Wheat salt-tolerant protein TaST1 as well as coding gene and application thereof

Abstract

The invention discloses a wheat salt-tolerant protein TaST1 as well as a coding gene and application thereof. Specifically disclosed are any one of the following applications: a protein, a substance for inhibiting or reducing or down-regulating the expression of a coding gene of the protein, or a substance for inhibiting or reducing or down-regulating the activity or content of the protein; a1) application in improving the salt tolerance of the wheat plants and / or application in preparing products for improving the salt tolerance of the wheat plants; a2) application in improving the survival rate of the wheat plants under the salt stress and / or application in preparing products for improving the survival rate of the wheat plants under the salt stress; a3) application in improving the biomass of the wheat plants under salt stress and / or application in preparing products for improving the biomass of the wheat plants under salt stress; the salt tolerance of the plant with the TaST1 protein coding gene knocked out is obviously higher than that of a wild plant, and the plant can be used for industrial production and plant breeding.

Description

Technical Field

[0001] The present invention specifically relates to wheat salt-tolerant protein TaST1 and its encoding gene and application. Background Art

[0002] Soil salinization seriously restricts the agricultural production of crops. Wheat is one of the most widely planted crops in the world, providing about 20% of human protein and carbohydrate intake. As one of the main staple crops in my country, wheat has relatively poor salt tolerance. However, due to industrial pollution, excessive use of chemical fertilizers and unreasonable irrigation, soil salinization is becoming more and more common in many cultivated lands. Soil salinization has become a global ecological problem, seriously reducing available land resources and causing huge losses to agricultural production of wheat. Therefore, it is of great significance to explore excellent salt-tolerant genes in wheat and analyze their molecular and genetic mechanisms for regulating salt tolerance, so as to cultivate new wheat varieties with stress resistance and stable yield. Summary of the invention

[0003] The invention provides a method for improving the salt tolerance of wheat plants based on TaST1 protein, which is used to improve the salt tolerance of wheat plants, including at least one of root length and survival rate.

[0004] In a first aspect, the present invention provides the following application.

[0005] Use of a protein, a substance that inhibits, reduces or down-regulates the expression of a gene encoding the protein, or a substance that inhibits, reduces or down-regulates the activity or content of the protein in any of the following;

[0006] A1) application in improving the salt tolerance of wheat plants and / or application in preparing products for improving the salt tolerance of wheat plants;

[0007] A2) application in improving the survival rate of Triticum plants under salt stress and / or application in preparing a product for improving the survival rate of Triticum plants under salt stress;

[0008] A3) application in increasing the biomass of Triticum spp. under salt stress and / or application in preparing a product for increasing the biomass of Triticum spp. under salt stress;

[0009] The protein is any of the following:

[0010] B1) contains a protein whose amino acid sequence is the protein shown in SEQ ID NO: 7;

[0011] B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1);

[0012] B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

[0013] In the above application, the protein is derived from wheat.

[0014] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0015] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0016] In the above proteins, the above 80% identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0017] In the above protein, sequence 2 (SEQ ID No. 2) consists of 874 amino acid residues. It is named TaST1 protein. Its encoding gene is TaST1 gene.

[0018] In the above, the amino acid sequence of the protein may be sequence 2, sequence 4 and / or sequence 6.

[0019] In the above, the salt tolerance index may be survival rate and / or biomass.

[0020] In the above, the biomass may be root length.

[0021] In the above application, the protein is derived from wheat.

[0022] In the above, the wheat may be the wheat variety Fielder.

[0023] In the above, the substance that inhibits, reduces or downregulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0024] In the above, the use comprises increasing the survival rate and / or biomass of Triticum plants.

[0025] In the above text, the biomass is the root length. In a specific embodiment, the root length is the length of the underground part.

[0026] In the above application, the amino acid sequence of the protein is sequence 2, sequence 4 and / or sequence 6.

