Application of VrWRKY40 gene in improving salt and alkali tolerance of mung bean

Abstract

The application discloses application of a VrWRKY40 gene in improving salt and alkali tolerance of mung beans and belongs to the technical field of plant stress resistance. The application aims to improve salt and alkali tolerance of mung beans. The application provides application of a VrWRKY40 protein in improving salt and alkali tolerance of mung beans, and the amino acid sequence of the VrWRKY40 protein is shown as SEQ ID NO. 10. The application provides a theoretical basis for salt and alkali resistance breeding of soybeans.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance technology, specifically involving the application of the VrWRKY40 gene in improving the salt and alkali tolerance of mung beans. Background Technology

[0002] my country has over 100 million hectares of saline-alkali land, representing a significant potential supplement to arable land resources. Mung beans, a legume that is tolerant of poor soil and helps fix nitrogen to improve soil quality, possess value for food, feed, and ecological restoration. Enhancing their salt and alkali resistance is crucial for expanding arable land, ensuring food security, and promoting ecological governance.

[0003] Salt and alkali stress can lead to osmotic imbalance, ion poisoning, and oxidative damage in mung beans. Traditional cultivation relies on freshwater leaching or chemical amendments, which are costly and ecologically damaging. Breeding salt-tolerant varieties is the core path for the sustainable use of saline-alkali land. However, traditional breeding methods are inefficient, and there is a need to provide an effective method to improve the salt and alkali tolerance of mung beans. Summary of the Invention

[0004] The purpose of this invention is to improve the salt and alkali tolerance of mung beans.

[0005] This invention provides the application of VrWRKY40 protein in improving the salt and alkali tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans. The amino acid sequence of the VrWRKY40 protein is shown in SEQ ID NO.10.

[0006] This invention provides an application of overexpressing the VrWRKY40 gene in improving the salt and alkali tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans, wherein the VrWRKY40 gene is shown in SEQ ID NO.9.

[0007] This invention provides the application of a recombinant vector containing the VrWRKY40 gene in improving the salt and alkali tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans, wherein the VrWRKY40 gene is shown in SEQ ID NO.9.

[0008] Further specifying, the originating vector for the recombinant vector is pCAMBIA3300.

[0009] This invention provides the application of recombinant host cells containing the VrWRKY40 gene in improving the salt and alkali tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans, wherein the VrWRKY40 gene is shown in SEQ ID NO.9.

[0010] Further specifying, the host cell is either Escherichia coli or Agrobacterium.

[0011] Further specifying the conditions for the salt-alkali mixture, the concentration of the salt-alkali mixture is 150 mmol / L (NaCl:Na2SO4:Na2CO3:NaHCO3=1:9:9:1, pH=8.5±0.1).

[0012] This invention provides a breeding method for improving the salt and alkali tolerance of mung beans. The specific steps of the breeding method are as follows: Step 1: Ligate the VrWRKY40 gene with an overexpression vector to obtain a recombinant vector; the VrWRKY40 gene is shown in SEQ ID NO.9; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 was transferred into mung beans to obtain mung bean hairy roots. Positive transgenic mung bean hairy roots were identified and screened.

[0013] To further specify, the primer pair used in step 1 to amplify the gene shown in SEQ ID NO.9 is SEQ ID NO.1 and SEQ ID NO.2.

[0014] Further specifying the conditions for the salt-alkali mixture, the concentration of the salt-alkali mixture is 150 mmol / L (NaCl:Na2SO4:Na2CO3:NaHCO3=1:9:9:1, pH=8.5±0.1).

[0015] Beneficial effects: After treatment with a mixed saline-alkali solution at a concentration of 150 mmol / L (NaCl:Na2SO4:Na2CO3:NaHCO3 = 1:9:9:1, pH = 8.5 ± 0.1) for 15 days, overexpression... VrWRKY40 The plant height, root surface area, root volume, and root length of the genetically modified strain with hairy roots were significantly higher than those of the control strain. Furthermore, the content of proline, soluble sugar, soluble protein, SOD, and POD activities were all significantly higher than those of the control strain. Attached Figure Description

