Wheat salt-tolerant auxin signal gene taaa25 and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a wheat salt-alkali tolerance auxin signal gene TaIAA25 and application thereof. The application discloses, for the first time, the application of the TaIAA25 gene in regulating plant salt-alkali tolerance. Knocking out or silencing the TaIAA25 gene can increase the salt-alkali tolerance of plants, increase the fresh weight and relative fresh weight of plants under salt-alkali stress, reduce the leaf wilting rate, and increase the total chlorophyll content. The application provides a good gene resource for wheat salt-alkali tolerance breeding, and has important significance for cultivating new wheat and promoting the commercialized breeding process of wheat.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the wheat salt-alkali tolerance auxin signaling gene TaIAA25 and its applications. Background Technology

[0002] Soil salinization severely restricts agricultural production and food security. Identifying important salt-tolerant genes and applying them to molecular design breeding and cultivating salt-tolerant crop varieties can help make full use of saline-alkali land and other sub-grade land, which is of great significance for ensuring food security.

[0003] wheat( Triticum aestivum As one of the world's staple foods, wheat faces severe challenges in its production and safety on saline-alkali land. Developing salt-tolerant wheat varieties is the best way to effectively utilize saline-alkali land and improve economic efficiency. Discovering wheat genes that respond to salt-alkali stress, elucidating the molecular mechanisms of wheat's salt-alkali tolerance, and developing salt-alkali tolerant wheat varieties are of great significance for increasing wheat yields and improving the utilization rate of saline-alkali land. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the purpose of this invention is to provide the wheat salt-alkali tolerance auxin signaling gene TaIAA25 and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an application of the TaIAA25 gene, said application being any of the following: (A1) Application in regulating plant salt and alkali tolerance; (A2) Application in cultivating salt-tolerant plants; (A3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (A4) Applications in increasing plant fresh weight and relative fresh weight; (A5) Application in reducing plant leaf mortality; (A6) Application in increasing the total chlorophyll content of plants; (A7) Application in the improvement of germplasm resources related to salt tolerance; The TaIAA25 gene includes TaIAA25-7A, TaIAA25-7B, and TaIAA25-7D. The nucleotide sequence of TaIAA25-7A is as shown in SEQ ID NO:1 or has 90% or more of the same identity. The nucleotide sequence of TaIAA25-7B is as shown in SEQ ID NO:3 or has 90% or more of the same identity. The nucleotide sequence of TaIAA25-7D is as shown in SEQ ID NO:5 or has 90% or more of the same identity.

[0006] Furthermore, the application is achieved by increasing or decreasing the expression level of the TaIAA25 gene.

[0007] A second aspect of the invention is the application of proteins, wherein the application is any of the following: (B1) Application in regulating plant salt and alkali tolerance; (B2) Application in cultivating salt-tolerant plants; (B3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (B4) Application in increasing plant fresh weight and relative fresh weight; (B5) Application in reducing plant leaf mortality; (B6) Application in increasing the total chlorophyll content of plants; (B7) Application in the improvement of germplasm resources related to salt tolerance; The protein is named TaIAA25 and includes protein 1, protein 2, and protein 3.

[0008] The name of protein 1 may be TaIAA25-7A, and may be any of the following: (D1) The amino acid sequence is that of the protein SEQ ID NO:2; (D2) A protein that has more than 80% identity with and has the same function as the protein shown in (D1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:2. (D3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of (D1) or (D2).

[0009] The protein 2 may be named TaIAA25-7B, and may be any of the following: (E1) The amino acid sequence is that of the protein SEQ ID NO:4; (E2) A protein that has more than 80% identity with and has the same function as the protein shown in (D1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:4. (E3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of (E1) or (E2).

[0010] The protein 3 may be named TaIAA25-7D, and may be any of the following: (F1) The amino acid sequence is that of the protein SEQ ID NO:6; (F2) A protein that has more than 80% identity with and has the same function as the protein shown in (D1) obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO:6. (F3) is a fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of (F1) or (F2).

[0011] In the above applications, the protein TaIAA25 can be derived from wheat ( Triticum aestivum ).

[0012] The connections described in (D3), (E3) and (F3) can be directly connected via peptide bonds or via adapters.

[0013] The substitution of amino acid residues described in (D2), (E2) and (F2) can be a conservative substitution of amino acid residues.

