Application of TaRHF2 protein and coding gene thereof in regulation and control of plant salt tolerance

Abstract

The invention discloses application of a TaRHF2 protein and a coding gene thereof in regulation and control of plant salt tolerance. The invention belongs to the technical field of biology, and particularly relates to application of TaRHF2 protein and a coding gene thereof to regulation and control of plant salt tolerance. The protein TaRHF2 or a substance for regulating and controlling the expression of the coding gene TaRHF2 of the protein or a substance for regulating and controlling the activity or content of the protein can be applied to any one of the following applications: 1) regulating and controlling the salt tolerance of plants; (2) preparing a product for regulating and controlling the salt tolerance of the plants; (3) cultivating plants with changed salt tolerance; (4) preparing a product for cultivating the plant with the changed salt tolerance; and 5) plant breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of TaRHF2 protein and its encoding gene in regulating plant salt tolerance. Background Technology

[0002] Salt stress is caused by high concentrations of Na in the soil. + and Cl － Salt stress, caused by oxidative stress, hinders the absorption of water and nutrients by plants and is one of the major abiotic stresses affecting plant development and yield. Salt stress causes oxidative stress, ion stress, osmotic stress, and secondary stress in plants; the adaptive response to salt stress can be divided into three processes: osmotic stress, ion stress, and detoxification response. Plant cell walls participate in the salt stress response. In Arabidopsis thaliana, leucine-rich repeat receptor kinases MIK2, FEI1, and FEI2 are key regulators of cell wall integrity maintenance and salt stress response. Glucuronyl transferase MOCA1, dependent on GIPC, senses environmental Na+. + Concentration changes, via GIPC sphingolipid binding of Na + Inducing cell membrane depolarization, via Ca 2+ Transport proteins AtANN1 and AtANN4 induce calcium influx. Genetic and biochemical analyses revealed several core signaling pathways involved in salt tolerance: the salt hypersensitivity (SOS) signaling pathway transports Na+... + Squeezed into the ectoplasm, it plays a key role in maintaining ion homeostasis; the mitogen-activated protein kinase (MAPK) cascade participates in the transduction of ion, osmotic and oxidative stress signals in response to salt stress in plants, participates in antioxidant defense responses and regulates reactive oxygen species (ROS) homeostasis to cope with salt stress; sucrose non-fermentative protein kinase (SnRK2) participates in maintaining osmotic homeostasis.

[0003] High salt content damages plants in five main ways: (1) It causes water stress. Excessive salt content leads to a drop in soil water potential, affecting the normal water absorption of plants, and in severe cases, it can lead to plant death. (2) It causes ion stress. For example, excessive Cl in the soil... － and Ca 2+ It can penetrate into plant cells, and the potassium inside the cells... + and H + Excreted into the soil, it inhibited K + and Ca 2+(3) Decreased photosynthetic rate. The biosynthesis of photosynthetic pigments is hindered, chlorophyll is destroyed, and the photosynthesis of plants decreases. (4) Inhibition of respiration. The disruption of ion balance leads to a decrease in enzyme activity, affecting respiration. (5) Accumulation of toxic substances. Excessive soil salt concentration leads to the accumulation of toxic metabolites in plants, such as peroxides and superoxides. These toxic substances damage the cell membrane system, inhibit plant biosynthesis, and disrupt ion balance.

[0004] To adapt to various adverse environmental stimuli, plants have evolved diverse mechanisms for responding to stress. Research has revealed that the ubiquitin-proteasome system plays a crucial role in a wide range of biological processes. The specificity of ubiquitination modification largely depends on the interaction between E3 ubiquitin ligases and their substrates; E3 ubiquitin ligases play a key role in substrate recognition within the ubiquitination pathway. RING E3 ubiquitin ligases play important roles in cellular processes, including regulating plant growth and development, hormone signal transduction, and stress responses. However, research on the role of RING E3 ubiquitin ligases (RHF) in abiotic stress responses in wheat is limited.

[0005] According to statistics from the Food and Agriculture Organization of the United Nations, more than 1 billion hectares of land are currently affected by salt. By 2050, global warming and freshwater scarcity will result in more than 50% of arable land being affected by salt, severely impacting global food security. Identifying and designing salt-tolerant crops is essential to addressing this challenge. Wheat is one of the world's most important crops, and ensuring stable wheat yields is crucial for ensuring food security. Therefore, discovering wheat salt-tolerant genes and elucidating the genetic basis of wheat salt tolerance are of great significance for the genetic improvement of wheat salt tolerance and molecular breeding. Summary of the Invention

[0006] The technical problem solved by this invention is how to regulate the salt tolerance of plants, especially wheat.

[0007] To address the aforementioned problems, the present invention provides applications related to proteins, substances that regulate the expression of genes encoding said proteins, or substances that regulate the activity or content of said proteins.

[0008] The use of the protein, the substance regulating the expression of the gene encoding the protein, or the substance regulating the activity or content of the protein provided by this invention in any of the following:

[0009] 1) Application in regulating plant salt tolerance;

[0010] 2) Application in the preparation of products that regulate plant salt tolerance;

[0011] 3) Application in cultivating plants with altered salt tolerance;

[0012] 4) Application in the preparation of products using plants with altered salt tolerance;

[0013] 5) Application in plant breeding.

[0014] The protein is any of the following proteins:

[0015] a1) Proteins with an amino acid sequence of SEQ ID No. 2, SEQ ID No. 5, or SEQ ID No. 8;

[0016] a2) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2, SEQ ID No. 5 or SEQ ID No. 8;

[0017] Proteins that have more than 80% identity and the same function of any of the amino acid sequences defined in (a3)a1)-(a2);

[0018] The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), and (a3).

[0019] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0020] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0021] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0022] Of the proteins mentioned above, SEQ ID No. 2 is the TARHF2D protein, which consists of 389 amino acid residues and is encoded by the TARHF2D gene.

[0023] SEQ ID No. 5 is the TARHF2A protein, which consists of 364 amino acid residues and is encoded by the TARHF2A gene.

[0024] SEQ ID No. 8 is the TARHF2B protein, which consists of 389 amino acid residues and is encoded by the TARHF2B gene.