[0027] In any of the above applications, the substance that inhibits, reduces or downregulates the expression of the gene encoding the protein is any of the following:

[0028] B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of the gene encoding the above protein or inhibits, reduces or down-regulates the activity or content of the protein;

[0029] B2), expressing the coding gene of the nucleic acid molecule described in B1);

[0030] B3), an expression cassette containing the gene described in B2);

[0031] B4, a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0032] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

[0033] B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4);

[0034] B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4);

[0035] B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0036] B1) In the nucleic acid molecule, a person skilled in the art can easily mutate the nucleotide sequence encoding the protein TaST1 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of the protein TaST1 isolated by the present invention are all 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 TaST1 and have the function of the protein TaST1.

[0037] The above 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0038] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0039] Herein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (ie, cosmid), Ti plasmid or viral vector.

[0040] In the above biological materials, the expression cassette described in B3) refers to a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription, but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoter from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolusin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters.All references cited herein are incorporated in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0041] In the above B4), the recombinant vector may be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector may be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85 or pCAMBIA1391-Xb. When using TaST1-A to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. As a specific embodiment, the present application uses the pBUE411 vector as an expression vector.

[0042] In the above application, the nucleic acid molecule target gene in B1) is the coding gene of the protein shown in Sequence 7.

[0043] In the above, the nucleic acid molecule target gene may be a gene encoding a protein shown in Sequence 2, Sequence 4 and / or Sequence 6.

[0044] In the above, the ORF of the gene encoding the protein shown in Sequence 2 may be that shown in Sequence 1.

[0045] In the above, the ORF of the gene encoding the protein shown in Sequence 4 may be shown in Sequence 3.

[0046] In the above, the ORF of the gene encoding the protein shown in Sequence 6 may be shown in Sequence 5.

[0047] In a second aspect, the present invention provides a method for cultivating a Triticum plant with high salt tolerance.

[0048] The method comprises inhibiting or reducing or down-regulating the expression level of a gene encoding any of the above proteins in a target wheat plant, and / or the activity and / or content of the protein to obtain a highly salt-tolerant wheat plant, wherein the highly salt-tolerant wheat plant has higher salt tolerance than the target wheat plant.

[0049] In a third aspect, the present invention provides a method for increasing the survival rate and / or biomass of Triticum plants under salt stress conditions.

[0050] The method comprises inhibiting, reducing or down-regulating the expression of the gene encoding the above-mentioned protein in the wheat plant, and / or the activity and / or content of any of the above-mentioned proteins to increase the survival rate and / or biomass of the wheat plant under salt stress conditions.

[0051] In the above method, the inhibition, reduction or down-regulation of the expression of the gene encoding any of the above proteins in the plant comprises introducing the nucleic acid molecule described in B1), the encoding gene described in B2), the expression cassette described in B3) or the recombinant vector described in B4) into the target plant.

[0052] In any of the above uses or any of the above methods, the plant is wheat.

[0053] In any of the above uses and any of the above methods, the Triticum plant is a Triticum plant in the seedling stage.

[0054] In the present application, the wheat may be the wheat variety Fielder.

[0055] In the present invention, the term "salt tolerance" refers to the ability of plants to adapt to and resist high salt stress. Specifically, it can be reflected in the effects of high salt stress on various indicators, including but not limited to photosynthesis, respiration, transpiration, osmotic regulation substance content, antioxidant system, plant hormones, cell ultrastructure, survival rate and / or biomass.

[0056] In the present invention, the term "use in improving the heat tolerance of wheat plants" includes but is not limited to preparing products for improving the heat tolerance of wheat plants, improving the heat tolerance of wheat plants and / or wheat plant breeding.

[0057] In the present invention, the terms "high salt stress" and "salt stress" have the same meaning and refer to a salt concentration that produces a harmful effect on plants.

[0058] In the above, high salt tolerance means higher survival rate and biomass under salt stress conditions.

[0059] In the present application, the salt stress may be 200 mM NaCl. The duration of the salt stress may be 7 days.

[0060] In the above, the wheat may be wheat in the seedling stage. The seedling stage is 7-14 days of growth. The 7-14 days of growth may be calculated from sowing.

[0061] In a fourth aspect, the present invention provides a kit.

[0062] The kit comprises the above-mentioned nucleic acid molecule and / or expression cassette and / or recombinant vector and / or recombinant microorganism and / or transgenic plant cell line and / or transgenic plant tissue and / or transgenic plant organ.

