CN115707713B

CN115707713B - Fruit-shape related protein, and coding gene and application thereof

Info

Publication number: CN115707713B
Application number: CN202110955318.5A
Authority: CN
Inventors: 李传友; 朱强; 邓磊; 杨天霞; 蒋红玲
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2024-03-01
Anticipated expiration: 2041-08-19
Also published as: CN115707713A

Abstract

The invention discloses a SlKRP7 protein related to plant fruit shape regulation and control and a related biological material and application thereof. The SlKRP7 protein may specifically be a protein of the following A1), A2) or A3): a1 Amino acid sequence is protein of sequence 1 in a sequence table; a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 90 percent of identity with the protein shown in A1) and has the activity of regulating the fruit shape of plants; a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2). The SlKRP7 protein and its related biomaterials can be used to transform long fruits into round fruits.

Description

Fruit-shape related protein, and coding gene and application thereof

Technical Field

The invention relates to a fruit-shaped related protein, a coding gene and application thereof in the field of biotechnology.

Background

Tomato is one of the most important vegetables and fruits worldwide. Tomato fruit is most commonly round and oblong in shape, round conforming to most consumer aesthetics, but round fruits are more easily crushed than oblong, oblong fruits provide consumers with new shape options, and oblong fruits are more resistant to pressure. Round is the most dominant shape, so it is very necessary to cultivate fruits of both shapes simultaneously.

Disclosure of Invention

The invention aims to solve the technical problem of simply and efficiently regulating and controlling the fruit shape of plants.

The invention provides a protein, named SlKRP7, which is a protein of the following A1), A2) or A3):

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

a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 90 percent of identity with the protein shown in A1) and has the activity of regulating the fruit shape of plants;

a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2).

Wherein the sequence 3 in the sequence table consists of 212 amino acid residues.

The protein may be derived from tomato (Solanum lycopersicum).

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.

In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, perresidue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biological materials related to the protein SlKRP7 are also within the scope of the invention.

The biological material related to the protein SlKRP7 provided by the invention is any one of the following B1) to B5):

b1 A nucleic acid molecule encoding said protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B1);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1), or a transgenic plant cell line comprising the expression cassette of B2), or a transgenic plant cell line comprising the recombinant vector of B3).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the nucleic acid molecule of B1) is a gene represented by B1) or B2) as follows:

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

b2 The nucleotide of the coding strand is a cDNA molecule or a DNA molecule of sequence 3 in the sequence table.

Wherein, the sequence 3 in the sequence table consists of 639 nucleotides, and codes the protein shown in the sequence 1 in the sequence table.

In the above biological material, the expression cassette (SlKRP 7 gene expression cassette) described in B2) containing the nucleic acid molecule refers to a nucleic acid molecule capable of expressing SlKRP7 in a host cell, and the nucleic acid molecule may include not only a promoter for initiating transcription of SlKRP7 gene but also a terminator for terminating transcription of SlKRP7. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiology 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

Recombinant vectors containing the gene encoding the protein SlKRP7 or the expression cassette for the gene encoding the protein SlKRP7 can be constructed using existing plant expression vectors. The plant expression vector may be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pGWB18, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When the SlKRP7 is used for constructing the recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like can be added before transcription initiation nucleotide thereof, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants.

In the above biological material, the recombinant microorganism may specifically be yeast, bacteria, algae and fungi; the bacteria may be Agrobacterium LBA4404 strain as described.

Use of the protein described above, or any of the following for the biological material described above:

c1 Regulating and controlling the fruit shape of the plant;

c2 Preparing a product for regulating and controlling the fruit shape of the plant;

c3 Changing the shape of the plant fruit from a long fruit to a round fruit;

c4 Preparing a product which converts the shape of the plant fruit from a long fruit to a round fruit.

In order to solve the technical problems, the invention also provides a plant reagent which is used for regulating and controlling the fruit shape of plants.

The plant reagent provided by the invention contains the protein or/and the protein related biological material.

The active ingredient of the plant agent may be the protein or/and the biological material related to the protein, and the active ingredient of the plant agent may further contain other biological components or/and non-biological components, and the other active components of the plant agent may be determined by those skilled in the art according to the nitrogen absorption effect of plants.

In order to solve the technical problems, the invention also provides a method for cultivating round fruit plants.

