wheat salt-tolerant protein TaST1, its encoding gene, and its applications

Abstract

This invention discloses the wheat salt-tolerant protein TaST1, its encoding gene, and its applications. Specifically, it discloses the application of the protein, substances that inhibit, reduce, or downregulate the expression of the gene encoding the protein, or substances that inhibit, reduce, or downregulate the activity or content of the protein in any of the following: A1) application in improving the salt tolerance of wheat species and / or application in the preparation of products that improve the salt tolerance of wheat species; A2) application in improving the survival rate of wheat species under salt stress and / or application in the preparation of products that improve the survival rate of wheat species under salt stress; A3) application in improving the biomass of wheat species under salt stress and / or application in the preparation of products that improve the biomass of wheat species under salt stress; plants with the TaST1 protein encoding gene knocked out exhibit significantly higher salt tolerance than wild-type plants and can be used for industrial production and plant breeding.

Description

Technical Field

[0001] This invention specifically relates to wheat salt-tolerant protein TaST1, its encoding gene, and its applications. Background Technology

[0002] Soil salinization severely restricts agricultural production. Wheat is one of the most widely cultivated crops globally, providing humans with approximately 20% of their protein and carbohydrate intake. As one of my country's main staple crops, wheat has relatively poor salt tolerance. However, due to industrial pollution, excessive use of chemical fertilizers, and improper irrigation, soil salinization is becoming increasingly prevalent in many arable lands. Soil salinization has become a global ecological problem, severely reducing usable land resources and causing significant losses to wheat production. Therefore, identifying superior salt-tolerant genes in wheat and elucidating their molecular mechanisms and genetic mechanisms regulating salt tolerance is of great significance for breeding new, stress-resistant, and high-yielding wheat varieties. Summary of the Invention

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

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

[0005] The use of a protein, a substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein, or a substance that inhibits, reduces, or downregulates the activity or content of the protein in any of the following.

[0006] A1) Application in improving the salt tolerance of wheat species and / or application in the preparation of products that improve the salt tolerance of wheat species;

[0007] A2) Application in improving the survival rate of wheat plants under salt stress and / or application in the preparation of products that improve the survival rate of wheat plants under salt stress;

[0008] A3) Application in improving the biomass of wheat under salt stress and / or application in the preparation of products that improve the biomass of wheat under salt stress;

[0009] The protein is any one of the following:

[0010] B1) A protein whose amino acid sequence is the same as that shown in Sequence 7;

[0011] B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues.

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

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

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

[0015] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

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

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

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

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

[0020] In the above text, the biomass may refer to root length.

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

[0022] The wheat mentioned above may be the wheat variety Fielder.

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

[0024] In the above text, the application includes improving the survival rate and / or biomass of wheat plants.

[0025] In the above text, the biomass refers to root length. In a specific embodiment, the root length refers to the length of the underground portion.

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

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

[0028] B1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of genes encoding the aforementioned proteins, or inhibit, reduce, or downregulate the activity or content of the aforementioned proteins;

[0029] B2) Expresses the gene encoding 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) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms 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] In the nucleic acid molecule described in B1), those skilled in the art can easily mutate the nucleotide sequence encoding the protein TaST1 of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more of the same nucleotide sequence as the protein TaST1 isolated in the present invention, as long as they encode and function as protein TaST1, are all derived from and equivalent to the nucleotide sequence of the present invention.

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

[0038] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0039] In this document, the vectors described are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0040] In the aforementioned biological materials, the expression cassette described in B3) refers to DNA capable of expressing the gene in a host cell. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this 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; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline inducible promoter (US Patent 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 beta-conglycin, napin, oleosin and soybean beta-conglycin (Beachy et al. (1985) EMBOJ.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited here are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: 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 B4) above, the recombinant vector can be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can 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 constructing a recombinant expression vector using TaST1-A, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. As a specific embodiment, this application uses the pBUE411 vector as the expression vector.