[0063] Beneficial Effects

[0064] The present invention discloses a wheat salt-tolerant protein TaST1 and its encoding gene and application. Specifically disclosed are the application of the protein, a substance that inhibits or reduces or down-regulates the expression of the encoding gene of the protein, or a substance that inhibits or reduces or down-regulates the activity or content of the protein in any of the following: A1) application in improving the salt tolerance of wheat plants and / or application in preparing products for improving the salt tolerance of wheat plants; A2) application in improving the survival rate of wheat plants under salt stress and / or application in preparing products for improving the survival rate of wheat plants under salt stress; A3) application in improving the biomass of wheat plants under salt stress and / or application in preparing products for improving the biomass of wheat plants under salt stress; plants with TaST1 protein encoding gene knocked out have significantly higher salt tolerance than wild-type plants and can be used for industrial production and plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The phenotype diagrams of the wild-type Fileder, KO#1 and KO#2 growing under salt stress and normal conditions. There is no significant difference in the growth of Fielder, KO#1 and KO#2 under normal conditions. Under salt stress, Fielder shows a salt-sensitive phenotype compared with KO#1 and KO#2.

[0066] Figure 2 This is a bar graph of the survival rates of wild-type Fileder, KO#1 and KO#2. The survival rate of Fielder under salt stress was significantly lower than that of KO#1 and KO#2.

[0067] Figure 3 The bar graph shows the root length of wild-type Fileder, KO#1 and KO#2. There is no significant difference in the root length between Fielder, KO#1 and KO#2 under normal conditions, but the root length of Fielder is significantly lower than that of KO#1 and KO#2 under salt stress. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0069] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0070] The following examples used SPSS11.5 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0071] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0072] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer regions) that are involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as intervening sequences (introns) between individual coding regions (exons).

[0073] The terms "include", "including", "have", "contain" and the like are all open terms, meaning including but not limited to.

[0074] The term "nucleic acid" refers to a polymer that exists in a single or double-stranded form and comprises at least two deoxynucleotides or nucleotides. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise noted, a specific nucleic acid sequence also implicitly encompasses variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences and sequences that are clearly noted. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassolet al. (1992); Rossolin et al., Mol. Cell. Probes 8:91-98 (1994)). "Nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked by the phosphate group. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that substitute new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and halogenated alkyls. The DNA may be present as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, products of polymerase chain reaction (PCR), vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The term nucleic acid is used interchangeably with gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules.

[0075] The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear, cyclic or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0076] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0077] The terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0078] When the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the local homology algorithm of Pearson et al. (1988) P. The similarity search method of Proc. Natl. Acad. Sci. 85: 2444; Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAS T-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0079] The term "expression cassette" refers to a DNA capable of expressing a target protein or nucleic acid in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing a nucleic acid molecule of any of the above proteins. The regulatory sequence can direct the coding sequence to express any of the above proteins in a suitable host cell under compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence includes a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation of the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence attached to the amino terminus of the protein that can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory systems are those that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can amplify genes. In these cases, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequences.

[0080] The term "promoter" is used according to its typical meaning to refer to a DNA sequence that regulates and / or initiates transcription of RNA from the DNA sequence.

[0081] Suitably, the promoter is operable in mammalian cells, such as human cells. The promoter may be capable of driving transgene expression in mammalian cells, such as human cells. The promoter may be a mammalian promoter, such as a human promoter.

[0082] The term "vector" generally refers to a vector that can carry foreign DNA or target gene into host cells for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into host cells by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cells. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cells. The vector includes, but is not limited to, plasmid, phage (e.g., lambda phage or M13 phage), cosmid, phagemid, shuttle vector (e.g., yeast expression vector), Ti plasmid, artificial chromosome (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC), or Ti plasmid artificial chromosome (TAC)), viral vector (e.g., baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), herpesvirus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequence, transcription initiation sequence, enhancer sequence, selection element, and reporter gene. In addition, the vector may also contain a replication initiation site.

[0083] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from Escherichia sp. (such as Escherichia coli), Erwinia sp., Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp. and Bacillus sp. (such as Bacillus), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from Saccharomyces sp. (such as Saccharomyces cerevisiae, methanol yeast, Pichia pastoris), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp., etc. The actinomycetes may be from Streptomyces sp. (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp., etc.

