Soybean advanced embryogenesis abundant protein gene GmLEA1 and application thereof in saline-alkaline tolerance

Abstract

The invention belongs to the technical field of plant heredity and genetic engineering, and particularly relates to a soybean late embryogenesis abundant protein gene GmLEA1 and application thereof in saline-alkaline tolerance. The invention discloses an existing plant gene engineering technology for the first time, GmLEA1 in Williams 82 is obtained through cloning and is expressed in soybean hairy roots, experiments prove that the salt tolerance of a soybean hairy root transgenic plant overexpressed by GmLEA1 is higher than that of a control plant, and the dry weight and fresh weight of the plant under salt stress are obviously higher than those of a no-load control plant; a haplotype analysis result shows that the GmLEA1 can be divided into four haplotypes, the Hap3 is a haplotype with excellent salt tolerance, the hundred-grain weight and the single-plant grain weight of the Hap3 under soybean salt are remarkably higher than those of other haplotypes, and it is fully proved that the soybean Williams 82 mid-late embryogenesis abundant protein GmLEA1 plays an important role in plant salt stress coping, so that the GmLEA1 has 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 late embryogenesis rich protein gene GmLEA1 and its application in salt and alkali tolerance. Background Art

[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] Soil salinization is a serious abiotic stress that causes metabolic disorders and growth inhibition in plants, even leading to plant death, through osmotic stress, ionic stress, and oxidative stress, severely impacting crop yield. Soybean, as an important food and economic crop, is significantly affected by salt stress in its growth and yield. Salt stress inhibits soybean plant growth and development, interferes with physiological metabolic processes, leading to reduced yield and deteriorated quality. Soybean plants are affected by salt stress at all stages of their life cycle, with the budding and seedling stages being the most severely affected. To cope with the severe challenges posed by salt stress, soybeans have evolved a series of defense and regulatory mechanisms. Late embryogenesis-abundant proteins (LEAs) are a class of small, hydrophilic proteins widely present in plants, involved in protecting plants from abiotic stress and playing an important role in improving plant stress resistance. However, the role of GmLEA1 in soybean salt tolerance has not yet been reported.

[0004] The creation of new crop varieties is highly dependent on expanding the genetic diversity of breeding materials. Wild plants and related genes serve as carriers for enhancing the innovation potential of crop varieties. The inventors discovered that the gene GmLEA1 in soybean Williams 82 encodes a protein abundant in late embryogenesis and has high homology with the LEA protein gene in wild soybean. However, its regulatory role and mechanism in plant salt / alkali tolerance have been rarely reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the soybean late embryogenesis-rich protein gene GmLEA1 and its application in salt and alkali tolerance. Specifically, the present invention isolated the late embryogenesis-rich protein gene GmLEA1 from soybean (Glycine max (L.) Merr) Williams 82, and experimental results have demonstrated that it plays an important role in the regulation of plant salt / alkali tolerance. Based on the above research results, the present invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a soybean late embryogenesis-rich protein gene, named GmLEA1, wherein the soybean late embryogenesis-rich protein gene is selected from:

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

[0009] (a2) and (a1) encode proteins with the same amino acid sequence, but are different nucleotide sequences due to the degeneracy of the genetic code.

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

[0011] A nucleotide sequence that is complementary to either (a4) or (a1)-(a3).

[0012] In a second aspect, the present invention provides a protein encoded by the aforementioned soybean late embryogenesis-rich protein gene, specifically a soybean late embryogenesis-rich protein.

[0013] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned soybean late embryogenesis-rich protein genes.

[0014] In a fourth aspect, the present invention provides a transgenic cell line, host bacterium, or transgenic plant containing the aforementioned gene for abundant late embryogenetic proteins in soybean.

[0015] A fifth aspect of the present invention provides the use of the above-mentioned soybean late embryogenesis-rich protein gene, soybean late embryogenesis-rich protein, recombinant expression vector containing the above-mentioned soybean late embryogenesis-rich protein gene, transgenic cell line, host bacterium or transgenic plant in any one or more of the following:

[0016] (b1) Regulation of plant salt / alkali tolerance;

[0017] (b2) Identify salt-tolerant / alkali-tolerant plants;

[0018] (b3) Improve and cultivate salt / alkali tolerant plants.