[0016] Figure 1 Figure 1 shows the results of the basic characteristic analysis of the VrWRKY40 gene; A. Amino acid composition; B. Hydrophilicity analysis. Figure 2 Figure showing the phosphorylation sites of VrWRKY40 protein; Figure 3 Figure showing the results of VrWRKY40 protein signal peptide analysis; Figure 4Figure A shows the results of secondary and tertiary structure analysis of the VrWRKY40 protein; Figure B shows the predicted confidence curves of secondary structure elements, with the horizontal axis representing amino acid residue sites (1~260 aa); Figure B shows the tertiary structure. Figure 5 This is a phylogenetic tree diagram of the VrWRKY40 system. Figure 6 Figure showing the classification and number of cis-regulatory elements in the VrWRKY40 gene promoter; Figure 7 Figure 1 shows the cloning results of the VrWRKY40 gene; Note: M: DL2000; 1-4: PCR products; A is the result of amplifying the VrWRKY40 gene ligated with the overexpression vector; B is the result of amplifying the VrWRKY40 gene ligated with the subcellular localization vector. Figure 8 Figure 1 shows the results of transforming DH5α Escherichia coli with plant expression vector; Note: M: DL2000; 1-5: PCR products; A is a positive clone containing pCAMBIA3300-VrWRKY40 recombinant plasmid, and B is a positive clone containing pCAMBIA1302-VrWRKY40 recombinant plasmid. Figure 9 A diagram showing the subcellular localization results of VrWRKY40 protein; Figure 10 The image shows the expression pattern of the VrWRKY40 gene in mung beans under salt and alkali stress. Note: CK = water control, Sa = salt and alkali stress treatment; different lowercase letters indicate significant differences at the P<0.05 level. Figure 11 Figure 1 shows the PCR results of Agrobacterium rhizogenes culture containing the recombinant plasmid pCAMBIA3300-VrWRKY40; M: DL2000; 1-5: PCR products. Figure 12 A quantitative fluorescence diagram of the positive strain overexpressing VrWRKY40; Figure 13 To investigate the regulation of the VrWRKY40 gene on the growth of transgenic hairy roots of mung beans under saline-alkali conditions; Figure 14 Figure showing the results of proline content determination in the hairy roots of mung bean regulated by the VrWRKY40 gene under saline-alkali conditions; Figure 15 Figure showing the results of soluble glycoprotein content determination in the hairy roots of mung bean regulated by the VrWRKY40 gene under saline-alkali conditions; Figure 16 Figure 1 shows the results of SOD enzyme activity assay in transgenic mung bean hairy roots regulated by the VrWRKY40 gene under saline-alkali conditions. Figure 17Figure 1 shows the results of POD enzyme activity assay in transgenic mung bean hairy roots regulated by the VrWRKY40 gene under saline-alkali conditions. Figure 18 Figure showing the results of soluble protein content determination in the hairy roots of mung bean transgenic mung bean regulated by the VrWRKY40 gene under saline-alkali conditions; Figure 19 The figure shows the results of malondialdehyde (MDA) content determination in the hairy roots of mung bean transgenic mung bean regulated by the VrWRKY40 gene under saline-alkali conditions. Detailed Implementation

[0017] Example 1. VrWRKY40 gene characterization analysis 1. Bioinformatics analysis was performed on the amino acid sequence of the VrWRKY40 gene from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) mung bean genome database. Basic physicochemical properties were analyzed using the Expasy online database (https: / / www.expasy.org / ). The analysis revealed that serine (S) constitutes the largest proportion of the VrWRKY40 protein's amino acid composition, approximately 10.8%, while tryptophan (W) constitutes the smallest proportion, at 0.8%. Pyrrolidone (O) and selenocysteine ​​(U) do not participate in the protein's biosynthesis. Figure 1 (A) The VrWRKY40 protein has 36 positively charged residues (Arg+Lys) and 30 negatively charged residues (Asp+Glu), and its molecular structure is C. 1273 H 2046 N 364 O 407 S 11 It has a relative molecular mass of 29.3 kDa, a theoretical isoelectric point of 8.97, a total number of atoms of 4101, an aliphatic index of 69.69, an average hydrophilicity index of -0.703, and an instability coefficient of 48.28, classifying it as an unstable protein. Based on protein hydrophilicity... Figure 1 In section B), it was found that the entire polypeptide chain of VrWRKY40 contains a majority of hydrophilic amino acids, indicating that it is an overall hydrophilic protein. Phosphorylation site prediction analysis was performed using the online website NetPhos 3.1 (https: / / mybiosoftware.com / netphos-3-1). The results showed that the VrWRKY40 protein contains 37 potential phosphorylation sites, including 25 serine phosphorylation sites, 9 threonine phosphorylation sites, and 3 tyrosine phosphorylation sites. Figure 2 Signal peptide prediction using SignalP 6.0 (SignalP 6.0 - DTU HealthTech) revealed that the VrWRKY40 protein lacks a signal peptide structure and is a non-secretory protein. Figure 3 Two-level and three-level structural models were constructed using the online websites SOPMA (https: / / npsa-prabi.ibcp.fr / ) and SWISS-MODEL (https: / / swissmodel.expasy.org / ), respectively. The results... Figure 4 Figure A shows that the 260 amino acids of the VrWRKY40 protein are composed of four elements: α-helix, β-sheet, random coil, and β-turn. The random coil accounts for 58.85%, the α-helix for 29.23%, and the β-sheet and β-turn together account for 11.92%. The protein's tertiary structure is predominantly random coil. Figure 4 The B in the figure is highly consistent with the results of the secondary structure prediction.

