Application of GmSRLK13 protein in improving plant salt stress sensitivity

Abstract

The present invention discloses the use of a GmSRLK13 protein in improving the salt stress sensitivity of a plant, belonging to the technical field of plant breeding. This invention provides a method for breeding salt-stress-sensitive plants. The present invention provides the use of a GmSRLK13 protein in improving the salt stress sensitivity of a plant, wherein the amino acid sequence of the GmSRLK13 protein is shown in SEQ ID NO. 5 or SEQ ID NO. 12. The GmSRLK13 gene is expressed in different tissues of soybean and responds to salt stress, providing a theoretical basis for research on soybean under salt stress.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and particularly relates to the application of GmSRLK13 protein in improving the salt stress sensitivity of plants. Background Art

[0002] Soybean (Glycine max), a leguminous crop, has enormous economic and nutritional demand worldwide. As a source of high-quality plant protein, it plays a vital role in human nutrition, animal feed, and oilseed production. Given its widespread cultivation, soybean growth and development are often affected by a variety of biotic and abiotic factors, which can alter soybean growth and development, as well as its symbiotic nitrogen fixation, ultimately leading to reduced soybean yields. Soil salinization is the accumulation of soluble salts, leading to a deterioration in basic soil properties and a decline in quality. Currently, the degree of soil salinization continues to rise globally, with the total area of ​​saline soils worldwide estimated to be approximately 1.1×10 9 hm 2 , of which 14% are woodlands, wetlands and nature reserves defined as unsuitable for production. High salt usually inhibits crop growth through osmotic effects, which reduce the amount of water absorbed by plants or ion toxicity effects, thereby inhibiting enzyme activity. Salt stress can reduce the yield of most crops. Due to factors such as global warming and insufficient drainage of irrigated land, the impact of salt stress on crop production has been seriously aggravated. As an important oil crop, soybeans will have a serious impact on my country's grain production if their yield and quality are reduced. Therefore, it is very important to maintain their stable production. It is very important to discover or identify the key salt-sensitive genes of plants to understand the salt-sensitive mechanism of plants and then regulate the salt tolerance and salt sensitivity of plants. Summary of the Invention

[0003] The purpose of the present invention is to provide a breeding method for salt stress sensitive plants.

[0004] The present invention provides an application of a GmSRLK13 protein in improving the salt stress sensitivity of a plant. The amino acid sequence of the GmSRLK13 protein is shown as SEQ ID NO.5 or SEQ ID NO.12.

[0005] The present invention provides an application of a GmSRLK13 gene in improving the salt stress sensitivity of a plant. The GmSRLK13 gene is shown as SEQ ID NO.6 or SEQ ID NO.11.

[0006] It is further defined that the plant is soybean or Arabidopsis thaliana.

[0007] It is further defined that the concentration of salt applied in the salt stress resistance is 100-175 mM.

[0008] The present invention provides a method for cultivating plants sensitive to salt stress, and the specific steps of the method are as follows:

[0009] (1) Amplify the GmSRLK13 gene and insert the gene sequence into a plant overexpression vector;

[0010] (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transform into Arabidopsis thaliana or soybean to obtain transgenic plants;

[0011] (3) Identify the transgenic soybeans obtained in step (2) to obtain positive transgenic plants.

[0012] It is further defined that the expression vector in step (1) is pCAM35S; the primers for amplifying the GmSRLK13 gene are SEQ ID NO.1 and SEQ ID NO.2; and the GmSRLK13 gene is as shown in SEQ ID NO.6 or as shown in SEQ ID NO.11.

[0013] The present invention provides a method for improving the salt stress sensitivity of plants, comprising placing transgenic plants overexpressing the GmSRLK13 gene in a 100-175 mM sodium chloride environment for stress treatment; the GmSRLK13 gene is shown in SEQ ID NO.6 or SEQ ID NO.11.

[0014] The present invention provides a gene for improving the salt stress sensitivity of plants. The gene sequence is shown as SEQ ID NO.6 or SEQ ID NO.11.

[0015] The present invention provides a plant over-expressing a GmSRLK13 gene, a recombinant vector containing the GmSRLK13 gene, or a recombinant microbial cell containing the GmSRLK13 gene for use in improving the salt stress sensitivity of plants.

[0016] It is further defined that the plant is soybean or Arabidopsis thaliana; and the GmSRLK13 gene is shown as SEQ ID NO.6 or SEQ ID NO.11.

[0017] Beneficial effects: The GmSRLK13 gene is expressed in different tissues of soybean and responds to salt stress; GmSRLK13 is localized in the cell membrane, cytoplasm and nucleus, while the kinase domain GmSRLK13-KD is localized in the cytoplasm and nucleus; GmSRLK13 overexpression increases the sensitivity of Arabidopsis to salt stress during the germination, seedling and seedling stages; overexpression of GmSRLK13 in soybean hairy roots also leads to increased sensitivity of soybean hairy root chimeras to salt stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the construction of the pCAM35-GmSRLK13-GFP vector are shown in Figure 2. Note: M: 5000 Marker; -: ddH2O control; +: positive plasmid control; 1: GmSRLK13 PCR amplification results; 2: PCR detection of DH5α bacterial solution transformed with pCAM35-GmSRLK13-GFP; 3: PCR detection of GV3101 Agrobacterium transformed with pCAM35-GmSRLK13-GFP;

[0019] Figure 2 Figure 1 shows the results of constructing the pCAM35-GmSRLK13-KD-GFP vector. Note: M: 2000 marker; -: ddH2O control; +: positive plasmid control; 1: GmSRLK13-KD PCR amplification results; 2: PCR detection of DH5α bacterial culture transformed with pCAM35-GmSRLK13-KD-GFP; 3: PCR detection of GV3101 Agrobacterium transformed with pCAM35-GmSRLK13-KD-GFP.

[0020] Figure 3 The figure shows the positive screening results of GmSRLK13 transgenic Arabidopsis T1 generation; Note: M: 5000 Marker; -: ddH2O control; +: positive plasmid control; 1-13: positive PCR detection of GmSRLK13 transgenic Arabidopsis T1 generation (35S-GmSRLK13-GFP-F, 35S-GmSRLK13-GFP-R);

[0021] Figure 4 The results of positive screening of GmSRLK13 transgenic Arabidopsis T2 generation; M: 2000 Marker; -: ddH2O control; +: positive plasmid control; 1-8: positive PCR detection of GmSRLK13 transgenic Arabidopsis T2 generation (HYG-F, HYG-R);

[0022] Figure 5 Schematic diagram of the subcellular localization of GmSRLK13 and GmSRLK13-KD. Note: Confocal laser scanning microscopy was used to observe the fluorescence signals of GmSRLK13::GFP, GmSRLK13-KD::GFP, and GFP in onion cells. Images from left to right are: GFP fluorescence image, bright field, and merged GFP fluorescence image. Green fluorescence was detected by confocal laser microscopy in onion epidermal cells infected with Agrobacterium. GmSRLK13 is localized in the nucleus, cytoplasm, and cell membrane; GmSRLK13-KD is distributed in both the nucleus and cytoplasm. Scale bar = 30 μm.

[0023] Figure 6Figure 1 shows the phenotypic analysis results of the germination level of GmSRLK13 overexpressing lines; Note: A: RT-PCR detection of the expression level of GmSRLK13 in the overexpressing lines; B: Germination of GmSRLK13 overexpressing Arabidopsis thaliana grown in 1 / 2 MS, 100 mM NaCl and 125 mM NaCl medium for 5 and 10 days; CE: Statistical analysis data of the germination rate of GmSRLK13 overexpressing lines; FH: Statistical analysis data of the leaf expansion rate of GmSRLK13 overexpressing lines;

[0024] Figure 7 Figure 1 shows the phenotypic analysis of root growth in GmSRLK13-overexpressing lines. Notes: A: Root growth of GmSRLK13-overexpressing Arabidopsis plants grown in 1 / 2 MS, 100 mM NaCl, and 125 mM NaCl media for 10 days; B: Statistical analysis of daily root elongation in GmSRLK13-overexpressing lines; E: Statistical analysis of lateral root number in GmSRLK13-overexpressing lines; HJ: Statistical analysis of root growth in GmSRLK13-overexpressing lines for 10 days. In the bar graphs, "*" indicates a significant difference (p < 0.05), and "**" indicates an extremely significant difference (p < 0.01). Scale bar = 1 cm.

[0025] Figure 8 Figure 2 shows the results of physiological index detection of GmSRLK13 overexpressing plants under salt stress; Note: A: Phenotype of GmSRLK13 overexpressing Arabidopsis and WT after salt stress treatment; B: DAB staining, NBT staining and trypan blue staining of GmSRLK13 overexpressing Arabidopsis and WT after salt stress treatment; CF: MDA, POD, SOD and CAT determination in GmSRLK13 overexpressing and WT; G: Chlorophyll content in GmSRLK13 overexpressing and WT; H: Na + / K + , (p<0.05);

[0026] Figure 9 Positive detection of chimeras in GmSRLK13 transgenic soybean hairy roots; Note: A: GUS staining results; B: GFP fluorescence signal detection of positive chimeras in transgenic soybean hairy roots. Scale bar = 100 μm;

[0027] Figure 10Figure 1 shows the phenotypic and physiological index test results of GmSRLK13 transgenic soybean hairy root chimeras under salt stress. Note: A: Phenotypic changes of GmSRLK13 and GmSRLK13-KD transgenic soybean hairy root chimeras after salt stress; B: Leaf phenotypes of GmSRLK13 and GmSRLK13-KD transgenic soybean hairy root chimeras after salt stress; CF: MDA, SOD, POD, and CAT measurements of GmSRLK13 and GmSRLK13-KD transgenic soybean hairy root chimeras after salt stress. (p < 0.05) Scale bar = 2 cm.

