Soybean casein kinase i gene gmSAL13 and application thereof

Abstract

This invention belongs to the field of plant genetics and genetic engineering technology, specifically relating to the soybean casein kinase I gene. GmSAL13 Its applications. This invention isolates the casein kinase I gene from soybean Williams 82. GmSAL13 ,Will GmSAL13 When the gene was expressed in the hairy roots of Arabidopsis and soybean, the germination rate of transgenic Arabidopsis seeds under salt stress was higher than that of the control, and the salt tolerance of transgenic soybean plants overexpressing the gene in their hairy roots was higher than that of the control plants. Simultaneously, this invention identified the gene in natural soybean populations... GmSAL13 The four haplotypes were analyzed, and significant differences were found in their salt tolerance, primarily manifested in differences in seedling emergence rates in saline-alkali soils. This fully demonstrates... GmSAL13 GmSAL13 Genes play an important role in the regulation of plant salt tolerance and can be used to regulate plant salt tolerance, thus having good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetics and genetic engineering technology, specifically relating to the soybean casein kinase I gene. GmSAL13 And its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Plants are frequently exposed to various environmental stresses in the natural environment, such as drought, extreme temperatures, nutrient deficiencies, and pests and diseases. Both biotic and abiotic stresses adversely affect the growth and development of plants facing these risks. To counteract these adversities, plants have developed a variety of stimulation and activation strategies.

[0004] Phosphorylation of serine, threonine, and tyrosine residues by cellular protein kinases plays a crucial role in the regulation of various cellular processes. Casein kinase 1 (CK1), a member of the serine / threonine protein kinase superfamily, is ubiquitous in eukaryotes. Previous studies have shown that CK1 plays a key role in fundamental eukaryotic cellular processes and signal transduction, including the regulation of the cell cycle, circadian rhythms, vesicle transport, DNA repair, and hormone signaling. In plants, CK1 family genes have also been reported to participate in developmental regulation and abiotic stress responses, but their functions vary across species, and functional annotations for specific family members remain incomplete.

[0005] Salt stress is one of the major abiotic stress factors limiting plant growth and crop yield. Soil salinization is becoming increasingly serious globally, posing a persistent threat to agricultural production. Soybean ( Glycine max As an important oilseed and food crop worldwide, soybean is highly sensitive to salt stress, and yield losses are severe under saline-alkali soil cultivation conditions. Therefore, exploring the salt tolerance genetic resources of soybean itself and elucidating its salt tolerance molecular mechanisms are of great significance for the breeding of salt-tolerant soybean varieties. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a soybean casein kinase I gene. GmSAL13 And its applications. Specifically, this invention isolated the casein kinase I gene from soybean Williams 82— GmSAL13The gene locus number of this gene is Glyma.06G087500, and the reference genome annotation version is Glycine maxWm82.a2.v1 (G.max Wm82.a2.v1: Phytozome). Experiments have demonstrated that it plays an important role in the regulation of plant salt tolerance. Furthermore, this invention identified [a specific gene] in natural soybean populations. GmSAL13 Four haplotypes were analyzed, and significant differences were found in their emergence rates in saline-alkali land over multiple years. Additionally, Arabidopsis thaliana... GmSAL13 The germination rate of overexpressing plants under 100 salt conditions was higher than that of the control. GmSAL13 Overexpression of hairy-root soybean plants enhances salt tolerance, while RNAi-treated soybeans show decreased salt tolerance, fully demonstrating the effectiveness of the Williams82 casein kinase I gene. GmSAL13 It plays an important role in regulating plant salt tolerance and can be used for plant salt tolerance regulation, thus having good practical application value. Based on the above research results, this invention is thus completed.

[0007] The present invention adopts the following technical solution: The first aspect of the present invention provides GmSAL13 Genes or containing GmSAL13 Application of genetic biomaterials in at least one of the following: (a1) Regulation of plant salt tolerance; (a2) To improve and cultivate plants; The nucleotide sequence of the GmSAL13 gene is selected from: (b1) The nucleotide sequence as shown in SEQ ID NO.1; (b2) and (b1) are nucleotide sequences that encode proteins with the same amino acid sequence, but are different in sequence due to the degeneracy of the genetic code; (b3) A nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes a nucleotide sequence that has the same or similar functional protein; (b4) is a complementary nucleotide sequence to any one of (b1)-(b3).

[0008] A second aspect of the present invention is an agent for improving plant alkaloid tolerance, comprising at least one of the following: (d1) contains GmSAL13 Gene expression vectors; (d2) contains a recombinant host of (d1); (d3) Enhancement GmSAL13 Promoters or enhancers of gene expression; (d4) Promote GmSAL13 Gene expression inducers; The GmSAL13The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] A third aspect of the present invention provides a method for improving the salt tolerance of plants, using the formulation described in the second aspect to improve the salt tolerance of plants. GmSAL13 Gene expression levels and / or activity, or making those without gene expression levels... GmSAL13 Gene or GmSAL13 The preparation comprises a nucleic acid molecule encoding the GmSAL13 protein; the amino acid sequence of the GmSAL13 protein is shown in SEQ ID NO.3; the plant is soybean.

[0010] A fourth aspect of the present invention provides a method for improving and cultivating salt-tolerant plants, comprising increasing the salt content of the target plant. GmSAL13 Gene expression levels and / or activity.