[0018] 2. A phylogenetic tree was constructed using MEGA 11 software. Analysis showed that the VrWRKY40 gene has homologous genes in multiple plants. Comparative analysis of 27 homologous genes from soybean, maize, Arabidopsis, and other plants revealed six main evolutionary branches, among which VrWRKY40 is most closely related to adzuki bean. Figure 5 ).

[0019] The 2000 bp upstream sequence of the VrWRKY40 gene ATG was selected from the NCBI database. The promoter region was analyzed using the Plant CARE online website (https: / / www.plantcare.co.uk / ). The analysis revealed that the core elements ABRE, GARE-motif, and CGTCA-motif are involved in abscisic acid, gibberellin, and methyl jasmonate responses, respectively. ARE is involved in anaerobic-induced gene regulation, and the G-box is a light-regulated element. These elements bind to the upstream gene and jointly participate in the regulatory expression of the VrWRKY40 gene. Therefore, it is speculated that this gene is involved in plant growth and development and resistance to various stresses. Figure 6 ).

[0020] CDS cloning of the target gene. The CDS sequence information of VrWRKY40 was obtained by searching the NCBI database using the mung bean gene number LOC106764381. Gene cloning primers were designed using SnapGene software. PCR was performed using cDNA as a template. The reaction system was set to 20 µL: VrWRKY40-3300-F (SEQ ID NO.1) 1 µL, VrWRKY40-3300-R (SEQ ID NO.2) 1 µL, template DNA 1 µL, KOD One™ PCR Master Mix (TOYOBO) 10 µL, ddH2O 7 µL. The PCR program was as follows: pre-denaturation 98℃, 2 min; denaturation 98℃, 10 s; annealing 60℃, 5 s; extension temperature: 68℃, 10 min, for a total of 35 cycles, stored at 4℃. After the reaction, the PCR product was analyzed by agarose gel electrophoresis and the target fragment was purified by gel extraction. Figure 7 A in the middle; PCR was performed using cDNA as a template. The reaction system consisted of 20 µL of: 1 µL of VrWRKY40-1302-F (SEQ ID NO.3), 1 µL of VrWRKY40-1302-R (SEQ ID NO.4), 1 µL of template DNA, 10 µL of KOD One™ PCR Master Mix (TOYOBO), and 7 µL of ddH2O. The PCR program was as follows: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s; annealing at 60 °C for 5 s; extension at 68 °C for 10 min, for a total of 35 cycles, and storage at 4 °C. After the reaction, the PCR products were analyzed by agarose gel electrophoresis and the target fragment was purified by gel extraction. Figure 7 (B in the middle).