[0014] To facilitate the isolation, purification, detection, and / or localization of the proteins described in (D1), (E1), and (F1), a tag protein may be attached to its amino or carboxyl terminus. Such tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA (influenza hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, phage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein. The use of tags does not alter the function of the target protein, and those skilled in the art know how to select appropriate tag proteins according to the desired purpose.

[0015] The application can be achieved by upregulating or downregulating the content and / or activity of the protein TaIAA25.

[0016] Furthermore, the application may include increasing the salt tolerance of plants by downregulating the content and / or activity of the protein TaIAA25 (e.g., knocking out or silencing the gene encoding the protein TaIAA25).

[0017] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein TaIAA25 using known methods, such as site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis) or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random recombination). Artificially modified nucleotide sequences that possess 75% or more identity with the nucleotide sequence encoding the protein TaIAA25, provided they encode the protein TaIAA25 and have the same function as the protein TaIAA25, are nucleotide sequences derived from and equivalent to those of the present invention.

[0018] A third aspect of the invention provides the use of a substance for reducing the activity and / or content of the protein described in the second aspect in any of the following: (C1) Application in improving the salt and alkali tolerance of plants; (C2) Application in cultivating salt-tolerant plants; (C3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (C4) Application in increasing plant fresh weight and relative fresh weight; (C5) Application in reducing plant leaf mortality; (C6) Application in increasing the total chlorophyll content of plants; (C7) Application in the improvement of germplasm resources related to salt tolerance.

[0019] The substance may be any substance that reduces the activity and / or content of the protein TaIAA25 through gene-level expression regulation or protein-level regulation.

[0020] The gene-level expression regulation may include expression regulation at the chromatin level (such as histone modification and chromatin remodeling), transcriptional level (such as regulation by promoters, transcription factors, and co-regulatory factors), post-transcriptional level (such as RNA splicing and microRNA regulation), and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.).

[0021] The regulation of protein levels may include regulating protein activity and / or content through protein degradation, protein interaction, or other methods that can modulate protein activity.

[0022] In the above applications, the substance includes substances that inhibit the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of nucleic acid molecules encoding the protein TaIAA25.

[0023] The nucleic acid molecule encoding the protein TaIAA25 may be the TaIAA25-7A, TaIAA25-7B, and / or TaIAA25-7D gene.

[0024] Furthermore, the substance may include a substance that causes the coding gene of the protein TaIAA25 to be deleted or inactivated by site-directed mutagenesis, gene knockdown, gene editing and / or gene knockout, or a substance that targets and binds to the protein TaIAA25 to reduce its content or inactivate its function.

[0025] It is well known to those skilled in the art to use site-directed mutagenesis (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis), gene knockout techniques (including RNA interference, Morpholino interference, antisense nucleic acid techniques, and ribozyme techniques), gene editing techniques (including zinc finger ribozyme gene editing, TALEN gene editing, and CRISPR gene editing), or gene knockout techniques (including complete gene knockout and conditional gene knockout) to inhibit gene expression, silence, or knock out genes. For example, shRNA, siRNA, or miRNA targeting the gene encoding the protein TaIAA25 can be used to inactivate or silence gene expression at the post-transcriptional or translational level. The CRISPR-Cas system containing gRNA (sgRNA) and Cas protein can also be used to knock out the target gene. Alternatively, site-directed mutagenesis can be used to mutate the gene encoding the protein TaIAA25 to induce a frameshift mutation or premature translation termination, thereby inactivating or weakening the gene. In some embodiments of the present invention, CRISPR / Cas9 gene editing technology is used to knock out the TaIAA25 gene in wheat.

[0026] Those skilled in the art will understand that nucleic acid molecules such as siRNA, miRNA, shRNA, or dsRNA can be designed by selecting target sequences based on the TaIAA25 gene sequence or the sequence of its transcribed mRNA. These nucleic acid molecules can inhibit or interfere with gene transcription, translation, or post-transcriptional and post-translational modifications, thereby affecting protein expression.

[0027] Furthermore, the substances may include nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells, and viral vectors (such as lentiviruses and adeno-associated viruses).

