Soybean stress-related protein gmSQLE1 and its coding gene in regulating plant stress resistance

Abstract

The application discloses a soybean stress resistance related protein GmSQLE1 and application of a coding gene thereof in regulation of plant stress resistance. Specifically disclosed is 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 GmSQLE1 gene derived from soybean into a receptor plant to obtain a homozygous transgenic GmSQLE1 plant. Experiments prove that, compared with a non-transgenic receptor control, the transgenic Arabidopsis and soybean both show stronger tolerance under drought stress and salt stress conditions, the survival rate is significantly higher than that of the receptor control, and the growth is significantly better than that of the receptor control, indicating that overexpression of the GmSQLE1 gene can significantly improve the drought stress resistance and salt tolerance of the plant (i.e. improve the stress resistance of the plant). The gene for regulating plant stress resistance 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 soybean stress resistance-related protein GmSQLE1 and its encoding gene in regulating plant stress resistance. Background Technology

[0002] During their growth and development, plants constantly face various abiotic stresses, such as drought, high salinity, and low temperature, which severely impact their growth and development. Research on plant stress resistance mechanisms and the breeding of stress-resistant new varieties have always been major needs for sustainable agricultural development. Abiotic stresses can induce changes in plant physiological traits and metabolism. Under abiotic stress, plants produce a series of responses, accompanied by many physiological, biochemical, and developmental changes. Clarifying the mechanisms of plant responses to stress will provide a scientific basis for research and application of stress-resistant genetic engineering.

[0003] Under environmental stress conditions such as drought, high salinity, and low temperature, plants can make corresponding adjustments at the molecular, cellular, and systemic levels to minimize environmental damage and survive. Over a long evolutionary process, from the perception of stress signals, intercellular transduction and transmission, to the final expression of various stress genes and the development of adaptations, a series of complex molecular mechanisms for stress signal transduction have formed. Many genes are induced to express under stress, and the products of these genes can not only directly participate in the plant's stress response but also regulate the expression of other related genes or participate in signal transduction pathways, thereby enabling plants to avoid or reduce damage and enhance their resistance to stressful environments. In addition to traditional breeding, research on plant stress resistance has gradually penetrated to the cellular and molecular levels. Therefore, identifying stress-related genes and applying them to crop genetic breeding will become an effective means of improving specific stress-resistant traits in crops, and has important theoretical guiding significance and practical application value for breeding new stress-resistant varieties and improving crop stress resistance.

[0004] Soybean (Glycine max (Linn.) Merr.) is an important food and oilseed crop. Its seeds are rich in protein, providing 70% of the world's edible protein, and are also an emerging raw material for biodiesel. With the increasing frequency of global climate change and natural disasters, soybeans are subjected to various environmental stresses during their growth and development. In particular, drought, high salinity, and low temperature are significant obstacles to soybean growth and development, severely impacting yield and quality. Therefore, studying the response and signal transduction mechanisms of soybeans to adverse conditions, and improving their resistance, has become a crucial task in soybean genetic research and variety improvement. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the stress resistance 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 GmSQLE1 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 protein GmSQLE1 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein GmSQLE1 isolated in this invention, provided they encode and function as protein GmSQLE1, 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 an identity search on a pair of amino acid sequences, the identity value (%) can then 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 GmSQLE1.

[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 soybean (Glycine max (Linn.) Merr.).

[0026] The present invention also provides applications of biomaterials related to the protein GmSQLE1, wherein the applications may be any of the following:

[0027] Application of biomaterials related to the protein GmSQLE1 in regulating plant stress resistance;

[0028] E2) Application of biomaterials related to the protein GmSQLE1 in the preparation of products that regulate plant stress resistance;

[0029] Application of biomaterials related to the protein GmSQLE1 in the cultivation of stress-resistant plants;

[0030] E4) Application of biomaterials related to the protein GmSQLE1 in the preparation of products for cultivating stress-resistant plants;

[0031] E5) Application of biomaterials related to the protein GmSQLE1 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 GmSQLE1;

[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 GmSQLE1 gene that regulates plant stress resistance) encodes the protein GmSQLE1, 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 gene encoding the protein GmSQLE1 (CDS). The protein GmSQLE1 gene (GmSQLE1 gene) described in this invention can be any nucleotide sequence capable of encoding the protein GmSQLE1. 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 a nucleic acid molecule 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 Peasyblunt vector, vector pBI121, and / or vector pTF101.

[0049] Recombinant expression vectors containing the GmSQLE1 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).

[0050] When constructing a recombinant plant expression vector using the GmSQLE1 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 GmSQLE1 gene or gene fragments provided in 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 GmSQLE1 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 the recombinant vector Peasyblunt-GmSQLE1, pBI121-GmSQLE1, and / or pTF101-GmSQLE1.

[0055] The recombinant vector Peasyblunt-GmSQLE1 is 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.

[0056] The recombinant vector pBI121-GmSQLE1 is a DNA molecule, as shown in SEQ ID No. 2, inserted into the SmaI restriction site of the vector pBI121.

[0057] The recombinant vector pTF101-GmSQLE1 is a DNA molecule, as shown in SEQ ID No. 2, inserted into the XbaI restriction site of the vector pTF101.

[0058] The recombinant microorganism may specifically be recombinant Agrobacterium GV3101 / pBI121-GmSQLE1 and / or GV3101 / pTF101-GmSQLE1.

[0059] The recombinant Agrobacterium GV3101 / pBI121-GmSQLE1 contains the GmSQLE1 gene with the coding sequence SEQ ID NO.2, and is a recombinant microorganism obtained by introducing the recombinant vector pBI121-GmSQLE1 into Agrobacterium GV3101.

[0060] The recombinant Agrobacterium GV3101 / pTF101-GmSQLE1 contains the GmSQLE1 gene encoding the sequence SEQ ID NO.2, and is a recombinant microorganism obtained by introducing the recombinant vector pTF101-GmSQLE1 into Agrobacterium GV3101. This invention also provides a method for cultivating stress-resistant plants, the method comprising increasing the content and / or activity of the protein GmSQLE1 in the target plant to obtain a stress-resistant plant with higher stress resistance than the target plant.

[0061] In the above method, the increase in the content and / or activity of the protein GmSQLE1 in the target plant is achieved by increasing the expression level of the gene encoding the protein GmSQLE1 in the target plant.