[0011] Compared with the prior art, the beneficial effects of the present invention are: This invention isolates and identifies the casein kinase I family gene, as shown in SEQ ID NO.1, from soybean Williams 82. GmSAL13 Experiments have shown that... GmSAL13 Overexpression of this gene significantly increased the germination rate of Arabidopsis thaliana and the fresh weight of soybean plants under salt stress, and reduced plant wilting. However, inhibiting gene expression through RNA interference led to increased plant sensitivity to salt stress, indicating that... GmSAL13 Genes can positively regulate the salt tolerance of plants. GmSAL14 Hap002 Soybeans have a high germination rate in saline-alkali soil. GmSAL14 Hap001 Conversely, this invention provides new genetic resources for improving plant salt tolerance. GmSAL13 The application of genes or their biological materials to the regulation of plant salt tolerance and the breeding of improved varieties can effectively enhance the salt tolerance of plants. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Picture 1 The plant expression vector in the embodiments of the present invention pTF101 Atlas; Picture 2 This is the entry carrier in the embodiments of the present invention. pDONR221 Atlas; Picture 3 Interference carrier in the embodiments of the present invention pB7GWIWGII Atlas; Picture 4 The Williams 82 gene in the embodiments of the present invention GmSAL13 The full-length CDS clone electrophoresis image was obtained by cloning soybean Williams 82 cDNA using it as a template. Picture 5 This invention provides an example of PCR verification of the Williams 82 gene at the DNA level. GmSAL13 Expression in transgenic Arabidopsis thaliana; where + represents a positive control, and the template used is [template name missing]. pTF101-GmSAL13-Flag Plasmid; - for Col-0 control; L1, L2, and L3 are three plasmids respectively. 35Spro::GmSAL13-Flag Transgenic homozygous lines; Picture 6 In this embodiment of the invention, the expression of GmSAL13-Flag protein in transgenic Arabidopsis thaliana was verified at the transcriptional level using Western blot; where - represents the Col-0 protein, and L1, L2, and L3 are three... 35Spro::GmSAL13-Flag Proteins from transgenic homozygous lines: primary antibody: Flag antibody; secondary antibody: mouse secondary antibody; antibody concentration for both was 1:5000. Picture 7 As described in the embodiments of the present invention 35Spro::GmSAL13-Flag Image showing the morphological results of salt-grown Arabidopsis thaliana; where L1, L2, and L3 are three... 35Spro::GmSAL13-Flag Transgenic homozygous lines; Col-0 is the wild-type control; The results of culturing L1, L2, L3, and Col-0 seeds on 0, 100, 150, and 200 mM NaCl medium for 16 hours (8 hours in the dark) at a temperature of 20-22℃ under light and a relative humidity of 70% for 5 days are shown in the figure. Picture 8 This is a graph showing the statistical results of germination rate over 10 days in an embodiment of the present invention; where L1, L2, and L3 are three... 35Spro::GmSAL13-Flag Transgenic homozygous lines; Col-0 is wild-type Arabidopsis thaliana, * indicates transgenic line from Student's... t -test, p<0.05, ** indicates P<0.01, *** indicates P<0.001; Picture 9 As described in the embodiments of the present invention GmSAL13 Photographs of soybean hairy root overexpression plants (OE) and their empty vector control (EV) after 48 h of treatment with water and 150 mM NaCl. Picture 10 As described in the embodiments of the present invention GmSAL13 Soybean hairy root overexpression plants (OE) and their empty vector control (EV) were treated with water and 150 mM NaCl for 48 h. GmSAL13Expression level detection data; Picture 11 As described in the embodiments of the present invention GmSAL13 Fresh weight data of aboveground and root parts of soybean hairy root overexpression plants (OE) and empty vector control (EV) after 48 h of water treatment and 150 mM NaCl treatment. The different letters were statistically significant after one-way ANOVA and Tukey post-hoc test, p<0.05 (n = 15). Picture 12 As described in the embodiments of the present invention GmSAL13 Photographs of gene-mediated soybean hairy root interference (RNAi) plants and their empty vector control (EV) after 36 h of treatment with water and 120 mM NaCl; Picture 13 As described in the embodiments of the present invention GmSAL13 The average fresh weight of soybean hairy root interference plants (RNAi) and their empty vector control (EV) after 36 h of water treatment and 120 mM NaCl treatment were analyzed. The different letters indicated significant differences after one-way ANOVA and Tukey post-hoc test, p < 0.05 (n = 15). Picture 14 As described in the embodiments of the present invention GmSAL13 The gene is classified into four main haplotypes in soybean cultivated populations based on SNPs (singlenucleotide polymorphism) and Indels (insertion and deletion mutations)—haplotype 1, haplotype 2, haplotype 3 and haplotype 4; Picture 15 In this embodiment of the invention, haplotypes 1, 2, and 3 showed significant differences in salt tolerance indicators (Student's). t -test). Detailed Implementation

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0017] In this invention, the term "identity" or "consistency" refers to sequence similarity to a nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0018] For sequence comparison, a sequence is typically used as a reference sequence and compared with the detection sequence. When using a sequence comparison algorithm, the detection and reference sequences are input into the computer, the coordinates of the subsequences are specified if necessary, and the parameters of the sequence algorithm program are specified. Then, based on the selected program parameters, the sequence comparison algorithm calculates the percentage sequence identity (consistency) of the detection sequence relative to the reference sequence.

[0019] Genes (nucleic acid molecules) can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0020] Furthermore, a large number of transformation vectors available for plant transformation are known to those skilled in the art, and the nucleic acid molecules of this invention can be used in conjunction with any vector. The choice of vector will depend on the preferred transformation technology and target plant species used for transformation, and is not specifically limited herein.

[0021] As described in the background section, environmental stress not only restricts plant growth and development but also leads to reduced yields of food crops. Higher plants achieve their growth and development and respond to environmental changes by regulating the expression of target genes. Since its first isolation and identification in the 1970s, the pleiotropic properties of plant CK1 genes have been widely validated in areas such as hormone signal transduction, stress response, and developmental regulation. For example, Arabidopsis thaliana CK1 is an important regulator of ethylene biosynthesis; AELs (plant-specific CK1 genes with different functions) promote ethylene biosynthesis by phosphorylating the transcription factor WRKY22, thereby promoting leaf senescence; CK1.3 and CK1.4 phosphorylate blue light receptors, negatively regulating blue light-mediated photomorphogenesis and flowering; rice EL1 / Hd16 negatively regulates gibberellin signaling-mediated flowering by phosphorylating DELLA protein; LTRPK1 participates in stress resistance by regulating cytoskeleton rearrangement and the formation of cold tolerance and adaptation; and LTG1 affects rice growth through auxin-dependent processes. In addition, salt stress, as one of the major abiotic stress factors that limit crop yield, has a wide range of effects on plant growth and development. Discovering salt tolerance-related genes and elucidating their functional mechanisms is of great significance for salt-tolerant crop breeding.

