Application of soybean malate dehydrogenase in regulating plant stress resistance

Abstract

The present invention belongs to the field of biological breeding technology, and specifically relates to the application of soybean malate dehydrogenase in regulating plant stress resistance. The technical problem to be solved by the present invention is: how to improve the stress resistance of soybean, for example, how to improve the salt tolerance of soybean. To solve this technical problem, the present invention provides the application of protein or a substance that regulates gene expression or a substance that regulates the activity or content of the protein in regulating plant salt tolerance, the gene encodes the protein, and the protein may be a GmMDH2 protein; the GmMDH2 protein is a protein whose amino acid sequence is shown in positions 1-352 of SEQ ID No.1. The results of the present invention show that GmMDH2 can significantly improve the salt resistance of transgenic plants, and GmMDH2 can be used as a salt-tolerant gene to cultivate soybean stress-resistant varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of biological breeding, and particularly relates to the application of soybean malate dehydrogenase in regulating plant stress resistance. Background Art

[0002] Malate dehydrogenase (MDH) is widely present in various organisms. MDHs in plants play a vital role in seed germination, plant growth, pollen and fruit development, and responses to abiotic stresses. Malate dehydrogenase is mainly involved in the tricarboxylic acid cycle (TCA), catalyzing the reversible conversion between malate and oxaloacetate. Different MDHs rely on NADH or NADPH as coenzymes to complete the reaction in this process. Therefore, MDH is generally divided into NAD-dependent malate dehydrogenase and NADP-dependent malate dehydrogenase. In plants, MDH is also involved in many other physiological and biochemical reactions, such as carbon fixation, nitrogen assimilation, and fatty acid oxidation in C4 plants. MDH in plants can be further subdivided according to the different metabolic pathways involved and the places where they play a role. [6] , which can generally be divided into five categories: (1) NAD-dependent MDH (cyMDH) in the cytoplasm; (2) mitochondrial NAD-dependent MDH (mMDH) as part of TCA; (3) NAD-dependent MDH in peroxisomes and MDH in uronidase involved in photorespiration, also known as microsomal malate dehydrogenase (gMDH); (4) NADP-dependent MDH in chloroplasts; and (5) chloroplast NAD-dependent MDH.

[0003] MDH can catalyze oxaloacetate to produce malate, making NAD + Generate NADH to provide reducing power for the nitrate reduction process [7]NADH produced by biological oxidation in the cytoplasm cannot pass through the inner membrane of mitochondria, but NADH can transfer hydrogen to oxaloacetate to generate malate under the action of MDH, which can be transported to the mitochondrial matrix through the malate-aspartate shuttle system. The malate in the mitochondrial matrix is ​​reoxidized to oxaloacetate and NADH, and then oxidized through the respiratory chain. With the deepening of the study of MDH genes, it has been found that MDH genes can also respond to abscisic acid (ABA) treatment and salt stress. Some studies have shown that MDH genes can be used to improve the low temperature and salt tolerance of plants. Guo et al. isolated SbNADP-ME from sweet sorghum (Sorghum dochna). Overexpression of SbNADP-ME in Arabidopsis thaliana can improve photosynthetic capacity and improve the salt tolerance of Arabidopsis thaliana by alleviating the photoinhibition of PS II and PS I under salt stress. The coding genes of NADP-dependent MDH in some plants are induced to express under low temperature treatment, and the content of malic acid decreases when the plants are subjected to low temperature. This shows that the main function of NADP-dependent MDH in response to low temperature stress in plants is not to synthesize malic acid, but there are other regulatory matrices. One possible mechanism is that NADP-dependent MDH, as a hydrophilic protein, combines with free water during low temperature stress in plants to reduce the degree of damage caused by water freezing, thereby reducing the phase change of some cell membrane structures at low temperatures and enhancing the stability of the membrane. At present, there are few reports on the MDH family in soybeans. Therefore, it is necessary to pay attention to the performance of the soybean MDH gene family under adversity. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to improve the stress resistance of soybeans, for example, how to improve the salt tolerance of soybeans.

