Use of soybean gm mate40 gene

Abstract

The application relates to the technical field of plant genetic engineering, and discloses application of a GmMATE40 gene in regulating acid aluminum stress tolerance of soybeans. Through cloning and analysis of a soybean GmMATE40 gene, it is found that heterologous overexpression of the GmMATE40 in Arabidopsis can effectively relieve the damage of plants under acid aluminum stress, and the acid aluminum tolerance of the overexpression plants is significantly enhanced, so that the research of the application can provide a theoretical basis and related genes for soybean aluminum tolerance molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to soybean GmMATE40 Application of genes in improving tolerance to acid-aluminum stress in Arabidopsis thaliana. Background Art

[0002] Studies have shown that soil acidification will directly reduce soil fertility and pose serious challenges to plant cultivation and production.

[0003] Under normal conditions, the soil is rich in aluminum, which does not cause stress to plant growth, but acidic soil can induce aluminum toxicity. In acidic soil (pH < 6), aluminum can be converted into Al 3+ 、Al(OH) 2+ Among them, Al³⁺ is the main toxic form. It binds to the cell walls of plant roots, interferes with cell division and elongation, inhibits the growth of plant roots, and thus hinders the plant's absorption of water and nutrients, thus seriously affecting plant growth and development.

[0004] Soybean ( Glycine max As a globally important dual-purpose grain and oil crop, soybean (L.) is rich in various nutrients and possesses symbiotic nitrogen fixation, playing a crucial role in the Second Green Revolution. However, soybeans are highly sensitive to aluminum toxicity and are susceptible to aluminum toxicity in acidic soils, resulting in stunted growth and development. Therefore, developing acid- and aluminum-tolerant soybean varieties has become an urgent need in agricultural research. Summary of the Invention

[0005] To achieve the above object, the present invention provides a soybean GmMATE40 Application of genes in improving tolerance to acid-aluminum stress in Arabidopsis, soybean GmMATE40 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1, or the soybean GmMATE40 The gene encodes the amino acid sequence shown in SEQ ID NO.2.

[0006] This application uses soybean overexpression in Arabidopsis GmMATE40 genes, thereby improving its tolerance to acid-aluminum stress.

[0007] The second object of the present invention is to provide a method for improving the tolerance of Arabidopsis thaliana to acid-aluminum stress, comprising the following steps:

[0008] S1, soybean GmMATE40 Insert the gene into the overexpression vector to construct the recombinant plasmid;

[0009] S2, using host bacteria-mediated technology to transform the recombinant plasmid into wild-type Arabidopsis thaliana;

[0010] S3, screening of transgenic Arabidopsis thaliana with functional expression;

[0011] The soybean GmMATE40 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1, or the soybean GmMATE40 The gene encodes an amino acid sequence as shown in SEQ ID NO. 2; the tolerance of the transgenic Arabidopsis thaliana to acid-aluminum stress is higher than that of the wild-type Arabidopsis thaliana.

[0012] Furthermore, the overexpression vector is pTF101.

[0013] Furthermore, the host bacterium is Agrobacterium.

[0014] This application is mainly studied through the following experiments:

[0015] S1, GmMATE40 Gene cloning and analysis: GmMATE40 The transcriptome data of overexpression plants (OE) and wild type plants (WT) under acid-aluminum stress were analyzed and the gene GmMATE40 It was significantly upregulated in transgenic plants, with the expression level increasing by more than 15 times; GmMATE40 Overexpression plants (OE) under acid-aluminum stress (+Al) and GmMATE40 The overexpression plants were compared and analyzed under non-acid aluminum stress conditions (-Al). GmMATE40 Under acid-aluminum stress conditions (+Al), the expression level in transgenic plants increased by more than 20 times, indicating that GmMATE40 This may be a response to acid-aluminum stress, which is consistent with the results of RNA-seq sequencing.

[0016] S2: GmMATE40 Analysis of gene expression patterns: Analyzed by fluorescence quantitative PCR technology, GmMATE40 The gene expression level in soybean roots was higher than that in stems, leaves, flowers and pods. GmMATE40 Gene responses to time and concentration of acid-aluminum stress, in the aluminum-sensitive soybean variety Zhonghuang 24, GmMATE40 The relative expression of the gene reached the maximum at 24 h under 50 μM AlCl3 treatment.