[0042] In the above applications, the nucleic acid target gene described in B1) is the gene encoding the protein shown in sequence 7.

[0043] In the above text, the nucleic acid molecular target gene may be the coding gene of the protein shown in sequence 2, sequence 4 and / or sequence 6.

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

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

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

[0047] Secondly, the present invention provides a method for cultivating highly salt-tolerant wheat plants.

[0048] The method includes inhibiting, reducing, or downregulating the expression level of the gene encoding any of the proteins described above in the target wheat plant, and / or, the activity and / or content of the proteins to obtain a salt-tolerant wheat plant, wherein the salt tolerance of the salt-tolerant wheat plant is higher than that of the target wheat plant.

[0049] Thirdly, the present invention provides a method for improving the survival rate and / or biomass of wheat plants under salt stress.

[0050] The method includes improving the survival rate and / or biomass of wheat plants under salt stress by inhibiting or reducing or downregulating the expression of genes encoding the proteins described above in wheat plants, and / or the activity and / or content of any of the proteins described above.

[0051] In the above methods, inhibiting, reducing, or downregulating the expression of the gene encoding any of the proteins described above in the plant includes introducing the nucleic acid molecule described in B1), the gene encoding the protein described in B2), the expression cassette described in B3), or the recombinant vector described in B4) into the target plant.

[0052] The plant wheat as described in any of the above applications or methods.

[0053] In any of the applications or methods described above, the wheat plant is a seedling wheat plant.

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

[0055] In this invention, the term "salt tolerance" refers to a plant's ability 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 content, antioxidant systems, plant hormones, cell ultrastructure, survival rate, and / or biomass.

[0056] In this invention, the term "use in improving the heat resistance of wheat plants" includes, but is not limited to, the preparation of products that improve the heat resistance of wheat plants, the improvement of the heat resistance of wheat plants, and / or wheat plant breeding.

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

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

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

[0060] In the above text, the wheat mentioned can be seedling wheat. The seedling stage is 7-14 days of growth. The 7-14 days of growth can be counted from sowing.

[0061] Fourthly, the present invention provides a reagent kit.

[0062] The kit includes the above-mentioned nucleic acid molecules and / or expression cassettes and / or recombinant vectors and / or recombinant microorganisms and / or transgenic plant cell lines and / or transgenic plant tissues and / or transgenic plant organs.

[0063] Beneficial effects

[0064] This invention discloses the wheat salt-tolerant protein TaST1, its encoding gene, and its applications. Specifically, it discloses the application of the protein, substances that inhibit, reduce, or downregulate the expression of the gene encoding the protein, or substances that inhibit, reduce, or downregulate the activity or content of the protein in any of the following: A1) application in improving the salt tolerance of wheat species and / or application in the preparation of products that improve the salt tolerance of wheat species; A2) application in improving the survival rate of wheat species under salt stress and / or application in the preparation of products that improve the survival rate of wheat species under salt stress; A3) application in improving the biomass of wheat species under salt stress and / or application in the preparation of products that improve the biomass of wheat species under salt stress; plants with the TaST1 protein encoding gene knocked out exhibit significantly higher salt tolerance than wild-type plants and can be used for industrial production and plant breeding. Attached Figure Description

[0065] Figure 1 Phenotypic diagrams of wild-type Fielder, KO#1, and KO#2 under salt stress and normal conditions; Fielder showed no significant difference in growth compared to KO#1 and KO#2 under normal conditions, but under salt stress, Fielder exhibited a salt-sensitive phenotype compared to KO#1 and KO#2.

[0066] Figure 2 Bar chart showing the survival rates of wild-type Fielder, KO#1, and KO#2; Fielder's survival rate under salt stress was significantly lower than that of KO#1 and KO#2.

[0067] Figure 3 The bar chart shows the root length of wild-type Fielder, KO#1, and KO#2. Under normal conditions, there was no significant difference in root length between Fielder and KO#1 and KO#2. Under salt stress, the root length of Fielder was significantly lower than that of KO#1 and KO#2. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0070] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0071] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0072] The term “gene” refers to a segment of DNA involved in the production of a polypeptide chain; it includes regions before and after the coding region (leader and tail regions) involved in the transcription / translation of the gene product and the regulation of said transcription / translation, as well as insertion sequences (introns) between individual coding regions (exons).