[0005] The technical problem to be solved by the present invention is: how to regulate the stress resistance of plants, for example, how to regulate the salt tolerance and / or alkali tolerance of soybeans.

[0006] To solve the above technical problems, in a first aspect, the present invention provides the use of a protein or a substance regulating gene expression or a substance regulating the activity or content of the protein in any of the following items, wherein the gene encodes the protein, and the protein may be a GmMDH2 protein;

[0007] A1) Application in regulating plant stress resistance;

[0008] A2) Application in the preparation of products for regulating plant stress resistance;

[0009] A3) Application in regulating plant salt tolerance;

[0010] A4) Application in the preparation of products for regulating plant salt tolerance;

[0011] A5) Application in plant breeding or plant assisted breeding;

[0012] The GmMDH2 is any one of the following proteins a1)-a3):

[0013] a1) the protein whose amino acid sequence is the protein shown in positions 1 to 352 of SEQ ID No. 1 or the protein whose amino acid sequence is the protein of SEQ ID No. 1;

[0014] a2) a protein having more than 80% identity with the amino acid sequence shown in a1) and related to plant soybean malate dehydrogenase, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1);

[0015] a3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

[0016] Furthermore, in the application, the substance regulating the activity or content of the protein may be a substance that upregulates, improves or promotes the gene encoding the protein and / or a substance that upregulates, improves or promotes the expression of the gene encoding the protein. The regulation of plant stress resistance or / and salt tolerance or / and alkali tolerance may be to improve plant stress resistance or / and salt tolerance or / and alkali tolerance.

[0017] The purpose of the plant breeding may be to cultivate target plants with improved stress resistance, such as cultivating plants with improved salt tolerance.

[0018] In the present invention, the plant with improved salt tolerance may be specifically manifested in that the root length and biomass of the underground part of soybean under salt stress conditions are significantly higher than those of wild-type plants.

[0019] Furthermore, in the application, the GmMDH2 protein is derived from soybean.

[0020] SEQ ID No. 1 consists of 595 amino acid residues.

[0021] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0022] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0023] Furthermore, in the application, the substance that regulates the expression of the protein encoding gene or the substance that regulates the activity or content of the protein is a biological material, and the biological material can be any of the following:

[0024] B1), nucleic acid molecule encoding the above protein

[0025] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0026] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0027] B4), a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0028] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0029] B6), transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0030] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0031] Furthermore, in the application, the expression cassette described in B2) refers to a DNA capable of expressing the GmMDH2 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of a gene encoding the GmMDH2 protein, but also a terminator for terminating transcription of a gene encoding the GmMDH2 protein.

[0032] Furthermore, in the application, B2) the expression cassette may further include an enhancer sequence.

[0033] Furthermore, in the application, the recombinant vector in B3) may contain a DNA molecule encoding the GmMDH2 protein as shown in SEQ ID No. 2.

[0034] Furthermore, in the application, the recombinant microorganism in B4) can specifically be yeast, bacteria, algae and fungi.

[0035] Furthermore, in the application, the plant tissue in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0036] Furthermore, in the application, the transgenic plant organ in B7) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0037] Furthermore, in the application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ described in B5) may or may not include propagation materials.

[0038] Furthermore, in the application, the nucleic acid molecule in B1) may be a DNA molecule as shown in any one of g1) to g3) below:

[0039] g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID No.2;

[0040] g2), a DNA molecule whose coding sequence of the coding strand is SEQ ID No.3;

[0041] g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and encodes soybean malate dehydrogenase.

[0042] Further, in the application, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gapcost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the identity value (%) can be obtained.

[0043] In the above applications, the above 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0044] Furthermore, in the application, the plant is selected from any one of the following:

[0045] C1), dicotyledonous plants;

[0046] C2), Leguminosae;

[0047] C3) Soybean

[0048] C4) soybeans.