[0022] Based on this, the present invention provides a soybean casein kinase I gene. GmSAL13 And its applications.

[0023] A typical embodiment of the present invention provides GmSAL13 Genes or containing GmSAL13 Application of genetic biomaterials in at least one of the following: (a1) Regulation of plant salt tolerance; (a2) To improve and cultivate plants; The GmSAL13 The nucleotide sequence of the gene was selected from: (b1) The nucleotide sequence as shown in SEQ ID NO.1; (b2) and (b1) are nucleotide sequences that encode proteins with the same amino acid sequence, but are different in sequence due to the degeneracy of the genetic code; (b3) A nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes a nucleotide sequence that has the same or similar functional protein; (b4) is a complementary nucleotide sequence to any one of (b1)-(b3).

[0024] In this invention, the GmSAL13The gene, derived from soybean Williams 82, showed that its expression was upregulated by NaCl under salt stress. Experiments confirmed that transgenic Arabidopsis exhibited higher salt tolerance than non-transgenic Arabidopsis, primarily reflected in a higher seed germination rate under salt stress. Transgenic soybean hairy-root plants also showed higher salt tolerance than control plants, mainly due to lower wilting under stress. Conversely, transgenic soybean hairy-root RNAi plants showed lower salt tolerance than control plants, primarily due to higher wilting under stress. As described above, this soybean casein kinase I gene can be used for regulating soybean salt tolerance and for variety improvement.

[0025] In one specific embodiment of the present invention, the biological material includes a recombinant expression vector, a transgenic cell line, a host bacterium, or a transgenic plant.

[0026] The recombinant expression vector may be any one or more of a viral vector, plasmid, phage particle, granule, or artificial chromosome; no specific limitation is made here.

[0027] The transgenic cell line is an isolated, in vitro, cultured plant cell or a part of a plant; wherein the plant cell is a cell of any one of Arabidopsis thaliana, soybean, tobacco, corn, rice, or wheat; no specific limitation is made here.

[0028] The host bacteria are eukaryotic or prokaryotic bacteria, including bacteria, fungi, and actinomycetes.

[0029] Furthermore, the bacteria may be derived from Escherichia coli, Flavobacterium, Agrobacterium, Pseudomonas, Bacillus, etc., and even further, they may be Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis, or Bacillus pumilus.

[0030] Furthermore, the fungus may be yeast. The fungus may originate from genera such as Fusarium, Verticillium, Aspergillus, and Cephalosporium. The actinomycetes may originate from genera such as Streptomyces, Nocardia, and Neurocystis.

[0031] The genetically modified plant can be any one of Arabidopsis thaliana, soybean, tobacco, corn, rice, or wheat, without any specific limitation.

[0032] In one specific embodiment of the present invention, in (a1), the regulation of plant salt tolerance manifests as: increasing GmSAL13 Gene expression levels, on the other hand, promote / enhance plant salt tolerance and reduce it. GmSAL13 Gene expression levels can inhibit / reduce plant salt tolerance.

[0033] Furthermore, the regulation of plant salt tolerance is specifically manifested in: [the following text is incomplete and requires further context to translate accurately] GmSAL13When the gene was expressed in the hairy roots of Arabidopsis and soybean, the transgenic Arabidopsis showed higher salt tolerance than the non-transgenic Arabidopsis, mainly reflected in a higher seed germination rate under salt stress than the control; the transgenic soybean hairy root plants showed higher salt tolerance than the control plants, mainly reflected in lower wilting under salt stress, i.e., promoting / enhancing plant salt tolerance; while the gene expression in the hairy roots of soybean showed higher salt tolerance than the control plants. GmSAL13 The expression of the gene (using RNAi interference technology) in soybean hairy roots resulted in a higher degree of wilting under salt stress, i.e., inhibiting / reducing plant salt tolerance.

[0034] Furthermore, GmSAL13 The RNAi target sequence of the gene is shown in SEQ ID NO.2.

[0035] In one specific embodiment of the present invention, the promotion / enhancement of plant salt tolerance includes one or more of the following: (c1) Improve seed germination rate; (c2) Reduce the degree of wilting; (c3) Increase plant fresh weight.

[0036] In one specific embodiment of the present invention, in (a2), the improvement and cultivation of plants specifically refers to the improvement and cultivation of salt-tolerant plant varieties. Preferably, the plants are Arabidopsis thaliana, soybean, tobacco, corn, rice, and wheat, and more preferably, the plants are soybean.

[0037] In another specific embodiment of the present invention, an agent for improving plant salt tolerance is provided, comprising at least one of the following: (d1) contains GmSAL13 Gene expression vectors; (d2) contains a recombinant host of (d1); (d3) Enhancement GmSAL13 Promoters or enhancers of gene expression; (d4) Promote GmSAL13 Gene expression inducers; The GmSAL13 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0038] In a third embodiment of the present invention, a method for improving the salt tolerance of plants is provided, using the above-mentioned preparation to improve the salt tolerance of plants. GmSAL13 Gene expression levels and / or activity, or making those without gene expression levels... GmSAL13 Gene or GmSAL13 The preparation comprises a nucleic acid molecule encoding the GmSAL13 protein; the amino acid sequence of the GmSAL13 protein is shown in SEQ ID NO.3; the plant is soybean.

[0039] In a fourth embodiment of the present invention, a method for improving and cultivating salt-tolerant plants is provided, comprising increasing the salt content of the target plant. GmSAL13 Gene expression levels and / or activity.

[0040] In the above method, the improvement of the target plant GmSAL13 Gene expression levels and / or activity can be increased by introducing a substance containing the aforementioned gene. GmSAL13 The gene plasmid, the strong promoter and the said GmSAL13 Genes can be operatively linked and can be achieved through methods such as introducing enhancers, but no specific limitations are made here.