[0073] The terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0074] The term "nucleic acid" refers to a polymer consisting of at least two deoxynucleotides or nucleotides, existing in single or double strands. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, having similar binding properties to reference nucleic acids, and being metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsukae et al., J. Biol. Chem. 260: 2605-2608 (1985); and Cassole et al. (1992); Rossolinie et al., Mol. Cell. Probes 8: 91-98 (1994)). A “nucleotide” contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, further including natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that replace new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates (esters), and alkyl halides. DNA can exist as antisense, plasmid DNA, portions of plasmid DNA, pre-compressed DNA, polymerase chain reaction (PCR) products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. The terms nucleic acid, gene, cDNA, mRNA encoded by a gene, and interfering RNA molecules may be used interchangeably.

[0075] The terms “polypeptide,” “peptide,” “protein,” and “protein protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes modified amino acid polymers, such as those modified by sulfation, glycosylation, esterification, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, isopreneation, racemization, selenoylation, transfer-RNA-mediated amino addition such as arginination, ubiquitination, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “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 construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0077] The terms “optional,” “optional,” or “optional” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0078] The term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule & Lar 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; and the local homology algorithm of Pearson et al. (1988) P… Similarity search methods are described in roc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAS. T-2 (AltschμL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, MountainView, California.

[0079] The term "expression cassette" refers to 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 the regulatory sequences necessary for the expression of any of the aforementioned proteins. These regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence 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 mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

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

[0081] Suitable of the promoter, it is operable in mammalian cells, such as human cells. The promoter can drive transgene expression in mammalian cells, such as human cells. The promoter can be a mammalian promoter, such as a human promoter.

[0082] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0083] The term "microorganism" typically includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria mentioned can be from genera such as *Escherichia* sp. (e.g., *Escherichia coli*), *Erwinia* sp., *Agrobacterium* sp. (e.g., *Agrobacterium tumefaciens*), *Flavobacterium* sp., *Alcaligenes* sp., *Pseudomonas* sp., and *Bacillus* sp. (e.g., *Bacillus*). The viruses mentioned can include rotaviruses, baculoviruses, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, papillomaviruses (e.g., SV40), and herpesviruses (e.g., herpes simplex virus). The fungi may originate from genera such as *Saccharomyces* sp. (e.g., *Saccharomyces cerevisiae*, *Methanolac*, *Pichia pastoris*), *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp. The actinomycetes may originate from genera such as *Streptomyces* sp. The algae may originate from phyla such as *Cyanophyta* (e.g., cyanobacteria), genera such as *Fucus* sp., *Achnanthes* sp., *Amphiprora* sp., *Amphora* sp., *Ankistrodesmus* sp., *Asteromonas* sp., and *Boekelovia* sp. .

[0084] The term "host cell," also known as the recipient cell, generally refers to any type of cell that can be used to introduce a vector, such as plant and animal cells. The term "host cell" can be understood not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, may not necessarily be identical to the original parent cell but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), and amphibian cells (e.g., Xenopus laevis cells or Andrias davidianus cells). These include, but are not limited to, davidianus cells, fish cells (e.g., grass carp, carp, rainbow trout, or catfish cells), insect cells (e.g., Sf21 cells, Sf-9 cells, or Hi-5 cells).

[0085] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating a foreign 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 foreign target gene into the recipient cell.

[0086] The goal is to provide the recipient cells with the ability to replicate, integrate, amplify, and / or express the exogenous target gene.

[0087] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign 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 whose genes have been manipulated and modified to obtain a functionally altered microorganism. This can be achieved by introducing a foreign target gene or recombinant vector into the target microorganism, or by directly editing the endogenous genes of the target microorganism.