[0049] In a second aspect, the present invention provides the protein in the above application and / or the biomaterial in the above application.

[0050] In a third aspect, the present invention provides a method for regulating plant stress resistance and / or salt tolerance, the method comprising regulating the stress resistance and / or salt tolerance of the recipient plant by regulating the expression of the GmMDH2 protein in the recipient plant or regulating the activity or content of the GmMDH2 protein.

[0051] Furthermore, the method comprises promoting or improving the stress resistance and / or salt tolerance of the recipient plant by up-regulating or increasing or promoting the expression of the GmMDH2 protein or the activity or content of the GmMDH2 protein in the recipient plant.

[0052] Furthermore, in the method, the plant is selected from any one of the following:

[0053] C1), dicotyledonous plants;

[0054] C2), Leguminosae;

[0055] C3) Soybean

[0056] C4) soybeans.

[0057] In a fourth aspect, the present invention provides a method for preparing stress-resistant and / or salt-tolerant soybeans, the method comprising promoting or improving the stress resistance and / or salt tolerance of the recipient soybeans by up-regulating or increasing or promoting the expression of the GmMDH2 protein or the activity or content of the GmMDH2 protein in the recipient soybeans, thereby obtaining the target soybeans having higher stress resistance and / or salt tolerance than the recipient plants.

[0058] Furthermore, the method includes introducing the encoding gene of the above-mentioned GmMDH2 protein into the recipient soybean to improve or promote the expression of the GmMDH2 protein encoding gene in the recipient soybean or to improve or promote the activity or content of the GmMDH2 protein in the recipient soybean, thereby obtaining the target soybean with higher stress resistance and / or salt tolerance than the recipient soybean.

[0059] In the present invention, the target soybean with improved stress resistance is specifically manifested in that the root length and the biomass of the underground part of the soybean under salt stress conditions are significantly higher than those of the wild type.

[0060] The technical effects achieved by the present invention are as follows:

[0061] In this study, GmMDH2 (Glyma.01G210400), a member of the soybean MDH gene family, was used as the research object, and DN50 was used as the receptor for hairy root transformation experiments. Two transgenic plants, OE-6 and OE-7, were selected for salt tolerance function identification. Using DN50 (CK) as the control, the changes in root length and fresh weight of GmMDH2 transgenic soybeans under salt stress were studied. The results showed that under salt stress conditions, the fresh weight of the roots of GmMDH2 transgenic soybeans was significantly higher than that of DN50, and the root length growth of the transgenic lines was significantly better than that of the control. These results indicate that GmMDH2 can significantly improve the salt resistance of transgenic plants, and GmMDH2 can be used as a salt tolerance gene to breed soybean stress-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Analysis of GmMDH2 gene expression pattern.

[0063] Figure 2 This is a map of the pSOY1-GmMDH2 fusion expression plasmid.

[0064] Figure 3 PCR verification of transgenic GmMDH2 hairy roots

[0065] Figure 4 These are photos of wild-type soybean and transgenic soybean plants being tested for stress tolerance.

[0066] Figure 5 Statistical results of root length of wild-type soybean and transgenic soybean under salt stress.

[0067] Figure 6 Statistical results of fresh weight of wild-type soybean and transgenic soybean under salt stress. DETAILED DESCRIPTION

[0068] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0069] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0070] The pSOY1 vector is preserved by this laboratory and is described in the document "Ying Zhao. etc. GmGPDH12, amitochondrial FAD-GPDH from soybean, increases salt and osmotic stress resistance by modulating redox state and respiration. The Crop Journal. Volume 9, Issue 1, February 2021, Pages 79-94." The public can obtain the above-mentioned biological material from the applicant. The obtained above-mentioned biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0071] Soybean seeds Dongnong 50 (also known as DN50) are kept in our laboratory and are described in the literature “Ying Zhao etc. Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD + -dependent glycerol-3-phosphate dehydrogenase in soybean. Journal of Integrative Plant Biology (IF9.106) Pub Date: 2021-03-26, DOI: 10.1111 / jipb.13094" (recorded as DN50 in the document). The public can obtain the above-mentioned biomaterial from the applicant. The obtained biomaterial can only be used for the verification of the embodiments of this application and cannot be used for other purposes.

