherein the 1 st to 320 th positions and the 729 th to 2364 th positions are exons), and the open reading frame in the cDNA is sequence 4 in the sequence table.

1. Construction of recombinant vectors

1. Selecting a target sequence: aiming at the SlGT-2 gene, selecting the 40 th to 59 th positions of a sequence 5 in a sequence table as a target sequence 1; aiming at the SlGTL1 gene, the 116 th to 136 th positions of the sequence 6 in the sequence table are selected as target sequence 2.

Two PCR primers 1 and 2 were synthesized, and then a target fragment containing two gRNA and U6 promoter sequences was amplified from the template plasmid pTX041 vector, and purified and recovered.

The nucleotide sequences of the two PCR primers F and R were as follows:

(the underlined sequence is specifically combined with the 40 th to 59 th positions of the sequence 5 in the sequence table, and the wavy line indicates that the sequence is BsaI enzyme recognition site);

(the underlined sequence specifically binds to the 116 th to 136 th positions of the sequence 6 in the sequence table, and the wavy line indicates the BsaI enzyme recognition site).

2. And (3) carrying out enzyme digestion on the target fragment recovered by the method by using restriction enzyme BsaI, and purifying and recovering to obtain the enzyme-digested target fragment. And (3) carrying out enzyme digestion on the CRISPR/Cas9 empty vector pTX041 by using restriction enzyme BsaI, purifying and recovering to obtain the linearized pTX041 vector skeleton.

3. And (3) connecting the digestion target fragment obtained in the step (2) with the pTX041 vector skeleton to obtain a recombinant plasmid obtained by inserting the digestion target fragment between digestion sites of the BsaI of the pTX041 vector and keeping other sequences of the pTX041 vector unchanged. Sequencing shows that the recombinant plasmid expresses sgRNA1 (aiming at target sequence 1) shown in sequence 7 in a sequence table and sgRNA2 (aiming at target sequence 2) shown in sequence 8 in the sequence table, and the recombinant plasmid is named pTX041-sgRNA1-sgRNA2.

The sgRNA1 is aimed at a target sequence 1 (40 th to 59 th positions of a sequence 5 in a sequence table), and a target sequence binding region in the sgRNA1 is shown as nucleotide numbers 2 to 21 of a sequence 7 in the sequence table.

The sgRNA2 is aimed at a target sequence 2 (116 th to 136 th nucleotides of a sequence 6 in a sequence table), and a target sequence binding region in the sgRNA1 is shown as the 2 nd to 21 nd nucleotides of the sequence 8 in the sequence table.

2. Preparation of Gene editing plants

1. The recombinant plasmid pTX041-sgRNA1-sgRNA2 prepared in the first step is taken and introduced into agrobacterium GV3101 to obtain recombinant agrobacterium which is named GV3101-sgRNA1-sgRNA2.

2. Taking recombinant agrobacterium GV3101-sgRNA1-sgRNA2, and carrying out genetic transformation on a wild tomato variety AC by an agrobacterium-mediated method to obtain a T0 generation plant, wherein the specific process is as follows:

(1) Selecting full and large seeds of a wild tomato variety AC, soaking the seeds in a 75% ethanol aqueous solution for 2min, then soaking the seeds in a 10% NaClO aqueous solution for 10min, washing the seeds with sterile water for 7 times, and sowing the seeds in a seed growth medium and culturing the seeds for 8 days. Taking cotyledons, cutting the cotyledons into small blocks under aseptic conditions, inoculating the cotyledon blocks into a preculture medium and culturing for 2 days, and taking the cotyledon blocks as explants.

(2) Recombinant Agrobacterium GV3101-sgRNA1-sgRNA2 was resuspended in 50mL liquid MS medium to give OD _600nm The bacterial suspension with the concentration of being=0.4 is added with 50 mu L of 0.074mol/L acetosyringone water solution, and the bacterial suspension is the infection liquid.

(3) Immersing the explant obtained in the step (1) in the immersion liquid obtained in the step (2) for 10min, and then inoculating the explant to a preculture medium for 2 days.

(4) After the completion of step (3), the explants were inoculated to the selection differentiation medium for 8 weeks (subculture every 2 weeks) at which time resistant shoots were grown on the explants.

(5) And (3) when the length of the resistant bud in the step (4) is 3cm, cutting the resistant bud, transferring the resistant bud to a rooting culture medium for culture, and obtaining a rooted plant which is a T0 generation plant.

Culture conditions of the whole process: 25 ℃, 16h light/8 h darkness.

3. And screening plants with homozygous mutation of the slGT-2 gene and the homologous gene slGTL1 thereof from the T0 generation plants.

The screening method comprises the following steps: and taking genomic DNA of plant leaves, respectively carrying out PCR amplification by using two pairs of primers, namely a primer pair 1 (consisting of F1 and R1) and a primer pair 2 (consisting of F2 and R2), and recovering PCR amplification products and sequencing.

F1：5′-GGTGTAATTGCTAACTTGCTTGG-3′；

R1：5′-GCATGATGAAACTGGTGCAGC-3′；

F2：5′-ATGCTTGGTGTTTCTTCAAG-3′；

R2：5′-AACTTCTTCCCATAACGGTCC-3′。

1 plant of homozygous mutation of the SLGT-2 gene and the homologous gene SLGTL1 thereof is selected and named as plant 1.

