CN113684225B - Application of tomato SlHMGA3 gene in cultivation of tomatoes with delayed fruit ripening - Google Patents

Abstract

The invention discloses an application of a tomato SlHMGA3 gene in cultivating tomatoes with delayed fruit ripening, belonging to the technical field of tomato genetic engineering, wherein a tomato SlHMGA3 gene CRISPR/Cas9 expression vector is constructed by the application method; designing 2 target sequences of the SlHMGA3 gene, then carrying out gene synthesis on the promoter and the sgRNA sequence containing 2 targets, and inserting the promoter and the sgRNA sequence between two enzyme cutting sites of the vector to obtain the SlHMGA3 gene mutation vector. The constructed sequencing correct vector is transferred into a host cell, then the cotyledon explant of the tomato is used for infecting tomato, and tomato plants with the mutation of the SlHMGA3 gene but without the gene knockout vector sequence are screened from the positive transgenic tomato offspring, so that the tomato plants with delayed mature fruits are obtained. To facilitate identification and selection of tomato plants, the vectors used may be processed, such as by the addition of plant selectable markers or antibiotic markers that are resistant.

Description

Application of tomato SlHMGA3 gene in cultivation of tomatoes with delayed fruit ripening

Technical Field

The invention belongs to the technical field of tomato genetic engineering, and particularly relates to application of a tomato SlHMGA3 gene in cultivation of tomatoes with delayed fruit ripening.

Background

The tomato is rich in multiple vitamins, has high nutritive value, special flavor and high agricultural economic value. Tomato is diploid and has small genome; the growth cycle is short, the growth and development stages of fruits are easy to distinguish, and the mature phenotype is easy to observe; tomato germplasm resources, mutant libraries, high-density genetic maps and EST resources are rich; maturation of the genetic transformation system; the whole genome fine sequencing of cultivated tomatoes has been completed, so tomatoes become model plants for studying the maturation of fleshy fruits. Tomatoes are respiratory fruits, and the color, taste, smell, fruit essence, physiological and biochemical metabolites and the like change in the ripening process. At the transcriptional level, the metabolic changes in fruit maturation are closely regulated by a variety of related transcription factors. Thus, the transcription factor regulation network becomes a hotspot for researching the mechanism of the mature molecules of fruits.

HMGA protein is a structural transcription factor, belongs to the family of HMG proteins (The high mobility groups), is a non-histone chromatin binding protein, and plays an important role in assembling and reconstructing chromosomes and regulating gene transcription, and comprises three subfamilies of HMGA, HMGB and HMGN. The structure and function of the HMG family have been studied extensively in mammals, but little in plants. HMGA proteins are ubiquitously expressed in various tissues and organs of plants, consisting of an N-terminal domain with a GH1 domain (histone H1 central globular domain) and a C-terminal domain comprising an AT-hook motif. The family of HMGA-like proteins in arabidopsis comprises three proteins: GH1-HMGA1, GH1-HMGA2 and GH1-HMGA3. These proteins typically have an additional highly conserved H1/H5 linking domain in histone H1, as compared to mammalian HMGA proteins. As a classical HMGA protein, GH1-HMGA3 has four well-recognized AT-hook motifs. The corn HMGA protein has strong binding force with AT-enriched DNA and can be strongly phosphorylated by SUC1 related kinase, thereby reducing the binding force with AT-enriched DNA in vitro. AtHMGA (recently more named GH1-HMGA 3) is located in the nucleus where it is extremely active. HMGA as a structural transcription factor, each protein has three conserved AT-hook DNA binding motifs that can be inserted into the minor groove of DNA and interact with AT base rich regions. HMGA proteins can interact specifically with a number of other proteins in vivo, inhibiting and activating transcription of these proteins.

The expression level of the transcription factor in the plant is changed by fully utilizing biotechnology means, and a foundation can be laid for the stable development of plant dominant breeding and agricultural production. Therefore, the cloning of the SlHMGA3 gene and the transgenic technology are adopted to cultivate the tomato material with the SlHMGA3 mutation, so that good genetic germplasm resources are provided in the aspect of the work of cultivating late-maturing tomato varieties, and the method has good application prospect.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides application of a tomato SlHMGA3 gene in cultivating tomatoes with fruits delayed to mature, which is realized by the following technology.