[0025] In the above applications, the protein is derived from wheat (Triticum aestivum L.).

[0026] In this article, the TaRHF2 protein may specifically refer to proteins TaRHF2A, TARHF2B, or TARHF2D.

[0027] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, and the gene TaRHF2A, TARHF2B or TARHF2D encodes the protein TaRHF2A, TARHF2B or TARHF2D.

[0028] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0029] In this invention, the regulation can be increased, enhanced, or improved; the regulation can also be decreased, weakened, or reduced.

[0030] In this article, the enhancement, increase or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0031] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0032] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence, including but not limited to zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a site in the genome containing multiple short repeat sequences, where the Cas9 protein, mediated by RNA, can cleave target sequences recognized by crRNA–tracrRNA.

[0033] In one specific embodiment, the gene knockout is achieved through CRISPR / Cas9 gene editing technology. The CRISPR / Cas9 system utilizes the Cas9 nuclease, guided by sgRNA, to cleave target sites, resulting in gene knockout during the repair of broken DNA, ultimately achieving targeted gene editing.

[0034] The gene editing vector used is a CRISPR vector containing two gRNAs. The intermediate vector pCBC-MT1T2 and the gene editing vector pBUE411 are used for vector construction. The pBUE411 vector is digested with BSAI enzyme.

[0035] In the above applications, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein can be a biological material related to the protein described above, and the biological material can be any of the following:

[0036] c1) The nucleic acid molecule that encodes the protein described above;

[0037] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0038] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0039] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0040] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0041] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0042] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0043] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0044] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0045] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0046] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0047] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0048] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0049] e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0050] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0051] d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No. 3, SEQ ID No. 6, or SEQ ID No. 9;

[0052] d2) The coding sequence is a DNA molecule shown in SEQ ID No. 1, SEQ ID No. 4, or SEQ ID No. 7;

[0053] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0054] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0055] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0056] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vectors pCAMBIA1302, pCBC-MT1T2, pBUE411, and pWMB110.

[0057] Recombinant expression vectors containing the TaRHF2 gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmids (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0058] When constructing recombinant plant expression vectors using the TaRHF2 gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0059] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0060] The present invention also provides a method for altering the salt tolerance of plants, the method comprising the following steps M or P:

[0061] Step M is to enhance, increase or upregulate the activity and / or content of the proteins mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the proteins mentioned above, so as to improve the salt tolerance of the plant.

[0062] The method includes step P, which is to inhibit or reduce or silence the activity and / or content of the aforementioned protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned protein, in order to reduce the plant's salt tolerance.

[0063] In the above method, reducing the expression level and / or activity of the gene encoding the protein TaRHF2 in the target plant can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown techniques to reduce or inactivate the gene encoding the protein TaRHF2 in the genome of the target plant.

[0064] The present invention also provides a method for cultivating highly salt-tolerant plants, comprising upregulating or enhancing or increasing the expression level of the coding gene of the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a highly salt-tolerant plant, wherein the salt tolerance of the highly salt-tolerant plant is higher than that of the target plant.

[0065] In one specific embodiment, the upregulation, enhancement, or increase of the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a highly salt-tolerant plant.

[0066] The present invention also provides a method for cultivating low-salt-tolerant plants, comprising inhibiting, reducing or silencing the expression level of the encoding gene of the protein described above in the target plant, and / or, the activity and / or content of the protein to obtain a low-salt-tolerant plant, wherein the low-salt-tolerant plant has weaker salt tolerance than the target plant.

[0067] In one specific embodiment, inhibiting, reducing, or silencing the expression of the gene encoding the protein described above in the plant includes introducing the nucleic acid molecule, expression cassette, or recombinant vector described above into the target plant to obtain a low-salt-tolerance plant.

[0068] In this article, the purpose of breeding includes cultivating plants with high salt tolerance; the purpose of breeding also includes cultivating plants with low salt tolerance.

[0069] The highly salt-tolerant plants showed increased salt tolerance compared to the target plants. The low-salt-tolerant plants showed decreased salt tolerance compared to the target plants.

[0070] The improved salt tolerance is manifested in any one of the following (1)-(3):

[0071] (1) Under salt stress, highly salt-tolerant plants grow better than recipient plants;

[0072] (2) Under salt stress, the chlorophyll content of highly salt-tolerant plants is higher than that of recipient plants;

[0073] (3) Under salt stress, highly salt-tolerant plants have a stronger ability to scavenge reactive oxygen species than receptor plants.

[0074] (4) Under salt stress, the ion permeability of highly salt-tolerant plants is lower than that of receptor plants.

[0075] The reduced salt tolerance manifests itself in any one of the following (1)-(3):

[0076] (1) Under salt stress, the growth of gene-edited plants was worse than that of recipient plants;

[0077] (2) Under salt stress, the chlorophyll content of gene-edited plants is lower than that of recipient plants;

[0078] (3) Under salt stress, the ion permeability of gene-edited plants is higher than that of recipient plants.

[0079] The proteins and / or the biological materials mentioned above are also within the scope of protection claimed in this invention.

[0080] In the above applications or methods, the plant may be any of the following:

[0081] C1) Monocotyledons;

[0082] C2) Plants of the order Poales;

[0083] C3) Gramineae plants;

[0084] C4) Plants of the genus Triticum;

[0085] C5) Wheat.

[0086] The wheat variety in question is Fielder.

[0087] Experiments of this invention demonstrate that transgenic Arabidopsis thaliana exhibits enhanced salt tolerance compared to wild-type Arabidopsis. Compared to the recipient wheat Fielder, wheat transgenic with the TaRHF2 gene shows enhanced salt tolerance; however, after editing the TaRHF2 gene, the salt tolerance of the mutant wheat is weakened compared to the recipient wheat Fielder. The proteins and genes provided by this invention will play an important role in cultivating plants with enhanced salt tolerance. Attached Figure Description

[0088] Figure 1 Analysis of the expression pattern of the TaRHF2 gene under salt stress.

[0089] Figure 2 To identify positive lines of Arabidopsis thaliana overexpressing TaRHF2. A: Positive detection, Marker: D2000, 1-6: Arabidopsis thaliana overexpressing lines; B: qRT-PCR analysis of expression levels in the overexpressing Arabidopsis thaliana lines. WT, Columbia-0.

