Wheat tagskb protein and application of the coding gene in regulating plant stress tolerance

Abstract

The application discloses wheat TaGSKB protein and application of a coding gene thereof in regulation of plant stress resistance. The application specifically discloses application of a protein with an amino acid sequence of SEQ ID No. 1 and a coding gene thereof in regulation of plant stress resistance. The application introduces TaGSKB gene from wheat into a receptor plant to obtain homozygous trans-TaGSKB gene plants. Experiments prove that, compared with a non-transgenic receptor control, transgenic Arabidopsis and wheat both show stronger tolerance under drought stress and salt stress conditions, the survival rates are both significantly higher than those of the receptor control, and the growth is also significantly better than that of the receptor control, indicating that overexpression of the TaGSKB gene can significantly improve the drought stress resistance and salt tolerance of the plants (i.e. improve the stress resistance of the plants). The stress resistance regulation gene of the application has important significance and application value for cultivating new plant stress resistance varieties.

Description

Technical Field

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

[0002] Plants inevitably suffer from various stresses during their growth and development, such as drought, salinity, high temperature, low temperature, and nutrient deficiency. Abiotic stresses limit the use of arable land globally and also seriously affect crop yield and quality, making them a significant adverse factor restricting agricultural development. Abiotic stresses cause various types of damage to plants: disrupting cell membrane systems, causing cell dehydration, and affecting enzyme activity, thereby disrupting plant growth and metabolism. Abiotic stresses affect many aspects of plant physiology and cause extensive changes in cellular processes. Over a long period of evolution, plants have developed a complex regulatory network to respond to different stress signals in order to resist abiotic stresses: first, plants sense external stress signals, transmit these signals, and regulate them at the cellular and molecular levels, ultimately enabling plants to produce adaptive responses that resist the stress and thus survive.

[0003] Wheat (Triticum aestivum L.) is one of the world's most important cereal crops and a major staple food for humans. However, abiotic stress severely restricts wheat production. Improving wheat's stress resistance is an effective way to ensure sustainable wheat production and a potential target for crop improvement. Therefore, identifying wheat stress-related genes, studying their functions, and using molecular breeding to improve wheat stress resistance are of significant economic and social importance for breeding stress-resistant wheat varieties and ensuring high and stable wheat yields.

[0004] In the process of plants resisting abiotic stress, the synergistic action of multiple genes is required, and proteins are the ultimate executors of cellular activities and functions, controlling and regulating many life activities within the cell. Normally, proteins cannot function independently in the cell; they typically interact with other proteins to perform specific functions at specific times and in specific spaces. Protein-protein interaction is a fundamental physiological function in cells, constituting the entire signaling network within the plant. Simultaneously, protein-protein interaction also serves as a bridge for various important physiological processes within the cell, such as gene replication, transcription, translation, cell cycle regulation, signal transduction, and immune responses. Numerous studies have shown that protein-protein interaction regulates the abiotic stress response in plants. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the stress resistance (i.e., stress tolerance) of plants. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To address the aforementioned technical problems, the present invention first provides the application of proteins or substances that regulate the activity and / or content of said proteins, wherein the application may be any of the following:

[0007] D1) The application of proteins or substances that regulate the activity and / or content of said proteins in regulating plant stress resistance;

[0008] D2) The use of proteins or substances that regulate the activity and / or content of said proteins in the preparation of products that regulate plant stress resistance;

[0009] D3) The application of proteins or substances that regulate the activity and / or content of said proteins in the cultivation of stress-resistant plants;

[0010] D4) The use of proteins or substances that regulate the activity and / or content of said proteins in the preparation of products for cultivating stress-resistant plants;

[0011] D5) The application of proteins or substances that regulate the activity and / or content of said proteins in plant breeding;

[0012] The protein is named TaGSKB and can be any of the following:

[0013] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0014] A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0015] A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0016] To facilitate the purification or detection of the protein in A1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0017] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0018] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaGSKB protein of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the TaGSKB protein isolated in this invention, as long as they encode and function the TaGSKB protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0019] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

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

[0021] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0022] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein TaGSKB.

[0023] 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).

[0024] The substance that regulates gene expression can be any of the biological materials described in B1)-B3).

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

[0026] The present invention also provides applications of biomaterials related to the protein TaGSKB, wherein the applications may be any of the following:

[0027] E1) Application of biomaterials related to the protein TaGSKB in regulating plant stress resistance;

[0028] E2) Application of biomaterials related to the protein TaGSKB in the preparation of products that regulate plant stress resistance;

[0029] E3) Application of biomaterials related to the protein TaGSKB in the cultivation of stress-resistant plants;

[0030] E4) Application of biomaterials related to the protein TaGSKB in the preparation of products for cultivating stress-resistant plants;

[0031] E5) Application of biomaterials related to the protein TaGSKB in plant breeding;

[0032] The biomaterial may be any one of the following B1) to B7):

[0033] B1) The nucleic acid molecule encoding the protein TaGSKB;

[0034] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0037] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0038] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0039] B7) Transgenic plant organs containing the nucleic acid molecules described in B1) or transgenic plant organs containing the expression cassette described in B2).

[0040] In the above applications, the nucleic acid molecule described in B1) can be any of the following:

[0041] C1) The coding sequence is a DNA molecule of SEQ ID No. 2;

[0042] C2) The nucleotide sequence is the DNA molecule of SEQ ID No.2.

[0043] The DNA molecule shown in SEQ ID No. 2 (the TaGSKB gene that regulates plant stress resistance) encodes the protein TaGSKB, whose amino acid sequence is the same as that in SEQ ID No. 1.

[0044] The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the protein TaGSKB encoding gene (CDS). The protein TaGSKB gene (TaGSKB gene) described in this invention can be any nucleotide sequence capable of encoding the protein TaGSKB. Considering codon degeneracy and codon preferences among different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0045] B1) The nucleic acid molecule may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2.

[0046] The nucleic acid molecules also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with that shown in SEQ ID No. 2 and originate from the same species.

[0047] 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.

[0048] 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, Peasyblunt vector, or vector pWMB110.

[0049] Recombinant expression vectors containing the TaGSKB 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) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0050] When constructing a recombinant plant expression vector using the TaGSKB 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 a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0051] 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.

[0052] By using any vector capable of guiding the expression of exogenous genes in plants, the TaGSKB gene or gene fragments provided by this invention can be introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with enhanced stress resistance. The expression vector carrying the TaGSKB gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0053] The microorganisms described in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*. Specifically, they may be *Escherichia coli* competent cells TOP10 and / or *Agrobacterium tumefaciens* GV3101.