[0072] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0073] The following examples were statistically analyzed using SPSS 16.0 (SPSS Inc, Chicago, IL, USA). One-way ANOVA was used to compare whether the differences between different strains (CK, OE-6 and OE-7) under the same treatment conditions were significant (one-way ANOVA), * indicates significant difference (P < 0.05), ** indicates extremely significant difference (P < 0.01), and *** indicates extremely significant difference (P < 0.001).

[0074] Example 1. Expression pattern of GmMDH2 gene in soybean leaves under abiotic stress

[0075] The nucleotide sequence of the soybean malate dehydrogenase gene (GmMDH2) genome is SEQ ID No. 3, its coding sequence is SEQ ID No. 2, and the amino acid sequence of the encoded soybean malate dehydrogenase is SEQ ID No. 1, positions 1 to 352 (Table 1).

[0076] Table 1 Sequence information of soybean malate dehydrogenase

[0077]

[0078]

[0079]

[0080]

[0081] 1.1 Test methods

[0082] DN50 seeds were sown on vermiculite and cultured in a culture room at 22°C for 4 days before being hydroponically cultured. After the first trifoliate leaf was fully expanded, the seeds were cultured with 120 mM NaCl, 100 mM NaHCO 3 Soybean (DN50) seedlings were subjected to stress treatment with 20% PEG and 200 mM mannitol, and leaves were sampled at different time points (0 h, 1 h, 3 h, 6 h, 12 h, 24 h) after treatment, frozen in liquid nitrogen, and stored at -80°C for RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen).

[0083] Reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). Real-time quantitative PCR was performed using the Bio-Rad Chromo4 real-time PCR system. The reaction mixture contained 12.5 μl of 2×SYBR Green Realtime PCR Master Mix (Toyobo), 0.5 μM of each upstream and downstream primer, 2 μl of cDNA template (equivalent to 100 ng of RNA), and a total volume of 15 μl. The reaction conditions were: 95°C, 30 s; 40 cycles: 95°C, 10 s; 60°C, 30 s; finally 95°C, 15 s; 60°C, 1 min; 95°C, 15 s. Soybean Actin4 was used as an internal reference to normalize the data. The relative expression level was measured using 2 -△△CT Methods Calculation. The expression level of GmMDH2 in 0h leaves was set as 1, and the other treatments were calculated accordingly. Three independent biological replicates were performed for each sample, and three technical replicates were performed each time. All primers are shown in Table 2.

[0084] Among them, hydroponics is hydroponics of soybean seedlings using distilled water;

[0085] Use 120 mM NaCl, 100 mM NaHCO 3 , 20% PEG and 200mM mannitol were used to stress soybean (DN50) seedlings:

[0086] 1) Replace distilled water with distilled water containing 120 mM NaCl by adding NaCl to distilled water to a final concentration of 120 mM NaCl;

[0087] 2) Replace distilled water with 100 mM NaHCO 3 The preparation method is as follows: add 100 mM NaHCO 3 To a final concentration of 100 mM NaHCO 3 ;

[0088] 3) replacing distilled water with distilled water containing 20% ​​PEG, the preparation method is: adding PEG to distilled water to a final concentration of 20% PEG (mass concentration);

[0089] 4) replacing distilled water with distilled water containing 200 mM mannitol, the preparation method is: adding mannitol to distilled water to a final concentration of 200 mM mannitol;

[0090] The time after the medium replacement was completed was set to 0.