[0090] Figure 3 Germination experiments of Arabidopsis thaliana overexpressing TaRHF2 under salt treatment. A represents seed germination phantom morphology; B represents germination rate. WT, Columbia-0.

[0091] Figure 4 Salt tolerance was assessed in Arabidopsis thaliana overexpressing TaRHF2. The following phenotypes were considered: A) root length; B) total root length; C) fresh weight; D) phenotype under soil salt stress; E) survival rate; and F) chlorophyll content. WT and Columbia-0 were used.

[0092] Figure 5To identify positive lines of wheat overexpressing TaRHF2. A: Positive detection, Marker: D2000, OE-1-OE-11: Overexpressing wheat lines, +: Positive plasmid, -: Fielder; B: qRT-PCR analysis of expression levels in overexpressing wheat lines. WT: Fielder.

[0093] Figure 6 Phenotypic identification and index determination of TaRHF2-overexpressing wheat under hydroponic salt stress. A. Phenotypic under salt treatment; B. Proline content; C. Malondialdehyde content; D. Hydrogen peroxide content. WT: Fielder.

[0094] Figure 7 Phenotypic identification and index determination of TaRHF2-overexpressing wheat under soil-cultured salt stress. A. Phenotyps under soil salt stress; B. Survival rate; C. Chlorophyll content; D. Ion permeability. WT: Fielder.

[0095] Figure 8 These are the mutation types in wheat edited with the TaRHF2 gene. Cri-TaRHF2-1, Cri-TaRHF2-2, and Cri-TaRHF2-3 represent triple-mutant lines with mutations in all three genomes.

[0096] Figure 9 Phenotypic identification and index determination of TaRHF2 gene-edited wheat under salt stress. A. Phenotyps under soil salt stress; B. Survival rate; C. Chlorophyll content; D. Ion permeability. WT: Fielder. Detailed Implementation

[0097] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0098] 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.

[0099] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0100] The wheat variety 'Xiaobaimai', Arabidopsis thaliana Columbia-0, and cloning vector pEASY-T1 used in the following examples are described in: Cui XY, Du YT, Fu JD, Yu TF, Wang CT, Chen M, Chen J, Ma YZ, Xu ZS. 2018. Wheat CBL-interacting protein kinase 23 positively regulates drought stress and ABA responses. BMC Plant Biology. doi:10.1186 / s12870-018-1306-5. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention; it may not be used for any other purpose.

[0101] The wheat variety Fielder described in the following examples is described in: Cui XY, Gao Y, Guo J, Yu TF, Zheng WJ, Liu YW, Chen J, Xu ZS, Ma YZ.2019.BES / BZR Transcription Factor TaBZR2Positively Regulates Drought Responses by Activation of TaGST1.PlantPhysiology.doi:10.1104 / pp.19.00100. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0102] The plant eukaryotic expression vector pCAMBIA1302 and subcellular localization vector p16318GFP used in the following examples were preserved in our laboratory and are documented in: Ru JN, Hou ZH, Zheng L, Zhao Q, Wang FZ, Chen J, Zhou YB, Chen M, Ma YZ, Xi YJ and Xu ZS. 2021. Genome-Wide Analysis of DEAD-box RNA Helicase Family in Wheat (Triticum aestivum) and Functional Identification of TaDEAD-box57 in Abiotic Stress Responses. Frontiers in Plant Science. doi:10.3389. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0103] The plant eukaryotic expression vector pWMB110 used in the following examples was preserved in our laboratory and is documented in: Liu Y, YuTF, Li YT, Zheng L, Lu ZW, Zhou YB, Chen J, Chen M, Zhang JP, Sun GZ, Cao XY, Liu YW, Ma YZ, Xu ZS. 2022. Mitogen-activated protein kinase TaMPK3 suppresses ABA response by destabilizing TaPYL4 receptor in wheat. New Phytologist. doi:10.1111 / nph.18326. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0104] The gene editing vectors pCBC-MT1T2 and pBUE411 used in the following examples were preserved in our laboratory and are documented in: Wang N, Chen J, Gao Y, Zhou YB, Chen M, Xu ZS, Fang ZW, Ma YZ. 2022. Genomic analysis of isopentenyltransferase genes and functional characterization of TaIPT8 indicates positive effects of cytokinins on drought tolerance in wheat. The Crop Journal. doi:10.1016 / j.cj.2022.04.010. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0105] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0106] Example 1: Cloning of the TaRHF2 gene and expression characteristics of TaRHF2

[0107] 1. Cloning of the TaRHF2 gene

[0108] Total RNA was extracted from wheat (wheat) that had grown normally for one week using a plant total RNA extraction kit from Tiangen Biotech. First-strand cDNA was synthesized using a TransGen first-strand cDNA synthesis kit from TransGen Biotech. Using the wheat cDNA as a template, PCR amplification was performed using the specific primer set TaRHF2-F / TaRHF2-R. After gel recovery, ligation into a cloning vector, bacterial culture PCR detection, and sequencing alignment, the correct TaRHF2 gene was obtained.

[0109] The specific primers for amplification are:

[0110] TaRHF2-F: 5'-GGTTGTGAACAGTCGATTCCA-3';

[0111] TaRHF2-R: 5'-GCCTTCTCAGAAACAGTAGCAT-3'.

[0112] The genomic sequence of the TaRHF2D gene is SEQ ID No. 3, and the coding sequence is SEQ ID No. 1. The protein encoded by this gene is named TaRHF2 protein, which consists of 389 amino acids. The amino acid sequence of this protein is SEQ ID No. 2 in the sequence listing.

[0113] The genomic sequence of the TaRHF2A gene is SEQ ID No. 6, and the coding sequence is SEQ ID No. 4. The protein encoded by this gene is named TaRHF2A protein, which consists of 364 amino acids. The amino acid sequence of this protein is SEQ ID No. 5 in the sequence listing.

[0114] The genomic sequence of the TaRHF2B gene is SEQ ID No. 9, and the coding sequence is SEQ ID No. 7. The protein encoded by this gene is named TaRHF2B protein, which consists of 389 amino acids. The amino acid sequence of this protein is SEQ ID No. 8 in the sequence listing.

