Wild soybean salt-tolerant gene and application thereof

Abstract

The invention belongs to the technical field of gene engineering and salt-tolerant plant cultivation, and particularly relates to a wild soybean salt-tolerant gene and application thereof. The gene GsWRKY53 responding to salt stress is identified by performing expression analysis on the gene of the wild soybean under salt treatment, the salt-tolerant gene GsWRKY53 in the wild soybean is transferred into a soybean cotyledon through an agrobacterium transformation method for the first time, and the gene function of the gene can be quickly and accurately verified by verifying the influence of the salt-tolerant gene GsWRKY53 on soybean hairy roots. Experiments prove that the fresh weight, the dry weight and the number of hairy roots of soybeans can be reduced by overexpression of the wild soybean salt-tolerant gene GsWRKY53, which indicates that the GsWRKY53 plays a negative regulation role in the salt tolerance of the soybeans. According to the technical scheme, a foundation is provided for screening and verifying the salt-tolerant functional gene and further screening or cultivating salt-tolerant plant varieties, especially salt-tolerant soybean varieties, so that the method has a good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and salt-tolerant plant breeding, and in particular relates to a wild soybean salt-tolerant gene and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Soil salinization is a global problem. The global salinized land area is about 1 billion hectares, accounting for 7% of the total land area. Soil salinity is one of the main environmental stresses that restrict plant growth and crop yield, and seriously hinders the sustainable development of modern agriculture. In addition to natural soil salinity, climate change and artificial irrigation have also aggravated soil salinization. Saline-alkali land is characterized by high soil salt content, low organic matter content, and unbalanced nutrients. It is not suitable for the growth of ordinary crops, and it is urgent to cultivate salt-tolerant economic crop varieties.

[0004] Wild soybean is a wild relative of cultivated soybean. It has high edible and medicinal value and rich excellent traits, such as strong adaptability, salt-alkali tolerance, drought resistance, disease and insect resistance. These excellent traits provide valuable genetic resources for the improvement of cultivated soybean germplasm. Soybean is an important oil crop and plays an important role in meeting the needs of human diet, animal feed and bio-oil. Soil salinization seriously restricts the growth and development of soybean, so it is urgent to screen and cultivate salt-tolerant soybean germplasm. Wild soybeans are generally more salt-tolerant than cultivated soybeans. Therefore, salt-tolerant genes can be introduced into soybeans through molecular breeding and other methods to improve the salt resistance of cultivated soybeans, which is of great significance for soybean cultivation in saline-alkali land and increasing soybean yields. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for identifying salt-tolerant genes in wild soybeans and their applications. Specifically, the present invention identifies genes that respond to salt stress by analyzing the expression of wild soybean genes under salt treatment, and transfers the salt-tolerant gene GsWRKY53 in wild soybeans into soybeans through soybean hairy root transformation. The experimental results show that overexpression of GsWRKY53 reduces the fresh weight, dry weight and number of hairy roots, indicating that GsWRKY53 plays a negative regulatory role in soybean salt tolerance. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0007] In a first aspect of the present invention, a wild soybean salt-tolerance gene is provided, which is named GsWRKY53. The wild soybean salt-tolerance gene has:

[0008] (a1) the nucleotide sequence shown in SEQ ID NO.1;

[0009] (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but having a different sequence due to the degeneracy of the genetic code;

[0010] (a3) a nucleotide sequence having ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encoding the same functional protein;

[0011] (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

[0012] The second aspect of the present invention provides a wild soybean salt-tolerance protein, wherein the wild soybean salt-tolerance protein is encoded by the wild soybean salt-tolerance gene.

[0013] The third aspect of the present invention provides a recombinant expression vector, which comprises the wild soybean salt-tolerance gene.

[0014] The fourth aspect of the present invention provides a transgenic cell line, a host bacteria or a transgenic plant containing the wild soybean salt-tolerance gene or the recombinant expression vector.

[0015] The fifth aspect of the present invention provides the use of the wild soybean salt-tolerance gene, wild soybean salt-tolerance protein, recombinant expression vector, transgenic cell line, host bacteria or transgenic plant in any one or more of the following:

[0016] (c1) regulating the number, fresh weight and / or dry weight of plant roots;

[0017] (c2) regulating plant salt tolerance;

[0018] (c3) Screening and / or breeding of salt-tolerant plants.