[0017] The Arabidopsis thaliana genetic transformation was achieved using Agrobacterium-mediated GmMATE40 The overexpression of the gene was detected in the plants. The phenotype of the genetically transformed strains was identified by the acid-aluminum stress experiment. The results showed that under normal growth conditions without aluminum, GmMATE40There was no significant difference between the overexpression strain and the wild type (WT). Under acid-aluminum stress, the growth of the overexpression strain was significantly better than that of the WT, and the performance was significant under 50μM AlCl3 treatment, further proving that GmMATE40 Genes are resistant to acid and aluminum.

[0018] At the same time, studies have shown that soybean GmMATE40 The gene was upregulated by acid-aluminum stress, and the expression level reached the highest under 50μM AlCl3 treatment, and with the increase of concentration, the expression level was higher than that of the control group; under the same 50μM AlCl3 treatment, GmMATE40 The expression level of the gene gradually increased with the treatment time and reached the maximum value after 24 hours. GmMATE40 The gene can alleviate the damage of aluminum toxicity to the root tip under acid-aluminum stress. GmMATE40 It has the ability to positively regulate plant root adaptation to acid-aluminum stress.

[0019] The transformation steps of transgenic Arabidopsis thaliana of the present application include: constructing pTF101- GmMATE40 Overexpression vector, the soybean GmMATE40 The gene is inserted into the overexpression vector to construct a recombinant plasmid, and then the target fragment is transferred into the recipient Arabidopsis thaliana using host bacteria-mediated technology, and functional transgenic plants are screened. Among them, the construction of the recombinant plasmid can be carried out by conventional methods, such as using the Gibson system, first using Xba I and Sac The pTF101 vector was double-digested with endonuclease I, and the double-digested product was ligated with the recombinant clone product. The host bacteria was Agrobacterium GV3101, and the genetic transformation material was Arabidopsis thaliana buds.

[0020] In summary, the beneficial effects of the present invention are as follows: GmMATE40 Gene cloning and analysis, verified by heterologous expression in Arabidopsis GmMATE40 The gene can alleviate the damage of aluminum toxicity to the root tip under acid-aluminum stress, indicating that GmMATE40 Positively regulate the ability of plant roots to adapt to acid-aluminum stress, and transform GmMATE40 The gene implanted into the plant body can significantly improve the plant's adaptability to acidic soil and aluminum stress. At the same time, this application also provides a theoretical basis and related genes for molecular breeding of soybean aluminum tolerance.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following preferred embodiments are specifically cited and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For soybeans GmMATE40 Promoter cis-acting element analysis;

[0023] Figure 2 This is the prediction of the functional domain of GmMATE40 protein, where part a is the protein functional domain predicted by SMART; part b is the protein transmembrane structure predicted by DeepTMHMM website; part c is the protein tertiary structure prediction of GmMATE40;

[0024] Figure 3 For soybeans GmMATE40 Schematic diagram of genetic transformation pTF101-GmMATE40 vector;

[0025] Figure 4 for GmMATE40 Protein subcellular localization map (Agrobacterium-mediated heterologous expression in tobacco epidermal cells), where "Light image" represents ordinary visible light image, "GFP" represents green fluorescent protein channel, "Marker" represents marker channel, and "merged" represents merged image.

[0026] Figure 5 for GmMATE40 Gene expression patterns in Zhonghuang 24 soybean seedlings;

[0027] Figure 6 For soybean seedlings GmMATE40 Relative expression levels of genes under 50 μM AlCl3 treatment;

[0028] Figure 7 for GmMATE40 Gene expression responses to acid-aluminum stress;

[0029] Figure 8 To verify the positive genetic transformation plants at the DNA level;

[0030] Figure 9 This is positive verification of genetic transformation;

[0031] Figure 10 To verify the use of homologous overexpression technology GmMATE40 The role of genes in the tolerance of Arabidopsis to aluminum; Part a shows the phenotypic differences of Arabidopsis grown on medium containing 0 μM, 50 μM, 100 μM, and 150 μM AlCl3 (pH < 4.5) for 14 days;