Compared with the wild tomato variety AC, the SlGT-2 gene in the plant 1 has a mutation of inserting one nucleotide, in particular a nucleotide T is inserted between the 56 th and 57 th positions of the sequence 5 in the sequence table, and the mutation is homozygous (namely the same mutation occurs on two chromosomes). The insertion causes a frame shift mutation of the gene CDS sequence from position 57, which leads to premature generation of a stop codon, and translation of the truncated protein which loses the DNA binding domain, so that the function of the SlGT-2 protein is lost (the plant does not contain the SlGT-2 protein). The mutated gene is named as a SlGT-2+T gene, the protein encoded by the SlGT-2+T gene is SlGT-2+T, and the amino acid sequence of the SlGT-2+T is sequence 9. Meanwhile, compared with a wild tomato variety AC, the SlGTL1 gene in the plant 1 is mutated by inserting two nucleotides, particularly, two nucleotides 'AG' are inserted between the 131 st and 132 th positions of the sequence 6 in the sequence table, and the strain is homozygous mutation (namely, the same mutation occurs on two chromosomes). The insertion causes a frame shift mutation of the gene CDS sequence from position 132, a stop codon is generated in advance, and truncated proteins losing the DNA binding domain are translated, so that the function of the SlGTL1 protein is lost (the plant does not contain the SlGTL1 protein). The mutated gene was designated as the slgtl1+ag gene, the protein encoded by the slgtl1+ag gene was slgtl1+ag, and the amino acid sequence of slgtl1+ag was sequence 10.

That is, plant 1 was compared with wild tomato variety AC, for the SlGT-2 gene, the SlGT-2 gene in both homologous chromosomes was mutated to the SlGT-2+T gene, the coding sequence of which was sequence 11; for the SlGTL1 gene, the SlGTL1 gene in both homologous chromosomes was mutated to the slgtl1+ag gene, and the coding sequence of the slgtl1+ag gene was sequence 12.

4. Normally culturing the plant 1, selfing to obtain seeds, namely T1 generation seeds, and culturing the T1 generation seeds into a plant group, namely a T1 generation plant 1 group.

5. Plants without exogenous DNA were selected from the T1 generation plant 1 population.

The screening method comprises the following steps: the leaves of each individual plant of the T1 generation plant 1 population are separately extracted with genomic DNA, and PCR amplification is carried out by adopting a primer pair consisting of F3 and R3 (the target sequence of the primer pair consisting of F3 and R3 is located in the Cas9 gene of the CRISPR/Cas9 vector pTX041, the amplified product is expected to be about 402 bp), if the amplified product indicates that the plant contains exogenous DNA, if the amplified product does not indicate that the plant does not contain exogenous DNA.

F3：5′-TTGACAAGCTGTTCATCCAG-3′；

R3：5′-CCTTCGTAATCTCGGTGTTC-3′。

About 1/4 of the individuals in the T1 generation plant 1 population do not contain exogenous DNA.

6. And (3) extracting genomic DNA of leaves from the plants which are obtained by screening in the step (5) and do not contain exogenous DNA, respectively adopting primer pairs consisting of F1, R1, F2 and R2 to carry out PCR amplification, recovering PCR amplification products and sequencing.

The plants which are screened from the T1 generation plant 1 population and do not contain exogenous DNA have homozygous insertion which is the same as that of the plant 1, wherein the gene slGT-2 and the homologous gene slGTL1 thereof. The results show that the mutation generated by introducing the recombinant plasmid pTX041-sgRNA1-sgRNA2 prepared in the first step into the wild tomato variety AC can be stably inherited from the T0 generation to the T1 generation.

Plants selected from the T1 generation plant 1 population and not containing exogenous DNA are named as 1-SlGT-2-SlGTL1 gene editing plants.

3. Fruit observation

The plants tested were: wild tomato variety AC, 1-SlGT-2-SlGTL1 gene editing plant.

1. The seedlings of the tested plants are cultivated under the conditions of 25 ℃ and 16h illumination/8 h darkness until the plants grow to 4-5 leaves.

2. After the step 1 is completed, the plants (10 plants of each tested plant and consistent growth vigor) are transplanted into a greenhouse. The plant spacing and the row spacing are above 60 cm; the plants tested are randomly distributed; and the normal water and fertilizer management ensures that the water and fertilizer conditions of all plants are basically consistent.

After 3 months of transplanting, the plants begin to enter a fruiting period, which lasts for about 6 months. Mature fruits were collected throughout the fruiting period.

The number of mature fruits harvested in the whole fruiting period of each plant is 55, and the mature fruits are square fruits.

The average number of mature fruits harvested in the whole fruiting period of each plant of the wild tomato variety AC is 58, and the fruits are round.

An exemplary fruit profile for a partial fruit is shown in fig. 1.

An exemplary fruit longitudinal cut of a partial fruit is shown in fig. 2.

The square calculation formula is as follows:

square = width at 5% of the longitudinal top of the fruit/width at the longitudinal middle of the fruit (see figure 3).

Fruit square statistics for each of the plants tested are shown in FIG. 4.

Compared with fruits of a wild tomato variety AC, fruits of a 1-SlGT-2-SlGTL1 gene editing plant are changed from round to square, and pectin water content is variable and small.