[0019] A sixth aspect of the present invention provides a method for reducing plant salt tolerance, the method comprising: transferring the wild soybean salt tolerance gene or recombinant expression vector into a plant, thereby reducing the fresh weight, dry weight and number of plant hairy roots.

[0020] A seventh aspect of the present invention provides a method for improving plant salt tolerance, the method comprising: inhibiting the expression of the wild soybean salt tolerance gene in the plant.

[0021] Beneficial technical effects of one or more of the above technical solutions:

[0022] The above technical scheme identified the gene GsWRKY53 that responds to salt stress in wild soybean for the first time, and used the Agrobacterium transformation method to transfer the salt-tolerant gene GsWRKY53 in wild soybean into soybean cotyledons. By verifying its effect on soybean hairy roots, its gene function could be quickly and accurately verified.

[0023] Experimental results show that overexpression of wild soybean salt-tolerance gene GsWRKY53 reduces the fresh weight, dry weight and number of soybean hairy roots, indicating that GsWRKY53 plays a negative regulatory role in soybean salt tolerance. The above technical solution provides a basis for screening and verifying salt-tolerance functional genes, and then screening or breeding salt-tolerant plant varieties, especially salt-tolerant soybean varieties, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is a map of the expression vector pCAMBIA1300-35S-sGFP in the examples of the present invention.

[0026] Figure 2 This is the chromosomal location of the GsWRKY gene family in the examples of the present invention.

[0027] Figure 3 This is the phylogenetic tree of the GsWRKY family and the AtWRKY family in the examples of the present invention.

[0028] Figure 4 The figure shows the alignment and cloning bands of GsWRKY53 and Arabidopsis proteins in the examples of the present invention.

[0029] Figure 5 This is the subcellular localization of GsWRKY53-GFP in the examples of the present invention.

[0030] Figure 6 This is a comparison of the hairy roots grown for 7 days with the mock and GsWRKY53-OE in the examples of the present invention.

[0031] Figure 7 The figure shows the statistics of the hairy roots of the mock and GsWRKY53-OE in the examples of the present invention; wherein A is the fresh weight of the hairy roots; B is the dry weight of the hairy roots; and C is the number of the hairy roots. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0035] In a typical embodiment of the present invention, a wild soybean salt-tolerance gene is provided, which is named GsWRKY53. The wild soybean salt-tolerance gene has:

[0036] (a1) the nucleotide sequence shown in SEQ ID NO.1;

[0037] (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but having a different sequence due to the degeneracy of the genetic code;

[0038] (a3) a nucleotide sequence having ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encoding the same functional protein;

[0039] (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

[0040] In another specific embodiment of the present invention, a wild soybean salt-tolerance protein is provided, and the wild soybean salt-tolerance protein is encoded by the wild soybean salt-tolerance gene mentioned above.

[0041] The wild soybean salt-tolerant protein has any one of the amino acid sequences (b1)-(b3):

[0042] (b1) the amino acid sequence shown in SEQ ID NO.2;

[0043] (b2) a protein derived from the amino acid sequence shown in SEQ ID NO.2 with the same function as that of the amino acid sequence shown in SEQ ID NO.2 after one or more amino acid residues are substituted and / or deleted and / or added;

[0044] (b3) proteins encoded by other genes that have a similarity of more than 90% with the amino acid sequence of SEQ ID NO.2 and have the activity of the protein of SEQ ID NO.2;

[0045] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the wild soybean salt-tolerance gene;

[0046] Furthermore, the recombinant expression vector is obtained by effectively connecting the above-mentioned wild soybean salt-tolerance gene to an expression vector, and the expression vector can be any one or more of a viral vector, a plasmid, a phagemid, a clay mid, or an artificial chromosome, and further a plasmid, and the plasmid can be a pCAMBIA series vector. In a specific embodiment of the present invention, the plasmid is pCAMBIA1300.

[0047] In another specific embodiment of the present invention, a transgenic cell line, a host bacteria or a transgenic plant containing the wild soybean salt-tolerance gene or the recombinant expression vector is provided.

[0048] The transgenic cell line may be isolated, ex vivo, in culture, or preferably, part of a plant.