[0062] In the above method, the expression level of the gene encoding the protein GmSQLE1 in the target plant is increased by introducing the gene encoding the protein GmSQLE1 into the target plant.

[0063] In the above method, the gene encoding the protein GmSQLE1 can be any of the following:

[0064] F1) The coding sequence is a DNA molecule of SEQ ID No. 2;

[0065] F2) The nucleotide sequence is the DNA molecule of SEQ ID No.2.

[0066] 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.

[0067] In one embodiment of the present invention, the method for cultivating stress-resistant plants includes the following steps:

[0068] (1) Construct a recombinant vector containing the DNA molecule shown in SEQ ID NO.2;

[0069] (2) Transfer the recombinant vector constructed in step (1) into the target plant (such as crop or soybean);

[0070] (3) Through screening and identification, stress-resistant plants with higher stress resistance than the target plant were obtained.

[0071] In the above method, the plant can be G1) or G2):

[0072] G1) Monocotyledonous or dicotyledonous plants;

[0073] G2) Legumes or cruciferous plants.

[0074] In this article, the plants mentioned can be crops (agricultural crops).

[0075] The present invention also provides the application of the protein GmSQLE1, or the biological material, or the nucleic acid molecule, or any of the methods for cultivating stress-resistant plants in the creation of stress-resistant plants and / or plant breeding.

[0076] The protein GmSQLE1, the biomaterial, and the nucleic acid molecule described herein are all within the scope of protection of this invention.

[0077] The protein GmSQLE1 described in this article may be a soybean stress resistance-related protein.

[0078] The plant breeding described in this article can be used for crop stress resistance breeding.

[0079] The regulation of plant stress resistance can be used to either increase or decrease plant stress resistance.

[0080] Increased expression and / or activity of the GmSQLE1 protein or its encoding gene in the target plant enhances the plant's stress resistance.

[0081] Furthermore, the improved stress resistance is manifested in:

[0082] (1) Improve the survival rate of the target plant;

[0083] (2) Increase the chlorophyll content of the target plant;

[0084] (3) Increase the proline content of the target plant;

[0085] (4) Reduce the malondialdehyde content of the target plant.

[0086] The aforementioned resistance includes, but is not limited to, salt tolerance, drought resistance, heat resistance, cold resistance and / or strong light resistance.

[0087] Specifically, the stress resistance may be salt tolerance and / or drought resistance.

[0088] 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 GmSQLE1 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.

[0089] This invention introduces the GmSQLE1 gene, which regulates plant stress resistance and is derived from soybean (Glycine max (Linn.) Merr.), into recipient control plants wild-type Arabidopsis thaliana (WT, Col) and wild-type soybean (Williams82), respectively, to obtain homozygous Arabidopsis thaliana lines (GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3) and homozygous soybean lines (GmSQLE1-OE1, GmSQLE1-OE2, and GmSQLE1-OE3). Experiments have shown that, compared with the non-transgenic recipient control, the survival rate of transgenic Arabidopsis thaliana and soybean overexpressing the GmSQLE1 gene was significantly higher than that of the recipient control under drought and salt stress conditions, and their growth was also significantly better than that of the recipient control. This indicates that overexpression of the GmSQLE1 gene can significantly improve the drought and salt tolerance of plants (i.e., improve the plant's stress resistance). Specifically, the survival rate of transgenic Arabidopsis thaliana and soybean overexpressing the GmSQLE1 gene was significantly improved, the chlorophyll content and proline content were significantly increased, and the malondialdehyde content was 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 soybean overexpressing the GmSQLE1 gene both showed stronger tolerance, indicating that the GmSQLE1 protein and its encoding gene GmSQLE1 of the present invention can regulate plant stress resistance (such as drought resistance and / or salt tolerance), and that increasing the content and / or activity of GmSQLE1 protein in the target plant (such as overexpressing the GmSQLE1 gene) can significantly improve the stress resistance of the target plant.

[0090] 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

[0091] Figure 1 Analysis for stress resistance identification in Arabidopsis thaliana transgenic with the GmSQLE1 gene. Figure 1 Figure A shows the phenotypic diagrams of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (Col-0) under salt conditions. Figure 1 Figure B shows the phenotypic diagrams of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (Col-0) under drought conditions. Figure 1 Figures C, D, and E show the results of measuring the proline (Pro), malondialdehyde (MDA), and chlorophyll (Chlorophyll) content in transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (Col-0) under salt and drought conditions.

[0092] Figure 2 Detection of GmSQLE1 gene-transgenic soybeans. Figure 2 In Figure A, the phenotypes of transgenic soybean and the wild-type recipient control are shown under normal conditions. Figure 2 Image B represents PCR detection of genetically modified soybeans; Figure 2 C represents the expression level analysis of the GmSQLE1 target gene in transgenic soybeans; Figure 2 D represents the total root length of transgenic soybeans and wild-type soybeans under normal growth conditions; Figure 2 E represents the hypocotyl length of transgenic soybeans and wild-type soybeans under normal growth conditions; Figure 2 F represents the fresh weight of genetically modified soybeans and wild-type soybeans under normal growth conditions.

[0093] Figure 3 To identify the stress resistance of transgenic GmSQLE1 soybeans during the seedling stage. Figure 3 Figure A shows the phenotypic diagrams of transgenic soybean seedlings and the recipient control after one week of treatment with solid medium containing 150 mM NaCl and 200 mM mannitol. Figure 3 In the study, biomass was measured in tissue-cultured transgenic soybean seedlings and recipient control seedlings after one week of treatment with solid culture media containing 150 mM NaCl and 200 mM mannitol, respectively. Figure 3 The figures in F represent the phenotypic figures of transgenic soybeans and recipient controls at the seedling stage after one week of drought and water control in soil culture (drought stress treatment) and after salt stress treatment. Figure 3 The graph in Figure GI shows the results of measuring the proline, malondialdehyde, and chlorophyll content in the leaves of transgenic soybean seedlings and recipient control seedlings after treatment with 200 mM NaCl aqueous solution for 3 days and drought stress treatment for 1 week.

[0094] Figure 4 To identify the stress resistance of transgenic GmSQLE1 soybeans during the flowering period. Figure 4 Image A shows the phenotypic figures of transgenic soybean seedlings at the flowering stage and the recipient control after one week of water control and 300 mM NaCl treatment, respectively. The first row represents normal conditions, the second row represents drought treatment, and the third row represents salt stress treatment. Figure 4 Figure B shows the survival rate of transgenic soybean seedlings and the recipient control after one week of water control treatment during the flowering stage. Figure 4 Figure C shows the biomass measurement results of transgenic soybean seedlings and recipient control after one week of treatment with 300mM NaCl aqueous solution during the flowering stage. Detailed Implementation

[0095] 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.