The result shows that the calyx seu fructus solani lycopersici gene calycinus and the homologous gene calgtl 1 thereof can regulate and control the fruit type of calycinus, and the circular succulent calyx lycopersici material can be converted into square succulent calycinus material by carrying out gene editing on the calycinus gene calycinus-2 and the homologous gene calgtl 1 thereof.

In summary, the inventors discovered for the first time that the SlGT-2 protein and its homologous protein SlGTL1, which controls the formation of square fruits, in tomato, can make round fruits square after knocking out these genes by non-transgenic gene editing technology, and at the same time, pectin state, especially water content, is significantly changed. The square fruits can be quickly cultivated by utilizing the gene editing technology on the target gene provided by the invention, and only half a year to one year are needed in tomatoes.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

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Ser Val Ser Gln Lys Ser Val Val Thr Gly Ala Ala Gln Pro Ala Val

210 215 220

Met Thr Ala Pro Ala Leu Pro Leu Ser Gln Val Pro Ile Gly Asn Asn

225 230 235 240

Asn Leu Asn Gln Met His Arg Pro Gln Gly Asn Thr Thr Thr Thr Lys

245 250 255

Thr Ser Phe Leu Ser Asn Ser Thr Ser Ser Ser Ser Ser Thr Ser Ser

260 265 270

Asp Glu Asp Ile Gln Arg Arg Gln Met Lys Lys Arg Lys Trp Lys Glu

275 280 285

Phe Phe Glu Ser Leu Met Lys Asp Val Ile Glu Lys Gln Glu Glu Leu

290 295 300

Gln Lys Lys Phe Leu Glu Thr Leu Glu Lys Arg Glu Arg Asp Arg Leu

305 310 315 320

Met Arg Glu Glu Ala Trp Arg Val Gln Glu Met Ala Arg Leu Asn Arg

325 330 335

Glu His Asp Leu Leu Val Gln Glu Arg Ser Met Ala Ala Ala Lys Asp

340 345 350

Ala Thr Ile Ile Ala Phe Leu Gln Lys Ile Thr Glu Gln Gln Asn Thr

355 360 365

Gln Thr Pro Asn Ser Thr Asn Asn Thr Ser Pro Ser Pro Phe Pro Ile

370 375 380

Ala Gln Ile Gln Leu Lys Leu Ser Glu Lys Pro Phe Ser Thr Pro Pro

385 390 395 400

Gln Pro Gln Pro Gln Pro Ser Ala Thr Ala Val Ser Leu Pro Met Thr

405 410 415

Ile His Thr Pro Thr Pro Ala Pro Pro Gln Thr Leu Thr Leu Pro Val

420 425 430

Val Ser Ser Lys Ser Leu Glu Pro Pro Lys Ser Asp Asn Gly Gly Glu

435 440 445

Asn Phe Ser Pro Ala Ser Ser Ser Arg Trp Pro Lys Glu Glu Ile Glu

450 455 460

Ala Leu Ile Ser Leu Arg Thr Cys Leu Asp Leu Lys Tyr Gln Glu Asn

465 470 475 480

Gly Pro Lys Gly Pro Leu Trp Glu Glu Ile Ser Ser Gly Met Arg Lys

485 490 495

Ile Gly Tyr Asn Arg Asn Ala Lys Arg Cys Lys Glu Lys Trp Glu Asn

500 505 510

Ile Asn Lys Tyr Phe Lys Lys Val Lys Glu Ser Asn Lys Lys Arg Pro

515 520 525

Glu Asp Ser Lys Thr Cys Pro Tyr Phe His Gln Leu Glu Ala Leu Tyr

530 535 540

Lys Glu Lys Ala Lys Leu Glu Pro Val Pro His Asn Thr Thr Phe Gly

545 550 555 560

Leu Thr Pro Gln Asn Asn Pro Pro Pro Pro Pro Pro Pro Ile Met Ala

565 570 575

Gln Pro Glu Gln Gln Trp Pro Ile Pro Gln Asn Gln Leu His Gln Gln

580 585 590

Asn Arg Asp His His His Asp Asn Glu Ser Asp Ser Met Asp His Asp

595 600 605

Leu Glu Glu Asp Glu Asp Glu Asp Glu Glu Asp Glu Gly Asn Gly Tyr

610 615 620

Glu Ile Ile Ile Thr Asn Lys Gln Gln Ser Ser Ser Met Ala Ala Thr

625 630 635 640

Pro Val Thr Thr Thr Thr Ser Ala Ala Ala Val

645 650

<210> 3

<211> 1965

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 3

atgctgggcg tttccggctt agtaagtagt gaaggtggtg gtgataatcc agaaagcggt 60

ggaggagctg gaagcggagg gagtagtgag attggattag gcggtggaag tggcggcggt 120

ggaggtagta gcggtggatt catgacggaa gatggagaaa gaaattcagg tggaaataga 180

tggccaagac aagaaacaat tgctttgctg aaaataaggt ctgaaatgga tgttattttt 240

agagactcaa gtcttaaagg acctttatgg gaagaagttt ccaggaaaat ggcagacctt 300

gggttccaca gaagttccaa gaaatgcaag gagaagttcg aaaatgtata caaatatcac 360

