Application of GsMYB10 gene in low phosphorus stress tolerance of plants

Abstract

The invention relates to the technical field of gene engineering, in particular to a plant low-phosphorus-resistant important gene GsMYB10 and application thereof. Researches show that the expression of the GsMYB10 gene is induced to be up-regulated under the stress of low phosphorus, and under different phosphorus concentration treatment conditions, the over-expression of the GsMYB10 can obviously increase the biomass of a transgenic plant, so that the plant growth is promoted under the low-phosphorus condition; meanwhile, overexpression of GsMYB10 can improve the tolerance of plants to low-phosphorus stress, and reduce the inhibition effect of low phosphorus on plant root growth.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically relates to the application of the GsMYB10 gene in plant tolerance to low phosphorus stress. Background Art

[0002] Soybean is a plant rich in nutrients. Its protein content is 2.5 to 8 times that of cereal and tuber foods. In addition, its essential substances for the human body such as fat, calcium, phosphorus, iron, vitamin B1, and vitamin B2 are also higher than those of cereal and tuber foods. Soybean has a very wide range of uses. In terms of food, soybeans can be processed into soy products such as tofu, soy milk, and yuba. Fermented soy products include fermented bean curd, stinky tofu, broad bean paste, soy sauce, fermented soybeans, natto, etc., while non-fermented soy products include fresh tofu, dried tofu, bean sprouts, and marinated soy products. In terms of feed, the stems, leaves, and soybean dregs of soybeans are good fertilizers and feeds, suitable for ruminant livestock such as chickens, pigs, and dairy cows.

[0003] Soybean is a phosphorus-loving crop, and phosphorus plays a key role in the entire life cycle of soybeans. If soybeans cannot absorb enough phosphorus, it will inhibit the growth of their root nodules, reduce the nitrogen fixation amount, and ultimately severely reduce the yield and quality of soybeans. China is the largest soybean consumer in the world, with an annual demand of more than 100 million tons. However, China's self-sufficiency rate of soybeans is only 18%-20%. China's soybeans are mainly produced in the Northeast region, with a production volume accounting for about 50%-60% of the country's total output. In the Huang-Huai-Hai region and the southern red soil region, although there are rich land resources, due to the lack of soil phosphorus, the growth of soybean plants is restricted and the number of pods is reduced, ultimately affecting the production and quality of soybeans.

[0004] Therefore, screening for phosphorus-efficient genes and improving the low-phosphorus tolerance of soybeans is one of the important ways to increase the yield and quality of soybeans. Summary of the Invention

[0005] To solve the problems in the above background art, a first object of the present invention is to provide the application of the gene GsMYB10 in improving the low-phosphorus stress tolerance of plants, so as to increase the yield and quality of soybeans in a low-phosphorus environment. The nucleotide sequence of the gene GsMYB10 is as shown in SEQ ID NO.1; or the gene GsMYB10 encodes an amino acid sequence as shown in SEQ ID NO.2.

[0006] This application overexpresses the gene GsMYB10 in plants, thereby improving the low-phosphorus stress tolerance of plants.

[0007] Further, the plant is soybean.

[0008] The second object of the present invention is to provide a method for improving the low phosphorus stress tolerance of plants, and the method is to increase the expression level of the amino acid sequence shown in SEQ ID NO.2 in the recipient plant; the specific steps include: introducing the gene GsMYB10 shown in SEQ ID NO.1 into the recipient plant to obtain transgenic soybean plants, and the low phosphorus stress tolerance of the transgenic plants is higher than that of the recipient plant; or overexpressing the amino acid sequence shown in SEQ ID NO.2 in the recipient plant.

[0009] Further, the improvement of the low phosphorus stress tolerance of the plant is reflected in the increase of its root length or the number of lateral roots.

[0010] The third object of the present invention is to provide the application of the recombinant plasmid, recombinant vector and transgenic plant cell line of the gene GsMYB10 in improving the low phosphorus stress tolerance of plants, and the nucleotide sequence of the gene GsMYB10 is shown in SEQ ID NO.1; or the gene GsMYB10 encodes the amino acid sequence shown in SEQ ID NO.2.

[0011] The present application studies the method for the soybean low phosphorus tolerance gene GsMYB10 to promote the growth of plants in a low phosphorus stress environment, including the following specific steps:

[0012] Using soybeans as materials, by means of Agrobacterium-mediated cotyledon node transformation of soybeans, the GsMYB10 gene was genetically transformed into soybeans by Agrobacterium tumefaciens-mediated cotyledon node method to obtain soybean plants overexpressing the GsMYB10 gene. By comparing the growth performance, phosphorus absorption efficiency and root system development and other indicators of transgenic plants and wild-type plants under low phosphorus stress conditions, the function and application potential of the GsMYB10 gene in improving soybean low phosphorus tolerance were verified.

[0013] In summary, the beneficial effects of the present invention are: through the cloning and analysis of the soybean gene GsMYB10, and by using the Agrobacterium-mediated cotyledon node transformation method of soybeans, the GsMYB10 gene can be successfully integrated into the soybean genome and stably expressed. At the same time, it is found that the low phosphorus tolerance of GsMYB10 overexpressing plants is significantly enhanced, which can lay a foundation for soybean low phosphorus tolerance molecular breeding.

[0014] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the description. And in order to make the above and other objects, features and advantages of the present invention more obvious and understandable, the following specific preferred embodiments are given, and in conjunction with the drawings, the details are described as follows. Brief Description of the Drawings

[0015] Figure 1 : Cloning of the GsMYB10 gene;

[0016] Figure 2 : Analysis of tissue expression pattern of GsMYB10 gene in wild soybean line BW69;

[0017] Figure 3 : Changes in expression levels of GsMYB10 gene at different times;

[0018] Figure 4 : Detection diagram of GsMYB10 transgenic soybean lines;

[0019] Figure 5 : Transcription activation activity diagram of GsMYB10 protein in yeast cells;

[0020] Figure 6 : Subcellular localization diagram of 35S::GsMYB10-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells (leaf epidermal cells transformed with 35S::GFP were used as a control, and RFP was used as a nuclear localization protein marker (PJIT-mCherry-Nuc));

[0021] Figure 7 : Hydroponic verification of GsMYB10 transgenic soybean seedlings;

[0022] Figure 8 : Soil cultivation verification and variety inspection data of GsMYB10 transgenic soybean during the whole growth period in the net room;

[0023] Figure 9 : Soil cultivation verification and variety inspection data of GsMYB10 transgenic soybean seedlings in the net room. Detailed implementation manners

[0024] In order to make the content of the present invention easier to be clearly understood, the following further illustrates the present invention according to specific embodiments in conjunction with the accompanying drawings. In the present invention, the equipment, raw materials, etc. adopted can all be purchased from the market or are commonly used in the art; the soybeans adopted are all provided by the Guangdong Branch of the National Center for Soybean Improvement. It should be understood that the implementation of the present invention is not limited to the following embodiments. Without special instructions, they are all within the protection scope of the present invention.