[0019] A sixth aspect of the present invention provides a method for improving and cultivating salt / alkali tolerant plants, the method comprising increasing the expression level and / or activity of the GmLEA1 gene in the target plant.

[0020] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0021] The above-mentioned technical solution is the first to disclose a plant genetic engineering technology that cloned GmLEA1 from Williams82 and expressed it in soybean hairy roots. Experiments confirmed that the transgenic soybean hairy root plants overexpressing GmLEA1 had higher salt tolerance than the control plants, and the dry and fresh weights of the plants under salt stress were significantly higher than their empty control. Haplotype analysis showed that GmLEA1 can be divided into five haplotypes, among which Hap3 is a haplotype with excellent salt tolerance. The 100-seed weight and single-plant seed weight of Hap3 soybeans under salt were significantly higher than those of other haplotypes, which fully demonstrates that the GmLEA1 protein, which is abundant in late embryogenesis of soybean Williams82, plays an important role in the plant's response to salt stress. This indicates that the gene can be widely used to improve the salt tolerance of plant varieties and therefore has good practical application value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 This is the spectrum of the pDONR221 introductory carrier in this invention.

[0024] Figure 2 This is a map of the plant overexpression vector pB7GWF2 used in this invention.

[0025] Figure 3 The phenotypic diagrams of the GmLEA1 soybean hairy root overexpression plant (OE) and its empty vector control (VC) after 10 days of treatment with water (Mock) and 150mM NaCl are shown.

[0026] Figure 4 The dry and fresh weight statistics of soybean hairy root overexpression plants (OE) and their empty vector control (VC) after 10 days of treatment with 150 mM NaCl in this invention are presented (Student's t-test, * indicates p<0.05, * indicates p<0.001).

[0027] Figure 5 This is a statistical chart showing the single-plant grain weight and 100-grain weight of soybean varieties Hap1-Hap5 under salt conditions in the GmLEA1 of this invention. The analysis method is one-way ANOVA, and different letters indicate significant differences. Detailed Implementation

[0028] 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.

[0029] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. 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. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0030] 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.

[0031] 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.

[0032] Genes (nucleic acid molecules) can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0033] 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.

[0034] As mentioned earlier, the gene GmLEA1 in soybean Williams 82 encodes a protein abundant in late embryogenesis and is highly homologous to the LEA protein gene in wild soybean. However, there are few reports on its regulatory role and mechanism in plant salt tolerance.

[0035] In view of this, a first aspect of the present invention provides a soybean late embryogenesis-rich protein gene, named GmLEA1, wherein the soybean late embryogenesis-rich protein gene is selected from:

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

[0037] (a2) and (a1) encode proteins with the same amino acid sequence, but are different nucleotide sequences due to the degeneracy of the genetic code.

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

[0039] A nucleotide sequence that is complementary to either (a4) or (a1)-(a3).

[0040] In this invention, GmLEA1 is located at position 55771363_55772209bp on soybean chromosome 1 (reference genome is Wm82.a2.v1, NCBI reference sequence is GCF_000004515.4(Glycine_max_v2.0)).

[0041] In another specific embodiment of the present invention, a protein is provided, which is encoded by the above-mentioned soybean late embryogenesis abundant protein gene, specifically soybean late embryogenesis abundant protein.

[0042] In another specific embodiment of the present invention, a recombinant expression vector is provided, the recombinant expression vector containing the above-mentioned soybean late embryogenesis abundant protein genes.

[0043] Furthermore, the recombinant expression vector is obtained by effectively linking a soybean late embryogenesis-rich protein gene to an expression vector. The expression vector can be any one or more of a viral vector, plasmid, phage particle, granule, or artificial chromosome. The expression vector can be a plant expression vector, which is not specifically limited here.

[0044] In another specific embodiment of the present invention, a transgenic cell line, host bacterium, or transgenic plant containing the aforementioned gene for abundant late embryogenetic proteins in soybean is provided.