[0096] 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.

[0097] The soybean varieties “Williams 82” and “Tiefeng 8” in the following examples are described in the following literature: Jiang Jinghan. Study on salt tolerance mechanism and salt tolerance gene localization in soybean seedlings [D]. Chinese Academy of Agricultural Sciences, 2013., which can be obtained by the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0098] The wild-type Arabidopsis thaliana (WT, Col) used in the following examples is the Colombian ecotype Arabidopsis thaliana (Col-0), purchased from SALK.

[0099] The Agrobacterium tumefaciens GV3101 used in the following examples was purchased from Beijing Bairddi Biotechnology Co., Ltd.

[0100] The vector pTF101 in the following examples is described in the following literature: Yu TF, Liu Y, Fu JD, Ma J, Fang ZW, Chen J, Zheng L, Lu ZW, Zhou YB, Chen M, Xu ZS and Ma YZ. (2021) The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stresstolerance. Plant Biotechnology Journal, (2021) 19, pp. 2589–2605.

[0101] The carrier pBI121 used in the following embodiments was purchased from Bayerdy.

[0102] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Two-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0103] Example 1: Obtaining the GmSQLE1 protein and its encoding gene

[0104] Under normal conditions, seedlings of Tiefeng No. 8 at the four-leaf stage, which have been growing for about two weeks, were treated with NaCl for 2 hours, flash-frozen with liquid nitrogen, and stored at -80℃ for later use.

[0105] Total RNA was extracted from soybean leaves using the Trizol method (TianGen), and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). ds cDNA was synthesized using the SMART method, and the PCR products were detected by 1.0% agarose gel electrophoresis.

[0106] The DNA molecule shown in SEQ ID No. 2 was obtained by 5' RACE and 3' RACE. The DNA molecule shown in SEQ ID No. 2 encodes the protein shown in SEQ ID No. 1.

[0107] The plant stress resistance-related protein gene isolated and cloned from the soybean variety "Tiefeng 8" was named the GmSQLE1 gene. The coding sequence (CDS) of the GmSQLE1 gene is SEQ ID No.2, and the protein encoding the amino acid sequence of SEQ ID No.1 is named the GmSQLE1 protein.

[0108] Example 2: Effect of GmSQLE1 protein on stress resistance in Arabidopsis thaliana

[0109] I. Construction of Recombinant Expression Vectors

[0110] 1. Total RNA was extracted from the leaves of Tiefeng No. 8 and cDNA was obtained by reverse transcription.

[0111] 2. Using the cDNA from step 1 above as a template, amplify the soybean GmSQLE1 gene using specific primers, and recover the PCR product. The specific amplification primers for the GmSQLE1 gene are as follows:

[0112] SQLE-F: 5'-CAAAGAGGTGTTCCAAGAAACTAAA-3',

[0113] SQLE-R: 5'-GAGTTCCCTCTCTTATTCTTCTTGG-3'.

[0114] 3. The amplified and recovered PCR products were ligated into the Peasyblunt vector (pEASY-Blunt Cloning Kit, catalog number CB101, TransGen, 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-GmSQLE1.

[0115] The Peasyblunt-GmSQLE1 plasmid is 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.

[0116] 4. Using the plasmid Peasyblunt-GmSQLE1 obtained in step 3 as a template, PCR amplification was performed using primer pairs composed of GmSQLE1-121F and GmSQLE1-121R to obtain PCR amplification products, and the PCR products were then recovered from the gel.

[0117] GmSQLE1-121F:5'-CTCTAGAGGATC CCCGGG ATGGATTATCCGTAC-3';

[0118] GmSQLE1-121R:5'-ACTAGTGGATCC CCCGGG TCAATGGACAGGAGG-3.

[0119] 5. Digest the vector pBI121 with the restriction endonuclease SmaI and recover the vector backbone.

[0120] 6. The PCR product from step 4 and the vector backbone from step 5 were ligated using In-Fusion technology (Liuhetong, Beijing) to obtain the recombinant plasmid pBI121-GmSQLE1. Based on the sequencing results, the structure of the recombinant plasmid pBI121-GmSQLE1 is described as follows: A DNA molecule as shown in SEQ ID No. 2 was inserted into the SmaI restriction site of the vector pBI121.

[0121] II. Obtaining Transgenic Arabidopsis

[0122] 1. The recombinant plasmid pBI121-GmSQLE1 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium GV3101 / pBI121-GmSQLE1.

[0123] 2. Inoculate the recombinant Agrobacterium GV3101 / pBI121-GmSQLE1 obtained in step 1 into liquid YEP medium and culture at 28°C and 3000 rpm for about 30 hours.

[0124] 3. 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.

[0125] 4. After completing step 3, collect the bacterial cells, centrifuge at 4000g for 10 minutes at 4℃, and dilute to OD500 with an aqueous solution containing 10% sucrose and 0.02% silwet. 600nm Approximately 1.0, which is the amount of the inoculum.

[0126] 5. Place the Colombian ecotype Arabidopsis thaliana (Col-0) planted in flowerpots upside down in a container filled with the infection solution, submerging the flowers for about 50 seconds. Then remove the flowerpots, place them on their sides in a tray, cover them with black plastic sheeting, and uncover them after 24 hours. Place the flowerpots upright and cultivate them under normal light conditions. Harvest the seeds, which are the seeds of the T0 generation Arabidopsis thaliana.

[0127] 6. Sow the seeds of T0 generation Arabidopsis thaliana on solid MS medium plates containing 50 mg / L kanamycin. The resulting plants are T1 generation Arabidopsis thaliana.

[0128] 7. Self-pollinate and harvest the seeds of T1 generation Arabidopsis thaliana to obtain T1 generation Arabidopsis thaliana seeds. Plants cultured from T1 generation Arabidopsis thaliana seeds are T2 generation Arabidopsis thaliana. Self-pollinate and harvest the seeds of T2 generation Arabidopsis thaliana to obtain T2 generation Arabidopsis thaliana seeds. Plants cultured from T2 generation Arabidopsis thaliana seeds are T3 generation Arabidopsis thaliana.