[0025] The full-length cDNA of the GsMYB10 gene of this application was cloned from wild soybean BW69 and encodes the same sequence as Huachun 6, a cultivated soybean. The GsMYB10 gene is located on chromosome 11, and the gene position is named Glyma11g183400, which contains 6 exons. The full length of the gene is 3695bp, the transcription sequence is 1867bp, the open reading frame is 1245bp, and it encodes 414 amino acids. The following are the experimental steps for obtaining the GsMYB10 gene:

[0026] S1. To obtain the full-length ORF (open reading frame) of GsMYB10, primers were designed based on the soybean genomic sequence information:

[0027] Forward Primer (SEQ ID NO.3): 5'-TGGCTTTGCAGGTTGA-3'

[0028] Reverse Primer (SEQ ID NO.4): 5'-AACTCATATTTGGCTAG-3'

[0029] S2. The GsMYB10 gene was amplified using the above primers to obtain an amplification product with a length of 1402 bp. The full-length CDS sequence of GsMYB10 was amplified according to the reaction system in Table 1 below. The reaction program was: pre-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; and stored in a refrigerator at 12°C.

[0030] Table 1 Fragment Amplification Reaction System

[0031]

[0032] S3. The DNA Fragment Purification Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to purify the amplified product by gel cutting and recovery, and a high-purity target gene fragment was obtained after recovery.

[0033] S4. The purified PCR fragment was cloned into the pLB vector (Zero Background Rapid Cloning Kit, Tiangen Biochemical Technology Co., Ltd.) to obtain a ligation product (pLB-GsMYB10). The reaction system is shown in Table 2 below, and the reaction program was: 20°C for 2 min; 70°C for 5 min; 22°C for 20 min.

[0034] Table 2 Ligation Reaction System

[0035]

[0036]

[0037] S5, Colonic transformation: Pipette 10 μL of the ligation product into 100 μL of DH5α Escherichia coli competent cells, gently pipette and mix well, then incubate on ice for 30 min, followed by heat shock at 42 °C for 45 s, and immediately transfer to ice for cooling for 2 min; then transfer the ligation product to the laminar flow hood, add 500 - 700 μL of LB liquid medium to the ligation product, and then culture in a shaker at 37 °C and 220 rpm for 1 h to obtain a bacterial solution. Spread the bacterial solution on a plate containing Amp and culture overnight at 37 °C. Pick a single colony for shaking culture and perform detection and sequencing.

[0038] Among them, the cloning of the GsMYB10 gene is as Figure 1 shown, M is DL2000 DNA marker; 1 is the amplified product size of the GsMYB10 gene, which is 1402 bp.

[0039] S6, Clone the coding sequence of the GsMYB10 gene in the correctly sequenced pLB - GsMYB10 plasmid into the pZY101 vector to construct the GsMYB10 - pZY101 overexpression vector.

[0040] Specific steps for vector construction:

[0041] 1. Recombinant ligation of the vector and the target fragment (refer to the instruction manual of Novoprotein OneStep Cloning Kit)

[0042] a. Primer design for the inserted fragment. Use the CE Design V1.03 software of Novoprotein Biotechnology Co., Ltd. in Nanjing to design primers.

[0043] GsMYB10 - pZY101 - CZ - F (SEQ ID NO.5): caggcatgcaagctcaagcttAGCTTGCATGCCTGCAGG

[0044] GsMYB10 - pZY101 - CZ - R (SEQ ID NO.6): gacctgcaggcatgcaagcttAAGCTTGCATGCCTGCAGG

[0045] b. Linearization of the cloning vector (refer to the instruction manual of the restriction enzyme kit of Guangzhou Ruizhen Biotechnology Co., Ltd.), and the enzyme digestion reaction system is shown in Table 3 below.

[0046] Table 3 Enzyme digestion reaction system

[0047] Reagent Volume Vector plasmid 3 μL 10*K Buffer 2 μL 10*BSA 2 μL SpeI 1 μL NcoI 1 μL Sterilized water 11 μL Total 20 μL

[0048] Mix the added digestion system thoroughly and centrifuge it briefly. Then place it in a PCR machine with the program set to 37°C for 1 hour and 4°C indefinitely. Purify and recover the digested vector fragment from the digested system using a PCR product purification kit.

[0049] c. PCR amplification of the inserted fragment.

[0050] d. Preparation of the reaction system and reaction: Measure the concentration of the digested vector and the inserted fragment using a Thermo Fisher NanoDrop 2000 micro UV-visible spectrophotometer and estimate it by running electrophoresis, then configure the reaction system. Configure the reaction system shown in Table 4 below on an ice box.

[0051] Table 4 Recombinant ligation reaction system

[0052]

[0053]

[0054] After the system preparation is completed, gently pipette up and down with a pipette to mix all components evenly. Then place each component in a centrifuge tube for brief centrifugation (to avoid air bubbles), and then place the centrifuge tube in a PCR machine for reaction with the program set to 37°C for 30 minutes and 4°C indefinitely. After the reaction is completed, promptly take out the centrifuge tube and place it in an ice-water bath for 5 minutes of cooling.

[0055] e. Transformation and plating of the reaction product.

[0056] f. Clone identification

[0057] g. Sequencing identification (sequenced by Shanghai Sangon Biotech Co., Ltd.)

[0058] S7. Use the Agrobacterium-mediated cotyledon node transformation method to transfer the GsMYB10-pZY101 constructed vector into the soybean cultivar Huachun 6;

[0059] 1. Preparation of electrocompetent Agrobacterium tumefaciens EHA101

[0060] a. Use a pipette to pick a single colony in a laminar flow hood and inoculate it into 1 ml of YEP liquid medium containing three antibiotics, Chl, and place it on a 28°C constant temperature shaker for 6 hours of cultivation at 240 rpm.

[0061] b. At 8 pm, use a pipette to transfer 1 ml of the bacterial solution into 50 ml of YEP liquid medium containing the corresponding antibiotics in a laminar flow hood and continue overnight cultivation until the OD value is approximately 0.6 the next day.