[0028] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA) and short hairpin RNA (shRNA) used in RNA interference technology; (2) antisense RNA (asRNA) and antisense oligonucleotides (AON) used in antisense nucleic acid technology; (3) gRNA and sgRNA used in gene editing technology; and (4) aptamers and ribozymes.

[0029] In the above applications, the substance may be sgRNA or a CRISPR / Cas9 system containing the sgRNA, wherein the sgRNA targets the gene encoding the protein TaIAA25.

[0030] Furthermore, the target sequence of the sgRNA may be as shown in SEQ ID NO:7 (target sequence of sgRNA1) and SEQ ID NO:8 (target sequence of sgRNA2).

[0031] A fourth aspect of the present invention provides a method for cultivating salt-tolerant plants, the method comprising reducing the content and / or activity of TaIAA25 protein in a target plant to obtain a plant with increased salt tolerance and / or reduced plant height, wherein the plant is wheat.

[0032] The reduction of the content and / or activity of TaIAA25 protein in the target plant is achieved by reducing the expression level of the gene encoding TaIAA25 protein in the target plant.

[0033] In the above method, the reduction of the expression level of the protein-coding gene in the target plant is performed using a CRISPR / Cas9 system, which includes sgRNA targeting the protein-coding gene described in the second aspect.

[0034] The target sequences of sgRNA are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0035] Furthermore, the CRISPR / Cas9 system also includes the Cas9 protein. Furthermore, the Cas9 protein described herein is not limited to any specific protein, as long as it can be used in conjunction with the sgRNA of this invention.

[0036] While the TaIAA25 gene is knocked out using CRISPR / Cas9 technology in one or more embodiments provided in this invention, the invention is not limited to this specific method. Those skilled in the art will recognize that other gene knockout, gene editing, gene mutation, gene knockdown, homologous recombination, and other techniques known in the art can be used to delete or inactivate the TaIAA25 gene in the plant genome. These methods can also be used in this invention. These alternative methods do not depart from the scope of this invention, and this invention should include these alternative methods.

[0037] In this article, the plant may be any one of the following (1)-(3): (1) Monocotyledons; (2) Gramineae; (3) Triticum.

[0038] In this article, the target plant may be a target plant containing the gene encoding the protein TaIAA25.

[0039] The method for cultivating salt-tolerant plants or plants with altered plant height described in this invention may further 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 a phenotype substantially consistent with the transgenic plants.

[0040] In this article, 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 gene encoding the protein TaIAA25 in the target plant, but also their progeny. The gene-edited plants include seeds, callus tissue, intact plants, and cells.

[0041] In this article, "TaIAA25 gene" refers to the TaIAA25-7A, TaIAA25-7B, and / or TaIAA25-7D genes.

[0042] In this article, “protein TaIAA25” refers to TaIAA25-7A, TaIAA25-7B and / or TaIAA25-7D proteins.

[0043] This invention utilizes CRISPR / Cas9 technology to knock out three alleles in wheat, namely the TaIAA25-7A, TaIAA25-7B, and TaIAA25-7D genes, successfully creating homozygous gene-edited plants with the TaIAA25 gene knockout. Experimental results show that knocking out the TaIAA25 gene significantly enhances the salt and alkali tolerance of wheat.

[0044] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention reveals for the first time the application of the TaIAA25 gene in regulating plant salt and alkali tolerance. Knocking out or silencing the TaIAA25 gene can increase plant salt and alkali tolerance, increase fresh weight and relative fresh weight under salt and alkali stress, reduce leaf mortality, and increase total chlorophyll content. This invention provides a valuable gene resource for salt and alkali tolerance breeding of wheat, and is of great significance for breeding new wheat varieties and promoting the commercialization of wheat breeding. Attached Figure Description

[0045] Figure 1 The expression level of TaIAA25 in the wheat variety Fielder changes at different times under alkaline salt treatment.

[0046] Figure 2 The gene editing site variations in the TaIAA25 wheat variety Fielder on chromosomes A, B, and D are shown.

[0047] Figure 3 The figures show the phenotypes of wheat varieties Fielder and iaa25 mutants under alkaline salt treatment or control conditions. A represents the phenotypes of wild-type wheat and mutant wheat under alkaline salt treatment and control conditions; B represents the fresh weight of wild-type wheat and mutant wheat under alkaline salt treatment and control conditions; C represents the relative fresh weight of wild-type wheat and mutant wheat under alkaline salt treatment and control conditions; D represents the proportion of necrotic leaves in wild-type wheat and mutant wheat under alkaline salt treatment; and E represents the total chlorophyll content of wild-type wheat and mutant wheat under alkaline salt treatment and control conditions. Detailed Implementation

[0048] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0050] 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.

