CN116120416A - SlWRKY57 gene and application of coding protein thereof in regulation and control of salt tolerance of tomatoes - Google Patents

Abstract

The invention discloses an application of a SlWRKY57 gene and a coding protein thereof in regulating and controlling salt tolerance of tomatoes. In particular to the application of a protein SlWRKY57 with an amino acid sequence of SEQ ID No.1 or a substance for regulating the activity and/or the content of the protein in regulating the salt tolerance of plants. According to the invention, the CRISPR-Cas9 system-based knockout is performed on the SlWRKY57 gene, so that the SlWRKY57 gene editing strain is obtained. Experiments prove that under the condition of salt stress, the wilting degree of the SlWRKY57 gene editing plant is obviously reduced, the fresh weight is obviously increased, and the tolerance of the plant to the salt stress is obviously increased. The identified SlWRKY57 gene and the coding protein thereof have the function of regulating and controlling the salt tolerance of plants, and have important significance and wide application prospect in cultivating new varieties of salt-tolerant tomatoes, promoting the breeding process of commercial tomatoes and ensuring the high and stable yield of tomatoes.

Description

SlWRKY57 gene and application of coding protein thereof in regulation and control of salt tolerance of tomatoes

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of a SlWRKY57 gene and a coding protein thereof in regulation and control of salt tolerance of tomatoes.

Background

Tomato (Solanum lycopersicum) is taken as one of fruits and vegetables with higher nutritive value and flavor quality, and is increasingly favored by people, and the planting area is increased. In recent years, the problem of salt stress of the facility tomatoes is increasingly serious due to unscientific water and fertilizer management measures in the cultivation processes of greenhouses, greenhouses and the like, the yield and the quality of the tomatoes are reduced, and the sustainable development of agriculture in China and the stability of vegetable baskets are severely restricted. Attempts have been made to remedy the problem of salt stress by reasonable irrigation, fresh water washing, and chemical modifier application, but the promotion is difficult due to high cost and slow effect. The salt tolerance of tomatoes is improved, important salt tolerance genetic resources are excavated, the key effect is played on cultivating salt tolerance tomato varieties, and the development and the utilization of the salt tolerance tomatoes have immeasurable ecological benefits, economic benefits and social benefits.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the salt tolerance of plants. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To solve the above technical problems, the present invention provides first an application of a protein or a substance regulating the activity and/or content of the protein, wherein the application may be any of the following:

a1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating the salt tolerance of a plant;

a2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating the salt tolerance of a plant;

a3 Use of a protein or a substance regulating the activity and/or content of said protein for growing salt tolerant plants;

a4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing salt tolerant plants;

a5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding or plant germplasm resource improvement;

the protein is named as SlWRKY57 and can be any one of the following:

b1 A protein having an amino acid sequence of SEQ ID No. 1;

b2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in B1) and has the same function;

b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).

In the above application, the protein may be derived from tomato (Solanum lycopersicum).

Further, the protein SlWRKY57 may be a tomato salt tolerance related protein.

In order to facilitate purification or detection of the protein of B1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing.

Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding the protein SlWRKY57 of the present invention can be easily mutated by a person of ordinary skill in the art using a known method, for example, directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein SlWRKY57 isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein SlWRKY57 and have the function of the protein SlWRKY57.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. 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, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gapcost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.

Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% identity or more may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 95% identity or more may be at least 95%, 96%, 97%, 98% or 99% identity.

Herein, the substance that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein SlWRKY57.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (including modification of transcription products of the gene, modification of splicing and/or processing); 3) Regulation of RNA transport of the gene (including regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (including regulation of the activity of the protein translated by the gene, such as processing of a protein precursor, transport of a protein, degradation of a protein, folding of a protein, and the like).

The substance regulating gene expression may specifically be a biological material as described in any one of E1) to E4) herein.

Further, the agent that modulates gene expression may be an agent (including a nucleic acid molecule or vector) that inhibits or reduces or down-regulates expression of the gene encoding the protein SlWRKY57. The agent that inhibits or reduces or down-regulates expression of the gene encoding the protein SlWRKY57 may be an agent that knocks out the gene (SlWRKY 57 gene), such as an agent that knocks out the gene by CRISPR-Cas9, or an agent that knocks out the gene by homologous recombination. The agent that inhibits or reduces expression of the gene may comprise a polynucleotide, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA, that targets the gene.

Further, the substance that regulates gene expression may also be a substance (including a nucleic acid molecule or a vector) that increases or upregulates expression of a gene encoding the protein SlWRKY57.

The invention also provides the use of a biological material associated with said protein SlWRKY57, said use being any of the following:

d1 Use of a biological material related to said protein SlWRKY57 for modulating salt tolerance in plants;

d2 The application of the biological material related to the protein SlWRKY57 in preparing a product for regulating and controlling the salt tolerance of plants;

d3 Use of biological material related to said protein SlWRKY57 for the cultivation of salt tolerant plants;

d4 Use of a biological material related to said protein SlWRKY57 for the preparation of a product for growing salt tolerant plants;

d5 Use of biological material related to said protein SlWRKY57 in plant breeding or plant germplasm resource improvement;

the biological material may be any of the following:

e1 A nucleic acid molecule encoding said protein SlWRKY 57;

e2 A nucleic acid molecule that inhibits or reduces expression of a gene encoding said protein SlWRKY 57;

e3 An expression cassette containing the nucleic acid molecule of E1) and/or E2);

e4 A recombinant vector comprising E1) and/or E2) said nucleic acid molecule, or a recombinant vector comprising E3) said expression cassette;

e5 A recombinant microorganism containing the nucleic acid molecule of E1) and/or E2), or a recombinant microorganism containing the expression cassette of E3), or a recombinant microorganism containing the recombinant vector of E4);

e6 A recombinant host cell containing the nucleic acid molecule of E1) and/or E2), or a recombinant host cell containing the expression cassette of E3), or a recombinant host cell containing the recombinant vector of E4);

e7 A) transgenic plant tissue containing E1) and/or E2) said nucleic acid molecule, or a transgenic plant tissue containing E3) said expression cassette;

e8 A transgenic plant organ comprising E1) and/or E2) said nucleic acid molecule, or a transgenic plant organ comprising E3) said expression cassette.

Further, E3) the expression cassette, E4) the recombinant vector, E5) the recombinant microorganism, E6) the recombinant host cell, E7) the transgenic plant tissue and E8) the transgenic plant organ each express E1) and/or E2) the nucleic acid molecule.

In the above application, the nucleic acid molecule of E1) may be any of the following:

f1 A DNA molecule whose coding sequence is positions 178-1161 of SEQ ID No. 3;

f2 A DNA molecule whose nucleotide sequence is SEQ ID No.3 at positions 178-1161 or SEQ ID No. 3;

f3 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

The DNA molecule shown at positions 178-1161 of SEQ ID No.3 may be the coding sequence (CDS) of the SlWRKY57 gene.

The DNA molecule shown at positions 178-1161 of SEQ ID No.3 encodes a protein SlWRKY57 whose amino acid sequence is SEQ ID No.1.

The DNA molecule shown in SEQ ID No.2 may be the genomic nucleotide sequence of the SlWRKY57 gene.

The DNA molecule shown in SEQ ID No.3 may be the cDNA sequence of the SlWRKY57 gene.

The SlWRKY57 gene may be a tomato salt tolerance related gene.