The method for cultivating the round fruit plant provided by the invention comprises the step of introducing nucleic acid molecules encoding the protein into a target plant to obtain the round fruit plant.

The plant of interest may be a monocot or dicot. The dicotyledonous plant may be any of F1) -F4):

f1 Tubular flower plants;

f2 Solanaceae plant;

f3 Plants of the genus Lycopersicon;

f4 Tomato (tomato).

In the above method, the nucleic acid molecule may be modified as follows before being introduced into the target plant to achieve better expression:

1) Modifying the gene sequence adjacent to the initiation methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) Ligating to promoters expressed by various plants to facilitate expression thereof in plants; the promoter may include constitutive, inducible, chronologically regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space of expression requirements and will also depend on the target species; for example, a tissue or organ specific expression promoter, depending on the desired time period of development of the receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots;

3) The expression efficiency of the gene of the invention can be improved by connecting with a proper transcription terminator; e.g., tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants may be ligated to the gene of the present invention;

4) Enhancer sequences such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.

The nucleic acid molecules can be introduced into plant cells by conventional biotechnological methods using Ti plasmids, plant virus cultivars, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,PlantMolecularBiology (2 nd Edition).

In the above method, the round fruit plant may be a transgenic plant, or a plant obtained by conventional breeding techniques such as crossing.

In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

Transgenic experiments in which the SlKRP7 coding sequence was introduced into a variety of long fruit tomatoes prove that transgenic tomatoes expressing the SlKRP7 protein are transformed from long fruits to round fruits compared with recipient tomatoes, which means that the SlKRP7 protein is a gene related to fruit shape and the SlKRP7 protein can be used to transform long fruits to round fruits. The method of the invention has simple operation and low cost, greatly accelerates the breeding process and has wide application prospect.

Drawings

FIG. 1 is a photograph of fruit phenotypes of wild type M82 tomato of a long fruit versus transgenic homozygous tomato of a round fruit.

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.

The long tomato variety M82 in the following examples is the American tomato genetic resource center product (TGRC, http:// TGRC. Ucdavis. Edu /), accession number LA3475.

The vectors pGWB18 and pQB used in the examples below are both the Addgene vector library products

(http://www.addgene.org/)。

The E.coli Top10 strain and the Agrobacterium LBA4404 strain used in the examples below were all produced by Beijing Hua Vietnam BioCo.

The MS solid medium containing 1.0mg/L indoleacetic acid, 1.75mg/L zeatin and having a pH of 5.9 used in the following examples was a basic medium comprising MS minimal medium (Beijing Wash Vietnam biological product), to which indoleacetic acid and zeatin nucleoside were added to give a medium having a concentration of 1.0mg/L indoleacetic acid, a concentration of zeatin nucleoside of 1.75mg/L and a pH of 5.9.

As the MS solid medium containing 1.0mg/L of indoleacetic acid, 1.75mg/L of zeatin, 200mg/L of timentin and 75mg/L of kanamycin and having a pH of 5.9, the MS solid medium used in the following examples was a basic medium comprising MS minimal medium (Beijing Hua Vietnam biological product), to which medium was added indoleacetic acid, zeatin, timentin and kanamycin to give a concentration of 1.0mg/L of indoleacetic acid, a concentration of zeatin of 1.75mg/L, a concentration of timentin of 200mg/L, a concentration of kanamycin of 75mg/L and a pH of 5.9.

The MS solid medium containing 200mg/L of timentin and 50mg/L of kanamycin and having a pH of 5.9 used in the following examples was a basal medium comprising MS minimal medium (Beijing Hua Vietnam biological product), to which the timentin and kanamycin were added to give a concentration of 200mg/L of timentin, a concentration of 50mg/L of kanamycin and a pH of 5.9.

The MS medium containing 150mg/L hygromycin used in the following examples was a medium in which MS minimal medium (Beijing Wash Vietnam biological product) was used as a basal medium, and hygromycin was added to the basal medium to give a hygromycin concentration of 150mg/L and a pH value of 5.9.

The 1/2MS medium containing 800mg/L hygromycin used in the following examples was a medium having a pH of 5.9 and a concentration of 800mg/L hygromycin obtained by adding hygromycin to a basic medium of 1/2 MS.