[0051] The pBUE411 vector in the following examples is described in the following literature: He, G., Zhang, Y., Liu, P., Jing, Y., Zhang, L., Zhu, Y. et al. (2021) The transcription factor TaLAX1 interacts with Q to antagonistically regulate grain threshability and spike morphogenesis in bread wheat. New Phytologist, 230, 988-1002.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1: Cloning of the wheat gene TaIAA25 The wheat gene TaIAA25 (TraesCS7A03G0794900) was obtained from a wheat salt-alkali sensitive mutant through exon sequencing and map-based cloning. Further cloning of the wheat gene TaIAA25 followed the steps below: 1. Wheat RNA extraction was performed according to the instructions for RNAiso Plus (Total RNA Extraction Reagent) (TaKaRa, #9109).

[0054] 2. Use the FastKing cDNA First-Strand Synthesis Kit (TIANGEN, #KR116) to reverse transcribe and synthesize cDNA according to its instructions.

[0055] 3. Obtain the TaIAA25 gene information using the wheat genome website WheatOmics (http: / / 202.194.139.32 / ), and design specific primers for gene amplification based on the gene sequence, as shown in SEQ ID NO:11-12.

[0056] The forward primer sequence used for amplifying the wheat gene TaIAA25 is as follows: TaIAA25-F: GCCACCACAAAAACACTTCCTG (SEQ ID NO: 11); TaIAA25-R: CCATATCTGCATGATCCACGAGAG (SEQ ID NO: 12).

[0057] 4. The TaIAA25 gene sequence was amplified using the Phanta Max Super-Fidelity DNA Polymerase kit (Vazyme, # P505). The amplification system is shown in Table 1.

[0058] Table 1 Amplification System

[0059] The PCR reaction conditions were as follows: pre-denaturation: 98℃, 4 min; (denaturation: 98℃, 15 sec; annealing: 57℃, 15 sec; extension: 72℃, 1 min) × 37 cycles; post-extension: 72℃, 10 min.

[0060] After the PCR reaction is completed, the PCR products are separated using agarose gel and the target band is detected.

[0061] 5. Cut the target band from the gel and place it in a 2.0 mL centrifuge tube. Use a universal DNA purification and recovery kit (TIANGEN, # DP214) to recover the DNA.

[0062] 6. After the amplified fragment is recovered, it is ligated into the pEASY-Blunt3 vector (TransGen Biotech, # CB301) and transformed into E. coli Trans1-T1 (TRANS, # CD501). Sanger sequencing is then performed to screen for E. coli containing the target gene plasmid, and the resulting fragments are stored for later use.

[0063] The coding region and amino acid sequence of the TaIAA25 gene on chromosome A are as follows: Nucleotide sequence of the coding region of wheat gene TaIAA25-7A: ATGTTGCAGACACAGGTAGTAGTGAAGTACCAGAGCTACAGCTTGGGAGAGGCCATGAAGAGTTCTTCAGTTGCACCAAGCCTGAAACAGAAGCAAGGAGATGCTAGCAAACTGCAGGATGGTGTCCCCAACAACCTTGAGCTTAGGCTTGGCATCTCTTCAGACAATGGCCTGAGTAGTGGTGGTGGTGGTGCTACTACTCCATGGCTTGGTGTTGGAGTGCACCCTTGGAGCCTGTCCGCCAGGCAAGACAAGGCAGCCCTGGAACAACCTCAGCAGAGGCCTAATGAGTGTCCTGCCCACAGAGAAGATCGACCTCAACTGGTCGGATGGCCACCGGTGCGCACATTCCGCAAGAACCTGTCTGCCACATCCACGAGGCCTGCATATTCTGGAGACCTGAGCAAAGTGGAACCATGCTATGAAGAAGAAGACCATGGAAACATAAACACTGGAGTTTCAGTACAAGAGAGGCCAGCCATGTTCGTGAAGGTGAACCTGGAAGGGTACGCTGTTGGGAGAAAGATTGACCTGAAGGCACACCACGGCTACGGCTCGCTGTCCGGTGCTCTGCAGAGCATGTTCCATGGCTTCTTGTCAGATGGCCATGGGAGGATTGCAACCAGAGAGGATGAAGAGCAGCTGGAATATCACAAGGGCAAGGGCACGATGAAGAACTACATCCTTCTGTACGAGGACAACGAGGGCGACCGCATGCTCGTCGGTGACGTTCCGTGGGAGCTGTTCGTCGCTTCGGTGAAGAGGCTCTACATTGCCAAGGACCCTAGAGCTGACAAAAGCAACAAGAAGTAG (SEQ ID NO: 1).

