Wheat salt-tolerant gene and application thereof

Abstract

The invention discloses a wheat salt-tolerant gene and application thereof. Specifically, the invention discloses a protein TaSGR (SEQ ID NO: 3, 6 and / or 9) and an application of a coding gene of the protein TaSGR in regulation and control of plant salt tolerance and / or plant height. According to the invention, a homozygous gene editing plant of which the TaSGR gene is knocked out is successfully created by utilizing a CRISPR / Cas9 technology. The result shows that after the TaSGR gene is knocked out, the plant height of the wheat is obviously reduced, and the salt tolerance is obviously enhanced. The invention discloses the application of the protein TaSGR and the coding gene thereof for the first time. The salt tolerance of the plant is increased and / or the plant height of the plant is reduced by reducing the content and / or activity of the protein TaSGR (for example, the TaSGR gene is knocked out or silenced). According to the invention, a good gene resource is provided for salt-tolerant breeding of wheat, a new field is developed for application of the TaSGR gene, and the TaSGR gene has important significance for cultivation of novel salt-tolerant wheat.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a wheat salt-tolerant gene and application thereof. Background Art

[0002] Salt stress refers to environmental stress caused by excessive salt content in the soil, which has adverse effects on plant growth and development. As common abiotic stresses, salt stress and saline-alkali stress seriously affect crop yields. Saline-alkali land is an important reserve arable land resource in my country. There are about 1.5 billion mu of saline-alkali land in my country, of which about 500 million mu has the potential for development and utilization. Therefore, identifying important salt-alkali tolerance genes and applying them to molecular design breeding and cultivating salt-alkali tolerant crop varieties will help make full use of saline-alkali land and other inferior land, which is of great significance to ensuring food security.

[0003] wheat( Triticum aestivum L.) is one of the most important food crops in the world, and its planting area, total output and trade volume are among the highest among food crops. However, wheat is difficult to survive in a high-salt environment, and its production is often seriously affected by soil salinization. Soil salinization is a major factor restricting crop production, especially in the context of climate change and seawater intrusion. Breeding salt-tolerant wheat varieties is the best way to effectively utilize saline-alkali land and improve economic benefits. Mining and utilizing salt-tolerant genes are of great practical significance not only for wheat, but also for other crops. Since common wheat is an allohexaploid, including three genomes, A, B and D, and its genome is too large and complex (about 40 times that of the rice genome), the mining of salt-tolerant genes and the analysis of its mechanism have been relatively lagging behind. Screening and identifying new salt-tolerant wheat germplasms and mining new salt-tolerant wheat genes are of great significance to increasing wheat yields, breeding new high-yield and high-quality wheat varieties, and ensuring food security and sustainable development. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to regulate plant salt tolerance and / or plant height. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] In order to solve the above technical problems, the present invention first provides an application of protein, which can be any of the following: A1) Application in regulating plant salt tolerance; A2) Application in regulating plant strains; A3) Application in breeding salt-tolerant plants or plants with altered plant height; A4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height; The protein (named TaSGR) may be protein 1, protein 2 and / or protein 3, wherein: The protein 1 may be named TaSGR-5A, and may be any of the following: B1) a protein having an amino acid sequence of SEQ ID NO: 3; 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 replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:3; B3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2); The protein 2 may be named TaSGR-5B, and may be any of the following: C1) a protein having an amino acid sequence of SEQ ID NO: 6; C2) a protein having more than 80% identity with the protein shown in C1) and having the same function as the protein shown in C1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:6; C3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of C1) or C2); The protein 3 may be named TaSGR-5D, and may be any of the following: D1) a protein having an amino acid sequence of SEQ ID NO: 9; D2) a protein having more than 80% identity with the protein shown in D1) and having the same function as the protein shown in D1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:9; D3) A fusion protein with the same function is obtained by connecting a tag to the N-terminus and / or D-terminus of D1) or D2).

[0006] In the above application, the proteins TaSGR-5A, TaSGR-5B and TaSGR-5D can be derived from wheat ( Triticum aestivum ).

[0007] The connections in B3), C3) and D3) can be directly connected via a peptide bond, or connected via a linker.

[0008] The substitution of the amino acid residues in B2), C2) and D2) may be conservative substitution of the amino acid residues.

[0009] In order to facilitate the separation, purification, detection and / or positioning of the protein in B1), C1) or D1), a label protein can be connected to its amino terminal or carboxyl terminal. The label includes but is not limited to: GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. The use of the label does not change the function of the target protein, and those skilled in the art know how to select a suitable label protein according to the desired purpose.

[0010] The application can be achieved by up-regulating or down-regulating the content and / or activity of the protein TaSGR.

[0011] Furthermore, the application may include increasing the salt tolerance of plants and / or reducing the plant height by down-regulating the content and / or activity of the protein TaSGR (eg, knocking out or silencing the gene encoding the protein TaSGR).