[0049] The host bacteria may be eukaryotic bacteria (such as fungi, and further such as yeast) or prokaryotic bacteria (such as bacteria, and further such as Escherichia coli).

[0050] In the present invention, the transgenic plant can be a crop, further an economic crop, such as soybean.

[0051] In another specific embodiment of the present invention, the use of the wild soybean salt-tolerance gene, wild soybean salt-tolerance protein, recombinant expression vector, transgenic cell line, host bacteria or transgenic plant in any one or more of the following is provided:

[0052] (c1) regulating the number, fresh weight and / or dry weight of plant roots;

[0053] (c2) regulating plant salt tolerance;

[0054] (c3) Screening and / or breeding of salt-tolerant plants.

[0055] Wherein, the plant roots may specifically be hairy roots.

[0056] The present invention has found through research that the salt-tolerance gene GsWRKY53 in wild soybean was transferred into soybean. The results showed that its overexpression would reduce the fresh weight, dry weight and number of hairy roots, indicating that GsWRKY53 plays a negative regulatory role in soybean salt tolerance.

[0057] In another specific embodiment of the present invention, a method for reducing plant salt tolerance is provided, the method comprising: transferring the wild soybean salt tolerance gene or recombinant expression vector into the plant, thereby reducing the fresh weight, dry weight and number of plant hairy roots.

[0058] Specifically, the specific method for transferring the recombinant expression vector into plants can be Agrobacterium transformation, specifically, transferring the recombinant expression vector into Agrobacterium, using the Agrobacterium to infect plant cotyledons, and culturing to obtain plant hairy roots.

[0059] In another specific embodiment of the present invention, a method for improving plant salt tolerance is provided, the method comprising: inhibiting the expression of the wild soybean salt tolerance gene in the plant.

[0060] Among them, the inhibition of the expression of wild soybean salt-tolerance gene can be carried out by existing known methods, such as T-DNA insertion mutagenesis, CRISPR / Cas9 gene editing or virus-mediated VIGS, etc., which are not specifically limited here.

[0061] The plant may be soybean.

[0062] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0063] Example

[0064] 1. Experimental Materials

[0065] Wild soybean (Glycine soja) seeds collected in the Yellow River Delta in Dongying City, Shandong Province, and soybean seeds provided by Dongying Academy of Agricultural Sciences.

[0066] 2. Experimental Methods

[0067] 2.1 Wild soybean seed treatment and cultivation

[0068] Select wild soybean seeds of uniform size, round and plump, soak them in concentrated sulfuric acid for 10-15 minutes, and rinse them with water several times. Sow the treated wild soybean seeds in a pot filled with clean sand, 15 seeds per pot, and place them in a greenhouse with 16h of light + 8h of darkness and a day and night temperature of 20℃ / 24℃. Water them before germination, and use Hoagland nutrient solution for watering after germination. After the first three-leaf compound leaf is extended, take the material and put it in a clean EP tube sterilized for 50 minutes.

[0069] 2.2 Bioinformatics analysis

[0070] The whole genome, protein sequence, CDS sequence and annotation files of wild soybean were downloaded from the JGIPhytozome website (http: / / phytozome-next.jpi.doe.gov). The protein sequence, genome and annotation files of the WRKY gene family of Arabidopsis were downloaded from the Arabidopsis Information Resource (TAIR) database (http: / / www.arabidopsis.org). Multiple sequence alignment was performed using DNAMAN (Version 7); the phylogenetic tree was constructed using the neighbor-joining method of MEGA (Version 7.0), with the bootstrap value set to 1000 times, and joint mapping was performed using TBtools.

[0071] 2.3 Wild soybean RNA extraction

[0072] RNA Kit( Plant Total RNA Isolation Kit, RC411-C1, Vazyme Biotech Co., Ltd) was used to extract total RNA from wild soybean.

[0073] 2.4 cDNA synthesis

[0074] RNA was reverse transcribed to synthesize cDNA using the Evo M-MLV RT Mix Kit with Gdna Clean for qPCR Ver.2 kit.