[0032] Figure 11 The difference in relative elongation between wild-type WT and OE-overexpressing plants after 14 days of treatment on acid-aluminum plates;

[0033] Figure 12The difference in fresh weight between wild-type WT and OE-overexpressing plants after 14 days of treatment on acid-aluminum plates;

[0034] Figure 13 The changes in flavonoid content in Arabidopsis lines. DETAILED DESCRIPTION

[0035] In order to make the content of the present invention more easily understood, the present invention will be further described below according to specific embodiments and in conjunction with the accompanying drawings. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any pro forma modification and / or change made to the present invention will fall within the scope of protection of the present invention. In the present invention, unless otherwise specified, all parts and percentages are weight units, and the equipment and raw materials used, etc. can be purchased from the market or are commonly used in this area. The methods in the following embodiments, unless otherwise specified, are conventional methods in this area.

[0036] Example 1: GmMATE40 Gene expression pattern analysis

[0037] Step 1:

[0038] S1. Planting Zhonghuang 24: Place four 10 cm square pots on an iron tray. Add 300 g of vermiculite as a substrate to each pot. Water the pots until the vermiculite is completely moist. Place 30 Zhonghuang 24 seeds in each pot, hilum-side down, to a depth of 1 cm. The pots are then placed in a culture room for incubation. Maintain an appropriate temperature (23-26°C) and humidity (60-70%), with 16 hours of light and 8 hours of darkness daily. Water regularly to ensure the substrate is moist. Pre-cultivation of Zhonghuang 24: After the seeds have grown cotyledons, clean the vermiculite from the roots. Select seedlings with intact roots and uniform length. Transfer them to a culture container containing a calcium solution containing 0.5 mM CaCl2 at a pH of 5.8. Pre-cultivate at 25°C for 24 hours to obtain soybean seedlings.

[0039] S2, roots, stems, leaves, flowers and pods of Zhonghuang No. 24 soybean seedlings were selected, and young leaves, 1 to 2 cm root tips, young stems, blooming flowers and pods without grains were taken as samples for testing. GmMATE40 Gene expression patterns in soybean seedlings of Zhonghuang 24.

[0040] S3, soybean seedlings with uniform root length and intact root tips after pre-culture were selected and grouped, and treated with 50 μM AlCl3 for 0, 3, 6, 9, 12, 24 and 36 hours respectively. Root tip samples were then taken and stored in a -80°C refrigerator to detect the presence of GmMATE40 Gene expression levels at different times under 50 μM AlCl3 treatment.

[0041] S4, soybean seedlings with uniform root length and intact root tips after pre-culture were selected and grouped, and treated with 0, 25, 50, 75, 100, and 150 μM AlCl3 for 24 h. Root tip samples were then taken and stored at -80 °C for detection. GmMATE40 Gene expression responses to acid-aluminum stress.

[0042] Detection method: Total RNA from different samples in steps S2, S3, and S4 was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing). The total RNA was reverse transcribed into cDNA, and real-time fluorescence quantitative PCR was performed using the cDNA as a template. The specific steps are as follows:

[0043] (1) RNA extraction

[0044] S1: Prepare a mortar, pestle, spoon, 1.5 mL centrifuge tube, and pipette tip, and sterilize them with high temperature and high pressure in advance.

[0045] S2 cools the mortar and grinding rod with liquid nitrogen, then puts the sample in, adds liquid nitrogen, and quickly grinds it into powder. Use a spoon to scoop 50-100 mg of powder into the EP tube;

[0046] S3: Add 500 μL of lysis buffer and 700 μL of diluent to the EP tube in sequence, then use a vortex mixer to mix until no obvious lumps remain. Then, heat the EP tube in a 70°C metal bath for 3 minutes. Then, transfer the EP tube to a centrifuge at 4°C and centrifuge at 12,000 rpm for 10 minutes.

[0047] S4: Take about 600 μL of the supernatant from the EP tube and transfer it to a new 1.5 mL centrifuge tube. Then, add 300 μL of anhydrous ethanol to the centrifuge tube and vortex the tube 20-25 times until the liquid in the centrifuge tube becomes turbid and contains white foam to allow for full reaction and obtain a reaction solution.