[0045] The transgenic cell line may be isolated, in vitro, cultured, or preferably part of a plant; wherein the plant cell may be a crop cell, the crop is a dicotyledonous crop, and legumes, especially soybeans, are preferred, with soybeans being the most preferred.

[0046] The host bacteria may be eukaryotic bacteria (such as fungi, further such as yeasts) or prokaryotic bacteria (such as bacteria, further such as Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis or Bacillus pumilus).

[0047] In this invention, the transgenic plant is a transgenic crop, more preferably a dicotyledonous crop, among which legumes, especially soybeans, are preferred, and soybeans are the most preferred.

[0048] In another specific embodiment of the present invention, the above-mentioned soybean late embryogenesis-rich protein gene, soybean late embryogenesis-rich protein, recombinant expression vector containing the above-mentioned soybean late embryogenesis-rich protein gene, transgenic cell line, host bacterium or transgenic plant are provided for use in any one or more of the following:

[0049] (b1) Regulation of plant salt / alkali tolerance;

[0050] (b2) Identify salt-tolerant / alkali-tolerant plants;

[0051] (b3) Improve and cultivate salt / alkali tolerant plants.

[0052] Specifically, in (b1), the regulation of plant salt tolerance is manifested as follows: when the gene for abundant late embryogenesis protein in soybean is overexpressed in soybean, the dry / fresh weight of overexpressed soybean increases in a salt environment compared with the control group, thus indicating that the gene can improve / promote plant salt tolerance.

[0053] In (b2), GmLEA1-hap3 is a superior salt-tolerant haplotype soybean.

[0054] In (b3), the improvement and cultivation of plants specifically refers to the improvement and cultivation of plant varieties with salt and alkali tolerance. The plant is a crop, more preferably a dicotyledonous crop, and even more preferably a dicotyledonous crop, among which legumes, especially soybeans, are preferred, and soybeans are the most preferred.

[0055] In another specific embodiment of the present invention, a method for improving and cultivating salt / alkali tolerant plants is provided, the method comprising increasing the expression level and / or activity of the GmLEA1 gene in the target plant.

[0056] In the above method, the increase in the expression level and / or activity of the GmLEA1 gene in the target plant can be achieved by means such as introducing a recombinant expression vector containing the GmLEA1 gene, operably linking a strong promoter to the GmLEA1 gene, and introducing an enhancer, etc., without specific limitations.

[0057] The target plant can be any plant at any developmental stage, in particular, the plant is a crop, more preferably a dicotyledonous crop, and even more preferably a dicotyledonous crop, of which legumes, especially soybeans, are preferred, and most preferably soybeans.

[0058] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the materials, reagents, carriers, strains, etc., used in the following embodiments are all commercially available. Unless otherwise specified, the experimental methods described are conventional methods in the art.

[0059] Example 1: Cloning of GmLEA1 and Construction of Plant Overexpression Vector

[0060] 1.1 Extraction of total RNA from Williams 82

[0061] (1) Take an appropriate amount of soybean Williams 82 leaf material, freeze it with liquid nitrogen and grind it or grind it into powder using a sampler (22 rpm). Apply it directly to the RNA extraction experiment or freeze it in an ultra-low temperature freezer at -80℃ for later use. Pre-cool the centrifuge by 4 degrees in advance.

[0062] (2) After the liquid nitrogen evaporates, immediately transfer 100-200mg of plant powder into a 1.5ml centrifuge tube, then quickly add 1ml of Trizol extract, vortex to fully dissolve the sample in the extract, and let stand at room temperature for 10min.

[0063] (3) Centrifuge at 4℃, 12,000 rpm for 10 min, transfer 0.9 ml of supernatant to a new 1.5 ml centrifuge tube, add 0.2 ml of chloroform, shake vigorously for 15 sec, and let stand at room temperature for 5-10 min.

[0064] (4) Centrifuge at 4℃, 12,000 rpm for 10 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 place at room temperature for 15 min. If the sample volume is small, it can be placed at -20℃ for 10-20 min to help sedimentation.