[0041] In one specific embodiment of the present invention, the target plant can be any plant at any developmental stage, such as Arabidopsis thaliana, soybean, tobacco, corn, rice, wheat, etc., without any specific limitation.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1 GmSAL13 Cloning and construction of plant expression vectors 1.1 Extraction of total RNA from Williams 82 (1) Take an appropriate amount of Williams 82 soybean (from the Institute of Crop Resources, Chinese Academy of Agricultural Sciences) leaf material, freeze it with liquid nitrogen, grind it into powder, and apply it directly to RNA extraction; (2) After the liquid nitrogen evaporates, immediately transfer 100-200 mg of plant powder into a 1.5 mL RNase-free centrifuge tube, then quickly add 1 mL of Trizol extraction solution, vortex to fully dissolve the sample in the extraction solution, place at room temperature for 10 min, then add 0.2 mL of chloroform and shake vigorously for 15 sec, place at room temperature for 5-10 min. (4) Centrifuge at 4℃, 12,000 rpm for 15 min, transfer 0.4 mL of supernatant to a new 1.5 mL centrifuge tube, add 0.4 mL of isopropanol, invert 15 times to mix the solution, and let stand at room temperature for 10 min. (5) Centrifuge at 4℃, 12,000 rpm for 10 min, discard the supernatant, wash the precipitate twice with 1 mL of 75% ethanol (prepared by diluting anhydrous ethanol with DEPC water), and centrifuge at 4℃, 12,000 rpm for 5 min. (6) Discard the supernatant, then air-free for 2 min and aspirate the excess liquid. Open the lid and place the RNA in a clean bench for about 5-7 min to dry. Add 40 µL of DEPC water and dissolve the RNA completely at 65℃ for 5 min. (7) Measure the OD value and concentration of RNA samples using a UV spectrophotometer. An A260 / A280 ratio of 1.7-2.0 is preferred. Detect quality using agarose gel electrophoresis.

[0044] 1.2 Reverse transcription of RNA (1) Add the following substances sequentially to an RNase-free centrifuge tube (40 µL reaction system):

[0045] (2) After gently mixing, denature at 65°C for 5 min, then immediately insert into ice and ice bath for at least 1 min; (3) Add the following substances to the centrifuge tube in sequence:

[0046] (4) After gently mixing, incubate in a constant temperature water bath at 42℃ for 1 h, denature at 65℃ for 10 min, and store at -20℃ for later use.

[0047] 1.3 GmSAL13 Gene cloning GmSAL13 CDS cloning primers: GmSAL13 CDS-F: 5'-agagaacacgggggactctagaATGGATGAGTTTGATAGTGGAGG-3'; GmSAL13 CDS-R: 5'-catggtctttgtagtccccgggTGACACTGTTCTCCCATAACAA-3'; GmSAL13 RNAi fragment cloning primers: GmSAL13 RNAi-F: 5'-CTTGGCCCTAGTCTGTGGGA- 3'; GmSAL13 RNAi-R: 5'-GAACTATCACGCCACCGAGT-3'.

[0048] The reaction system for amplifying gene fragments with the high-fidelity enzyme Phanta is as follows (50 µL system):

[0049] The amplification conditions are as follows:

[0050] After the reaction was completed, the reaction solution was detected by 1% TAE agarose gel electrophoresis.

[0051] (2) Purification and recovery of cloned gene fragments (Novozymes gel recovery kit) 1) Place the gel containing the target fragment into a 1.5 mL centrifuge tube and weigh the gel. Add an equal volume of sol solution and dissolve the gel at 65 °C for 5-10 min, turning it continuously during the dissolution process until it is completely dissolved. 2) After the gel has completely melted, take ≤700 μL of solution back into the column and let stand for 1 min; 3) Centrifuge at 12,000 rpm for 30 seconds at room temperature, then discard the solution; 4) Add 300 µL of sol to the recovery column, let stand for 1 min, then centrifuge at 12000 rpm for 1 min and discard the waste liquid; 5) Add 700 µL of wash buffer to the column, centrifuge at 12000 rpm for 1 min, discard the wash buffer, and repeat the cleaning step once more.

[0052] 6) Empty column, centrifuge at 12000 rpm for 2 min; 7) After the recovery column is opened and dried for 3-5 minutes, place it into a new clean 1.5 mL centrifuge tube, add 30 µL of preheated sterile water or EB buffer at 65℃, and let it stand for 2 minutes. 8) Centrifuge at 12000 rpm for 1 min. The resulting solution is the recovered fragment. After measuring the concentration, it can be used.

[0053] 1.4 Connection (1) Construction of overexpression vectors—homological recombination ligation 1) Vector linearization. First, linearize the vector... pTF101 The plasmid (purchased from Baosai Plasmid and Strains Company) underwent enzyme digestion. pTF101 The enzyme digestion reaction system is as follows (20 µL system):

[0054] The reaction was carried out at 37℃ for 1 hour and 30 minutes, followed by treatment at 65℃ for 20 minutes to terminate the reaction. The enzyme digestion results were then verified by gel electrophoresis.

[0055] 2) Recombination reaction. The optimal amount of cloning vector used in the ClonExpress II recombination reaction system is 0.03 pmol, and the optimal amount of insert fragment is 0.06 pmol (the molar ratio of vector to insert fragment is 1:2). The DNA mass corresponding to these molar numbers can be roughly calculated using the following formula.

[0056] Optimal cloning vector usage = [0.02 × number of cloning vector base pairs] ng (0.03 pmol) Optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol) The recombination reaction system is as follows (10 μL system):

[0057] Gently pipette and mix (do not shake to mix), then briefly centrifuge to collect the reaction solution at the bottom of the tube. React at 37°C for 30 min, then cool to 4°C or immediately place on ice to cool.

[0058] (2) RNAi vector construction – Gateway ligation, step one The BP reaction (Gateway system) is as follows (2.5 µL system):

[0059] React overnight at 25°C.

[0060] 1.5 Plasmid transformation of Escherichia coli (aseptic technique) (1) Add the above ligation product to 50 µL of commercial DH5α competent cells, gently tap the centrifuge tube to mix, and incubate on ice for 30 min. (2) 42℃, warm water bath heat shock for 90 seconds, then immediately ice bath for 2-3 minutes; (3) Add 0.8 mL of antibiotic-free LB medium and incubate at 37°C with shaking at 200 rpm for 45-60 min; (4) Centrifuge at 5000 rpm for 3 min at room temperature and collect the bacterial cells; (5) Spread the bacteria on a culture plate containing plasmids carrying resistance and incubate it upside down at 37°C overnight.