[0021] VrWRKY40-3300-F: acgggggactctagaggatccATGGAACCAACATGCTTGG (SEQ IDNO.1); VrWRKY40-3300-R: gcccttgctcaccataagcttTCACCATTTGGCTTCTAGAATTTTTC (SEQID NO.2); VrWRKY40-1302-F: ggactcttgaccatggATGGAACCAACATGCTTGG (SEQ ID NO.3); VrWRKY40-1302-R: cttctcctttactagtCCATTTGGCTTCTAGAATTTTTCCT (SEQ ID NO.4); VrWRKY40-qRT-F: GATCCCAATTTCACCACAGCAC (SEQ ID NO.5); VrWRKY40-qRT-R: CTATGACTCTGCAGCCTCTGAG (SEQ ID NO.6); VrActin 101-F: GGTGGTTCTATCTTGGCATC (SEQ ID NO.7); VrActin 101-R: CTTTCGCTTCAATAACCCTA (SEQ ID NO.8); The CDS sequence of the VrWRKY40 gene (SEQ ID NO.9): ATGGAACCAACATGCTTGGATACTTCACTCAATCTCAACGTTGATCCTTCTTCTGTTCACACGGATATGGTGTTGGAGGAAGAGTTGCAGAGGCTGAGACGTGAGAACAAGAGGTTGACTGAGATGTTGACCCACTTGTGTGACGGCTACATGGTTTTGCAGAAGCAATTGACCCAATTGAAGAACACAAATTTTGAGCAAGACCAACTCGAATCACGCAAGAGAAAAGCAGTTAACAATAACAATGGCGAGTGCAGCTCCATAACAGAGGATTCCTTCAAAAGGTACAGCTACAAGGATTTCAGCTCCTCCCCAAAGGTTTCCAAGGTTCTTGTAAAGACACAAGCTTCTAATAACAGTTTATATGTGATGGATGGATATCAATGGAGGAAATACGGTCAAAAGGTGACCAGGGATAACCCTTCTCCTAGAGCTTACTTCAAGTGTTCCTTTGCTCCAAGCTGCCCCGTTAAAAAAAAGGTGCAAAGGAGTTTAGAAGACCCTACAATACTTGTTACAACATATGAAGGAGAGCATAATCATGGTAATGAAAGAGGTGAAATTGAAGGAGGAAAAGGGTCAAGTCCAGTTTCTTCACCAAAAGCCAGAATTGGAAGTGTAACGCTTGATTTGGTCAAATCAGGATTGTTTGAAACTGCCCAAAAGTCATCTATCCAACAATTTTTGGTTCAACAAATGGCTACTTCTTTGACAAGGGATCCCAATTTCACCACAGCACTTGCCACTGCCATTTCAGGAAAAATTCTAGAAGCCAAATGGTGA; Amino acid sequence of VrWRKY40 gene (SEQ ID NO.10): MEPTCLDTSLNLNVDPSSVHTDMVLEEELQRLRRENKRLTEMLTHLCDGYMVLQKQLTQLKNTNFEQDQLESRKRKAVNNNNGECSSITEDSFKRYSYKDFSSSPKVSKVLVKTQASNNSLYVMDGYQWR KYGQKVTRDNPSPRAYFKCSFAPSCPVKKKVQRSLEDPTILVTTYEGEHNHGNERGEIEGGKGSSPVSSPKARIGSVTLDLVKSGLFETAQKSSIQQFLVQQMATSLTRDPNFTTALATAISGKILEAKW.

[0022] Example 2. Vector construction and coliform transformation Using a homologous recombination kit, linearized pCAMBIA3300 and pCAMBIA1302 vector plasmids were homologously ligated with the target gene VrWRKY40 to obtain pCAMBIA3300-VrWRKY40 and pCAMBIA1302-VrWRKY40 recombinant plasmids, respectively. The ligation system was 20 µL: 2 µL plasmid, 1 µL VrWRKY40 fragment, 2 µL ligase, 2 µL 5×CE Buffer, and 13 µL ddH2O. The incubation program was 37℃ for 30 min. The ligation product was further transformed into DH5α competent cells, and single-clone cells were cultured. Positive clones obtained from transformation with the pCAMBIA3300-VrWRKY40 recombinant plasmid were further identified using specific primers (SEQ ID NO.1 and SEQ ID NO.2), and positive clones obtained from transformation with the pCAMBIA1302-VrWRKY40 recombinant plasmid were further identified using specific primers (SEQ ID NO.3 and SEQ ID NO.4). Using the cultured bacterial culture as a template, PCR identification was performed (PCR procedure was the same as the VrWRKY40 cloning procedure). After the reaction was complete, detection was performed by agarose gel electrophoresis, and the target fragment size was found to be 783 bp. Figure 8 ), Figure 8 In the diagram, A represents a positive clone containing the pCAMBIA3300-VrWRKY40 recombinant plasmid. Figure 8B in the result represents a positive clone containing the pCAMBIA1302-VrWRKY40 recombinant plasmid. The correctly identified bacterial cultures were further sequenced by Sangon Biotech (Shanghai) Co., Ltd., and sequence alignment was performed using DNAMAN software. The correctly sequenced E. coli cultures were then amplified, and plasmid DNA was extracted from the amplified E. coli using the FastPure Plasmid Mini Kit.