[0028] Figure 11 The results of positive screening of GmOTSa and GmOTSa(C486S) transgenic Arabidopsis T1 generation are shown in the figure; Note: M: 2000 Marker; -: ddH2O control; +: positive plasmid control; WT: wild-type Arabidopsis; 1-6: positive PCR detection of GmOTSa transgenic Arabidopsis T1 generation; 7-11: positive PCR detection of GmOTSa(C486S) transgenic Arabidopsis T1 generation;

[0029] Figure 12 Figure 2 shows the phenotypic analysis results of the germination level of GmOTSa and GmOTSa(C486S) overexpressing lines. Note: A: Germination of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSa OE#1, and GmOTSa(C486S) OE#1 after 4 and 9 days of growth in 1 / 2 MS, 100 mM NaCl, and 125 mM NaCl medium. BD: Statistical analysis data of germination rate of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSaOE#1, and GmOTSa(C486S) OE#1. EG: Statistical analysis data of green leaf rate of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSa OE#1, and GmOTSa(C486S) OE#1.

[0030] Figure 13 Figure 1 shows the response of Arabidopsis root growth to salt stress by overexpressing GmOTSa and GmOTSa(C486S); Note: A: Root length phenotype of GmSRLK13, GmOTSa, and GmOTSa(C486S) overexpressing lines grown on 1 / 2 MS, 100 mM NaCl, and 125 mM NaCl medium for 6 days; B: Statistical analysis data of root growth of Arabidopsis overexpressing GmSRLK13, GmOTSa, and GmOTSa(C486S), (p<0.05); Scale bar = 1 cm.

[0031] Figure 14The results show the response of GmOTSa and GmOTSa(C486S) overexpressing Arabidopsis to salt stress at the seedling stage; Note: A: Phenotypes of GmSRLK13, GmOTSa, and GmOTSa(C486S) overexpressing lines at the seedling stage before and after treatment with 175 mM NaCl; BE: Determination of MDA, SOD, POD, and chlorophyll contents in GmSRLK13, GmOTSa, and GmOTSa(C486S) overexpressing Arabidopsis after salt stress treatment (p<0.05);

[0032] Figure 15 Figure 1 shows the results of positive screening of T1 plants co-expressing GmSRLK13 and GmOTSa. Note: M1: 2000 Marker; M2: 5000 Marker; -: ddH2O control; +: positive plasmid control. A: PCR detection of T1 positive plants co-expressing GmSRLK13 and GmOTSa using bar primers; B: PCR detection of T1 positive plants co-expressing GmSRLK13 and GmOTSa using HYG primers; C: PCR detection of T1 positive plants co-expressing GmSRLK13 and GmOTSa using GmOTSa-specific primers; D: PCR detection of T1 positive plants co-expressing GmSRLK13 and GmOTSa using GmSRLK13-specific primers.

[0033] Figure 16 Figure 2 shows the phenotypic analysis results of the germination level of Arabidopsis thaliana co-expressing GmSRLK13 and GmOTSa. Note: A: Germination of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSa OE#1, and GmSRLK13 GmOTSa OE#4 after 4 and 8 days of growth in 1 / 2 MS, 100 mM NaCl, and 125 mM NaCl medium; B: Statistical analysis data of the germination rate of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSa OE#1, and GmSRLK13 GmOTSa OE#4; E: Statistical analysis data of the green leaf rate of Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9, GmOTSa OE#1, and GmSRLK13 GmOTSa OE#4.

[0034] Figure 17The results of phenotypic and physiological index analysis of GmSRLK13 and GmOTSa co-expressing lines under salt stress treatment; Note: A: Phenotypes of GmSRLK13 and GmOTSa over-expressing and co-expressing Arabidopsis lines at the seedling stage after treatment with 175 mM NaCl; B: Chlorophyll content determination of GmSRLK13 and GmOTSa over-expressing and co-expressing Arabidopsis lines after salt stress treatment; C: MDA content determination of GmSRLK13 and GmOTSa over-expressing and co-expressing Arabidopsis lines after salt stress treatment;

[0035] Figure 18 The results of the protein expression and SUMOylation analysis of GmSRLK13 and GmOTSa co-expressed Arabidopsis; Note: M: protein marker; 1: untreated GmSRLK13 overexpressed Arabidopsis; 2: untreated GmSRLK13 and GmOTSa co-expressed Arabidopsis; 3: salt-treated GmSRLK13 overexpressed Arabidopsis; 4: salt-treated GmSRLK13 and GmOTSa co-expressed Arabidopsis;

[0036] Figure 19 Figure 1 shows the results of constructing the p2300-GmSRLK13-CeGFP vector. Note: M: 5000 markers; -: ddH2O control; +: positive plasmid control; 1: GmSRLK13 PCR amplification results; 2: PCR detection of p2300-GmSRLK13-CeGFP transformed into DH5α bacteria; 3: Identification of p2300-GmSRLK13-CeGFP transformed into GV3101.

[0037] Figure 20 Figure 1 shows the results of constructing the p2300-GmSRLK13-KD-CeGFP vector. Note: M: 2000 marker; -: ddH2O control; +: positive plasmid control; 1: GmSRLK13-KD PCR amplification results; 2: PCR detection of p2300-GmSRLK13-KD-CeGFP transformed into DH5α bacteria; 3: Identification of p2300-GmSRLK13-KD-CeGFP transformed into GV3101.

[0038] Figure 21 Figure 1. BiFC validation of the interaction between GmSRLK13 and GmOTSa. Note: Fluorescence signals were observed using confocal laser scanning microscopy in onion inner epidermal cells co-infected with GmSRLK13::CeGFP, GmSRLK13-KD::CeGFP, and GmOTSa::NeGFP. Images from left to right: GFP fluorescence image, bright field image, merged GFP fluorescence image; scale bar = 100 μm.

[0039] Figure 22Figure 1 shows the results of constructing the pET32a-GmSRLK13-KD vector. Note: M: 2000 marker; -: ddH2O control; +: positive plasmid control; 1: GmSRLK13-KD PCR amplification results; 2: PCR detection of pET32a-GmSRLK13-KD transformed into DH5α bacteria; 3: identification of pET32a-GmSRLK13-KD transformed into BL21.

[0040] Figure 23 This is a diagram showing the interaction between GmSRLK13 and GmOTSa verified by GST pull-down assay. Note: Protein expression was induced at low temperature, and successful protein expression was verified using GST and His tag antibodies. GST pull-down assays were then performed to verify the interaction between GmSRLK13 and GmOTSa.

[0041] Figure 24 Figure 2 shows the in vivo validation of the interaction between GmSRLK13-KD and SUMO in tobacco. Note: GmSRLK13-KD-GFP was co-expressed with either SUMO2GG or SUMO2AA in tobacco leaves. Protein expression was verified using GFP and FLAG antibodies, respectively. Proteins were then purified using GFP magnetic beads and analyzed by Western blotting using FLAG antibodies. DETAILED DESCRIPTION

[0042] Example 1. Construction of plants overexpressing the GmSRLK13 gene

[0043] 1. GmSRLK13, GmSRLK13-KD Gene amplification

[0044] Soybean cDNA was used as template and GmSRLK13-F and GmSRLK13-R were used as primers to amplify GmSRLK13 Gene, with GmSRLK13-KD -F and GmSRLK13-KD -R is primer amplification GmSRLK13-KD Target gene, primer sequence ( GmSRLK13 -F:ATGCATTTAAGTACTAGTAGAGAGG(SEQ ID NO.1); GmSRLK13 -R: ACGAGGGAGCACATTATTTTCATGG (SEQ ID NO.2), GmSRLK13-KD -F:ATGTTCAGAACCAGCAAGAACTCTG (SEQ ID NO.3), GmSRLK13- KD-R, ACGAGGGAGCACATTATTTTCATGG (SEQ ID NO. 4)), the optimal PCR amplification conditions were explored according to the annealing temperature and fragment length during primer design. The PCR reaction system (2×Phanta Master Mix, 10 μL; cDNA template, 1.5 μL; upstream primer (10 μM), 1 μL; downstream primer (10 μM), 1 μL; RNase-free H2O, 6.5 μL) and PCR conditions (98°C, 5 min; 98°C, 15 sec; 57°C, 15 sec; 72°C, 1 min; 72°C, 7 min; 4°C, ∞) are shown as follows.