[0062] c. Transfer the bacterial solution to a 50 ml sterile centrifuge tube in a laminar flow hood, place it in an ice box containing ice water for ice bath for 30 min, then place it in an ultracentrifuge, set the rotation speed to 4000 rpm, the temperature to 4 °C, and the time to 10 min for centrifugation.

[0063] d. Discard the supernatant in a laminar flow hood, then resuspend the cells with a small amount of cold sterile water to completely dissolve the bacterial solution, and then supplement it to 50 ml with cold sterile water, and centrifuge it again in the centrifuge set in step c.

[0064] e. Discard the supernatant again in a laminar flow hood, then resuspend the cells with a small amount of cold sterile water to completely dissolve the bacterial solution, and then supplement it to 25 ml with cold sterile water, and centrifuge it in the centrifuge set in step c.

[0065] f. Repeat discarding the supernatant in a laminar flow hood, then resuspend the cells with a small amount of cold sterile water to completely dissolve the bacterial solution, and then supplement it to 5 ml with cold sterile water, and centrifuge it in the centrifuge set in step c.

[0066] g. Finally, discard the supernatant in a laminar flow hood, and suspend the precipitate with 10% glycerol to completely dissolve the bacterial solution into the solution, and store it at -80 °C in aliquots of 100 μl each for later use.

[0067] 2. Electroporation transformation of Agrobacterium tumefaciens EHA101 competent cells

[0068] a. Take out the electroporation cuvette stored in 70% ethanol in a laminar flow hood, place it in the laminar flow hood to dry, and then perform ultraviolet sterilization for 20 min and blow dry for use.

[0069] b. Take out the prepared Agrobacterium tumefaciens EHA101 competent cells from the -80 °C ultra-low temperature refrigerator and place them in an ice box to thaw. At the same time, take out the plasmid to be transformed from the -20 °C refrigerator and melt it at room temperature. Use a pipette to aspirate the completely melted plasmid and add it to the thawed competent cells. Gently pipette and mix it in a laminar flow hood, and then transfer it to the sterilized and blow-dried electroporation cuvette.

[0070] c. Then place the electroporation cuvette in the preheated electroporator, and set the parameters as: voltage 1800 V, time 5 ms, and perform electroporation.

[0071] d. After electroporation, place it in a laminar flow hood, use a pipette to aspirate 1 ml of YEP liquid medium without antibiotics and slowly add it to the electroporation cuvette, and pipette several times to suspend and mix the cells.

[0072] e. Aspirate the suspended and mixed bacterial solution from the electroporation cuvette and place it in a centrifuge tube, then place it on a constant temperature shaker, set the temperature to 28 °C, the rotation speed to 240 rpm, and the time to 3 h for cultivation.

[0073] f. Using a pipette, aspirate 50 μl of the bacterial solution in a sterile laminar flow hood, and use a spreader sterilized by alcohol burning to spread it onto a YEP solid plate containing three antibiotics, str, spe, and chl. Place it in an incubator at 28 °C and incubate for about 36 h until colonies grow.

[0074] 3. Genetic transformation of soybean cotyledon nodes

[0075] a. Seed disinfection: Soybean seeds are disinfected using the method of dry surface disinfection. First, take out the pre-screened and stored Huachun 6 seeds from the -20 °C refrigerator and arrange them in a single layer in a petri dish. Then place it in a desiccator in the fume hood, and also place a 250 mL beaker containing 100 mL of sodium hypochlorite in the desiccator. Use a 5 ml pipette to slowly add 4.2 mL of concentrated hydrochloric acid along the wall of the beaker, and immediately cover the lid of the desiccator. After chlorine sterilization for 13.5 h, open the fume hood, cover the lid of the petri dish in the desiccator, and quickly place it in a laminar flow hood that has been sterilized by ultraviolet light and turn on the blower. After about 40 min, proceed to the next step.

[0076] b. Germination: Place the pre-prepared germination medium (GM) in the laminar flow hood as well. Then use forceps sterilized by alcohol flame to place about 12 disinfected soybean seeds with the hilum facing down in the germination medium. After inoculation, seal it with medical tape, and place the petri dish in the tissue culture room. The environment of the tissue culture room is set as follows: 16 h light / 8 h dark, temperature set at 24 °C, and light intensity set at 140 μmoles / m2 / sec, and culture for 5 days.

[0077] c. Bacterial solution preparation: Take out 1 ml of the Agrobacterium tumefaciens strain EHA101 containing GsMYB10-pZY101 that has been identified by bacterial solution PCR, place it in a sterile laminar flow hood, and use a pipette to aspirate and add it to a 250 ml sterile Erlenmeyer flask containing 50 ml of YEP liquid medium with spe 100 mg / L, chl 25 mg / L, and str 100 mg / L. Then place it in a constant temperature shaker at a temperature of 28 °C and a rotation speed of 240 rpm for overnight culture. The next day, measure the absorbance value OD600 of the bacterial solution until it is between 1.0 and 1.2. Then add it to a sterile 50 ml centrifuge tube, place it in a centrifuge with a rotation speed of 5000 rpm, a temperature of 24 °C, and a time of 10 min. After centrifugation, remove the supernatant in the laminar flow hood and collect the bacterial solution precipitate. Then resuspend the bacterial solution precipitate with the liquid co-culture medium and adjust the absorbance value until OD600 is between 0.6 and 0.8. Store it at 4 °C and wait for the next experiment (note that the placement time cannot be too long, and it needs to be prepared and used immediately).

[0078] d. Preparation and transformation of explants: For the germinated seedlings that have grown in the culture room for 5 days, carefully check the culture dishes for contamination. If there is contamination, discard them immediately. Place the uncontaminated germinated dishes into the sterilized laminar flow hood. Cut the soybean cotyledons and 0.5 cm hypocotyl from the seedlings using forceps and a scalpel. Then, vertically cut open the seeds along the cotyledons and also vertically separate the hypocotyl. Remove the young buds and epicotyls on the cotyledons. Use a scalpel to gently scratch about 10 times parallel to the hypocotyl in the cotyledon node region. The wounds should be approximately 0.5 mm deep and 3 - 4 mm long.

[0079] e. Infection and co - cultivation: Pour the prepared bacterial solution separately into other sterile 150 ml Erlenmeyer flasks in the laminar flow hood. Place the scratched explants into the flasks and soak them until the explants are just covered. Infect for 30 min, and shake the flask frequently to ensure that the explants can come into contact with fresh bacterial solution. Then pour out the bacterial solution, and use forceps to transfer the explants from the flask to a clean sterile culture dish. Then place them flat on the solid medium with the wound facing down (a layer of sterilized filter paper is placed on the medium). Place about 18 explants on each medium. Then carefully seal the dish with medical tape and transfer it to an incubator at a temperature of 22°C for 3 days of dark culture.