Example 1 transformation of elongated fruits into round fruits Using transgenic methods

The inventor discovers a gene SlKRP7 related to tomato fruit shape, and the sequence of the gene is shown as a sequence 2 and consists of 990 nucleotides; the coding sequence of the SlKRP7 is sequence 3 in a sequence table, consists of 639 nucleotides, codes the protein shown in the sequence 1, and is named as protein SlKRP7.

1. Construction of recombinant vectors

The genomic DNA of the wild tomato M82 plants was amplified by PCR using the following primer sequences:

forward primer: 5'-ATGAGAAGAAAGTATAAGTGC-3';

reverse primer: 5'-TCATTGTCGAACCCATTCGTAGCG-3'.

The coding sequence of the SlKRP7 (the nucleotide sequence is shown as a sequence 3 in a sequence table and the protein shown as a coding sequence 1) is obtained through amplification, and is recovered for standby.

The linearized pQB was obtained by cleavage of the gate vector pQB with EcoRV enzyme and ligation of the above-described SlKRP7 coding sequence to the linearized pQB with T4 ligase gave a ligation product.

And (3) transforming the ligation product into the escherichia coli Top10 strain to be competent, extracting plasmids, sequencing, and identifying clones with correct sequences to obtain an intermediate plasmid pQB-SlKRP7.

The coding sequence of SlKRP7 in the intermediate plasmid pQB-SlKRP7 is recombined into the final vector pGWB18 by LR reaction to obtain a recombinant vector pGWB18-SlKRP7. The recombinant vector pGWB18-SlKRP7 contains a SlKRP7 coding sequence shown in a sequence 3 in a sequence table, can transcribe mRNA shown in a sequence 4 in the sequence table, and further expresses protein shown in a sequence 1 in the sequence table.

2. Acquisition and identification of transgenic tomato

First), transgenic tomato obtaining:

and (3) introducing the recombinant plasmid pGWB18-SlKRP7 obtained in the step (A) into an agrobacterium tumefaciens LBA4404 strain to obtain recombinant bacteria pGWB18-SlKRP7/LBA4404.

Infecting a bacterial solution of recombinant bacteria pGWB18-SlKRP7/LBA4404 on a long tomato M82 explant with a few juice varieties (the cut cotyledons are used as the explants), and then co-culturing for 48 hours in an MS solid culture medium containing 1.0mg/L indoleacetic acid, 1.75mg/L zeatin nucleoside and pH of 5.9 at 25+/-1.5 ℃ and illumination intensity of 100-200 lx; transferring into MS solid culture medium containing 1.0mg/L indoleacetic acid, 1.75mg/L zeatin, 200mg/L timentin and 75mg/L kanamycin, and pH 5.9, and under conditions of 25+ -1.5deg.C, 16h/d illumination, 8h/d darkness, and 800-1200lx illumination intensityCulturing until regeneration buds grow out; cutting off the regenerated bud when the regenerated bud grows to 2-3cm, transferring into MS solid culture medium containing 200mg/L of timentin and 50mg/L of kanamycin and pH of 5.9, culturing at 25+ -1.5deg.C under illumination for 16h/d, darkness for 8h/d and illumination intensity of 800-1200lx until rooting to obtain T ₀ And (5) replacing tomato plants.

Two) identification of transgenic tomato:

seedlings capable of growing roots are transgenic positive seedlings on MS culture medium containing 150mg/L hygromycin. Will T ₀ Cutting generation positive tomato and wild type M82 tomato in MS culture medium containing 150mg/L hygromycin, and comparing with wild type M82 tomato, T ₀ The generation of tomato grows root while the wild M82 tomato does not root, T ₀ The generation positive tomato is T transferred with the SlKRP7 gene ₀ Transgenic tomato.

Will T ₀ Transgenic tomato is planted in a greenhouse, and T is obtained through selfing ₁ Transgenic tomato seed. Will T ₁ Sowing the transgenic tomato seeds on a 1/2MS culture medium containing 800mg/L hygromycin, transplanting 10 seedlings with normal growth to soil, and harvesting T from single plant ₂ Transgenic tomato seed. 30 grains T of each sowing ₂ Transgenic tomato seeds are on 1/2MS culture medium of 800mg/L hygromycin, and the single plant with all seeds having resistance is transgenic homozygous tomato.

Gene expression assays showed about 20-fold overexpression of SlKRP7 in transgenic tomato.

Thirdly, observing fruits

The plants to be tested are transgenic homozygous tomatoes and wild type M82 tomatoes (as control plants) obtained in the second step).