[0115] 2. Real-time quantitative PCR analysis of TaRHF2 expression characteristics

[0116] 1) Different stress treatments on wheat

[0117] White wheat seedlings were planted in nutrient soil and grown at 22℃ for one week. Different stress treatments were then applied to the seedlings. The roots of the wheat seedlings were immersed in a 250mM NaCl solution to induce salt stress. Samples were taken at 0, 0.5, 1, 2, 4, 6, 12, and 24 hours after treatment and stored at -80℃ for later use.

[0118] 2) Real-time quantitative PCR

[0119] Total RNA was extracted from wheat using the plant total RNA extraction kit from Tiangen Biotech, and first-strand cDNA was synthesized using the first-strand cDNA synthesis kit from TransGen Biotech. The cDNA from different stress treatments was appropriately diluted and used as a template. Wheat Actin was used as an internal reference gene, and the relative expression level of the TaRHF2 gene under different stress treatments was analyzed using real-time quantitative PCR.

[0120] Amplification was performed using an ABI 7500 Real-Time PCR instrument, employing a three-step PCR reaction program: 95℃ for 15 min pre-denaturation (to fully activate the hot-start enzyme); 95℃ for 10 s, 55℃ for 30 s, and 72℃ for 32 s (to collect fluorescence signals), for 40 cycles. Data processing was performed after the reaction using 2... -ΔΔCt The algorithm was used to create a bar chart using Excel. Three biological replicates were performed.

[0121] The primers used to detect Actin gene expression are as follows:

[0122] Actin-F: 5'-GCCATGTACGTCGCAATTCA-3';

[0123] Actin-R: 5'-AGTCGAGAACGATACCAGTAGTACGA-3'.

[0124] The primers used to detect TaRHF2 gene expression are as follows:

[0125] RT-TaRHF2-F: 5'-TCCTGCTCTTGGTGATTTCG-3';

[0126] RT-TaRHF2-R: 5'-ATGTTGGTGTGACCTTCCCAT-3'.

[0127] See results Figure 1 After salt stress treatment, the expression level of TaRHF2 gene showed a trend of first increasing and then decreasing, with the highest expression level being upregulated by more than 5 times.

[0128] Example 2: Obtaining and salt tolerance identification of Arabidopsis thaliana overexpressing TaRHF2.

[0129] I. Construction of Recombinant Expression Vectors

[0130] 1. Adapter primers were designed based on the restriction enzyme sites of the pCAMBIA1302 vector to amplify the target gene with the adapter from the constructed pEASY-T1 cloning vector. PCR amplification was performed using primers 1302-TaRHF2-F and 1302-TaRHF2-R to obtain the amplification products, which were then recovered from the gel.

[0131] 1302-TaRHF2-F:5'-GGACTCTTGA CCATGG ATGGCATCTGGAACT-3';

[0132] 1302-TaRHF2-R: 5'-TCAGATCTAC CCATGG ATTTGAACCAGACGT-3'.

[0133] 2. Digest the vector pCAMBIA1302 with the restriction endonuclease NcoI and recover the digested vector.

[0134] 3. The PCR product recovered in step 1 and the vector digested in step 2 were subjected to homologous recombination using In-Fusion technology. After correct sequencing, the recombinant plasmid pCAMBIA1302-TaRHF2 was obtained.

[0135] The recombinant plasmid pCAMBIA1302-TaRHF2 is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 1 into the NcoI restriction site of the pCAMBIA1302 vector while keeping the other sequences of the pCAMBIA1302 vector unchanged. It is named recombinant plasmid pCAMBIA1302-TaRHF2.

[0136] II. Obtaining Arabidopsis thaliana through overexpression

[0137] 1. The recombinant plasmid pCAMBIA1302-TaRHF2 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

[0138] 2. After completing step 2, transfer the bacterial culture to liquid YEP medium containing 50 μg / L rifampin and 50 μg / L kanamycin, and incubate at 28°C with shaking at 300 rpm until OD reaches 100%. 600nm =1.5-3.0.

[0139] 3. After completing step 3, collect the bacterial cells by centrifugation at 4℃ and 4000g for 10 min, resuspend the bacterial cells in the infection solution, and adjust the OD600 value to 0.6-0.8 (infection solution formula: 1 / 2 MS, 10mM MgCl2, 1.0g / L MES, 5% sucrose, 100μl / L Silwet L-77; prepare in advance and let stand at room temperature for 3h).

[0140] 4. Lay the Arabidopsis flowerpot down so that the inflorescence is completely submerged in the infection solution for 3 minutes. Then lay the Arabidopsis plant flat, cover it with a black plastic bag, and let it grow in the dark for 24 hours. The next day, remove the plastic bag, place the Arabidopsis upright, and let it grow under normal light conditions. After one week, immerse it in the solution again.

[0141] 5. Sow the seeds of T0 generation Arabidopsis thaliana on solid MS medium plates containing 50 mg / L hygromycin. Select Arabidopsis thaliana seedlings that can grow normally and transfer them to soil for growth. Harvest individual plants after maturity to obtain the T1 generation. Continue screening until the T3 generation homozygous Arabidopsis thaliana is obtained.

[0142] 6. The expression levels of different strains were detected by real-time quantitative PCR. The strain with the highest expression level was selected for subsequent phenotypic experiments.

[0143] See results Figure 2 In sections A and B: Specific bands were detected in both Arabidopsis thaliana positive lines, and the expression levels of the overexpressing Arabidopsis thaliana lines were more than 2 times higher than those of wild-type Arabidopsis thaliana (WT, Columbia-0). The Arabidopsis thaliana lines with the highest expression levels were selected and named OE-1, OE-2, and OE-3 for subsequent experimental analysis.

[0144] III. Salt Tolerance Identification of Arabidopsis thaliana

[0145] 1. For the germination experiment, wild-type and transgenic Arabidopsis seeds harvested from the same batch and with plump grains were selected. After sterilization, the seeds were sown separately on MS medium and MS medium containing 125 mM or 150 mM NaCl, with aseptic operation throughout the process. After vernalization at 4℃ for 3 days, they were transferred to a 22℃ light incubator with 16h light / 8h darkness and 60% relative humidity for growth. Germination was defined as the emergence of white seeds, and the germination rate was calculated over the next 6 days.