[0045] The gene sequence of GmSRLK13 is shown in SEQ ID NO. 5; the amino acid sequence of GmSRLK13 is shown in SEQ ID NO. 6;

[0046] GmSRLK13-KD gene sequence: SEQ ID NO.11

[0047] TTCAGAACCAGCAAGAACTCTGAGACGGTGGATCAACAACGGCACCTTCTTTCCGCCACTGGGTTTCAGAGGTTCACCTACGCAGAGCTCAAAAGCGCAACAAAAGGGTTCAAAGAAGAAATTGGACGAGGAGCGGGTGGGGTTGTTTACAAAGGAGTACTCTATGATGACCGTGTTGCGGCTATTAAACGCTTAGGAGAAGCTACACAAGGAGAAGCTGAGTTTTTGGCGGAGATAAGCACAATAGGCATGTTGAACCACATGAATTTGATTGACATGTGGGGGTACTGCGTTGAAGGGAAGCATAGGATGTTGGTGTATGAGTACATGGAACATGGGTCCTTAGCGGGCAATCTTTTTTCTAATACCCTTGATTGGAAGAAGAGGTTTAACGTTGCTGTGGGTACAGCGAAAGGTTTGGCTTATTTGCACGAAGAGTGTTTGGAGTGGATTTTGCATTGCGATGTGAAGCCTCAAAACATTCTCCTTGACTCGGATTTCCAACCCAAGGTGGCGGATTTCGGTTTGTCAAAACTTTTAAACAGAGACGAGCGTGGGAATTCTACTTTCTCAAGAATAAGAGGGACACGAGGTTACATGGCTCCGGAGTGGGTTTACAATCTGCCCATAACTTCCAAGGTGGATGTTTATAGTTATGGGATTGTGGTGTTGGAAATGGTGACTGGAAGGAGCCCTATGGAAATTCACAGCCTTGAGAACAGTAGGGGCATAGAGCAGCGACGGTTGGTGATGTGGGTAACGGACAAGATAAACGATGCACCCACGAGTGGGTTTTGGATTGAGGAGATACTTGATCCCAACTTGGAAGGCCAATGCCAAGTTTCGCAAGTTGAGGTTTTGGTTAAGGTGGCTTTGCAGTGTGTGCAAGATGACATGAATCAAAGACCCTCCATGAGCCAAGTAGTGGAGATGCTTCTGTCCCATGAAAATAATGTGCTCCCTCGT;

[0048] GmSRLK13-KD protein: SEQ ID NO.12

[0049] FRTSKNSETVDQQRHLLSATGFQRFTYAELKSATKGFKEEIGRGAGGVVYKGVLYDDRVAAIKRLGEATQGEAEFLAEISTIGMLNHMNLIDMWGYCVEGKHRMLVYEYMEHGSLAGNLFSNTLDWKKRFNVAVGTAKGLAYLHEECLEWILHCDVKPQNI LLDSDFQPKVADFGLSKLLNRDERGNSTFSRIRGTRGYMAPEWVYNLPITSKVDVYSYGIVVLEMVTGRSPMEIHSLENSRGIEQRRLVMWVTDKINDAPTSGFWIEEILDPNLEGQCQVSQVEVLVKVALQCVQDDMNQRPSMSQVVEMLLSHENNVLPR.

[0050] 2. Restriction enzyme digestion reaction

[0051] To a 200 μL centrifuge tube, add 5 μL of CutSmart restriction enzyme buffer and 10 μg of plasmid, depending on the plasmid concentration. Add 1 μL of the appropriate restriction enzyme and bring the total volume to 50 μL with ddH₂O. Gently mix with a pipette tip and centrifuge briefly to concentrate all the liquid at the bottom of the tube. Place the enzyme digestion reaction system in a 37°C incubator overnight. After digestion, perform agarose gel electrophoresis using the plasmid as a control to determine if the enzyme has been digested. The migration speed of the plasmid during the gel electrophoresis indicates whether the enzyme has been digested.

[0052] 3. Ligation reaction between target fragment and expression vector

[0053] (1) Ligation reaction between target fragment and plant expression vector

[0054] Using the plasmid containing the target gene fragment as a template, 35S- GmSRLK13 -GFP-F: GAGCTCGGTACCCGGGGATCCATGCATTTAAGTACT (SEQ ID NO.7); 35S- GmSRLK13 -GFP-R:GGTGTCGACTCTAGAGGATCCACGAGGGAGCACATT (SEQ ID NO. 8); 35S- GmSRLK13-KD -GFP-F: GAGCTCGGTACCCGGGGATCCATGTTCAGAACCAGC (SEQ ID NO.9); 35S- GmSRLK13-KD- GFP-R: GGTTGCGACTCTAGAGGATCCCACGAGGGAGCACATT (SEQ ID NO. 10), recovered GmSRLK13 、 GmSRLK13-KD Gene fragment, the plasmid target gene fragment was connected with the cut plasmid by homologous recombination method, and the homologous recombination reaction system (insert fragment recovery product, 1 μL; linearized vector pCAM35S-GFP / p2300-CeGFP, 1.5 μL; 5×CE II Buffer, 1 μL; Exnase II, 0.5 μL; ddH2O, 1 μL) was used.

[0055] Overexpression vector plant expression vector pCAM35S-eGFP (PCG3301, hygromycin resistance) and GmSRLK13 Gene ligation to obtain pCAM35S- GmSRLK13 -eGFP recombinant vector;

[0056] Overexpression vector pCAM35S-eGFP (PCG3301, hygromycin resistance) and GmSRLK13-KD The gene fragments were connected to obtain pCAM35S-GmSRLK13- KD -GFP recombinant vector;

[0057] After ligation at 37°C for 30 min, the cells were transformed into competent E. coli DH5α. Single colonies were picked for PCR identification, and the PCR-positive bacterial liquid was sent for sequencing.

[0058] Results: Figure 1 、 2 As shown, the full-length gene of GmSRLK13 was used as a template and primers 35S- GmSRLK13 -GFP-F, 35S- GmSRLK13 -GFP-R and 35S- GmSRLK13-KD -GFP-F, 35S- GmSRLK13-KD -GFP-R pair GmSRLK13 The target band was obtained by PCR amplification of the gene, and then restriction endonucleases were used to Bam The plant expression vector pCAM35S-GFP was digested, recovered and identified by H Ⅰ, and the homologous recombination method was used to transform GmSRLK13 and GmSRLK13-KD The full-length gene was connected with the linearized pCAM35S-GFP expression vector and transformed into Escherichia coli DH5α; 35S- GmSRLK13 -GFP-F, 35S- GmSRLK13 -GFP-R and 35S- GmSRLK13-KD -GFP-F, 35S- GmSRLK13-KD-GFP-R were detected by PCR. The target fragments amplified by PCR were about 2500 bp and 1000 bp respectively, indicating that the vector was successfully constructed. Positive transformants were obtained and the correct recombinant plasmid pCAM35S- GmSRLK13 -GFP, pCAM35S- GmSRLK13-KD -GFP was transformed into Agrobacterium tumefaciens GV3101 to obtain a recombinant plasmid containing the target gene tagged with GFP, which was then used for transient transformation in the inner epidermis of onions. Agrobacterium containing the target plasmid was first activated twice and used to infect the inner epidermis of onions. The localization of GmSRLK13 and GmSRLK13-KD was analyzed by observing the distribution of GFP green fluorescence signals in the inner epidermis of onions under a laser confocal microscope, using an empty pCAM35S-GFP vector as a control.

[0059] 4. Obtain transgenic plants by Agrobacterium-transfected plants containing overexpression recombinant vectors:

[0060] 1. Obtaining Overexpressed Soybean Transgenic Materials

[0061] Using the hypocotyl cutting method, Agrobacterium rhizogenes K599 and K599 empty bacteria transformed with pCAM35S-GmSRLK13-GFP and pCAM35S-GmSRLK13-KD-GFP were smeared on the wound after soybean hypocotyl cutting to induce the production of soybean hairy roots.

[0062] In order to detect whether GmSRLK13 and GmSRLK13-KD are normally expressed in soybean hairy roots, GUS staining was performed at the early growth stage of soybean hairy roots, and the root tips of the hairy roots were detected by fluorescence microscopy using the GFP tag fused to the target gene in the plant expression vector ( Figure 9 A in Figure 9 In B, green fluorescence was observed in the GmSRLK13 and GmSRLK13-KD transgenic soybean hairy roots, indicating that the GmSRLK13, GmSRLK13-KD and GFP fusion proteins were successfully expressed in the soybean hairy roots.

[0063] 2. Acquisition and Screening of Overexpressing Arabidopsis

[0064] 1. Infecting Arabidopsis thaliana by floral dipping

[0065] The plant expression vector containing the target gene was transferred into Agrobacterium tumefaciens GV3101 and transfected into Arabidopsis thaliana.

[0066] 2. Positive screening of overexpressed Arabidopsis: Molecular biological detection of overexpressed Arabidopsis

[0067] (1) After crude extraction of Arabidopsis leaf DNA, PCR detection of overexpressed Arabidopsis

[0068] Using crude DNA as template and wild type as control, GmSRLK13 PCR detection was performed using specific primers.

[0069] Crude DNA was extracted from Arabidopsis leaves, and the wild type was used as a negative control. HYG, bar ) primers and GmSRLK13 PCR identification was performed using specific primers. HYG, bar Amplification program (95°C, 7 min; 95°C, 30 sec; 57°C, 15 sec; 72°C, 2 min; 72°C, 30 sec; 4°C, ∞), HYG -F: AGACGTCGCGGTGAGTTCAGGCTTTTTCAT (SEQID NO.13); HYG -R: ATACCTACGCTAGCGACGCCGGCTAGAATC (SEQ ID NO.14); Bar -F: TGCCAGTTCCCGTGCTTGAA (SEQ ID NO.15); Bar -R: CTGCACCATCGTCAACCACTA (SEQ ID NO. 16).