Culturing the plant to be tested in a greenhouse for 20 days under the conditions of 25 ℃ and 16 h/8 h darkness. 10 seedlings with consistent growth vigor of transgenic homozygous tomatoes and wild M82 tomatoes are selected and transplanted into a greenhouse, the plant spacing and the row spacing are over 60cm, and the two materials are randomly distributed. And the normal water and fertilizer management ensures that the water and fertilizer conditions of all plants are basically consistent. Fruits of the transgenic homozygous tomatoes were compared to fruits of the wild type M82 tomatoes of the control plants after the fruits became completely red and photographed for recording. 15 tomato fruits were randomly picked for each line and the length and width of the fruits were measured.

The definition and formula for tomato shape are as follows:

fruits with aspect ratios greater than 1 are defined as long fruits.

Fruits with aspect ratios approximately equal to 1 are defined as round fruits.

Aspect ratio refers to the ratio of length to width.

Length refers to the longest diameter of the fruit perpendicular to the pedicel portion.

Width refers to the longest diameter of the fruit parallel to the pedicel portion.

The results are shown in FIG. 1, where the aspect ratio of the fruits of transgenic homozygous tomatoes changed from elongated to round M82 tomato fruits was about 1.2 compared to the wild type M82 tomato of the control plant; the aspect ratio of the transgenic homozygous tomato fruit is about 1.0.

The above shows that the SlKRP7 gene and the protein encoded by the same can regulate the shape of tomato, and can change the tomato material of long fruits into the tomato material of round fruits.

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

<110> institute of genetic and developmental biology of national academy of sciences

<120> fruit-shape related protein, and coding gene and application thereof

<130> GNCSY212245

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 212

<212> PRT

<213> tomato (Lycopersicon esculentum)