aagagaacca aggatggccg agcatcgaaa gcggatggaa agaattatag gtttttcgag 420

caattggaag ccctggagaa cattacatct catcattctc taatgccagt accgtcgtct 480

aatacgcgtc ctccaccccc tccgttggaa gctactccaa taaatatggc tatgccaatg 540

gcatcatcaa atgtacaagt cacggcttca caaggtacta ttcctcatca tgttactatt 600

tcatcagcac caccgccacc gaatagcctt tttgctcctt ctcatcaaaa tgctccgtca 660

agttcacccg tgccactacc accaccgcca tcacagcaac catcaccgca gccagctgtc 720

aatccgatta ataatattcc tcaacaagtg aacgcttcag caatgtcgta ttcaacttct 780

tcgtctactt cctcggatga ggatatacaa agaaggcata agaagaagag gaaatggaag 840

gattattttg agaagttcac caaggatgtg attaataagc aggaggaatc gcacaggagg 900

ttcttggaga agcttgagaa gcgggaacat gatcggatgg ttcgagaaga agcatggaaa 960

gtagaggaaa tggcaaggat gaatagggag catgatcttt tagttcaaga aagagcaatg 1020

gcggcagcca aggatgcagc tgttatttct tttttacaaa agataactga acagcaaaac 1080

attcaaattc caaatagtat caacgttggc cctccatcag cacaagtaca aatacaattg 1140

cctgaaaacc cactatccgc gcctgtacca acacaaatac aaccgacgac tgttacagca 1200

gcagcaccac ctcaaccagc accggtccca gtatcgttgc cagtaacaat accagctcca 1260

gtaccagcat taataccatc attgtcgcta ccactgacac caccagtgcc atccaagaac 1320

atggagttag taccaaaaag cgataacgga ggtgatagtt acagtccagc aagctcttca 1380

aggtggccaa aagcagaagt tgaagcattg attaaacttc gtacaaattt agatgtcaaa 1440

taccaagaga acggacctaa aggtccactt tgggaagaga tatcatctgg aatgaagaaa 1500

attggataca atcggaatgc aaagagatgc aaagaaaaat gggaaaacat caacaaatac 1560

ttcaagaagg tgaaggagag caacaaaaaa cgacccgaag attccaaaac ttgcccatat 1620

ttccaccagc tcgatgcact gtacaaggag aaagccaaaa accccgaaac agcttcttca 1680

acgtcttcgt tcaatccttc attcgcttta aaccccgata acaaccaaat ggctcccatc 1740

atggctcgtc cagaacagca atggccactt ccacaacacc atgaaagcac cacccgtatc 1800

gaccacgaaa acgagagcga caacatggat gaagatgatc acgatgatga ggaggatgaa 1860

gatgacgagg acgaaaacaa cgcttatgag atagtagcaa acaagcaaca atcctcaatg 1920

gcggccgcaa acaccactac cagcaccgca acaacaacag tttga 1965

<210> 4

<211> 1956

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 4

atgcttggtg tttcttcaag tttaatagct agcagtaata ctagtattac tgctggtgct 60

gcaggtgatg gagctgccat ttcggcagct ccatcacagt tagcaccgcc accacaagaa 120

gctccggaga gtggtgggag tagtgaaggt ggtggcggtg gaggagattt gtcgattggc 180

ggtgaagatg gagaaaggaa ctcaggtgga aatcgatggc caaggcaaga aactttagct 240

ttactgaaaa ttagatcgga aatggatgtt gttttcaaag attcaagtct taaaggaccg 300

ttatgggaag aagtttccag aaaactcgcg gagttgggtt atcatcgaag tgctaagaaa 360

tgtaaagaga aattcgagaa tgtttacaag tatcacagga gaaccaaaga tggtcgtgct 420

tcgaaagcag atggaaaaac ttatcgattc tttgatcagt tacaggcttt ggaaaacaat 480

ccatcttctc attctaacat accgccacct ccattagcag caacacccat aacaatggca 540

atgccaatgc gatcaggaaa caattcagca aatcctccaa tgccgacgcc aacgccaact 600

ccacaaaatc ataatcattt ttttagtgtt tcgcagaaaa gtgttgtgac aggagcagcg 660

cagcctgctg ttatgactgc acctgcgctg ccactgtcac aagtgccgat aggtaataat 720

aacttgaacc agatgcatcg gcctcaaggt aatactacta ctacaaaaac aagtttcctg 780

tcgaattcaa cttcatcatc atcttcaact tcgtcggatg aggatataca aaggaggcag 840

atgaagaagc ggaaatggaa ggaattcttt gagagtttaa tgaaggatgt gattgagaag 900

caagaggaat tgcagaagaa gtttttggaa acgctcgaga agcgcgagag ggataggttg 960

atgagagagg aggcatggag agtgcaagag atggctagat tgaataggga acatgatctt 1020

ttagtccaag agagatcaat ggcagcagct aaagacgcaa caatcatcgc cttcttgcaa 1080

aaaataactg aacagcaaaa cacacaaacc ccgaatagta caaataacac ttctccttct 1140

ccttttccaa ttgctcaaat tcaattaaaa ttgtccgaaa agccattcag tacaccacca 1200

caaccacaac cacaaccatc agctaccgcg gtatcactgc caatgacaat acatacacca 1260

acaccagcac caccacagac actgacatta cctgtagtat catcaaaatc acttgaacct 1320

ccaaaatccg ataatggtgg tgagaatttc tctccagcaa gctcgtcaag atggccgaaa 1380

gaagaaatcg aagcattgat aagtctccga acctgtttag atctaaaata ccaagaaaat 1440