[0054] The recombinant vector may specifically be Peasyblunt-TaGSKB, pCAMBIA1302-TaGSKB, and / or pWMB110-TaGSKB.

[0055] The recombinant vector Peasyblunt-TaGSKB is obtained by using blunt-end cloning, where the DNA fragment with the nucleotide sequence of SEQ ID No. 2 in the sequence listing is ligated into the Peasyblunt vector while keeping the other sequences of the Peasyblunt vector unchanged.

[0056] The recombinant plasmid pCAMBIA1302-TaGSKB is formed by inserting the DNA molecule shown in SEQ ID No. 2 into the NcoI site of the vector pCAMBIA1302.

[0057] The recombinant vector pWMB110-TaGSKB is a DNA molecule, as shown in SEQ ID No. 2, inserted into the BamHI restriction site of the vector pWMB110.

[0058] The recombinant microorganisms may specifically be GV3101 / pCAMBIA1302-TaGSKB and / or EHA105 / pWMB110-TaGSKB.

[0059] The recombinant Agrobacterium GV3101 / pCAMBIA1302-TaGSKB contains the TaGSKB gene with the coding sequence SEQ ID NO.2, and is a recombinant microorganism obtained by introducing the recombinant vector pCAMBIA1302-TaGSKB into Agrobacterium GV3101.

[0060] The recombinant Agrobacterium EHA105 / pWMB110-TaGSKB contains the TaGSKB gene with the coding sequence SEQ ID NO.2, and is a recombinant microorganism obtained by introducing the recombinant vector pWMB110-TaGSKB into Agrobacterium EHA105.

[0061] The present invention also provides a method for cultivating stress-resistant plants, the method comprising increasing the content and / or activity of the protein TaGSKB in the target plant to obtain a stress-resistant plant with higher stress resistance than the target plant.

[0062] In the above method, the increase in the content and / or activity of the protein TaGSKB in the target plant is achieved by increasing the expression level of the gene encoding the protein TaGSKB in the target plant.

[0063] In the above method, the expression level of the gene encoding the protein TaGSKB in the target plant is increased by introducing the gene encoding the protein TaGSKB into the target plant.

[0064] In the above method, the gene encoding the protein can be any of the following:

[0065] F1) The coding sequence is a DNA molecule of SEQ ID No. 2;

[0066] F2) The nucleotide sequence is the DNA molecule of SEQ ID No.2.

[0067] Specifically, in one embodiment of the present invention, the increase in the expression level of the gene encoding the protein in the target plant is achieved by introducing the DNA molecule shown in SEQ ID No. 2 into the target plant.

[0068] In one embodiment of the present invention, the method for cultivating stress-resistant plants includes the following steps:

[0069] (1) Construct a recombinant vector containing the DNA molecule shown in SEQ ID NO.2;

[0070] (2) Transfer the recombinant vector constructed in step (1) into the target plant (such as wheat);

[0071] (3) Through screening and identification, stress-resistant plants with higher stress resistance than the target plant were obtained.

[0072] In the above method, the plant can be G1) or G2):

[0073] G1) Monocotyledonous or dicotyledonous plants;

[0074] G2) Grasses or cruciferous plants.

[0075] In this article, the plants mentioned can be crops (agricultural crops).

[0076] Furthermore, the plant described in this article may specifically be Arabidopsis thaliana or wheat.

[0077] This invention also provides the application of the protein TaGSKB, or the biological material, or the nucleic acid molecule, or any of the methods for cultivating stress-resistant plants described herein in the creation of stress-resistant plants and / or plant breeding.

[0078] The protein TaGSKB, the biomaterial, and the nucleic acid molecule described herein are all within the scope of protection of this invention.

[0079] The protein TaGSKB described in this article may be a stress-related protein in wheat.

[0080] The plant breeding described in this article can be used for crop stress resistance breeding.

[0081] The regulation of plant stress resistance can be used to either increase or decrease plant stress resistance.

[0082] Increased expression and / or activity of the TaGSKB protein or its encoding gene in the target plant leads to increased plant stress resistance.

[0083] Furthermore, the improved stress resistance is manifested in:

[0084] (1) Improve the survival rate of the target plant;

[0085] (2) Increase the chlorophyll content of the target plant;

[0086] (3) Increase the proline content of the target plant;

[0087] (4) Reduce the malondialdehyde content of the target plant.

[0088] The aforementioned resistance includes, but is not limited to, salt tolerance, drought resistance, heat resistance, cold resistance and / or strong light resistance.

[0089] Specifically, the stress resistance (resistance) can be salt tolerance and / or drought resistance.

[0090] In this invention, the term "stress-resistant plant" is understood to include not only the first-generation transgenic plants obtained by transforming the target plant with the TaGSKB gene, but also its progeny. The gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The stress-resistant plant includes seeds, callus tissue, intact plants, and cells.

[0091] This invention introduces the TaGSKB gene, which regulates plant stress resistance and is derived from wheat (Triticum aestivum L.), into recipient control plants wild-type Arabidopsis thaliana (WT, Col) and wild-type wheat (Fielder), respectively, to obtain homozygous Arabidopsis thaliana lines (#3, #5, #8) and homozygous wheat lines (OE-1, OE-2, OE-4) with the TaGSKB gene. Experiments have shown that, compared with the non-transgenic recipient control, transgenic Arabidopsis and wheat overexpressing the TaGSKB gene exhibited significantly higher survival and germination rates under drought and salt stress conditions, and their growth was also significantly better than the recipient control. This indicates that overexpression of the TaGSKB gene can significantly improve the plant's resistance to drought and salt stress (i.e., improve the plant's stress resistance). Specifically, the survival rate of transgenic Arabidopsis and wheat overexpressing the TaGSKB gene was significantly improved, the seed germination rate was significantly improved, the chlorophyll content and proline content were significantly increased, the catalase activity was increased, and the malondialdehyde and H2O2 content and relative conductivity were significantly reduced. All physiological indicators were significantly better than those of the recipient control. Therefore, under drought and high salt stress conditions, Arabidopsis thaliana and wheat overexpressing the TaGSKB gene showed stronger tolerance, indicating that the TaGSKB protein and its encoding gene TaGSKB of the present invention can regulate plant stress resistance (such as drought resistance and / or salt tolerance), and that the stress resistance of the target plant can be significantly improved by increasing the content and / or activity of TaGSKB protein in the target plant (such as by overexpressing the TaGSKB gene).

[0092] This invention has discovered a new gene that can regulate plant stress resistance under drought and salt stress conditions, which is of great significance and application value for breeding new stress-resistant plant varieties and improving plant stress resistance. Attached Figure Description

[0093] Figure 1 Analysis of the expression patterns of the TaGSKB gene under different abiotic stresses.