D1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated at positions 178 to 1161 of SEQ ID No. 3.

D1 The nucleic acid molecules also include nucleic acid molecules which have more than 95% identity to the nucleotide sequence shown in SEQ ID No.3 at positions 178-1161, SEQ ID No.3 or SEQ ID No.2 and are derived from the same species.

The coding sequence (CDS) of the protein SlWRKY57 gene of the present invention may be any nucleotide sequence capable of encoding the protein SlWRKY57. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.

Further, the E2) may be a nucleic acid molecule that reduces the expression level of the SlWRKY57 gene.

E2 The nucleic acid molecule may be sgRNA, microRNA, siRNA, shRNA and/or antisense oligonucleotides.

Further, the sgRNA, microRNA, siRNA, shRNA and/or antisense oligonucleotides are useful for inhibiting expression of the SlWRKY57 gene.

Further, E2) the nucleic acid molecule may be sgRNA.

Further, the target sequences of the sgrnas may be SEQ ID No.4 and SEQ ID No.5.

Further, the sgrnas are used to knock out the SlWRKY57 gene.

It is well known to those skilled in the art that in addition to inhibiting expression of the SlWRKY57 gene using gene editing techniques, gene knockdown techniques can also be used to inactivate or silence expression of the SlWRKY57 gene from the post-transcriptional or translational level. Such gene knockdown techniques include, but are not limited to, RNA interference, morpholino interference, antisense nucleic acids, ribozymes, or dominant negative mutations. In addition, the silencing of the SlWRKY57 gene by inhibiting the expression of the SlWRKY57 gene using shRNA or siRNA expressed by viruses (e.g., lentiviruses, adeno-associated viruses) is also well known to those skilled in the art.

The expression cassettes described herein include a promoter, which may be a CaMV35S promoter, a NOS promoter, or an OCS promoter, a nucleic acid molecule encoding the protein SlWRKY57, and a terminator, which may be a NOS terminator or an OCS polyA terminator.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, viral vectors (e.g., retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of the invention, the vectors are pCBSG012 and pSG-SlU.

The microorganism described herein may be a bacterium, fungus, actinomycete, protozoan, algae or virus. Wherein the bacteria may be derived from Escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, alcaligenes sp, pseudomonas sp, bacillus sp, etc., but are not limited thereto, and for example, the bacteria may be Escherichia coli, bacillus subtilis Bacillus subtilis, or Bacillus pumilus. The fungus may be a yeast, which may be from the genera Saccharomyces (e.g., saccharomyces cerevisiae Saccharomyces cerevisiae), kluyveromyces (e.g., kluyveromyces lactis Kluyveromyces lactis), pichia (e.g., pichia pastoris), schizosaccharomyces (e.g., schizosaccharomyces pombe Schizosaccharomyces pombe), hansenula (e.g., hansenula polymorpha Hansenula polymorpha), etc., but is not limited thereto. The fungus may also be from Fusarium sp, rhizoctonia sp, verticillium sp, penicillium sp, aspergillus sp, cephalosporium sp, and the like, but is not limited thereto. The actinomycetes may be derived from Streptomyces sp, nocardia sp, micromonospora sp, neurospora sp, actinoplanes sp, thermoactinomyces sp, etc., but are not limited thereto. The algae may be from, but not limited to, fucus sp, aspergillus sp, coccoli sp, amphizora sp, double-eyebrow sp, cellulomella sp, astrocaryus sp, boekelovisopsis sp, boekelovisalpis sp, etc. The virus may be rotavirus, herpes virus, influenza virus, adenovirus, etc., but is not limited thereto. In one or more embodiments of the invention, the microorganism is agrobacterium tumefaciens GV3101.

The host cell (also referred to as a recipient cell) described herein may be a plant cell or an animal cell. The host cell is understood to mean not only the particular recipient cell, but also the progeny of such a cell, and such progeny may not necessarily correspond, in their entirety, to the original parent cell, but are included in the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, wherein: the plant cell may be, but is not limited to, plant cells such as arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryzasativa), wheat (Triticum aestivum), etc.; the animal cells may be mammalian cells (e.g., chinese hamster ovary cells (CHO cells), african green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK 293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., xenopus laevis) cells or giant salamander (andrias davidianus) cells), fish cells (e.g., grass carp, rainbow trout or catfish cells), insect cells (e.g., sf21 cells or Sf-9 cells), etc., but are not limited thereto.

The recombinant vector refers to a recombinant DNA molecule constructed by connecting an exogenous target gene or nucleic acid molecule with the vector in vitro.

As used herein, a recombinant microorganism (or recombinant host cell) refers to a recombinant microorganism (or recombinant host cell) whose function has been altered by manipulation and modification of the genes of the microorganism (or host cell) of interest. Such as a recombinant microorganism (or recombinant host cell) obtained by introducing an exogenous gene of interest or a recombinant vector into a microorganism (or host cell) of interest, or a recombinant microorganism (or recombinant host cell) obtained by directly gene editing an endogenous gene of a microorganism (or host cell) of interest. The recombinant microorganism (or recombinant host cell) is understood to mean not only the particular recombinant microorganism (or recombinant host cell), but also the progeny of such a cell, and the progeny may not necessarily correspond exactly to the original parent cell, but are included in the scope of the recombinant microorganism (or recombinant host cell), due to natural, accidental, or deliberate mutations and/or alterations.

E4 The recombinant vector may be a recombinant vector CRISPR/Cas9-SlWRKY57.

The recombinant vector CRISPR/Cas9-SlWRKY57 contains two editing targets (SEQ ID No.4 and SEQ ID No. 5) and coding genes of Cas9 proteins, after the gene is introduced into a receptor, two transcribed guide RNAs can target sequences near the receptor genome PAM through base complementary pairing, namely target SlWRKY57 genes, the Cas9 proteins enable DNA double strand breaks at the upstream and downstream of the SlWRKY57 genes, and sequences at the upstream and downstream ends of the breaks are connected through a DNA damage repair response mechanism of an organism, so that the knockout of the SlWRKY57 genes is realized.

The preparation method of the recombinant vector CRISPR/Cas9-SlWRKY57 comprises the following steps:

and (3) taking an intermediate vector pSG-SlU as a template, performing PCR amplification by using a primer F (5'-caatggtctcatgattgcttgattcagtagtagcgcgttttagagctagaaata-3') and a primer R (5'-ttggggtctctaaacgcgcgaacatctctatttccaaactacactgttagattt-3'), cloning an obtained PCR product (containing double targets of SEQ ID No.4 and SEQ ID No. 5) into a vector pCBSG012, and obtaining a SlWRKY57 gene editing vector, namely the recombinant vector CRISPR/Cas9-SlWRKY57, which is used for gene knockout.

E5 The recombinant microorganism may be recombinant agrobacterium tumefaciens GV3101/CRISPR/Cas9-SlWRKY57.

The GV3101/CRISPR/Cas9-SlWRKY57 is a recombinant microorganism obtained by introducing the recombinant vector CRISPR/Cas9-SlWRKY57 into the Agrobacterium tumefaciens GV3101.

The introduction may be by recombinant means including, but not limited to, agrobacterium-mediated transformation, biolistic (biolistic) methods, electroporation, in planta techniques, freeze thawing methods, and the like.

The present invention also provides a method of growing a salt tolerant plant, which may comprise reducing the content and/or activity of the protein SlWRKY57 in a plant of interest, resulting in a salt tolerant plant having a salt tolerance higher than the plant of interest.