[0080] f. Induction of young buds: Place the prepared bud induction medium and the dark - cultured explants together on the sterile laminar flow hood. Use forceps to insert the explants obliquely into the bud induction medium. Note that the cut surface of the explant should be completely buried in the medium to ensure full contact between the wound and the medium. Place 10 explants in each culture dish. Then seal the dish with medical tape and place it in the tissue culture room. Culture for 14 days. After 14 days of culture, carefully check the medium and discard the contaminated plates. Place the sterile plates back into the laminar flow hood, transfer them to a new bud induction medium, and discard the explants that have not grown clustered buds and those that have only grown a single large bud. Place 7 explants on each plate of the replaced medium. Seal the dish with medical tape and place it in the tissue culture room. Continue to induce for 14 days.

[0081] g. Growth of young buds: Carefully check the bud induction medium and discard the contaminated medium. Place the sterile medium into the sterilized laminar flow hood. At the same time, place the bud elongation medium into the laminar flow hood. Discard the undifferentiated materials. Cut off the cotyledons of the explants with clustered buds and make a new cut at the base of the growing node using a scalpel. Transfer them to the bud elongation medium. Place 9 explants in each culture dish. Culture in the tissue culture room for 2 - 8 weeks. Check carefully every 2 weeks for contaminated culture dishes and discard the contaminated ones. Replace the non - contaminated culture dishes with a new bud elongation medium.

[0082] h. Rooting: During the process of changing the bud elongation medium, seedlings with buds elongated by at least 3 cm were placed in a laminar flow hood. The buds were cut from the roots of the tissue using a scalpel, soaked in auxin for 2 minutes, and then transferred to a triangular flask containing the rooting medium. Subsequently, the flask was sealed with a sealing film and placed in the tissue culture room to continue growing until several main roots began to grow and lateral roots also emerged. The plants were taken out of the tissue culture room, gently removed from the rooting medium, and the medium on the roots was carefully rinsed off with tap water. The seedlings were transplanted into a pot filled with nutrient soil prepared by mixing vermiculite that had been autoclaved at high temperature and pressure and genes in a ratio of 1:2. The pot was covered with a plastic film to reduce water evaporation. The film was removed after the plants were fully viable. Subsequently, nutrient solution was applied for cultivation until pod formation.

[0083] S8, Identify transgenic positive plants with specific primers (GsMYB10-JD);

[0084] Forward Primer (SEQ ID NO.7): 5'-GGGCTTACCTTGTATCAT-3'

[0085] Reverse Primer (SEQ ID NO.8): 5'-GAGCCATTTCCTGTTTG-3'

[0086] The experimental results are as Figure 4 shown in part A of the figure. M is DL2000 plus DNA marker; - is ddH2O; + is pZY101-GsMYB10 plasmid; WT is wild-type Huachun 6 soybean; L1-L3 are GsMYB10 transgenic lines.

[0087] S9, Detect the gene expression of transgenic soybean lines by qRT-PCR. The experimental results are as Figure 4 shown in part B of the figure. Compared with wild-type soybeans, the expression level of the GsMYB10 gene in transgenic soybeans is higher. The data in the figure are the mean ± standard deviation of three biological replicates (t-test P<0.05);

[0088] S10, Detect the glyphosate tolerance of transgenic plants; The experimental results are as Figure 4 shown in part C of the figure. Compared with wild-type soybeans, transgenic soybeans have stronger glyphosate tolerance.

[0089] Experiment 1, Analysis of the tissue expression pattern of the GsMYB10 gene in wild soybean BW69:

[0090] Collect roots, stems, leaves, flowers, pods and apical meristems from wild soybean BW69 (flowering stage) for total RNA extraction. Use TRIzol, a plant total RNA extraction reagent, to reverse transcribe the RNA into cDNA. Then, using cDNA as a template, employ qRT-PCR technology to measure the expression levels of the GsMYB10 gene in the above-mentioned different tissues.

[0091] Step 1: RNA Extraction (refer to Tiangen Biotech Co., Ltd.)

[0092] a. Take about 100 mg of the above sample, quickly and thoroughly grind it in liquid nitrogen, transfer it into a 2 ml centrifuge tube containing RNase-free water, and quickly add 1 ml of TRIzol Universal reagent using a pipette. Then place it on a multi-functional tissue cell grinder and shake it thoroughly for 5 min. Let the well-mixed sample stand at room temperature for 5 min.

[0093] b. Place the sample in a centrifuge for centrifugation. Set the centrifuge temperature to 4°C, the rotation speed to 12000 rpm, and the time to 10 min. After centrifugation, take about 1 ml of the supernatant and transfer it to a new RNase-free centrifuge tube.

[0094] c. Add 0.2 ml of chloroform pre-cooled in an ice-water bath to the centrifuge tube, cover the tube cap, shake it vigorously for 30 s, and then let it stand at room temperature for 3 min.

[0095] d. Place the sample in a centrifuge for centrifugation. Set the centrifuge temperature to 4°C, the rotation speed to 12000 rpm, and the centrifugation time to 15 min. After centrifugation, the sample will be layered, and the RNA is mainly in the colorless aqueous phase. Take 400 μL of the aqueous phase and transfer it to a new centrifuge tube.

[0096] e. Add 400 μL of isopropanol to the centrifuge tube containing the RNA aqueous phase, mix it thoroughly on a vortex shaker, and then let it stand at room temperature on a centrifuge plate for 10 min.

[0097] f. Centrifuge under the same conditions of temperature, rotation speed, and time as in step b, discard the supernatant, and the RNA will precipitate on the side and bottom of the tube.

[0098] g. Add 1 ml of 75% ethanol prepared with RNase-free ddH2O to wash the precipitate.

[0099] h. With the centrifuge settings unchanged, set the time to 5 min. After centrifugation, pour out the liquid in a laminar flow hood and slowly remove the remaining small amount of liquid using a pipette.

[0100] i. Let it dry in a laminar flow hood at room temperature for 5 min, add 100 μL of RNase-Free ddH2O, and shake it thoroughly on a vortex shaker to fully dissolve the RNA.

[0101] Step 2: Reverse transcription (refer to the TaKaRa instruction manual)

[0102] a. Genomic DNA removal reaction

[0103] Prepare the following system:

[0104] Table 5 DNA removal reaction system

[0105]

[0106]

[0107] Incubate at 42°C for 2 min, then place on ice for later use.

[0108] b. Reverse transcription reaction

[0109] Prepare the reaction solution on ice.