[0012] The present invention also provides an application of the biomaterial, which may be any of the following: E1) Application in regulating plant salt tolerance; E2) Application in regulating plant strains; E3) Application in breeding salt-tolerant plants or plants with altered plant height; E4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height; The biological material may be any of the following: F1) a nucleic acid molecule encoding the protein TaSGR; F2) an expression cassette containing the nucleic acid molecule described in F1); F3) a recombinant vector containing the nucleic acid molecule described in F1), or a recombinant vector containing the expression cassette described in F2); F4) a recombinant microorganism containing the nucleic acid molecule described in F1), or a recombinant microorganism containing the expression cassette described in F2), or a recombinant microorganism containing the recombinant vector described in F3); F5) a recombinant host cell containing the nucleic acid molecule described in F1), or a recombinant host cell containing the expression cassette described in F2), or a recombinant host cell containing the recombinant vector described in F3); F6) transgenic plant tissue containing the nucleic acid molecule described in F1), or transgenic plant tissue containing the expression cassette described in F2); F7) A transgenic plant organ containing the nucleic acid molecule described in F1) or a transgenic plant organ containing the expression cassette described in F2).

[0013] In the above application, the nucleic acid molecule F1) may be a nucleic acid molecule encoding protein 1, a nucleic acid molecule encoding protein 2 and / or a nucleic acid molecule encoding protein 3, wherein: The nucleic acid molecule encoding the protein 1 may be any of the following: G1) a DNA molecule whose coding sequence is SEQ ID NO: 2; G2) a DNA molecule whose nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2; The nucleic acid molecule encoding the protein 2 may be any of the following: H1) a DNA molecule whose coding sequence is SEQ ID NO: 5; H2) a DNA molecule whose nucleotide sequence is SEQ ID NO: 4 or SEQ ID NO: 5; The nucleic acid molecule encoding the protein 3 may be any of the following: J1) a DNA molecule whose coding sequence is SEQ ID NO: 8; J2) A DNA molecule whose nucleotide sequence is SEQ ID NO: 7 or SEQ ID NO: 8.

[0014] The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0015] The nucleotide sequence shown in SEQ ID NO:2 may be TaSGR-5A The coding sequence (CDS) of the gene encodes the protein TaSGR-5A with the amino acid sequence shown in SEQ ID NO: 3. The nucleotide sequence shown in SEQ ID NO: 1 may be TaSGR- 5A The genomic sequence of a gene.

[0016] The nucleotide sequence shown in SEQ ID NO:5 may be TaSGR-5B The coding sequence (CDS) of the gene encodes the protein TaSGR-5B with the amino acid sequence shown in SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 4 may be TaSGR- 5B The genomic sequence of a gene.

[0017] The nucleotide sequence shown in SEQ ID NO:8 may be TaSGR-5D The coding sequence (CDS) of the gene encodes the protein TaSGR-5D with an amino acid sequence as shown in SEQ ID NO: 9. The nucleotide sequence shown in SEQ ID NO: 7 may be TaSGR- 5D The genomic sequence of a gene.

[0018] The nucleic acid molecules described herein may also include nucleic acid molecules obtained by modifying the codon preference based on the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 and / or SEQ ID NO:8.

[0019] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein TaSGR by using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.) or directed evolution (including error-prone PCR, DNA shuffling and in vitro random priming recombination, etc.). Those artificially modified nucleotide sequences that have 75% or more identity with the nucleotide sequence encoding the protein TaSGR 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 TaSGR and have the same function as the protein TaSGR.

[0020] The present invention also provides the use of a substance for reducing the activity and / or content of the protein TaSGR in any of the following: K1) Application in regulating plant salt tolerance; K2) Application in regulating plant strains; K3) Application in breeding salt-tolerant plants or plants with altered plant height; K4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height.

[0021] The substance may be any substance that reduces the activity and / or content of the protein TaSGR by regulating expression at the gene level or regulating the protein level.

[0022] The expression regulation at the gene level may include expression regulation at the chromatin level (such as histone modification, chromatin remodeling), transcription level (such as regulation of promoters, transcription factors, co-regulators), post-transcriptional level (such as RNA splicing, microRNA regulation) and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.).

[0023] The regulation of protein levels may include regulating the activity and / or content of the protein by protein degradation, protein interaction or other methods capable of regulating protein activity.

[0024] In the above application, the substance includes a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of the nucleic acid molecule encoding the protein TaSGR.

[0025] The nucleic acid molecule encoding the protein TaSGR may be TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0026] Furthermore, the substance may include a substance that causes the gene encoding the protein TaSGR to be deleted or inactivated by site-directed mutagenesis technology, gene knockdown technology, gene editing technology and / or gene knockout technology, or a substance that targets and binds to the protein TaSGR to reduce its content or inactivate its function.