[0064] Protein sequence of wheat gene TaIAA25-7A: MLQTQVVVKYQSYSLGEAMKSSSVAPSLKQKQGDASKLQDGVPNNLELRLGISSDNGLSSGGGGATTPWLGVGVHPWSLSARQDKAALEQPQQRPNECPAHREDRPQLVGWPPVRTFRKNLSATSTRPAYSGDLSKV EPCYEEEDHGNINTGVSVQERPAMFVKVNLEGYAVGRKIDLKAHHGYGSLSGALQSMFHGFLSDGHGRIATREDEEQLEYHKGKGTMKNYILLYEDNEGDRMLVGDVPWELFVASVKRLYIAKDPRADKSNKK* (SEQ ID NO:2).

[0065] The coding region and amino acid sequence of the TaIAA25 gene on chromosome B are as follows: Nucleotide sequence of the coding region of wheat gene TaIAA25-7B: ATGAAGAGCTCTTCAGTTGCACCAAGCCTGAAACAGGAGCAAGGAGATGCTAGCAAACTGCAGGAAGGTGTCCTCAACAACCTTGAGCTTAGGCTTGGCATCTCTTCAGACAATGGCCTGAGTAGTGGTGGTGGTGGTGCTACTACTCCATGGCTTGGTGTTGGAGTGCACCCTTGGAGCCTGTCCGCCAGGCAAGACAAGGCGGCCCTGGAACAACCTCAGGAGAGGCCTAATGAGTGTCCTGCCCACAGAGAAGATCGCCCTCAACTGGTCGGATGGCCACCGGTGCGCACATTCCGCAAGAACCTGTCCGCCACATCCACAAGGCCTGCATATTCTGAAGACCTGAGTAAAGTGGAACACTGCTATGAAGAAGAAGACCATGGAAACATAAACACTGGAGTTGCAGTACAAGAGAGGCCGGCCATGTTCGTGAAGGTGAACCTGGAAGGGTACGCCGTTGGGAGAAAGATTGACCTGAATGCACACCGCGGCTACGGTTCGCTGTCTGGTGCTCTGCAGAGCATGTTCCATGGCTTCTTGTCAGATGGCCACGGGAGGATTGCAACCAGAGGGGGTGAAGAGCAGCTGGAATATCACAAGGGCAAGGGCACGATGAAGAACTACATCCTTCTGTACGAGGACAACGAGGGCGACCGCATGCTCGTCGGCGACGTTCCATGGGAGCTGTTCGTCGCTTCGGTAAAGAGGCTCTACATTGCCAGGGACCCTAGAGCTGACAAAAGCAATAAGAAGTAG (SEQ ID NO:3).

[0066] Protein sequence of wheat gene TaIAA25-7B: MKSSSVAPSLKQEQGDASKLQEGVLNNLELRLGISSDNGLSSGGGGATTPWLGVGVHPWSLSARQDKAALEQPQERPNECPAHREDRPQLVGWPPVRTFRKNLSATSTRPAYSEDLSKVEHCYEEEDH GNINTGVAVQERPAMFVKVNLEGYAVGRKIDLNAHRGYGSLSGALQSMFHGFLSDGHGRIATRGGEEQLEYHKGKGTMKNYILLYEDNEGDRMLVGDVPWELFVASVKRLYIARDPRADKSNKK* (SEQ ID NO:4).