[0110] Table 6 Reverse transcription reaction solution system

[0111] Reagent Dosage Reaction solution of Step 1 10.0 μL PrimeScript RT Enzyme Mix I 1.0 μL RT Primer Mix 1.0 μ 5×PrimeScript Buffer 2 4.0 μL <![CDATA[RNaseFreedH2O]]> 4.0 μL Total 20 μL

[0112] Put the reaction system into a PCR instrument, set at 37°C / 15 min, 85°C / 5 sec, and store the obtained cDNA in a 4°C refrigerator for later use.

[0113] Step 3: Design of quantitative PCR primers

[0114] Search for the gene sequence in the Phytozome database, download the genomic CDS sequence of GsMYB7 as shown in SEQ ID NO.1 and the protein sequence of GsMYB7 as shown in SEQ ID NO.2 from the website; and design primers and synthesize specific quantitative amplification primers according to the genomic sequence on the NCBI website:

[0115] GsMYB10_qF (SEQ ID NO.9): 5’-GACTAAATGAGCAACTTCAGGTGC-3’;

[0116] GsMYB10_qR (SEQ ID NO.10): 5’-CTGAGAGGACACTTTTGACACTAGC-3’.

[0117] Step 4: Real-time fluorescence quantitative PCR

[0118] ① Use Actin3 as the internal reference gene;

[0119] ② Dilute the cDNA of all samples 1-fold with ddH2O as the template for the quantitative PCR reaction;

[0120] ③ The real-time fluorescence quantitative PCR system was carried out according to the reaction system shown in Table 7 below. The reaction procedure was: pre-denaturation at 95 °C for 30 s; 39 cycles (denaturation at 95 °C for 5 s; annealing at 60 °C for 30 s); melting curve at 95 °C for 10 s; 54.3 °C for 5 s; 94.3 °C for 5 s;

[0121] Table 7 Real-time fluorescence quantitative PCR reaction system

[0122]

[0123] ④ Process the data, and the relative expression analysis adopted the 2^-△△CT method.

[0124] The experimental results were as Figure 2 shown. In soybeans, the expression level of the GsMYB10 gene was higher in roots, flowers and pods compared to stems, leaves and apical meristems.

[0125] Experiment 2, Analysis of the expression pattern of the GsMYB10 gene in soybean roots under low phosphorus stress

[0126] Specific experimental steps:

[0127] S1. Prepare BX13 soybean seeds, disinfect their surfaces with alcohol, sow the disinfected seeds in moist vermiculite, and germinate them in an incubator (temperature: 26 °C, light: 16 h / dark: 8 h). Subsequently, transfer the seedlings with the same growth and expanded cotyledons to normal phosphorus nutrition (Normal Phosphorus, NP, 500 μM KH2PO4) and low phosphorus nutrition (Low Phosphorus, LP, 5 μM KH2PO4) culture solutions, and collect root samples at 0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 7 d respectively. Extract RNA and reverse transcribe it into cDNA, and use cDNA as a template for real-time fluorescence quantitative PCR. Fluorescent quantitative qPCR primers:

[0128] GsMYB10_qF: 5’-GACTAAATGAGCAACTTCAGGTGC-3’;

[0129] GsMYB10_qR: 5’-CTGAGAGGACACTTTTGACACTAGC-3’.

[0130] Observe the change in the expression level of this gene at the above time periods (time), and the experimental results were as Figure 3 shown. Low phosphorus stress induced the expression of the GsMYB10 gene.

[0131] Experiment 3, Determination of the transcriptional activity of the GsMYB10 protein

[0132] S1, Amplify the target fragment: Clone the target fragment of the GsMYB10 gene using the pLB vector plasmid (the cloning reaction is the same as above), and amplify the full-length coding region of GsMYB10 with special primers to obtain the amplification product. Among them, the sequences of the special primers are as follows:

[0133] GsMYB10-pGBKT7-F (SEQ ID NO.11): atggccatggaggccgaattcATGTACAGTACTCATCAGCAACATCAA

[0134] GsMYB10-pGBKT7-R (SEQ ID NO.12): ccgctgcaggtcgacggatccTTCAGTTCCAACTGAATCCATTCAA

[0135] S2, Insert the amplification product into the NcoI and BamHI sites of the pGBKT7 vector to form the pGBKT7-GsMYB10 construct (Clontech).

[0136] S3, To verify whether the GsMYB10 protein has transcriptional activity, transform the plasmid pGBKT7 (control) and the plasmid pGBKT7-GsMYB10 into yeast Y2HGold respectively. Yeast cells grow on the control medium plate (SD / -Trp) or the selective medium plate (SD / -Trp / -X-α-Gal).

[0137] Perform the transformation of Y2H yeast cells and the detection of transcriptional self-activation effect (refer to the product manual of Y2HGold Chemically Competent Cell of Shanghai Weidi Biotechnology Co., Ltd.)

[0138] a. Take 100 μL of Y2HGold competent cells in each tube from the -80 °C ultra-low temperature freezer and quickly place them in an ice box containing an ice-water mixture for thawing. PEG / LiAc is also placed in the ice box for pre-cooling. Carrier DNA is placed in a boiling water bath for 5 min, and quickly placed in an ice bath for 3 min. After repeating the operation once, it is placed in the ice box for standby. Then, use a pipette to add 2 μg of the target plasmid that has been pre-cooled, measured for concentration, and calculated for volume, 10 μL of Carrier DNA that has been treated in a boiling water bath, and 500 μL of pre-cooled PEG / LiAc in sequence, and pipette several times to mix well.

[0139] b. Place the well-mixed competent cells in a pre-prepared 30 °C constant temperature water bath for 30 min, and turn them upside down 6 - 8 times by hand at the 15th minute for mixing.

[0140] c. Transfer the competent cells that have been in a 30°C water bath for half an hour to another pool in a constant temperature water bath set at 42°C for a 15-minute water bath, and invert the tube 6 - 8 times again at the 7.5-minute mark to mix well.

[0141] d. Take out the tube that has completed the water bath and place it in a microcentrifuge. Set the rotation speed to 5000 rpm and the time to 1 minute. After centrifugation, discard the supernatant in a laminar flow hood, resuspend with 400 μL of sterile ddH2O at room temperature, and centrifuge again to discard the supernatant.

[0142] e. Resuspend again with 50 μL of ddH2O, spread the cells on an SD plate using a spreader that has been sterilized by flaming with alcohol, then seal the plate with parafilm and incubate it in a 29°C constant temperature incubator for 48 - 96 hours.