[0027] It is well known to those skilled in the art to inhibit gene expression, silence or knock out genes by using site-directed mutagenesis technology (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis and cassette mutagenesis, etc.), gene knockdown technology (including RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology, etc.), gene editing technology (including zinc finger ribozyme gene editing technology, TALEN gene editing technology and CRISPR gene editing technology, etc.) or gene knockout technology (including complete gene knockout and conditional gene knockout). For example, shRNA, siRNA or miRNA targeting the protein TaSGR encoding gene can be used to inactivate gene expression or silence genes at the post-transcriptional level or translation level. The target gene can also be knocked out using a CRISPR-Cas system containing gRNA (sgRNA) and Cas protein. Alternatively, the protein TaSGR encoding gene is mutated by site-directed mutagenesis technology to produce a frameshift mutation or premature translation termination, thereby inactivating the gene or weakening its function. In some embodiments of the present invention, CRISPR / Cas9 gene editing technology is used to knock out the protein TaSGR in wheat. TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0028] Those skilled in the art know that TaSGR-5A , TaSGR-5B and / or TaSGR-5D The gene sequence or the sequence of the mRNA generated by its transcription is used to select the target sequence to design nucleic acid molecules such as siRNA, miRNA, shRNA or dsRNA. The nucleic acid molecule can inhibit or interfere with the transcription, translation or post-transcription and translation modification of the gene, thereby affecting the expression of the protein.

[0029] Furthermore, the substance may include nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells and viral vectors (such as lentivirus and adeno-associated virus).

[0030] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), micro RNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotide (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; (4) aptamer and ribozyme, etc.

[0031] In the above application, the substance may be sgRNA or a CRISPR / Cas9 system containing the sgRNA, and the sgRNA targets the gene encoding the protein TaSGR.

[0032] Furthermore, the target sequence of the sgRNA may be as shown in SEQ ID NO: 10 (target sequence of sgRNA1) and / or SEQ ID NO: 11 (target sequence of sgRNA2). Wherein: sgRNA1 may target TaSGR-5A , TaSGR-5B and TaSGR-5D Genes, sgRNA2 can also target TaSGR-5A , TaSGR-5B and TaSGR-5D Gene, sgRNA1 and / or sgRNA2 can be used to knock out the gene in cooperation with Cas9 protein TaSGR-5A , TaSGR-5B and TaSGR-5D Gene.

[0033] The present invention also provides a method for cultivating salt-tolerant plants or plants with altered plant height, which comprises reducing the content and / or activity of the protein TaSGR in the target plant to obtain plants with improved salt tolerance and / or reduced plant height.

[0034] In the above method, reducing the content and / or activity of the protein TaSGR in the target plant can be achieved by reducing the expression level of the gene encoding the protein TaSGR (TaSGR-5A, TaSGR-5B and / or TaSGR-5D) in the target plant.

[0035] The nucleotide sequence of the gene encoding the protein TaSGR-5A may be as shown in SEQ ID NO:2; the nucleotide sequence of the gene encoding the protein TaSGR-5B may be as shown in SEQ ID NO:5; the nucleotide sequence of the gene encoding the protein TaSGR-5D may be as shown in SEQ ID NO:8.

[0036] In the above method, reducing the expression level of the gene encoding the protein in the target plant can be performed using the CRISPR / Cas9 system, and the CRISPR / Cas9 system includes an sgRNA targeting the gene encoding the protein TaSGR.

[0037] In the above method, the target sequence of sgRNA may be shown as SEQ ID NO:10 and / or SEQ ID NO:11.

[0038] Furthermore, the CRISPR / Cas9 system also includes a Cas9 protein. Furthermore, the Cas9 protein described herein is not limited to a specific protein, as long as it can be used in conjunction with the sgRNA of the present invention.

[0039] Further, the Cas9 protein described herein includes Streptococcus pyogenes ( Streptococcus pyogenes )Cas9 (spCas9, II-A subtype), spCas9 HF (high fidelity), nickase Cas9 (nCas9), Staphylococcus aureus ( Staphylococcus aureus )Cas9 (saCas9, subtype II-A), Neisseria meningitidis ( Neisseria meningitidis )Cas9 (NmCas9, subtype II-C), Francisella novicida ( Francisella novicida )Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus ( Streptococcus thermophilus )Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni ( Campylobacter jejuni )Cas9 (CjCas9) and Treponema dendriticum ( Treponema sp.) Cas9, and Cas9 orthologs of other organisms but not limited thereto. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut™ HiFi Cas9 protein).

[0040] The method of the present invention can be implemented with any Cas9 protein known in the art. Those skilled in the art can make appropriate selections for the coding sequence of the Cas9 protein without departing from the principles of the embodiments of the present invention.

[0041] Furthermore, reducing the expression level of the gene encoding the protein TaSGR in the target plant using the CRISPR / Cas9 system can be achieved by contacting the gene encoding the protein TaSGR in the target plant cells with any of the sgRNAs described herein (such as sgRNA1 and / or sgRNA2) and the Cas9 protein.

[0042] Furthermore, the contacting step may be performed by the following (1) and (2): (1) directly introducing any of the sgRNAs described herein into the target plant cells, or first constructing a DNA molecule encoding any of the sgRNAs described herein into an expression vector and then introducing it into the target plant cells; (2) directly introducing the Cas9 protein or the mRNA of the Cas9 protein into the target plant cells, or first constructing the DNA molecule encoding the Cas9 protein into an expression vector and then introducing it into the target plant cells, or fusing the Cas9 protein with a cell-penetrating peptide and then introducing it into the target plant cells via the cell-penetrating peptide.