Use of a tomato SlHMGA3 gene in cultivating tomatoes with delayed fruit ripening, said tomato SlHMGA3 gene comprising any one of the following nucleotide sequences:

(1) As set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

(2) From SEQ ID NO:1, and has a nucleotide sequence which does not change the function of the original nucleotide sequence and has the same function;

(3) And SEQ ID NO:1 and encodes a nucleotide sequence having more than 90% homology as set forth in SEQ ID NO:2, and a DNA molecule having an amino acid sequence shown in the specification.

The four tomato SlHMGA3 genes listed above, species (2) refers to the sequence of SEQ ID NO:1, but still can normally encode and express a sequence which realizes the actual function of the original tomato SlHMGA3 gene. The (3) th means that the same protein as the original tomato SlHMGA3 gene can be obtained by normal transcription and translation, although there is a difference in part (not more than 10%) of bases (nucleotides).

A method for cultivating tomatoes with delayed ripening fruits, comprising the following steps:

s1, constructing a host cell engineering bacterium containing a tomato SlHMGA3 gene knockout vector;

s2, in the tomato leaf explant transfected by the agrobacterium tumefaciens engineering bacteria obtained in the step S1, screening tomato plants which are subjected to the mutation of the tomato SlHMGA3 gene and do not contain the tomato SlHMGA3 gene knockout carrier sequence in the step S1;

s3, planting the mutant tomato plants obtained in the step S2 in a greenhouse, and culturing to obtain tomatoes with delayed mature fruits;

the tomato SlHMGA3 gene comprises any one of the following nucleotide sequences:

(1) As set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

(2) From SEQ ID NO:1, and has a nucleotide sequence which does not change the function of the original nucleotide sequence and has the same function;

(3) And SEQ ID NO:1 and encodes a nucleotide sequence having more than 90% homology as set forth in SEQ ID NO:2, and a DNA molecule having an amino acid sequence shown in the specification.

According to the cultivation method of the tomato with the fruit delayed ripening, a tomato SlHMGA3 gene CRISPR/Cas9 expression vector is constructed; 2 target sequences of the SlHMGA3 gene are designed by using a CRISPR-P website, and then AtU d and AtU b promoters and a sgRNA sequence AtU d-sgRNA1-AtU b-sgRNA2 containing 2 targets are subjected to gene synthesis and then inserted between SbfI and SmaI cleavage sites of a vector 2300GN-Ubi-Cas9, so that the SlHMGA3 gene mutation vector is obtained. The constructed sequencing correct vector is transferred into a host cell (such as agrobacterium tumefaciens EHA 105), then a cotyledon explant of a tomato (a variety such as Micro-Tom) infected by the vector is utilized, and tomato plants with the mutation of the SlHMGA3 gene but without the gene knockout vector sequence are screened from positive transgenic tomato offspring, so that tomato plants with delayed mature fruits are obtained. In order to facilitate the identification and screening of genetically mutated tomato plants, the vectors used may be processed, for example by adding plant selectable markers or antibiotic markers with resistance, etc.

The cultivation method of the tomato with the delayed maturation of the fruits comprises the steps of knocking out part of bases (nucleotides) of the tomato SlHMGA3 gene through a gene editing technology to obtain a mutated vector, and then transfecting to obtain a tomato plant with the corresponding mutation of the genes, wherein the tomato plant contains the tomato SlHMGA3 gene mutant, so that the purpose of delaying the maturation of the fruits is achieved.

Preferably, in the tomato cultivation method with fruit delayed ripening, the tomato SlHMGA3 gene knockout in the step S1 is implemented by using a CRISPR/Cas9 gene editing technology, and the tomato SlHMGA3 gene knockout vector is 2300GN-Ubi-Cas9-AtU3d-sgRNA1-AtU3b-sgRNA2.

Preferably, in the above cultivation method, step S1 specifically includes the following steps:

s11, designing 2 target sequences on an exon of a SlHMGA3 gene, wherein the nucleotide sequences of the 2 target sequences are shown as SEQ ID No.3 and SEQ ID No. 4; then, carrying out gene synthesis on a AtU d promoter and a AtU b promoter together with an sgRNA sequence AtU3d-sgRNA1-AtU3b-sgRNA2 containing 2 target sequences, and then inserting the obtained product between SbfI and SmaI cleavage sites of a vector 2300GN-Ubi-Cas9 to obtain a SlHMGA3 gene mutation vector;

s12, transfecting the SlHMGA3 gene mutation vector in the step S11 into a host cell to obtain a host cell engineering bacterium.