[0094] Figure 2 Analysis of drought resistance in wild-type and TaGSKB transgenic Arabidopsis thaliana. Figure 2 A: Germination experiments of wild-type and transgenic Arabidopsis thaliana under normal and PEG treatments; Figure 2 B: Germination rate statistics of wild-type and transgenic Arabidopsis thaliana under normal and PEG treatments; Figure 2 C: Phenotypic analysis of wild-type and transgenic Arabidopsis thaliana under normal and drought treatments; Figure 2D: Survival rates of wild-type and transgenic Arabidopsis thaliana under normal and drought treatments. #3, #5, and #8 represent the three transgenic TaGSKB lines TaGSKB-3, TaGSKB-5, and TaGSKB-8, respectively. ** and * indicate significant differences (p<0.01 and P<0.05).

[0095] Figure 3 Salt tolerance analysis of wild-type and TaGSKB transgenic Arabidopsis thaliana. Figure 3 A: Germination experiments of wild-type and transgenic Arabidopsis thaliana under normal and NaCl treatments; Figure 3 B: Germination rate statistics of wild-type and transgenic Arabidopsis thaliana under normal and NaCl treatments; Figure 3 C: Phenotypic analysis of wild-type and transgenic Arabidopsis thaliana under normal and high-salt treatments; Figure 3 D: Survival rates of wild-type and transgenic Arabidopsis thaliana under normal and high-salt treatments. #3, #5, and #8 represent the three transgenic TaGSKB lines TaGSKB-3, TaGSKB-5, and TaGSKB-8, respectively. ** and * indicate significant differences (p<0.01 and P<0.05).

[0096] Figure 4 Mannitol stress analysis for the receptor and transgenic TaGSKB wheat. Figure 4 A: Hydroponic experiments of recipient and transgenic wheat under normal and mannitol treatments; Figure 4 BE: Analysis of proline, malondialdehyde, H2O2 content and catalase activity in recipient and transgenic wheat under normal and mannitol treatments; Figure 4 F: Phenotypic analysis of recipient and transgenic wheat under normal and mannitol treatments; Figure 4 G: NBT staining of recipient and transgenic wheat under normal and mannitol treatments; Figure 4 H: O in recipient and transgenic wheat under normal and mannitol treatments 2- Content analysis. OE-1, OE-2, and OE-4 represent three transgenic TaGSKB lines: TaGSKB-1, TaGSKB-2, and TaGSKB-4, respectively. ** and * indicate significant differences (p<0.01 and P<0.05).

[0097] Figure 5 Analysis of drought resistance in recipient and transgenic TaGSKB wheat. Figure 5 A: Soil culture experiments of recipient and transgenic wheat under normal and drought treatments; Figure 5BD: Survival rate, water loss rate, and relative moisture content of recipient and transgenic wheat under normal and drought treatments. OE-1, OE-2, and OE-4 represent three transgenic TaGSKB lines, namely TaGSKB-1, TaGSKB-2, and TaGSKB-4. ** and * indicate significant differences (p<0.01 and P<0.05, respectively).

[0098] Figure 6 Salt tolerance analysis for the recipient and transgenic TaGSKB wheat. Figure 6 A: Hydroponic experiments of recipient and transgenic wheat under normal and high-salt treatments; Figure 6 B: Phenotypic analysis of recipient and transgenic wheat under normal and high salt treatments; Figure 6 CD: Analysis of proline and malondialdehyde (MDA) content in recipient and transgenic wheat under normal and high salt treatments. OE-1, OE-2, and OE-4 represent three transgenic TaGSKB lines: TaGSKB-1, TaGSKB-2, and TaGSKB-4, respectively. ** and * indicate significant differences (p < 0.01 and P < 0.05).

[0099] Figure 7 Salt tolerance analysis for the recipient and transgenic TaGSKB wheat. Figure 7 A: Soil culture experiments of recipient and transgenic wheat under normal and high salt treatments; Figure 7 BD: Survival rate, chlorophyll content, and relative electrical conductivity of recipient and transgenic wheat under normal and high salt treatments. OE-1, OE-2, and OE-4 represent three transgenic TaGSKB lines, namely TaGSKB-1, TaGSKB-2, and TaGSKB-4. ** and * indicate significant differences (p<0.01 and P<0.05, respectively).

[0100] Figure 8 Expression analysis of abiotic stress-related genes in wheat with the receptor and the transgenic TaGSKB gene. Detailed Implementation

[0101] 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.

[0102] 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.

[0103] In the quantitative experiments described below, all experiments were repeated three times, and the results were averaged. Unless otherwise specified, all percentages in the following examples refer to mass percentages.

[0104] The wheat variety “Xiaobaimai” in the following examples is described in the following literature: 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. “Xiaobaimai” in this literature is the “Xiaobaimai” described in this invention.

[0105] The wheat variety “Fielder” in the following examples is described in the following literature: Cui XY, Gao Y, Guo J, YuTF, Zheng WJ, Liu YW, Chen J, Xu ZS, Ma YZ. BES / BZR Transcription Factor TaBZR2 Positively Regulates Drought Responses by Activation of TaGST1. Plant Physiology. doi:10.1104 / pp.19.00100.

[0106] The pEASY-Blunt blunt-end cloning vector used in the following examples is a product of Beijing TransGen Biotech Co., Ltd.

[0107] The E. coli competent cells TOP10 in the following examples are products of Beijing Bomaide Gene Technology Co., Ltd.

[0108] The Agrobacterium tumefaciens GV3101 and EHA105 used in the following examples are products of Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0109] The wild-type Arabidopsis thaliana (WT, Col) used in the following examples is the Colombian ecotype Arabidopsis thaliana (Col-0), purchased from SALK.

[0110] The wheat Fielder and plant binary expression vector pWMB110 used in the following examples were provided by Professor Ye Xingguo's laboratory at the Chinese Academy of Agricultural Sciences. The plant eukaryotic expression vector pWMB110 is described in the following literature: Cui XY, Gao Y, Guo J, Yu TF, Zheng WJ, Liu YW, Chen J, Xu ZS, Ma YZ. BES / BZR Transcription Factor TaBZR2 Positively Regulates Drought Responses by Activation of TaGST1. Plant Physiology. doi:10.1104 / pp.19.00100.