[0023] Example 3. Subcellular localization and expression pattern analysis 1. Subcellular localization The recombinant plasmid pCAMBIA1302-VrWRKY40 was transformed into GV3101 (psoup-p19) competent cells to further clarify the functional location of the VrWRKY40 gene in cells. Agrobacterium tumefaciens pCAMBIA1302-VrWRKY40 was infected into young tobacco leaves, with Agrobacterium tumefaciens pCAMBIA1302-GFP serving as a control. The functional location of the VrWRKY40 gene in cells was observed under a laser confocal microscope. The results showed that pCAMBIA1302-GFP was expressed in the cell membrane, nucleus, and cytoplasm of young tobacco leaves, while pCAMBIA1302-VrWRKY40 was expressed in the nucleus, indicating that this gene functions in the nucleus. Figure 9 ).

[0024] 2. Expression pattern analysis of VrWRKY40 under salt-alkali stress RNA was extracted from fresh mung bean root samples, and reverse transcription was performed using the First-Strand cDNA Synthesis and One-Step gDNA Removal Kit. Real-time quantitative PCR was performed using the Green qPCR MasterMix kit, with VrActin101 from mung bean as the internal control gene. The primer sequences for the internal control gene are shown in SEQ ID NO.7 and SEQ ID NO.8. Real-time quantitative PCR was performed using VrWRKY40-qRT-F (SEQ ID NO.5) and VrWRKY40-qRT-R (SEQ ID NO.6). The results showed that the VrWRKY40 gene responded rapidly in the early stages of salt-alkali stress, and then maintained a sustained response as the stress progressed. Figure 10 ).

[0025] Example 4. Transformation of Agrobacterium rhizogenes with recombinant vector Transformation method of Agrobacterium rhizogenes K599: Take out K599 competent cells from the -80℃ ultra-low temperature freezer and place them on ice. After the cells are in an ice-water mixed state on ice, add 1 μg of the constructed pCAMBIA3300-VrWRKY40 recombinant plasmid and mix rapidly and vigorously. Then place the cells in ice for 5 min, liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 700 μL of liquid YEP was added, and the mixture was incubated at 28°C and 200 rpm for 2 h in a shaker. After centrifugation at 5000 rpm for 1 min, 700 μL of the supernatant was discarded. The remaining liquid was mixed by pipetting, and 100 μL was evenly spread onto solid YEP medium (containing Kan antibiotic). The medium was then incubated upside down at 28°C for 24 h. Single colonies were then picked for further verification by PCR (SEQ ID NO.1 and SEQ ID NO.2). The target band of 783 bp was obtained, confirming that pCAMBIA3300-VrWRKY40 was successfully transformed into Agrobacterium rhizogenes K599. Figure 11 ).

[0026] Example 5. Obtaining Agrobacterium rhizogenes-mediated transgenic lines Select plump, smooth, and disease-free Lvfeng No. 2 mung bean seeds and sow them in pots with a vermiculite:nutrient soil ratio of 4:1. Cultivate the seeds at 25 ℃ under 16 h light / 8 h dark conditions. Once the true leaves are fully expanded and the seedlings have reached the two-leaf-one-heart stage, cut the stem 1 cm below the cotyledon node using sterile scissors. Incubate 300 μL of Agrobacterium on YEP medium with shaking for 24 h, adjusting the OD... 600=1.0. Using a 1 mL sterile syringe, make 8 small holes by making cross-shaped perforations at the cotyledon node and 2–3 mm above the cotyledon node; inject 0.5 mL of bacterial solution through the hypocotyl incision, allowing the bacterial solution to seep out from the small holes. Insert the seedlings into the substrate, ensuring close contact between the incision and the substrate, seal with plastic wrap to maintain moisture, and culture at 25 ℃ under 6 h light / 18 h dark conditions. When the seedling leaves push up the plastic wrap, remove the film and restore the light conditions to 16 h light / 8 h dark. Seven days after removing the film, subject the seedlings to salt and alkali treatment with a salt-alkali mixture of 150 mmol / L (molar ratio as follows: NaCl: Na2SO4: Na2CO3: NaHCO3 = 1:9:9:1, pH = 8.5±0.1) to simulate a moderate salt and alkali stress environment. Apply 300 mL of the salt-alkali mixture to each pot in two applications. Root samples were collected on day 15 post-treatment, and positive lines were identified by PCR. RNA was extracted from transgenic and control hairy roots to determine the expression level of the VrWRKY40 gene in overexpressing hairy roots. The results showed that the expression level of the VrWRKY40 gene in the hairy roots of the overexpressing lines (OE1 and OE3) was significantly higher than that in the control line. The expression level of VrWRKY40 gene in the overexpressing line OE1 was approximately 2 times higher than that in the control line, and the expression level of VrWRKY40 gene in OE3 was approximately 1.4 times higher than that in the control line. Figure 12 This indicates that the construction of the overexpression line was successful. The roots of the identified transgenic positive lines were then flash-frozen in liquid nitrogen for subsequent physiological assays.