ggaccgaaag gaccactgtg ggaagaaatt tcatctggaa tgagaaagat aggatacaac 1500

aggaatgcaa agagatgcaa ggaaaaatgg gagaacatca acaagtactt caagaaggta 1560

aaagaaagca acaaaaaaag accagaagat tccaaaactt gcccatattt ccaccagctg 1620

gaagcactgt acaaagaaaa agccaagctc gaacctgtac cacacaacac taccttcgga 1680

ttaacacccc aaaacaatcc tcctcctcct cctcctccca tcatggctca acccgagcaa 1740

caatggccaa ttcctcaaaa tcaacttcac cagcaaaatc gtgatcatca tcacgataat 1800

gaaagcgaca gcatggatca cgatttggaa gaggacgagg atgaggacga agaagatgaa 1860

ggtaatggct atgaaataat aatcacaaat aaacaacaat catcatcaat ggcggctacc 1920

ccagtaacaa caacaacttc tgctgctgca gtttaa 1956

<210> 5

<211> 2717

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 5

atgctgggcg tttccggctt agtaagtagt gaaggtggtg gtgataatcc agaaagcggt 60

ggaggagctg gaagcggagg gagtagtgag attggattag gcggtggaag tggcggcggt 120

ggaggtagta gcggtggatt catgacggaa gatggagaaa gaaattcagg tggaaataga 180

tggccaagac aagaaacaat tgctttgctg aaaataaggt ctgaaatgga tgttattttt 240

agagactcaa gtcttaaagg acctttatgg gaagaagttt ccaggtaatt aaattcaatt 300

tcattattcc aatttcttca cctgaccttc tcaatcatta ttaagctgca ccagtttcat 360

catgcataaa taaaaattga tagaaatgga atctttattt aatttttttt ttcaatttct 420

acttttggga aaaaaataat taatagaatg atttttattt tttgggaaat gaaaagatag 480

atctatggat cagaattcca ttgatttatt gcttttttga ttaaaagggt tattgttttt 540

cagttcattt cactacaaac aatacaacaa aaatacaatt gttgaggaaa ttcagattcc 600

ctccttccgg gttttgagcc aaattcagtt ttgctttttt ggcgtttttt ctttctctgc 660

caattccagc aacaaatttt ggaaactaat ttactcatct tttttgtatt agagttccaa 720

ctttatgaac tacctttttt taaatttagc aaataaataa gtttggtaat catcaaatct 780

aataattaag caagtaaaaa aacaagattt atgattgaga aaaatgtggt ttccatagag 840

tgtttcaatt gtctcctact tgtttaatta attgatttct taattacctt aatcttgatt 900

aataatctca tttttatttt atgtggtgaa tagtatttta ctattgaatt caattaccaa 960

ggatttaaat tattgtactt gtttatttac taccattttt tctaatactt atgccaactg 1020

ttgttatcat gagcaggaaa atggcagacc ttgggttcca cagaagttcc aagaaatgca 1080

aggagaagtt cgaaaatgta tacaaatatc acaagagaac caaggatggc cgagcatcga 1140

aagcggatgg aaagaattat aggtttttcg agcaattgga agccctggag aacattacat 1200

ctcatcattc tctaatgcca gtaccgtcgt ctaatacgcg tcctccaccc cctccgttgg 1260

aagctactcc aataaatatg gctatgccaa tggcatcatc aaatgtacaa gtcacggctt 1320

cacaaggtac tattcctcat catgttacta tttcatcagc accaccgcca ccgaatagcc 1380

tttttgctcc ttctcatcaa aatgctccgt caagttcacc cgtgccacta ccaccaccgc 1440

catcacagca accatcaccg cagccagctg tcaatccgat taataatatt cctcaacaag 1500

tgaacgcttc agcaatgtcg tattcaactt cttcgtctac ttcctcggat gaggatatac 1560

aaagaaggca taagaagaag aggaaatgga aggattattt tgagaagttc accaaggatg 1620

tgattaataa gcaggaggaa tcgcacagga ggttcttgga gaagcttgag aagcgggaac 1680

atgatcggat ggttcgagaa gaagcatgga aagtagagga aatggcaagg atgaataggg 1740

agcatgatct tttagttcaa gaaagagcaa tggcggcagc caaggatgca gctgttattt 1800

cttttttaca aaagataact gaacagcaaa acattcaaat tccaaatagt atcaacgttg 1860

gccctccatc agcacaagta caaatacaat tgcctgaaaa cccactatcc gcgcctgtac 1920

caacacaaat acaaccgacg actgttacag cagcagcacc acctcaacca gcaccggtcc 1980

cagtatcgtt gccagtaaca ataccagctc cagtaccagc attaatacca tcattgtcgc 2040

taccactgac accaccagtg ccatccaaga acatggagtt agtaccaaaa agcgataacg 2100

gaggtgatag ttacagtcca gcaagctctt caaggtggcc aaaagcagaa gttgaagcat 2160

tgattaaact tcgtacaaat ttagatgtca aataccaaga gaacggacct aaaggtccac 2220

tttgggaaga gatatcatct ggaatgaaga aaattggata caatcggaat gcaaagagat 2280

gcaaagaaaa atgggaaaac atcaacaaat acttcaagaa ggtgaaggag agcaacaaaa 2340

aacgacccga agattccaaa acttgcccat atttccacca gctcgatgca ctgtacaagg 2400

agaaagccaa aaaccccgaa acagcttctt caacgtcttc gttcaatcct tcattcgctt 2460

taaaccccga taacaaccaa atggctccca tcatggctcg tccagaacag caatggccac 2520