[0146] See results Figure 3 In A and B: Under normal conditions, there was no significant difference in germination between TaRHF2 overexpression and wild-type Arabidopsis thaliana; after salt treatment, the germination of both TaRHF2 overexpression and wild-type Arabidopsis thaliana was inhibited, but the germination rate of TaRHF2 overexpression Arabidopsis thaliana was significantly higher than that of wild-type Arabidopsis thaliana.

[0147] 2. After sterilizing the Arabidopsis seeds harvested from the same batch, they were sown on MS medium. Then, Arabidopsis with uniform growth were selected and transferred to MS medium and MS medium containing 100 mM NaCl or 125 mM NaCl, and allowed to grow vertically. The root system was scanned using an Expression 11000XL instrument, and the total root length was counted. The fresh weight of the plants was measured using an electronic balance. Three-week-old Arabidopsis were used for soil salt tolerance assessment. After 7 days of growth on MS medium, the sterilized seeds were transplanted into pots with a 1:1 ratio of potting soil and vermiculite (watered to saturation) and placed in a controlled greenhouse at 22°C for 2 weeks. Arabidopsis grown in the soil for 2 weeks were then watered with a 200 mM NaCl solution for salt tolerance assessment until significant differences were observed between transgenic and wild-type Arabidopsis, and the survival rate was calculated. Chlorophyll content was measured during the stress period.

[0148] See results Figure 4 In the AF study: Under normal growth conditions, there was no significant difference in total root length and fresh weight between TaRHF2-overexpressing and wild-type Arabidopsis thaliana; after salt treatment, the root growth of TaRHF2-overexpressing Arabidopsis thaliana was stronger than that of wild-type Arabidopsis thaliana. Under normal growth conditions, there was no significant difference in growth vigor between TaRHF2-overexpressing and wild-type Arabidopsis thaliana; after salt treatment, wild-type Arabidopsis thaliana showed more leaf yellowing, while TaRHF2-overexpressing Arabidopsis thaliana showed relatively better growth. After salt treatment, the survival rate and chlorophyll content of TaRHF2-overexpressing Arabidopsis thaliana were significantly higher than those of wild-type Arabidopsis thaliana.

[0149] Example 3: Obtaining and Salt Tolerance Identification of Wheat Overexpressing TaRHF2

[0150] I. Construction of Recombinant Expression Vectors

[0151] 1. Adapter primers were designed based on the restriction enzyme sites of the pWMB110 vector to amplify the target gene with the adapter from the constructed zero-background cloning vector. PCR amplification was performed using 110-TaRHF2-F and 110-TaRHF2-R primers to obtain the amplification products, which were then recovered from the gel.

[0152] 110-TaRHF2-F:5'-CGACTCTAGA GGATCC ATGGCATCTGGAACT-3';

[0153] 110-TaRHF2-R:5'-GGGTACCCGG GGATCC CTAATTTGAACCAGA-3'.

[0154] 2. Digest the vector pWMB110 with the restriction endonuclease BamHI and recover the digested vector.

[0155] 3. The PCR product recovered in step 1 and the vector digested in step 2 were subjected to homologous recombination using In-Fusion technology. After correct sequencing, the recombinant plasmid pWMB110-TaRHF2 was obtained.

[0156] The recombinant plasmid pWMB110-TaRHF2 is a recombinant vector obtained by inserting the DNA molecule shown in SEQ ID No. 1 into the BamHI restriction site of the pWMB110 vector while keeping the other sequences of the pWMB110 vector unchanged. It is named recombinant plasmid pWMB110-TaRHF2.

[0157] II. Agrobacterium-mediated wheat transformation

[0158] Prepare wheat embryos in advance and use Agrobacterium-mediated transformation technology to transform recombinant plasmids into the recipient Fielder.

[0159] 1. Transform Agrobacterium EHA105 with the constructed recombinant plasmid pWMB110-TaRHF2.

[0160] 2. Transfer the bacterial culture to liquid YEP medium containing 50 μg / L rifampin and 50 μg / L kanamycin, and incubate at 28°C with shaking at 300 rpm until OD reaches zero. 600nm =1.5-3.0.

[0161] 3. Sterilize the surface of wheat seeds, remove the wheat embryos under a microscope, incubate them at room temperature with Agrobacterium infection solution (containing acetylsuccinone) carrying pWMB110-TaRHF2 for 5 minutes, and grow them on WLS-AS medium in the dark at 25°C for 2 days with the scutellum facing upwards.

[0162] 4. The radicle was surgically removed and transferred to WLS-Res medium to resume growth. After 5 days, the tissue was transferred to callus medium to induce growth on WLS-P5 medium. After 2 weeks, the callus was transferred to WLS-P10 medium for 3 weeks of growth.

[0163] 5. Transfer the regenerated shoots to cups containing MSF-P5 medium for elongation and rooting. After 9 days, transplant the well-developed rooted plants into flowerpots to continue growing.

[0164] III. Obtaining wheat overexpression

[0165] Overexpressing wheat lines were identified using a combination of PCR positive detection and qRT-PCR analysis.

[0166] 1. DNA was extracted using the CTAB method. PCR positive detection was performed using 2×Rapid Taq Master Mix. Recipient wheat and water served as negative controls, and the plasmid as a positive control. Single plants with correct sequencing were harvested as different lines. T1 generation seeds were planted according to these lines, and positive detection was performed again to harvest positive T2 generation seeds. T2 generation seeds were then planted again, and after positive detection, homozygous positive lines were obtained.

[0167] The primers used for positive detection are as follows:

[0168] 110-JC-F: 5'-TTTAGCCCTGCCTTTCATACG-3';

[0169] 110-JC-R: 5'-CATCTCATAAATAACGTCATGC-3'.

[0170] 2. RNA was extracted from different positive lines and reverse transcribed into cDNA. The expression levels of different lines were detected by real-time quantitative PCR, and the three positive lines with the highest expression levels (OE-1, OE-2, and OE-3) were selected for further experiments.