[0089] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to obtain a recombinant host cell with altered function. This can include introducing a foreign target gene or recombinant vector into the host cell, or directly editing the host cell's endogenous genes.

[0090] Example 1: Obtaining and Identifying 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, the DNA fragment containing the vector adapter and target site was amplified using the above four primers, and purified by gel extraction to obtain the PCR product. The pMT1T2 plasmid is described 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 this literature is pCBC-MT1T2.

[0098] 3. The vector pBUE411 was digested with Bsa1 enzyme and PCR product from step 2 was ligated overnight with T4 ligase and transformed into Escherichia coli Mach1-T1.

[0099] The vector pBUE411 is described 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. https: / / doi.org / 10.1186 / s12870-014-0327-y, where it is named pBUE411.

[0100] 4. Screen positive clones by PCR reaction, sequence them, and name the correctly sequenced clones as pBUE411-TaST1 knockout vector. The pBUE411-TaST1 knockout vector is obtained by opening the BsaI restriction endonuclease 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 containing the target adapter sequence MT1T2 (sequence 8), while keeping the other sequences of the pBUE411 vector unchanged. The resulting recombinant vector is named pBUE411-TaST1 knockout vector (also known as pBUE411-TaST1 knockout vector, pBUE411-TaST1 knockout plasmid, or knockout plasmid pBUE411-TaST1 or knockout vector pBUE411-TaST1). Simultaneously, the pBUE411-TaST1 knockout vector was extracted, transformed into Agrobacterium GV3101, and 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] Simultaneously, the pBUE411-TaST1 knockout vector was transformed into Agrobacterium EHA105 and sent to a wheat transgenic platform for genetic transformation of wheat (the recipient wheat variety was Fielder). Six TaST1 gene knockout T0 generation transgenic wheat plants were obtained, and they were self-crossed to obtain TaST1 gene knockout T2 generation homozygous transgenic wheat plants, which were named KO#1 and KO#2.

[0104] 5. Perform PCR and sequencing identification on the obtained T0 generation plants.

[0105] DNA was extracted from T0 generation plants, and specific primers for three subgenomes were designed near the target site. The DNA was then amplified and sequenced to determine whether the area near the target site had been edited or altered.

[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 harvesting the identified positive lines, they were further propagated in a greenhouse, and the T1 generation plants were identified using PCR sequencing. Further edited T1 generation plants were selected, planted, and identified, resulting in two TaST1 knockout T2 generation homozygous lines: TaST1-KO1 and TaST1-KO2. The T2 generation plants were self-pollinated to obtain T3 generation seeds (also known as TaST1 knockout T3 generation homozygous seeds): TaST1-KO1 and TaST1-KO1 T3 generation homozygous seeds.

[0114] The genomes of homozygous TaST1-KO1 T2 generation plants contain the following mutations:

[0115] In TaST1-KO1 T2 generation homozygous plants, a mutation occurred in the region of the TaST1 gene in the genome compared to the wheat variety Fielder:

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

[0117] In the TaST1-B subgenome, a frameshift mutation occurs between positions 118 and 119 of sequence 3 in the sequence listing, resulting in premature termination of the encoded protein (sequence 4) at amino acid 61, thus knocking out the TaST1-B gene.

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

[0119] The genome of the TaST1-KO2 T2 generation homozygous plants contains the following mutations:

[0120] In TaST1-KO2 T2 generation homozygous plants, a mutation occurred in the region of the TaST1 gene in the genome compared to the wheat variety Fielder:

[0121] In the TaST1-A subgenome, four deoxyribonucleotide residues are deleted between positions 116 and 120 of sequence 1 in the sequence listing. In other words, one thymine deoxyribonucleotide residue is inserted between positions 66 and 67 of sequence 1 in the sequence listing. This causes a frameshift mutation in the TaST1-A gene, which results in the premature termination of the encoded protein (sequence 2) at amino acid position 57, thereby knocking out the TaST1-A gene.