[0084] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like plant cells but are not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), Chinese hamster ovary cell substrain (CHO-K1 cell), African green monkey kidney cell (Vero cell), SV40-transformed African green monkey kidney cell (COS cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc., but are not limited thereto.

[0085] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. The recombinant vector can be constructed in any suitable manner as long as it can carry the exogenous target gene into the recipient cell.

[0086] It can be used to transfer genes into recipient cells and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cells.

[0087] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0088] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0089] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0090] Example 1. Acquisition and identification of transgenic plants

[0091] Obtaining TaST1 transgenic knockout wheat

[0092] 1. TaST1 knockout primers:

[0093] TaST1-p414-F:aataatggtctcAAGCgGGGGCTGCTGTACGACCAG;

[0094] TaST1-p414-F0:gGGGGCTGCTGTACGACCAG gttttagagctagaaatagc;

[0095] TaST1-p414-R0: CACCTCCGCTTCTTTCCACGCTTCTTGGTGCC;

[0096] TaST1-p414-R:ATTATTGGTCTCTAAACCACCTCCGCTTCTCTCCA.

[0097] 2. Using plasmid pMT1T2 as a template, use the above 4 primers to amplify the DNA fragment containing the vector linker and the target, and gel recovery and purification to obtain the PCR product. The pMT1T2 plasmid is recorded in the following literature: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, & Chen, QJ (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC plant biology, 14, 327.3, and its name in the literature is pCBC-MT1T2.

[0098] 3. Use Bsa1 to digest the vector pBUE411 and the PCR product in step 2, and ligate them overnight using T4 ligase to transform Escherichia coli Mach1-T1.

[0099] The vector pBUE411 is described in the following document: Xing, HL, Dong, L., Wang, ZP, Zhang, HY, Han, CY, Liu, B., Wang, XC, & Chen, QJ (2014). A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC plant biology, 14, 327. https: / / doi.org / 10.1186 / s12870-014-0327-y, and its name in the document is pBUE411.

[0100] 4. Screen positive clones by PCR reaction and sequence them. The correctly sequenced clones are named pBUE411-TaST1 knockout vector. The pBUE411-TaST1 knockout vector is obtained by opening the restriction endonuclease BsaI recognition site of the pBUE411 vector, replacing the sequence between fragment 1 (5'-TGCAGATGATCCGTGGC-3') and fragment 2 (5'-ATTTCTAGCTCTAAAAC-3') of the pBUE411 vector with a DNA fragment of the sequence MT1T2 with a target linker (sequence 8), while keeping other sequences of the pBUE411 vector unchanged. The resulting recombinant vector is named pBUE411-TaST1 knockout vector (also known as pBUE411-TaST1 knockout vector or pBUE411-TaST1 knockout plasmid or knockout plasmid pBUE411-TaST1 or knockout vector pBUE411-TaST1). At the same time, the pBUE411-TaST1 knockout vector was extracted and transformed into Agrobacterium GV3101, and then sent to the wheat transgenic platform for genetic transformation of wheat (the recipient plant was the wheat Fielder variety).

[0101] Sequence 8 is as follows:

[0102]

[0103] At the same time, the pBUE411-TaST1 knockout vector was transformed into Agrobacterium EHA105 and sent to the wheat transgenic platform for wheat (the recipient wheat was the Fielder variety) genetic transformation. 0 Six transgenic wheat plants were self-pollinated to obtain TaST1 gene knockout T 2 The homozygous transgenic wheat lines were named KO#1 and KO#2.

[0104] 5. For the T 0 PCR was performed in the plants of the next generation and sequenced for identification.

[0105] To T 0 Samples were taken from the first-generation plants and DNA was extracted. Specific primers for three subgenomes were designed near the target site, and amplified for first-generation sequencing to determine whether the vicinity of the target site was edited and changed.