[0143] f. When the colonies on the SD plate grow to 1 mm in size, pick single colonies of pGBKT7 and pGBKT7 - GsMYB10 and spread them separately in zones on an SD / -Trp plate supplemented with X - a - Gal. Seal the plate with parafilm and incubate it upside down at 29°C for 2 - 3 days to observe whether the colonies on the medium turn blue for self - activation detection.

[0144] The results are as Figure 5 shown. The Y2H strain containing pGBKT7 - GsMYB10 grew and showed blue on the selective medium, indicating that the GsMYB10 protein has transcriptional activation activity.

[0145] Experiment 4. Verification of the subcellular localization of the GsMYB10 protein

[0146] S1. Amplify the complete open reading frame (ORF) without a stop codon and insert it into the NcoI and SpeI sites of pCAMBIA1302, which contains a GFP reporter gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, to form the construct 35S::GsMYB10 - GFP. For vector construction, please refer to the construction of overexpression vectors. The following are the recombinant cloning primers:

[0147] GsMYB10 - 1302CZ - F (SEQ ID NO.13): acgggggactcttgaccatggCTATGTACAGTACTCATCAGCAACATCAA,

[0148] GsMYB10 - 1302CZ - R (SEQ ID NO.14): aagttcttctcctttactagtGTTCCAACTGAATCCATTCAAGTCC

[0149] The experimental steps are as follows

[0150] 1. Agrobacterium tumefaciens GV3101 transformation (refer to the instruction manual of GV3101 Chemically Competent Cell of Shanghai Weidi Biotechnology Co., Ltd.)

[0151] a. Take out the preserved Agrobacterium tumefaciens GV3101 competent cells from the -80°C ultra-low temperature freezer and place them on the room-temperature workbench. Transfer them to an ice box when they are partially melted.

[0152] b. Take the plasmid to be transformed. First, measure the concentration of the plasmid. Calculate the volume of the plasmid to be added based on adding 0.01 - 1 μg of plasmid DNA to every 100 μL of competent cells. Then, use a pipette to aspirate the corresponding volume of the plasmid and add it to the competent cells. Gently tap the bottom of the tube with your hand to mix evenly. Then, perform the following experimental steps in sequence: let it stand in an ice-water bath for 5 minutes, place it in liquid nitrogen for 5 minutes, then place it on a floating plate and keep it in a water bath at 37°C for 5 minutes (be careful that the centrifuge tube does not burst during this period), and finally place it in an ice-water bath for another 5 minutes.

[0153] c. In a sterilized laminar flow hood, use a pipette to aspirate 700 μL of antibiotic-free YEB liquid medium and add it to the transformed competent cells. Then, place it on a shaker at 28°C and shake it for 3 hours.

[0154] d. Take out the resuscitated bacterial liquid in which the resistance plasmid has been expressed and place it in a microcentrifuge. Set the rotation speed to 6000 rpm and the time to 1 min for centrifugation. Spread it on a plate and culture it for 2 - 3 days.

[0155] e. When a single colony grows to a diameter of 1 mm in the laminar flow hood, use a sterile pipette tip to pick out the single colony and place it into YEP liquid medium containing the corresponding antibiotic. Then, place it on a shaker at 28°C and culture it for 6 hours. Then, shake the bacteria in the laminar flow hood and perform PCR in a PCR instrument. Finally, run electrophoresis for identification.

[0156] 2. Cultivation of Nicotiana benthamiana

[0157] a. The proportion of each component in the substrate is as follows: flower nutrient soil: vermiculite = 2:1, 26°C, 16 h / 8 h (light / dark), 70% humidity (cover with a transparent white box for moisturizing for 2 weeks)

[0158] b. After thoroughly watering the substrate, use a pipette to spot the tobacco seeds into the substrate. Finally, leave 4 seeds in each pot. Water with 1 / 6 Hoagland nutrient solution every 5 days.

[0159] 3. Agrobacterium injection into tobacco leaves

[0160] a. Select tobacco leaves (4 - leaf stage) that have been growing for about one month. Choose healthy tobacco leaf plants with good growth status (young, fully expanded, 4 - week - old) for Agrobacterium infection. Water thoroughly before transformation and do not water until after photographing is completed.

[0161] b. Preparation of Agrobacterium: Pre - culture, inoculate monoclonal into a 2 - ml centrifuge tube containing 1 ml of YEP with the corresponding antibiotics. Culture: Inoculate 1 mL of the pre - culture medium into 50 ml of YEB liquid medium containing three antibiotics, Rif (20 mg / mL), Gen (50 mg / mL), and Kan (50 mg / mL), to activate Agrobacterium (GV3101). Check that the bacterial solution has grown to the late exponential phase with OD600 = 0.6, then perform centrifugation at a speed of 4000 rpm at room temperature for 10 min to harvest the bacterial solution.

[0162] c. Prepare the suspension: Mix well and store at 4°C.

[0163] d. Resuspend to OD600 using the resuspension solution (10 mM MES - KOH, pH 5.6; 10 mM MgCl2; 100 μM acetosyringone). Prepare the resuspension solution freshly each time.

[0164] e. Similarly suspend the Agrobacterium containing P19 that has been cultured in the same way and adjust the concentration of the suspension to OD600 = 1.

[0165] f. According to the experimental design, mix the above two kinds of Agrobacterium in a 1:1 volume ratio and let it stand at room temperature for 3 - 4 h.

[0166] g. Place the tobacco under white fluorescent lights for 1 h before injection to fully open the stomata.

[0167] h. Agrobacterium injection: Resuspend the bacterial cells, suck the bacterial solution with a 1 - mL syringe without a needle, gently pierce a small hole in the middle of the leaf with the syringe needle, then use the left hand to hold the front of the leaf, and gently press the injection tube vertically against the back of the leaf with the right hand. Slowly and evenly push with the right thumb to observe the slow movement of the bacterial solution under the leaf epidermis (it is best if the bacterial solution forms bubbles in the lower epidermis for the best injection effect until the entire leaf is infected. Also, pay attention to selecting the best - growing leaf (do not use cotyledons) on one plant for injection, and inject at most half of the leaves per plant. Cover with plastic film and culture at 24°C for 2.5 - 3 days, then tear off the lower epidermis of the leaf and observe under a laser confocal microscope.

