阅读说明：本技术 一种早花高产番茄材料的制备方法 (Preparation method of early-flowering high-yield tomato material ) 是由 李传友 朱强 邓磊 李常保 于 2019-03-06 设计创作，主要内容包括：本发明公开了一种早花高产番茄材料的制备方法。本发明公开的早花高产番茄材料的制备方法包括：降低受体植物中SlTOE1的活性、降低受体植物中SlTOE1的含量、抑制受体植物中SlTOE1的编码基因的表达或敲除受体植物中SlTOE1的编码基因,得到与受体植物相比开花时间缩短且产量提高的目的植物。本发明可以在1年以内得到一个早花高产植物新品种,而利用传统育种手段需要连续回交、自交,至少需要3-4年时间,本发明的方法操作简单,成本低,大大加快了育种进程,并且克服了早花与高产难以兼备的不足,极大满足了生产需求,具有广泛的应用前景。(The invention discloses a preparation method of a high-yield early-flowering tomato material. The invention discloses a preparation method of a high-yield early-flowering tomato material, which comprises the following steps: the activity of SlTOE1 in a receptor plant is reduced, the content of SlTOE1 in the receptor plant is reduced, the expression of a coding gene of SlTOE1 in the receptor plant is inhibited or the coding gene of SlTOE1 in the receptor plant is knocked out, and a target plant with shortened flowering time and improved yield compared with the receptor plant is obtained. The method can obtain a new early-flowering high-yield plant variety within 1 year, and the traditional breeding means needs continuous backcross and selfing, and at least needs 3-4 years.)

1. Use of a protein or a substance modulating the content and/or activity of said protein of any one of the following:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 3;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

2. Use of a biological material related to a protein according to claim 1, wherein the biological material is selected from the group consisting of:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the content and/or activity of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following b1) -b 5):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b2) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b3) DNA molecule shown in sequence 1 in the sequence table;

b4) a cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3) and encoding the protein of claim 1;

b5) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) or b4) and encodes the protein of claim 1;

B8) the nucleic acid molecule is any one of c1) -c4) as follows:

c1) RNA molecule shown in sequence 4 in the sequence table;

c2) transcribing a DNA molecule of RNA shown in a sequence 4 in a sequence table;

c3) an RNA molecule or DNA molecule with 75 percent or more than 75 percent of identity with the nucleotide sequence defined by c1) or c2) and the same function;

c4) RNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by c1) or c2) or c3) under strict conditions and has the same function.

4. Any one of the following methods:

x1) a method for reducing flowering time in plants comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time as compared to the recipient plant;

x2) method for cultivating plants with reduced flowering time comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time as compared to the recipient plant;

x3) method for increasing plant yield, comprising: reducing the activity of a protein according to claim 1 in a recipient plant, reducing the content of a protein according to claim 1 in a recipient plant, inhibiting the expression of a gene encoding a protein according to claim 1 in a recipient plant, or knocking out a gene encoding a protein according to claim 1 in a recipient plant, resulting in a plant of interest having an increased yield as compared to the recipient plant;

x4) method for growing plants with increased yield, comprising: reducing the activity of a protein according to claim 1 in a recipient plant, reducing the content of a protein according to claim 1 in a recipient plant, inhibiting the expression of a gene encoding a protein according to claim 1 in a recipient plant, or knocking out a gene encoding a protein according to claim 1 in a recipient plant, resulting in a plant of interest having an increased yield as compared to the recipient plant;

x5) method for reducing flowering time and increasing plant yield in plants comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time and increased yield as compared to said recipient plant;

x6) method for cultivating plants with reduced flowering-time and increased yield, comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time and increased yield as compared to said recipient plant.

5. The method of claim 4, wherein: knocking out a gene encoding the protein of claim 1 in a recipient plant is achieved using the CRISPR/Cas9 method.

6. The method of claim 6, wherein: the target sequence used in the CRISPR/Cas9 method is 317-336 th site of sequence 1 in the sequence table.

7. A product having the function of shortening flowering-time and/or increasing plant yield, comprising the biomaterial as claimed in claim 2 or 3.

8. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, or a product according to claim 7, wherein: the plant is any one of c1) -c 4):

c1) tubular plants of the order florida;

c2) a plant of the Solanaceae family;

c3) a plant of the genus Lycopersicon;

c4) tomato.