[0075] The first-strand cDNA synthesis was performed according to the kit instructions, and the specific system was as follows: the first step was to eliminate residual genomic DNA, 42°C, 2 min;

[0076]

[0077]

[0078] The second step is to obtain the first strand of cDNA, 37℃, 15min; 85℃, 5s; 4℃, ∞. The specific system is as follows:

[0079]

[0080] 2.5 Primer design

[0081] Clone the sequence from ATG to the stop codon in the expression vector

[0082] The forward / reverse primers of GsWRKY53 were designed based on the SalI restriction site of pCAMBIA1300-35S-sGFP, the stop codon was removed, and 15-20 bp of vector sequence was added to both ends of the forward / reverse primers by seamless cloning.

[0083] Table 1 Primer sequences

[0084]

[0085] 2.6 Cloning of full-length genes

[0086] The full-length gene of GsWRKY53 was cloned using the cDNA of wild soybean as a template. PCR amplification system:

[0087]

[0088] PCR reaction procedure:

[0089]

[0090] The PCR products were detected by 1% agarose gel electrophoresis. The PCR stock solution with the correct band length was selected for sequencing according to the electrophoresis results. The PCR stock solution with the correct sequence was sequenced according to Fast Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China) was used to recover and purify the product.

[0091] 2.7 Ligation of target fragment and vector, transformation and screening of positive clones

[0092] 2.7.1 Vector DNA preparation

[0093] The vector pCAMBIA1300-35S-sGFP was digested with SalI enzyme. The reaction system and procedure are as follows:

[0094]

[0095] Reaction conditions: 37℃ for 3h

[0096] The PCR products were detected by 1% agarose gel electrophoresis, and the fragments with the correct band length were selected according to the electrophoresis results and analyzed according to Fast Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China) was used to recover and purify the product, and the recovered product was named Vector DNA.

[0097] 2.7.2 Ligation of target fragment and vector

[0098] use IIOne Step Cloning Kit was used to construct the GsWRKY53 recombinant vector. The reaction system and procedure are as follows:

[0099]

[0100]

[0101] Reaction procedure: 37℃30min

[0102] 2.7.3 Transformation of recombinant vector into E. coli, screening and sequencing of positive clones

[0103] According to the transformation method of DH5α competent cells (Qingke Biotechnology DH5α Chemically Competent Cell), the above-mentioned ligation product was transformed into Escherichia coli.

[0104] The conversion steps are as follows:

[0105] (1) After taking out the DH5α competent cells from -80°C, place them on ice to thaw. Pipette 2 μL of the ligation product and mix with 50 μL of competent cells, and place them on ice for 5 min.

[0106] (2) Heat shock at 42℃ for 45s.

[0107] (3) Ice bath for 2 minutes.

[0108] (4) Under a sterile environment, add 700 μL of LB liquid culture medium to the centrifuge tube and incubate in a shaker at 37°C for 1 h.

[0109] (5) Centrifuge at 5000 rpm for 1 min. Aspirate 600 μL of supernatant under sterile conditions. Gently blow off the remaining supernatant and precipitate with a gun. Pipette 30 μL of bacterial solution and spread it on LB solid medium containing kanamycin.

[0110] (6) Culture in a 37°C incubator overnight.

[0111] (7) Select positive monoclonal colonies, streak them, and perform colony PCR. The PCR system is as follows:

[0112]

[0113] PCR reaction procedure:

[0114]

[0115]

[0116] According to the electrophoresis results, colonies with the correct band length were selected for sequencing.

[0117] (8) Mix 700 μL of 50% sterile glycerol and 700 μL of bacterial solution (sequencing was correct) in a 2 mL EP tube by shaking, freeze in liquid nitrogen for 1-2 min, and store in a -80°C refrigerator.

[0118] 2.7.4 Extraction of plasmid

[0119] The correct bacterial solution was sequenced according to The plasmid was extracted using the Plasmid Mini Kit DC201 (China, Vazyme).

[0120] 2.7.5 Transformation of Agrobacterium, Screening of Positive Clones and Preservation of Strains

[0121] The recombinant plasmid was transferred into K599 competent cells according to the K599 Chemically Competent Cell method. The transformation steps are as follows:

[0122] (1) After taking out the K599 competent cells from -80°C, place them on ice to thaw, and pipette 2 μL of the ligation product and mix it with 50 μL of competent cells.

[0123] (2) Ice bath for 5 minutes.