More preferably, in the above cultivation method, the sequence AtU d-sgRNA1-AtU b-sgRNA2 of step S11 is shown in SEQ ID No. 5.

Preferably, the host cell used in step S1 is an E.coli strain or an Agrobacterium tumefaciens strain. Agrobacterium tumefaciens is typically employed as EHA105.

The cultivation method can be widely applied to cultivation of tomatoes with fruits delayed to mature.

Compared with the prior art, the invention has the following advantages:

1. the invention constructs tomato SlHMGA3 gene knockout plants for the first time and performs functional research. Through phenotype observation, data statistics, measurement of fruit maturation related indexes, ethylene related gene expression analysis and maturation related transcription factor gene expression analysis, the mutation of the knockout SlHMGA3 gene is found to play a role in delaying tomato fruit maturation, and meanwhile plant growth is not obviously affected.

2. The SlHMGA3 gene provided by the invention provides gene resources for cultivating new varieties of late-maturing tomatoes, has a good potential application value, and lays a theoretical foundation for researching a tomato plant maturation related transcription factor regulation network.

Drawings

FIG. 1 shows the analysis of the expression pattern of the SlHMGA3 gene in different tissues (Root, stem Leaf, flower) of wild type tomato (Micro Tom) and different stages of fruit development (fruit IMG 5 days after flowering, fruit IMG 15 days after flowering, fruit MG 30 days after flowering, broken fruit BR 39 days after flowering, yellow ripe fruit O, red ripe fruit RR) in example 1;

FIG. 2 shows target 1 (Taget 1) and target 2 (Taget 2) for the SLHMGA3 gene knockout in example 2;

FIG. 3 shows the sequencing results of the SLhmga3 mutant target of the tomato homozygous mutant line of example 3;

FIG. 4 is a delayed ripening phenotype of tomato of the wild type and the slhmga3 mutants (slhmga 3-1 and slhmga 3-2) of example 4;

FIG. 5 is a graph showing fruit ripening time statistics for wild type and slhmga3-1, slhmga3-2 of example 4;

FIG. 6 is a color record of fruit ripening for wild type and slhmga3-1, slhmga3-2 of example 4; in fig. 6, hue represents the phase angle of the color, 180 ° represents pure green, 90 ° represents orange, 45 ° represents orange, and 0 ° represents pure red;

FIG. 7 shows the variation of chlorophyll content during fruit ripening of wild type and slhmga3-1, slhmga3-2 mutants during fruit ripening in example 5;

FIG. 8 shows the variation of carotenoid content during fruit ripening of wild type and slhmga3-1, slhmga3-2 mutants during fruit ripening in example 5;

FIG. 9 shows the change in ethylene content during fruit ripening of wild type and slhmga3-1, slhmga3-2 mutants during fruit ripening in example 6;

FIG. 10 is an analysis of the expression pattern of ethylene synthesis genes of wild type and slhmga3-1, slhmga3-2 mutants during fruit ripening in example 7;

FIG. 11 is an analysis of the expression pattern of ethylene signal response genes of wild type and slhmga3-1, slhmga3-2 mutants during fruit ripening in example 7;

FIG. 12 is a schematic representation of transcriptional gene expression patterns of wild type and slhmga3-1, slhmga3-2 mutants involved in regulatory maturation during fruit ripening in example 7;

FIG. 13 shows that the synthesized sgRNA comprises two 20bp oligonucleotide target sites (darkened and bolded) and a conserved structural sequence (underlined), and the corresponding promoter sequence, atU d, precedes the first target site and At3Ub precedes the second target site (unbroken and light);

FIG. 14 is a map of a 2300GN-Ubi-Cas9 vector.

Detailed Description

The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, tissue culture, molecular biology, biophysical biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are well explained in the prior art literature.