[0043] The cell-penetrating peptide is used to promote the Cas9 protein fused thereto to be taken up and absorbed by the cell and to exert biological functions in the cell. Suitable cell-penetrating peptides are not limited to specific types, as long as they can achieve the purpose of carrying the Cas9 protein through the membrane and internalization. For example, the cell-penetrating peptide can be the transcriptional transactivator Tat (Tat peptide) of the human immunodeficiency virus HIV.

[0044] It is known to those skilled in the art that Cas9 protein, Cas9 protein mRNA, Cas9 expression vector (a vector containing and expressing a DNA molecule encoding Cas9 protein), sgRNA, and sgRNA expression vector (a vector containing and expressing a DNA molecule encoding sgRNA) can be transferred into plant cells by various methods known in the art, such as chemical stimulation (including PEG, calcium phosphate, calcium chloride treatment, etc.), electric shock, liposome-mediated method, microinjection, gene gun method (also known as microparticle bombardment method), laser microbeam method, pollen tube channel method, ultrasonic method, air gun method, and vortex method, etc., and the target gene can also be transferred into plant receptor cells using a vector as a medium, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method.

[0045] When using an expression vector to deliver sgRNA and Cas9 protein, sgRNA and Cas9 protein can be linked to different expression vectors for expression, or linked to the same expression vector for expression.

[0046] Furthermore, the method for cultivating salt-tolerant plants or plants with altered plant height described herein may include the following steps: (1) constructing a sgRNA (such as sgRNA1 and / or sgRNA2) targeting the gene encoding the protein TaSGR into a Cas9 expression vector to obtain a CRISPR / Cas9 gene editing vector; (2) introducing the CRISPR / Cas9 gene editing vector into the target plant; (3) After screening and identification, salt-tolerant plants or plants with reduced plant height in which the gene encoding the protein TaSGR is knocked out are obtained.

[0047] Furthermore, the Cas9 expression vector described in step (1) contains the Cas9 gene and can express the Cas9 protein. The Cas9 expression vector may also contain one or more of the following elements: a replication origin (ori), a promoter (such as a U6 promoter, a U6-2 promoter of the present invention), an enhancer (such as a CAG enhancer), a tag (such as a FLAG tag), a terminator (such as a bGH poly(A) terminator), a resistance gene (such as a Kana antibiotic resistance gene, an ampicillin resistance gene), a promoter of a resistance gene, a screening gene (such as a bar gene), a promoter of a screening gene, and a promoter of a Cas9 gene (such as an Ubi promoter).

[0048] The Cas9 expression vector can be obtained commercially. After designing the sgRNA targeting the target gene, the DNA molecule encoding the sgRNA can be easily inserted into the commercial Cas9 expression vector, and the Cas9 protein and sgRNA are expressed at the same time, thereby editing the target gene. In addition, the Cas9 expression vector can also be constructed by conventional methods in the art. For example, the genome of Streptococcus pyogenes can be used as a template to amplify the Cas9 gene, and then the Cas9 gene can be cloned into a backbone expression vector (such as pET28a, pET32a, etc.) to obtain a Cas9 expression vector.

[0049] Furthermore, the introduction in step (2) can be introduced by Agrobacterium-mediated method, which may include the following steps: introducing the CRISPR / Cas9 gene editing vector constructed in step (1) into Agrobacterium (such as Ca ion-induced transformation method, polyethylene glycol-mediated transformation method, metal cation-mediated transformation method, electroporation transformation method, phage transduction method, etc.) to obtain recombinant Agrobacterium; infecting the callus tissue or explant of the target plant with the recombinant Agrobacterium; and inducing and culturing the positive callus tissue or explant obtained after identification to obtain regenerated plants.

[0050] The explants include, but are not limited to, seeds, roots, leaves, petioles, cotyledons, cotyledon petioles, hypocotyls, stem segments, stem apical meristems, epidermal parenchyma cells, tubers, stolon segments, embryonic suspension cells, and protoplasts.

[0051] The screening and identification methods are known to those skilled in the art. For example, gene-edited plants (including offspring materials of gene-edited plants) can be identified by PCR detection, Sanger sequencing, high-throughput sequencing, Western Blot, Southern blot hybridization, etc.

[0052] Although in one or more embodiments provided by the present invention, CRISPR / Cas9 technology is used to TaSGR- 5A , TaSGR-5B and TaSGR-5D The present invention is not limited to this specific method. It is well known to those skilled in the art that other techniques known in the art such as gene knockout, gene editing, gene mutation, gene knockdown, homologous recombination, etc. can be used to knock out the gene in the plant genome. TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene deletion or inactivation. These methods can also be used in the present invention. These alternative methods do not depart from the scope of the present invention, and the present invention should include these alternative methods.