[0168] As Figure 6As shown in the figure, eGFP in the figure corresponds to the GFP channel; RFP corresponds to the RFP channel; Merge represents the merged image of GFP and RFP; Bright field is the bright field. Analysis by a laser confocal microscope showed that 35S-GFP was distributed throughout the cell, while the 35S-GsMYB10-GFP fusion protein was only localized in the nucleus. The results indicated that GsMYB10 is a nuclear localization protein. Experiment Five, Hydroponic Verification of GsMYB10

[0169] After disinfecting the GsMYB10 transgenic seeds, after germinating in vermiculite for 6 days, when the cotyledons were fully expanded and the seedlings had consistent growth, they were transferred to 1 / 2 Hoagland solution for cultivation and divided into two treatment groups: the experimental group and the control group. Among them, the experimental group was cultivated with a low phosphorus concentration (5 μmol / L K2HPO4), and the control group was cultivated with a normal phosphorus concentration (500 μmol / L K2HPO4). Among them, each group included the wild type (WT) and transgenic lines. WT was used as the within-group control (CK). The transgenic lines specifically included two lines, Gm10A and Gm10B. Each group was set with 3 replicates. After 14 days, the hydroponic phenotypes were observed, the seeds were examined, and the total phosphorus content was measured (the total phosphorus content was the total phosphorus content multiplied by the dry weight of the upper and lower parts of the plant).

[0170] ① Method for the determination of total phosphorus in plants by molybdenum antimony anti-colorimetry:

[0171] I. All reagents used in this experiment are of analytical purity, and the water is deionized water or distilled water or water of equivalent purity.

[0172] (1) Sulfuric acid;

[0173] (2) 30% hydrogen peroxide;

[0174] (3) 10% sodium hydroxide;

[0175] (4) 0.2% dinitrophenol indicator;

[0176] (5) 0.5% potassium antimony tartrate solution: Weigh 0.5 g of chemically pure potassium antimony tartrate and dissolve it in 100 mL of water;

[0177] (6) Sulfuric acid molybdenum antimony stock solution: Measure 126 mL of concentrated sulfuric acid and slowly add it to 100 mL of water, stirring constantly and cooling. Separately weigh 10 g of finely ground ammonium molybdate and dissolve it in about 300 mL of water at 60 °C, and cool. Then slowly pour the sulfuric acid solution into the ammonium molybdate solution. Then add 100 mL of 0.5% potassium antimony tartrate solution. After cooling, dilute with water to 1000 mL, shake well, and store in a brown reagent bottle. This stock solution contains 1% ammonium molybdate and 2.25 mol / L sulfuric acid;

[0178] (7) Molybdenum-antimony anti-color reagent: Weigh 1.5 g of ascorbic acid and dissolve it in 100 mL of molybdenum-antimony stock solution. This solution has a short shelf life and should be prepared freshly before use.

[0179] (8) Phosphorus standard stock solution: Accurately weigh 0.4390 g of potassium dihydrogen phosphate dried at 105 °C for 2 h. Dissolve it in water, add 5 mL of concentrated sulfuric acid, and then make up the volume to 1000 mL with water. This solution contains 100 mg / L of phosphorus and can be stored in the refrigerator for long-term use.

[0180] (9) 5 mg / L phosphorus standard solution: Pipette 5 mL of the phosphorus stock solution into a 100 mL volumetric flask and make up the volume with water. This solution should be prepared freshly before use.

[0181] II. Experimental instruments: Analytical balance (0.0001 g), 100 ml volumetric flask, 50 ml volumetric flask, funnel, several small test tubes, graphite digestion instrument (SH220F), spectrophotometer (UV-1800PC), marker pen, etc.

[0182] III. Experimental procedures

[0183] (1) Preparation of sample solution

[0184] Weigh an appropriate amount of the sample, accurate to 0.001 g, and place it in a 500 ml digestion tube (do not let the sample adhere to the bottleneck). First, drip a little water to moisten the sample, then add 6 mL of sulfuric acid, shake gently and let it stand overnight. Place a bent-neck funnel at the mouth of the tube and digest it on a digestion furnace at 250 °C first (start timing after the temperature stabilizes, about 30 min). After a large amount of white smoke emerges from the decomposition of H2SO4, raise the temperature to 400 °C. When the solution becomes uniformly dark brown, remove it.

[0185] After cooling slightly, add 10 drops of H2O2, shake well, and heat to just boiling. Digest for about 5 min, remove it and let it cool slightly. Repeat adding 5 - 10 drops of H2O2 and digesting. Repeat this 3 - 5 times. The amount of H2O2 added each time should be gradually reduced. Digest until the solution becomes colorless or clear and the bottom is grayish-white, then heat for about 5 - 10 min to remove the remaining H2O2.

[0186] Remove the digestion tube and let it cool. Rinse the bent-neck funnel with a small amount of water and let the washing solution flow into the digestion tube. Transfer the digestion solution into a 100 mL volumetric flask without loss, make up the volume with water, and shake well. Filter or let it stand for clarification for the determination of phosphorus.

[0187] (2) Preparation of blank solution

[0188] Except for not adding the sample, use the same reagents and operation steps as above.

[0189] (3) Plotting the standard curve

[0190] Respectively pipette 0, 2, 4, 6, 8, 10, 14, 20 mL of 5 mg / L phosphorus standard solution into 50 mL volumetric flasks. At the same time, add the same volume of blank solution as the sample solution used in the colorimetric determination and 2 - 3 drops of dinitrophenol indicator. Then adjust the solution to just turn slightly yellow with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony anti - color reagent, shake well, add water to make up the volume to the mark, and thus obtain a series of standard solutions with phosphorus contents of 0.0, 0.2, 0.4, 0.8, 1.0, 1.4, 2.0 mg / L respectively. Shake well, after standing at a temperature above 15 °C for 30 min, measure the absorbance at a wavelength of 880 nm. On the square coordinate paper, use the absorbance as the ordinate and the phosphorus concentration (mg / L) as the abscissa to draw the calibration curve.

[0191] (4) Determination of phosphorus in the sample solution

[0192] Pipette 2 - 10 mL of the sample solution to be measured (containing 0.04 - 1.0 g of phosphorus) into a 50 mL volumetric flask. Add 2 - 3 drops of dinitrophenol indicator, and adjust the solution to just turn slightly yellow with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony anti - color reagent, shake well, add water to make up the volume to the mark. Place it for 30 min under the condition of room temperature above 15 °C.

[0193] For the colored sample solution on the spectrophotometer, use a 1 cm light path cuvette at 880 nm, adjust the instrument zero point with the blank test as the reference solution, conduct colorimetric determination, and read the absorbance. Check the corresponding phosphorus content from the standard curve.

[0194] IV. Result calculation

[0195] Total sample

[0196] In the formula:

[0197] C——The mass concentration of phosphorus in the colored solution obtained from the calibration curve or regression equation, μg / L;

[0198] C0——Blank value

[0199] V——Volume of the colored solution;

[0200] D——Dilution factor, volume of the digested solution made up to the mark / volume of the digested solution taken;

[0201] m——Mass of the sample taken, g;

[0202] 1000——Conversion factor for converting the unit mL to L. The obtained result should be retained to three decimal places.