[0129] 8. Perform PCR identification on T2 generation Arabidopsis and sampled T3 generation Arabidopsis. If both a T2 generation Arabidopsis and its self-pollinated T3 generation are positive for PCR, then the T2 generation Arabidopsis and its self-pollinated offspring constitute a homozygous transgenic Arabidopsis line carrying the GmSQLE1 gene. The PCR identification method is as follows: Genomic DNA is extracted from leaves and amplified by PCR using primers consisting of GmSQLE1-JCF and GmSQLE1-JCR. A positive PCR identification is indicated by an amplification product of approximately 610 bp. The PCR primers are:

[0130] GmSQLE1-JCF:5'-CCTCTCGCCAATCTTATTCTA-3',

[0131] GmSQLE1-JCR: 5'-CAACAGCAAAGAAATGGAGAACCAA-3'.

[0132] Three homozygous transgenic Arabidopsis thaliana lines were identified by PCR and named as GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3.

[0133] III. Identification of stress resistance in Arabidopsis thaliana

[0134] 1. Drought stress treatment (drought resistance assessment)

[0135] The seeds tested included: seeds from the T3 generation of the GmSQLE1-1 strain, seeds from the T3 generation of the GmSQLE1-2 strain, seeds from the T3 generation of the GmSQLE1-3 strain, and seeds of wild-type Arabidopsis thaliana (Col-0). Among these, the GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 strains were all homozygous transgenic Arabidopsis thaliana strains carrying the GmSQLE1 gene.

[0136] Drought group: The seeds to be tested were sown, and the number of days from the start of seed germination was counted. After 20 days, drought treatment (i.e., no watering for one week) was started, followed by re-watering treatment (i.e., normal watering resumed for one week). Then, photos were taken and the survival rate was counted.

[0137] Normal group: The seeds to be tested were sown, and the number of days from the start of seed germination was counted. After 20 days, normal watering and management continued for two weeks, and then photos were taken.

[0138] Three replicate experiments were set up, with 10 plants of each type of seed observed and statistically analyzed in each replicate experiment. Survival rate was recorded, and physiological indicators such as proline (Pro), malondialdehyde (MDA), and chlorophyll content were measured.

[0139] See results Figure 1 . Figure 1 Col-0 represents wild-type Arabidopsis thaliana; OE-1 represents Arabidopsis thaliana of the GmSQLE1-1 strain; OE-2 represents Arabidopsis thaliana of the GmSQLE1-2 strain; and OE-3 represents Arabidopsis thaliana of the GmSQLE1-3 strain.

[0140] Under normal conditions, the growth of Arabidopsis thaliana in the GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 strains was no different from that in the wild type.

[0141] One week after drought treatment, wilting and death occurred in the Arabidopsis thaliana plants. The GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 lines all showed significantly better growth than the wild-type Arabidopsis. The survival rate of the GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 lines was significantly higher than that of the wild-type Arabidopsis. This indicates that overexpression of the GmSQLE1 gene can significantly improve the drought resistance of plants.

[0142] Furthermore, under normal conditions, there was no significant difference in chlorophyll content between Arabidopsis thaliana strains GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 and wild-type Arabidopsis thaliana (Col-0). After drought treatment, the chlorophyll content of transgenic Arabidopsis thaliana strains overexpressing the GmSQLE1 gene (GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3) was significantly higher than that of wild-type Arabidopsis thaliana (Col-0). This indicates that under drought treatment, transgenic Arabidopsis thaliana with the GmSQLE1 gene showed stronger tolerance compared to non-transgenic wild-type Arabidopsis thaliana (Col-0).

[0143] Under normal conditions, there was no significant difference in proline (Pro) content between Arabidopsis thaliana lines GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 and wild-type Arabidopsis thaliana (Col-0). After drought treatment, the proline (Pro) content of transgenic Arabidopsis thaliana lines overexpressing the GmSQLE1 gene (GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 lines) was significantly higher than that of wild-type Arabidopsis thaliana (Col-0). Proline, as an important osmotic regulator, plays a crucial role in maintaining cellular osmotic potential, protecting cells, and maintaining osmotic balance. Proline content can indirectly reflect a plant's ability to resist abiotic stress. Therefore, the increase in proline (Pro) content after drought treatment indicates that overexpression of the GmSQLE1 gene enhances the drought resistance of transgenic Arabidopsis thaliana.

[0144] Under normal conditions, there was no significant difference in malondialdehyde (MDA) content between Arabidopsis thaliana lines GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 and wild-type Arabidopsis thaliana (Col-0). After drought treatment, the MDA content of transgenic Arabidopsis thaliana lines overexpressing the GmSQLE1 gene (GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 lines) was significantly lower than that of wild-type Arabidopsis thaliana (Col-0). MDA is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of abiotic stress suffered by plants. Accumulation of MDA can also cause damage to membranes and cells. MDA content is a commonly used indicator in plant resistance physiology studies, and it can be used to understand the degree of membrane lipid peroxidation. Therefore, after drought treatment, Arabidopsis thaliana overexpressing the GmSQLE1 gene reduced the accumulation of MDA, thereby alleviating the oxidative damage caused by drought stress to transgenic Arabidopsis thaliana and improving the drought stress resistance of transgenic Arabidopsis thaliana with the GmSQLE1 gene.

[0145] 2. High-salt stress treatment (salt tolerance assessment)

[0146] The seeds tested included: seeds from the T3 generation of the GmSQLE1-1 strain, seeds from the T3 generation of the GmSQLE1-2 strain, seeds from the T3 generation of the GmSQLE1-3 strain, and seeds of wild-type Arabidopsis thaliana (Col-0). Among these, the GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 strains were all homozygous transgenic Arabidopsis thaliana strains carrying the GmSQLE1 gene.

[0147] Salt stress group (Salt): The test seeds were sown, and the number of days from the start of seed germination was counted. After 20 days, salt stress treatment was started (i.e., watering with 200mM NaCl aqueous solution and treating for one week), and then the survival rate was recorded by taking pictures.

[0148] Normal group: The seeds to be tested were sown, and the number of days from the start of seed germination was counted. After 20 days, normal watering and management continued for one week, and then photos were taken.

[0149] Three replicate experiments were set up, with 10 plants of each type of seed observed and statistically analyzed in each replicate experiment. Survival rate was recorded, and physiological indicators such as proline (Pro), malondialdehyde (MDA), and chlorophyll content were measured.