In the above method, the reduction of the content and/or activity of the protein SlWRKY57 in the target plant may be achieved by reducing the expression amount and/or activity of a gene encoding the protein SlWRKY57 in the target plant.

In the above method, the reducing the expression level and/or activity of the gene encoding the protein SlWRKY57 in the target plant may be reducing or inactivating the activity of the gene encoding the protein SlWRKY57 in the target plant genome by using a gene mutation, gene knockout, gene editing or gene knockdown technique.

In the above method, the decreasing or inactivating of the activity of the gene encoding the protein of claim 1 or 2 in the genome of the plant of interest using gene editing techniques may be performed using a CRISPR/Cas9 system, the CRISPR/Cas9 system comprising a vector expressing a sgRNA targeting the gene encoding the protein, the target sequence of the sgRNA may be SEQ ID No.4 and SEQ ID No.5.

The method for cultivating salt-tolerant plants can comprise the following steps: inhibiting expression of nucleic acid molecules capable of expressing SlWRKY57 protein in target plants to obtain transgenic plants; the transgenic plants have improved salt tolerance compared to the plant of interest. Wherein the inhibition of expression of a nucleic acid molecule capable of expressing a SlWRKY57 protein in a plant of interest can be achieved by any means that achieves this objective, such as by specific cleavage of the nucleic acid molecule by a sequence specific nuclease (e.g., CRISPR/Cas9 nuclease) to reduce its expression in the plant of interest. The method for cultivating the salt-tolerant plants can be realized by hybridization means or transgenic means.

In one embodiment of the invention, the method of growing a salt tolerant plant comprises the steps of:

(1) Constructing CRISPR/Cas9 gene editing vectors targeting SEQ ID No.4 and SEQ ID No. 5;

(2) Introducing the CRISPR/Cas9 gene editing vector constructed in the step (1) into a target plant;

(3) And screening and identifying to obtain the salt-tolerant plant with salt tolerance higher than that of the target plant.

In the above method, the CRISPR/Cas9 gene editing vector may be the recombinant vector CRISPR/Cas9-SlWRKY57.

In the above method, the plant of interest may be tomato, and in particular may be tomato variety Solanum lycopersicum CvCastlemart (CM).

In the above method, the introducing includes, but is not limited to: the plant cells or tissues are transfected by using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium-mediated methods, etc., and the transfected plant cells or tissues are grown into plants.

In the above method, the introduction may specifically be an Agrobacterium-mediated method.

The present invention also provides a method for preparing tomatoes with improved salt tolerance, which may comprise the steps of: the method comprises the steps of (1) mutating a SlWRKY57 gene shown in SEQ ID No.2 in a sequence table in a tomato genome into a SlWRKY57/+1bp gene or a SlWRKY57/-1bp gene to obtain tomatoes with improved salt tolerance; the nucleotide sequence of the SlWRKY57/+1bp gene is shown in SEQ ID No.6, and the nucleotide sequence of the SlWRKY57/-1bp gene is shown in SEQ ID No. 7.

The invention also provides the application of the SlWRKY57/+1bp gene or the SlWRKY57/-1bp gene or the SlWRKY57/+1bp gene or the SlWRKY57/-1bp gene in improving the salt tolerance of tomatoes.

The nucleotide sequence of the SlWRKY57 gene mutant SlWRKY57/+1bp gene is shown as SEQ ID No.6, and is unchanged at the first target point, and a base "C" is inserted at the second target point.

The nucleotide sequence of the SlWRKY57 gene mutant SlWRKY57/-1bp gene is shown as SEQ ID No.7, which is a first target point deleted one base "G", and a second target point unchanged.

Herein, the plant may be a crop (e.g., a crop).

Herein, the plant may be any one of the following:

g1 Monocotyledonous or dicotyledonous plants;

g2 Solanaceae plant;

g3 Solanum plants;

g4 Tomato (tomato).

Further, the tomato may specifically be tomato variety Solanum lycopersicum cv Castlemart (CM).

The invention also provides application of the method for cultivating the salt-tolerant plant in creating the salt-tolerant plant and/or plant breeding and/or plant germplasm resource improvement.

The SlWRKY57 gene described herein may be a tomato salt tolerance related gene.

The plant breeding described herein may be crop salt tolerance breeding, in particular tomato salt tolerance breeding, the purpose of which may be to seed tomatoes with improved salt tolerance.

The plant breeding described herein may be molecular breeding that utilizes the SlWRKY57 genes and/or proteins SlWRKY57 of the invention to improve salt tolerance in crops.

Modulating plant salt tolerance as described herein may be increasing (up-regulating) plant salt tolerance or decreasing (down-regulating) plant salt tolerance. Further, the improvement (upregulation) of salt tolerance in plants is manifested by: increasing the fresh weight of the plant and/or reducing the wilting degree of the plant and/or increasing the fresh weight of the plant under salt stress conditions. The reduction (downregulation) of plant salt tolerance is manifested by: reducing the fresh weight of the plant and/or increasing the wilting degree of the plant and/or reducing the fresh weight of the plant under salt stress conditions.

In this context, the salt tolerant plant is understood to include not only the first generation transgenic plant from which the SlWRKY57 gene was knocked out, but also its progeny. The salt tolerant plants include seeds, calli, whole plants and cells.

The salt tolerant plant described herein may be a salt tolerant tomato.

The salt tolerant plants have increased tolerance to salt stress as compared to the recipient plant (plant of interest). The improvement in tolerance to salt stress may be manifested as: after the expression quantity and/or activity of the SlWRKY57 protein in the receptor plant are reduced, the fresh weight of the plant is higher than that of the wild type, and the wilting degree is lower than that of the wild type.

According to the invention, a tomato SlWRKY57 gene is taken as a research object, a CRISPR/Cas9-SlWRKY57 expression vector is constructed in vitro, and introduced into a receptor plant tomato wild CM by an agrobacterium-mediated method to obtain tomato SlWRKY57 gene editing plants slwry 57-cr-1 and slwry 57-cr-2. Plant phenotypes were observed after 30 days of treatment with 300mmol NaCl in the "four leaves one heart" period, fresh weights were weighed, and differences in tolerance to salt stress of tomato wild type CM plants before and after gene knockout were analyzed. The results show that: after 30 days of treatment with 300mmol NaCl, the tolerance of the slwrky57-cr-1 and slwrky57-cr-2 lines to salt stress increased and the fresh plant weight increased significantly. The invention enriches related genes of tomatoes in salt resistance regulation and control, and provides references for tomato breeding and yield improvement in abiotic stress.

The identified SlWRKY57 gene and the SlWRKY57 protein coded by the gene can regulate and control the salt tolerance of plants, and the salt tolerance of the target plants can be obviously improved by reducing the content and/or activity of the SlWRKY57 protein in the target plants (such as gene knockout of the SlWRKY57 gene). The salt tolerance of the plant of interest can be significantly reduced by increasing the amount and/or activity of the SlWRKY57 protein in the plant of interest (e.g., over-expressing the SlWRKY57 gene). The invention discovers the application of the SlWRKY57 gene and the coding protein thereof in improving the salt tolerance of tomatoes for the first time, and has important significance and wide application prospect in cultivating new varieties of salt-tolerant tomatoes, overcoming the short plates of traditional breeding, promoting the breeding process of commercial tomatoes and ensuring the high and stable yield of tomatoes.