[0048] S5: Prepare a centrifuge column and a collection tube, place the centrifuge column in the collection tube, transfer the reaction solution to the centrifuge column, and then place the centrifuge column and collection tube containing the reaction solution in a centrifuge for centrifugation at a temperature of 4°C, a speed of 12,000 rpm, and a centrifugation time of 1 minute.

[0049] S6: Remove the centrifuge column and collection tube, discard the filtrate, add 600 μL of RNA washing solution, and then place the centrifuge column and collection tube in a centrifuge for centrifugation at 4°C, 12,000 rpm, and 45 s.

[0050] S7: Remove the centrifuge column and collection tube, discard the filtrate, and prepare incubation solution (5 μL of 10× DNase I buffer; 5 μL of DNase I; 40 μL of nuclease-free water). Pour 50 μL of the prepared incubation solution into the center of the spin column adsorption membrane and let it stand for 15 minutes.

[0051] S8 Add 600 μL RNA wash solution to the centrifuge column and wash the precipitate twice. Then place the centrifuge column and collection tube in a centrifuge for centrifugation at a temperature of 4°C and a speed of 12,000 rpm for 45 seconds, and discard the filtrate.

[0052] S9: Move the centrifuge column to the elution tube, then add 50-200 μL of nuclease-free water to the centrifuge column, let it stand for 2 minutes, and then place the centrifuge column and elution tube in a centrifuge for centrifugation at 4°C, 12,000 rpm, and centrifuge for 1 minute.

[0053] The RNA concentration was measured using S10 and stored at -80°C until use.

[0054] (2) Reverse transcription

[0055] Measure the OD value of the extracted RNA by UV spectrophotometer or microplate spectrophotometry, and calculate the RNA dosage (0.1ng-1μg) based on the OD value of the extracted RNA. The specific calculation formula is: The reaction system is shown in Table 1 below. The reaction procedure is: incubate at 50°C for 15 minutes, followed by heating at 85°C for 5 seconds. All operations must be completed on ice. After the reaction, the cDNA should be stored in a refrigerator at -20°C.

[0056] Table 1 Reverse transcription reaction system

[0057]

[0058] (3) Quantitative PCR primer design

[0059] Search gene sequences from the Phytozome database and download from the website GmMATE40 The genomic CDS sequence is shown in SEQ ID NO. 1, and the GmMATE40 protein sequence is shown in SEQ ID NO. 2. Primers were designed and synthesized based on the genomic sequence on the NCBI website for specific quantitative amplification:

[0060] GmMATE40-qF (SEQ ID NO.3): 5'-GTTAGGGTTTGGGATCATGGGT-3';

[0061] GmMATE40-qR (SEQ ID No. 4):

[0062] 5'-GAGTCCAGAGAATGCCTCAACA-3'.

[0063] (4) Real-time fluorescence quantitative PCR

[0064] S1 was detected using SYBR blue luciferase complex (Vazyme, Nanjing) and CFX Connect PCR instrument (BIO-RAD, USA). Actin3 As an internal reference gene;

[0065] S2: cDNA of all samples was diluted 1-fold with ddH2O and used as template for quantitative PCR reaction;

[0066] The S3 real-time fluorescence quantitative PCR system was used according to the reaction system in Table 2 below. The reaction program was as follows: initial denaturation at 95°C for 30 seconds; 39 cycles of denaturation at 95°C for 5 seconds; annealing at 60°C for 30 seconds; melting curve at 95°C for 10 seconds; 54.3°C for 5 seconds; and 94.3°C for 5 seconds.

[0067] Table 2

[0068]

[0069] Experimental results:

[0070] like Figure 5 As shown, in Zhonghuang 24 soybean, compared with the stems, leaves and pods, GmMATE40 The gene was expressed at higher levels in roots and flowers.

[0071] like Figure 6 As shown, the soybean seedlings GmMATE40 The gene expression level reached the highest level at 24 h under 50 μM AlCl 3 treatment.

[0072] like Figure 7 As shown, GmMATE40 The expression level of the gene increased under acid-aluminum stress.