[0111] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. ANOVA test was used, and P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0112] Example 1: Obtaining the TaGSKB protein and its encoding gene

[0113] Total RNA was extracted from wheat (white wheat) that had grown normally for one week using the plant total RNA extraction kit from Tiangen Biotech. First-strand cDNA was synthesized using the first-strand cDNA synthesis kit from TransGen Biotech. Using the wheat cDNA as a template, PCR amplification was performed with specific primers. Following gel recovery, ligation into a cloning vector, bacterial culture PCR detection, and sequencing alignment, the correct TaGSKB gene was obtained.

[0114] The plant stress resistance-related protein gene isolated and cloned from the wheat variety "Xiaobaimai" was named the TaGSKB gene. The coding sequence (CDS) of the TaGSKB gene is SEQ ID No.2, and the protein encoding the amino acid sequence of SEQ ID No.1 is named the TaGSKB protein.

[0115] Example 2: Real-time quantitative PCR analysis of the expression characteristics of the TaGSKB gene

[0116] I. Different stress treatments for wheat

[0117] White wheat was planted in nutrient soil and grown at 22℃ for one week. Then, the seedlings were subjected to different stress treatments.

[0118] 1. Drought treatment: Place wheat seedlings on absorbent filter paper to simulate drought conditions.

[0119] 2. Salt treatment: The roots of wheat seedlings were immersed in a 200mM NaCl solution to simulate salt stress.

[0120] 3. High temperature and low temperature treatment: Wheat seedlings were treated in incubators at 42℃ (high temperature) and 4℃ (low temperature).

[0121] II. Real-time quantitative PCR

[0122] 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 control gene. The relative expression level of the TaGSKB gene under different stress treatments was analyzed using real-time quantitative PCR.

[0123] 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. -ΔΔCt The algorithm was used to create a bar chart using Excel. Three biological replicates were performed.

[0124] The primers used to detect the Actin gene are as follows:

[0125] Actin-F: 5'-GCCATGTACGTCGCAATTCA-3',

[0126] Actin-R: 5'-AGTCGAGAACGATACCAGTAGTACGA-3'.

[0127] The primers used to detect the expression level of the TaGSKB gene under different stresses are as follows:

[0128] RT-TaGSKB-F: 5'-TTCAACTTCAAGCATGAACTGG-3',

[0129] RT-TaGSKB-R: 5'-AACAATAAGGTGCAAGGTTGAG-3'.

[0130] See results Figure 1The TaGSKB gene responded to abiotic stresses to varying degrees. TaGSKB gene expression was significantly upregulated after drought, high salinity (NaCl), and high temperature treatments, with the strongest response observed in drought. This suggests that the TaGSKB gene may be involved in multiple abiotic stress responses.

[0131] Example 3: Effect of TaGSKB protein on stress tolerance in Arabidopsis thaliana

[0132] I. Construction of Recombinant Expression Vectors

[0133] 1. Total RNA was extracted from wheat leaves and reverse transcribed to obtain cDNA.

[0134] 2. Using the cDNA from step 1 above as a template, amplify the wheat TaGSKB gene using specific primers, and recover the PCR product. The specific amplification primers for the TaGSKB gene are as follows:

[0135] TaGSKB-F: 5'-AGATAGGCGTAGGTGGATG-3',

[0136] TaGSKB-R: 5'-AAGAATGGTGCAATGTTGAG-3'.

[0137] 3. The amplified and recovered PCR products were ligated into the Peasyblunt vector (pEASY-Blunt Cloning Kit, catalog number CB101, TransGen Biotech, Beijing). The ligation product was transformed into *E. coli* competent cells TOP10 and plated on solid LB agar plates containing 50 μg / L kanamycin, and incubated overnight at 37°C. Colony PCR screening of *E. coli* clones was performed. Positive clones were sent to the company for sequencing. Colonies with correct sequencing were preserved, and plasmids were extracted. The plasmid with the correct sequence was named Peasyblunt-TaGSKB.

[0138] The Peasyblunt-TaGSKB plasmid is a recombinant vector obtained by using blunt-end cloning, where the DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence listing is ligated into the Peasyblunt vector while keeping the other sequences of the Peasyblunt vector unchanged.

[0139] 4. Using the plasmid Peasyblunt-TaGSKB obtained in step 3 as a template, PCR amplification was performed using primer pairs composed of TaGSKB-1302-F and TaGSKB-1302-R to obtain PCR amplification products, and the PCR products were then recovered from the gel.

[0140] TaGSKB-1302-F:5'-GGGACTCTTG ACCATG ATGGAGCATCCGGCG-3';

[0141] TaGSKB-1302-R: 5'-TCAGATCTAC CCATGG GCTCCCCGCATGCAC-3'.

[0142] 5. Digest the vector pCAMBIA1302 with the restriction endonuclease NcoI and recover the digested vector.

[0143] 6. The PCR product from step 4 and the digested vector from step 5 were subjected to homologous recombination using In-Fusion technology (Liuhetong, Beijing). After successful sequencing, the recombinant plasmid pCAMBIA1302-TaGSKB was obtained.

[0144] The recombinant plasmid pCAMBIA1302-TaGSKB is formed by inserting the DNA molecule shown in SEQ ID No. 2 into the NcoI site of the vector pCAMBIA1302.

[0145] II. Obtaining Transgenic Arabidopsis

[0146] 1. The recombinant plasmid pCAMBIA1302-TaGSKB was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium GV3101 / pCAMBIA1302-TaGSKB.

[0147] 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.

[0148] 3. After completing step 2, collect the bacterial cells by centrifugation at 4℃ and 4000g for 10 minutes, resuspend the bacterial cells in the infection solution, and adjust the OD. 600 Adjust the pH to 0.6-0.8 to obtain a bacterial resuspension (infection solution formulation: 1 / 2 MS, 10 mM MgCl2, 1.0 g / L MES, 5% sucrose, 100 μl / L Silwet L-77; prepared in advance and incubated at room temperature for 3 h).

[0149] 4. Pour the bacterial suspension into a petri dish, place the pot containing the Colombian ecotype Arabidopsis thaliana (Col-0) on its side, ensuring the inflorescence is completely submerged in the infection solution for 3 minutes. Then, lay the Arabidopsis plants flat, cover them with a black plastic bag, and grow them in the dark for 24 hours. The next day, remove the plastic bag and place the Arabidopsis plants upright under normal light conditions. After one week, reinfect them and allow them to grow normally until the T0 generation seeds are harvested.

[0150] 5. Seeds of T0 generation Arabidopsis thaliana were sown on solid MS medium plates containing 50 mg / L kanamycin. Normally growing Arabidopsis thaliana seedlings were selected and transferred to soil for further growth. Mature seedlings were harvested individually to obtain different T1 generation lines. The same method was then used to continue screening until homozygous T3 generation Arabidopsis thaliana was obtained.