[0124] (3) Liquid nitrogen for 5 minutes.

[0125] (4) Heat shock at 37°C for 5 min.

[0126] (5) Ice bath for 5 minutes.

[0127] (6) Under sterile conditions, add 700 μL of YEB liquid culture medium into the centrifuge tube and incubate in a shaker at 28 °C for 3 h.

[0128] (7) Centrifuge at 6000 rpm for 1 min. Aspirate 600 μL of supernatant under sterile conditions. Gently blow away the remaining supernatant and precipitate with a gun. Then, aspirate 30 μL of bacterial solution and spread it on YEB solid culture medium containing Kan and Rif antibiotics.

[0129] (8) Culture in a 28°C incubator for 2-3 days.

[0130] (9) Select positive monoclonal colonies, streak them, and perform colony PCR. The PCR system and reaction procedure are the same as those for E. coli colony PCR. Based on the electrophoresis results, select colonies with the correct band length for subsequent experiments and preservation of bacterial strains.

[0131] (10) Mix 700 μL of 50% sterile glycerol and 700 μL of bacterial solution in a 2 mL EP tube by vortexing, freeze in liquid nitrogen for 1-2 min, and store in a -80 °C refrigerator.

[0132] 2.8 Soybean hairy root experiment

[0133] 2.8.1 Drugs and culture media

[0134] Drugs: Sucrose, Murashige and Skoog Vitamin, Murashige and Skoog Basal Salt Mixture (MS), Gamborg's B5 Vitamin, Gamborg's B-5 Basic Salt, DTT, Cef (Cefotaxime sodium), CB (Carbenicillin disodium), AS (Acetosyringone), MES (2-morpholineethanesulfonic acid), dimethyl sulfoxide (DMSO), agar

[0135]

[0136] The germination medium GM (1 L, pH = 5.8) was configured as follows:

[0137]

[0138] The co-culture medium CCM (1 L, pH = 5.6) was prepared as follows: AS and DTT were added according to the required mass,

[0139]

[0140] The root induction medium ICM (1L, pH = 5.8) was prepared as follows: Cef and CB were added according to the required mass.

[0141]

[0142]

[0143] 2.8.2 Disinfection of soybean seeds

[0144] 1. Sterilize seeds with chlorine: Place a single layer of seeds in an open Petri dish and place it in a vacuum desiccator under a fume hood; put 100ml of sodium hypochlorite solution into a 100ml beaker in the desiccator, then slowly add 4ml of concentrated hydrochloric acid, immediately cover and seal the desiccator, and place the seeds in chlorine for 16-20h.

[0145] 2. Sterilize seeds with H2O2 / ethanol: Place seeds in a 50ml centrifuge tube. Add the hydrogen peroxide / ethanol mixture (half each) to a 40ml label and gently invert to rinse the seeds for 2 minutes. Pour off the hydrogen peroxide solution and rinse the seeds 5-6 times with excess sterile water.

[0146] 3. Disinfect seeds with ethanol and sodium hypochlorite: Wash the dust on the surface of the seeds with clean water, soak the seeds in 75% ethanol for 5 minutes in a clean bench, and wash them with sterile water. Then soak the seeds in 20% sodium hypochlorite solution for 5 minutes and wash them three times with sterile water.

[0147] 2.8.3 Soybean seed culture

[0148] The sterilized soybean seeds were planted in germination medium (GM) and grown in a tissue culture room at 28°C (16h / 8h, 27°C / 25°C, light / dark conditions) for one week. The whole cotyledons were then harvested from the green seedlings as explants.

[0149] 2.8.4 Agrobacterium infection of soybean cotyledons

[0150] The infection experiment was carried out using Agrobacterium with the successfully constructed pCAMBIA1300-GsWRKY53 expression vector.

[0151] (1) Take the stored pCAMBIA1300-GsWRKY53 expression vector Agrobacterium tumefaciens from the -80°C refrigerator and place it in an ice-water mixture to melt.

[0152] (2) The thawed bacterial solution was added to YEB medium containing Kan (100 μg / mL) and Rif (50 μg / mL) and cultured in a shaking incubator at 29°C for activation.