[0171] See results Figure 5 In both A and B, specific bands were detected in the positive wheat lines, but no specific bands were detected in the recipient wheat (WT, Fielder). Furthermore, the expression levels of the overexpressing wheat lines were more than 2 times higher than those of the recipient wheat (WT, Fielder). The wheat lines with the highest expression levels were selected and named OE-1, OE-2, and OE-3 for subsequent experimental analysis.

[0172] IV. Salt tolerance assessment of wheat overexpression

[0173] Test samples: seeds of OE-1, OE-2 and OE-3, and wheat Fielder (WT) seeds as the control.

[0174] 1. Hydroponic salt treatment

[0175] Wheat seeds harvested from the same batch and with plump grains were selected, soaked in 0.7% H2O2 solution for 12 hours, rinsed three times with water, and placed in a 4℃ refrigerator for 2 days. They were then grown in a 22℃ light incubator with 16 hours of light / 8 hours of darkness and 60% relative humidity. Three-day-old overexpressing and recipient wheat seeds were transferred to 96-well boxes containing Hoagland's nutrient solution for growth, followed by treatment with 300 mM NaCl, and phenotype was observed. Relevant physiological indicators, including proline (Pro), malondialdehyde (MDA), and hydrogen peroxide (H2O2) content, were measured according to the kit instructions from Suzhou Keming Biotechnology Co., Ltd. Three replicates were set up.

[0176] See results Figure 6 In the study of TaRHF2 overexpression and recipient wheat, under normal growth conditions, there was no significant difference in growth vigor between the two wheat species. Salt treatment inhibited the growth of both, but the overexpressing wheat showed stronger growth than the recipient wheat. Indicator measurements showed that under normal conditions, there were no significant differences in proline, malondialdehyde (MDA), and hydrogen peroxide content between the overexpressing and recipient wheat species. After salt treatment, the proline content of the overexpressing wheat was higher than that of the recipient wheat, while the MDA and hydrogen peroxide contents were lower.

[0177] 2. Phenotypic analysis under soil salt stress

[0178] First, select small red pots of uniform size and fully saturate them with potting soil, weighing out an equal amount of potting soil for each pot. Then, select wheat seeds from the same batch, ensuring plump grains for both the overexpression and recipient crops, and sow the same number of seeds in each pot. After a period of normal growth, irrigate with a 250 mM NaCl solution for salt stress treatment and observe the phenotype. During the stress treatment, samples were taken to measure chlorophyll content and ion permeability, and the survival rate (%) was recorded. Three replicates were set up.

[0179] Chlorophyll content determination: Weigh 0.1g of leaf, cut into small pieces, place in a 10ml EP tube, add 10ml of 80% acetone solution, and extract in the dark for 12h until the tissue turns completely white, indicating complete extraction. Take 200μl of the extract into a 96-well plate, zero the extract, and measure the absorbance at 663nm and 645nm, recording them as A663 and A645. Chlorophyll content (mg / g fresh weight) = (20.21×A645+8.02×A663)×Vextract×D÷m÷1000 = 0.01×(20.21×A645+8.02×A663)×D÷m. Vextract: extraction volume, 10ml; D: dilution factor; m: sample mass, 0.1g.

[0180] Ion permeability determination: Weigh 0.1g of leaf, cut it into small pieces, put it into a 10ml EP tube, add 5ml of distilled water, let it stand at room temperature for 30min, and measure it with a conductivity meter, which is recorded as V0; after soaking the leaf for 10h, measure it with a conductivity meter, which is recorded as V1; after boiling it in boiling water for 30min, cool it to room temperature, measure it with conductivity, which is recorded as V2; Ion permeability (%) = (V1-V0) / (V2-V0)×100.

[0181] See results Figure 7 In the study of TaRHF2 overexpression and recipient wheat, under normal growth conditions, there was no significant difference in growth between the two wheat species. After 7 days of salt treatment, all plants exhibited chlorosis and wilting, but the recipient wheat showed more severe chlorosis and wilting, and the overexpression wheat showed stronger growth than the recipient wheat. The phenotypes became more pronounced after 9 days of salt treatment, and the survival rate of the overexpression wheat was significantly higher than that of the recipient wheat. The results of index measurements showed that under normal conditions, there was no significant difference in chlorophyll content and ion permeability between the overexpression and recipient wheat. After salt treatment, the chlorophyll content of the overexpression wheat was significantly higher than that of the recipient wheat, while the ion permeability was significantly lower.

[0182] Example 4: Obtaining CRISPR gene-edited wheat and identifying its salt tolerance

[0183] I. Construction of gene-editing wheat vector

[0184] By using the CRISPR / Cas9 system to directionally edit the wheat genome, gene-edited wheat material can be obtained.

[0185] 1. Design two GuideRNAs (gRNAs) on the website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR / CRISPR. Note that the gRNAs should not have any variation among the three genomic sequences and the CDS sequence of wheat, the gRNAs should not cross introns, the sequences with higher fractions should be selected, and the GC content should be 50%-60%.

[0186] The target sequences of sgRNA1 are: 5'-TTCTCAGTGCCCTATGTGC-3', with the target site located in the 4th exon region of the TaRHF2A gene (nucleotides 7332-7350 of SEQ ID No. 6, corresponding to nucleotides 201-219 of SEQ ID No. 4); the target site is located in the 5th exon region of the TaRHF2B gene (nucleotides 3038-3056 of SEQ ID No. 9, corresponding to nucleotides 201-219 of SEQ ID No. 7); and the 3rd exon region of the TaRHF2D gene (nucleotides 1747-1765 of SEQ ID No. 3, corresponding to nucleotides 201-219 of SEQ ID No. 1).

[0187] The target sequence of sgRNA2 is 5'-TAGCTGCTGCTGCAGCAAT-3', with the target site located in the 6th exon region of the TaRHF2A gene (nucleotides 7835-7853 of SEQ ID No. 6, corresponding to nucleotides 413-431 of SEQ ID No. 4); the target site is located in the 6th exon region of the TaRHF2B gene (nucleotides 3865-3883 of SEQ ID No. 9, corresponding to nucleotides 413-431 of SEQ ID No. 7); and the target site is located in the 5th exon region of the TaRHF2D gene (nucleotides 2305-2323 of SEQ ID No. 3, corresponding to nucleotides 413-431 of SEQ ID No. 1).