[0091] Table 2 Real-time quantitative PCR primer sequences

[0092]

[0093] 1.2 Expression pattern analysis

[0094] Real-time quantitative PCR was used to analyze the expression of GmMDH2 gene in response to salt, alkali, drought and osmotic stress (120 mM NaCl, 100 mM NaHCO 3 , 20% PEG and 200 mM mannitol). Figure 1 The results show that GmMDH2 responds to different stresses in leaves, such as NaCl, NaHCO 3 After 3 h of mannitol stress treatment and 1 h of PEG6000 stress treatment, the GmMDH2 gene was significantly upregulated in leaves. 3The expression intensity of GmMDH2 gene reached the maximum value after 12h treatment; after 6h treatment with PEG6000; after 3h treatment with mannitol. The above results show that GmMDH2 can be induced to express in leaves to varying degrees by salt, alkali, drought and osmosis, indicating that GmMDH2 is an important regulatory factor in soybean adaptation to abiotic stress.

[0095] Example 2: Experimental method for transforming soybean hairy roots with GmMDH2

[0096] 2.1 Test methods

[0097] The coding sequence of the GmMDH2 gene cloned from soybean (SEQ ID No. 2) was constructed into the pSOY1 vector to form the pSOY1-GmMDH2 fusion expression plasmid. The pSOY1-GmMDH2 fusion expression plasmid was introduced into the Agrobacterium K599 strain by Agrobacterium transformation, and the resistant clone plasmid DNA was confirmed as an expression vector containing the target gene by restriction enzyme digestion map.

[0098] The map of the pSOY1-GmMDH2 fusion expression plasmid is as follows Figure 2 As shown, its structure is: a recombinant expression vector is obtained by replacing the BlsI and EcoRI restriction recognition sites of the pSOY1 vector with a DNA molecule having a nucleotide sequence of SEQ ID No.2, while keeping the other nucleotide sequences of the pSOY1 vector unchanged. The pSOY1-GmMDH2 fusion plasmid can express the fusion protein GmMDH2-GFP having an amino acid sequence of SEQ ID No.1. Among them, the 1st to 352nd positions of SEQ ID No.1 are the amino acid sequence of soybean malate dehydrogenase (GmMDH2), and the 357th to 595th positions are the amino acid sequence of GFP.

[0099] (1) Preparation of Agrobacterium tumefaciens culture solution

[0100] Pick a single colony of the Agrobacterium strain containing the target gene, spread it on a solid TY plate containing spectinomycin, and culture it in the dark at 28℃ for 2-3 days. Pick a single colony and inoculate it in 3ml of TY liquid medium containing 50μg / ml spectinomycin, 200rpm, 28℃, and culture it overnight. Take out the bacterial solution with an inoculum volume of 1:100 for secondary activation. When the bacterial solution concentration reaches OD600nm=0.8, centrifuge it at 5000rpm for 10min and discard the supernatant. Resuspend the bacteria in liquid co-culture medium to OD600nm=0.6, and use it as the bacterial solution for transformation.

[0101] The formula of TY liquid medium is: 5g tryptone, 3g yeast extract, CaCl 20.4 g, dilute to 1 L with water. Add 15 g agar to the TY liquid medium to prepare the TY solid medium.

[0102] (2) Soybean sterilization method

[0103] 100 seeds of Dongnong 50 harvested that year with full color and no disease spots were selected and placed in a 150mL conical flask. The surface was sterilized with 75% ethanol for 5 minutes. The ethanol was removed and 15% hydrogen peroxide was added for sterilization. The conical flask was placed in a 28℃ shaker at 100rpm for 15 minutes. In the clean bench, it was repeatedly washed five times with sterilized water to wash away the residual liquid.

[0104] Add 100 mL of sterilized water to the sterilized Dongnong 50 seeds and culture them in a dark room at 4°C for 2 days.

[0105] (3) Soybean hairy root transformation method

[0106] 1) Explant preparation: In an ultra-clean workbench, use a scalpel to cut off 1 / 3 of the hypocotyl of the soybean and remove the outer skin of the soybean to complete the explant preparation.