9. The use, method or product according to claim 8, wherein: said yield is represented on the fruit of said plant.

10. Use of a plant of interest prepared by the method of any one of claims 4-6 in plant breeding.

Technical Field

The invention relates to a preparation method of a high-yield early-flowering tomato material, belonging to the technical field of agricultural biology.

Background

Flowering time is a key factor influencing yield, and generally, late flowering can prolong the growth period, increase biomass and yield; premature flowering shortens the growth period, but biomass decreases and yield decreases. How to realize early flowering and high yield is always a difficult problem in breeding. At present, the varieties of crops with late flowers and high yield in the market are more, and the varieties with early flowers and high yield are few. Compared with the late high-yield variety, the early high-yield variety has the advantages of shortening the growth period, appearing on the market and the like on the premise of keeping high yield, so that the early high-yield variety is more popular with farmers and is also the target pursued by breeders.

The Arabidopsis TOE1 transcription factor can inhibit flowering, and after the function of the Arabidopsis TOE1 gene is completely lost, flowering is advanced, but the plant is thin and small, and the seed yield is obviously reduced.

Genome editing technology (genome editing) is a genetic manipulation technology that can engineer DNA sequences at the genome level. The CRISPR/Cas9system (clustered regulated short tandem repeats/Cas9endonuclease) is one of the most widely used genome editing tools at present. The working principle of the technology is that the endonuclease Cas9 protein cuts a target gene under the guide of guide RNA (short guide RNA, sgRNA) to cause DNA double strand break, at the moment, a DNA repair mechanism of a cell is induced, and variation such as insertion, deletion or replacement of a base is inevitably introduced in the repair process, so that site-directed mutation of the target gene is realized. Then removing the transgenic marker, and the obtained variety is completely the same as the variety cultivated by the traditional breeding method.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the yield of the plant and shorten the flowering time of the plant.

In order to solve the technical problem, the invention firstly provides any one of the following applications of the protein or the substance for regulating the content and/or activity of the protein:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the protein is named SlTOE1 and is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 3;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 3 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein in A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 3 in the sequence table is attached with the tags shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The SlTOE1 protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 3 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The SlTOE1 protein in A2) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the SlTOE1 protein in a2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in sequence No. 2, and/or by performing missense mutation of one or several base pairs, and/or by attaching to its 5 'end and/or 3' end a coding sequence of the tag shown in the above table. Wherein the DNA molecule shown in the sequence 2 encodes SlTOE1 protein shown in the sequence 3.

The invention also provides any one of the following applications of the biological material related to SlTOE 1:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding SlTOE 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) nucleic acid molecules that reduce the content and/or activity of SlTOE 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

In the above application, the nucleic acid molecule of B1) may be any one of the following B1) -B5):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b2) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b3) DNA molecule shown in sequence 1 in the sequence table;

b4) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2) or b3) and encoding SlTOE 1;

b5) a cDNA molecule or a DNA molecule which is hybridized with a nucleotide sequence defined by b1) or b2) or b3) or b4) under strict conditions and codes for SlTOE 1;

B8) the nucleic acid molecule is any one of c1) -c4) as follows:

c1) RNA molecule shown in sequence 4 in the sequence table;

c2) transcribing a DNA molecule of RNA shown in a sequence 4 in a sequence table;

c3) an RNA molecule or DNA molecule with 75 percent or more than 75 percent of identity with the nucleotide sequence defined by c1) or c2) and the same function;

c4) RNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by c1) or c2) or c3) under strict conditions and has the same function.

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.

The nucleotide sequence encoding SLTOE1 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the SLTOE1 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the SLTOE1 protein and have the function of the SLTOE1 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridizing with 1mM EDTA, rinsing in 2 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.5 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 65 deg.C, hybridizing in 6 × SSC, 0.5% SDS at 65 deg.C, washing with 2 × SSC, 0.1% SDS, 1 × SSC, 0.1% SDS once, hybridizing in 2 × SSC, 0.1% SDS at 68 deg.C for 5min, hybridizing in 0.5 × SSC, 0.1% SDS at 68 deg.C for 15min, or hybridizing in 0.1 × SSPE (or 0.1 SSC 0.1 ×) and 0.1% SDS for 2 minIn solution, hybridization was carried out at 65 ℃ and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding SLTOE1 protein (SLTOE1 gene expression cassette) described in B2) refers to a DNA capable of expressing SLTOE1 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of SLTOE1 gene but also a terminator for terminating transcription of SLTOE1 gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the SLTOE1 gene expression cassette can be constructed by using the existing expression vector.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

B9) The recombinant vector can be a recombinant vector which is prepared by utilizing a crisper/cas 9system and can reduce the SLTOE1 content. The recombinant vector can express a sgRNA targeted to B1) the nucleic acid molecule. The target sequence of the sgRNA can be 317-336 th of the sequence 1 in the sequence table.