Drawings

Fig. 1 is a graph of different editing types of a SlWRKY57 gene editing line obtained by constructing a CRISPR/Cas9-SlWRKY57 expression vector by CRISPR/Cas9 double-target knockout with tomato CM wild type as a background. T1 and T2 are respectively a target point 1 and a target point 2, wherein the target point 1 is positioned at 74-96bp downstream of the ATG, the base sequence is 5'-CCGGCGCTACTACTGAATCAAGC-3', the target point 2 is positioned at 211-233bp downstream of the ATG, and the base sequence is 5'-GGAAATAGAGATGTTCGCGCCGG-3', PAM, and the sequences are CCG and CGG respectively.

FIG. 2 shows the plant phenotype observations after 30 days of 300mmol NaCl treatment during the "four leaves one heart" period for different tomato lines. CM is a tomato wild type plant; slwry 57-cr-1 and slwry 57-cr-2 were SlWRKY57 gene editing plants.

FIG. 3 shows the fresh weight statistics of plants after 30 days of treatment with 300mmol NaCl in the "four leaves one heart" period of different tomato lines. CM is a tomato wild type plant; slwry 57-cr-1 and slwry 57-cr-2 were SlWRKY57 gene editing plants.

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 experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The following examples use ANOVA statistical software to process the data, the experimental results are expressed as mean ± standard deviation, P < 0.05 (x) represents significant differences, P < 0.01 (x) represents very significant differences, and different letters represent differences that are statistically significant. The quantitative experiments in the following examples were performed in triplicate, and the results were averaged unless otherwise indicated.

Tomato variety Solanum lycopersicum cv Castlemart (CM) in the following examples is described in the following documents: huang Huang, wenchao Zhao, hui Qiao, chongghua Li, lulu Sun, rui Yang, xuechun Ma, jilin Ma, susheng Song, and Shaohui Wang. SlWRKY45 interacts withjasmonate-ZIM domain proteins to negatively regulate defense against the root-knotnematode Meloidogyne incognita in, technical Research, 2022. Horticulure Research,9:uhac197, the public may obtain the biomaterial from the applicant, which is used only for duplicate experiments of the invention, but not as other uses.

KT in the following examples is Kinetin (Kinetin), 2,4-D is 2, 4-dichlorophenoxyacetic acid, IAA is Indole-3-acetic acid (Indole-3-acetic acid), and ZT is zetin (Zeatin).

Kan in the examples below is kanamycin, rif is rifamycin (rifampicin), and Hyg is hygromycin B (Hygromycin B).

The A1 medium in the following examples was an MS solid medium containing 1mg/L IAA and 1.75mg/L ZT, the A2 resistant medium was an MS solid medium containing 1.0mg/L IAA, 1.75mg/L ZT and 5mg/L Hyg, the A3 medium was an MS solid medium containing 1.0mg/L IAA, 1.75mg/L ZT and 5mg/L Hyg, and the A4 medium was an MS solid medium containing 5mg/L Hyg.

The pCBSG012 vector and plasmid pSG-SlU6 in the examples below are described in the following documents: huang, wenchao Zhao, hui Qiao, chonghua Li, lulu Sun, rui Yang, xuechun Ma, jilin Ma, suseng Song, and Shaohui Wang. SlWRKY45 interacts with jasmonate-ZIMdomain proteins to negatively regulate defense against the root-knot nematodeMeloidogyne incognita in, technical Research,9:uhac197. The biological material is available to the public from the applicant and is only used for repeated experiments of the invention and is not available for other uses.

Example 1, slWRKY57 Gene and protein encoded thereby

Through extensive and intensive studies, the inventors of the present invention have found that the SlWRKY57 gene and its encoded protein can regulate and control salt tolerance of plants. The nucleotide sequence of the SlWRKY57 genome is shown as SEQ ID No.2, the full-length 3383bp,SlWRKY57 cDNA sequence is shown as SEQ ID No.3, the length is 1306bp, the nucleotide sequence of the coding sequence (CDS) of the SlWRKY57 gene is shown as positions 178-1161 of SEQ ID No.3, the length is 984bp, 327 amino acids are coded, and the coded amino acid sequence is shown as SEQ ID No.1. The protein coded by the SlWRKY57 gene is named as SlWRKY57 protein, and the amino acid sequence of the SlWRKY57 protein is SEQ ID No.1.

Example 2 construction and identification of Gene-edited tomato

1. Construction of CRISPR/Cas9-SlWRKY57 expression vector

Two targets of about 20bp are selected from CDS (178-1161 bits of SEQ ID No. 3) region of the SlWRKY57 gene by taking tomato CM wild type as background to perform CRISPR/Cas9 double-target knockout, so as to construct the CRISPR/Cas9-SlWRKY57 expression vector. Two target sequences were designed as follows:

target 1:5'-CCGGCGCTACTACTGAATCAAGC-3' (SEQ ID No. 4),

target 2:5'-GGAAATAGAGATGTTCGCGCCGG-3' (SEQ ID No. 5).

Target 1 is located 74-96bp downstream of ATG, target 2 is located 211-233bp downstream of ATG, and PAM sequences are CCG and CGG respectively.

The CRISPR/Cas9-SlWRKY57 expression vector is constructed as follows:

two sgRNAs targeting the first exon of SlWRKY57 were selected using CRISPR-P. PCR amplification was performed using the plasmid pSG-SlU6 as a template and the following primer pairs.

F：5’-caatggtctcatgattgcttgattcagtagtagcgcgttttagagctagaaata-3’，

R：5’-ttggggtctctaaacgcgcgaacatctctatttccaaactacactgttagattt-3’。

The PCR product contained Bsa I cleavage site, two targets, gRNA scaffold, TTTTTTTT termination signal and tomato U6 promoter. The pCBSG012 vector and the PCR product are simultaneously subjected to enzyme digestion by using Bsa I, are connected after purification and recovery, and are recombined to obtain the CRISPR/Cas9-SlWRKY57 expression vector. The vector construction procedure and related vectors are described in the literature Huang Huang, wenchao Zhao, hui Qiao, chonghua Li, lulu Sun, rui Yang, xuechun Ma, jilin Ma, suseng Song, and Shaohui Wang. SlWRKY45 interacts withjasmonate-ZIM domain proteins to negatively regulate defense against the root-knotnematode Meloidogyne incognita in tool.2022. Horticulture Research,9:uhac197.

The obtained SlWRKY57 gene editing vector (i.e. recombinant vector for gene knockout) is named as CRISPR/Cas9-SlWRKY57.

The recombinant vector CRISPR/Cas9-SlWRKY57 contains two editing targets (SEQ ID No.4 and SEQ ID No. 5) and coding genes of Cas9 proteins, after the gene is introduced into a receptor, two transcribed guide RNAs can target sequences near the receptor genome PAM through base complementary pairing, namely target SlWRKY57 genes, the Cas9 proteins enable DNA double strand breaks at the upstream and downstream of the SlWRKY57 genes, and sequences at the upstream and downstream ends of the breaks are connected through a DNA damage repair response mechanism of an organism, so that the knockout of the SlWRKY57 genes is realized.

2. Transformation of Agrobacterium

The recombinant vector CRISPR/Cas9-SlWRKY57 with correct sequencing is transferred into the agrobacterium tumefaciens GV3101 strain by an electric shock method to obtain the recombinant agrobacterium tumefaciens GV3101/CRISPR/Cas9-SlWRKY57. And (5) after the bacterial liquid PCR is verified to be correct, preserving bacteria for later use.