[0067] The coding region and amino acid sequence of the TaIAA25 gene on chromosome D are as follows: Nucleotide sequence of the coding region of wheat gene TaIAA25-7D: ATGAAGAGCTCTTCAGTTGCACCAAGCCTGAAACAGAAGCAAGGAGATGCTAGCAAACTGCAGGAAGGTGTCCCCAACAACCTTGAGCTTAGGCTTGGCATCTCTTCAGACAATGGCCTGAGTAGTGGTGGTGGTGGTGCTACTACTCCATGGCTTGGTGTCGGAGTGCACCCTTGGAGCCTGTCCGCCAGGCAAGACAAGGCGGCCCTGGAACAACCTCAGCAGAGGCCTAATGAGTGTCCTGCCCACAGAGAAGATCGCCCTCAACTGGTCGGATGGCCACCGGTGCGCACATTCCGCAAGAACCTGTCCGCCACATCCACAAGGCCTGCATATTCTGAAGACCTGAGCAAAGTGGAACCCTGCTATGAAGAAGAAGACCATGGAAACATAAACACTGGAGTTTCAGTTCAAGAGGGGCCGGCCATGTTCGTGAAGGTGAACCTGGAAGGGTATGCCGTCGGGAGAAAGATTGACCTGAACGCACACTGTGGCTACGGCTCGCTGTCCGGTGCTCTGCAGAGCATGTTCCATGGCTTCTTGTCAGATGGCCATGGGAGGATTGTAACAAGAGAGGATGAAGAGCAGCTGGAATATCACAAGGGCAAGGGCACGGTGAAGAACTACATCCTTCTGTACGAGGACAACGAGGGCGACCGCATGCTCGTCGGCGACGTTCCATGGGAGCTGTTCGTCGCTTCGGTGAAGAGGCTCTACATTGCCAAGGACCCTAGAGCTGACAAAAGCAACAAGAAGTAG (SEQ ID NO:5).

[0068] Protein sequence of wheat gene TaIAA25-7D: MKSSSVAPSLKQKQGDASKLQEGVPNNLELRLGISSDNGLSSGGGGATTPWLGVGVHPWSLSARQDKAALEQPQQRPNECPAHREDRPQLVGWPPVRTFRKNLSATSTRPAYSEDLSKVEPCYEEEDH GNINTGVSVQEGPAMFVKVNLEGYAVGRKIDLNAHCGYGSLSGALQSMFHGFLSDGHGRIVTREDEEQLEYHKGKGTVKNYILLYEDNEGDRMLVGDVPWELFVASVKRLYIAKDPRADKSNKK* (SEQ ID NO:6).

[0069] Example 2: Analysis of Gene Expression Patterns 1. Select wheat seeds of the Fielder variety that are of uniform size and condition, disinfect them by soaking them in 1% sodium hypochlorite for 5 minutes, and then rinse them 5 times with clean water; after soaking them in 1 / 8 Hoagland culture solution for 12 hours, place them on moist filter paper; after 2-3 days of growth, transfer the wheat seedlings to black hydroponic boxes and culture them in 1 / 8 Hoagland culture solution under 16 hours of light / 8 hours of darkness for 7 days.

[0070] 2. The wheat seedlings cultured above were treated with 50 mM (Na2CO3:NaHCO3 = 1:9) alkaline salt. Sampling time points were 0, 1, 3, 6, 12, 24, 48, and 72 hours after treatment.

[0071] 3. RNA was extracted from wheat roots and then reverse transcribed to synthesize cDNA.

[0072] 4. Real-time quantitative PCR (RT-qPCR) was performed using the PerfectStart Green qPCR SuperMix kit (TransGen Biotech, #AQ601). The RT-qPCR primers for TaIAA25 are shown in SEQ ID NO:9-10. The TaGAPDH gene was used as an internal control.

[0073] TaIAA25-RT-F: ACGAGGACAACGAGGGCGAC (SEQ ID NO: 9); TaIAA25-RT-R:GCTTTTGTCAGCTCTAGGGTC (SEQ ID NO: 10).

[0074] The RT-qPCR reaction system is shown in Table 2.

[0075] Table 2 Reaction System

[0076] RT-qPCR reaction procedure: Pre-denaturation: 94℃, 4 min; (denaturation: 94℃, 10 sec; annealing: 56℃, 10 sec; extension: 72℃, 10 sec) × 40 cycles, with fluorescence numerical detection performed before each extension cycle.