[0151] 6. RNA was extracted from the different transgenic lines obtained through screening and reverse transcribed into cDNA. The cDNA obtained by reverse transcription was appropriately diluted and used as a template for real-time quantitative PCR to detect the expression levels of different lines. Suitable lines were selected for subsequent phenotypic experiments. The PCR identification primers were:

[0152] RT-TaGSKB-F: 5'-TTCAACTTCAAGCATGAACTGG-3',

[0153] RT-TaGSKB-R: 5'-AACAATAAGGTGCAAGGTTGAG-3'.

[0154] Three homozygous transgenic Arabidopsis thaliana lines were identified by PCR and named: #3 (i.e., TaGSKB-3), #5 (i.e., TaGSKB-5), and #8 (i.e., TaGSKB-8).

[0155] III. Identification of stress tolerance in Arabidopsis thaliana

[0156] The seeds to be tested were: seeds of the #3 strain T3 generation plants, seeds of the #5 strain T3 generation plants, seeds of the #8 strain T3 generation plants, and seeds of wild-type Arabidopsis thaliana (Col-0).

[0157] 1. Germination rate under drought stress: Wild-type and transgenic Arabidopsis seeds harvested at the same time were selected, sterilized, and sown on MS medium and stress treatment medium supplemented with PEG (10% and 12% PEG were set up as experimental groups, respectively). After vernalization at 4℃ for 3 days, the seeds were transferred to a 22℃ incubator with 16h light / 8h darkness and 60% relative humidity. The germination rate was recorded every 24 hours. Germination was defined as the emergence of white seeds, and the germination rate was recorded until it no longer changed. Three biological replicates were performed.

[0158] See results Figure 2In sections A and B: On MS medium, there was no difference in germination rate between wild-type and transgenic Arabidopsis thaliana, with all seeds germinating by day 4. On media containing 10% and 12% PEG, the germination rate of both wild-type and transgenic Arabidopsis thaliana was slowed, but the germination rate of transgenic Arabidopsis thaliana was higher than that of wild-type, indicating that transgenic TaGSKB gene (i.e., overexpression of the TaGSKB gene) improved the germination rate of Arabidopsis thaliana seeds under drought stress.

[0159] 2. Growth and survival rate under drought stress treatment

[0160] When wild-type and transgenic Arabidopsis thaliana had grown four leaves on MS medium, they were transferred to pots with a 1:1 ratio of potting soil to vermiculite (watered to saturation to ensure sufficient moisture) and placed in a 22°C culture room with 16h light / 8h darkness and 60% relative humidity.

[0161] Drought group: Wild-type and transgenic Arabidopsis thaliana with normal growth for 3 weeks and similar growth were subjected to drought treatment, i.e., water was withheld for two weeks and then rehydrated for one week. Phenotyps were observed, photos were taken, and survival rate was counted.

[0162] Control group: Wild-type and transgenic Arabidopsis thaliana with normal growth for 3 weeks and the same growth rate were continued to be watered normally, and their phenotypes were observed, photographed and the survival rate was counted.

[0163] Three biological replicate experiments were set up, and in each replicate experiment, 36 plants of each type of seed were observed and statistically analyzed. The results are shown below. Figure 2 In sections C and D: Under normal growth conditions (Before treatment), there was no significant difference between wild-type and transgenic Arabidopsis thaliana; under drought treatment, the growth of transgenic Arabidopsis thaliana was significantly stronger than that of wild-type Arabidopsis thaliana; after rehydration, the survival rate of transgenic Arabidopsis thaliana was significantly higher than that of wild-type Arabidopsis thaliana. This indicates that overexpression of the TaGSKB gene can significantly improve the drought resistance of plants.

[0164] 3. Germination rate under salt stress: Wild-type and transgenic Arabidopsis seeds harvested at the same time were selected, disinfected, and sown on MS medium and stress treatment medium (MS medium) supplemented with 125 mM and 150 mM NaCl. After vernalization at 4℃ for 3 days, the seeds were transferred to a 22℃ incubator with 16 h light / 8 h darkness and 60% relative humidity. Germination rate was recorded every 24 h. Germination was defined as the emergence of white seeds, and the germination rate was recorded until it no longer changed. Three biological replicates were performed.

[0165] See results Figure 3In sections A and B: On MS medium, there was no difference in germination rate between wild-type and transgenic Arabidopsis thaliana, with all seeds germinating by day 4. On media containing 125 mM and 150 mM NaCl, the germination rate of both wild-type and transgenic Arabidopsis thaliana slowed down, but the germination rate of transgenic Arabidopsis thaliana was higher than that of wild-type Arabidopsis thaliana, indicating that transgenic TaGSKB gene (i.e., overexpression of the TaGSKB gene) improved the germination rate of Arabidopsis thaliana seeds under salt stress.

[0166] 4. Growth and survival rate under high salt stress treatment

[0167] When wild-type and transgenic Arabidopsis thaliana had grown four leaves on MS medium, they were transferred to pots with a 1:1 ratio of potting soil to vermiculite (watered to saturation to ensure sufficient moisture) and placed in a 22°C culture room with 16h light / 8h darkness and 60% relative humidity.

[0168] Salt treatment group: Wild-type and transgenic Arabidopsis thaliana with normal growth for 3 weeks and similar growth were treated with 200mM NaCl for 1 week until a significant difference in growth was observed between wild-type and transgenic Arabidopsis thaliana. The survival rate was then recorded by taking pictures.

[0169] Control group: Wild-type and transgenic Arabidopsis thaliana with normal growth for 3 weeks and the same growth status were continued to be watered normally, and their phenotypes were observed, photographed and the survival rate was counted.

[0170] Three biological replicate experiments were set up, and in each replicate experiment, 36 plants of each type of seed were observed and statistically analyzed. The results are shown below. Figure 3 In sections C and D: Under normal growth conditions (Before treatment), there was no significant difference between wild-type and transgenic Arabidopsis thaliana; under salt treatment, the growth of transgenic Arabidopsis thaliana was significantly stronger than that of wild-type Arabidopsis thaliana; statistical analysis of survival rates showed that the survival rate of transgenic Arabidopsis thaliana was significantly higher than that of wild-type Arabidopsis thaliana. This indicates that overexpression of the TaGSKB gene can significantly improve the salt tolerance of plants.

[0171] In summary, the TaGSKB protein and its encoding gene TaGSKB can regulate the stress resistance (such as drought resistance and / or salt tolerance) of Arabidopsis thaliana. Increasing the content and / or activity of TaGSKB protein in the target plant Arabidopsis thaliana (such as overexpressing the TaGSKB gene) can significantly improve the stress resistance of the target plant Arabidopsis thaliana.