[0073] Step 2: GmMATE40 Genetic information analysis

[0074] S1, soybean GmMATE40 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1. GmMATE40The CDS of the gene is 1518 bp long and encodes a protein sequence containing 506 amino acid residues. The gene contains 7 exons and 6 introns. The promoter region cis-acting elements were predicted using the PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) website and the image was drawn using TBtools software. The GmMATE40 promoter cis-acting elements are as follows: Figure 1 shown.

[0075] S2, the SMART (http: / / smart.embl-heidelberg.de / ) and Deep TMHMM (https: / / services.healthtech.dtu.dk / services / DeepTMHMM-1.0 / ) websites were used to compare GmMATE40 The protein functional domains were predicted and analyzed on the SWISS-MODEL (https: / / swissmodel.expasy.org / ) website. GmMATE40 The tertiary structure of the protein is predicted based on the amino acids encoded by it. Figure 2 As shown, GmMATE40 The protein contains two Pfam domains MATE_eukaryotic. It belongs to the MATE family, a branch of the multidrug efflux transporter family. It mainly transports macromolecular substrates such as organic acids, plant hormones, and secondary metabolites. It is predicted to have 12 transmembrane domains (TMDs). The protein tertiary structure prediction shows GmMATE40 The encoded protein has a transmembrane transport structure.

[0076] S3, transient expression experiments were performed using tobacco leaf epidermal cells to construct Figure 3 The 35S::GmMATE40-GFP fusion expression vector was expressed and the co-localization of GmMATE40 protein and cell membrane marker protein in leaves was performed. The epidermal cells of tobacco leaves were observed by laser confocal microscopy. Figure 4 As shown, the green fluorescence signal of GmMATE40-GFP completely overlaps with the red fluorescence signal of the cell membrane marker, indicating that the GmMATE40 protein is localized on the cell membrane.

[0077] Example 2: Verification of gene overexpression in Arabidopsis GmMATE40 Gene function

[0078] Step 1: Construction of overexpression vector

[0079] S1. Extract RNA from soybean Zhonghuang No. 24 and reverse transcribe it into cDNA (refer to RNA extraction in Example 1). Design primers for overexpression of the GmMATE40 gene and amplify the CDS region of the target gene using Zhonghuang No. 24 cDNA as a template. Use NCBI Primer-BLAST to design specific primers. The specific primer sequences are:

[0080] GmMATE40 -CDS-F (SEQ ID NO.5): 5'-ATGATGGAATCCCAGAACCAGAAC-3';

[0081] GmMATE40 -CDS-R (SEQ ID NO. 6): 5'-ACTCTGAATCTTAGGTTCCCTTCTTGTACC-3'.

[0082] S2, amplify according to the reaction system in Table 3 below GmMATE40 The reaction procedure for the full-length CDS sequence was as follows: initial denaturation at 95°C for 3 min; 34 cycles (denaturation at 95°C for 15 s; annealing at 55°C for 15 s; extension at 72°C for 1 min / kb); final extension at 72°C for 5 min; storage at 12°C;

[0083] Table 3 Fragment amplification reaction system

[0084]

[0085] S3, the method for constructing overexpression vector is Gibson. Sac I and Xba I. Perform double restriction enzyme digestion on pTF101 plasmid DNA to linearize the vector. The Gibson recombination reaction system is shown in Table 4. The reaction procedure is: 37°C, 30 min;

[0086] Table 4

[0087]

[0088] S4, colon transformation: Pipette 10 μL of the ligation product into 100 μL of DH5α competent E. coli, gently pipette to mix, then place on ice for 30 minutes, heat shock at 42°C for 45 seconds, and immediately transfer to ice to cool for 2 minutes; then move the ligation product to a clean bench, add 500 μL of LB liquid medium to the ligation product, and then culture at 37°C on a shaker at 220 rpm for 1 hour to obtain a bacterial solution. The bacterial solution was spread on a plate containing spectinomycin (100 μg / mL) and cultured at 37°C overnight. Single clones were selected for shaking, detection, and sequencing. Single clones with correct sequencing were expanded, and plasmids were extracted and stored. The plasmid was named pTF101- GmMATE40 -OE.