[0188] 2. Design specific primers based on the two gRNAs for subsequent vector construction.

[0189] 3. Using TaRHF2-F0 and TaRHF2-R0, PCR amplification was performed with pCBC-MT1T2 as a template, and amplification product 1 was recovered. Amplification product 1 was then amplified by PCR using TaRHF2-F1 and TaRHF2-R1, and amplification product 2 was recovered.

[0190] 4. The amplification product 2 and the PUBE411 vector were digested with the restriction endonuclease BSAI, and the digestion products 3 and 4 were recovered. The recovered fragments and the digestion vector (digestion products 3 and 4) were ligated with T4 ligase.

[0191] 5. Transformed into competent intestinal Trans-T1 cells, and PCR detection was performed using TaRHF2-F2 and TaRHF2-R2 primers. After sequencing, the wheat editing vector PUBE411-sgRNA1-sgRNA2 containing two gRNAs was finally obtained.

[0192] The structure of the vector PUBE411-sgRNA1-sgRNA2 is described as follows: The recombinant vector obtained by inserting the DNA molecule of SEQ ID No. 10 into the BSAI site of the vector PUBE411 while keeping the other sequences of the PUBE411 vector unchanged is named recombinant plasmid PUBE411-sgRNA1-sgRNA2.

[0193] The primers used for constructing CRISPR gene editing vectors are as follows:

[0194] TaRHF2-F0: 5'-GTTCTCAGTGCCCTATGTGCGTTTTAGAGCTAGAAATAGC-3';

[0195] TaRHF2-R0: 5'-ATTGCTGCAGCAGCAGCTACGCTTCTTGGTGCC-3';

[0196] TaRHF2-F1: 5'-AATAATGGTCTCAGGCGTTCTCAGTGCCCTATGTGC-3';

[0197] TaRHF2-R1: 5'-ATTATTGGTCTCTAAACATTGCTGCAGCAGCAGCTA-3';

[0198] TaRHF2-F2: 5'-TTCTCAGTGCCCTATGTGC-3';

[0199] TaRHF2-R2: 5'-ATTGCTGCAGCAGCAGCTA-3'.

[0200] II. Agrobacterium-mediated wheat transformation

[0201] Prepare wheat embryos in advance and use Agrobacterium-mediated transformation technology to transform recombinant plasmids into the recipient Fielder.

[0202] III. Obtaining Gene-Edited Wheat

[0203] 1. DNA was extracted from the obtained T0 generation transformed seedlings using the CTAB method. PCR positive detection was performed using 2×Rapid Taq Master Mix with primers Cas9-F and Cas9-R. Wheat and water served as controls. Specific primers containing gRNA from the A, B, and D genomes were designed. PCR amplification was performed on positive plants, and the specific amplification products were sequenced. The gene editing status of the wheat was assessed based on the sequencing results, and T1 generation seeds were harvested.

[0204] The primers used for positive detection are as follows:

[0205] Cas9-F: 5'-GGCCTCGATATTGGGACTAACT-3';

[0206] Cas9-R: 5'-TGGCCCCTGAACTTAATCATGT-3'.

[0207] The three genomic primers used for specific amplification are as follows:

[0208] Crispr-TaRHF2A-F: 5'-AGAGCTTCTTGAGGCAGTACAGT-3';

[0209] Crispr-TaRHF2A-R: 5'-GGCGGCCATGAGAGCCAGAA-3';

[0210] Crispr-TaRHF2B-F: 5'-TCCCTCCGTCCCATATTAGGGCT-3';

[0211] Crispr-TaRHF2B-R: 5'-GTCAGCACCTTCACCCTGAGCA-3';

[0212] Crispr-TaRHF2D-F: 5'-TGTATGTATTCTGGAACTAATTG-3';

[0213] Crispr-TaRHF2D-R: 5'-CTGAATTGCATAAAAGGTTAAGCC-3'.

[0214] 2. The edited wheat materials were propagated, and DNA was extracted from a small number of leaves. First, Cas9 primers were used for detection, followed by amplification using specific primers containing gRNA from the three genomes. The amplified genomes were then sequenced to check the gene editing status. The genome of each generation of plants was amplified and sequenced until homozygous single-mutant, double-mutant, and triple-mutant wheat materials were obtained. Due to the redundancy of wheat homologous genes, subsequent experiments used homozygous triple-mutant materials (Cri-1, Cri-2, and Cri-3) for verification.

[0215] See results Figure 8 Cri-TaRHF2-1, Cri-TaRHF2-2, and Cri-TaRHF2-3 (Cri-1, Cri-2, and Cri-3) represent triple-mutant lines in wheat genomes A, B, and D, where mutations have occurred in all three genomes. The gene editing type involves base deletions in all three wheat genomes (A, B, and D).

[0216] Compared with wild-type wheat, the mutant Cri-TaRHF2-1 exhibits the following mutations: The target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” in the TaRHF2A gene is mutated to “5'-TAGCTGCTGCTGCAGAAT-3'”, a deletion of one base causing a frameshift; the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 7 and positions 3865-3883 of SEQ ID No. 9)” in the TaRHF2B gene is mutated to “5'-TAGCTGCTGCTGAAT-3'”, a deletion of four bases causing a frameshift; the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 7 and positions 3865-3883 of SEQ ID No. 9)” in the TaRHF2D gene is mutated to “5'-TAGCTGCTGCTGCAGCAAT-3'”, a deletion of four bases causing a frameshift; the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” in the TaRHF2D gene is mutated to “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” in the TaRHF2B gene is mutated to “5'-TAGCTGCTGCTGCAG The mutation (positions 413-431 of ID No. 1 and positions 2305-2323 of SEQ ID No. 3) is changed to "5'-TAGCTGCTGCTGCAGAAT-3'", which is a deletion of one base. This deletion causes a frameshift, which alters the amino acid composition of TaRHF2, resulting in the loss of TaRHF2 protein function and thus knocking out the entire TaRHF2 gene.