[0107] 2) Preparation of Agrobacterium infection solution: Take out the bacterial solution from the 4°C refrigerator and centrifuge it at 25°C, 4000 rpm for 10 min. Remove the supernatant and resuspend the bacterial block with the prepared LCCM medium. The concentration of the bacterial solution is controlled at OD600 of about 0.5 and stand at room temperature for use.

[0108] The formula of LCCM medium is MES 3.9g; B5 salt powder 0.32g; Sucrose 30g; adjust pH to 5.4. Make up to 1L with water. Sterilize in an autoclave at 121℃ for 20min. When using, add 1mL of B5 vitamin (1mg / mL); 1.67mL of 6-BA (1mg / mL); 250μL of GA3 (1mg / mL); 1mL of acetosyringone (AS 40mg / mL); 4mL of cysteine ​​(L-cys 100mg / mL); 1mL of dithiothreitol (DTT 154.2mg / mL) to each liter of medium.

[0109] 3) Explant infection: Place the prepared explants in a conical flask, add Agrobacterium infection solution, and let stand at room temperature for 30-60 minutes.

[0110] 4) Explant co-culture: Use sterilized absorbent paper to absorb the residual liquid on the surface of the infected explants. Spread the sterilized filter paper on the LCCM solid culture medium and evenly place the explants on the LCCM culture medium in the order of 3, 4, 4, 3. Culture at 23℃ in the dark for 4-5 days.

[0111] Among them, LCCM solid culture medium is supplemented with 6.5g / L agar on the basis of LCCM liquid culture medium.

[0112] 5) Inducing transgenic hairy roots: Select the explants after co-cultivation, select the explants with elongated hypocotyls and obvious swell at the incision, insert the hypocotyls of the explants into SIM solid culture medium, and culture the tissue culture bottles in an environment of 25°C, 16h light and 8h dark.

[0113] The formula of SIM solid culture medium is MES 3.9g; B5 salt powder 0.32g; Sucrose 30g; adjust pH to 5.4. Make up to 1L and add Agar 6g. Sterilize in an autoclave at 121℃ for 20min. When using, add B5 vitamin (5.6mg / mL) 1mL; 6-BA (1mg / mL) 1.67mL; GA3 (1mg / mL) 250μL; acetosyringone (40mg / mL) 1mL; cysteine ​​(L-cys 100mg / mL) (100mg / mL) 4mL; dithiothreitol (DTT 154.2mg / mL) 1mL to each liter of culture medium.

[0114] 6) Verification of transgenic hairy roots: Select hairy roots with better growth (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7 and OE-8), cut part of the hairy roots and grind them, extract DNA and select specific primers for verification.

[0115] Table 3 PCR detection primer sequences

[0116]

[0117] 2.2 Experimental results The PCR verification results showed that the 1056 bp GmMDH2 specific fragment was amplified in the hairy roots of the eight transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7 and OE-8), while the WT plant did not amplify the specific fragment ( Figure 3 ).

[0118] Example 3: Root length experiment and root fresh weight experiment

[0119] 3.1 Test methods

[0120] The GmMDH2 overexpressing plants and DN50 transformed with hairy roots were transferred from the culture medium to a hydroponic box for hydroponics. After 7 days of culture in a culture room at 22°C, a control group (also called Control or 0Mm NaCl) and a salt stress treatment group (also called NaCl or 120Mm NaCl) were set up. The control group was treated with water and 120mM NaCl to replace the hydroponic solution and continue to be cultured for 4 days; the salt stress treatment group replaced the hydroponic solution with 120mM NaCl and continued to be hydroponics for 4 days. After 4 days of treatment, the root length of each strain under NaCl treatment conditions was photographed and measured, and the fresh weight of the underground part of different strains was counted. The experiment was repeated three times independently, and 3 plants of each plant were planted in each repetition.