[0106] The detection primers are:

[0107] TaST1-A-CRI-F:GTGGCATTTGTCCCAGAC;

[0108] TaST1-A-CRI-R:AGGCCTGACTAGAGCGATC;

[0109] TaST1-B-CRI-F:CAGCTATAGTTGTGCCGTGG;

[0110] TaST1-B-CRI-R:TGGAGGTCTGACTAGAGCGATA;

[0111] TaST1-D-CRI-F:GGCATTTGTCTTAGACTTAAACG;

[0112] TaST1-D-CRI-R:GTCAGCACTCAGGATCGG,

[0113] 6. After the positive strains were harvested, they were grown in the greenhouse and sequenced by PCR. 1 The T1 generation plants were selected for further editing and planted and identified, and a total of 2 TaST1 knockout T 2 The homozygous strain, TaST1-KO1 T 2 Homozygous strains and TaST1-KO2 T 2 The homozygous strain was generated. 2 The plants of the first generation were self-pollinated to obtain T 3 Generation seeds (also known as TaST1 knockout T 3homozygous seeds of the same generation), namely TaST1-KO1 T 3 Generation of homozygous seeds and TaST1-KO1 T 3 Generation of homozygous seeds.

[0114] TaST1-KO1 T 2 The genome of the homozygous plants of the first generation contained the following mutations:

[0115] In TaST1-KO1 T 2 In the homozygous plants of the first generation, the region corresponding to the TaST1 gene in the genome mutated compared to the wheat variety Fielder:

[0116] In the TaST1-A subgenome, an adenine deoxyribonucleotide residue was inserted between positions 119 and 120 of sequence 1 in the sequence list, resulting in a frameshift mutation in the TaST1-A gene and causing the encoded protein (sequence 2) to terminate prematurely at amino acid position 57, thereby knocking out the TaST1-A gene.

[0117] In the TaST1-B subgenome, an adenine deoxyribonucleotide residue is missing between positions 118 and 119 of sequence 3 in the sequence list, resulting in a frameshift mutation in the TaST1-B gene, causing the encoded protein (sequence 4) to terminate prematurely at amino acid position 61, thereby knocking out the TaST1-B gene.

[0118] In the TaST1-D subgenome, an adenine deoxyribonucleotide residue was inserted between positions 119 and 120 of sequence 5 in the sequence list, resulting in a frameshift mutation in the TaST1-D gene and causing the encoded protein (sequence 6) to terminate prematurely at amino acid position 57, thereby knocking out the TaST1-D gene.

[0119] TaST1-KO2 T 2 The genome of the homozygous plants of the first generation contained the following mutations:

[0120] In TaST1-KO2 T 2 In the homozygous plants of the first generation, the region corresponding to the TaST1 gene in the genome mutated compared to the wheat variety Fielder:

[0121] In the TaST1-A subgenome, four deoxyribonucleotide residues are missing between positions 116 and 120 of sequence 1 in the sequence list, that is, one thymine deoxyribonucleotide residue is inserted between positions 66 and 67 of sequence 1 in the sequence list, resulting in a frameshift mutation in the TaST1-A gene, causing the encoded protein (sequence 2) to terminate prematurely at amino acid position 57, thereby knocking out the TaST1-A gene.

[0122] In the TaST1-B subgenome, an adenine deoxyribonucleotide residue is missing between positions 118 and 119 of sequence 3 in the sequence list, resulting in a frameshift mutation in the TaST1-B gene, causing the encoded protein (sequence 4) to terminate prematurely at amino acid position 61, thereby knocking out the TaST1-B gene.

[0123] In the TaST1-D subgenome, an adenine deoxyribonucleotide residue was inserted between positions 119 and 120 of sequence 5 in the sequence list, resulting in a frameshift mutation in the TaST1-D gene and causing the encoded protein (sequence 6) to terminate prematurely at amino acid position 57, thereby knocking out the TaST1-D gene.

[0124] 5. After the positive strains (KO#1 and KO#2) were harvested, they were grown in the greenhouse and sequenced by PCR. 3 The plants were positively identified.