[0070] (2) Semi-quantitative PCR identification

[0071] Take 0.1 g pCAM35S- GmSRLK13 -GFP transgenic Arabidopsis, use RNA extraction kit to extract total RNA of plants, and then reverse transcribe RNA into cDNA using reverse transcription kit. GmSRLK13 PCR identification was performed using specific primers and Arabidopsis thaliana internal reference gene Atactin primers. According to the conventional PCR method, the optimal cycle number was selected by agarose gel electrophoresis, and the grayscale measurement of the band brightness was performed to ensure that the sample amount remained consistent. GmSRLK13 -F: CAATAGAAAGACTCCCAGAA (SEQ ID NO. 17); RT-PCR- GmSRLK13 -R:AAGTGAATCGTCCAAAGG(SEQID NO.18); Atactin -F: CTGTTCTCTCCTTGTACGCCAGT (SEQ ID NO.19); Atactin -R: CGGGTAATTCATAGTTCTTCTCGAT (SEQ ID NO. 20).

[0072] Results: Gene expression patterns were analyzed GmSRLK13In order to further explore the function of GmSRLK13 in salt stress response, the successfully constructed pCAM35S- GmSRLK13 -GFP was transformed into Agrobacterium GV3101, and Arabidopsis was infected by the floral dip method. Transgenic Arabidopsis was screened on a culture medium containing 25 mg / L hygromycin. When the Arabidopsis seedlings in the culture medium grew to the four-leaf stage, the positive seedlings were transplanted to nutrient soil for cultivation. Figure 3 、 4 As shown, primers 35S- GmSRLK13 -GFP-F, 35S- GmSRLK13 -GFP-R and primers HYG -F, HYG PCR testing of the overexpressing plants was performed using WT as a negative control and the recombinant vector plasmid as a positive control. The PCR results showed eight positive plants. The positive Arabidopsis plants screened in the T1 generation were designated as lines OE#1 to 8. Further screening was performed to obtain T2 generation seeds. Lines OE#1, OE#2, OE#4, OE#6, and OE#8 were then selected and inoculated into 1 / 2 MS medium supplemented with 25 mg / L hygromycin. Positive T3 generation seeds were harvested for subsequent studies of the phenotypic and physiological parameters of the overexpressing Arabidopsis lines after salt stress.

[0073] Example 2. Subcellular localization of GmSRLK13 and GmSRLK13-KD

[0074] The subcellular localization of GmSRLK13 and GmSRLK13-KD was analyzed, and the plant expression vector pCAM35S- GmSRLK13 -GFP, pCAM35S- GmSRLK13-KD -GFP. The green fluorescence signals of GmSRLK13-GFP and GmSRLK13-KD-GFP fusion proteins in the onion inner epidermis were observed under a laser confocal microscope. Figure 5 The results showed that GmSRLK13 was expressed in the nucleus, cytoplasm and cell membrane; while GmSRLK13-KD was expressed in the cytoplasm and nucleus.

[0075] Example 3. Phenotypic and physiological index detection of transgenic materials

[0076] 1. Arabidopsis seedling germination and root length statistics

[0077] Germination experiment: Prepare culture medium treated with different concentrations of NaCl. After disinfection and vernalization, WT and overexpressing Arabidopsis seeds were inoculated onto the culture medium in a clean bench and placed in a light incubator. The germination rate was counted daily, and the phenotype was recorded by photographing.

[0078] Root length experiment: Arabidopsis seeds were sown on 1 / 2 MS medium and grown vertically in a light incubator for 2 days. In a clean bench, seedlings of uniform growth were selected using pointed tweezers and transferred to medium treated with different NaCl concentrations for a further 5–7 days. Root lengths were then measured using Digimizer software.

[0079] 2. Salt stress experiment on Arabidopsis seedlings

[0080] After disinfection, Arabidopsis seeds were placed at 4℃ for vernalization for 2-3 days, first inoculated on 1 / 2 MS medium, grown to the four-leaf stage in a light incubator, and then moved into soil (vermiculite: imported nutrient soil = 2:1) with tweezers. The same weight of soil was weighed in each black square, and 4 seedlings were transplanted into it. The plastic wrap was covered to keep it moist for 2 days and then uncovered. After the Arabidopsis thaliana grew for 14 days, 1 L of water and 175 mM NaCl were poured into the tray respectively to allow the soil to fully absorb water so that the soil moisture content of the seedlings was consistent. Then, water was applied every 4 days. During this process, photos were taken and recorded until the leaves lost their green color. The samples were then collected for testing of physiological indicators.

[0081] 3. DAB, NBT and Trypan Blue Staining

[0082] After transplanting Arabidopsis into soil, once salt treatment has occurred and phenotypic differences have emerged, the leaves are stained with DAB, NBT, and Trypan Blue. The leaf surface is cleaned with distilled water, dried with filter paper, and then soaked in 1 mg / mL DAB, 0.5 mg / mL NBT, and Trypan Blue, respectively. Stain for 12 hours at room temperature in the dark. For DAB and NBT staining, carefully pick up the leaf with tweezers and place it in a boiling water bath with 95% ethanol for decolorization. Fresh ethanol can be replaced several times during decolorization. After decolorization, remove any remaining ethanol with distilled water. Trypan Blue staining is decolorized with 2.5 g / mL chloral hydrate, and the image is photographed.

[0083] 4. Determination of chlorophyll content

[0084] Collect 0.5 g of Arabidopsis leaves after salt stress treatment and place them in a 10 mL centrifuge tube. Soak them in 95% ethanol at 4°C for 2-3 days. Use a 1 mL cuvette to measure the absorbance at wavelengths of 665 nm, 649 nm, and 470 nm. For specific steps, refer to the Plant Physiology Experiment Tutorial.

[0085] 5. Determination of MDA, SOD, POD and CAT

[0086] After salt stress treatment, 0.2 g of Arabidopsis leaves were collected and placed in a centrifuge tube. After quick freezing in liquid nitrogen, the leaves were cryogenically ground using a tissue disruptor. 2 mL of crude enzyme extract (MDA crude enzyme extract: 10% trichloroacetic acid solution; SOD, POD, and CAT crude enzyme extract: 0.2 M PBS solution containing 1% PVP, pH 7.8) was added. The tubes were centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant was the enzyme solution. Detailed measurement procedures were referred to the "Plant Physiology Experimental Tutorial." Data were statistically analyzed using Excel and SPSS 26.0 software for one-way analysis of variance (ANOVA) at a significance level of 0.05. Prism software was used for plotting.

[0087] Results: (1) Response of GmSRLK13 overexpressing Arabidopsis to NaCl during germination: GmSRLK13 Transgenic T3 generation Arabidopsis positive lines were subjected to salt stress treatment. First, lines OE#1-11, OE#2-1, and OE#6-9 were selected for RT-PCR detection. Actin As an internal reference gene, GmSRLK13 Gene-specific detection primers RT-PCR- GmSRLK13 -F, RT-PCR- GmSRLK13 -R for RT-PCR detection. Figure 6 As shown in A, Actin During internal control amplification, the amount of cDNA template sample was adjusted, and the brightness of the final amplified samples was basically the same. Actin Internal reference band; GmSRLK13 When RT-PCR was performed using specific primers for detection of non-Arabidopsis endogenous genes, no band was amplified for WT, but GmSRLK13 Overexpressing Arabidopsis OE#1-11, OE#2-1, and OE#6-9 were all positive lines, and OE#1-11 and OE#6-9 were selected for subsequent studies.

[0088] Plants are extremely susceptible to adverse stress during the seed germination period, which is a critical period in plant growth. GmSRLK13 Effects of genes on Arabidopsis germination, WT and GmSRLK13 After sterilization and vernalization for 2 days, overexpressing Arabidopsis seeds were sown on 1 / 2 MS medium and then cultured in a light incubator. GmSRLK13 To investigate the function of the gene, the germination rates of the overexpression lines OE#1-11 and OE#6-9 were statistically analyzed under the same light cycle culture conditions. Figure 6 As shown in B, under salt stress conditions, GmSRLK13The seeds of the two overexpression lines were sown simultaneously with the WT on 1 / 2 MS medium treated with NaCl stress, and the germination rate and leaf expansion rate of the seeds were calculated within 9 days ( Figure 6 It was found that the germination of Arabidopsis seedlings of different strains was inhibited on 100 mM NaCl and 125 mM NaCl media, and GmSRLK13 The germination rate and leaf expansion rate of the overexpression strain Arabidopsis were significantly lower than those of WT. The difference in germination rate was most obvious on the 4th day. In 1 / 2 MS medium, both WT and overexpression Arabidopsis germinated completely. In 100 mM NaCl medium, the germination rate of WT was as high as 85%, while that of overexpression Arabidopsis was only 65%. In 125 mM NaCl medium, the germination rate of WT reached 75%, while that of overexpression Arabidopsis was only 30%. GmSRLK13 The difference in leaf expansion rate of overexpressed plants became more obvious with the increase of NaCl concentration. The difference in leaf expansion rate was most obvious on the 6th day in 100 mM NaCl medium, while the difference in leaf expansion rate was the largest on the 8th day in 125 mM NaCl medium. GmSRLK13 Overexpression of α-terminal locus reduces the tolerance of Arabidopsis to salt stress.