[0053] Herein, the plant may be any one of the following (1)-(3): (1) Monocots; (2) Poaceae; (3) Triticum.

[0054] Herein, the target plant may be a target plant containing a gene encoding the protein TaSGR.

[0055] The method for cultivating salt-tolerant plants or plants with altered plant height described in the present invention may also include the step of hybridizing the salt-tolerant plants or plants with altered plant height obtained by any of the methods described above with the plant to be improved to obtain offspring transgenic plants; the offspring transgenic plants have basically the same phenotype as the transgenic plants.

[0056] Herein, the salt-tolerant plants or plants with altered plant height are understood to include not only the first-generation gene-edited plants obtained by knocking out the protein TaSGR encoding gene in the target plant, but also their progeny. The gene-edited plants include seeds, callus tissues, complete plants and cells.

[0057] In this article, TaSGR Gene TaSGR-5A , TaSGR-5B and / or TaSGR-5D Gene.

[0058] Herein, "protein TaSGR" means TaSGR-5A, TaSGR-5B and / or TaSGR-5D protein.

[0059] The present invention utilizes CRISPR / Cas9 technology to knock out three alleles in wheat, namely TaSGR-5A , TaSGR-5B and TaSGR-5D Gene, successfully created TaSGR Homozygous gene-edited plants with gene knockout. The experimental results show that TaSGR After the gene was introduced, the plant height of wheat was significantly reduced and its salt tolerance was significantly enhanced.

[0060] The present invention discloses for the first time the application of the protein TaSGR and its encoding gene in regulating plant salt tolerance and / or plant growth. TaSGR Genes) to increase the salt tolerance of plants, and / or reduce the plant height (to better resist lodging). The present invention provides a good gene resource for salt tolerance breeding of wheat, TaSGR The application of genes has opened up new areas and is of great significance for cultivating new types of wheat and promoting the commercial breeding process of wheat.

[0061] Definition of terms 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. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0062] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a target gene. A typical expression cassette contains a promoter, an MCS (multiple cloning site) and a terminator. An expression cassette may also include a target gene, a marker gene (such as TK gene, DHFR gene, CAT gene and NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected through a linker.

[0063] The term "vector" generally refers to a vector that can carry exogenous 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 the host cell by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cell. The vector includes, but is not limited to, plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e., Cosmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (such as SV40), herpes virus (such as herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including but not limited to promoter sequence, transcription start sequence, enhancer sequence, selection element and reporter gene. In addition, the vector may also contain a replication start site.

[0064] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from the genus Escherichia ( Escherichia sp. ) (such as Escherichia coli), Erwinia spp. ( Erwinia sp. ), Agrobacterium ( Agrobacterium sp. ) (such as Agrobacterium tumefaciens), Flavobacterium ( Flavobacterium sp. )、Alcaligenes spp. Alcaligenes sp. ), Pseudomonas ( Pseudomonas sp. ) and Bacillus ( 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 the genus Saccharomyces ( Saccharomyces sp. ) (such as Saccharomyces cerevisiae, methanol yeast, Pichia pastoris), Fusarium spp. ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. ), Verticillium ( Verticillium sp. )、Penicillium( Penicillium sp. )、Aspergillus( Aspergillus sp. ) and Cephalosporium ( Cephalosporium sp. ) etc. The actinomycetes may be from the genus Streptomyces (Streptomyces sp. ) (such as Streptomyces). The algae may be from Cyanophyta (such as cyanobacteria), Fucus ( Fucus sp. ), the genus A. Achnanthes sp. ), Cocoon algae ( Amphiprora sp. ), Diplocoriaceae ( Amphora sp. ), Fibrocystis spp. Ankistrodesmus sp. ), Astrophytum ( Asteromonas sp. ) and Chromophytes ( Boekelovia sp. )wait.

[0065] 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 offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring 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, among which: the plant cell can be Arabidopsis thaliana ( Arabidopsis thaliana ),tobacco( Nicotiana tabacum ),corn( Zea mays )、Rice( Oryza sativa ),wheat( Triticum aestivum ) and other plant cells, but not limited thereto; the animal cells may be mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell substrain (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby 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), amphibian cells (e.g., African clawed frogs ( Xenopus laevis ) cells 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The term "link" generally refers to the association of two or more molecules. The link can be covalent or non-covalent. The link described herein can be directly connected by a peptide bond, or connected by a linker (joint).

[0070] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is generally expressed as a percentage. The identity described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. It is known to those skilled in the art that the identity of an amino acid sequence or a nucleotide sequence can be determined using an identity search site on the Internet, such as the BLAST webpage of 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 performing a search to calculate the identity of an amino acid sequence, then the value (%) of the identity can be obtained. In addition, sequence analysis software (such as CLC MainWorkbench and MegAlign TM ), for example using the computer program BLAST, in particular BLASTP or TBLASTN, with default parameters. The 75% or more identity described herein may be at least 75%, 80%, 85%, 90% or more than 95% identity. The 80% or more identity described herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity.