[0125] Sequence 1 is as follows:

[0126]

[0127] sequence 2 as follows:

[0128] MAAAAGTSRQAGAAPRVGLLYDQRMLKHAPAGKKERETPERLRAIWRKLAAEGVTSRCVGMRAKEAKEKYIASVHGPNHIDLIRNISSKDSSERKNTAEKLDSIYFNKGSSESAFLAAGSVIEVAEKVAAGELSSAIALVRPPGHHAEHSKPMGFCLFNNVAIAANYLLNERPDLGISKILIVDWDVHHGNGTQNMFYN DPRVLFFSVHRYDDGSFYPYEADASHVFIGDETGRGYNINVPWEHAECGDADYVAAWDHVLLPVAEAFDPDIILLSAGFDAALGDDMGNCCITPNGYALLLTKLLGFAKGRIVMALEGGYNPESIANSVCACAKVLLGDKFTLTSPEMQPFESTWRVIQMVRDELKTYWPVLSSKLPENVSLRSTPSYIQPCTSSGSE。

[0129] sequence 3 as follows:

[0130]

[0131] sequence 4 specific as follows:

[0132] MAAAAGTARQEGAAPRVGLLYDQRMLAHAPALNTEKETPERLRAIWRKLAAEGVTSRCV

[0133] GMRAKEAKEKYIASVHARKHVDLIRKISSKDSSDLKKTAKKFDSVYFSKGSSESAFLAAGS

[0134] VIEVAEKVAAGELSSAIALVRPPGHHAEHSHPMGYCLFNNVAIAANYLLNERPDLGINKILIV

[0135] DWDVHHGNGTQNMfyNDPRVLFFSVHRYGYGSFYPYEADASHVFIGDETGRGYNINVPW

[0136] EHAKCGDADYVAAWDHVLLPVAEFDPDIILLSAGFDAAALGDDMGDCCITPNGYALLLTKL

[0137] LGFAKGRIVMALEGGYNPESIANSVCACAKVLLGDKFTNLSPEMQPFESTWRVIQMVRDELKTYWPVLSSKLPENVSLRSTPSYIQK。

[0138] sequence 5 as follows:

[0139]

[0140] The specific sequence 6 is as follows:

[0141] MAAAAGTARQAGAAPRVGLLYDLRMLAHAPAGKKEKETPERLRAIWRKLAAEGVTSRCVGMRAKVAKEKYIASVHARKHVDLIRKISSKDSSDLKKTAKKFDSVYFSKGSSESAFLAAGSVIEVAEKVAAGELSSAIALVRPPGHHAEHSKPMGFCLFNNVAIAANYLLNERPDLGINKILIVDWDVHHGNGTQNMFYSDPRVLFFSVHRYDYGRFYPYEADASHVFIGDETGRGYNINVPWEHAKCGDADYVAAWDHVLLPVAEAFDPDIILLSAGFDAARGDHMGDCCITPNGYALLLTKLLGFAKGRIVMALEGGYNPESIANSVCACAKVLLGDKFTLNSPEMQPFESTWRVIQMVRDELKAYWPVLSSKLPENVSLRSTPLSLRSTPSY。

[0142] The specific sequence 7 is as follows:

[0143] MAAAAGTXRQXGAAPRVGLLYDXRMLXHAPAXXXEXETPERLRAIWRKLAAEGVTSRCVGMRAKXAKEKYIASVHXXXHXDLIRXISSKDSSXXKXTAXKXDSXYFXKGSSESAFLAAGSVIEVAEKVAAGELSSAIALVRPPGHHAEHSXPMGXCLFNNVAIAANYLLNERPDLGIXKILIVDWDVHHGNGTQNMFYXDPRVLFFSVHRYXXGXFYPYEADASHVFIGDETGRGYNINVPWEHAXCGDADYVAAWDHVLLPVAEAFDPDIILLSAGFDAAXGDXMGXCCITPNGYALLLTKLLGFAKGRIVMALEGGYNPESIANSVCACAKVLLGDKFTLXSPEMQPFESTWRVIQMVRDELKXYWPVLSSKLPENVSLRSTP. X is any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, O, S, U, T, W, Y, or V.