[0089] (2) GmSRLK13 Response of overexpressed Arabidopsis thaliana seedlings to NaCl

[0090] The seedling stage is still very important for the plant growth process. The nutrients accumulated during the seedling stage will directly affect the plant's ability to resist stress in the later stage. GmSRLK13 The root phenotype experiment under salt stress was conducted on the T3 generation of transgenic Arabidopsis. WT, OE#1-11, and OE#6-9 were sterilized and vernalized for 2 days and then inoculated into 1 / 2 MS medium for 2 days of germination. Seedlings with consistent growth conditions (root length about 1 cm) were selected and inoculated into 1 / 2 MS medium containing 0 mM NaCl, 100 mM NaCl, and 125 mM NaCl for vertical growth. The root growth of each strain of Arabidopsis was observed. Figure 7 As shown in Figure A, in 1 / 2 MS medium, there was no significant difference in the growth status between WT and the two overexpression lines. However, after salt stress treatment with 100 mM NaCl and 125 mM NaCl, GmSRLK13 The roots of transgenic Arabidopsis seedlings were significantly shortened, severely curled, and had poor growth. The elongation of the main root and the number of lateral roots were statistically analyzed ( Figure 7 (BG in the GmSRLK13 Compared with WT, the difference between the main root elongation speed and the number of lateral roots of overexpressed Arabidopsis and WT became more obvious with the increase of NaCl concentration and the number of days. GmSRLK13The root elongation rate of overexpressed Arabidopsis was significantly lower than that of WT. GmSRLK13 The root length of overexpressing Arabidopsis thaliana grown on different treatment media for 10 days was statistically analyzed ( Figure 7 The length of the primary root of overexpressing Arabidopsis thaliana was significantly shorter than that of the wild type, further explaining GmSRLK13 Overexpression leads to salt sensitivity in Arabidopsis.

[0091] (3) GmSRLK13 Detection of salt stress phenotypes and physiological indicators in overexpressed Arabidopsis thaliana seedlings

[0092] Found in the seedling stage GmSRLK13 Overexpression of Arabidopsis thaliana can lead to salt sensitivity. GmSRLK13 Are overexpressed Arabidopsis equally sensitive to salt stress during the seedling stage? GmSRLK13 Overexpressing Arabidopsis seedlings were treated with 175 mM NaCl for 5 days, and Arabidopsis seedlings grown under normal conditions were used as controls to observe the phenotypes. Figure 8 As shown in Figure A, the normally grown WT, OE#1-11, and OE#6-9 Arabidopsis all grew well, with no significant difference. After 5 days of NaCl treatment, the leaves of the overexpressing Arabidopsis showed more severe wilting and yellowing compared with the WT.

[0093] To verify GmSRLK13 Does overexpressing Arabidopsis thaliana eliminate hydrogen peroxide (H2O2) and superoxide anions (O) in the body through the ROS system in a timely manner under salt stress? 2- ) et al., used diaminobenzidine (DAB), tetrazolium blue chloride (NBT), and trypan blue staining to detect the degree of oxidative damage and the number of dead cells in Arabidopsis leaves. Hydrogen peroxide reacts rapidly with DAB under the catalysis of peroxidase to produce a brown compound, and the plant will be stained with DAB at the site where hydrogen peroxide is produced. 2- It can reduce NBT into a water-insoluble blue compound, thus making the oxidatively damaged tissues of plants appear dark blue. Figure 8 As shown in Figure B, the leaves of WT, OE#1-11 and OE#6-9 strains that were not treated with salt stress showed a light color in most areas after staining, even close to the color of the leaves themselves. However, in the leaves treated with salt stress, it can be seen that both WT and GmSRLK13 In overexpressed Arabidopsis, leaves were stained and the blue and brown colors were darker than those of untreated Arabidopsis leaves. GmSRLK13 Overexpression in Arabidopsis revealed that GmSRLK13 The dark blue and dark brown areas of the overexpressing Arabidopsis leaves were significantly more than those of the WT, indicating that under salt stress GmSRLK13The ROS level in overexpressed Arabidopsis was higher than that in WT. GmSRLK13 The number of dead cells in overexpressed Arabidopsis increased significantly compared to WT. POD and SOD can prevent the damage of reactive oxygen species and other peroxide free radicals to the membrane system, and CAT can decompose H2O2 produced by stress in plants, thereby reducing the accumulation of reactive oxygen species. Figure 8 As shown in CE, it can be seen that the activities of POD, SOD and CAT in the overexpressed Arabidopsis thaliana without salt treatment remained at normal levels and had no significant difference compared with WT. However, they all increased after salt stress treatment. GmSRLK13 The activities of POD, SOD and CAT in overexpressed Arabidopsis were significantly lower than those in WT. The MDA content reflects the degree of membrane lipid peroxidation. Malondialdehyde is one of the final products of membrane lipid peroxidation decomposition, and its content can reflect the degree of membrane lipid peroxidation. The accumulation of malondialdehyde in the body will also cause further damage to the cell membrane, so the MDA content can reflect the degree of aging and adverse damage suffered by the organism. Therefore, the MDA content was detected ( Figure 8 F in the figure), the MDA content increased after salt treatment, and GmSRLK13 The MDA content in overexpressed Arabidopsis was significantly higher than that in WT. GmSRLK13 Overexpression of α-aminobutyric acid aggravated the degree of oxidative damage in Arabidopsis and reduced the plant's tolerance to salt.

[0094] The chlorophyll content in plant leaves can often reflect the photosynthetic capacity of plant leaves and objectively reflect the strength of plant stress resistance. Therefore, the WT and GmSRLK13 Overexpression of chlorophyll content in Arabidopsis, e.g. Figure 8 The statistical analysis of G in the results showed that there was no significant difference in chlorophyll content between the overexpressed Arabidopsis and the WT without stress, but the chlorophyll content of the overexpressed Arabidopsis was much lower than that of the wild type after salt stress. + / K + It is an important indicator to measure the salt tolerance of plants. Excessive Na + / K + It will cause ion poisoning, affect various enzymatic reactions, and thus affect the normal growth of plants. Figure 8 As shown in H, after salt stress treatment, the Na + The contents of Na + The accumulation was much higher than that of WT; after salt stress treatment GmSRLK13 Overexpression of Arabidopsis Na + / K + The increase in the ratio further indicates that GmSRLK13 Overexpression of Arabidopsis thaliana is sensitive to salt stress.

[0095] (4)GmSRLK13 Analysis of phenotypes and physiological parameters of transgenic soybean hairy roots treated with salt

[0096] Heterologous expression in Arabidopsis GmSRLK13 With salt-sensitive phenotype, for further study GmSRLK13 function under salt stress in soybean, and also on the kinase domain GmSRLK13-KD To explore its function under salt stress conditions in soybean, the correct recombinant plasmid pCAM35S- GmSRLK13 -GFP, pCAM35S- GmSRLK13-KD -GFP was transformed into Agrobacterium rhizogenes K599 to obtain the recombinant plasmid pCAM35S- GmSRLK13 -GFP, pCAM35S- GmSRLK13-KD -GFP Agrobacterium.

[0097] Soybean hairy roots were induced by Agrobacterium-mediated genetic transformation, and the pCAM35S- GmSRLK13 -GFP and pCAM35S- GmSRLK13-KD -GFP-expressing Agrobacterium rhizogenes K599 and K599 empty bacteria were applied to the wound after soybean hypocotyl cutting to induce soybean hairy roots.

[0098] To detect GmSRLK13, GmSRLK13-KD To determine whether the gene is normally expressed in soybean hairy roots, GUS staining was performed at the early growth stage of soybean hairy roots, and fluorescence microscopy was performed on the root tips of the hairy roots using the GFP tag fused to the target gene in the plant expression vector ( Figure 9 A in Figure 9 B in GmSRLK13, GmSRLK13-KD The green fluorescence observed in transgenic soybean hairy roots indicated that GmSRLK13, GmSRLK13-KD and GFP fusion proteins were successfully expressed in soybean hairy roots.

[0099] The transgenic K599 soybean hairy roots were used as the control. GmSRLK13, GmSRLK13-KD Transgenic soybean hairy root chimera plants were treated with 175 mM NaCl in water for 24 h. Phenotypes were observed, photographed, and samples collected every 12 h. Figure 10 As shown in A and B, GmSRLK13, GmSRLK13-KD Compared with K599 empty bacteria plants, the transgenic plants GmSRLK13, GmSRLK13-KD The transgenic plants were significantly more severely damaged, with a higher degree of leaf wilting and darker leaf color, and showed greater salt sensitivity in the early stages of salt stress. The MDA content in soybean leaves of transgenic soybean hairy root chimeras was measured before and after salt treatment. The results are as follows Figure 10As shown in C, before salt treatment, K599 empty bacteria and GmSRLK13, GmSRLK13-KD There was no significant difference in MDA content among transgenic hairy root chimeras, but after salt treatment, GmSRLK13, GmSRLK13- KD The MDA content of transgenic soybean hairy root chimeras was significantly increased, indicating that GmSRLK13, GmSRLK13-KD The cell membrane of transgenic soybean hairy root chimera plants was severely damaged. GmSRLK13, GmSRLK13-KD SOD, POD, and CAT in transgenic soybean hairy roots were analyzed ( Figure 10 DF in the figure), the results showed that under normal conditions, K599 empty bacteria plants and GmSRLK13, GmSRLK13-KD There was no significant difference in the transgenic plants. After salt treatment, K599, GmSRLK13, GmSRLK13-KD The activities of POD, SOD and CAT in transgenic hairy roots were increased. GmSRLK13, GmSRLK13-KD The activities of SOD, POD and CAT in transgenic plants were lower than those in K599 empty bacteria plants, indicating that the transgenic plants GmSRLK13, GmSRLK13-KD The gene increases the level of oxidative damage in plants, thereby reducing their ability to tolerate salt.