[0150] See results Figure 1 . Figure 1Col-0 represents wild-type Arabidopsis thaliana; OE-1 represents Arabidopsis thaliana of the GmSQLE1-1 strain; OE-2 represents Arabidopsis thaliana of the GmSQLE1-2 strain; and OE-3 represents Arabidopsis thaliana of the GmSQLE1-3 strain.

[0151] Under normal conditions, the growth of Arabidopsis thaliana in the GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 strains was not significantly different from that in the wild-type Arabidopsis thaliana.

[0152] After salt stress treatment, the Arabidopsis thaliana lines GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 all showed significantly better growth than the wild-type Arabidopsis. The survival rate of these three lines was also significantly higher than that of the wild-type Arabidopsis. This indicates that overexpression of the GmSQLE1 gene can significantly improve the salt tolerance of plants.

[0153] Furthermore, under normal conditions, there were no significant differences in proline (Pro) content, malondialdehyde (MDA) content, and chlorophyll content between Arabidopsis thaliana strains GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3 and wild-type Arabidopsis thaliana (Col-0). After salt stress treatment, the proline (Pro) content and chlorophyll content of transgenic Arabidopsis thaliana strains overexpressing the GmSQLE1 gene (GmSQLE1-1, GmSQLE1-2, and GmSQLE1-3) were significantly lower. The content of Chlorophyll was significantly higher than that of wild-type Arabidopsis thaliana (Col-0), while the content of malondialdehyde (MDA) was significantly lower than that of wild-type Arabidopsis thaliana. This indicates that under salt stress treatment, transgenic Arabidopsis thaliana with the GmSQLE1 gene showed stronger tolerance than non-transgenic wild-type Arabidopsis thaliana (Col-0). Overexpression of the GmSQLE1 gene in Arabidopsis thaliana reduced the accumulation of MDA and increased the content of proline, thus mitigating the oxidative damage caused by salt stress to transgenic Arabidopsis thaliana and improving the salt tolerance of transgenic Arabidopsis thaliana with the GmSQLE1 gene.

[0154] In summary, the GmSQLE1 protein and its encoding gene GmSQLE1 can regulate plant stress resistance (such as drought resistance and / or salt tolerance). Increasing the content and / or activity of GmSQLE1 protein in target plants (such as overexpressing the GmSQLE1 gene) can significantly improve the stress resistance of target plants.

[0155] The methods for determining the proline (Pro), malondialdehyde (MDA), and chlorophyll (Chlorophyll) content in the above-mentioned Arabidopsis thaliana stress resistance identification steps are as follows:

[0156] (1) Chlorophyll content determination: Weigh 0.1g of Arabidopsis thaliana leaves, cut them into approximately 1mm fragments, and place them in a solution containing 20mL of 95% ethanol (80% acetone). Soak in the dark at room temperature for 36-48h. Filter the extract into a 50mL brown volumetric flask, dilute to 50mL with 95% ethanol (80% acetone), shake well, and store in the dark for later testing. Take a 1cm cuvette, pour in the above chlorophyll alcohol solution, use 95% ethanol as the reference solution, and the 1cm cuvette as the absorption cell. Measure the absorbance at wavelengths of 663nm and 645nm, respectively. Read three times at each wavelength, and use the average value to calculate the chlorophyll content. Formula: Chlorophyll a content (Ca) = 12.7A 663 -2.69A 645 Chlorophyll b content (Cb) = 22.9A 645 -4.68A 663 Total chlorophyll content (Ct) = (Ca + Cb).

[0157] (2) Proline content determination: The proline content was determined according to the instructions of the proline content detection kit (Beijing Solarbio Technology Co., Ltd.).

[0158] (3) Malondialdehyde content determination: The malondialdehyde content was determined according to the instructions of the malondialdehyde (MDA) content detection kit (Beijing Solarbio Technology Co., Ltd.).

[0159] Example 3: Effects of GmSQLE1 protein on soybean stress resistance

[0160] I. Construction of Recombinant Expression Vectors

[0161] 1. Using the plasmid (Peasyblunt-GmSQLE1) obtained in step 3 of Example 2 as a template, PCR amplification was performed using primer pair composed of GmSQLE1-101F and GmSQLE1-101R to obtain PCR amplification products, and the PCR products were then recovered from the gel.

[0162] GmSQLE1-101F:5'-GAGAACACGGGGGACTCTAGAATGGATTATCCGTAC-3';

[0163] GmSQLE1-101R:5'-GCCCTTGCTCACCATTCTAGAATGGACAGGAGG-3.

[0164] 2. Digest the vector pTF101 with the restriction endonuclease XbaI and recover the vector backbone.

[0165] 3. The PCR product recovered in step 1 and the vector backbone from step 2 were ligated using In-Fusion technology to obtain the recombinant plasmid pTF101-GmSQLE1. After confirmation by the company's sequencing, the plasmid was extracted from the positive bacterial culture for later use.

[0166] The recombinant plasmid pTF101-GmSQLE1 is formed by inserting the DNA molecule shown in SEQ ID No. 2 into the XbaI restriction site of the vector pTF101.

[0167] II. Obtaining Genetically Modified Soybeans

[0168] 1. The recombinant plasmid pTF101-GmSQLE1 was introduced into Agrobacterium GV3101 to obtain Agrobacterium GV3101 / pTF101-GmSQLE1 containing the recombinant plasmid.

[0169] (1) Agrobacterium GV3101 / pTF101-GmSQLE1 containing recombinant plasmid was inoculated into YEP liquid medium and cultured at 28℃ and 3000rpm for about 18 hours.

[0170] (2) Draw the bacterial culture obtained in step (1) into YEP solid medium (containing 50 μg / L streptomycin and 50 μg / L kanamycin) and incubate at 28°C for about 2 days.

[0171] 2. Sterilize soybean seeds (Williams 82) with chlorine for 8 hours, then sow them evenly in sterilized B5 medium and culture until the radicle forms.

[0172] 3. After completing step 2, remove the soybean cotyledons and the radicle below the cotyledon node, and then culture the soybean callus tissue until it grows. Infect the callus tissue with Agrobacterium GV3101 (GV3101 / pTF101-GmSQLE1) containing the recombinant vector. After culturing, obtain GmSQLE1 gene-transgenic soybean (T0 generation transgenic soybean).