[0027] Example 6. Phenotypic and physiological identification of transgenic mung bean hairy roots under salt-alkali stress Observe the growth of hairy roots after 15 days of salt and alkali stress. Figure 13 As shown in the figure, the plants overexpressing VrWRKY40 showed better growth than the control without treatment. After treatment with a 150 mmol / L saline-alkali mixture, plant growth was inhibited, and the plants overexpressing VrWRKY40 showed significantly better growth than the control. After 15 days of saline-alkali stress, the plant height, stem diameter, root surface area, root volume, and root length of the transgenic hairy roots were measured. CK was the control treatment with water, Sa was the saline-alkali stress treatment, EV-3300 was mung bean hairy roots transformed with the empty vector pCAMBIA3300, and OE-VrWRKY40 was mung bean hairy roots overexpressing the VrWRKY40 gene. After sampling, the contents of soluble sugar, proline (Pro), and soluble protein were determined using the anthrone colorimetric method, the sulfosalicylic acid method, and the Coomassie Brilliant Blue G-250 staining method, respectively. SOD, POD activity and MDA were determined using the nitroblue tetrazolium (NBT) method, the guaiacol method and the thiobarbituric acid (TBA) method, respectively.

[0028] The root samples used in this experiment were all mixed samples of three independently transgenic hairy roots. Data analysis was performed using GraphPadPrism software. The results showed that the plant height, root surface area, root volume, and root length of the overexpression line hairy roots were significantly higher than those of the control. Furthermore, the proline, soluble sugar, and soluble protein contents, as well as the SOD and POD activities, of the overexpression line hairy roots were significantly higher than those of the control line. Figure 14-18 The MDA content was significantly lower than that of the control strain ( Figure 19 ).

[0029] Table 1. Phenotypic data of WT, EV-3300, and OE-VrWRKY40 under 15 days of salt-alkali stress.

[0030] Note: CKl = water control; Sa = salt-alkali stress treatment; EV-3300 is mung bean hairy root transformed with the empty vector pCAMBIA3300; OE-VrWRKY40 is mung bean hairy root overexpressing the VrWRKY40 gene. Different lowercase letters in the same column indicate significant differences at the P<0.05 level; n=3, representing three replicates of three independent transgenic hairy root samples.

Claims

1. The application of overexpression of the VrWRKY40 gene in improving salt tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity in mung beans, characterized in that... The amino acid sequence of the VrWRKY40 protein is shown in SEQ ID NO.

10.

2. The application of a recombinant vector containing the VrWRKY40 gene in improving the salt tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans, characterized in that... The VrWRKY40 gene is shown in SEQ ID NO.

9.

3. The application according to claim 2, characterized in that, The starting vector for the recombinant vector was pCAMBIA3300.

4. The application of recombinant host cells containing the VrWRKY40 gene in improving the salt tolerance, proline content, soluble sugar content, soluble protein content, SOD enzyme activity, and POD enzyme activity of mung beans, characterized in that... The VrWRKY40 gene is shown in SEQ ID NO.

9.

5. The application according to claim 4, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

6. The application according to claim 5, characterized in that, The host cells are Escherichia coli or Agrobacterium.

7. The application according to any one of claims 1-6, characterized in that, The saline-alkali conditions are NaCl:Na2SO4:Na2CO3:NaHCO3 = 1:9:9:1, pH = 8.5 ± 0.

1.

8. A breeding method for improving the salt and alkali tolerance of mung beans, characterized in that, The specific steps of the breeding method are as follows: Step 1: Ligate the VrWRKY40 gene with an overexpression vector to obtain a recombinant vector; the VrWRKY40 gene is shown in SEQ ID NO. 9; Step 2: Transform the recombinant vector described in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: The recombinant Agrobacterium described in Step 2 was transferred into mung beans to obtain mung bean hairy roots. Positive transgenic mung bean hairy roots were identified and screened.

9. The breeding method according to claim 8, characterized in that, The primer pair used to amplify the gene shown in SEQ ID NO.9 in step 1 is SEQ ID NO.1 and SEQ ID NO.

2.

10. The breeding method according to claim 8, characterized in that, The conditions for the salt-alkali mixture are: the concentration of the salt-alkali mixture is NaCl:Na2SO4:Na2CO3:NaHCO3 = 1:9:9:1, and the pH is 8.5 ± 0.1.

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