[0100] Example 4. Screening of GmOTSa and GmOTSa(C486S)-overexpressing Arabidopsis

[0101] Construction of overexpression vectors of GmOTSa and GmOTSa(C486S)

[0102] (1) GmOTSa Preparation of linearized fragments

[0103] Vazyme high-fidelity enzyme was used to amplify the target fragment, and primers 35S-GmOTSa-GFP-F (GAGCTCGGTACCCGGGGATCCATGGAGGAACAACAA, SEQ ID NO. 21) and 35S-GmOTSa-GFP-R (GGTGTCGACTCTAGAGGATCCCGTCACAGAATCCTG, SEQ ID NO. 22) were used to amplify GmOTSa.

[0104] (2) Mutation of the active site of the target gene

[0105] The insert fragment GmOTSa(C486S)-N was amplified using primers GmOTSa-32a-S (ATCGGATCCGAATTCGAGCTCATGGAGGAACAACAACAACA, SEQ ID NO. 23) and SER-S2 (AAGAATGAATATGACTCTGGTCTTTTTGTATTG, SEQ ID NO. 24), and the insert fragment GmOTSa(C486S)-C was amplified using primers SER-S1 (TCTGGTCTTTTTGTATTG, SEQ ID NO. 25) and GmOTSa-32a-AS (CTCGAGTGCGGCCGCAAGCTTTTACGTCACAGAATCCTGGG, SEQ ID NO. 26). The fragments were ligated with the plant expression vector pCAM35S-eGFP to obtain the plant expression vector pCAM35S- GmOTSa(C486S) -GFP recombinant vector; the obtained GmOTSa gene sequence was connected with pCAM35S-eGFP to obtain the plant expression vector pCAM35S- GmOTSa -GFP recombinant vector, and transform the above two recombinant vectors into Arabidopsis and soybean to obtain transgenic materials.

[0106] Results: GmOTSa is a SUMO protease with deSUMOylation function, and GmOTSa (C486S) is a mutant form of the active site of GmOTSa. GmOTSa To investigate the tolerance of CAM35S to salt stress and whether the active site mutation affects its physiological function, a plant expression vector pCAM35S- GmOTSa -GFP, pCAM35S- GmOTSa(C486S) -GFP. First, 35S -GmOTSa -GFP-F, 35S -GmOTSa -GFP-R was used to amplify the target fragment by PCR, and then the target fragment was connected to the vector pCAM35S-GFP after enzyme digestion by homologous recombination. After transformation into DH5α, PCR identification was performed, and the amplified target fragments were all about 1800 bp, indicating that the plant expression vector pCAM35S- GmOTSa -GFP, pCAM35S- GmOTSa(C486S) -GFP was successfully constructed. Then the constructed pCAM35S- GmOTSa -GFP, pCAM35S- GmOTSa(C486S) -GFP was transformed into Agrobacterium GV3101, which was then infected into Arabidopsis thaliana by the floral dip method, and transgenic positive Arabidopsis thaliana were screened on the culture medium.

[0107] The seedlings that can grow normally in the medium containing 25 mg / L hygromycin resistance were transplanted into nutrient soil for further growth. -GmOTSa -GFP-F, 35S -GmOTSa -GFP-R was used for PCR detection of T1 generation overexpressing Arabidopsis, with WT Arabidopsis as negative control and the plasmid of the recombinant vector as positive control. Figure 11 As shown, PCR identification results showed that 3 strains were pCAM35S- GmOTSa -GFP overexpressing Arabidopsis positive lines, denoted as OE#1~3; 4 lines were pCAM35S- GmOTSa(C486S) -GFP overexpressing Arabidopsis positive lines were recorded as OE#1~4. The culture was continued until harvest, and the harvested T2 generation seeds were used for research GmOTSa 、 GmOTSa(C486S) Phenotypic and physiological index determination of overexpressing Arabidopsis lines under salt treatment.

[0108] Example 5. Response of GmOTSa and GmOTSa(C486S)-overexpressing Arabidopsis to NaCl

[0109] one, GmOTSa as well as GmOTSa(C486S) Response of overexpressed Arabidopsis to NaCl during germination

[0110] Under the same conditions, Arabidopsis thaliana overexpressing GmSRLK13 OE#6-9 was used as control and detection GmOTSa, GmOTSa (C486S) Effects of genes on Arabidopsis germination under salt stress conditions. GmSRLK13 Overexpression Arabidopsis line OE#6-9 and T2 generation GmOTSa OE#1 , GmOTSa(C486S) The OE#1 overexpressing Arabidopsis strain was sterilized and vernalized for 2 days before being sown on 1 / 2 MS medium and then cultured in a light incubator. GmSRLK13, GmOTSa, GmOTSa(C486S) The germination rate of overexpressed Arabidopsis was counted. GmSRLK13 OE#6-9 、 GmOTSa OE#1 , GmOTSa(C486S) The seeds of OE#1 overexpressing strain were sown simultaneously with WT on 1 / 2MS medium containing different concentrations of NaCl. The germination rate and green leaf rate of the seeds were calculated within 9 days. Figure 12 As shown in Figure 2, it was found that the germination of Arabidopsis seedlings with different overexpression genes was inhibited in 100 mM NaCl and 125 mM NaCl media, but GmSRLK13 The germination rate and green leaf rate of overexpressed Arabidopsis were significantly lower than those of WT.GmOTSa, GmOTSa(C486S) The germination rate and green leaf rate of overexpressed Arabidopsis were significantly higher than those of WT. The phenotype was most obvious on the 4th day of germination under 125 mM NaCl, and this trend became more obvious as the NaCl concentration increased. GmOTSa and GmOTSa(C486S) The overexpression of Arabidopsis thaliana showed the same phenotypic trend during the germination period and showed GmOTSa(C486S) The green leaf rate of overexpressed Arabidopsis was slightly lower than GmOTSa Overexpression in Arabidopsis thaliana showed that the GmOTSa active site mutation did not significantly affect the physiological function of the plant in the overall trend. GmSRLK13 Overexpression of α significantly reduced the tolerance of Arabidopsis to NaCl treatment. GmOTSa, GmOTSa(C486S) Overexpression of α-amylase significantly improved the tolerance of Arabidopsis thaliana to NaCl treatment.

[0111] II. GmOTSa as well as GmOTSa(C486S) Response of overexpressed Arabidopsis thaliana seedlings to NaCl

[0112] Next, we overexpressed Arabidopsis GmSRLK13 OE#6-9 are control pairs GmOTSa, GmOTSa(C486S) Phenotypic experiments on root growth under salt stress were conducted at the seedling stage of overexpressing Arabidopsis thaliana. GmSRLK13 OE#6-9 , GmOTSa OE#1 、 GmOTSa(C486S) After sterilization and vernalization for 2 days, Arabidopsis seeds overexpressing OE#1 were transferred to 1 / 2 MS medium for germination for 1 day. Seedlings with consistent germination conditions were selected and inoculated into 1 / 2 MS medium containing 0 mM NaCl, 100 mM NaCl, and 125 mM NaCl for salt stress treatment. 1 / 2 MS was used as a blank control to observe the root growth of Arabidopsis overexpressing different genes. Figure 13 As shown in A, in 1 / 2 MS medium, WT and GmSRLK13, GmOTSa, GmOTSa(C486S) There was no significant difference in root length between the overexpression lines, and after 6 days of NaCl treatment, GmSRLK13 The root length of overexpressing Arabidopsis seedlings was significantly shortened, the leaves were curled, and the growth was poor; GmOTSa, GmOTSa(C486S) The roots of overexpressing Arabidopsis seedlings were longer and the curling of leaves was lower. The main root length was measured and statistically analyzed, such as Figure 13 As shown in BD, GmSRLK13 The main root length of overexpressing Arabidopsis was significantly shorter than that of WT. GmOTSa, GmOTSa(C486S) The main root length of overexpressed Arabidopsis was significantly longer than that of WT. GmOTSa The main root length of overexpressing Arabidopsis is slightly longer than GmOTSa(C486S) Overexpression in Arabidopsis thaliana. GmSRLK13Overexpression of Arabidopsis thaliana resulted in salt sensitivity compared to GmOTSa, GmOTSa(C486S) Overexpression can enhance salt tolerance in Arabidopsis.

[0113] 3. Phenotype and physiological index detection of GmOTSa and GmOTSa(C486S) overexpressing Arabidopsis thaliana during salt treatment at the seedling stage

[0114] according to GmSRLK13, GmOTSa Phenotypic analysis of Arabidopsis thaliana seedlings after salt stress treatment revealed GmSRLK13 and GmOTSa Under salt stress conditions, the phenotype was opposite. To further verify this result, 21-day-old WT and GmSRLK13 OE#6-9 , GmOTSa OE#1 , GmOTSa(C486S) OE#1 overexpressing Arabidopsis seedlings were treated with 175 mM NaCl for 8 days, and normal watering was used as a control to observe the phenotype. Figure 14 As shown in A, without treatment, WT, GmSRLK13, GmOTSa, GmOTSa(C486S) All Arabidopsis plants grew well, with no significant difference; after 8 days of treatment with 175 mM NaCl, GmSRLK13 Compared with WT, the leaves of overexpressing Arabidopsis thaliana were wrinkled, wilted and yellowed seriously; GmOTSa, GmOTSa(C486S) Compared with WT, the growth state of overexpressed Arabidopsis plants was significantly stronger and the degree of leaf yellowing was lower. GmSRLK13 Overexpressing Arabidopsis is salt sensitive; GmOTSa, GmOTSa(C486S) Overexpression of Arabidopsis can improve plant salt tolerance. Then, with no treatment as the control, the physiological indicators MDA, chlorophyll content, SOD, and POD activity of Arabidopsis leaves after salt stress treatment were measured. Under the condition of no treatment, there was no significant difference in MDA, chlorophyll content, SOD, and POD activity between WT and different overexpressed Arabidopsis; however, the activity of SOD and POD increased after salt treatment, the content of MDA increased, and the chlorophyll content decreased significantly. GmSRLK13 The MDA and content in overexpressed Arabidopsis were much higher than those in WT, and the activities of SOD and POD and the chlorophyll content were significantly reduced compared with those in WT; GmOTSa, GmOTSa(C486S) The MDA content of overexpressed Arabidopsis was lower than that of WT, while the activities of SOD and POD and the chlorophyll content were significantly higher than those of WT. GmSRLK13 Overexpression of the gene in Arabidopsis thaliana makes it sensitive to high salt. GmOTSa Genes improve plant salt tolerance and GmOTSa The active sites did not affect its salt tolerance.