[0122] In the TaST1-B subgenome, a frameshift mutation occurs between positions 118 and 119 of sequence 3 in the sequence listing, resulting in premature termination of the encoded protein (sequence 4) at amino acid 61, thus knocking out the TaST1-B gene.

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

[0124] 5. After harvesting the identified positive lines (KO#1 and KO#2), the plants were multiplied in a greenhouse, and the T3 generation plants were identified positively by PCR sequencing.

[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 text, "TaST1-KO1 T3 generation homozygous seedlings" refers to seedlings grown from TaST1-KO1 T3 generation homozygous seeds. "TaST1-KO1 T3 generation homozygous seed plants" refers to plants grown from TaST1-KO1 T3 generation homozygous seeds. "TaST1-KO2 T3 generation homozygous seedlings" refers to seedlings grown from TaST1-KO2 T3 generation homozygous seeds. "TaST1-KO2 T3 generation homozygous seed plants" refers to plants grown from TaST1-KO2 T3 generation 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, the remainder is water.

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

[0148] 1. TaST1 knockout wheat salt tolerance phenotype identification experiment

[0149] Seeds of two lines of knockout wheat material, TaST1-KO1 and TaST1-KO2 (KO#1 and KO#2), which have been identified as pure lines, were selected (TaST1-KO1 T3 generation homozygous seeds and TaST1-KO2 T3 generation homozygous seeds), as well as seeds of wild-type Fielder control. The seeds were disinfected with 1% hydrogen peroxide for 10 min, washed 3-4 times with distilled water, placed in petri dishes with two layers of filter paper, a small amount of distilled water added, and left at room temperature for 48 h.

[0150] Select seedlings with uniform germination and transplant them into culture boxes containing Hogland's nutrient solution. Two planting methods were used: 1. Each pot was divided into two parts: one part containing control seeds (wild-type Fielder seedlings) and the other part containing TaST1-KO1 T3 generation homozygous seedlings, resulting in planting box one; 2. Each pot was divided into two parts: one part containing control seeds (wild-type Fielder seedlings) and the other part containing TaST1-KO2 T3 generation homozygous seedlings, resulting in planting box two. A total of four replicates were used.

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

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

[0153] After being planted in the culture boxes (planting boxes one and two were randomly selected), the seedlings were grown in Hogland nutrient solution for 7 days (22℃-18℃, 16 hours light / 8 hours darkness, 60%-70% humidity). Afterwards, the salt tolerance of the seedlings was assessed.

[0154] Planting boxes 1 and 2 were treated with 200mM NaCl solution for 7 days (at 22℃-18℃, 16 hours of light / 8 hours of darkness), and then transferred to Hogland nutrient solution for 7 days (at 22℃-18℃, 16 hours of light / 8 hours of darkness, 60%-70% humidity). Phenotypic data were taken and the survival rate and root length were recorded.

[0155] The control group underwent the following procedures:

[0156] After being planted in the culture boxes (planting boxes one and two were randomly selected), the plants were cultured in Hogland nutrient solution throughout the process (22℃-18℃, 16 hours of light / 8 hours of darkness, 60%-70% humidity). After the salt-tolerant experimental group recovered for 7 days, they were photographed together, and the survival rate and root length were recorded.

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

[0158] The criteria for survival are: the stem remains green, the leaves are partially or completely green again, or new leaves have unfolded.

[0159] The criteria for determining death are: leaves turning yellow and dying, and the stem completely dying with no signs of regrowth.

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

[0161] Phenotypic results are as follows Figure 1 As shown ( Figure 1 In the study, Fielder was a wild-type wheat variety, KO#1 was a homozygous seedling of the TaST1-KO1 T3 generation, KO#2 was a homozygous seedling of the TaST1-KO2 T3 generation, Normal comdition was the control group, and Salt treatment was the salt tolerance experimental group. Under normal conditions, there were no significant differences between the different knockout and control lines. However, after salt stress treatment, significant differences were observed. The knockout lines (TaST1-KO#2 being the offspring of KO#2, i.e., homozygous seedlings of the TaST1-KO1 T3 generation and TaST1-KO2 T3 generation) showed significantly better growth and recovery than the wild-type plants.