[0061] 1.6 PCR verification of Escherichia coli Single colonies of bacteria were picked from the culture dish and shaken for PCR verification using the Polymermax 2×M5 Hiper plus Taq HiFi PCR mix. The reaction volume is as follows (20 µL):

[0062] The amplification conditions are as follows:

[0063] After the reaction was completed, the reaction solution was examined by 1% TAE agarose gel electrophoresis to check for the presence of the correct target band.

[0064] 1.7 Recombinant plasmid sequencing Select a single positive colony with a correct PCR band and inoculate it into 0.8 mL of solution containing the appropriate antibiotic ( pDONR221 The plasmid is kanamycin resistant (50 mg / mL). pTF101 The plasmid containing spectinomycin resistance (60 mg / mL) was cultured in LB liquid medium at 37°C and 200 rpm for 4-6 h, and then sent to Ribo Biotech for sequencing. Bidirectional sequencing was performed using universal primers for the corresponding vectors to obtain the sequencing results.

[0065] If the sequencing results are compared with the standard sequence and the overlap is 100%, it indicates that the vector construction was successful and the gene... GmSAL13 The nucleotide sequence of the cDNA is shown in SEQ ID NO.1, and the gene... GmSAL13 The target sequence for RNAi is shown in SEQ ID NO.2, wherein the constructed pDONR221-GmSAL13RNAi The intermediate carrier can then be used for the second step of the subsequent Gateway connection – the LR reaction.

[0066] 1.8 Extraction of E. coli plasmid DNA (1) Select the successfully sequenced positive single colonies and inoculate them into 10 mL of LB liquid medium containing plasmids carrying resistance, and incubate overnight at 37°C and 200 rpm in a shaker; (2) Centrifuge at 12,000 rpm for 1 min at room temperature, collect the bacterial cells, and extract plasmids using the Novizan FastPure PlasmidMini Kit. (3) Discard the supernatant, add 250 µL of pre-cooled solution I, and vortex to completely resuspend the cells; (4) Add 250 µL of solution II and quickly invert to mix 6-8 times; (5) Add 350 µL of solution III and mix by inverting 6-8 times; (6) Centrifuge at 12000 rpm for 10 min; (7) Place the FastPure DNA Mini Columns adsorption column into a 2 mL collection tube. Carefully transfer the supernatant from step 6 into the adsorption column using a pipette, being careful not to aspirate any precipitate. Centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. (8) Add 600 μL of Buffer PW2 to the adsorption column. Centrifuge at 12,000 rpm for 30-60 seconds. Discard the waste liquid and return the adsorption column to the collection tube; (9) Repeat step 8; (10) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 1 min; (11) Place the adsorption column in a new sterile 1.5 mL centrifuge tube, add 30-100 μL of sterile water or EB buffer preheated to 65℃ to the center of the adsorption column membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min to elute DNA, and store at -20℃ for later use.

[0067] 1.9 RNAi Vector Construction – Gateway Ligation Step 2 The LR reaction (Gateway system) structure is as follows:

[0068] React overnight at 25°C.

[0069] 1.10 E. coli plasmid transformation (same as 1.5) 1.11 PCR verification of E. coli (same as 1.6) 1.12 Recombinant plasmid sequencing (same as 1.7) 1.13 Extraction of E. coli plasmid DNA (same as 1.8) 1.14 Plasmid transformation of Agrobacterium (aseptic technique) (1) Add 1 µL of plasmid DNA to 50 µL of... GV3101 In Agrobacterium competent cells, gently tap the centrifuge tube to mix, and incubate on ice for 5 min; (2) Quick freeze in liquid nitrogen for 5 min; then bath in water at 37°C for 5 min; then immediately ice bath for 3-5 min; (3) Add 0.8 mL of antibiotic-free LB medium and incubate at 28℃ for 2-4 h; (4) Centrifuge at 5,000 rpm for 3 min at room temperature and collect the bacterial cells; (5) Spread the bacteria on YEP culture plates containing plasmids carrying resistance and 50 μg / mL rifampicin, and incubate upside down at 28°C for 48 h.

[0070] 1.15 PCR Validation of Agrobacterium The reaction system is as follows (20 µL system):

[0071] The amplification conditions are as follows:

[0072] After the reaction was completed, the reaction solution was detected by 1% TAE agarose gel electrophoresis.

[0073] Example 2: Verification of the salt tolerance function of the protein gene GmSAL13 expressed in Arabidopsis thaliana 2.1 Transformation of Arabidopsis thaliana by inflorescence infection method (1) When Arabidopsis thaliana (Col-0 wild type) grows to 1 cm in length, the top is cut off to induce the formation of lateral inflorescences; (2) One day before transformation, take 1 mL of activated Agrobacterium GV3101 containing the expression vector plasmid and add it to 40 mL of YEP medium containing the corresponding antibiotic and 50 μg / mL rifampin. Incubate at 28°C with shaking until the OD600 is about 0.8-1.2. (3) Centrifuge at 5000 rpm for 10 min at room temperature, collect the bacterial cells, and resuspend the cells in infection solution (5% sucrose, 0.02% Silwet L-77) to adjust OD. 600 Approximately 0.8; (4) Before infection, cut off the pods and flowers that have already grown in Col-0. Use a pipette to drop Agrobacterium onto the inflorescence for infection. Make sure the Agrobacterium infection droplets completely cover the inflorescence. After all the inflorescences are infected, let it stand for about 10 minutes to ensure complete infection and prevent the droplets from slipping off. (5) Cover the inflorescence with a black plastic bag and place it at 20-22℃ overnight in the dark. After another day of cultivation, remove the plastic bag and continue cultivation until the seeds mature.