[0153] (3) Pipette 1 mL of activated bacterial solution into YEB medium containing Kan (100 μg / mL) and Rif (50 μg / mL) and incubate in a shaker at 29°C for 16-18 h for secondary activation.

[0154] (4) When the OD value of the bacterial liquid after secondary activation reached 0.6, centrifuge at 5000 rpm at room temperature for 10 min, remove the supernatant, and resuspend the bacteria in liquid CCM medium to obtain the infection liquid, which is used to infect soybean cotyledons in the next step.

[0155] (5) Cut off the cotyledons including the 0.5 cm hypocotyl from the seedlings. Separate a pair of cotyledons into individual cotyledons, and use a blade to injure the attachment points of the cotyledons and the lower cotyledons, leaving seven to eight scars on one cotyledon.

[0156] (6) Move the cotyledons into a conical flask containing the infection solution and soak for about 30 minutes (shake for 30 minutes at 28°), pour out the bacterial solution, wash twice with CCM liquid culture medium, 3 minutes each time (use a new conical flask for the second time), absorb the remaining liquid with sterile filter paper, and spread the subhypocotyl downward on the solid CCM culture medium with sterile filter paper to fully soak. Cultivate at 25°C, 16h / 8h light / dark conditions for 4-5 days.

[0157] (7) Collect the co-cultivated cotyledons in a sterile Erlenmeyer flask, wash once with sterile water, wash and sterilize the liquid ICM medium three times (2 min each time), dry the liquid with filter paper, and insert the cotyledons with the back side facing down into the solid ICM medium at a 45° angle. Cultivate and root at 25°C, 16h / 8h light / dark conditions.

[0158] 2.8.5 Identification of positive plants

[0159] 1. CTAB method to extract DNA

[0160] (1) Weigh 0.1 g of soybean hairy roots, place in a 2 mL centrifuge tube with a small steel ball, freeze in liquid nitrogen, and grind into powder.

[0161] (2) Pipette the required CTAB solution into a centrifuge tube, heat it in a 65°C water bath, then pipette 800 mL of CTAB solution into the centrifuge tube in step (1), heat it in a 65°C water bath for 30 min, and centrifuge it at 12,000 rpm for 5 min.

[0162] (3) Pipette the supernatant from step (2) into a new centrifuge tube, add 400 mL of chloroform, shake gently, let stand at room temperature for 15 min until stratification is obvious, and centrifuge at 12,000 rpm for 5 min.

[0163] (4) Pipette the supernatant from step (3) into a new centrifuge tube, add isopropanol in a volume ratio of 1:1 to the supernatant, mix by inversion, place in a -20°C refrigerator for 2 h, centrifuge at 12,000 rpm for 10 min at 4°C, and discard the supernatant.

[0164] (5) Wash with 70% ethanol two to three times and dry in a 37°C oven.

[0165] (6) Add 50 μL of sterile water to dissolve the precipitate. After complete dissolution, measure the concentration using Nanodrop and store in a -20°C refrigerator.

[0166] 2. PCR detection

[0167] PCR detection was performed using soybean hairy root DNA as a template, pCAMBIA1300 vector universal primers 1300-S, 1300-A, and 2×TaqMaster Mix (P213) enzyme. The PCR reaction system was as follows:

[0168]

[0169] The PCR program is as follows:

[0170]

[0171] The PCR products were detected by 1% agarose gel electrophoresis, and the fragments with the correct band length were selected according to the electrophoresis results, which were the positive plants identified.

[0172] 3. Experimental results

[0173] 3.1 GsWRKY bioinformatics analysis

[0174] 3.1.1 Chromosome localization

[0175] Based on the protein sequences of 72 WRKY members in Arabidopsis, 114 GsWRKY genes were identified from the wild soybean genome. The TBtools software was used for visualization and the members were renamed according to their positions on the chromosome (e.g. Figure 2 shown).

[0176] 3.1.2 Phylogenetic tree

[0177] The conserved domain of WRKY family members consists of 60-70 amino acids, including a highly conserved seven benzo ring sequence (WRKYGQK) and a zinc finger structure (C2H2 or C2HC). According to the number of conserved domains and the type of zinc finger structure, WRKY gene family members can be divided into three categories: Class I members contain two WRKY domains and C2H2-type zinc finger structures; Class II members contain one WRKY domain and C2H2-type zinc finger structures, and Class II can be further divided into five subclasses, namely, IIa, IIb, IIc, IId and IIe; Class III members contain one WRKY domain and C2HC-type zinc finger structures.