[0065] (5) Centrifuge at 4℃, 12,000 rpm for 10 min, discard the supernatant, wash the precipitate twice with 1 ml of 75% ethanol, and centrifuge at 4℃, 8,000 rpm for 5 min.

[0066] (6) Discard the supernatant, open the lid and place the RNA in a clean bench for about 2-5 minutes to dry. Add 40 μl of RNase-Free water and dissolve the RNA completely at 60°C for 10 minutes.

[0067] (7) The OD value and concentration of the RNA sample were measured using a UV spectrophotometer.260 / A 280 A value of 1.7-2.0 is ideal; quality is determined by agarose gel electrophoresis.

[0068] 1.2 Reverse transcription of RNA

[0069] (1) Add the following substances sequentially to a centrifuge tube (40 μl reaction system):

[0070]

[0071] (2) After gently mixing, denature at 65°C for 5 minutes, then immediately insert into ice and ice bath for at least 1 minute;

[0072] (3) Add the following substances to the centrifuge tube in sequence.

[0073]

[0074] (4) After gently mixing, incubate at 42℃ for 1 hour, denature at 65℃ for 10 minutes, and store at -20℃ for later use.

[0075] 1.3 G mLEA1 CDS Cloning

[0076] GmLEA1 CDS-F: 5'-AAAAAAGCAGGCTCGATGGCGCCAATTCGAGTAGC-3'

[0077] GmLEA1 CDS-R: 5'-AGAAAGCTGGGTTTTAAGGAAGGGTGTGAGGCTT-3'(1) The reaction system for the first step amplification of the Gateway system using the high-fidelity enzyme Phanta is as follows (50 μl system):

[0078]

[0079] The amplification conditions are as follows:

[0080]

[0081] After the reaction was completed, the reaction solution was detected by 1% TAE agarose gel electrophoresis.

[0082] (2) Purification and recovery of cloned gene fragments (Novozymes kit)

[0083] 1) Place the gel containing the target fragment into a 1.5ml centrifuge tube and weigh the gel. Add an equal volume of sol solution and dissolve the gel at 60℃ for 5-10 minutes, turning it continuously during the dissolution process until it is completely dissolved.

[0084] 2) After the gel has completely melted, take ≤700uL of solution back into the column and let stand for 1 min;

[0085] 3) Centrifuge at 12,000 rpm for 30 seconds at room temperature, then discard the solution;

[0086] 4) Add 300 μL of sol solution to the recovery column, let stand for 1 min, then centrifuge at 12000 rpm for 1 min and discard the waste liquid;

[0087] 5) Add 700 μl of washing solution to the column, centrifuge at 12000 rpm for 1 min, discard the washing solution, and repeat the cleaning step once more.

[0088] 6) Empty column, centrifuge at 12000 rpm for 2 min;

[0089] 7) Open the recovery column and let it air dry for 1-2 minutes. Place it in a new, clean 1.5ml centrifuge tube, add 40μl of preheated sterile water or EB buffer at 60℃, and let it stand for 2 minutes.

[0090] 8) Centrifuge at 12000 rpm for 1 min. The resulting solution is the recovered fragment. After measuring the concentration, it can be used.

[0091] (3) The reaction system for the second step amplification of the Gateway system using the high-fidelity enzyme Phanta is as follows (50 μl system):

[0092] GmLEA1-F2: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCG-3'

[0093] GmLEA1-R2: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTT-3'

[0094]

[0095]

[0096] The amplification conditions are as follows:

[0097]

[0098] After the reaction was completed, the reaction solution was examined by 1% TAE agarose gel electrophoresis, and the correct target bands were cut and recovered.

[0099] 1.4 BP response of cloned gene fragments

[0100] The BP reaction (Gateway system) is as follows:

[0101]

[0102] React overnight at 25°C.

[0103] 1.5 Plasmid transformation of Escherichia coli (aseptic technique)

[0104] (1) The BP reaction ligation product was added to 50 μl of competent cells (Novozymes commercial competent cells).