[0089] In step S5, pipette 4 μL of plasmid into 100 μL of GV3101 competent Agrobacterium. Place the plasmid on ice for 5 minutes, then freeze it in liquid nitrogen for 5 minutes. Heat shock the plasmid at 37°C for 5 minutes, and then cool the plasmid on ice for 5 minutes. Transfer the plasmid to a clean bench and add 500 μL of YEP liquid medium. Incubate the culture at 28°C in a shaker at 220 rpm for 2-3 hours to obtain a bacterial suspension. Plate the suspension onto YEB solid medium containing rifampicin (50 μg / mL) and spectinomycin (100 μg / mL) and incubate at 28°C for 40-48 hours. Pick a single colony, shake it, and test it. Add an equal volume of 50% glycerol to the successful suspension and store it at -80°C.

[0090] Step 2: Arabidopsis genetic transformation

[0091] S1. Preparation of genetic transformation materials

[0092] The Arabidopsis floral dipping method was used for genetic transformation. Wild-type Arabidopsis seeds were vernalized at 4°C for 3 days and sown in small planting pots containing moist nutrient soil and an appropriate amount of vermiculite. The pots were covered with plastic wrap, leaving a certain height. The plastic wrap was removed after 3 days, leaving 4-5 seedlings in each pot. When there were many wild-type Arabidopsis flower buds, infection began, and the blooming flowers were cut off before infection.

[0093] S2, Agrobacterium infection

[0094] The pTF101- GmMATE40 GV3101 bacteria containing the vector were cultured to an OD of 0.6, then centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded. 50 mL of fresh suspension (5% sucrose, 1 / 2 MS, and 0.02% Silwet L-77) was then added to the suspension. Note that the suspension should be stirred on a magnetic stirrer until foamy. After adding the fresh suspension, pipette and mix thoroughly to resuspend the bacteria. Wild-type Arabidopsis buds were then completely immersed in the suspension and infected for 2 minutes. The infected plants were placed in a plastic bag for preservation and then incubated in the dark for 24 hours.

[0095] Step 3: Select training

[0096] S1: Vernalize all harvested seeds at 4°C for 3 days. Place seeds in trays filled with moistened soil and sow them on the soil surface. The seeds are then incubated in an incubator (16 h light / 8 h dark, 24°C). Once both cotyledons of the Arabidopsis thaliana plants have fully expanded, prepare a 20 µg / mL glyphosate solution and spray every two days. Keep the soil moist during this period. Approximately 7 days after spraying, non-positive seedlings will cease to grow normally. Those that remain normally are considered positive seedlings and transplanted individually into small pots. Each individual plant is harvested as a single strain, and positive seedlings are subsequently screened and identified until homozygous T4 generations are established.

[0097] Step 4: Verification of transgenic plants

[0098] The leaves of transgenic plants were selected and DNA was extracted. The wild type WT was used as negative control and pTF101- GmMATE40 The plasmid was used as a positive control to verify whether the vector carrying the target fragment was transferred into the Arabidopsis genome. Figure 8 As shown in the figure, M (marker) is the DNA molecular weight standard, H (H2O Control) is the water control, and the rest are positive plants. After cultivation to the T4 generation, RNA was extracted and reverse transcribed from the aerial parts and roots, and identified by fluorescence quantification. The results are shown in the figure. Figure 9 As shown, 3 independent GmMATE40 Gene overexpression lines (OE-1, OE-2, and OE-3).

[0099] Select strains with high expression levels and multiply them in large quantities for subsequent experiments.

[0100] The DNA verification primers for overexpression plants are:

[0101] pTF101-R (SEQ ID No. 7): 5'-TCATCGCAAGACCGGCAAC-3';

[0102] OX- GmMATE40 -F (SEQ ID No. 8): 5'-AATTCTTCTCTTGCTGGGGGAG-3'.

[0103] Step 5: GmMATE40 Phenotypic identification of overexpression

[0104] Arabidopsis wild type WT, GmMATE40 The acid-aluminum phenotype of the overexpressing plants was identified, and the growth, plant height, root length, biomass, and flavonoid content of each strain were observed and measured.