<400> 1

Met Arg Arg Lys Tyr Lys Cys Lys Ser Lys Gly Ile Ile Gly Glu Val

1 5 10 15

Val Ile Met Glu Val Ala Glu Val Ala Val Lys Met Thr Arg Glu Arg

20 25 30

Glu Val Leu Glu Val Val Asp Thr Arg Lys Arg Lys Lys Arg Asp Gly

35 40 45

Asp Leu Glu Met Ser Pro Thr Val Ala Arg Val Arg Arg Asn Ser Val

50 55 60

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

65 70 75 80

Leu Cys Glu Pro Ala Val Ser Ser Asn Phe Asp Asn Val Ser Ala Ser

85 90 95

Gly Phe Gly Asp Asn Glu Ser Ser Asn Val Thr Lys Gly Ser Ser Lys

100 105 110

Phe Leu Asp Leu Asp Glu Asp Ser Val Glu Ile Ala Thr Ser Tyr Ser

115 120 125

Glu Leu Arg Glu Ser Arg Glu Ile Ile Leu Ser Ser Ser Lys Phe Lys

130 135 140

Val Glu Phe Gln Ser Thr Pro Lys Pro Gln His Ala Lys Ser Cys Arg

145 150 155 160

Arg Arg Leu Thr Glu Ala Thr Met Pro Ser Glu Ala Glu Leu Asp Val

165 170 175

Phe Phe Ala Ala Ala Glu Lys Asp Leu His Lys His Phe Ala Glu Lys

180 185 190

Tyr Asn Phe Asp Phe Ala Lys Glu Glu Pro Leu Glu Gly Arg Tyr Glu

195 200 205

Trp Val Arg Gln

210

<210> 2

<211> 990

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 2

atgagaagaa agtataagtg caagtcgaag ggaattattg gagaagttgt gattatggag 60

gttgcggagg tagcagtgaa gatgacaaga gaacgggagg ttttagaggt ggttgacaca 120

aggaagagga agaagcgtga tggtgatttg gagatgtcac caactgtagc tcgtgttaga 180

aggaattccg taatctcaga atctccagca agcgagctta gttcccaggg aaatactgtg 240

ttgtgtgaac cggctgtgag ttcgaacttt gataacgttt cagcatctgg ttttggagat 300

aatgaatcaa gcaatgtcac aaaaggaagc tcgaaatttt tagatctgga tgtatgtgaa 360

attaaactct ttgaatttta ctagtttcgc tcttccattt ttctgctttg ttattttcag 420

gtctgatagt aatgcattag aattggagcg aattgttatg ctgttctata actgttcatt 480

tatggcggac tgatatttga atttcgaatt tcgaattttt caggaggaca gtgtggaaat 540

tgcaaccagt tattcagagt tgagagaaag gtaaatatgc attctatatg taattttaat 600

ggcgtttcaa ttcactttca gatccgcctt ttctttcaac gtttattgaa aacaaagaat 660

cattctgcag tagagagata atactatcat ccagcaaatt caaagttgaa tttcaatcca 720

caccgaaacc acaacatgct aaatcttgcc gccgccgatt gacagaggca actatgccct 780

ccgaggctga gcttgatgtg ttctttgctg cagccgagaa agatctccat aaacattttg 840

cagaaaagta caaaaactct ctctctctcc ctatctcctt ttttttcaaa attttgcatt 900

actcatgaaa gtgcgtgctg taacagatac aactttgact ttgcaaaaga ggagccattg 960

gaaggtcgct acgaatgggt tcgacaatga 990

<210> 3

<211> 639

<212> DNA

<213> tomato (Lycopersicon esculentum)

<400> 3

atgagaagaa agtataagtg caagtcgaag ggaattattg gagaagttgt gattatggag 60

gttgcggagg tagcagtgaa gatgacaaga gaacgggagg ttttagaggt ggttgacaca 120

aggaagagga agaagcgtga tggtgatttg gagatgtcac caactgtagc tcgtgttaga 180

aggaattccg taatctcaga atctccagca agcgagctta gttcccaggg aaatactgtg 240

ttgtgtgaac cggctgtgag ttcgaacttt gataacgttt cagcatctgg ttttggagat 300

aatgaatcaa gcaatgtcac aaaaggaagc tcgaaatttt tagatctgga tgaggacagt 360

gtggaaattg caaccagtta ttcagagttg agagaaagta gagagataat actatcatcc 420

agcaaattca aagttgaatt tcaatccaca ccgaaaccac aacatgctaa atcttgccgc 480

cgccgattga cagaggcaac tatgccctcc gaggctgagc ttgatgtgtt ctttgctgca 540

gccgagaaag atctccataa acattttgca gaaaaataca actttgactt tgcaaaagag 600

gagccattgg aaggtcgcta cgaatgggtt cgacaatga 639

<210> 4

<211> 639

<212> RNA

<213> tomato (Lycopersicon esculentum)

<400> 4

augagaagaa aguauaagug caagucgaag ggaauuauug gagaaguugu gauuauggag 60

guugcggagg uagcagugaa gaugacaaga gaacgggagg uuuuagaggu gguugacaca 120

aggaagagga agaagcguga uggugauuug gagaugucac caacuguagc ucguguuaga 180

aggaauuccg uaaucucaga aucuccagca agcgagcuua guucccaggg aaauacugug 240

uugugugaac cggcugugag uucgaacuuu gauaacguuu cagcaucugg uuuuggagau 300

aaugaaucaa gcaaugucac aaaaggaagc ucgaaauuuu uagaucugga ugaggacagu 360

guggaaauug caaccaguua uucagaguug agagaaagua gagagauaau acuaucaucc 420

agcaaauuca aaguugaauu ucaauccaca ccgaaaccac aacaugcuaa aucuugccgc 480

cgccgauuga cagaggcaac uaugcccucc gaggcugagc uugauguguu cuuugcugca 540

gccgagaaag aucuccauaa acauuuugca gaaaaauaca acuuugacuu ugcaaaagag 600

gagccauugg aaggucgcua cgaauggguu cgacaauga 639

Claims

1. Use of a protein, or a biological material associated with said protein, in any of the following:

c1 Regulating tomato shape;

c2 Preparing a product for regulating and controlling the shape of the tomato fruits;

c3 Changing tomato shape from long shape to round shape;

c4 Preparing a product which converts tomato fruit shape from long fruit to round fruit;

the protein is the protein of A1) or A2) as follows:

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

a2 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1);

the biomaterial is any one of the following B1) to B4):

b1 A nucleic acid molecule encoding said protein;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

2. The use according to claim 1, wherein the coding sequence of the nucleic acid molecule B1) is a DNA molecule of sequence 3 in the sequence listing.

3. A method of growing round tomatoes, comprising introducing a nucleic acid molecule encoding a protein according to claim 1 into a tomato of interest to obtain round tomatoes.