[0074] 2.2 Surface disinfection of Arabidopsis seeds Place an appropriate amount of Arabidopsis thaliana seeds to be sterilized into a 1.5 mL centrifuge tube, add 1 mL of 75% ethanol (containing 0.05% Triton X-100 by volume) and shake for 3 min (1-3 times, depending on the seed quality). Then shake with anhydrous ethanol for 1 min (twice). Finally, use a pipette to transfer the seeds to sterile filter paper and blow them dry. Then, use a sterile toothpick to spot them into the culture medium or sprinkle the sterilized seeds evenly on the culture medium.

[0075] 2.3 Screening of transgenic homozygous plants After infection, the harvested T1 generation seeds of Arabidopsis thaliana were surface-sterilized and then evenly sown on 1 / 2 MS medium (containing Basta). After vernalization for 3 days, they were transferred to a light incubator. Approximately 10 days after exposure to light, positive plants were selected. Plants with dark green cotyledons and normal root growth were identified as transgenic positive plants, while plants with light green or even yellow cotyledons and inhibited root growth were identified as non-transgenic plants. The transgenic positive plants were then transplanted into nutrient soil and individually numbered. T2 generation seeds were harvested (single-plant seed collection). A portion of the harvested T2 generation seeds were further selected on 1 / 2 MS medium containing Basta, with a segregation ratio of 3:1. 1 (positive) Transplant positive plants with a single copy of the negative (negative) insertion, about 10 single plants per line, and culture until the T3 generation seeds are harvested (single plant seed harvest). The T3 generation seeds harvested from the single plants are then evenly sown on 1 / 2 MS medium (containing Basta). If all offspring are positive, then pure line T3 generation seeds are obtained. Transplant pure line T3 generation seeds for propagation.

[0076] 2.4 Extraction of DNA from transgenic homozygous plants (1) Take an appropriate amount of Arabidopsis thaliana leaf material into a 1.5 mL centrifuge tube, freeze it with liquid nitrogen, grind it or grind it into powder using a sampler (22 rpm), apply it directly to the DNA extraction experiment or freeze it in an ultra-low temperature freezer at -80℃ for later use, and pre-cool the centrifuge by 4 degrees in advance. (2) Add 600 µL of CTAB extraction solution to the centrifuge tube, vortex to fully dissolve the sample in the extraction solution, and place at 65℃ for 15-30 min; (3) Add 600 µL of chloroform and shake vigorously to mix. Centrifuge at 4°C, 12,000 rpm for 15 min. (4) Transfer 0.4 mL of supernatant to a new 1.5 mL centrifuge tube, add 0.4 mL of isopropanol, invert thoroughly to mix the solution, and place at -20℃ for 15 min. (5) Centrifuge at 12,000 rpm for 10 min at 4℃, discard the supernatant, wash the precipitate twice with 600 µL of 70% ethanol, and centrifuge at 12,000 rpm for 5 min at 4℃. (6) Discard the supernatant, then air-dry for 2 min and remove excess liquid. Open the lid and dry the DNA in a clean bench for about 5-7 min. Add 100 µL of water and dissolve the DNA completely in 65℃ for 5 min. (7) Measure the OD value and concentration of the DNA sample using a UV spectrophotometer. 260 / A 280 A value of 1.7-2.0 is ideal.

[0077] 2.5 PCR detection of transgenic plants PCR screening of transgenic positive plants: DNA from resistant plants is used... GmSAL13 Gene identification primers were used for PCR detection.

[0078] GmSAL13 Gene identification PCR primer sequences: TF101-F :5'-GAGAACACGGGGGACTCTAGA-3' GmSAL13CDS-R :5'-catggtctttgtagtccccgggTGACACTGTTTCCCATAACAA-3' PCR amplification was performed using 2×M5 HiPer plus Taq HiFi PCR mix, and the reaction system is as follows (20 µL system):

[0079] The amplification conditions are as follows:

[0080] After the reaction was completed, the reaction solution was detected by 1% TAE agarose gel electrophoresis.

[0081] See results Picture 5 .

[0082] 2.6 Western blot detection of protein expression in transgenic homozygous plants (1) Take an appropriate amount of fresh plant tissue and grind it with liquid nitrogen. Weigh the ground powder and transfer it to a 1.5 mL centrifuge tube. Add protein extract at a weight-volume ratio of 1:1 between powder and protein extract. Shake to mix well and let stand on ice for 30 min. (2) Centrifuge at 12000 rpm for 10 min at 4℃ and collect the supernatant; (3) Repeat (2) until no precipitate remains in the supernatant; (4) Mix the supernatant and 4× protein loading buffer at a volume ratio of 3:1, denature at 95℃ for 10 min, add 10 μL of sample to the loading well of a 10% PAGE gel, and perform electrophoresis at a constant voltage of 100 V. (5) Cut a PVDF membrane of appropriate size and activate it in methanol for 1 min, then soak it thoroughly in Transfer Buffer along with the electrophoresis gel and transfer sponge pad; (6) Place the transfer sponge pad, electrophoresis gel, PVDF membrane and transfer sponge pad in order from bottom to top from the black side of the transfer clamp. After assembly, transfer the membrane in the electrophoresis tank at a constant current of 150 mA. (7) The PVDF membrane was sealed in 10 mL of blocking solution for 1 h; (8) Discard the blocking solution, add 10 mL of primary antibody blocking solution, and incubate on a shaker for 1 h; (9) Recover the primary antibody blocking solution and clean the PVDF membrane with 1 × TBS-T for 10 min for a total of 3 times; (10) Add 10 mL of secondary antibody blocking solution and incubate on a shaker for 1 h; (11) Recover the secondary antibody blocking solution and clean the PVDF membrane with 1×TBS-T for 10 min for a total of 3 times; (12) The chemiluminescent liquid is evenly covered on the PVDF film, wrapped with an outer plastic film, and imaged in a chemiluminescence imager and saved as an image.

[0083] Result: Among the 3 selected... 35Spro::GmSAL13-Flag In transgenic Arabidopsis pure lines L1, L2, and L3, the target band appeared around 70 KD (the protein size of GmSAL13-Flag is approximately 70 KD), while no corresponding band was observed in the control wild-type Arabidopsis Col-0. The results indicate that the GmSAL13-Flag protein, in the measured range... 35Spro::GmSAL13- Flag Successful expression in transgenic Arabidopsis thaliana pure lines, results are shown in [link to results]. Picture 6 .