Example 1: wild tomato SlHMGA3 Gene expression Pattern

The RNA of plant tissue of wild tomato root, stem, leaf, flower and fruit of different development stages is extracted by taking 5 tissues of tomato (variety Micro-Tom) of the same tissue or development stage as biological repetition. Fruits at different developmental stages are: fruit IMG (immature green) 5 days after flowering, fruit IMG (immature green) 15 days after flowering, fruit MG (mature green) 30 days after flowering, broken color BR (break), yellow ripe O (orange), red ripe RR (red ripe). Reverse transcription of the plant tissue RNA into cDNA as template, qRT-PCR to detect the expression level of the SlHMGA3 gene in tomato fruit, and Actin as reference gene and 2 ^-ΔCT The method and primer information are shown in Table 1 below.

TABLE 1 primer information for use in the test

Gene name	Gene numbering	Upstream primer (5 '-3')	Downstream primer (5 '-3')
				ACTIN	Solyc11g005330	TGTCCCTATCTACGAGGGTTAT	AGTTAAATCACGACCAGCAAGA
SlHMGA3	Solyc04g007890	GCAAGCTGGGCAATTAGTTATG	ACTGAACTTTTCGGCTTCGG
				SlACS2	Solyc01g095080	TGTTAGCGTATGTATTGACAAC	TCATAACATAACTTCACTTTTGC
SlACO1	Solyc07g049530	GCCAAAGAGCCAAGATTTGA	TTTTTAATTGAATTGGGATCTAA
				SlACS4	Solyc05g050010	CTCCTCAAATGGGGAGTACG	TTTTGTTTGCTCGCACTACG
SlE4	Solyc03g111720	GACCACTCTAAATCGCCAGG	TTCCTGAGCGGTATTGCTTT
				SlE8	Solyc09g089580	TGGCTCCGAATCCTCCCAGTCT	GTCCGCCTCTGCCACTGAGC
SlETR3	Solyc09g075440	TGCTGTTCGTGTACCGCTTT	TCATCGGGAGAACCAGAACC
				SlTAGL1	Solyc07g055920	ACTTTCTGTTCTTTGTGATGCT	TTGGATGCTTCTTGCTGGTAG
SlETR4	Solyc06g053710	TGGAGGAGTGAGTGTGGATGC	ATGGCTGTCGTTCTTGGGC
				SlEIN2	Solyc09g007870	GTGTGCTGAATAAGTTTAGTGG	TGCTGTACAATAGAAGAATGGA
SlEIL3	Solyc01g096810	ACAGGACTTCAAGAAACAACC	GTGTTGTGCTCATAGTTGATCTG

As a result, according to FIG. 1, it was found that SlHMGA3 was expressed in each tissue of tomato by Qrt-PCR analysis, in which the expression level of flowers and fruits was high and the expression level was highest when the tomato fruits reached the red ripe stage as the fruits ripened, as shown in FIG. 1.

Example 2: construction of SlHMGA3 gene knockout vector and corresponding host cell engineering bacterium

S11, designing 2 target sequences on the exon of the SlHMGA3 gene by using a CRISPR-P website, wherein the nucleotide sequences of the 2 target sequences are shown as SEQ ID No.3 (GGTACACTACCACCAGCGCA) and SEQ ID No.4 (GATGGGTATCCTAGACCACG), and the figure is shown as figure 2; then, carrying out gene synthesis on a AtU d promoter and a AtU b promoter together with a sgRNA sequence AtU d-sgRNA1-AtU3b-sgRNA2 (shown as SEQ ID No. 5) containing 2 target sequences, and then inserting the obtained product between SbfI and SmaI cleavage sites of a vector 2300GN-Ubi-Cas9 to obtain a SlHMGA3 gene mutation vector; the specific operation of the method is that the sgRNA and the corresponding promoter fragment thereof are synthesized in Nanjing Jinsrui company, and cloned between the Sbf I and Sma I double cleavage sites of 2300GN-Ubi-Cas9 double-element vector, and the obtained SlHMGA3 gene mutation vector is sent to the sequencing and confirmation of the Optimus of the Nanjing;

s12, transfecting the SlHMGA3 gene mutation vector which is confirmed to be correct by sequencing into the host cell agrobacterium tumefaciens EHA105 to obtain the host cell engineering bacterium.