[0115] Example 6. GmSRLK13 and GmOTSa Screening of co-expression Arabidopsis

[0116] 1. Proven through preliminary experimentsGmSRLK13 The tolerance of overexpressing Arabidopsis to salt stress was reduced. GmOTSa Overexpression of Arabidopsis thaliana will make it salt resistant. GmOTSa -3×FLAG (pFGC5941 plasmid inserted GmOTSa Gene sequence) plasmid Agrobacterium GV3101 infection stable inheritance T3 generation GmSRLK13 OE#6-9 overexpressed Arabidopsis. The infected seeds were first screened in 1 / 2 MS medium containing 25 mg / L hygromycin. After 10 days, the well-grown Arabidopsis were transplanted into moist nutrient soil (soil: vermiculite = 1:2). When they grew to the four-leaf stage, positive Arabidopsis were selected by spraying basta (1:2000).

[0117] The leaves of overexpressed Arabidopsis thaliana that were still able to grow normally were collected and the crude DNA was used as a template. Bar -F and Bar -R, HYG -F and HYG -R,35S- GmOTSa -GFP-F and 35S- GmOTSa -GFP-R,35S- GmSRLK13 -GFP-F and 35S- GmSRLK13 -GFP-R for T1 generation GmSRLK13 and GmOTSa Co-expression of Arabidopsis was positively detected, and the PCR identification results were as follows Figure 15 , a total of 4 positive strains were detected and recorded as GmSRLK13GmOTSa OE#1-4. Continue to cultivate until harvesting T2 generation seeds for research GmSRLK13 and GmOTSa Phenotypes of co-expressing Arabidopsis thaliana after salt treatment.

[0118] 2. GmSRLK13 and GmOTSa Response of co-expressing Arabidopsis seedlings to NaCl

[0119] Through GmSRLK13 Overexpression of Arabidopsis GmOTSa Analysis of phenotypic and physiological indicators of overexpressed Arabidopsis under salt stress revealed that GmSRLK13 and GmOTSa Overexpression of these two genes leads to opposite salt resistance phenotypes in plants. To explore the response of co-expression of these two genes to salt stress, we first GmSRLK13 OE#6-9 , GmOTSa OE#1 , GmSRLK13 GmOTSa OE#4 overexpressing Arabidopsis seeds were sterilized and vernalized for 2 days before being sown on 1 / 2 MS medium containing 100 mM NaCl and 125 mM NaCl respectively.GmSRLK13 OE#6-9 , GmOTSa OE#1 , GmSRLK13 GmOTSa The germination rate of OE#4 overexpressed Arabidopsis was counted. GmSRLK13 OE#6-9 , GmOTSa OE#1 、 GmSRLK13 GmOTSa OE#4 overexpressing Arabidopsis seeds and WT were cultured on 1 / 2 MS medium containing different concentrations of NaCl, and the germination rate and green leaf rate of the seeds were counted within 9 days. Figure 16 As shown in Figure 2, the germination of Arabidopsis seedlings with different overexpression genes was inhibited to varying degrees in 100 mM NaCl and 125 mM NaCl media. GmSRLK13 The germination rate and green leaf rate of overexpressed Arabidopsis were significantly lower than those of WT. GmOTSa The germination rate and green leaf rate of Arabidopsis thaliana overexpressing GmSRLK13 and GmOTSa There was no significant difference in the germination rate of co-expressed Arabidopsis compared to the WT. The green leaf rate statistics were most obvious at 100 mM NaCl and 125 mM NaCl, and this trend became more obvious as the NaCl concentration increased. The above results show that GmSRLK13 Overexpression of GmOTSa Overexpression of increased the tolerance of Arabidopsis to NaCl treatment; GmSRLK13 and GmOTSa Co-expression in Arabidopsis thaliana, under salt stress conditions, GmOTSa Positive regulation can offset GmSRLK13 Negative regulatory effects under salt stress.

[0120] 3. GmSRLK13 and GmOTSa Salt treatment phenotype and physiological index detection of co-expressed Arabidopsis thaliana seedlings

[0121] according to GmSRLK13 and GmOTSa Phenotypic analysis of co-expressed Arabidopsis thaliana seedlings after salt stress treatment revealed GmSRLK13 and GmOTSa The phenotype of co-expressed Arabidopsis thaliana under salt stress was similar to that of WT. To further verify this result, 21-day-old WT and GmSRLK13 OE#6-9 , GmOTSa OE#1 ​ OE#4 overexpressing Arabidopsis seedlings were treated with 175 mM NaCl for 5 days, and normal watering was used as a control to observe the phenotype. ​ As shown in A, in the untreated case, WT, ​ OE#6-9 ​OE#1 ​ All Arabidopsis plants overexpressing OE#4 grew well, with no significant difference; after 5 days of treatment with 175 mM NaCl, ​ Compared with WT, the leaves of overexpressing Arabidopsis thaliana were wrinkled, wilted, and severely yellowed; ​ The growth state of overexpressing Arabidopsis plants was significantly more robust, and the degree of leaf yellowing was less; ​ and ​ Compared with WT, the co-expressing Arabidopsis had similar leaf chlorosis and similar growth status. ​ Overexpression of Arabidopsis thaliana is salt sensitive; ​ Overexpression of Arabidopsis thaliana can improve plant salt tolerance and ​ Can eliminate ​ The effect of overexpression on salt sensitivity of Arabidopsis thaliana. Then, with normal growth conditions as the control, the physiological indicators MDA and chlorophyll content of Arabidopsis thaliana leaves after salt stress treatment were measured, such as ​ As shown in Figures B and C, there was no significant difference in the MDA and chlorophyll content between WT and different overexpressed Arabidopsis plants without treatment; however, the MDA content increased and the chlorophyll content decreased significantly after salt treatment. ​ The MDA and content in overexpressed Arabidopsis were much higher than those in WT, and the chlorophyll content was significantly reduced compared with WT. ​ The MDA content of overexpressed Arabidopsis was lower than that of WT, and the chlorophyll content was significantly higher than that of WT; ​ and ​ The MDA and chlorophyll contents in the co-expressed Arabidopsis thaliana were basically the same as those in the WT. ​ Overexpression of the gene in Arabidopsis thaliana makes it sensitive to high salt. ​ Genes improve plant salt tolerance and ​ Able to recover ​ Overexpression of Arabidopsis thaliana results in salt-sensitive phenotypes.

[0122] 4. ​ and ​ Changes in protein levels after salt treatment in co-expressed Arabidopsis

[0123] Through ​ and ​ Phenotypic analysis of co-expressed Arabidopsis thaliana after salt stress treatment showed that it was similar to WT. ​ Overexpression of Arabidopsis thaliana showed salt-sensitive phenotype and no ​ Overexpression of Arabidopsis thaliana salt resistance phenotype. SUMOylation in plants can respond to environmental stress by changing protein function. Previous experiments have verified that SUMO protease GmOTSa has a deSUMO function. Therefore, in protein level analysis ​ and ​The stability and SUMOylation level of co-expressed Arabidopsis thaliana proteins were investigated under salt stress. ​ Overexpression of Arabidopsis ​ and ​ Co-expressed Arabidopsis was cultured in 175 mM NaCl for 24 h. Arabidopsis samples were collected from normal growth and salt-stressed Arabidopsis plants and the Arabidopsis protein was extracted using the TCA / acetone method. The protein loading was first adjusted using an Actin antibody, and then Western Blot verification was performed using FLAG and SUMO antibodies. The results are shown in Figure 2. ​ , the expression of GmOTSa increased after salt stress treatment; and the SUMOylation level of GmSRLK13 increased after salt stress, while ​ and ​ After co-expression of Arabidopsis thaliana under salt stress, SUMO conjugates decreased and free SUMO increased, indicating that GmOTSa deSUMOylated GmSRLK13 under salt stress.