[0121] 3.2 Root length test results

[0122] The results are as follows Figure 4 and Figure 5 As shown, Figure 4 These are the stress tolerance test results of wild-type soybean and genetically modified soybean. Figure 5 The statistical results of root length of wild type soybean and transgenic soybean under salt stress are shown in Figure 2. Figure 4 and Figure 5 In the figure, WT represents wild-type Dongnong 50, and OE6 and OE7 represent two different overexpression transgenic lines OE-6 and OE-7. The results showed that there was no difference in root growth between the transgenic lines and the control lines under normal conditions. However, under stress conditions, the root length of the transgenic lines was significantly longer than that of the control, indicating that GmMDH2 is involved in regulating the growth of plant roots under salt stress. The root length experiment further showed that GmMDH2 can improve soybean tolerance to salt stress.

[0123] 3.3 Changes in fresh weight of underground parts of GmMDH2 transgenic lines under salt stress

[0124] The results are as follows Figure 6 As shown, Figure 6 The fresh weight statistics of wild-type soybean and transgenic soybean under salt stress are shown in Figure 2. Figure 6 WT represents the wild type Dongnong 50, and OE6 and OE7 represent the overexpression transgenic lines OE-6 and OE-7 prepared in Example 2. The results showed that there was no difference in the root growth of the transgenic lines and the control lines under normal conditions. However, under stress conditions, the root fresh weight and growth of the transgenic lines were significantly higher than those of the control (DN50), from which we can conclude that the overexpression of the GmMDH2 gene improves the salt tolerance of soybean in the seedling growth stage.

[0125] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance regulating gene expression or a substance regulating the content of the protein in any of the following items, wherein the gene encodes the protein, and the protein is GmMDH2 protein; A1) Application in regulating soybean salt tolerance; A2) Application in the preparation of products for regulating the salt tolerance of soybeans; A3) Use in soybean breeding or soybean assisted breeding, wherein the purpose of soybean breeding is to cultivate soybeans with improved salt tolerance; The GmMDH2 protein is a protein whose amino acid sequence is shown in positions 1 to 352 of SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The substance that regulates the expression of the protein encoding gene or the substance that regulates the protein content is a biological material, and the biological material is any one of the following: B1), a nucleic acid molecule encoding the protein according to claim 1 B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4), a recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6), transgenic plant tissue containing the nucleic acid molecule described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7), a transgenic plant organ containing the nucleic acid molecule described in B1), a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is a DNA molecule as shown in any one of g1) to g3) below: g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID No.2; g2), a DNA molecule whose coding sequence of the coding strand is SEQ ID No.3; g3) A DNA molecule that has more than 80% identity with the DNA molecule described in g1) or g2) and encodes soybean malate dehydrogenase.

4. A method for regulating soybean salt tolerance, characterized in that: The method comprises regulating the salt tolerance of the recipient soybean by regulating the expression of the GmMDH2 protein in the recipient soybean or regulating the content of the GmMDH2 protein, wherein the GmMDH2 protein is a protein whose amino acid sequence is shown in positions 1 to 352 of SEQ ID No.

1.

5. The method according to claim 4, characterized in that: The method comprises promoting or increasing the salt tolerance of the recipient soybean by up-regulating, increasing or promoting the expression of the GmMDH2 protein or the content of the GmMDH2 protein in the recipient soybean.

6. A method for preparing salt-tolerant target soybeans, the method comprising promoting or improving the salt tolerance of the recipient soybeans by up-regulating, increasing or promoting the expression of the GmMDH2 protein or the content of the GmMDH2 protein in the recipient soybeans, and obtaining target soybeans with higher salt tolerance than the recipient soybeans, wherein the GmMDH2 protein is a protein whose amino acid sequence is shown in positions 1-352 of SEQ ID No.

1.

7. The method according to claim 6, characterized in that: The method comprises introducing the encoding gene of the GmMDH2 protein into a recipient soybean to increase or promote the expression of the encoding gene of the GmMDH2 protein in the recipient soybean or to increase or promote the content of the GmMDH2 protein in the recipient soybean, thereby obtaining target soybeans with higher salt tolerance than that of the recipient soybeans.

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