3. Genetic transformation of tomato

1. Sowing seeds

The seeds were sterilized with 75% ethanol for 4 minutes, then with sterile water for 4-5 times, sterilized with 3% sodium hypochlorite solution for about 8 minutes, then with sterile water for 8-10 times, and the seeds were sown in 1/2MS medium and cultured in a tissue culture chamber for 5-7d until cotyledons developed without true leaves.

2. Explant shearing

The cotyledon tips and end portions were cut off with sterilized forceps and scissors to form explants of about 1cm, the cut explants were immersed in MS liquid (1 mg/L KT and 1 mg/L2, 4-D were added) for 0.5h, transferred to A1 medium (MS solid medium +1mg/L IAA +1.75mg/L ZT) and precultured for 1-2D with the right side facing upward.

3. Preparation of infectious microbe liquid

Monoclonal positive colonies (recombinant Agrobacterium tumefaciens GV3101/CRISPR/Cas9-SlWRKY 57) were selected, cultured using 2ml of YEB liquid medium containing antibiotics (50 mg/L Kan and 50mg/L Rif), and on the day of infection, 20ml of YEB liquid medium containing antibiotics (50 mg/L Kan and 50mg/L Rif) was added to the above small shaking bacterial liquid (bacterial liquid approximately OD600 = 1), and the culture was expanded at 28℃shaker at 200rpm for about 4 hours to obtain Agrobacterium bacterial liquid, OD600 = 1-2.

4. Infection explant

The agrobacterium solution obtained in the step 3 is centrifuged at 4000rpm for 10 minutes at room temperature, the supernatant is discarded, the YEB liquid culture medium is used for suspending after the bacterial cells are collected, the centrifugation is carried out at 4000rpm for 10 minutes, the bacterial cells are resuspended by using an MS salt solution after the supernatant is discarded, the OD600 = 0.3-0.5 is regulated, the explant is soaked in the resuspension and cultured for 15 minutes, after the sterile filter paper absorbs the excessive liquid, the explant is transferred to the A1 culture medium, the back surface of the explant faces upwards, and the dark culture is carried out for 2 days.

5. Culturing

After two days of co-cultivation (dark cultivation), cotyledons were transferred to A2 resistant medium (MS solid medium+1.0 mg/LIAA+1.75mg/L ZT+5mg/L Hyg) and cultivated at 26 ℃ (16 h light)/18 ℃ (8 h dark). Fresh A2 resistant medium was changed weekly until callus formation. After callus formation, it was transferred to A3 medium (MS solid medium +1.0mg/L IAA +1.75mg/L ZT +5mg/L Hyg) to induce sprouting. Seedlings were transferred to A4 medium (MS solid medium +5mg/L Hyg) for rooting screening. Finally, T0 generation transgenic tomato plants (namely, the SlWRKY57 gene editing tomato) are obtained.

6. Authentication

And (3) taking T0 generation transgenic tomato plant leaves to extract DNA, carrying out PCR amplification by taking genomic DNA as a template, and delivering amplified products to a biological sequencing screen for positive transformation seedlings, wherein the identification primers are as follows.

SlWRKY57-F：5’-TTCAATTTTTTCTCTCTCCGC-3’；

SlWRKY57-R：5’-GTGATTTTCTCCGTCACCGTC-3’。

The amplified products were sequenced and compared to the wild-type sequence, and effective mutant lines were identified and designated as slwrky57-cr-1 and slwrky57-cr-2, respectively. Wherein:

SlWRKY57-cr-1 is a mutant line obtained by knocking out the SlWRKY57 gene by inserting a base "C" into a second target point and terminating translation in advance, so that a mutant of the SlWRKY57 gene is obtained correspondingly, and the mutant is named as SlWRKY57/+1bp gene. The nucleotide sequence of the SlWRKY57/+1bp gene is shown in SEQ ID No. 6.

The slwry 57-cr-2 is a mutant line obtained by knocking out the SlWRKY57 gene by deleting one base "G" from the first target point and leading to early termination of translation due to unchanged second target point, and a mutant of the SlWRKY57 gene is correspondingly obtained and named as SlWRKY57/-1bp gene. The nucleotide sequence of the SlWRKY57/-1bp gene is shown in SEQ ID No. 7.

The identification results of the SlWRKY57 gene mutant strain are shown in FIG. 1.

Example 3 salt tolerance functional verification of SlWRKY57 Gene-edited plants of different lines

Test tomato: control (CM) tomatoes and PCR-identified positive test groups obtained in example 2, slwrky57-cr-1 strain and slwrky57-cr-2 strain tomatoes, 20 strains each.

1. Salt stress treatment of plants of different lines to observe phenotypes

The different tomatoes tested were grown to a period of 'four leaves one heart', treated with 300mmol NaCl, irrigated 30mL per 48h of single plant, and observed for plant phenotypes after 30 days.

As a result, as shown in FIG. 2, the slwrky57-cr-1 and slwrky57-cr-2 gene-edited lines were significantly less wilted than the wild-type (FIG. 2). Under the condition of salt stress, the tomato edited by the SlWRKY57 gene shows stronger tolerance compared with wild tomato, which indicates that inhibiting or reducing the expression of the SlWRKY57 gene (such as knocking out the SlWRKY57 gene) can improve the salt tolerance of the tomato.

2. Salt stress treatment is carried out on plants of different strains to measure fresh weight

The fresh weights of the different tomatoes tested were measured after 30 days by treating them with 300mmol NaCl (the method is the same as the treatment in step 1) until they were grown to a period of "four leaves one heart".

As shown in FIG. 3, the fresh weight of the plants of the slwrky57-cr-1 and slwrky57-cr-2 gene-editing lines was significantly higher than that of the wild type plants. The results further demonstrate that inhibiting or reducing expression of the SlWRKY57 gene (e.g., knocking out the SlWRKY57 gene) increases salt tolerance of tomato.

In summary, the SlWRKY57 gene and the SlWRKY57 protein encoded by the SlWRKY57 gene identified by the invention can regulate and control the salt tolerance of plants (such as improving or reducing the salt tolerance of plants), the salt tolerance of target plants can be obviously reduced by reducing the content and/or activity of the SlWRKY57 protein in target plants (such as inhibiting the expression of the SlWRKY57 gene), and the salt tolerance of target plants can be obviously improved by improving the content and/or activity of the SlWRKY57 protein in target plants (such as over-expressing the SlWRKY57 gene).

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.