[0178] The 114 GsWRKY family members were compared with the Arabidopsis AtWRKY gene family to construct a phylogenetic tree and were classified according to the number of conserved domains and the type of zinc finger structure, e.g. Figure 3 shown.

[0179] 3.1.3 Screening genes

[0180] According to the phylogenetic tree of wild soybean and Arabidopsis, multiple candidate WRKY genes were screened out, and compared with the proteins of salt-tolerant genes in Arabidopsis, GsWRKY53 with high homology was finally screened out, with a homology of 46.01%. The full length of GsWRKY53 gene is 1119bp, encoding 372 amino acids. The electrophoresis band detected by agarose gel electrophoresis is consistent with the length of the target fragment.

[0181] 3.2 Subcellular localization of GsWRKY53

[0182] The GsWRKY53 gene was connected to the pCAMBIA1300-35S-sGFP overexpression vector by homologous recombination, transformed into Agrobacterium GV3101 (the transformation method is the same as that of K599 Agrobacterium), and transiently transformed into tobacco. The empty vector pCAMBIA1300-35S-sGFP was used as a control. The specific operation is as follows:

[0183] (1) Agrobacterium containing the expression vector pCAMBIA1300-35S-sGFP-GsWRKY53, and GV3101 Agrobacterium containing the cell membrane, nucleus, endoplasmic reticulum, and Golgi Maker were inoculated into 5 mL of YEB liquid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, respectively, and cultured at 29°C overnight.

[0184] (2) The activated Agrobacterium culture solution was added to 50 mL of YEB liquid medium containing rifampicin and kanamycin. At the same time, 5 μL (20 μM) acetosyringone (AS) was added to the medium and the culture was shaken until the OD 600 =1.00;

[0185] (3) Collect 20 mL of the shaken bacterial solution, centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells with 20 mL of MMA (10 mM MgCl2·6H2O; 10 mM MES; 40 mL AS; pH 5.5) and let stand at room temperature for 3 h. At the same time, place the tobacco in the dark to close the stomata.

[0186] (4) Mix the Agrobacterium containing the target expression vector and the corresponding Maker Agrobacterium in a volume ratio of 1:1 and inject into the back of the pre-dark treated tobacco leaves using a 1 mL syringe. After the injection, place the tobacco leaves in a 25°C light incubator and culture them for 36-48 hours.

[0187] Two-photon laser scanning confocal microscopy observation:

[0188] (1) Use tweezers to tear off the lower epidermis of tobacco leaves to make a temporary slide;

[0189] (2) Observe the slides under a laser scanning confocal microscope.

[0190] like Figure 5 As shown in the figure, the subcellular localization results showed that the signal expressed by the empty vector pCAMBIA1300-35S-sGFP was distributed in the nucleus, cell membrane and part of the cytoplasm of tobacco epidermal cells, while the green fluorescence signal of the recombinant protein 35S::GsWRKY53-GFP was mainly located in the cell nucleus.

[0191] 3.3 Functional verification of GsWRKY53 overexpression

[0192] After the infected soybean cotyledons began to grow roots, the cotyledons of the unloaded and GsWRKY53-OE plants with the same growth potential were placed on the control (CK) and rooting induction medium (ICM) containing 150 mM NaCl, respectively, and grown for 7 days at 25°C and 16h / 8h light and dark conditions. The phenotypes were recorded by taking photos on the first and seventh days ( Figure 6 ), with at least 5 replicates per treatment group. After 7 days, the fresh weight, dry weight and number of hairy roots were measured.

[0193] Fresh weight: Take out the soybean hairy roots from the culture medium, remove the cotyledons, wash and dry them, measure the fresh weight and record it.

[0194] Dry weight: Mark the soybean hairy roots after measuring their fresh weight, put them in an envelope, sterilize them at 105℃ for 10 min, dry them at 70℃ to constant weight, and weigh their dry weight.

[0195] Number of hairy roots: The number of taproots of hairy roots in each treatment group was recorded.