B9) The recombinant vector can be specifically a recombinant vector which is obtained by inserting double-stranded DNA of a target sequence into a pTX041 vector by using a restriction endonuclease BsaI and can transcribe the sgRNA shown in a sequence 4 in a sequence table.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes LBA 4404.

In the above application, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

The invention also provides any one of the following methods:

x1) a method for reducing flowering time in plants comprising: reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant to obtain a target plant with shortened flowering time compared with the receptor plant;

x2) method for cultivating plants with reduced flowering time comprising: reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant to obtain a target plant with shortened flowering time compared with the receptor plant;

x3) method for increasing plant yield, comprising: reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant to obtain a target plant with improved yield compared with the receptor plant;

x4) method for growing plants with increased yield, comprising: reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant to obtain a target plant with improved yield compared with the receptor plant;

x5) method for reducing flowering time and increasing plant yield in plants comprising: reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant to obtain a target plant with shortened flowering time and improved yield compared with the receptor plant;

x6) method for cultivating plants with reduced flowering-time and increased yield, comprising: the method comprises the steps of reducing the activity of SlTOE1 in a receptor plant, reducing the content of SlTOE1 in the receptor plant, inhibiting the expression of a coding gene of SlTOE1 in the receptor plant or knocking out the coding gene of SlTOE1 in the receptor plant, and obtaining a target plant with shortened flowering time and improved yield compared with the receptor plant.

The acceptor plant contains a coding gene of SlTOE 1.

In the method, the coding gene of SlTOE1 in the receptor plant is knocked out by using a CRISPR/Cas9 method.

The target sequence used in the CRISPR/Cas9 method is 317-336 th site of sequence 1 in a sequence table.

The sgRNA used in the CRISPR/Cas9 method can be specifically RNA shown as a sequence 4 in a sequence table.

In the above method, the knockout of the gene encoding SlTOE1 in the recipient plant can be specifically achieved by introducing the recombinant vector described in B9) into a plant.

The method may further comprise the step of selecting a plant of interest that does not contain the exogenous DNA sequence.

Compared with the acceptor plant, the target plant has a mutation of a coding gene of SlTOE1, so that the content of SlTOE1 is reduced and/or the function of SlTOE1 is lost. The mutation may be a deletion, insertion and/or alteration of one to more nucleotides.

In one embodiment of the invention, the nucleotide corresponding to position 320-323 of sequence 2 in the sequence listing of the gene encoding SlTOE1 is deleted in the plant of interest compared to the recipient plant.

The invention also provides products having the function of shortening the flowering time of plants and/or improving the yield of plants, which contain the biological material.

The product can use the biological material as the active component, and can also combine the biological material and substances with the same functions together as the active component.

The application of the target plant prepared by the method in plant breeding also belongs to the protection scope of the invention.

In the present invention, the plant may be any one of c1) -c 4):

c1) tubular plants of the order florida;

c2) a plant of the Solanaceae family;

c3) a plant of the genus Lycopersicon;

c4) tomato.

Said specific producibility is embodied on the fruit of said plant.

Experiments prove that after the TOE1 gene in the plant is knocked out by using a CRISPR/Cas9 method, the flowering time is shortened, the yield is improved, and the early-flowering high-yield plant is successfully cultured. The method can obtain a new early-flowering high-yield plant variety within 1 year, and the traditional breeding means needs continuous backcross and selfing, and at least needs 3-4 years.

Drawings

FIG. 1 shows the sequence mutation of plant 2.

FIG. 2 shows the flowering time phenotype of wild type tomato and plant 2, with the number of leaves before the first-order flower representing the flowering time. Wild type refers to wild type tomato.

FIG. 3 shows the yield phenotype of wild type tomato and plant 2, wherein A is a phenotype photo and B is a yield statistic result, and the wild type is wild type tomato.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Tomato is of tomato conventional variety M82 (hereinafter also referred to as wild type tomato) and is derived from the American tomato genetic resource center (TGRC, http:// TGRC. ucdavis. edu. /).