[0105] In a centrifuge tube, gently tap to mix, then incubate on ice for 30 minutes.

[0106] (2) 42℃, warm water bath heat shock for 90 seconds, then immediately ice bath for 2-3 minutes;

[0107] (3) Add 1 ml of LB medium and incubate at 37°C with shaking at 200 rpm for 45-60 min;

[0108] (4) Centrifuge at 4000 rpm for 3 min at room temperature and collect the bacterial cells;

[0109] (5) Spread the bacteria on a culture plate containing the corresponding antibiotic and incubate it upside down at 37°C overnight.

[0110] 1.6 PCR verification of Escherichia coli

[0111] Single colonies of bacteria were picked from the culture dish and shaken for PCR verification using the Polymerex 2×M5 Hiper plus Taq HiFi PCR mix. The reaction system is as follows (20 μl system):

[0112]

[0113] The amplification conditions are as follows:

[0114]

[0115] 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.

[0116] 1.7 Recombinant plasmid sequencing

[0117] Positive single colonies with correct PCR bands were selected and cultured overnight in liquid LB medium containing kanamycin (25 mg / L) at 37°C and 200 rpm in a shaker. The colonies were then sent for sequencing using universal primers M13-F and M13-R. The sequencing results were obtained as shown in SEQ ID NO.1 of the sequence listing.

[0118] If the sequencing results are compared with the standard sequence and the overlap is 100%, it indicates that the recombinant intermediate vector has been successfully constructed. The nucleotide sequence of the cDNA of the gene GmLEA1 is shown in SEQ ID NO.1. The intermediate vector can then be used for subsequent LR experiments.

[0119] 1.8 Extraction of E. coli plasmid DNA

[0120] (1) Select the successful positive single colony and inoculate it into 10 ml of LB liquid medium containing the corresponding antibiotic kanamycin, and incubate it in a shaker at 37℃ and 200 rpm for 8 h.

[0121] (2) Centrifuge at 12,000 rpm for 1 min at room temperature, collect the bacterial cells, and extract plasmids using the Novizan FastPure Plasmid MiniKit plasmid mini-prep kit;

[0122] (3) Discard the supernatant, add 250 μl of pre-cooled solution I, and vortex to completely resuspend the cells;

[0123] (4) Add 250 μl of solution II and quickly invert to mix 6-8 times;

[0124] (5) Add 350 μl of solution III and mix by inverting the container 6-8 times;

[0125] (6) Centrifuge at 12000 rpm for 10 min;

[0126] (7) Place the FastPure DNA Mini Columns adsorption column into a ColLEAtion Tube 2ml 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.

[0127] (8) Add 600 μl of Buffer PW2 (please check that it has been diluted with anhydrous ethanol) 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;

[0128] (9) Repeat step 7;

[0129] (10) Place the adsorption column back into the collection tube. Centrifuge at 12,000 rpm for 1 min to dry the adsorption column, the purpose of which is to completely remove the residual washing solution in the adsorption column;

[0130] (11) Place the adsorption column into a new sterile 1.5 ml centrifuge tube. Add 30-100 μL of Elution Buffer to the center of the membrane of the adsorption column. 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.

[0131] 1.9 LR response of cloned gene fragments

[0132] The LR reaction (Gateway system) structure is as follows:

[0133]

[0134] React at 25℃ for 8 hours.

[0135] 1.10 E. coli plasmid transformation (same as 1.5)

[0136] 1.11 PCR verification of E. coli (same as 1.6)

[0137] 1.12 Extraction of E. coli plasmid DNA (same as 1.8)

[0138] Verify the cloning region of the positive pB7GWF2::GmLEA1 plasmid transformed into the pB7GWF2 vector.

[0139] 1.13 Plasmid transformation of Agrobacterium (aseptic technique)

[0140] (1) Add 2 μl of plasmid DNA to 50 μl of commercially available K599 Agrobacterium competent cells, gently tap the centrifuge tube to mix, and incubate on ice for 5 min;

[0141] (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;

[0142] (3) Add 1 ml of YEP medium and incubate at 28℃ for 2-4 h;

[0143] (4) Centrifuge at 5,000 rpm for 3 min at room temperature and collect the bacterial cells;

[0144] (5) Spread the bacteria on LB culture plates containing the corresponding antibiotics and incubate them upside down at 28°C for 48 hours.