[0217] Compared to wild-type wheat, the mutant Cri-TaRHF2-2 exhibits the following mutations: In the TaRHF2A gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” is mutated to “5'-TAGCTGCTGCTGCA-3'”, a deletion of 5 bases, which causes a frameshift; in the TaRHF2B gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 7 and positions 3865-3883 of SEQ ID No. 9)” is mutated to “5'-TAGCTGCTGCTGCAGCAT-3'”, a deletion of 1 base, which causes a frameshift; in the TaRHF2D gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” is mutated to “5'-TAGCTGCTGCTGCAGCAAT ... The sequence (positions 413-431 of SEQ ID No. 1 and positions 2305-2323 of SEQ ID No. 3) is mutated to "5'-TAGCTGCTGCTGCAGAAT-3'", which is a deletion of one base. This deletion causes a frameshift. The frameshift results in an alteration of the TaRHF2 amino acid profile, leading to the loss of function of the TaRHF2 protein, thus completely knocking out the TaRHF2 gene.

[0218] Compared with wild-type wheat, the mutant Cri-TaRHF2-3 exhibits the following mutations: In the TaRHF2A gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 4 and positions 7835-7853 of SEQ ID No. 6)” is mutated to “5'-TAG-3'”, a deletion of 19 bases, which causes a frameshift; in the TaRHF2B gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 7 and positions 3865-3883 of SEQ ID No. 9)” is mutated to “5'-TAGCTGCTGCTGCAGAT-3'”, a deletion of 2 bases, which causes a frameshift; in the TaRHF2D gene, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 1 and positions 7835-7853 of SEQ ID No. 6)” is mutated to “5'-TAG-3'”, a deletion of 19 bases, which causes a frameshift; in the wild-type wheat, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 1 and positions 7835-7853 of SEQ ID No. 6)” is mutated to “5'-TAGCTGCTGCTGCAGCAAT-3'”, a deletion of 2 bases, which causes a frameshift; in the wild-type wheat, the target site “5'-TAGCTGCTGCTGCAGCAAT-3' (corresponding to positions 413-431 of SEQ ID No. 1 and positions 7 The mutation at position 2305-2323 of ID No. 3 is "5'-TAGCTGCTGCTGAAT-3'", which is a deletion of 4 bases. This deletion causes a frameshift. The frameshift results in an alteration of the TaRHF2 amino acid profile, leading to the loss of function of the TaRHF2 protein, thus completely knocking out the TaRHF2 gene.

[0219] 3. The selected three-protrusion materials Cri-TaRHF2-1, Cri-TaRHF2-2, and Cri-TaRHF2-3 were propagated, and homozygous seeds were harvested for subsequent experiments. The same batch of recipient wheat seeds was used for propagation.

[0220] IV. Salt Tolerance Identification of Gene-Edited Wheat

[0221] Test samples: Cri-1, Cri-2 and Cri-3 seeds, and control recipient wheat Fielder (WT) seeds.

[0222] For the analysis of soil salt stress tolerance, small red pots of uniform size were first selected, and the nutrient soil was fully saturated with water. An equal amount of nutrient soil was weighed into each pot. Then, gene-edited and recipient wheat seeds from the same batch, with plump grains, were sown in each pot with the same number of seeds. After a period of normal growth, a 250 mM NaCl solution was applied for salt stress treatment, and the phenotype was observed. During the stress treatment, samples were taken to measure chlorophyll content and ion permeability, and the survival rate was recorded.

[0223] See results Figure 9 In the study of AD: Under normal growth conditions, there was no significant difference in growth between TaRHF2 mutant and recipient wheat; after 5 days of salt treatment, mutant wheat showed more severe chlorosis and wilting phenotypes and its growth was weaker than that of recipient wheat; after 6 days of salt treatment, the phenotypes were more obvious, and the survival rate of mutant wheat after salt treatment was lower than that of recipient wheat.

[0224] The results of the index measurements showed that under normal conditions, there were no significant differences in chlorophyll content and ion permeability between mutant and recipient wheat. After salt treatment, the chlorophyll content of mutant wheat was significantly lower than that of recipient wheat, while the ion permeability was significantly higher than that of recipient wheat.

[0225] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating plant salt tolerance; 2) Application in the preparation of products that regulate plant salt tolerance; 3) Application in cultivating plants with altered salt tolerance; 4) Application in the preparation of products using plants with altered salt tolerance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) Proteins with an amino acid sequence of SEQ ID No. 2, SEQ ID No. 5, or SEQ ID No. 8; a2) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2, SEQ ID No. 5 or SEQ ID No. 8; a3) is a protein that has more than 80% identity with the amino acid sequence defined by a1) or a2) and has the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

2. The application according to claim 1, characterized in that, The protein is derived from wheat.

3. The application according to claim 1 or 2, characterized in that, The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) The nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is a DNA molecule shown in SEQ ID No. 3, SEQ ID No. 6, or SEQ ID No. 9; d2) The coding sequence is a DNA molecule shown in SEQ ID No. 1, SEQ ID No. 4, or SEQ ID No. 7; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.

5. A method for cultivating highly salt-tolerant plants, characterized in that, This includes enhancing, increasing, or upregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein to obtain a highly salt-tolerant plant, wherein the salt tolerance of the highly salt-tolerant plant is higher than that of the target plant.

6. The method according to claim 5, characterized in that, The enhancement, improvement, or upregulation of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule of claim 4c1) into the target plant to obtain a highly salt-tolerant plant.

7. A method for cultivating low-salt-tolerant plants, characterized in that, This includes inhibiting, reducing, or silencing the expression level of the gene encoding the protein of claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein, to obtain a low-salt-tolerant plant, wherein the low-salt-tolerant plant has lower salt tolerance than the target plant.

8. A method for altering the salt tolerance of plants, characterized in that: The method includes step M or P, wherein step M is to inhibit or reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit or reduce or downregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to reduce the plant's salt tolerance. The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the plant's salt tolerance.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

10. The method according to any one of claims 5-8, characterized in that, The plant is any one of the following: C1) Monocotyledons; C2) Plants of the order Poales; C3) Gramineae plants; C4) Plants of the genus Triticum; C5) Wheat.

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