[0071] The term "conservative substitution" generally refers to replacing an amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein and do not substantially change the biological activity of the protein. Conservative substitutions in the protein sequence that are expected to have little or no effect on the protein structure or function can be easily designed by a person of ordinary skill in the art.

[0072] The term "introduction" generally refers to the transfer of foreign genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the target gene (such as the DNA molecule of the present invention) into the recipient cell. The method of introduction may include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) transferring the target gene into plant recipient cells by physical or chemical methods, such as gene gun method (also known as microprojectile bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method and vortex method, etc. (4) Using vectors as a medium to transfer the target gene into plant recipient cells, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium mediated method), plant virus vector mediated transformation method, etc. (5) Introducing the target gene into in vitro animal cells (transfection) through calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrotransfection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Introducing the target gene into in vivo animal cells through microinjection method, retrovirus vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, etc., and further preparing transgenic animals.

[0073] The term "Cas9 protein" generally refers to the Cas endonuclease of the type II CRISPR system that forms a complex with crRNA and tracrRNA or with a guide RNA, which is used to specifically recognize and cut all or part of the DNA target sequence. The Cas9 protein has two different domains: the HNH domain and the RuvC domain. The HNH domain is responsible for cutting the DNA strand (target strand) that is complementary to the crRNA (or gRNA), while the RuvC domain is responsible for cutting the non-complementary strand (non-target strand). The Cas9 protein is not limited to a specific protein, as long as it can be used in conjunction with sgRNA (gRNA). The Cas9 protein can be derived from a bacterial species.

[0074] The term "sgRNA (single-guide RNA)" generally refers to the structure of a single RNA formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, connecting crRNA and tracrRNA directly (or through a linker). sgRNA is a component of the CRISPR-Cas9 system, responsible for guiding the Cas9 protein to recognize and cut the target nucleic acid molecule. In actual gene editing applications, sgRNA can be directly synthesized or obtained through plasmid expression or in vitro transcription. sgRNA includes a recognition region and a framework region (scaffold). The framework region is known to those skilled in the art and is responsible for binding to the Cas protein, and the recognition region is responsible for binding to the target site of the target gene and guiding the Cas protein to the target site.

[0075] The term "explant" generally refers to a part of a plant body used as an in vitro culture material in plant tissue culture, which can be regenerated into a whole plant under appropriate conditions after appropriate treatment. In actual operation, those skilled in the art select appropriate explants for transformation according to different plants.

[0076] The term "callus" usually refers to the newly formed tissue on the wound surface after the original plant body is partially stimulated by trauma. It is composed of living thin-walled cells and can originate from living cells of various tissues in any organ of the plant body. In plant tissue culture, it can refer to a group of disorderly growing thin-walled cells with vigorous division ability formed by explants. Cultivating callus on an appropriate culture medium can induce it to form a whole plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 For Example 2 TaSGR Phenotypes and mutation sequences of gene-edited wheat materials.

[0078] Figure 2 For Example 3 TaSGR Results of identification of salt-tolerant phenotype of gene-edited mutants.

[0079] Figure 3 1 is a structural diagram of the 964 bp PCR product fragment in Example 1. DETAILED DESCRIPTION

[0080] 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.

[0081] 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.

[0082] The following examples used Excel statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. , s t -test method, P < 0.05 (*) indicates statistical difference, P < 0.01 (**) indicates statistically significant difference, and P < 0.001 (***) indicates extremely significant statistical difference. The quantitative experiments in the following examples, unless otherwise specified, were performed with three biological replicates, and the results were averaged.

[0083] The pCBC-DT1T2 and pBUE411 vectors in the following examples are described in the following literature: He, G., Zhang, Y., Liu, P., Jing, Y., Zhang, L., Zhu, Y. et al. (2021) The transcriptionfactor TaLAX1 interacts with Q to antagonistically regulate grain threshability and spike morphogenesis in bread wheat. New phytologist, 230, 988-1002.

[0084] Embodiment 1, wheat TaSGR Gene editing vector construction Through transcriptome analysis of salt-tolerant wheat varieties, we found that TraesCS5B03G0809200 The transcriptional expression level of genes (STAYGREEN, TaSGR) was significantly lower than that of the control variety. TaSGR Three alleles of a geneTaSGR-5A , TaSGR-5B and TaSGR-5D The sequence of its encoded protein is as follows: TaSGR-5A The genomic nucleotide sequence of the gene is shown in SEQ ID NO:1. TaSGR-5A The nucleotide sequence of the coding region (CDS) of the gene is shown in SEQ ID NO: 2, and the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO: 3. The protein is named TaSGR-5A.

[0085] TaSGR-5B The genomic nucleotide sequence of the gene is shown in SEQ ID NO:4. TaSGR-5B The nucleotide sequence of the coding region (CDS) of the gene is shown in SEQ ID NO:5, and the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO:6. The protein is named TaSGR-5B.

[0086] TaSGR-5D The genomic nucleotide sequence of the gene is shown in SEQ ID NO:7. TaSGR-5D The nucleotide sequence of the coding region (CDS) of the gene is shown in SEQ ID NO:8, and the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO:9. The protein is named TaSGR-5D.