The sequences referred to herein are as follows:

MDENDKQVDNDPLPGGGGGGAAEFTGATTESSWSLGGGDDESDNVYFFGTSSTDRESSILTEFGWNFQPHGNRDVRAGGDGSRFDRIDEDLAGNSTTTTASVSASVSITEPTVTEKITDEPVSSSCSDDPPEKSTASGSSSASRPPSDTASKVKKKGQKRIRQPRFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPFPRSYYRCTNTKCTVKKRVERSSEDSSIVITTYEGQHCHHTVGFPRGGLINHEAFTSQLTPLPSQFYHPSGVQYPHELVPMSSPAAPESHTMPGETGPEPIRLPETSQPDATQQPTDEGLLGDIVPPGMRSR(SEQ ID No.1)

tctatactatttttctgagtgtcttctcaccaattgccattgacacgacaaagctttcaaatcccccaaaaaagtttctaattaaatattccaaactctttacgcgtttatcttctccttctctataaaccctagcttcaattttttctctctccgcgtaaaattcactctgtaactatggatgaaaacgataagcaggttgataatgatccattacctggtggtggtggtggtggtgcagcggagttcaccggcgctactactgaatcaagctggtcactcggtggtggagatgatgaatcggataatgtttacttcttcggtactagtagtactgatagagagagcagtatattaaccgaattcggctggaattttcaaccgcacggaaatagagatgttcgcgccggtggtgacggtagtcgatttgatcggatcgatgaggatttggcgggaaatagtactacaactactgcttctgtttctgcttctgtttctattactgaaccgacggtgacggagaaaatcaccgacgaacctgtttcatctagctgttctgatgatcccccggagaaatctacagcttccggtagctcctccgcctctcgaccgccgtccgatacagcgtgagtcatgtgtttatcatcgacttctctctaagtatgtctaaattcgtaattaactgcgatttgaaatggtaaaaataaggagtaaaacgccattaacgtgtccgtacgtaggtgtagaaaaaatgtcaagtggttcacgcaagttgagggaagagaagagaggtattatcgtaattttaatcatcaaggatgggcttttcggtatttgctcacttttctgtggctttaaaacagccgtcgttgcggcgggaatggcgtggatgaattttttcctctaaaattaaatatcgtaataataattaaaattaactgttaaggaaaagtattttttttaaaatttctttttatataaaaaagtaacttgcgataactcttccctaataatggtttaggtgtaatctcttacaaaattagtttcttctacttcattatttcgtggtccattgcttcgtacattagtatttaaagaaagaaaaaaggaataggtacaatgtggaaacttttaactgttaaagcaataataaagttatcttagtgtgatagatctgctaaaattgtaaaattagtcatcaatatagcttttagtccgttttcatatgaatacagaatactttgtgcaaattactgataaataaatgaattttggtttgtgtgaactctcaaggttcattgcatgcatttctcttcgttgtggtctgcattgttcttgtagtataacctaattatacaagcccaagtgaacttttgtttttttgttttttaaacatttaatatatttcgttaataataaattgaactaaggtagagttacttaaagcaaacaaacatgaagagaatttggatacgtttttataagtattaagttcaactgcagactcttcaattttttatgttgcatccgtatcggattcttcaaaaatacactatgtttggcgaatccaatacgcacctattgacctttttgaagagttcgagcaactgtactactattttgatgttgtttagtagtattacgctgcccactatatttataagggtttctaaatttcaactaagtttgagcagctagataaacataacttgttacttatcaatatataacaagaatcagaggtatataataccaggtaatttctttctatctgttatagtcttggatatggtaagatagatattctgtgaaactagccccaggggttggatacccaaaaatataaaaccaatttataatattcttcatgtctataacttcattaaaagaataaatacccattctttttcaattgaaagatattttgtttattacaaagatttactaattggaaactagattggtttgtattaaatgatgcattgagtttcttagttataaagaaaat gcaagtgtaccaggaattataattttttaaaatattttctagtactatagtatatatattatgtaatataagatgaaggaatcacattcctaatgttactttaggttgaatattccacagaagcaaggttaaaaagaagggtcagaaacgaatcaggcagccccgctttgcattt atgaccaaaagtgaagttgatcatcttgaagatggctatagatggaggaaatatggccaaaaagctgttaaaaacagtccatttccaaggtatgttggaatgaatttaaattttatgaatttctaacgaattatttgaagtatatttagttattatgttcaaaattcaacattgagaaaattgtttaataaaacaaaccattagatgtacatgtacttaactaaattactcttaattctaccttatatacctgagt aattatttgttatatgcctttttggaatagtatggaagttgttttcaagaaatttttttttttcttcttggggattttagaagtgatagattaaatatttggacaaaaattgtttgactaaagtgatggtataataaataggatagggtagtacttttcgtatatctaatcctaattcctgtccttatatcctattgaaacatatacatgtatcttaacgagttatgtatatcttgattaattttatgaattactt tattacaatcaccaataaataggtatcagataattattcacaaagatttaaacagataaaatcatgtatttcttcatttcaatttgtatgatgtagtctgggttgataaaagttgaaataattactcatgatgaatatatatgacttgtcaggagttattatcgttgtacaaatacaaagtgtacagtaaagaaaagagtggagagatcctctgaagattcctcaattgtaatcacaacatatgaaggacaacatt gtcaccatacagttggatttcctagaggtggacttatcaatcacgaagcatttacatctcaattaacacctttaccctcacaattctatcatccatccggtgttcaataccctcatgaattagttcctatgagtagccccgctgcaccagaatcacatacaatgcctggtgaaaccggacccgaacctattagattgccagaaacaagtcaaccagatgctacacaacaaccaactgatgaaggattgcttggagatattgtacctcctgggatgcgaagcagataatataaagggtaatatattccgtttcttaatttgcattaatttacatatatttttttatttcatcaatgacatagtatttgaacgttacacgagtgcgcctacacaagtgtaagtgtatgtgttttatttaaaaagaaatttctgt(SEQ ID No.2)

tctatactatttttctgagtgtcttctcaccaattgccattgacacgacaaagctttcaaatcccccaaaaaagtttctaattaaatattccaaactctttacgcgtttatcttctccttctctataaaccctagcttcaattttttctctctccgcgtaaaattcactctgtaactatggatgaaaacgataagcaggttgataatgatccattacctggtggtggtggtggtggtgcagcggagttcaccggcgctactactgaatcaagctggtcactcggtggtggagatgatgaatcggataatgtttacttcttcggtactagtagtactgatagagagagcagtatattaaccgaattcggctggaattttcaaccgcacggaaatagagatgttcgcgccggtggtgacggtagtcgatttgatcggatcgatgaggatttggcgggaaatagtactacaactactgcttctgtttctgcttctgtttctattactgaaccgacggtgacggagaaaatcaccgacgaacctgtttcatctagctgttctgatgatcccccggagaaatctacagcttccggtagctcctccgcctctcgaccgccgtccgatacagcaagcaaggttaaaaagaagggtcagaaacgaatcaggcagccccgctttgcatttatgaccaaaagtgaagttgatcatcttgaagatggctatagatggaggaaatatggccaaaaagctgttaaaaacagtccatttccaaggagttattatcgttgtacaaatacaaagtgtacagtaaagaaaagagtggagagatcctctgaagattcctcaattgtaatcacaacatatgaaggacaacattgtcaccatacagttggatttcctagaggtggacttatcaatcacgaagcatttacatctcaattaacacctttaccctcacaattctatcatccatccggtgttcaataccctcatgaattagttcctatgagtagccccgctgcaccagaatcacatacaatgcctggtgaaaccggacccgaacctattagattgccagaaacaagtcaaccagatgctacacaacaaccaactgatgaaggattgcttggagatattgtacctcctgggatgcgaagcagataatataaagggtaatatattccgtttcttaatttgcattaatttacatatatttttttatttcatcaatgacatagtatttgaacgttacacgagtgcgcctacacaagtgtaagtgtatgtgttttatttaaaaagaaatttctgt(SEQ ID No.3)

CCGGCGCTACTACTGAATCAAGC(SEQ ID No.4)