[0077] RT-qPCR results are as follows Figure 1 As shown, the expression level of the TaIAA25 gene was significantly reduced under saline-alkali treatment compared to the control group.

[0078] Example 3: Construction of TaIAA25 gene knockout wheat lines To verify the function of the TaIAA25 gene, a gene editing vector, TaIAA25-7A / 7B / 7D (TraesCS7A03G0794900 / TraesCS7B03G0633300 / TraesCS7D03G0759100), was constructed using pBUE411. Due to the high homology among the three genes, a CRISPR / Cas9 dual-target design was employed targeting the conserved sequence of the TaIAA25 gene. The specific steps are as follows: 1. Design sgRNA targeting sequences on the first CDS of TaIAA25, the two sgRNA targeting sequences being shown in SEQ ID NO:7-8.

[0079] sgRNA target sequence 1: AACAACCTTGAGCTTAGGCTTGG (SEQ ID NO:7); sgRNA targeting sequence 2: CTTGGCATCTCTTCAGACAATGG (SEQ ID NO:8).

[0080] The sgRNA sequence was constructed into the plant expression vector pBUE411 using the NEB ridge Golden Gate assembly kit (NEW ENGLANGD Biolabs, #1602) to obtain pBUE411-TaIAA25.

[0081] 2. The pBUE411-TaIAA25 recombinant vector carrying sgRNA and the CRISPR / Cas9 system was transformed into Agrobacterium strain EHA105 for wheat genetic transformation. The transformation steps are as follows: a. Take 100 µL of Agrobacterium competent cells, place them at room temperature until they partially melt and leave small solids, then immediately place them on ice until they completely melt; b. Add 1 µL of the above plasmid to competent cells, tap it with your fingertip to mix, incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C metal bath for 5 min, and on ice for 5 min.

[0082] c. Add 1 mL of YEP medium and incubate at 28°C in a shaker for 2 h; d. Take 100 µL of the above bacterial suspension and spread it evenly on solid YEP medium containing rifampicin and kanamycin antibiotics; e. After incubating in an inverted incubator at 28℃ for 48 h, the single colonies that grew were identified by PCR, and then the positive clones were propagated and preserved.

[0083] 3. Agrobacterium containing the pBUE411-TaIAA25 recombinant vector, preserved as described above, was used to infect and genetically transform immature embryos of the wheat variety Fielder. Subsequently, wheat regenerated plants were obtained through callus culture, antibiotic screening, and differentiation. The obtained wheat plants were initially screened by PCR to select positive plants containing the transgenic fragment. Then, the target editing status was detected using the Hi-TOM platform, and mutants with premature termination of TaIAA25 were selected for subsequent Cas-free mutant screening.

[0084] 4. The transgenic vector fragment and TaIAA25 genomic mutation sites were identified through continuous self-crossing and PCR. Finally, homozygous loss-of-function mutants of TaIAA25 from chromosomes A, B, and D without transgenic components were screened and named iaa25-1. iaa25-2 was used for subsequent phenotypic identification. The mutation types of the knockout sites in the mutants are as follows: Figure 2 As shown.

[0085] Example 4: Phenotypic Identification of TaIAA25 Loss-of-Function Mutants 1. Select wheat varieties Fielder and iaa25 mutant seeds with uniform plumpness, wait for them to completely break dormancy, disinfect them by soaking in 1% sodium hypochlorite for 5 min, and then rinse them 5 times with clean water; after soaking in 1 / 8 Hoagland culture solution for 12 h, place them on moist filter paper; after 2-3 days of growth, transfer the wheat seedlings to black hydroponic boxes and culture them in 1 / 8 Hoagland culture solution under 16 h light / 8 h darkness conditions for 7 days.

[0086] 2. The wheat seedlings cultured above were treated with 50 mM (Na2CO3:NaHCO3 = 1:9) alkaline salt and a control treatment without alkaline salt were added. The light and temperature conditions were kept constant and the treatment solution was changed every day during the period. The alkaline treatment phenotype of the wheat seedlings was then statistically analyzed.