[0162] Survival results as follows Figure 2 As shown ( Figure 2In the figure, the bar chart represents the survival rate of the salt tolerance experimental groups. Fielder is the wild-type wheat Fielder, KO#1 is the TaST1-KO1 T3 generation homozygous seedling, and KO#2 is the TaST1-KO2 T3 generation homozygous seedling. The vertical axis represents the survival rate. The results show that the survival rate of the knockout lines (the offspring of KO#1 and KO#2, i.e., the TaST1-KO1 T3 generation homozygous seedlings and the TaST1-KO2 T3 generation homozygous seedlings) was significantly higher than that of the wild-type plants after recovery.

[0163] Root length statistics are as follows Figure 3 As shown ( Figure 3 In the figure, the bar chart represents root length, the normal treatment is the control group, the salt treatment is the salt tolerance experimental group, Fielder is the wild-type wheat Fielder, KO#1 is the TaST1-KO1 T3 generation homozygous seedling, and KO#2 is the TaST1-KO2 T3 generation homozygous seedling. The vertical axis represents root length (cm). The root length of each knockout transgene in the treatment groups was significantly higher than that of the wild-type plants. Figure 2 This indicates that the knockout strain has significantly better salt tolerance than the wild-type strain, suggesting that inhibiting TaST1 expression significantly improves the plant's salt tolerance.

[0164] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein, or a substance that inhibits, reduces, or downregulates the activity or content of the protein, in any of the following; A1) Application in improving the salt tolerance of wheat species and / or application in the preparation of products that improve the salt tolerance of wheat species; A2) Application in improving the survival rate of wheat plants under salt stress and / or application in the preparation of products that improve the survival rate of wheat plants under salt stress; A3) Application in increasing the biomass of wheat under salt stress and / or application in the preparation of products that increase the biomass of wheat under salt stress; The protein is any one of the following: B1) Proteins with amino acid sequences of sequences 2, 4, and 6; B2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1).

2. The application according to claim 1, characterized in that, The applications include improving the survival rate and / or biomass of wheat plants.

3. The application according to claim 1 or 2, characterized in that, The substance that inhibits, reduces, or downregulates the expression of the gene encoding the protein is any one of the following: B1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1 or 2, or inhibit, reduce, or downregulate the activity or content of the protein; B2) Expresses the gene encoding 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) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms 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).

4. The application according to claim 3, characterized in that, B1) The target genes of the nucleic acid molecules are the genes encoding proteins of sequences 2, 4 and 6.

5. A method for cultivating highly salt-tolerant wheat plants, characterized in that, This includes inhibiting, reducing, or downregulating the expression level of the gene encoding the protein of any one of claims 1 or 2 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 salt tolerance of the highly salt-tolerant wheat plant is higher than that of the target wheat plant.

6. A method for improving the survival rate and / or biomass of wheat plants under salt stress, characterized in that, This includes improving the survival rate and / or biomass of wheat plants under salt stress by inhibiting or reducing or downregulating the expression of the gene encoding any of the proteins described in claim 1 or 2 in wheat plants, and / or the activity and / or content of any of the proteins described in claim 1 or 2.

7. The method as described in claim 4 or 5, characterized in that, The inhibition, reduction, or downregulation of the expression of the gene encoding any of the proteins described in claim 1 or 2 in the plant comprises introducing into the target plant the nucleic acid molecule described in claim 3B1), the gene encoding the protein described in claim 3B2), the expression cassette described in claim 3B3), or the recombinant vector described in claim 3B4).

8. The application as described in any one of claims 1-4 or the method as described in any one of claims 5-7, characterized in that, The plant in question is wheat.

9. The application as described in any one of claims 1-4 or the method as described in any one of claims 5-7, characterized in that, The wheat species mentioned are wheat seedlings.

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