[0173] 4. Harvesting soybean seeds and screening homozygous lines: RNA was extracted from the plants and reverse transcribed into cDNA. Transgenic positive plants (containing the GmSQLE1 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: GmSQLE1-OE1 line, GmSQLE1-OE2 line, and GmSQLE1-OE3 line.

[0174] III. Detection of the relative expression level of the GmSQLE1 gene

[0175] After completing step two or three, soybean leaves were taken, total RNA was extracted and reverse transcribed to obtain cDNA, and qRT-PCR was performed using cDNA as a template. The Actin gene was used as an internal reference gene to detect the relative expression level of the GmSQLE1 gene.

[0176] The primers used to detect the GmSQLE1 gene are as follows:

[0177] GmSQLE1ygdl-F:5'-GCAAAGAAGGTCACAGATTCAAGTT-3',

[0178] GmSQLE1ygdl-R:5'-CGTCCATCCTTTCCAAGAGTGT-3'.

[0179] The primers used to detect the Actin gene are as follows:

[0180] Actin-F: 5'-CTGTTGGAAGGTGCTGAG-3',

[0181] Actin-R: 5'-ACATTGTTCTTAGTGGTGGCT-3'.

[0182] The identification of GmSQLE1 transgenic soybeans and the determination of the relative expression level of the GmSQLE1 gene, total root length, hypocotyl length, and fresh weight of the plants are shown in the table below. Figure 2 . Figure 2 In the diagram, WT represents wild-type soybean; OE1 represents soybean from the GmSQLE1-OE1 line; OE2 represents soybean from the GmSQLE1-OE2 line; and OE3 represents soybean from the GmSQLE1-OE3 line. The vector represents a positive control for the transgenic soybean test.

[0183] The results showed that the expression level of the GmSQLE1 gene in transgenic soybean was 5-17 times higher than that in wild-type (recipient control) soybean, with a significant difference. The exogenous GmSQLE1 gene not only successfully integrated into the soybean genome but also was able to be normally transcribed and expressed in transgenic soybean.

[0184] IV. Identification of stress resistance in soybeans

[0185] 1. Identification of stress resistance in genetically modified soybean seedlings

[0186] The drought resistance and salt tolerance of transgenic GmSQLE1 soybeans under tissue culture and soil culture conditions during the seedling stage were identified.

[0187] The test lines were: five-week-old transgenic GmSQLE1 soybean lines obtained in step two (GmSQLE1-OE1, GmSQLE1-OE2 and GmSQLE1-OE3 soybean lines) and wild-type soybean (WT, Williams 82).

[0188] 150mM NaCl group (150mM NaCl): Seeds of the test lines were sown in B5 medium containing 150mM NaCl and cultured for 1 week. Hypocotyl length and fresh weight of the whole plant were measured.

[0189] 200mM Mannitol group: Seeds of the test lines were sown in B5 medium containing 200mM mannitol and cultured for 1 week. Hypocotyl length and fresh weight of the whole plant were measured.

[0190] The 150mM NaCl group and the 200mM mannitol group were identical in all experimental conditions except for the culture medium. The experiment was repeated three times, with 20 seeds of each test line in each replicate.

[0191] Drought group: The test lines were subjected to water control treatment (specifically, the same mass of soil was weighed and sown on the same number of transgenic soybeans and the recipient control). After the seedlings grew to the four-leaf stage, water control treatment was applied, i.e., no watering for one week, followed by re-watering treatment (i.e., resuming normal watering for one week). The phenotype was observed. Five days after water control, the fresh weight of the aboveground parts of 10 plants and the content of malondialdehyde, proline, and chlorophyll in the leaves were measured.

[0192] Normal group: The same mass of soil was weighed and sown on the same number of transgenic soybeans and the recipient control. After the seedlings grew to the four-leaf stage, they were watered once. One week after watering, their phenotype was observed, and the fresh weight of the above-ground parts, malondialdehyde, proline and chlorophyll content of the leaves were measured.

[0193] Salt stress group (Salt, 200mM NaCl): Equal amounts of soil were weighed and sown on transgenic soybeans and the recipient control with the same number of seeds. After the seedlings grew to the four-leaf stage, they were irrigated once with a 200mM NaCl aqueous solution. One week after the irrigation, their phenotype was observed, and the fresh weight of the aboveground parts, malondialdehyde, proline, and chlorophyll content of the leaves were measured.

[0194] The drought stress group, normal group, and salt stress group were all subjected to the same experimental conditions except for irrigation. Three replicate experiments were set up, with 15 plants of each test line observed and statistically analyzed in each replicate. Simultaneously, various physiological indicators, such as leaf proline (Pro), malondialdehyde (MDA), and chlorophyll content, were measured.

[0195] Determination of chlorophyll content in samples: 0.2 g of leaves from both treated and untreated transgenic soybeans and the control group (three replicates per sample, leaves treated with 200 mM NaCl for 3 days and leaves treated with 25% PEG stress for 1 week) were taken from the same mass and at the same time. The leaves were cut into strips approximately 1 cm long and placed in corresponding 10 mL centrifuge tubes. 5 mL of 80% acetone was added to each tube, and the tubes were incubated overnight in the dark at room temperature. The chlorophyll content was measured using a microplate reader.

[0196] The determination methods for proline and malondialdehyde (MDA) in the samples should be performed according to the specific operating procedures of the kits: MDA content detection kit and proline content detection kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing).

[0197] The results are as follows Figure 3 As shown, Figure 3 In the diagram, WT represents the recipient control wild-type soybean; OE1 represents soybean from the GmSQLE1-OE1 line; OE2 represents soybean from the GmSQLE1-OE2 line; and OE3 represents soybean from the GmSQLE1-OE3 line. Wherein:

[0198] Figure 3 Photograph A shows the phenotypic images of transgenic soybean seedlings and the recipient control after one week of treatment with solid culture media containing 150 mM NaCl and 200 mM mannitol, respectively. Figure 3 (A)

[0199] Figure 3 F shows phenotypic images of transgenic soybean seedlings and recipient controls after one week of irrigation with 200 mM NaCl and after soil cultivation with drought and water control, respectively. Figure 3 (F).

[0200] Figure 3 Biomass was measured in the BE (biological study) after tissue-cultured transgenic soybean seedlings and recipient control seedlings were treated for one week with solid culture media containing 150 mM NaCl and 200 mM mannitol, respectively.