GGAAATAGAGATGTTCGCGCCGG(SEQ ID No.5)

tctatactatttttctgagtgtcttctcaccaattgccattgacacgacaaagctttcaaatcccccaaaaaagtttctaattaaatattccaaactctttacgcgtttatcttctccttctctataaaccctagcttcaattttttctctctccgcgtaaaattcactctgtaactatggatgaaaacgataagcaggttgataatgatccattacctggtggtggtggtggtggtgcagcggagtt caccggcgctactactgaatcaagctggtcactcggtggtggagatgatgaatcggataatgtttacttcttcggtactagtagtactgatagagagagcagtatattaaccgaattcggctggaattttcaaccgcacggaaatagagatgttcgccgccggtggtgacggtagtcgatttgatcggatcgatgaggatttggcgggaaatagtactacaactactgcttctgtttctgcttctgtttctattactgaaccgacggtgacggagaaaatcaccgacgaacctgtttcatctagctgttctgatgatcccccggagaaatctacagcttccggtagctcctccgcctctcgaccgccgtccgatacagcgtgagtcatgtgtttatcatcgacttctctctaagtatgtctaaattcgtaattaactgcgatttgaaatggtaaaaataaggagtaaaacgccattaacgtgtccgtacgtaggtgtagaaaaaatgtcaagtggttcacgcaagttgagggaagagaagagaggtattatcgtaattttaatcatcaaggatgggcttttcggtatttgctcacttttctgtggctttaaaacagccgtcgttgcggcgggaatggcgtggatgaattttttcctctaaaattaaatatcgtaataataattaaaattaactgttaaggaaaagtattttttttaaaatttctttttatataaaaaagtaacttgcgataactcttccctaataatggtttaggtgtaatctcttacaaaattagtttctt ctacttcattatttcgtggtccattgcttcgtacattagtatttaaagaaagaaaaaaggaataggtacaatgtggaaacttttaactgttaaagcaataataaagttatcttagtgtgatagatctgctaaaattgtaaaattagtcatcaatatagcttttagtccgttttcatatgaatacagaatactttgtgcaaattactgataaataaatgaattttggtttgtgtgaactctcaaggttcattgcatgcatttctcttcgttgtggtctgcattgttcttgtagtataacctaattatacaagcccaagtgaacttttgtttttttgttttttaaacatttaatatatttcgttaataataaattgaactaaggtagagttacttaaagcaaacaaacatgaagagaatttggatacgtttttataagtattaagttcaactgcagactcttcaattttttatgttgcatccgtatcggattcttcaaaaatacactatgtttggcgaatccaatacgcacctattgacctttttgaagagttcgagcaactgtactactattttgatgttgtttagtagtattacgctgcccactatatttataagggtttctaaatttcaactaagtttgagcagctagataaacataacttgttacttatcaatatataacaagaatcagaggtatataataccaggtaatttctttctatctgttatagtcttggatatggtaagatagatattctgtgaaactagccccaggggttggatacccaaaaatataaaaccaatttataatattcttcatgtctataacttcattaaaagaataaatacccattctttttcaattgaaagatattttgtttattacaaagatttactaattggaaactagattggtttgtattaaatgatgcattgagtttcttagttat aaagaaaatgcaagtgtaccaggaattataattttttaaaatattttctagtactatagtatatatattatgtaatataagatgaaggaatcacattcctaatgttactttaggttgaatattccacagaagcaaggttaaaaagaagggtcagaaacgaatcaggcagccccgctttgcatttatgaccaaaagtgaagttgatcatcttgaagatggctatagatggaggaaatatggccaaaaagctgttaaaaacagtccatttccaaggtatgttggaatgaatttaaattttatgaatttctaacgaattatttgaagtatatttagttattatgttcaaaattcaacattgagaaaattgtttaataaaacaaaccattagatgtacatgtacttaactaaattactcttaattctaccttatatacctgagtaattatttgttatatgcctttttggaatagtatggaagttgttttcaagaaatttttttttttcttcttggggattttagaagtgatagattaaatatttggacaaaaattgtttgactaaagtgatggtataataaataggatagggtagtacttttcgtatatctaatcctaattcctgtccttatatcctattgaaacatatacatgtatcttaacgagttatgtatatcttgattaattttatgaattactttattacaatcaccaataaataggtatcagataattattcacaaagatttaaacagataaaatcatgtatttcttcatttcaatttgtatgatgtagtctgggttgataaaagttgaaataattactcatgatgaatatatatgacttgtcaggagttattatcgttgtacaaatacaaagtgtacagtaaagaaaagagtggagagatcctctgaagattcctcaattgtaatcacaacatatgaaggacaacattgtcaccatacagttggatttcctagaggtggacttatcaatcacgaagcatttacatctcaattaacacctttaccctcacaattctatcatccatccggtgttcaataccctcatgaattagttcctatgagtagccccgctgcaccagaatcacatacaatgcctggtgaaaccggacccgaacctattagattgccagaaacaagtcaaccagatgctacacaacaaccaactgatgaaggattgcttggagatattgtacctcctgggatgcgaagcagataatataaagggtaatatattccgtttcttaatttgcattaatttacatatatttttttatttcatcaatgacatagtatttgaacgttacacgagtgcgcctacacaagtgtaagtgtatgtgttttatttaaaaagaaatttctgt(SEQ ID No.6)

tctatactatttttctgagtgtcttctcaccaattgccattgacacgacaaagctttcaaatcccccaaaaaagtttctaattaaatattccaaactctttacgcgtttatcttctccttctctataaaccctagcttcaattttttctctctccgcgtaaaattcactctgtaactatggatgaaaacgataagcaggttgataatgatccattacctggtggtggtggtggtggtgcagcggagttcaccggcctactactgaatcaagctggtcactcggtggtggagatgatgaatcggataatgtttacttcttcggtactagtagtactgatagagagagcagtatattaaccgaattcggctggaattttcaaccgcacggaaatagagatgttcgcgccggtggtgacggtagtcgatttgatcggatcgatgaggatttggcgggaaatagtactacaactactgcttctgtttctgcttctgtttctattactgaaccgacggtgacggagaaaatcaccgacgaacctgtttcatctagctgttctgatgatcccccggagaaatctacagcttccggtagctcctccgcctctcgaccgccgtccgatacagcgtgagtcatgtgtttatcatcgacttctctctaagtatgtctaaattcgtaattaactgcgatttgaaatggtaaaaataaggagtaaaacgccattaacgtgtccgtacgtaggtgtagaaaaaatgtcaagtggttcacgcaagttgagggaagagaagagaggtattatcgtaattttaatcatcaaggatgggcttttcggtatttgctcacttttctgtggctttaaaacagccgtcgttgcggcgggaatggcgtggatgaattttttcctctaaaattaaatatcgtaataataattaaaattaactgttaaggaaaagtattttttttaaaatttctttttatataaaaaagtaacttgcgataactcttccctaataatggtttaggtgtaatctcttacaaaattagtttcttctacttcattatttcgtggtccattgcttcgtacattagtatttaaagaaagaaaaaaggaataggtacaatgtggaaacttttaactgttaaagcaataataaagttatcttagtgtgatagatctgctaaaattgtaaaattagtcatcaatatagcttttagtccgttttcatatgaatacagaatactttgtgcaaattactgataaataaatgaattttggtttgtgtgaactctcaaggttcattgcatgcatttctcttcgttgtggtctgcattgttcttgtagtataacctaattatacaagcccaagtgaacttttgtttttttgttttttaaacatttaatatatttcgttaataataaattgaactaaggtagagttacttaaagcaaacaaacatgaagagaatttggatacgtttttataagtattaagttcaactgcagactcttcaattttttatgttgcatccgtatcggattcttcaaaaatacactatgtttggcgaatccaatacgcaccta