[0144] Example 2 Identification of the salt tolerance phenotype of transgenic plants

[0145] In the following, TaST1-KO1 T 3 The homozygous seed seedlings of the generation are TaST1-KO1 T 3 Generation of seedlings grown from homozygous seeds. 3 The homozygous seed plants of the first generation are TaST1-KO1 T 3 Plants grown from homozygous seeds. 3 The homozygous seed seedlings of the generation are TaST1-KO2 T 3 Generation of seedlings grown from homozygous seeds. 3 The homozygous seed plants are plants grown from TaST1-KO2 T3 homozygous seeds.

[0146] 200mM NaCl solution: calcium nitrate 945mg / L, potassium nitrate 607mg / L, ammonium phosphate 115mg / L, magnesium sulfate 493mg / L, iron salt solution 2.5ml / L, sodium chloride 11.7g / L, and the rest is water.

[0147] Hoagland nutrient solution: calcium nitrate 945mg / L, potassium nitrate 607mg / L, ammonium phosphate 115mg / L, magnesium sulfate 493mg / L, iron salt solution 2.5ml / L, and the rest is water.

[0148] 1. Experiment on identification of salt tolerance phenotype of TaST1 knockout wheat

[0149] Seeds of two knockout wheat lines TaST1-KO1 and TaST1-KO2 (KO#1 and KO#2) that have been confirmed to be pure lines were selected (TaST1-KO1 T 3 Generation of homozygous seeds and TaST1-KO2 T 3 The seeds of the first generation homozygous seeds) and the control wild-type Fielder were sterilized with 1% hydrogen peroxide for 10 min, washed 3-4 times with distilled water, placed in a culture dish, covered with two layers of filter paper, added with a small amount of distilled water, and left at room temperature for 48 h.

[0150] Select seedlings with consistent germination (seed roots about 1 cm) and transplant them into a culture box filled with Hoagland nutrient solution. Two planting methods: First, each pot is divided into two parts, one part is control seeds (seed seedlings of wild-type Fielder), and the other part is TaST1-KO1 T 3 The homozygous seed seedlings were obtained by planting box 1; 2. Each pot was divided into two parts, one part was control seeds (wild-type Fielder seed seedlings), and the other part was TaST1-KO2 T 3 The homozygous seed seedlings were then obtained to plant box 2. There were four replicates in total.

[0151] The experiment was divided into a salt tolerance experimental group and a control group.

[0152] The salt tolerance experimental group underwent the following operations:

[0153] After planting in the culture boxes (randomly selecting planting box 1 and planting box 2), they were grown under Hoagland nutrient solution for 7 days (22°C-18°C, 16 hours of light / 8 hours of darkness, 60%-70% humidity conditions), and then the salt tolerance of the seedlings was identified:

[0154] Planting box 1 and planting box 2 were placed in 200 mM NaCl solution for 7 days (at 22°C-18°C, 16 h light / 8 h dark), and then transferred to Hoagland nutrient solution for recovery for 7 days (at 22°C-18°C, 16 h light / 8 h dark, 60%-70% humidity). The phenotypes were recorded by taking photos, and the survival rate and root length were calculated.

[0155] The control group underwent the following operations:

[0156] After planting in the culture boxes (planting box one and planting box two were randomly selected), the whole process was cultured in Hoagland nutrient solution (22℃-18℃, 16 hours light / 8 hours dark, 60%-70% humidity conditions). When the salt-tolerant experimental group recovered for 7 days, they were photographed together with it to calculate the survival rate and root length.

[0157] Survival rate = number of surviving seedlings / total number of seedlings.

[0158] Survival was determined when the stem remained green, leaves partially or fully turned green, or new leaves unfolded.

[0159] The criteria for determining death are: the leaves turn yellow and die, and the stems completely die with no signs of greening again.

[0160] The root length before and after stress was the length of wheat root below the seed and the length was measured and recorded.

[0161] Phenotypic results Figure 1 As shown ( Figure 1 Fielder in the middle is wild-type wheat Fielder, KO#1 is TaST1-KO1 T 3 Generation of homozygous seedlings, KO#2 is TaST1-KO2 T 3 The normal condition was the control group, and the salt treatment was the salt tolerance experimental group). Under normal conditions, there was no significant difference between the knockout and control lines. However, after salt stress treatment, there were significant differences. The knockout line (TaST1-KO#2 is the offspring of KO#2, i.e., TaST1-KO1 T 3Generation of homozygous seedlings and TaST1-KO2 T 3 The growth and recovery degree of the homozygous seed seedlings of the first generation were significantly better than those of the wild-type plants.