[0172] Example 4: Effects of TaGSKB protein on wheat stress resistance

[0173] I. Construction of Recombinant Expression Vectors

[0174] 1. Using the plasmid (Peasyblunt-TaGSKB) obtained in step 3 of Example 3 as a template, PCR amplification was performed using primer pair composed of TaGSKB-110-F and TaGSKB-110-R to obtain PCR amplification products, and the PCR products were recovered by gel extraction.

[0175] TaGSKB-110-F:5'-CGACTCTAGA GGATCC ATGGAGCATCCGGCG-3';

[0176] TaGSKB-110-R:5'-GGGTACCCGG GGATCC TTAGCTCCCCGCATG-3'.

[0177] 2. Digest the vector pWMB110 with the restriction endonuclease BamHI and recover the digested vector.

[0178] 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-TaGSKB was obtained.

[0179] The recombinant plasmid pWMB110-TaGSKB is a DNA molecule, as shown in SEQ ID No. 2, inserted into the BamHI site of the vector pWMB110.

[0180] II. Obtaining Genetically Modified Wheat

[0181] Wheat embryos were prepared in advance, and recombinant plasmids were transformed into spring wheat Fielder using Agrobacterium-mediated gene transformation technology. The main steps were performed according to the reference "Wang K, Shi L, Liang X, Zhao P, Wang W, Liu J, Chang Y, Hiei Y, Yanagira C, Du L, Ishida Y, Ye X (2022). The gene TaWOX5 overcomes genome dependency in wheat genetic transformation. Nat Plants. doi:10.1038 / s41477-021-01085-8." The details are as follows:

[0182] 1. The recombinant plasmid pWMB110-TaGSKB was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pWMB110-TaGSKB.

[0183] 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.

[0184] 3. Sterilize the surface of wheat seeds, remove the wheat embryos under a microscope, incubate them for 5 minutes at room temperature with Agrobacterium infection solution (containing acetylsuccinone) carrying pWMB110-TaGSKB, and grow them on WLS-AS medium in the dark at 25°C for 2 days with the scutellum facing upwards.

[0185] 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.

[0186] 5. The regenerated shoots were transferred to cups containing MSF-P5 medium for elongation and rooting. Nine days later, the plants with well-developed root systems were transplanted into flowerpots to continue growing. After cultivation, transgenic wheat (T0 generation transgenic wheat) was obtained.

[0187] 6. Harvesting wheat seeds and screening homozygous lines: RNA was extracted from the plants and reverse transcribed into cDNA. Transgenic positive plants (containing the TaGSKB gene with the coding sequence SEQ ID NO.2) were verified by PCR. Stable homozygous lines that could grow normally were screened in the third generation and named as follows: OE-1 line (i.e., TaGSKB-1 line), OE-2 line (i.e., TaGSKB-2 line), and OE-4 line (i.e., TaGSKB-4 line).

[0188] III. Detection of positive transgenic wheat lines

[0189] For the detection of genetically modified wheat, PCR and qRT-PCR analyses were used to identify transgenic wheat lines simultaneously at the DNA and transcriptional levels. DNA was extracted using the CTAB method, and PCR detection was performed. Individual plants with the correct band size were sent for sequencing, and those with correct sequencing results were harvested as different lines.

[0190] RNA was extracted from different transgenic lines obtained from positive tests and reverse transcribed into cDNA. The cDNA obtained from reverse transcription was appropriately diluted and used as a template for real-time quantitative PCR to detect the expression levels of different lines.

[0191] The primers used for positive detection of the TaGSKB gene are as follows:

[0192] TaGSKByxjc-F:5'-TTTAGCCCTGCCTTTCATACG-3',

[0193] TaGSKByxjc-R:5'-CCCATCTCATAAATAACGTCATGC-3'.

[0194] The primers used to detect the Actin gene are as follows:

[0195] Actin-F: 5'-GCCATGTACGTCGCAATTCA-3',

[0196] Actin-R: 5'-AGTCGAGAACGATACCAGTAGTACGA-3'.

[0197] The qRT-PCR primers used to detect the expression level of TaGSKB positive lines are as follows:

[0198] RT-TaGSKB-F:5'-TTCAACTTCAAGCATGAACTGG-3'

[0199] RT-TaGSKB-R: 5'-AACAATAAGGTGCAAGGTTGAG-3'.

[0200] IV. Identification of wheat's stress resistance

[0201] The seeds to be tested were: seeds of the T3 generation of transgenic wheat TaGSKB-1 strain, seeds of the T3 generation of TaGSKB-2 strain, seeds of the T3 generation of TaGSKB-4 strain, and seeds of wild-type wheat (recipient wheat, Fielder).

[0202] 1. Physiological indicators under drought stress (mannitol treatment): Seeds of both recipient and transgenic wheat were germinated in petri dishes and placed in an incubator at 22℃ (16h light / 8h dark, 60% humidity). After 3 days, the emerging seeds were transferred to Hoagland's nutrient solution for further growth for 3-4 days, followed by treatment with 200mM and 300mM mannitol. Root length was measured after 5 days. Four days after treatment, the contents of proline, malondialdehyde (MDA), H2O2, and catalase (CAT) in both recipient and transgenic wheat were measured. The methods for measuring the physiological indicators of the samples were performed according to the specific operating procedures of the kit (Suzhou Keming Biotechnology Co., Ltd.). Wheat roots were stained with NBT, and O2 content was measured. 2- Content. Three biological replicates were performed, with 32 seeds of each type tested in each replicate.

[0203] See results Figure 4Under normal growth conditions, there was no significant difference in growth between the recipient and transgenic wheat. The proline, malondialdehyde (MDA), H2O2 content, and catalase activity in transgenic wheat were not significantly different from those in recipient wheat. Under mannitol treatment, the growth of both recipient and transgenic wheat was inhibited, but the growth of transgenic wheat was stronger than that of recipient wheat. The proline content and catalase activity of transgenic wheat were higher than those of recipient wheat, while the MDA and H2O2 content of transgenic wheat were lower than those of recipient wheat. NBT staining of the root tips of transgenic and recipient wheat showed that the color depth correlated with the amount of O2 in the root tips. 2- The content was positively correlated; under normal growth conditions, there was no difference in the color of root tips among different wheat varieties; after mannitol treatment, the root tip color of overexpressing wheat lines was significantly lighter than that of the recipient, and the O content of transgenic wheat was... 2- The content is lower than that of the recipient wheat.