ttgacctttttgaagagttcgagcaactgtactactattttgatgttgtttagtagtattacgctgcccactatatttataagggttt

ctaaatttcaactaagtttgagcagctagataaacataacttgttacttatcaatatataacaagaatcagaggtatataataccagg

taatttctttctatctgttatagtcttggatatggtaagatagatattctgtgaaactagccccaggggttggatacccaaaaatata

aaaccaatttataatattcttcatgtctataacttcattaaaagaataaatacccattctttttcaattgaaagatattttgt ttatt

acaaagatttactaattggaaactagattggtttgtattaaatgatgcattgagtttcttagttataaagaaaatgcaagtgtaccag

gaattataattttttaaaatattttctagtactatagtatatatattatgtaatataagatgaaggaatcacattcctaatgttactt

taggttgaatattccacagaagcaaggttaaaaagaagggtcagaaacgaatcaggcagccccgctttgcatttatgaccaaaagtga

agttgatcatcttgaagatggctatagatggaggaaatatggccaaaaagctgttaaaaacagt ccatttccaaggtatgttggaatg

aatttaaattttatgaatttctaacgaattatttgaagtatatttagttattatgttcaaaattcaacattgagaaaattgtttaata

aaacaaaccattagatgtacatgtacttaactaaattactcttaattctaccttatatacctgagtaattatttgttatatgcctttt

tggaatagtatggaagttgttttcaagaaatttttttttttcttcttggggattttagaagtgatagattaaatatttggacaaaaat

tgtttgactaaagtgatggtataataaataggatagggtagtacttttcgtatatctaatcctaattcctgtccttatatcctattga

aacatatacatgtatcttaacgagttatgtatatcttgattaattttatgaattacttt attacaatcaccaataaataggtatcaga

taattattcacaaagatttaaacagataaaatcatgtatttcttcatttcaatttgtatgatgtagtctgggttgataaaagttgaaa

taattactcatgatgaatatatatgacttgtcaggagttattatcgttgtacaaatacaaagtgtacagtaaagaaaagagtggagag

atcctctgaagattcctcaattgtaatcacaacatatgaaggacaacattgtcaccatacagttggatttcctagaggtggacttatc

aatcacgaagcatttacatctcaattaacacctttaccct cacaattctatcatccatccggtgttcaataccctcatgaattagttc

ctatgagtagccccgctgcaccagaatcacatacaatgcctggtgaaaccggacccgaacctattagattgccagaaacaagtcaacc

agatgctacacaacaaccaactgatgaaggattgcttggagatattgtacctcctgggatgcgaagcagataatataaagggtaatat

attccgtttcttaatttgcattaatttacatatatttttttatttcatcaatgacatagtatttgaacgttacacgagtgcgcctaca

caagtgtaagtgtatgtgttttatttaaaaagaaatttctgt(SEQ ID No.7)。

Claims (10)

1. use of a protein or a substance regulating the activity and/or content of said protein, characterized in that said use is any of the following:

a1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating the salt tolerance of a plant;

a2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating the salt tolerance of a plant;

a3 Use of a protein or a substance regulating the activity and/or content of said protein for growing salt tolerant plants;

a4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing salt tolerant plants;

a5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding or plant germplasm resource improvement;

the protein is any one of the following:

b1 A protein having an amino acid sequence of SEQ ID No. 1;

b2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in B1) and has the same function;

b3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of B1) or B2).

2. The use according to claim 1, wherein the protein is derived from tomato.

3. Use of a biological material related to a protein as claimed in claim 1 or 2, characterized in that the use is any of the following:

d1 Use of biological material related to the protein of claim 1 or 2 for regulating salt tolerance of plants;

d2 Use of a biological material related to the protein of claim 1 or 2 for the preparation of a product for modulating salt tolerance in a plant;

d3 Use of biological material related to the protein of claim 1 or 2 for growing salt tolerant plants;

d4 Use of a biological material related to the protein of claim 1 or 2 for the preparation of a product for growing salt tolerant plants;

d5 Use of biological material related to the protein of claim 1 or 2 in plant breeding or plant germplasm resource improvement;

the biological material is any one of the following:

e1 A nucleic acid molecule encoding a protein as claimed in claim 1 or 2;

e2 A nucleic acid molecule which inhibits or reduces the expression of a gene encoding a protein as claimed in claim 1 or 2;

e3 An expression cassette containing the nucleic acid molecule of E1) and/or E2);

e4 A recombinant vector comprising E1) and/or E2) said nucleic acid molecule, or a recombinant vector comprising E3) said expression cassette;

e5 A recombinant microorganism containing the nucleic acid molecule of E1) and/or E2), or a recombinant microorganism containing the expression cassette of E3), or a recombinant microorganism containing the recombinant vector of E4);

e6 A recombinant host cell containing the nucleic acid molecule of E1) and/or E2), or a recombinant host cell containing the expression cassette of E3), or a recombinant host cell containing the recombinant vector of E4);

e7 A) transgenic plant tissue containing E1) and/or E2) said nucleic acid molecule, or a transgenic plant tissue containing E3) said expression cassette;

e8 A transgenic plant organ comprising E1) and/or E2) said nucleic acid molecule, or a transgenic plant organ comprising E3) said expression cassette.

4. The use according to claim 3, wherein E1) the nucleic acid molecule is any one of the following:

f1 A DNA molecule whose coding sequence is positions 178-1161 of SEQ ID No. 3;

f2 A DNA molecule whose nucleotide sequence is SEQ ID No.3 at positions 178-1161 or SEQ ID No. 3;

f3 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

5. A method of growing a salt tolerant plant, the method comprising reducing the content and/or activity of a protein according to claim 1 or 2 in a plant of interest to obtain a salt tolerant plant having a salt tolerance higher than the plant of interest.

6. The method according to claim 5, wherein said reducing the amount and/or activity of the protein according to claim 1 or 2 in the plant of interest is achieved by reducing the expression amount and/or activity of a gene encoding the protein in the plant of interest.

7. The method according to claim 6, wherein the reduction of the expression level and/or activity of the gene encoding the protein of claim 1 or 2 in the genome of the plant of interest is by means of gene mutation, gene knockout, gene editing or gene knockdown techniques.

8. The method of claim 7, wherein the decreasing or inactivating the activity of the gene encoding the protein of claim 1 or 2 in the genome of the plant of interest using gene editing techniques is performed using a CRISPR/Cas9 system comprising a vector expressing sgrnas targeting the gene encoding the protein, the target sequences of the sgrnas being SEQ ID No.4 and SEQ ID No.5.

9. A method for preparing tomatoes with improved salt tolerance, said method comprising the steps of: the method comprises the steps of (1) mutating a SlWRKY57 gene shown in SEQ ID No.2 in a sequence table in a tomato genome into a SlWRKY57/+1bp gene or a SlWRKY57/-1bp gene to obtain tomatoes with improved salt tolerance; the nucleotide sequence of the SlWRKY57/+1bp gene is shown in SEQ ID No.6, and the nucleotide sequence of the SlWRKY57/-1bp gene is shown in SEQ ID No. 7.

10. The use of the SlWRKY57/+1bp gene or SlWRKY57/-1bp gene of claim 9, or the SlWRKY57/+1bp gene or SlWRKY57/-1bp gene for improving salt tolerance of tomato.

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