[0087] Further, in order to verify TaSGR The function of genes, we constructed TaSGR-5A / 5B / 5D ( TraesCS5A02G319900 / TraesCS5B02G320200 / TraesCS5D02G325900 )CRRISPR / Cas9 gene editing vector, the vector used is pBUE411.

[0088] Since the three genes are highly homologous, TaSGR The CRISPR / Cas9 dual target sites were designed based on the conserved gene sequence. The designed sgRNA target sequences are as follows: sgRNA1 target sequence: 5'-TCACGTCGCTGTGGGTGAG-3' (SEQ ID NO: 10), sgRNA2 target sequence: 5′-TGAGCTTGGACGCCTCGAA-3′ (SEQ ID NO: 11).

[0089] The target sequence of sgRNA1 is located at TaSGR-5A , TaSGR-5B and TaSGR-5D gene; the target sequence of sgRNA2 is also located at TaSGR-5A , TaSGR-5B and TaSGR-5D Genetically; using sgRNA1 and sgRNA1 dual targetsTaSGR-5A , TaSGR-5B and TaSGR-5D Gene (also called TaSGR-5A / 5B / 5D Gene ） Knockout.

[0090] The PCR amplification primers are shown in Table 1.

[0091]

[0092] The gene editing vector construction process is as follows: 1. PCR amplification Dissolve and mix the above four primers, where the concentration of TaSGR-F / R primers is 10 µM, and the concentration of TaSGR-F0 / R0 primers is 0.5 µM. The PCR reaction system is as follows:

[0093] In Table 2, the components of KOD Plus PCR reaction solution are as follows: KOD Plus buffer 5 µl, 2 mM dNTP 5 µl, 25 mM MgSO4 5 µl, KOD-Plus 1 µl.

[0094] 2. Recover the above PCR product fragment (964 bp, nucleotide sequence is SEQ ID NO: 12, structure is Figure 3 As shown in Table 3), after BsaI digestion, the vector was connected to pBUE411 by T4 Ligase. The digestion-ligation reaction system is shown in Table 3:

[0095] The knockout TaSGR-5A , TaSGR-5B and TaSGR-5D The recombinant vector of the gene was named pBUE411-TaSGR.

[0096] The recombinant vector pBUE411-TaSGR contains two editing target sites (SEQ ID NO: 10 and SEQ ID NO: 11) and the coding gene of the Cas9 protein on the vector. After being introduced into the receptor, the two transcribed guide RNAs can target the target sequence near the PAM of the receptor genome through base complementary pairing, that is, targeting TaSGR-5A , TaSGR-5B and TaSGR-5D Gene, Cas9 protein TaSGR-5A , TaSGR-5B and TaSGR-5DThe double-stranded DNA break at the gene target site causes a gene mutation in the sheared region through the organism's own DNA damage repair response mechanism during the repair process, resulting in a frameshift mutation in the coding gene or premature termination of translation, thereby achieving TaSGR-5A , TaSGR-5B and TaSGR-5D Knockout of genes.

[0097] Embodiment 2, TaSGR Construction of gene-edited wheat materials Will TaSGR The gene editing vector (pBUE411-TaSGR) was transformed into Fielder wheat cultivar by Agrobacterium-mediated method, and two genes were obtained after PCR sequencing. TaSGR-5A / 5B / 5D Homozygous gene-edited strains (named Tasgr- aabbdd #1 and Tasgr-aabbdd #2), planted in the spring in the wheat experimental field of the Chinese Academy of Agricultural Sciences. The gene-edited strains showed a phenotype of reduced plant height ( Figure 1 b and c). TaSGR-5A / 5B / 5D Gene editing mutant mutation sequence Figure 1 As shown in a.

[0098] Embodiment 3, TaSGR Identification of salt-tolerance phenotypes of gene-edited mutants The tested plants were the third generation. First, Fielder and Tasgr-aabbdd #1 and Tasgr-aabbdd The seeds of #2 material were germinated. After the seeds turned white, they were transplanted into a 96-well hydroponic box and cultured with Hoagland's nutrient solution. After 7-10 days of culture, when the material grew to the stage of two leaves and one heart, the above materials were photographed before treatment, and then the Hoagland's nutrient solution was replaced with 150 mM NaCl salt solution (8.766 grams of sodium chloride were added to the Hoagland's nutrient solution). After the material was cultured in the salt solution for at least two weeks, the salt solution was replaced with Hoagland's nutrient solution for rehydration, and the phenotype after rehydration was collected 7 days after treatment. Among them, the nutrient solution for hydroponics was changed every 2-3 days. The above materials were all cultured in a wheat incubator. The incubator conditions were 22°C, 16 hours of light; 19°C, 8 hours of darkness.

[0099] The results are as follows Figure 2 As shown, compared with the control variety Fielder, TaSGR The salt tolerance of the gene-edited mutant was significantly enhanced.