[0162] The survival results were as follows Figure 2 As shown ( Figure 2 The bar graph shows the survival rate of the salt tolerance experimental group, where Fielder is the wild-type wheat Fielder, and KO#1 is TaST1-KO1 T 3 Generation of homozygous seedlings, KO#2 is TaST1-KO2 T 3 The results showed that the knockout line (the offspring of KO#1 and KO#2, namely TaST1-KO1T 3 Generation of homozygous seedlings and TaST1-KO2 T 3 The survival rate of the first generation homozygous seed seedlings was significantly higher than that of the wild-type plants after recovery.

[0163] The root length statistics are as follows Figure 3 As shown ( Figure 3 In the figure, the bar graph shows root length, Normal condition is the control group, Salt treatment is the salt tolerance experimental group, Fielder is the wild type wheat Fielder, KO#1 is TaST1-KO1 T 3 Generation of homozygous seedlings, KO#2 is TaST1-KO2 T 3 The root length of the knockout transgenic plants in the treatment group was significantly higher than that of the wild-type plants ( Figure 2 ). It can be seen that the salt tolerance of the knockout strain is significantly better than that of the wild-type strain, indicating that inhibiting the expression of TaST1 significantly improves the salt tolerance of the plant.

[0164] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a protein, a substance that inhibits, reduces or down-regulates the expression of a gene encoding the protein, or a substance that inhibits, reduces or down-regulates the activity or content of the protein in any of the following; A1) application in improving the salt tolerance of wheat plants and / or application in preparing products for improving the salt tolerance of wheat plants; A2) application in improving the survival rate of Triticum plants under salt stress and / or application in preparing a product for improving the survival rate of Triticum plants under salt stress; A3) application in increasing the biomass of Triticum spp. under salt stress and / or application in preparing a product for increasing the biomass of Triticum spp. under salt stress; The protein is any of the following: B1) contains a protein whose amino acid sequence is the protein shown in SEQ ID NO: 7; B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the protein shown in B1); B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that: The use comprises increasing the survival rate and / or biomass of Triticum plants.

3. The use according to claim 1, characterized in that: The amino acid sequence of the protein is sequence 2, sequence 4 and / or sequence 6.

4. The use according to any one of claims 1 to 3, characterized in that: The substance that inhibits, reduces or downregulates the expression of the gene encoding the protein is any of the following: B1), a nucleic acid molecule that inhibits, reduces or down-regulates the expression of a gene encoding the protein of claim 1 or 2, or inhibits, reduces or down-regulates the activity or content of the protein; B2), expressing the coding gene of the nucleic acid molecule described in B1); B3), an expression cassette containing the gene described in B2); B4, a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

5. The use according to claim 4, characterized in that: B1) The nucleic acid molecule target gene is a gene encoding the protein shown in Sequence 7.

6. A method for cultivating a highly salt-tolerant Triticum plant, characterized in that: The method comprises inhibiting, reducing or down-regulating the expression level of the gene encoding the protein of any one of claims 1 to 3 in the target wheat plant, and / or the activity and / or content of the protein to obtain a highly salt-tolerant wheat plant, wherein the highly salt-tolerant wheat plant has higher salt tolerance than the target wheat plant.

7. A method for increasing the survival rate and / or biomass of Triticum plants under salt stress conditions, characterized in that: The method comprises inhibiting, reducing or down-regulating the expression of a gene encoding a protein described in any one of claims 1 to 3 in a wheat plant, and / or increasing the survival rate and / or biomass of a wheat plant under salt stress conditions.

8. The method according to claim 5 or 6, characterized in that The inhibition, reduction or down-regulation of the expression of the gene encoding the protein of any one of claims 1 to 3 in the plant comprises introducing the nucleic acid molecule described in B1) of claim 4, the encoding gene described in B2), the expression cassette described in B3) or the recombinant vector described in B4) into the target plant.

9. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8, characterized in that: The plant is wheat.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 8, characterized in that: The wheat plant is a wheat plant in the seedling stage.

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