[0087] 3. Phenotypic characteristics of wheat seedlings after alkali treatment, as follows: Figure 3 As shown, under conditions without alkali treatment, there was no significant difference in fresh weight and total chlorophyll level between the iaa25 mutant and the control line. However, under 50 mM (Na2CO3:NaHCO3 = 1:9) alkaline salt treatment, the iaa25 mutant showed a significant increase in fresh weight and relative fresh weight compared to the control line, a significant decrease in leaf mortality, and a significant increase in total chlorophyll content. These results indicate that the iaa25 mutant exhibits stronger resistance to alkaline salt treatment compared to the control line, suggesting that knocking out TaIAA25 or its homologous genes (with nucleotide or protein sequences possessing 90% or more identity and functional consistency) or inhibiting their expression in wheat or other crops can enhance their salt tolerance.

[0088] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the TaIAA25 gene, wherein the application is any one of the following: (A1) Application in regulating plant salt and alkali tolerance; (A2) Application in cultivating salt-tolerant plants; (A3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (A4) Application in increasing plant fresh weight and relative fresh weight; (A5) Application in reducing plant leaf mortality; (A6) Application in increasing the total chlorophyll content of plants; (A7) Application in the improvement of germplasm resources related to salt tolerance; The TaIAA25 gene includes TaIAA25-7A, TaIAA25-7B, and TaIAA25-7D. The nucleotide sequence of TaIAA25-7A is as shown in SEQ ID NO:1 or has 90% or more of the same identity. The nucleotide sequence of TaIAA25-7B is as shown in SEQ ID NO:3 or has 90% or more of the same identity. The nucleotide sequence of TaIAA25-7D is as shown in SEQ ID NO:5 or has 90% or more of the same identity. The application is achieved by increasing or decreasing the expression level of the TaIAA25 gene.

2. Applications of proteins, wherein the application is any one of the following: (B1) Application in regulating plant salt and alkali tolerance; (B2) Application in cultivating salt-tolerant plants; (B3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (B4) Application in increasing plant fresh weight and relative fresh weight; (B5) Application in reducing plant leaf mortality; (B6) Application in increasing the total chlorophyll content of plants; (B7) Application in the improvement of germplasm resources related to salt tolerance; The proteins are protein 1, protein 2, and protein 3, wherein protein 1 is a protein with the amino acid sequence SEQ ID NO:2; protein 2 is a protein with the amino acid sequence SEQ ID NO:4; and protein 3 is a protein with the amino acid sequence SEQ ID NO:

6. The application can be achieved by upregulating or downregulating the content and / or activity of the protein.

3. The use of a substance for reducing the activity and / or content of the protein of claim 2 in any of the following: (C1) Application in improving the salt and alkali tolerance of plants; (C2) Application in cultivating salt-tolerant plants; (C3) Application in the preparation of products that improve the salt and alkali tolerance of plants; (C4) Application in increasing plant fresh weight and relative fresh weight; (C5) Application in reducing plant leaf mortality; (C6) Application in increasing the total chlorophyll content of plants; (C7) Application in the improvement of germplasm resources related to salt tolerance.

4. The application as described in claim 3, characterized in that, The application is achieved by downregulating the content and / or activity of the protein in the plant, the substance being achieved by causing the deletion or inactivation of the gene encoding the protein of claim 2 in the plant.

5. The application as described in claim 4, characterized in that, The substance is a nucleic acid molecule in which the coding gene of the protein described in claim 2 is deleted or inactivated through site-directed mutagenesis, gene knockdown, gene editing and / or gene knockout techniques.

6. The application as described in claim 5, characterized in that, The substance is a CRISPR / Cas9 system containing sgRNA, wherein the sgRNA targets the gene encoding the protein described in claim 2.

7. A method for cultivating salt-tolerant plants, characterized in that, The method includes reducing the content and / or activity of the protein described in claim 2 in the target plant to obtain a plant with increased salt tolerance and / or reduced plant height, wherein the plant is wheat.

8. The method as described in claim 7, characterized in that, The reduction of the content and / or activity of the protein of claim 2 in the target plant is achieved by reducing the expression level of the gene encoding the protein in the target plant.

9. The method as described in claim 8, characterized in that, The reduction of the expression level of the protein-coding gene in the target plant is performed using a CRISPR / Cas9 system, which includes sgRNA targeting the protein-coding gene of claim 2.

10. The method as described in claim 9, characterized in that, The target sequences of sgRNA are shown in SEQ ID NO:7 and SEQ ID NO:8.

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