[0145] 1.14 PCR verification of Agrobacterium

[0146] The reaction system is as follows (20 μl system):

[0147]

[0148] The amplification conditions are as follows:

[0149]

[0150] After the reaction was completed, the reaction solution was detected by 1% TAE agarose gel electrophoresis.

[0151] Example 2: Verification of salt tolerance function by overexpressing GmLEA1 in hairy roots

[0152] 2.1 Methods for transforming soybean hairy roots

[0153] (1) Wild-type soybean W82 is planted in the soil and grows for 4-5 days;

[0154] (2) Make a cut at a 45° angle at the hypocotyl about 2 cm away from the cotyledon to form a wound;

[0155] (3) Spread Agrobacterium rhizogenes K599 containing the target recombinant plasmid on a solid culture medium plate, spread Agrobacterium rhizogenes on the cut soybean hypocotyl, and then place it on a moistened filter paper and co-culture overnight in the dark.

[0156] (4) The explants are transferred into vermiculite. Non-positive roots are removed once a week. Callus tissue grows at the wound site in about ten days. Soybean hairy roots are formed after two weeks. Subcellular localization and other signals can be observed. After about twenty days, composite plants with similar growth in the above-ground and underground parts can be selected for subsequent phenotypic experiments.

[0157] (5) Hairy root transformation was carried out according to the above method. Hairy roots of pB7GWF2::GmLEA1 overexpression plants (OE) and their empty vector control (EV) were transformed respectively. When the hairy roots grew vigorously, groups of 3 hairy roots with consistent aboveground parts and good growth were selected and transplanted into vermiculite. Nutrient solution was irrigated. The composition of the nutrient solution is shown in the table below:

[0158] Preparation of soybean hairy root nutrient solution (1L):

[0159]

[0160] Add 500 μl of the above solution to each liter of nutrient solution.

[0161] 2.2 Salt tolerance phenotype experiment of soybean hairy roots

[0162] Once the compound plants have developed trifoliate leaves, they can be treated with saline solution. The phenotypes of the pB7GWF2::GmLEA1 overexpressing plants (OE) and their empty control (EV) plants are observed after treatment with water and 150mM NaCl solution, respectively.

[0163] Results: Under water treatment, soybean hairy root complex plants overexpressing pB7GWF2::GmLEA1 showed good growth and no significant phenotypic difference compared to the unloaded control. After 7 days of treatment with 150mM NaCl, the wilting degree of pB7GWF2::GmLEA1-overexpressing soybean hairy root complex plants (OE) was lower than that of the unloaded control (EV) (see...). Figure 3 The fresh and dry weights of the plants were also significantly higher than those of the control group (see...). Figure 4 (* indicates that the result was obtained from Student's t-test, p<0.05, n=3), proving that this gene positively regulates plant salt tolerance during the plant's salt stress response.

[0164] Example 3: Haplotype Analysis and Phenotyping of Salt Tolerance Phenotyping

[0165] 3.1 Haplotype Analysis

[0166] To further investigate the function of GmLEA1, haplotype analysis was performed on the gene and its promoter region's SNPs and InDel values ​​from a germplasm population comprising 371 cultivated soybean accessions, 111 local soybean accessions, and 56 wild soybean accessions. The analysis revealed five main haplotypes, Hap1-Hap5. Specifically, SNP 1 showed a C / T substitution at 55,771,363 bp; SNP 2 showed an A / G substitution at 55,771,516 bp; SNP 3 showed an A / G substitution at 55,771,522 bp; SNP 4 showed a T / A substitution at 55,771,647 bp; SNP 5 showed an A / T substitution at 55,771,689 bp; and SNP 6 showed an A / G substitution at 55,771,697 bp. SNP 7 shows a G / A substitution at 55,771,748 bp; SNP 8 shows an A / G substitution at 55,771,752 bp; SNP 9 shows a G / A substitution at 55,771,792 bp; SNP 10 shows a C / G substitution at 55,771,856 bp; SNP 11 shows a C / T substitution at 55,771,872 bp; SNP 12 shows a G / C substitution at 55,772,086 bp; SNP 13 shows a T / A substitution at 55,772,106 bp; SNP 14 shows a G / A substitution at 55,772,132 bp; SNP 15 shows a T / C substitution at 55,772,140 bp; SNP... At bp 55, 772, 209, a G / T substitution occurred. The results are shown in Table 1.