[0204] 2. Growth, survival rate, water loss rate, and relative water content under drought stress treatment

[0205] The potting soil was watered to saturation. Equal amounts of soil were weighed, and the same number of recipient and transgenic wheat seedlings were planted in flowerpots. Water control treatment was applied in a 22℃ (16h light / 8h dark, 60% humidity) incubator. After 12 days of water control treatment, leaf samples from both the recipient and transgenic wheat were taken to determine water loss rate and relative water content. Drought treatment continued until the recipient Fielder and transgenic wheat seedlings wilted with significant differences. Watering was then resumed, and survival rates were calculated 2 days later. Three biological replicates were performed, with 60 seeds of each type tested in each replicate.

[0206] See results Figure 5 Under normal growth conditions, there was no significant difference in growth between the recipient and transgenic wheat. After drought treatment, the growth of both recipient and transgenic wheat was inhibited, but the growth of transgenic wheat was significantly stronger than that of wild-type wheat. After drought treatment, the water loss rate of transgenic wheat was lower than that of recipient wheat, while its relative water content (RWC) was higher. After rehydration, the survival rate of transgenic wheat was significantly higher than that of recipient wheat. This indicates that overexpression of the TaGSKB gene can improve the drought resistance of transgenic wheat.

[0207] 3. Physiological indicators under salt stress: Fielder recipient and transgenic wheat seeds were germinated in petri dishes and placed in an incubator at 22℃ (16h light / 8h dark, 60% humidity). After 3 days, the emerging seeds were transferred to Hogrange nutrient solution for further growth for 3-4 days, followed by treatment with 300mM NaCl. Root length was measured after 5 days. Proline and malondialdehyde (MDA) content in the recipient and transgenic wheat were measured 4 days after treatment. Three biological replicates were performed, with 60 seeds of each type tested in each replicate.

[0208] See results Figure 6Under normal growth conditions, there was no significant difference in growth between the recipient and transgenic wheat, and the proline and malondialdehyde (MDA) contents of transgenic wheat were not significantly different from those of recipient wheat. After high salt treatment, the growth of both recipient and transgenic wheat was inhibited, but the growth of transgenic wheat was significantly stronger than that of recipient wheat; the proline content of transgenic wheat was higher than that of recipient wheat, while the MDA content was lower than that of recipient wheat.

[0209] 4. Growth, survival rate, chlorophyll content, and electrical conductivity under high salt stress treatment

[0210] The potting soil was watered to saturation. Equal amounts of soil were then used to plant the same number of recipient wheat (Fielder) and transgenic wheat pure lines in flowerpots. The plants were grown in a 22℃ (16h light / 8h dark, 60% humidity) incubator. Wheat plants that had grown normally for 10 days were subjected to a high-salt treatment with 250mM NaCl. Chlorophyll content and relative conductivity were measured 7 days after the salt treatment. Photographs were taken and survival rates were recorded when a significant difference was observed between the recipient and transgenic wheat. Three biological replicates were performed, with 60 seeds of each type tested in each replicate.

[0211] See results Figure 7 Under normal growth conditions, there was no significant difference in growth between the recipient and transgenic wheat. The chlorophyll content and relative electrical conductivity of transgenic wheat were not significantly different from those of the recipient wheat. After high salt treatment, the growth of both recipient and transgenic wheat was inhibited, but the growth of transgenic wheat was significantly stronger than that of the recipient wheat. The chlorophyll content of transgenic wheat was higher than that of the recipient wheat, while its relative electrical conductivity was lower. The survival rate of transgenic wheat was higher than that of the recipient wheat. This indicates that overexpression of the TaGSKB gene can improve the salt tolerance of transgenic plants.

[0212] In summary, TaGSKB protein and its encoding gene TaGSKB can regulate the stress resistance of wheat (such as drought resistance and / or salt tolerance). Increasing the content and / or activity of TaGSKB protein in the target plant wheat (such as overexpressing the TaGSKB gene) can significantly improve the stress resistance of the target plant wheat.

[0213] V. Molecular Mechanisms of Enhanced Stress Tolerance in Transgenic Wheat

[0214] We identified several genes associated with abiotic stress and detected their expression in transgenic and recipient wheat. The NPF2.3 gene is involved in nitrate transport under salt stress, enhancing the plant's ability to adapt to salt stress; the Lea7 gene is involved in the synthesis of osmotic substances, thereby improving the plant's resistance to stress; the APX1 gene regulates peroxide oxidase levels, clearing excess peroxides accumulated under stress and reducing oxidative stress caused by excessive peroxides; the RD29A gene is a key marker gene corresponding to drought stress. Detecting the expression of genes associated with abiotic stress can help understand the mechanisms of plant stress response.

[0215] See results Figure 8 The expression of TaNPF2.3, TaLea7, TaAPX1, and TaRD29A genes was detected in transgenic wheat and recipient wheat. The results showed that the expression of these genes was increased to varying degrees in wheat transgenic with the TaGSKB gene, indicating that the TaGSKB gene improves the drought resistance and salt tolerance of plants by directly or indirectly regulating the expression of genes related to abiotic stress.

[0216] Therefore, the experimental results of both wheat and Arabidopsis thaliana overexpressing the TaGSKB gene show that the TaGSKB protein and its encoding gene TaGSKB of the present invention can regulate the stress resistance of plants (such as drought resistance and / or salt tolerance), and the stress resistance of the target plants can be significantly improved by increasing the content and / or activity of TaGSKB protein in the target plants (such as by overexpressing the TaGSKB gene).