ttccacaaca ccatgaaagc accacccgta tcgaccacga aaacgagagc gacaacatgg 2580

atgaagatga tcacgatgat gaggaggatg aagatgacga ggacgaaaac aacgcttatg 2640

agatagtagc aaacaagcaa caatcctcaa tggcggccgc aaacaccact accagcaccg 2700

caacaacaac agtttga 2717

<210> 6

<211> 2364

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 6

atgcttggtg tttcttcaag tttaatagct agcagtaata ctagtattac tgctggtgct 60

gcaggtgatg gagctgccat ttcggcagct ccatcacagt tagcaccgcc accacaagaa 120

gctccggaga gtggtgggag tagtgaaggt ggtggcggtg gaggagattt gtcgattggc 180

ggtgaagatg gagaaaggaa ctcaggtgga aatcgatggc caaggcaaga aactttagct 240

ttactgaaaa ttagatcgga aatggatgtt gttttcaaag attcaagtct taaaggaccg 300

ttatgggaag aagtttccag gtactgtttt tttttggtca ttttgattaa ctctttcatc 360

atcatcatat gcatagaatt cagaaaatta aaagatctat tatatttgga attaaaattc 420

gtatttgatg gaaagtgtta ttttttttta gttttgttgt tataccattt tctgctgatt 480

ccagcaagaa atttaggatg agtttaattt ctctactcat cttcaacact ttttgtgctt 540

ttccctattt tccagaaaat tcaagctaag atgttgatga ttgatggttt attatgtttt 600

attttatgta tataaaaata gtatgagatt ttgttttttt attactaatg aatgatgaat 660

atgagatgag ataattaaga caaggtgttt ttctttttgt atccattttg aactttgttg 720

tttatcagaa aactcgcgga gttgggttat catcgaagtg ctaagaaatg taaagagaaa 780

ttcgagaatg tttacaagta tcacaggaga accaaagatg gtcgtgcttc gaaagcagat 840

ggaaaaactt atcgattctt tgatcagtta caggctttgg aaaacaatcc atcttctcat 900

tctaacatac cgccacctcc attagcagca acacccataa caatggcaat gccaatgcga 960

tcaggaaaca attcagcaaa tcctccaatg ccgacgccaa cgccaactcc acaaaatcat 1020

aatcattttt ttagtgtttc gcagaaaagt gttgtgacag gagcagcgca gcctgctgtt 1080

atgactgcac ctgcgctgcc actgtcacaa gtgccgatag gtaataataa cttgaaccag 1140

atgcatcggc ctcaaggtaa tactactact acaaaaacaa gtttcctgtc gaattcaact 1200

tcatcatcat cttcaacttc gtcggatgag gatatacaaa ggaggcagat gaagaagcgg 1260

aaatggaagg aattctttga gagtttaatg aaggatgtga ttgagaagca agaggaattg 1320

cagaagaagt ttttggaaac gctcgagaag cgcgagaggg ataggttgat gagagaggag 1380

gcatggagag tgcaagagat ggctagattg aatagggaac atgatctttt agtccaagag 1440

agatcaatgg cagcagctaa agacgcaaca atcatcgcct tcttgcaaaa aataactgaa 1500

cagcaaaaca cacaaacccc gaatagtaca aataacactt ctccttctcc ttttccaatt 1560

gctcaaattc aattaaaatt gtccgaaaag ccattcagta caccaccaca accacaacca 1620

caaccatcag ctaccgcggt atcactgcca atgacaatac atacaccaac accagcacca 1680

ccacagacac tgacattacc tgtagtatca tcaaaatcac ttgaacctcc aaaatccgat 1740

aatggtggtg agaatttctc tccagcaagc tcgtcaagat ggccgaaaga agaaatcgaa 1800

gcattgataa gtctccgaac ctgtttagat ctaaaatacc aagaaaatgg accgaaagga 1860

ccactgtggg aagaaatttc atctggaatg agaaagatag gatacaacag gaatgcaaag 1920

agatgcaagg aaaaatggga gaacatcaac aagtacttca agaaggtaaa agaaagcaac 1980

aaaaaaagac cagaagattc caaaacttgc ccatatttcc accagctgga agcactgtac 2040

aaagaaaaag ccaagctcga acctgtacca cacaacacta ccttcggatt aacaccccaa 2100

aacaatcctc ctcctcctcc tcctcccatc atggctcaac ccgagcaaca atggccaatt 2160

cctcaaaatc aacttcacca gcaaaatcgt gatcatcatc acgataatga aagcgacagc 2220

atggatcacg atttggaaga ggacgaggat gaggacgaag aagatgaagg taatggctat 2280

gaaataataa tcacaaataa acaacaatca tcatcaatgg cggctacccc agtaacaaca 2340

acaacttctg ctgctgcagt ttaa 2364

<210> 7

<211> 97

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gggugauaau ccagaaagcg gguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60

ccguuaucaa cuugaaaaag uggcaccgag ucggugc 97

<210> 8

<211> 97

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gaagaagcuc cggagagugg uguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60

ccguuaucaa cuugaaaaag uggcaccgag ucggugc 97

<210> 9

<211> 28

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Met Leu Gly Val Ser Gly Leu Val Ser Ser Glu Gly Gly Gly Asp Asn