[0105] Flavonoid content was determined using a flavonoid kit from Suzhou Keming Biotechnology Co., Ltd. Samples were collected from 0 μmol / L and 50 μmol / L aluminum-acid-treated Arabidopsis culture plates, dried to constant weight, crushed, and passed through a 40-mesh sieve. Approximately 0.02 g of the sample was then added to 2 mL of the extract. The sample was shaken and extracted at 60°C for 2 h. After extraction, the sample was centrifuged at 10,000 g for 10 min at 25°C. The supernatant was collected for subsequent analysis. Using a microplate reader with the wavelength adjusted to 510 nm and zeroed with distilled water, the absorbance of the sample supernatant at 510 nm was measured to calculate the flavonoid content.

[0106] Experimental results

[0107] The results of transgenic Arabidopsis root phenotype identification are as follows Figure 10 As shown, the root growth of transgenic Arabidopsis was better than that of wild-type Arabidopsis (WT), indicating that heterologous overexpression of GmMATE40 It can alleviate the inhibitory effect of acid-aluminum stress on the growth and development of Arabidopsis thaliana. The statistical results of its biomass index are as follows: Figure 11 and Figure 12 As shown, compared with the wild type, when plants were subjected to acid-aluminum stress, Arabidopsis heterologous overexpression GmMATE40 The relative root elongation and fresh weight of the strain were higher, which could alleviate the aluminum toxicity in the root tip of Arabidopsis thaliana.

[0108] like Figure 13 As shown in the results, without aluminum treatment, the flavonoid content of transgenic lines and wild type was similar. Under aluminum treatment, the secretion of flavonoids in Arabidopsis increased, and the secretion of transgenic lines was significantly higher than that of wild type. The above results prove that GmMATE40 Genes are regulating soybean's tolerance to acid and aluminum.

[0109] In summary, soybean GmMATE40 The cloning and analysis of the gene were combined with homologous overexpression to verify its function in Arabidopsis WT. GmMATE40 Overexpression significantly enhances the acid and aluminum resistance of the plant. The invention provides a theoretical basis and related genes for molecular breeding of acid and aluminum resistance in soybean.

[0110] The data in this application are the average of 3 repeated experiments. "*", "**", "***" and "****" indicate significant differences between the treatment and the control (* P ≤0.05), extremely significant (** P ≤0.001), extremely significant (*** P ≤0.001) and extremely significant (**** P ≤0.0001)).

[0111] The embodiments described above are only preferred implementations of the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention fall within the scope of protection of the present invention.

[0112] GmMATE40 CDS sequence of the gene

[0113] SEQ ID NO.1:

[0114]

[0115] Amino acid sequence

[0116] SEQ ID NO.2

[0117] MMESQNQNLLRQPLINSTHHHHHSADSRLEEVLSDPTLPWSKRILSATWIELNLLFPLAAPAILVYVFNNLMSNVTRAFAGHLGNLELAAANLGNSGIQLFAYGLMLGMGSAVETLCGQAYGANKYEMLGIYMQRAIIVLTITGIPLTVVYIFCKPILLLLGEPPEVASVAAMFVYGLIPQIFAYAVNFPIQKFLQAQSVVAPSTYISAATLVLHVALSWVVVYKLGFGIMGSSLMLSLSWWIIVGAQFLYVVSASKFKDTWSGFSVEAFSGLWDFVKLSAASAVMLCLETWYFQVLVLITGLLDNPQLSLDSISVCMAITGLTMHIGIGFNAAASVRVSNELGAEHPKSAAFSVIVVNMISFIIAVIEAVVVLALRRVVSYAFTDGETVANAVSDLCPYLAVTLILNGIQPVLSGVAVGCGWQAIVAYVNVGCYYGIGIPLGCVLGFTFGLGVQGIWSGMIGGTMLQTLILLWITLRTDWNKEVNTAKKRLNKWGYKKEPKIQS