[0124] Example 7. Verification of the interaction between GmSRLK13 and GmOTSa

[0125] 1. Construction of vector: The candidate interacting protein GmSRLK13 was obtained through the SUMO protease GmOTSa yeast two-hybrid screening library. ​ Sequence analysis revealed a conserved kinase domain and named it ​ In order to further verify the interaction between GmSRLK13 and GmOTSa and to explore whether the kinase domain GmSRLK13-KD also interacts with GmOTSa, the plant expression vector p2300 was constructed. ​ -CeGFP, p2300- ​ -CeGFP. First, ​ The full-length gene was used as a template and primers P2300- ​ -F and P2300- ​ -R, P2300- ​ -F and P2300- ​ -R was used for PCR amplification to obtain the target band, and then restriction endonucleases were used to ​ The plant expression vector p2300-CeGFP was digested, recovered and identified by R Ⅰ, and the plant expression vector p2300-CeGFP was transformed into ​ The target fragment was connected with the p2300-CeGFP expression vector and transformed into Escherichia coli DH5α; the recombinant plasmid was used as a template for PCR detection to obtain positive transformants and sequence them; the correctly constructed recombinant plasmid pCAMBIA2300- ​ -CeGFP, pCAMBIA2300- ​-CeGFP was transformed into Agrobacterium tumefaciens GV3101 to obtain BiFC recombinant vector (such as ​ 、 20 shown), for transient transformation in onion inner epidermis, P2300- ​ -CeGFP-F: GCCATGGAGGCCAGTGAATTCATGCATTTAAGTACT, SEQ ID NO. 27; P2300- ​ -CeGFP-R:ATGCCCACCCGGGTGGAATTCACGAGGGAGCACATT, SEQ ID NO. 28; P2300- ​ -CeGFP-F: GCCATGGAGGCCAGTGAATTCATGTTCAGAACCAGC, SEQ ID NO. 29; P2300- ​ -CeGFP-R:ATGCCCACCCGGGTGGAATTCACGAGGGAGCACATT, SEQ ID NO. 30.

[0126] 2. BiFC Verification of Protein Interactions

[0127] The GFP fluorescence signal was observed under a laser confocal microscope. ​ , in p2300-CeGFP+p2300-NeGFP, p2300-CeGFP+p2300- ​ -NeGFP and p2300-NeGFP+p2300- ​ -CeGFP, p2300-NeGFP+p2300- ​ -CeGFP empty load control under the microscope did not observe the fluorescence signal, through the detection found that in p2300- ​ -CeGFP+p2300- ​ -NeGFP, p2300- ​ -CeGFP+p2300- ​ -NeGFP can observe GFP green fluorescence, such as ​ As shown in the figure, the interaction signals between GmSRLK13 and GmOTSa are distributed in the cytoplasm and cell membrane, while the interaction signals between GmSRLK13-KD and GmOTSa are mainly distributed in the cytoplasm and cell nucleus, indicating that both GmSRLK13 and GmSRLK13-KD have an interaction relationship with GmOTSa.

[0128] 3. GST Pull-down Verification of Protein Interaction

[0129] The target fragment is connected with the prokaryotic expression vector using the plasmid containing the target gene fragment as a template. ​ -F: GCTGATATCGGATCCGAATTCATGTTCAGAACCAGC (SEQ ID NO. 31); 32a- ​ -R:TTGTCGACGGAGCTCGAATTCACGAGGGAGCACATT (SEQ ID NO.32), recovered ​ -KD gene fragment, the plasmid target gene fragment was ligated with the cut plasmid using homologous recombination. After ligation at 37°C for 30 minutes, the plasmid was transformed into competent E. coli DH5α. Single colonies were picked for PCR identification, and the PCR-positive bacterial liquid was sent for sequencing.

[0130] Positive PCR detection of transformants: ligated and transformed E. coli DH5α, cultured overnight on LB solid medium containing the corresponding resistance, picked up a smooth single colony with a sterilized 10 μL pipette tip and shaken overnight, using this bacterial solution as a template, PCR amplification using gene-specific primers, and extracted plasmids from the correctly sequenced E. coli bacterial solution, and transformed into E. coli BL21 ​ (DE3) and Agrobacterium GV3101 and K599 were used for the next experiments.

[0131] First, construct the prokaryotic expression vector pET32a- ​ However, the expression of GmSRLK13 protein was not induced successfully. Therefore, the prokaryotic expression vector pET32a- ​ .like ​ As shown, first ​ The full-length gene was used as a template and primers 32a- GmSRLK13-KD -F, 32a- GmSRLK13-KD -R was amplified by PCR to obtain GmSRLK13-KD The target band was then cleaved with restriction endonucleases Eco R Ⅰ digested, recovered and identified the prokaryotic expression vector pET32a, and constructed the expression vector pET32a- GmSRLK13-KD , the correct recombinant plasmid pET32a- GmSRLK-KD and pEGX4T-1- GmOTSa (Preserved in the laboratory) were transformed into Escherichia coli BL21 for low temperature induction expression to obtain GmSRLK-KD and GmOTSa proteins. Meanwhile, His tag protein purification and GST tag purification were performed on GmSRLK13-KD-His and GST-GmOTSa, respectively. Figure 23As shown in the figure, the fusion proteins GmSRLK13-KD-His and GST-GmOTSa were observed at 57 kDa and 95 kDa, respectively. The GST pull-down experiment verified that the two proteins interacted with each other.

[0132] 4. Interaction between GmSRLK13 and SUMO molecules

[0133] In the early stage, BiFC and GST pull-down experiments have verified that GmSRLK13 interacts with GmOTSa. OTSa, as a SUMO protease, is a type of protease involved in the deSUMOylation of SUMOylated proteins during post-translational modification. Prediction analysis has found that the sites where GmSRLK13 can undergo SUMOylation are mainly concentrated in the kinase domain GmSRLK13-KD. Therefore, in order to verify the relationship between GmSRLK13 and SUMO molecules, the plant expression vector pCAM35S- GmSRLK13-KD -GFP and the plant expression vector pFGC5941-3×FLAG- GmSUMO2GG and the control vector pFGC5941-3×FLAG- GmSUMO2AA Transform tobacco into pCAM35S- GmSRLK13-KD -EGFP and pFGC5941-3×FLAG- GmSUMO2GG , and its control pFGC5941-3×FLAG- GmSUMO2AA Agrobacterium GV3101 was transformed and transformed into Nicotiana benthamiana leaves for transient expression. Total tobacco protein was extracted and Western Blot detection was performed using GFP antibody. Figure 24 In the input group, a signal consistent with the GFP-tagged GmSRLK13-KD was seen at 64 kDa, and the FLAG-tagged GmSUMO2GG and its control group GmSUMO2AA were successfully detected at 25 kDa using a FLAG antibody. These results indicate that the target proteins GmSRLK13-KD and GmSUMO2GG or GmSUMO2AA have been successfully expressed in tobacco leaves.

[0134] pCAM35S- GmSRLK13-KD -GFP were respectively combined with pFGC5941-3×FLAG- GmSUMO2GG and pFGC5941-3×FLAG- GmSUMO2AA Co-expressed in tobacco leaves, after protein extraction, the co-expressed protein was purified using GFP magnetic beads and detected by Western Blot using GFP antibody, such as Figure 24 IP group, pCAM35S- GmSRLK13-KD-GFP+pFGC5941-3×FLAG- GmSUMO2GG Compared with the control group pCAM35S- GmSRLK13-KD -GFP+pFGC5941-3×FLAG- GmSUMO2AA The successful expression of GmSRLK13-KD can be detected; Figure 24 In the co-IP group, simultaneous detection with a FLAG antibody revealed that GmSUMO2GG, fused to the FLAG tag, was successfully detected at 25 kDa in samples transiently co-expressing GmSRLK13-KD and GmSUMO2GG. In the control group transiently co-expressing GmSRLK13-KD and GmSUMO2AA, a GmSUMO2AA signal was also detected at the same position, but weaker. This suggests that GmSRLK13-KD not only interacts with SUMO in tobacco but may also undergo SUMOylation, which requires further verification in vitro.

Claims

1. Application of GmSRLK13 protein in improving the salt stress sensitivity of plants, characterized in that: The amino acid sequence of the GmSRLK13 protein is shown in SEQ ID NO.6 or SEQ ID NO.12; and the plant is soybean or Arabidopsis thaliana.

2. Application of the GmSRLK13 gene in improving the salt stress sensitivity of plants, characterized in that: The GmSRLK13 gene is shown as SEQ ID NO.5 or SEQ ID NO.11; and the plant is soybean or Arabidopsis thaliana.

3. The use according to claim 1 or 2, characterized in that The concentration of salt applied in salt stress resistance was 100-175 mM.

4. A method for cultivating plants sensitive to salt stress, characterized in that: The specific steps of the method are as follows: (1) amplifying the GmSRLK13 gene and inserting the gene sequence into a plant overexpression vector; the GmSRLK13 gene is shown in SEQ ID NO.5 or SEQ ID NO.11; (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transform into Arabidopsis thaliana or soybean to obtain a transgenic plant; (3) Identify the transgenic soybeans obtained in step (2) to obtain positive transgenic plants.

5. The method according to claim 4, characterized in that The expression vector in step (1) is pCAM35S; the primers for amplifying the GmSRLK13 gene are SEQ ID NO.1 and SEQ ID NO.

2.

6. A method for improving plant salt stress sensitivity, characterized in that: A transgenic plant overexpressing the GmSRLK13 gene is placed in a 100-175 mM sodium chloride environment for stress treatment; the GmSRLK13 gene is shown in SEQ ID NO.5 or SEQ ID NO.11; and the plant is soybean or Arabidopsis thaliana.

7. A gene for improving the salt stress sensitivity of a plant, characterized in that: The gene sequence is shown as SEQ ID NO.5 or SEQ ID NO.11; the plant is soybean or Arabidopsis thaliana.

8. Use of a plant overexpressing the GmSRLK13 gene, a recombinant vector containing the GmSRLK13 gene, or a recombinant microbial cell containing the GmSRLK13 gene in improving plant salt stress sensitivity; the plant is soybean or Arabidopsis thaliana; the GmSRLK13 gene is shown in SEQ ID NO.5 or SEQ ID NO.11.

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