[0167] Table 1

[0168]

[0169] 3.2 Salt tolerance phenotypic data of soybean varieties

[0170] Salt tolerance phenotypic data of soybeans in this germplasm population were collected in the saline-alkali land of Dongying. This study focused on the 100-grain weight and single-plant grain weight under salt stress, which are related to soybean yield.

[0171] 3.3 Association analysis between different soybean haplotypes and salt tolerance phenotypes

[0172] The results showed that the grain weight per plant and the 100-grain weight of Hap3 soybean under salt conditions were significantly higher than those of other haplotype soybeans. Figure 5The presence of a haplotype with excellent salt tolerance further illustrates the role of GmLEA1 in regulating soybean salt tolerance.

[0173] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A soybean late embryogenesis-rich protein gene, named GmLEA1, characterized in that, The soybean late embryogenetic abundant protein gene is selected from: (a1) The nucleotide sequence shown in SEQ ID NO.1; (a2) and (a1) encode proteins with the same amino acid sequence, but are different nucleotide sequences due to the degeneracy of the genetic code. (a3) has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence that has the same or similar functional protein; A nucleotide sequence that is complementary to either (a4) or (a1)-(a3).

2. A protein, characterized in that, The protein is encoded by the soybean late embryogenesis-rich protein gene as described in claim 1, specifically the soybean late embryogenesis-rich protein.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the soybean late embryogenesis-rich protein gene as described in claim 1.

4. A transgenic cell line, host bacterium, or transgenic plant containing the soybean late embryogenesis-rich protein gene as described in claim 1.

5. The transgenic cell line, host bacterium, or transgenic plant as described in claim 4, characterized in that, The transgenic cell line is isolated, in vitro, cultured, or preferably part of a plant; wherein the plant cell is a crop cell, the crop is a dicotyledonous crop, and legumes, especially soybeans, are preferred, with soybeans being the most preferred. The host bacteria are eukaryotic bacteria (including fungi) or prokaryotic bacteria (including bacteria). The genetically modified plant is a genetically modified crop, more preferably a dicotyledonous crop, among which legumes, especially soybeans, are preferred, with soybeans being the most preferred.

6. The use of the soybean late embryogenesis-rich protein gene of claim 1, the protein of claim 2, the recombinant expression vector of claim 3, or the transgenic cell line, host bacterium, or transgenic plant containing the soybean late embryogenesis-rich protein gene of any one of claims 4-5, in any one or more of the following: (b1) Regulation of plant salt / alkali tolerance; (b2) Identify salt-tolerant / alkali-tolerant plants; (b3) Improve and cultivate salt / alkali tolerant plants.

7. The application as described in claim 6, characterized in that, In (b1), the regulation of plant salt tolerance is specifically manifested as follows: overexpression of the soybean late embryogenesis-rich protein gene in soybeans will improve / promote the plant's salt and alkali tolerance.

8. The application as described in claim 6, characterized in that, In (b3), the improvement and cultivation of plants specifically refers to the improvement and cultivation of plant varieties with salt and alkali tolerance.

9. A method for improving and cultivating salt / alkali-tolerant plants, characterized in that, The method includes increasing the expression level and / or activity of the GmLEA1 gene as described in claim 1 in the target plant.

10. The method as described in claim 9, characterized in that, The target plant is a crop, more specifically a dicotyledonous crop, with legumes, especially soybeans, being preferred, and soybeans being the most preferred.

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