Example 3: construction and detection of tomato SlHMGA3 mutant material

The host cell engineering bacteria prepared in the example 2 are used for infecting cotyledon explants of a tomato variety Micro-Tom, tissue culture seedlings are obtained through inducing callus, resistance induced differentiation and rooting culture, and positive SlHMGA3 gene mutation tomato plants are screened out by using PCR and sequencing technology verification.

Sequencing shows that in the mutant tomato plant, the slhmga3-1 lacks 6 bases at the target point 1 and lacks 4 bases at the target point 2, the slhmga3-2 inserts 1 base at the target point 1 and lacks 2 bases at the target point 2 (shown in figure 3).

Example 4: delayed maturation trait observation of SlHMGA3 gene mutant material

The tomato SlHMGA3 mutant material prepared in example 3 (i.e., tomato plant with delayed fruit ripening) was subjected to marking during flowering, the flowering time was noted, the fruits were photographed at the time when the fruits were about to enter ripening, the ripening process was recorded (as shown in fig. 4), the time to fruit burst for wild-type and mutant materials was counted (as shown in fig. 5), and the color change of the fruits was measured using a konikama-merida color difference meter CR-400 (as shown in fig. 6).

Fruit ripening of mutant tomatoes was significantly delayed compared to wild type, with slhmga3-1 delayed for 3 days and slhmga3-2 delayed for 6 days.

Example 5: determination of total chlorophyll content and total carotenoid content of tomato fruits

The color of tomato fruits is mainly related to the accumulation and relative proportion of chlorophyll, carotenoid and flavonoid and other pigment substances in the fruit peel and pulp, and the decrease of chlorophyll content and the increase of carotenoid content lead to the change of tomato from green to red.

Taking 5 tomato peel at the same stage as biological repetition, weighing 0.75g of sample, adding a small amount of quartz sand, calcium carbonate powder and 2mL of 95% ethanol, grinding into homogenate, adding 10mL of ethanol, continuously grinding until the tissue becomes white, and standing for 5min. Filtering into 25mL brown volumetric flask, metering with ethanol to 25mL, shaking, pouring chloroplast pigment extractive solution into cuvette with optical path of 1cm, and measuring absorbance at wavelength of 665nm and 649nm with 95% ethanol as blank.

Carotenoids absorbance was measured at 470 nm. Substituting the measured absorbance into the following formula:

Ca＝13.95A ₆₆₅ -6.88A ₆₄₉ ；

Cb＝24.96A ₆₄₉ -7.32A ₆₆₅ ；

C(X.C)＝(1000A ₄₇₀ -2.05Ca-114.8Cb)/245。

the concentration (mg/L) of chlorophyll a, chlorophyll b and carotenoid can be obtained by the method, and the sum of chlorophyll a and chlorophyll b is the total concentration of chlorophyll. Finally, the chlorophyll or carotenoid content in the plant tissue can be further determined according to the following formula:

chlorophyll content (mg/L) = (concentration of chlorophyll x volume of extract x dilution factor)/fresh weight (or dry weight) of sample.

The results showed that chlorophyll degradation (as shown in fig. 7) and carotenoid accumulation (as shown in fig. 8) processes in the fruits were delayed after knocking out SlHMGA3, further demonstrating that fruit ripening processes were delayed.

Example 6: determination of ethylene Release from tomato fruit

And (3) measuring the ethylene release amount by adopting a Thermo Trace Ultra GC gas chromatograph, picking fresh fruits, standing at room temperature for 2 hours, avoiding stress-induced ethylene release caused by picking, weighing 5 fruits at the same stage as a group of biological repetition, putting the fruits into a 50mL centrifuge tube after weighing respectively, sealing by a sealing film, and standing for 8 hours for measurement. Chromatographic conditions: the sample injection temperature is 130 ℃, the column temperature is 80 ℃, the FID temperature is 230 ℃, and the N is the same as the FID ₂ 0.2Mpa, 0.2Mpa of air, H ₂ The sample injection amount is 10 μl under 0.2 Mpa. After the base line is stabilized, 10 mu L of ethylene standard substance is injected, a standard curve is prepared, after the standard curve is prepared, a gas injection machine in a sealed centrifuge tube bottle is extracted by a 1ml syringe for measurement, and each sample is subjected to 3 times of technical repetition.