[0084] 2.7 Salt tolerance test of transgenic Arabidopsis thaliana (1) Surface disinfection of Arabidopsis seeds (same as 2.2).

[0085] (2) NaCl treatment Sterile Col-0, OE-L1 , OE-L2, OE-L3 Purebred seeds were evenly sown on 0, 100, 150, and 200 mM NaCl culture medium plates, respectively. After vernalization treatment for 3 days, the plants were transferred to a light incubator for growth. The germination rate of each transgenic plant at each concentration was counted every 24 hours for a total of 10 days.

[0086] Results: On normal 1 / 2 MS medium, Col-0, cultured under the same growth conditions, OE-L1 , OE- L2 and OE-L3 The germination rate of the seeds reached almost 100% after 24 hours of exposure to light, indicating that the transgenic seeds and wild-type seeds were of high quality; in 1 / 2 MS medium containing 100 mM, 150 mM, and 200 mM NaCl, OE-L1 , OE-L2 , OE-L3 The germination rate is higher than that of Col-0, proving that... GmSAL13It plays a certain role in the process of plants coping with salt stress. Results are shown below. Picture 7 Statistical data can be found Picture 8 (Data are expressed as mean ± standard deviation of three biological replicates).

[0087] Example 3: Overexpression of protein genes in hairy roots GmSAL13 Salt tolerance verification 3.1 Methods for transforming soybean hairy roots (1) Select about 300 soybean seeds with relatively intact seed coats and no discoloration. Spread the soybean seeds evenly on vermiculite with the hilum facing down, and cover the seeds with about 1 cm of vermiculite. Incubate them at 25℃ for 16 h in light and 22℃ for 8 h in darkness until the soybean seedlings emerge and the cotyledons are not fully open (about 5 days). Observe the condition of the soybean seedlings during this period. (2) During the 5-day period of seed germination, Agrobacterium rhizogenes was cultured by adding 1 µL of plasmid DNA to 50 µL of commercially available... GV3101 In Agrobacterium competent cells, gently tap the centrifuge tube to mix, incubate on ice for 5 min; flash freeze in liquid nitrogen for 5 min; then incubate in water at 37°C for 5 min; immediately incubate on ice for 3-5 min; add 0.8 mL of antibiotic-free LB medium, and shake in a shaker at 28°C for 2-4 h; centrifuge at 5,000 rpm for 3 min at room temperature to collect the cells; spread the cells on TY solid medium containing plasmid-carrying antibiotics and 100 μg / mL streptomycin, and incubate upside down at 28°C for 48 h. After PCR identification of single colonies, select multiple positive colonies and inoculate them into 5 mL of TY liquid medium containing plasmid-carrying antibiotics and 100 μg / mL streptomycin, and incubate overnight at 28°C with a shaker at 200 rpm until turbidity appears; (3) Take 400 μL of bacterial culture and spread it on TY solid medium containing plasmids carrying resistance and streptomycin. Incubate upside down in a constant temperature incubator at 28℃ for 12-24 h. During this period, observe the growth of the bacteria until a bacterial film is formed. (4) Select healthy soybean seedlings that have successfully emerged, are growing uniformly, and whose cotyledons are not fully open. Remove the soybean seed coat with tweezers. Cut off the root of the seedling at the hypocotyl 2-4 cm away from the cotyledon with a blade, keeping the cut clean and retaining the upper half containing the cotyledons. Scrape an appropriate amount of Agrobacterium rhizogenes from the cut of the seedling. K599 Bacterial cells, ensuring the entire incision is contaminated with Agrobacterium; (5) Arrange the Agrobacterium-infected seedlings neatly in a square dish containing moistened filter paper, ensuring that the cotyledons are not exposed or crowded. After infection, place the square dish in a dark environment at 25°C overnight.

[0088] (6) The next day, the infected seedlings were placed in a tray containing vermiculite, covered, and cultured at 25°C for 16 h under light and 22°C in the dark for 8 h.

[0089] (7) After the soybean seedlings have grown for about a week, remove the adventitious roots from the non-cut ends of each seedling, place them in the seedling tray as before, cover them, and continue to grow under the original growing conditions until the first trifoliate compound leaf is fully unfolded.

[0090] (8) Without damaging the hairy roots, gently remove the soybean seedlings from the soil and wash them. Place them in a hydroponic container for 1-2 days to allow them to adapt to the hydroponic environment. Select 15 seedlings with uniform above-ground parts and healthy hairy roots and transplant them into a 15-cell hydroponic container. Water them with Hoagland nutrient solution. The composition of Hoagland nutrient solution is shown in the table below: Hoagland nutrient solution preparation (1L):

[0091] * Trace elements: H3BO3, MnSO4, ZnSO4·7H2O, CuSO4·5H2O, Na2MoO4·2H2O, with final concentrations of 25 μmol / L, 2 μmol / L, 2 μmol / L, 0.5 μmol / L, and 0.5 μmol / L, respectively.

[0092] (9) Perform hairy root transformation according to the above method, and transform them respectively. 35S::GmSAL13 Hairy roots of overexpressing plants (OE) and their empty vector control (EV(OE)), pB7GWIWGII::GmSAL13 RNAi plants and their hairy roots (EV(RNAi)) were compared with those of empty vector control plants.

[0093] 3.2 Salt tolerance phenotype experiment of soybean hairy roots 3.2.1 Overexpression of hairy root salt tolerance phenotype experiment Once the soybean seedlings in the hydroponic box have grown their second trifoliate compound leaf, they can be treated with saline solution by irrigating with 150 mM NaCl solution to overexpress the phenotype of the hairy roots of the plant (OE) and its empty control (EV(OE)).

[0094] Results: After treatment with 150 mM NaCl for 48 h... 35S::GmSAL13 Overexpressing soybean hairy root plants (OE) showed less wilting than their empty control (EV), exhibiting a salt-insensitive phenotype (see [link to original text]). Picture 9 ), the root system of hairy roots GmSAL13 Data on expression levels (see) Picture 10 ) and data on the fresh weight of a single plant (see Picture 11This conclusion is also supported by the fact that (the differences between the different letters were statistically significant according to one-way ANOVA and Tukey post-hoc test, p<0.05, n=15), which proves that this gene plays a certain role in the regulation of plant salt tolerance, and may play a positive role in regulating salt tolerance.