1 5 10 15

Pro Glu Ser Arg Trp Arg Ser Trp Lys Arg Arg Glu

20 25

<210> 10

<211> 79

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Met Leu Gly Val Ser Ser Ser Leu Ile Ala Ser Ser Asn Thr Ser Ile

1 5 10 15

Thr Ala Gly Ala Ala Gly Asp Gly Ala Ala Ile Ser Ala Ala Pro Ser

20 25 30

Gln Leu Ala Pro Pro Pro Gln Glu Ala Pro Glu Arg Val Val Gly Val

35 40 45

Val Lys Val Val Ala Val Glu Glu Ile Cys Arg Leu Ala Val Lys Met

50 55 60

Glu Lys Gly Thr Gln Val Glu Ile Asp Gly Gln Gly Lys Lys Leu

65 70 75

<210> 11

<211> 1966

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

atgctgggcg tttccggctt agtaagtagt gaaggtggtg gtgataatcc agaaagtcgg 60

tggaggagct ggaagcggag ggagtagtga gattggatta ggcggtggaa gtggcggcgg 120

tggaggtagt agcggtggat tcatgacgga agatggagaa agaaattcag gtggaaatag 180

atggccaaga caagaaacaa ttgctttgct gaaaataagg tctgaaatgg atgttatttt 240

tagagactca agtcttaaag gacctttatg ggaagaagtt tccaggaaaa tggcagacct 300

tgggttccac agaagttcca agaaatgcaa ggagaagttc gaaaatgtat acaaatatca 360

caagagaacc aaggatggcc gagcatcgaa agcggatgga aagaattata ggtttttcga 420

gcaattggaa gccctggaga acattacatc tcatcattct ctaatgccag taccgtcgtc 480

taatacgcgt cctccacccc ctccgttgga agctactcca ataaatatgg ctatgccaat 540

ggcatcatca aatgtacaag tcacggcttc acaaggtact attcctcatc atgttactat 600

ttcatcagca ccaccgccac cgaatagcct ttttgctcct tctcatcaaa atgctccgtc 660

aagttcaccc gtgccactac caccaccgcc atcacagcaa ccatcaccgc agccagctgt 720

caatccgatt aataatattc ctcaacaagt gaacgcttca gcaatgtcgt attcaacttc 780

ttcgtctact tcctcggatg aggatataca aagaaggcat aagaagaaga ggaaatggaa 840

ggattatttt gagaagttca ccaaggatgt gattaataag caggaggaat cgcacaggag 900

gttcttggag aagcttgaga agcgggaaca tgatcggatg gttcgagaag aagcatggaa 960

agtagaggaa atggcaagga tgaataggga gcatgatctt ttagttcaag aaagagcaat 1020

ggcggcagcc aaggatgcag ctgttatttc ttttttacaa aagataactg aacagcaaaa 1080

cattcaaatt ccaaatagta tcaacgttgg ccctccatca gcacaagtac aaatacaatt 1140

gcctgaaaac ccactatccg cgcctgtacc aacacaaata caaccgacga ctgttacagc 1200

agcagcacca cctcaaccag caccggtccc agtatcgttg ccagtaacaa taccagctcc 1260

agtaccagca ttaataccat cattgtcgct accactgaca ccaccagtgc catccaagaa 1320

catggagtta gtaccaaaaa gcgataacgg aggtgatagt tacagtccag caagctcttc 1380

aaggtggcca aaagcagaag ttgaagcatt gattaaactt cgtacaaatt tagatgtcaa 1440

ataccaagag aacggaccta aaggtccact ttgggaagag atatcatctg gaatgaagaa 1500

aattggatac aatcggaatg caaagagatg caaagaaaaa tgggaaaaca tcaacaaata 1560

cttcaagaag gtgaaggaga gcaacaaaaa acgacccgaa gattccaaaa cttgcccata 1620

tttccaccag ctcgatgcac tgtacaagga gaaagccaaa aaccccgaaa cagcttcttc 1680

aacgtcttcg ttcaatcctt cattcgcttt aaaccccgat aacaaccaaa tggctcccat 1740

catggctcgt ccagaacagc aatggccact tccacaacac catgaaagca ccacccgtat 1800

cgaccacgaa aacgagagcg acaacatgga tgaagatgat cacgatgatg aggaggatga 1860

agatgacgag gacgaaaaca acgcttatga gatagtagca aacaagcaac aatcctcaat 1920

ggcggccgca aacaccacta ccagcaccgc aacaacaaca gtttga 1966

<210> 12

<211> 1958

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atgcttggtg tttcttcaag tttaatagct agcagtaata ctagtattac tgctggtgct 60