The results (shown in FIG. 9) are similar to example 5, with the slhmga3-1 and slhmga3-2 mutants having a delayed ethylene release peak time and a lower peak-to-peak than the wild type. This result demonstrates that the SlHMGA3 gene can regulate fruit ripening through ethylene.

Example 7: expression profiling of ethylene synthesis genes, ethylene signal response genes and maturation-associated transcription factors

Extracting fruit RNA of wild type and slhmga3-1, slhmga3-2 mutant tomato fruit at different time points (32 dap, 36dap, 38dap, 42dap, 46dap, 50 dap) in the ripening process, reverse transcribing into cDNA as template, detecting ethylene synthesis gene, signal response gene and change of expression quantity of ripening related transcription factor in tomato fruit by qRT-PCR (figure 10-12), using action as internal reference gene, using 2 ^-ΔCT The method and primer information are shown in Table 1. The expression of ethylene synthesis genes ACS2, ACS4 and ACO1 in the slhmga3-1 and slhmga3-2 mutants is delayed and the highest expression level is lower than the highest expression level of WT (shown in FIG. 10); ethylene signal response genes ETR3, ETR4, CTR1, EI in slhmga3-1 and slhmga3-2 mutantsThe expression of L3 and EIN2 was delayed and consistent with the expression trend of ethylene synthesis genes (as shown in FIG. 11); the expression of the maturation-associated transcription factors E4 and E8 increased dramatically starting from the wild-type fruit 36dpa, whereas in the slhmga3 mutant this increase was delayed to 38dpa, the induction of the maturation-associated transcription factor in the mutant fruit was delayed for about 3 to 6 days, and the maximum difference in WT and mutant occurred at 38dpa (as shown in FIG. 12). These results indicate that SlHMGA3 regulates ethylene conductance and some maturation-related key transcription factors by regulating ethylene synthesis and signal-related genes, thereby regulating fruit maturation.

In addition, as is common knowledge in the art, the sequence set forth in SEQ ID NO:1, and the nucleotide sequence having the same function as the original nucleotide sequence, by changing the nucleotide sequence by a conventional means without affecting the functional function thereof, for example, by substituting, deleting and/or adding one or more nucleotides; or a nucleotide sequence having more than 90% homology and encoding the same functional protein.

Sequence listing

<110> Nanjing agricultural university

<120> application of tomato SlHMGA3 Gene in cultivation of tomato with delayed fruit ripening

<141> 2021-05-28

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Claims (4)

1. The application of knocking out the tomato SlHMGA3 gene in cultivating tomatoes with delayed fruit ripening is characterized in that the tomato SlHMGA3 gene is any one of the following nucleotide sequences:

(1) As set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

(2) And SEQ ID NO:1 and encodes a nucleotide sequence having more than 90% homology as set forth in SEQ ID NO:2, and a DNA molecule having an amino acid sequence shown in the specification.

2. A method for cultivating tomatoes with delayed ripening fruits, which is characterized by comprising the following steps:

s1, constructing a host cell engineering bacterium containing a tomato SlHMGA3 gene knockout vector;

s2, in the tomato leaf explant transfected by the agrobacterium tumefaciens engineering bacteria obtained in the step S1, screening tomato plants which are subjected to the mutation of the tomato SlHMGA3 gene and do not contain the tomato SlHMGA3 gene knockout carrier sequence in the step S1;

s3, planting the mutant tomato plants obtained in the step S2 in a greenhouse, and culturing to obtain tomatoes with delayed mature fruits;

the tomato SlHMGA3 gene is any one of the following nucleotide sequences:

(1) As set forth in SEQ ID NO:1, and a nucleotide sequence shown in the specification;

(2) And SEQ ID NO:1 and encodes a nucleotide sequence having more than 90% homology as set forth in SEQ ID NO:2, and a DNA molecule having an amino acid sequence shown in the specification.

3. The method of cultivation of tomatoes with delayed ripening of fruits according to claim 2, wherein the host cell used in step S1 is escherichia coli strain or agrobacterium tumefaciens strain.

4. Use of a cultivation method according to claim 2 or 3 for cultivating tomatoes whose fruits are delayed from ripening.