[0196] Depend on Figure 7 It can be seen that compared with the empty load, the fresh weight, dry weight and number of hairy roots of GsWRKY53-OE were decreased, indicating that GsWRKY53 negatively regulates the salt tolerance of soybean hairy roots.

[0197] The nucleotide and amino acid sequences involved in the present invention are:

[0198] CDS Sequence

[0199] >GsWRKY53

[0200]

[0201] Protein sequence

[0202] >GsWRKY53

[0203] MSVEPKELYYQDLVLDHNIINNQHQGTEAGSNTTMYEKHFSSTLIPSVSAAYDSSQAFDPSYMSFTDCLQGSMDYNSLATSFGLSPSSSEVFSSVEGNQKPAEGGGDGGGGGGGGSGGSETLATLNSSISSSSSEAGAEEDSGKSKKERQVKTEEGGENSKKGNKEKKKGEKKQKEPRFAFMTKSEVD HLEDGYRWRKYGQKAVKNSPYPRSYYRCTTQKCTVKKRVERSFQDPTTVITTYEGQHNHPVPTSLRGNAAAGMFTPSSLLATPTHPLAAGSNFPQDLFLHMHYPRHHHQYHHIHNNLFATQSMTNATTATTTTTTATIAPSSFYSSYNNINNSLLNNQFLPPEYGLLQDIVPSMLHNKSHHNQN(SEQ ID NO.2)

[0204] Matters not covered by the present invention are known technologies.

[0205] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A wild soybean salt tolerance gene, named GsWRKY53, characterized in that: The wild soybean salt-tolerance gene has: (a1) the nucleotide sequence shown in SEQ ID NO.1; (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but differing in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence having ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encoding the same functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

2. A wild soybean salt-tolerant protein, characterized in that The wild soybean salt-tolerance protein is encoded by the wild soybean salt-tolerance gene according to claim 1.

3. The wild soybean salt-tolerant protein according to claim 2, characterized in that The wild soybean salt-tolerant protein has any one of the amino acid sequences (b1)-(b3): (b1) the amino acid sequence shown in SEQ ID NO.2; (b2) a protein derived from the amino acid sequence shown in SEQ ID NO.2 with the same function as that of the amino acid sequence shown in SEQ ID NO.2 after one or more amino acid residues are substituted and / or deleted and / or added; (b3) proteins encoded by other genes that have an amino acid sequence composition that is more than 90% similar to that of SEQ ID NO. 2 and have the activity of the protein of SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the wild soybean salt-tolerance gene according to claim 1; Furthermore, the recombinant expression vector is obtained by effectively connecting the wild soybean salt-tolerance gene to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phagemid, a clay mid, or an artificial chromosome, and further is a plasmid, and the plasmid is a pCAMBIA series vector, and further, the plasmid is pCAMBIA1300.

5. A transgenic cell line, host bacteria or transgenic plant containing the wild soybean salt-tolerance gene according to claim 1 or the recombinant expression vector according to claim 4. Furthermore, the transgenic plant is a crop, further an economic crop, including soybean.

6. Use of the wild soybean salt-tolerance gene according to claim 1, the wild soybean salt-tolerance protein according to any one of claims 2-3, the recombinant expression vector according to claim 4, the transgenic cell line, host bacteria or transgenic plant according to claim 5 in any one or more of the following: (c1) regulating the number, fresh weight and / or dry weight of plant roots; (c2) regulating plant salt tolerance; (c3) Screening and / or breeding of salt-tolerant plants.

7. The use according to claim 6, characterized in that The plant roots are hairy roots.

8. A method for reducing salt tolerance of plants, characterized in that: The method comprises: transferring the wild soybean salt-tolerance gene of claim 1 or the recombinant expression vector of claim 4 into a plant, thereby reducing the fresh weight, dry weight and number of hairy roots of the plant.

9. The method according to claim 8, characterized in that The specific method for transferring the recombinant expression vector into plants is the Agrobacterium transformation method, specifically, transferring the recombinant expression vector into Agrobacterium, using the Agrobacterium to infect plant cotyledons, and culturing to obtain plant hairy roots.

10. A method for improving plant salt tolerance, characterized in that: The method comprises: inhibiting the expression of the wild soybean salt-tolerance gene of claim 1 in a plant.

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