[0217] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Institute of Crop Science, Chinese Academy of Agricultural Sciences <120> Application of wheat TaGSKB protein and its encoding gene in regulating plant stress tolerance <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 402 <212> PRT <213> Wheat (Triticum aestivum L.) <400> 1 Met Glu His Pro Ala Pro Ala Pro Glu Pro Met Leu Leu Asp Glu Gln 1 5 10 15 Pro Pro Thr Ala Val Ala Cys Glu Lys Lys Gln Gln Asp Gly Glu Ala 20 25 30 Pro Tyr Ala Glu Gly Asn Asp Ala Met Thr Gly His Ile Ile Ser Thr 35 40 45 Thr Ile Gly Gly Lys Asn Gly Glu Pro Lys Gln Thr Ile Ser Tyr Met 50 55 60 Ala Glu Arg Val Val Gly Thr Gly Ser Phe Gly Ile Val Phe Gln Ala 65 70 75 80 Lys Cys Leu Glu Thr Gly Glu Thr Val Ala Ile Lys Lys Val Leu Gln 85 90 95 Asp Arg Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met Arg Ser Met Ile 100 105 110 His Ser Asn Val Val Ser Leu Lys His Cys Phe Phe Ser Thr Thr Ser 115 120 125 Arg Asp Glu Leu Phe Leu Asn Leu Val Met Glu Tyr Val Pro Glu Thr 130 135 140 Leu Tyr Arg Val Leu Lys His Tyr Ser Asn Ala Lys Gln Gly Met Pro 145 150 155 160 Leu Ile Tyr Val Lys Leu Tyr Thr Tyr Gln Leu Phe Arg Gly Leu Ala 165 170 175 Tyr Ile His Thr Val Pro Gly Val Cys His Arg Asp Val Lys Pro Gln 180 185 190 Asn Val Leu Val Asp Pro Leu Thr His Gln Val Lys Ile Cys Asp Phe 195 200 205 Gly Ser Ala Lys Val Leu Val Ala Gly Glu Pro Asn Ile Ser Tyr Ile 210 215 220 Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu 225 230 235 240 Tyr Thr Thr Ser Ile Asp Ile Trp Ser Ala Gly Cys Val Leu Ala Glu 245 250 255 Leu Leu Leu Gly Gln Pro Leu Phe Pro Gly Glu Ser Ala Val Asp Gln 260 265 270 Leu Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Glu Ile 275 280 285 Arg Cys Met Asn Pro Asn Tyr Thr Glu Phe Arg Phe Pro Gln Ile Lys 290 295 300 Ala His Pro Trp His Lys Val Phe His Lys Lys Met Pro Pro Glu Ala 305 310 315 320 Ile Asp Leu Ala Ser Arg Leu Leu Gln Tyr Ser Pro Ser Leu Arg Cys 325 330 335 Thr Ala Leu Asp Ala Cys Ala His Pro Phe Phe Asp Glu Leu Trp Glu 340 345 350 Pro Asn Ala Arg Leu Pro Asn Gly Arg Pro Phe Pro Pro Leu Phe Asn 355 360 365 Phe Lys His Glu Leu Ala Asn Ala Ser Gln Asp Leu Ile Asn Arg Leu 370 375 380 Val Pro Glu His Val Arg Arg Gln Ala Gly Leu Ala Phe Val His Ala 385 390 395 400 Gly Ser <210> 2 <211> 1209 <212> DNA <213> Wheat (Triticum aestivum L.) <400> 2 atggagcatc cggcgccggc gccggagccg atgctgctcg acgagcagcc ccccaccgca 60 gtcgcctgcg agaagaagca gcaggatggc gaggcgccgt atgcggaggg gaacgacgcc 120 atgaccggtc acatcatctc caccaccatc ggcggcaaga acggcgagcc caagcagacg 180 attagctaca tggcggagcg cgttgtgggc actggttcgt ttggcatcgt cttcaggct 240 aaatgcctgg agaccgggga gacagtggcc attaagaagg tactgcagga ccgacggtac 300 aagaatcgtg agctgcagct tatgcgttcg atgatccatt ccaatgttgt ctccctcaag 360 cactgcttct tctcaaccac aagtagagat gagctgtttc tgaaccttgt catggagtat 420 gtcccggaga cactctaccg cgtgcttaag cactacagta atgccaaaca ggggatgcca 480 cttatctacg tcaagcttta cacctatcag ctattcaggg ggctggcgta cattcatact 540 gttccaggag tctgtcacag ggatgtgaag ccacaaaatg ttttggttga tcctttaaca 600 catcaagtta agatctgcga ctttggaagc gcgaaagttc tggtggcggg tgagcccaat 660 atatcataca tatgctcacg ctactaccgt gctccggagc ttatatttgg tgcgactgaa 720 tatacaacat cgatagatat atggtcagct ggttgtgttc ttgcagaact gctccttggt 780 cagccattat ttccaggcga gagtgcagtc gatcaacttg tagagataat caaggttctt 840 ggaacgccaa ctcgggagga atacgttgt atgaacccga attatacgga gtttaggttt 900 ccacagataa aagctcatcc ttggcacaag gttttccaca agaaaatgcc tcctgaagcc 960 atagatcttg cttcacgtct tcttcaatat tcaccaagcc tccgttgcac tgctcttgac 1020 gcatgtgcgc atcctttctt tgatgagcta tgggagccta acgcgcgcct gccaaatgga 1080 cgcccgttcc cacctctgtt caacttcaag catgaactgg caatgcttc acaagacctc 1140 atcaacaggc ttgtgcctga acatgttcgc cgacaagctg gtcttgcttt cgtgcatgcg 1200 gggagctaa 1209

Claims

1. The application of proteins, characterized in that, The application is any one of the following: D1) Application in improving plant salt tolerance and drought resistance; D2) Application in the preparation of products that improve the salt tolerance and drought resistance of plants; Application of D3 in the cultivation of salt-tolerant and drought-resistant plants; D4) Application in the preparation of products for cultivating salt-tolerant and drought-resistant plants; The protein in question is the protein whose amino acid sequence is SEQ ID No.

1. The plant is wheat or Arabidopsis thaliana; the application is achieved by increasing the expression level of the gene encoding the protein.

2. The application of biomaterials related to the protein described in claim 1, characterized in that, The application is any one of the following: E1) Application in improving plant salt tolerance and drought resistance; E2) Application in the preparation of products that improve the salt tolerance and drought resistance of plants; E3) Application in the cultivation of salt-tolerant and drought-resistant plants; E4) Application in the preparation of products for cultivating salt-tolerant and drought-resistant plants; The plant is wheat or Arabidopsis thaliana; The biomaterial is any one of B1) to B7) below: B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); The application is achieved by increasing the expression level of the gene encoding the protein described in claim 1.

3. The application according to claim 2, characterized in that, B1) The nucleic acid molecule is a DNA molecule whose coding sequence is SEQ ID No.

2.

4. A method for cultivating salt-tolerant and drought-resistant plants, characterized in that, The method includes increasing the content of the protein described in claim 1 in the target plant to obtain a plant with higher salt tolerance and drought resistance than the target plant; the plant is wheat or Arabidopsis thaliana.

5. The method according to claim 4, characterized in that, The increase in the content of the protein described in claim 1 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

6. The method according to claim 5, characterized in that, The improvement in the expression level of the protein-coding gene in the target plant is achieved by introducing the protein-coding gene of claim 1 into the target plant.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the gene encoding the protein is shown in SEQ ID No.

2.

8. The use of the protein of claim 1, or the biomaterial of claim 2, or the nucleic acid molecule of claim 3, or the method of any one of claims 4-7 in the creation of salt-tolerant and drought-resistant plants, wherein the plant is wheat or Arabidopsis thaliana.

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