[0201] Figure 3 The GI method involves measuring the malondialdehyde, proline, and chlorophyll content in the leaves of transgenic soybean seedlings and recipient control seedlings after treatment with 200 mM NaCl aqueous solution for 3 days and drought stress for 1 week.

[0202] The results showed that after one week of salt treatment and one week of drought treatment, the recipient control exhibited severe wilting. However, the soybean plants transgenic with the GmSQLE1 gene showed significantly better growth than the control, and their physiological indicators under drought and high salt treatments were significantly superior to those of the recipient control. These results further demonstrate that overexpression of the GmSQLE1 gene can significantly improve the drought resistance and salt tolerance of plants.

[0203] 2. Identification of stress resistance during flowering period in genetically modified soybeans

[0204] To further verify whether overexpression of the GmSQLE1 gene under soil cultivation can improve the stress resistance of transgenic soybeans, the drought resistance and salt tolerance of GmSQLE1 transgenic soybeans during the flowering period under soil cultivation conditions were identified.

[0205] The plant lines to be tested were: five-week-old transgenic GmSQLE1 soybean plants obtained in step two (GmSQLE1-OE1, GmSQLE1-OE2 and GmSQLE1-OE3 lines of soybeans) and wild-type soybeans (WT, Williams 82).

[0206] The test plants were subjected to water control treatment (drought treatment) and NaCl treatment (salt stress treatment) (specific treatment method: weigh the same mass of soil and sow the same number of transgenic soybeans and recipient controls. After the seedlings grew to the flowering stage, they were subjected to normal treatment, water control treatment (drought treatment - 10 plants per line, watering stopped for 2 weeks, then watering resumed for 1 week, and their phenotypes were observed and survival rates were counted), and 300mM NaCl treatment (salt stress treatment). Their phenotypes were observed one week after treatment). All three treatments were identical except for the irrigation method. The experiment was repeated three times, with 15 seeds of each test line in each replicate.

[0207] Drought treatment (water control treatment): No watering for two consecutive weeks, then re-watering (i.e., resume normal watering for one week), then take photos and count the survival rate.

[0208] Salt stress treatment: Irrigate once with a 300mM NaCl aqueous solution. Observe the phenotype and measure the fresh weight of the aboveground parts one week after irrigation.

[0209] Normal treatment: Irrigate once with water, observe its phenotype one week after irrigation, and measure the fresh weight of the above-ground parts.

[0210] Three replicate experiments were set up, and phenotypic observations and statistics were performed on 10 plants of each test line in each replicate experiment. At the same time, the survival rate and biomass were recorded.

[0211] The results are as follows Figure 4 As shown, Figure 4 In the diagram, WT represents the recipient control wild-type soybean; OE1 represents soybean from the GmSQLE1-OE1 line; OE2 represents soybean from the GmSQLE1-OE2 line; and OE3 represents soybean from the GmSQLE1-OE3 line. Wherein:

[0212] Figure 4Image A shows phenotypic photos of transgenic soybean seedlings at the flowering stage and the recipient control after one week of water control and 300 mM NaCl treatment, respectively. The first row represents normal conditions, the second row represents drought treatment, and the third row represents salt stress treatment.

[0213] Figure 4 Figure B shows the survival rate of transgenic soybean seedlings and the recipient control at the flowering stage after one week of water control treatment.

[0214] Figure 4 In Figure C, the biomass of transgenic soybean seedlings and the recipient control at the flowering stage was measured after one week of treatment with a 300 mM NaCl aqueous solution.

[0215] The results showed that after one week of salt stress treatment and one week of drought treatment, the recipient control showed severe wilting. The growth of the GmSQLE1 gene-transformed soybean plants was better than that of the control. Their survival rate after drought treatment was significantly higher than that of the recipient control, and their physiological indicators under high salt treatment were also significantly better than those of the recipient control.

[0216] Therefore, the experimental results of both soybeans and Arabidopsis thaliana overexpressing the GmSQLE1 gene show that the GmSQLE1 protein and its encoding gene GmSQLE1 of this invention can regulate the stress resistance of plants (such as drought resistance and / or salt tolerance). By increasing the content and / or activity of GmSQLE1 protein in the target plant (such as by overexpressing the GmSQLE1 gene), the stress resistance of the target plant can be significantly improved.