[0118] Met Met Glu Ser Gln Asn Gln Asn Leu Leu Arg Gln Pro Leu Ile Asn SerThr His His His His His Ser Ala Asp Ser Arg Leu Glu Glu Val Leu Ser Asp ProThr Leu Pro Trp Ser Lys Arg Ile Leu Ser Ala Thr Trp Ile Glu Leu Asn Leu LeuPhe Pro Leu Ala Ala Pro Ala Ile Leu Val Tyr Val Phe Asn Asn Leu Met Ser AsnVal Thr Arg Ala Phe Ala Gly His Leu Gly Asn Leu Glu Leu Ala Ala Ala Asn LeuGly Asn Ser Gly Ile Gln Leu Phe Ala Tyr Gly Leu Met Leu Gly Met Ser AlaVal Glu Thr Leu Cys Gly Gln Ala Tyr Gly Ala Asn Lys Tyr Glu Met Leu Gly IleTyr Met Gln Arg Ala Ile Val Leu Thr Ile Thr Gly Ile Pro Leu Thr Val ValTyr Ile Phe Cys Lys Pro Ile Leu Leu Leu Gly Glu Pro Pro Glu Val Ala SerVal Ala Ala Met Phe Val Tyr Gly Leu Ile Pro Gln Ile Phe Ala Tyr Ala Val AsnPhe Pro Ile Gln Lys Phe Leu Gln Ser Val Val Ala Pro Ser Thr Tyr IleSer Ala Ala Thr Leu Val Leu His Val Ala Leu Ser Trp Val Val Val Tyr Lys LeuGly Phe Gly Ile Met Gly Ser Ser Leu Met Leu Ser Leu Ser Trp Trp Ile Ile ValGly Ala Gln Phe Leu Tyr Val ValSer Ala Ser Lys Phe Lys Asp Thr Trp Ser GlyPhe Ser Val Glu Ala Phe Ser Gly Leu Trp Asp Phe Val Lys Leu Ser Ala Ala SerAla Val Met Leu Cys Leu Glu Thr Trp Tyr Phe Gln Val Leu Val Leu Ile Thr GlyLeu Leu Asp Asn Pro Gln Leu Ser Leu Asp Ser Ile Ser Val Cys Met Ala Ile ThrGly Leu Thr Met His Ile Gly Ile Gly Phe Asn Ala Ala Ala Ser Val Arg Val SerAsn Glu Leu Gly Ala Glu His Pro Lys Ser Ala Ala Phe Ser Val Ile Val Val AsnMet Ile Ser Phe Ile Ile Ala Val Ile Glu Ala Val Val Val Leu Ala Leu Arg ArgVal Val Ser Tyr Ala Phe Thr Asp Gly Glu Thr Val Ala Asn Ala Val Ser Asp LeuCys Pro Tyr Leu Ala Val Thr Leu Ile Leu Asn Gly Ile Gln Pro Val Leu Ser GlyVal Ala Val Gly Cys Gly Trp Gln Ala Ile Val Ala Tyr Val Asn Val Gly Cys TyrTyr Gly Ile Gly Ile Pro Leu Gly Cys Val Leu Gly Phe Thr Phe Gly Leu Gly ValGln Gly Ile Trp Ser Gly Met Ile Gly Gly Thr Met Leu Gln Thr Leu Ile Leu LeuTrp Ile Thr Leu Arg Thr Asp Trp Asn Lys Glu Val Asn Thr Ala Lys Arg LeuAsn Lys Trp Gly Tyr Lys Lys Glu Pro Lys Ile Gln Ser

Claims

1. Soybeans GmMATE40 The application of the gene in improving the tolerance of Arabidopsis thaliana to acid and aluminum stress is characterized by: soybeans GmMATE40 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1, or the soybean GmMATE40 The gene encodes the amino acid sequence shown in SEQ ID NO.

2.

2. A method for improving the tolerance of Arabidopsis thaliana to acid-aluminum stress, characterized in that: The following steps are involved: S1, soybean GmMATE40 Insert the gene into the overexpression vector to construct the recombinant plasmid; S2, using host bacteria-mediated technology to transform the recombinant plasmid into wild-type Arabidopsis thaliana; S3, screening of transgenic Arabidopsis thaliana with functional expression; The soybean GmMATE40 The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1, or the soybean GmMATE40 The gene encodes the amino acid sequence shown in SEQ ID NO.2; The tolerance of the transgenic Arabidopsis thaliana to acid-aluminum stress is higher than that of the wild-type Arabidopsis thaliana.

3. The method according to claim 2, wherein The overexpression vector is pTF101.

4. The method according to claim 2, wherein The host bacteria is Agrobacterium.

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