gcaggtgatg gagctgccat ttcggcagct ccatcacagt tagcaccgcc accacaagaa 120

gctccggaga gagtggtggg agtagtgaag gtggtggcgg tggaggagat ttgtcgattg 180

gcggtgaaga tggagaaagg aactcaggtg gaaatcgatg gccaaggcaa gaaactttag 240

ctttactgaa aattagatcg gaaatggatg ttgttttcaa agattcaagt cttaaaggac 300

cgttatggga agaagtttcc agaaaactcg cggagttggg ttatcatcga agtgctaaga 360

aatgtaaaga gaaattcgag aatgtttaca agtatcacag gagaaccaaa gatggtcgtg 420

cttcgaaagc agatggaaaa acttatcgat tctttgatca gttacaggct ttggaaaaca 480

atccatcttc tcattctaac ataccgccac ctccattagc agcaacaccc ataacaatgg 540

caatgccaat gcgatcagga aacaattcag caaatcctcc aatgccgacg ccaacgccaa 600

ctccacaaaa tcataatcat ttttttagtg tttcgcagaa aagtgttgtg acaggagcag 660

cgcagcctgc tgttatgact gcacctgcgc tgccactgtc acaagtgccg ataggtaata 720

ataacttgaa ccagatgcat cggcctcaag gtaatactac tactacaaaa acaagtttcc 780

tgtcgaattc aacttcatca tcatcttcaa cttcgtcgga tgaggatata caaaggaggc 840

agatgaagaa gcggaaatgg aaggaattct ttgagagttt aatgaaggat gtgattgaga 900

agcaagagga attgcagaag aagtttttgg aaacgctcga gaagcgcgag agggataggt 960

tgatgagaga ggaggcatgg agagtgcaag agatggctag attgaatagg gaacatgatc 1020

ttttagtcca agagagatca atggcagcag ctaaagacgc aacaatcatc gccttcttgc 1080

aaaaaataac tgaacagcaa aacacacaaa ccccgaatag tacaaataac acttctcctt 1140

ctccttttcc aattgctcaa attcaattaa aattgtccga aaagccattc agtacaccac 1200

cacaaccaca accacaacca tcagctaccg cggtatcact gccaatgaca atacatacac 1260

caacaccagc accaccacag acactgacat tacctgtagt atcatcaaaa tcacttgaac 1320

ctccaaaatc cgataatggt ggtgagaatt tctctccagc aagctcgtca agatggccga 1380

aagaagaaat cgaagcattg ataagtctcc gaacctgttt agatctaaaa taccaagaaa 1440

atggaccgaa aggaccactg tgggaagaaa tttcatctgg aatgagaaag ataggataca 1500

acaggaatgc aaagagatgc aaggaaaaat gggagaacat caacaagtac ttcaagaagg 1560

taaaagaaag caacaaaaaa agaccagaag attccaaaac ttgcccatat ttccaccagc 1620

tggaagcact gtacaaagaa aaagccaagc tcgaacctgt accacacaac actaccttcg 1680

gattaacacc ccaaaacaat cctcctcctc ctcctcctcc catcatggct caacccgagc 1740

aacaatggcc aattcctcaa aatcaacttc accagcaaaa tcgtgatcat catcacgata 1800

atgaaagcga cagcatggat cacgatttgg aagaggacga ggatgaggac gaagaagatg 1860

aaggtaatgg ctatgaaata ataatcacaa ataaacaaca atcatcatca atggcggcta 1920

ccccagtaac aacaacaact tctgctgctg cagtttaa 1958

Claims (5)

1. A method for controlling the shape of a plant fruit, characterized by: the method comprises the following steps: inhibiting the expression of the SlGT-2 gene and the SlGTL1 gene in the receptor round fruit plant to obtain a target plant with square fruits; the plant is tomato;

the SlGT-2 gene is shown in the following B1) or B2):

b1 A coding sequence of the coding chain is a DNA molecule of a sequence 3 in a sequence table;

b2 A nucleotide of the coding chain is a DNA molecule of a sequence 5 in a sequence table;

the SlGTL1 gene is a gene shown in the following B3) or B4):

b3 A coding sequence of the coding chain is a DNA molecule of a sequence 4 in a sequence table;

b4 The nucleotide of the coding strand is a DNA molecule of a sequence 6 in a sequence table.

2. The method according to claim 1, characterized in that: the expression of the SlGT-2 gene of claim 1 and the SlGTL1 gene of claim 1 in the recipient circular fruit plant is achieved by performing gene editing on the SlGT-2 gene and the SlGTL1 gene in the plant by means of a CRISPR/Cas9 system, wherein the CRISPR/Cas9 system comprises a plasmid expressing Cas9 and sgrnas, the sgrnas are sgrnas 1 and 2 for target sequence 1 and target sequence 2, the target sequence 1 is 40-59 of sequence 5 in the sequence table, and the target sequence 2 is 116-136 of sequence 6 in the sequence table.

3. The method according to claim 2, characterized in that: the nucleotide sequence of the sgRNA1 is shown as a sequence 7 in a sequence table, and the nucleotide sequence of the sgRNA2 is shown as a sequence 8 in the sequence table.

4. A method according to claim 3, characterized in that: the expression of the SlGT-2 gene of claim 1 and the SlGTL1 gene of claim 1 in the recipient round fruit plant is the following X1 and X2:

x1 mutates the SlGT-2 gene shown in a sequence 5 in a sequence table into a SlGT-2+T gene, wherein the coding sequence of the SlGT-2+T gene is shown in a sequence 11;

and X2 mutates the SlGTL1 gene shown in a sequence 6 in the sequence table into a SlGTL1+AG gene, wherein the coding sequence of the SlGTL1+AG gene is shown in a sequence 12.

5. Use of the method according to any one of claims 1-4 for controlling the water content of pectin in a plant, said plant being tomato.