[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 soybean stress resistance-related protein GmSQLE1 and its encoding gene in regulating plant stress resistance <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 525 <212> PRT <213> Soybean (Glycine max) <400> 1 Met Asp Tyr Pro Tyr Ile Leu Gly Gly Ile Met Ala Cys Ser Phe Ala 1 5 10 15 Phe Leu Tyr Val Val Tyr Ser Phe Gly Ala Lys Lys Val Thr Asp Ser 20 25 30 Ser Ser Ile His Val Lys Ser Asn Glu Cys Ala Lys Thr Ser Ser Glu 35 40 45 Gly Gly Ile Cys Ser Ser Asp Glu Asp Ala Gly Ser Ala Asp Ile Ile 50 55 60 Ile Val Gly Ala Gly Val Ala Gly Ala Ala Leu Ala Tyr Thr Leu Gly 65 70 75 80 Lys Asp Gly Arg Arg Val His Val Ile Glu Arg Asp Leu Ser Glu Pro 85 90 95 Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Gly Tyr Leu Arg Leu 100 105 110 Ile Glu Leu Gly Leu Gln Asp Cys Val Asp Glu Ile Asp Ser Gln Gln 115 120 125 Val Phe Gly Tyr Ala Leu Tyr Met Asp Gly Lys Asn Thr Lys Leu Ser 130 135 140 Tyr Pro Leu Glu Lys Phe Ser Ser Asp Ile Ser Gly Arg Ser Phe His 145 150 155 160 Asn Gly Arg Phe Ile Gln Arg Met Arg Glu Lys Ala Ser Ser Leu Pro 165 170 175 Asn Val Lys Leu Glu Gln Gly Thr Val Thr Ser Leu Leu Glu Glu Lys 180 185 190 Gly Thr Ile Thr Gly Val His Tyr Lys Ile Lys Ser Gly Gln Glu Phe 195 200 205 Thr Ala Lys Ala Pro Leu Thr Ile Val Cys Asp Gly Cys Phe Ser Asn 210 215 220 Leu Arg Arg Ser Leu Cys Asn Pro Lys Val Asp Val Pro Ser His Phe 225 230 235 240 Val Gly Leu Val Leu Glu Asn Cys Asn Leu Pro Tyr Ala Asn His Gly 245 250 255 His Val Ile Leu Gly Asp Pro Ser Pro Val Leu Phe Tyr Pro Ile Ser 260 265 270 Ser Thr Glu Ile Arg Cys Leu Val Asp Val Pro Gly Gln Lys Leu Pro 275 280 285 Ser Leu Gly Gly Gly Glu Met Ala Cys Tyr Leu Lys Thr Val Val Ala 290 295 300 Pro Gln Val Pro Pro Glu Leu Tyr Asp Ser Phe Ile Ala Ala Ile Asp 305 310 315 320 Lys Gly Asn Ile Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ser Pro 325 330 335 Tyr Pro Thr Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg 340 345 350 His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp Ile Val 355 360 365 Val Leu Arg Asp Leu Leu Lys Pro Leu His Asp Leu His Asp Ala Ser 370 375 380 Ala Leu Cys Arg Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val 385 390 395 400 Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Cys 405 410 415 Ala Ser Pro Asp Pro Ala Arg Lys Glu Met Arg Gln Ala Cys Phe Asp 420 425 430 Tyr Leu Ser Leu Gly Gly Val Phe Ser Asp Gly Pro Ile Ala Leu Leu 435 440 445 Ser Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Leu His Phe Phe Ala 450 455 460 Val Ala Ile Tyr Gly Val Gly Arg Leu Leu Ile Pro Phe Pro Ser Pro 465 470 475 480 Lys Arg Met Trp Ile Gly Ala Arg Leu Ile Ser Gly Ala Ser Gly Ile 485 490 495 Ile Phe Pro Ile Ile Lys Ala Glu Gly Val Arg Gln Met Phe Phe Pro 500 505 510 Ala Thr Val Pro Ala Tyr Tyr Arg Thr Pro Pro Val His 515 520 525 <210> 2 <211> 1578 <212> DNA <213> Glycine max <400> 2 atggattatc cgtacattct aggagggatc atggcttgta gttttgcatt tttgtatgtt 60 gtgtacagtt ttggagcaaa gaaggtcaca gattcaagtt caatacatgt aaagagtaat 120 gagtgtgcaa agacatcatc agaaggtgga atatgttctt cagatgagga tgcaggaagt 180 gctgacatca tcattgtggg tgctggggtt gctggtgcag ctcttgctta cactcttgga 240 aaggatggac ggcgagtgca tgtaattgaa agggatttga gtgaacctga caggatcgtg 300 ggtgaattgc tacaacctgg gggctatctg aggttaattg agttgggcct tcaggattgt 360 gtggatgaaa ttgattcaca gcaagtcttt ggctatgctc tgtatatgga tggcaaaaat 420 accaagctgt cttatccttt ggaaaaattc agctctgata tttctgggag aagctttcac 480 aatggccgtt tcatacagag aatgccagaa aaggcttcgt ctcttccaaa tgtaaaatta 540 ctgtcacatc tctacttgaa ccatcactgg ggtgcactac 600 aaaatcaaaa gtggacaaga gttcacagca aaggctcccc tcaccatagt atgtgatggt 660 tgtttttcca acttgaggcg ttctctttgc aatcctaagg ttgacgttcc ctctcatttt 720 gttggtctgg tcttggagaa ttgtaatctt ccatatgcaa atcatggtca cgtcatctta 780 ggtgatcctt cacccgtttt gtttatccc atcagtagca ctgagattcg ctgtttggtt 840 gatgtgcccg gccaaaaatt accttcccta ggtggtggtg aaatggcctg ttatttgaaa 900 actgtggtgg ctcctcaggt tcctccagag ctgtatgatt cttttatagc agctattgac 960 aaaggaaaca taagaaccat gccaaataga agcatgcctg cctcacctta tcccacacca 1020 ggtgcacttc taatgggaga cgccttcaat atgcgtcacc ctttaactgg aggaggaatg 1080 actgtggctt tatctgacat tgttgtgcta agggatttac ttaaacctct tcatgatctg 1140 catgatgctt ctgctctttg cagatacctt gaatcattct acaccctacg caagccagtg 1200 gcatctacaa taaacacact agctggggca ttgtacaagg tcttttgtgc atctcctgat 1260 ccagctagaa aggaaatgcg ccaagcatgt ttcgattact tgagccttgg aggtgttttc 1320 tcagatggac caatagctct actctctggt ctaaatcctc gtccattgag tttggttctc 1380 catttcttcg cagtcgctat atacggtgtt ggccgcctgc tcataccatt tccttctcca 1440 aaacggatgt ggattggagc tagattgatt tcaggtgcat caggcatcat tttccccatt 1500 attaaggctg aaggagtgag acaaatgttc ttcccagcaa ctgtgccagc atattacaga 1560 acccctcctg tccattga 1578

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 / or drought resistance; D2) Application in the preparation of products that improve the salt tolerance and / or drought resistance of plants; D3) Application in the cultivation of salt-tolerant and / or drought-resistant plants; D4) Application in the preparation of products for cultivating salt-tolerant and / or drought-resistant plants; The amino acid sequence of the protein is shown in SEQ ID No. 1; the plant is soybean or Arabidopsis thaliana.

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 / or drought resistance; E2) Application in the preparation of products that improve the salt tolerance and / or drought resistance of plants; E3) Application in the cultivation of salt-tolerant and / or drought-resistant plants; E4) Application in the preparation of products for cultivating salt-tolerant and / or drought-resistant plants; The plant in question is soybean or Arabidopsis thaliana; The biomaterial is any one of B1) to B4) 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).

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 plants, characterized in that, The method includes increasing the content of the protein described in claim 1 in the target plant to obtain a salt-tolerant plant with higher salt tolerance than the target plant, wherein the plant is soybean or Arabidopsis thaliana.

5. A method for cultivating 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 drought-resistant plant with higher drought resistance than the target plant, wherein the plant is soybean or Arabidopsis thaliana.

6. The method according to claim 4 or 5, 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.

7. The method according to claim 6, 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.

8. The method according to claim 7, characterized in that, The gene encoding the protein is a DNA molecule with the coding sequence SEQ ID No.

2.

9. 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, 6-8, in the creation of salt-tolerant soybeans or Arabidopsis thaliana.

10. 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 5, 6-8, in the creation of drought-resistant soybeans or Arabidopsis thaliana.

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