[0095] 3.2.2 Interference with the salt tolerance phenotype of hairy roots Once the soybean seedlings in the hydroponic box have grown their second trifoliate compound leaf, they can be treated with saline solution using 120 mM NaCl solution. The phenotypes of the interfering plants (RNAi) and their empty control (EV) hairy roots can then be observed.

[0096] Results: After treatment with 120 mM NaCl for 36 h... pB7GWIWGII::GmSAL13 The wilt degree of soybean hairy root interference (RNAi) plants was higher than that of their empty control (EV), exhibiting a salt-sensitive phenotype (see...). Picture 12 ), data on the average fresh weight of the root system (see Picture 13 This conclusion is also supported by the fact that (the differences between the different letters were statistically significant according to one-way ANOVA and Tukey post-hoc test, p<0.05, n=15), which proves that this gene plays a certain role in the regulation of plant salt tolerance, and may play a positive role in regulating salt tolerance.

[0097] Example 4: Haplotype Analysis and Phenotyping of Salt Tolerance Phenotyping 4.1 Haplotype Analysis GmSAL13 In soybean cultivars, four main haplotypes—Hap001, Hap002, Hap003, and Hap004—were identified based on SNPs (single nucleotide polymorphism) and Indels (insertion / deletion mutations). The number of varieties in these haplotypes were 428, 56, 56, and 33, respectively. Hap001 had the highest proportion and was the dominant haplotype (see...). Picture 14 A total of 7 mutation sites were found, all of which were base substitutions; 6 of these mutation sites were located within a 2.0 kb region upstream of the promoter, and 1 mutation site was located in the 5'-UTR region. No polymorphic sites were detected in the coding region, indicating that this region may be under strong functional constraints. The conservation of the GmSAL13 protein sequence may play an important role in maintaining soybean survival.

[0098] 4.2 Analysis of haplotype salt tolerance phenotype The Hap001, Hap002, and Hap003 varieties showed significant differences in germination rates across multiple locations in saline-alkali soils (salinity approximately 0.3%) over many years (Student's...). t -test) (see Picture 15Three-year germination rate data in saline-alkali land showed that Hap002 had a significantly higher germination rate than Hap001 and Hap003. Analysis of the differences in salt tolerance phenotypic indices among the different haplotypes indicated that Hap002 has the advantage of a high germination rate in saline-alkali land. GmSAL13 Excellent haplotype with salt and alkali resistance.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. GmSAL13 Genes or containing GmSAL13 Application of genetic biomaterials in at least one of the following: (a1) Regulation of plant salt tolerance; (a2) To improve and cultivate plants; The nucleotide sequence of the GmSAL13 gene is selected from: (b1) The nucleotide sequence as shown in SEQ ID NO.1; (b2) and (b1) are nucleotide sequences that encode proteins with the same amino acid sequence, but are different in sequence due to the degeneracy of the genetic code; (b3) A nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes a nucleotide sequence that has the same or similar functional protein; (b4) is a complementary nucleotide sequence to any one of (b1)-(b3).

2. The application as described in claim 1, characterized in that, The biological materials include recombinant expression vectors, transgenic cell lines, host bacteria, or transgenic plants.

3. The application as described in claim 2, characterized in that, The recombinant expression vector is any one or more of the following: viral vector, plasmid, phage particle, visceral particle, or artificial chromosome. The transgenic cell line is an isolated, in vitro, cultured plant cell or a part of a plant; wherein the plant cell is a cell of any one of Arabidopsis thaliana, soybean, tobacco, corn, rice, or wheat; The host bacteria are eukaryotic or prokaryotic bacteria, including bacteria, fungi, and actinomycetes; The genetically modified plant is any one of Arabidopsis thaliana, soybean, tobacco, corn, rice, and wheat.

4. The application as described in claim 1, characterized in that, In (a1), the regulation of plant salt tolerance is specifically manifested in: increasing GmSAL13 Gene expression levels, on the other hand, promote / enhance plant salt tolerance and reduce it. GmSAL13 Gene expression levels can inhibit / reduce plant salt tolerance.

5. The application as described in claim 4, characterized in that, The promotion / enhancement of plant salt tolerance includes one or more of the following: (c1) Improve seed germination rate; (c2) Reduce the degree of wilting; (c3) Increase plant fresh weight.

6. The application as described in claim 1, characterized in that, In (a2), the improvement and cultivation of plants specifically refers to the improvement and cultivation of salt-tolerant plant varieties. Preferably, the plants are Arabidopsis thaliana, soybean, tobacco, corn, rice, and wheat. More preferably, the plant is soybean.

7. A preparation for improving the alkaloid tolerance of plants, characterized in that, Includes at least one of the following: (d1) contains GmSAL13 Gene expression vectors; (d2) contains a recombinant host of (d1); (d3) Enhancement GmSAL13 Promoters or enhancers of gene expression; (d4) Promote GmSAL13 Gene expression inducers; The GmSAL13 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

8. A method for improving the salt tolerance of plants, characterized in that, The formulation according to claim 7 improves the plant GmSAL13 Gene expression levels and / or activity, or making those without [the gene] GmSAL13 Gene or GmSAL13 The preparation comprises a nucleic acid molecule encoding the GmSAL13 protein; the amino acid sequence of the GmSAL13 protein is shown in SEQ ID NO.3; the plant is soybean.

9. A method for improving and cultivating salt-tolerant plants, characterized in that, Including improving the target plant GmSAL13 Gene expression levels and / or activity.

10. The method as described in claim 9, characterized in that, The plant mentioned in the description of improving the target plant GmSAL13 Gene expression levels and / or activity are controlled by introducing a substance containing the aforementioned gene. GmSAL13 The gene plasmid, the strong promoter and the said GmSAL13 Genes can be operatively linked and through the introduction of enhancers.

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