[0100] The above results show that knocking out wheat TaSGR-5A , TaSGR-5B and TaSGR-5DAfter the gene was inserted, the height of the gene-edited plants was significantly reduced, and they showed better growth than wild-type plants under salt stress conditions, indicating that TaSGR Genes can be used to regulate plant height and salt tolerance in wheat.

[0101] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. The use of protein, characterized in that The application is any of the following: A1) Application in regulating plant salt tolerance; A2) Application in regulating plant strains; A3) Application in breeding salt-tolerant plants or plants with altered plant height; A4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height; The protein is protein 1, protein 2 and / or protein 3, wherein: The protein 1 is any one of the following: B1) a protein having an amino acid sequence of SEQ ID NO: 3; 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 replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:3; B3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2); The protein 2 is any one of the following: C1) a protein having an amino acid sequence of SEQ ID NO: 6; C2) a protein having more than 80% identity with the protein shown in C1) and having the same function as the protein shown in C1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:6; C3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of C1) or C2); The protein 3 is any one of the following: D1) a protein having an amino acid sequence of SEQ ID NO: 9; D2) a protein having more than 80% identity with the protein shown in D1) and having the same function as the protein shown in D1) obtained by replacing, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:9; D3) A fusion protein with the same function is obtained by connecting a tag to the N-terminus and / or D-terminus of D1) or D2).

2. Application of biomaterials, characterized in that The application is any of the following: E1) Application in regulating plant salt tolerance; E2) Application in regulating plant strains; E3) Application in breeding salt-tolerant plants or plants with altered plant height; E4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height; The biological material is any of the following: F1) A nucleic acid molecule encoding the protein according to claim 1; F2) an expression cassette containing the nucleic acid molecule described in F1); F3) a recombinant vector containing the nucleic acid molecule described in F1), or a recombinant vector containing the expression cassette described in F2); F4) a recombinant microorganism containing the nucleic acid molecule described in F1), or a recombinant microorganism containing the expression cassette described in F2), or a recombinant microorganism containing the recombinant vector described in F3); F5) a recombinant host cell containing the nucleic acid molecule described in F1), or a recombinant host cell containing the expression cassette described in F2), or a recombinant host cell containing the recombinant vector described in F3); F6) transgenic plant tissue containing the nucleic acid molecule described in F1), or transgenic plant tissue containing the expression cassette described in F2); F7) A transgenic plant organ containing the nucleic acid molecule described in F1) or a transgenic plant organ containing the expression cassette described in F2).

3. The use according to claim 2, characterized in that: F1) The nucleic acid molecule is a nucleic acid molecule encoding protein 1 according to claim 1, a nucleic acid molecule encoding protein 2 according to claim 1, and / or a nucleic acid molecule encoding protein 3 according to claim 1, wherein: The nucleic acid molecule encoding the protein 1 in claim 1 is any one of the following: G1) a DNA molecule whose coding sequence is SEQ ID NO: 2; G2) a DNA molecule whose nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2; The nucleic acid molecule encoding the protein 2 in claim 1 is any one of the following: H1) a DNA molecule whose coding sequence is SEQ ID NO: 5; H2) a DNA molecule whose nucleotide sequence is SEQ ID NO: 4 or SEQ ID NO: 5; The nucleic acid molecule encoding the protein 3 in claim 1 is any one of the following: J1) a DNA molecule whose coding sequence is SEQ ID NO: 8; J2) A DNA molecule whose nucleotide sequence is SEQ ID NO: 7 or SEQ ID NO:

8.

4. Use of a substance for reducing the activity and / or content of the protein in claim 1 in any of the following: K1) Application in regulating plant salt tolerance; K2) Application in regulating plant strains; K3) Application in breeding salt-tolerant plants or plants with altered plant height; K4) Application in molecular breeding for improving plant salt tolerance and / or plant height or in improving germplasm resources related to salt tolerance and / or plant height.

5. The use according to claim 4, characterized in that: The substance includes a substance that inhibits the replication, transcription, translation, post-transcriptional modification and / or post-translational modification of a nucleic acid molecule encoding the protein of claim 1.

6. The use according to claim 4 or 5, characterized in that: The substance is sgRNA or a CRISPR / Cas9 system containing the sgRNA, and the sgRNA targets the gene encoding the protein of claim 1.

7. A method for cultivating salt-tolerant plants or plants with altered plant height, characterized in that: The method comprises reducing the content and / or activity of the protein of claim 1 in the target plant, thereby obtaining a plant with improved salt tolerance and / or reduced plant height.

8. The method according to claim 7, characterized in that The reduction of the content and / or activity of the protein in claim 1 in the target plant is achieved by reducing the expression level of the gene encoding the protein in the target plant.

9. The method according to claim 8, characterized in that The reducing the expression level of the gene encoding the protein in the target plant is performed using the CRISPR / Cas9 system, wherein the CRISPR / Cas9 system comprises an sgRNA targeting the protein encoding gene in claim 1.

10. The method according to claim 9, characterized in that The target sequence of sgRNA is shown in SEQ ID NO:10 and / or SEQ ID NO:11.

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