[0203] Fluorescent quantitative qPCR primers:

[0204] GsMYB10_qF: 5'-GACTAAATGAGCAACTTCAGGTGC-3';

[0205] GsMYB10_qR: 5'-CTGAGAGGACACTTTTGACACTAGC-3'.

[0206] The results are as Figure 7 shown. Compared with WT, the primary root length and the number of lateral roots of transgenic plants increased, the aboveground fresh weight and the underground fresh weight of transgenic plants increased significantly, and the total phosphorus content of transgenic lines was significantly higher than that of the control group.

[0207] Experiment 6, Verification of GsMYB10 Tolerance to Low Phosphorus during the Whole Growth Period in Soil Culture

[0208] The transgenic soybean was planted in the greenhouse of the experimental network of South China Agricultural University in August 2024, and the soybean variety was Huachun 6.

[0209] The soybeans were set up into the following treatment groups: low phosphorus (LP) treatment experimental group, normal phosphorus (NP) treatment control group. Among them, each group included wild type (WT) and transgenic lines. WT was used as the within-group control (CK). The transgenic lines specifically included two lines, Gm10A and Gm10B. Each group was set with 3 replicates.

[0210] Treatment methods for treatment groups

[0211] Low phosphorus treatment experimental group: Uniformly spray 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the seedling stage, and irrigate the roots with 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the flowering stage and podding stage respectively. Normal phosphorus treatment group: Uniformly spray 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the seedling stage, and irrigate the roots with 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the flowering stage and podding stage respectively. A total of three treatments were carried out, one spraying and two irrigations. The spraying dosage of 1 / 2 Hoagland solution with low phosphorus and normal phosphorus at the seedling stage was about 7 L respectively, and the irrigation dosage of 1 / 2 Hoagland solution with low phosphorus and normal phosphorus at each time during the flowering stage and podding stage was about 15 L respectively.

[0212] After the soybeans matured, their growth conditions were photographed and recorded. The experimental results are as Figure 8As shown in the figure, in the LP and NP treatment groups, the growth of GsMYB10 transgenic soybeans was better than that of the WT control within the same group. Through yield trait analysis, it was found that the number of pods per plant and the height of the bottom pod of transgenic plants were higher than those of the control group under LP treatment. Under LP treatment, the plant height of transgenic plants of Gm10A was higher than that of the control group, and there were no obvious changes in other plant lines. There was no obvious change in 100-seed weight. The above results can help prove the application of this gene in plant tolerance to low phosphorus. Experiment 7: Verification of GsMYB10 tolerance to low phosphorus at the seedling stage in soil culture

[0213] The transgenic soybeans were planted in the experimental greenhouse of South China Agricultural University, and the soybean variety was Huachun 6.

[0214] The soybeans were set up into the following treatment groups: low phosphorus (LP) treatment experimental group and normal phosphorus (NP) treatment control group. Among them, each group included wild type (WT) and transgenic lines. WT was used as the control within the group (CK). The transgenic lines specifically included two lines, Gm10A and Gm10B. Each group was set with 3 replicates.

[0215] Treatment methods for treatment groups

[0216] Low phosphorus treatment experimental group: Uniformly spray 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the seedling stage. Normal phosphorus treatment group: Uniformly spray 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the seedling stage.

[0217] When the soybeans were cultured to the V2 stage, the growth conditions were photographed and recorded. The experimental results are as Figure 9 shown. Under low phosphorus (LP) and normal phosphorus (NP) treatment conditions, the above-ground weight of transgenic lines was significantly higher than that of the control group (CK).

[0218] The present invention provides the application of the GsMYB10 gene in regulating the tolerance of soybeans to low phosphorus stress. The GsMYB10 gene can enhance the tolerance of soybeans to low phosphorus stress. Overexpression of the GsMYB10 gene enhances the low phosphorus tolerance of plants.

[0219] GsMYB10 gene sequence and amino acid sequence

[0220] The CDS sequence of GsMYB10 (Glyma.11g183400) is shown in SEQ ID NO.1

[0221]

[0222] The amino acid sequence encoded by GsMYB10 (Glyma.11g183400) is shown in SEQ ID NO.2:

[0223] MYSTHQQHQGKNIHSSSSSSRMPIPSERHMFLQTGNGSGDSGLVLSTDAKPRLKWTPDLHARFIEAVQQLGGADKATPKTVMKLMGIPGLTLYHLKSHLQKYRLSKSLHGQSNNATHKITINSGSATDERLRENNETHVMNNLNLAPQSINKDLHISEALQMQIEVQRRLNEQLQVQRLLQLRIEAQGKYLQAVLEKAQETLGRQNLGVVGLEAAKLQLSELVSKVSSQCLNSAFSELKEIQGFSPHHQKQTQTNNNQPINANDCSMDSCLTSCEGSSQKDQQEIQNRGMNLIPFNVHTFMEGPNLNNLPNTDLKWCDPVKKNNTFLTRLSMHAERSPSNLSMSIGLLEGETTENRSTIVRTESIKPAVAEKVSQDYGLPSNYFAASKLDQTTEDNKDTKTSCKQLDLNGFSWN*

[0224] The above-described embodiments are only preferred embodiments of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of the gene GsMYB10 in enhancing the low phosphorus stress tolerance of plants, characterized in that: The nucleotide sequence of the gene GsMYB10 is shown in SEQ ID NO.1; or the gene GsMYB10 encodes an amino acid sequence shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The plant is soybean.

3. A method for improving the low phosphorus stress tolerance of plants, characterized in that, The method is to increase the expression level of the amino acid sequence shown in SEQID NO.2 in the recipient plant; The specific steps include: introducing the gene GsMYB10 shown in SEQ ID NO.1 into the recipient plant to obtain a transgenic plant, and the low-phosphorus stress tolerance of the transgenic plant is higher than that of the recipient plant; or overexpressing the amino acid sequence shown in SEQ IDNO.2 in the recipient plant.

4. The method according to claim 3, characterized in that, The improvement of the low-phosphorus stress tolerance of the plant is manifested as an increase in its root length or the number of lateral roots.

5. Application of the recombinant plasmid, recombinant vector, and transgenic plant cell line of the gene GsMYB10 in improving the low-phosphorus stress tolerance of plants, wherein the nucleotide sequence of the gene GsMYB10 is shown in SEQ ID NO.1; or the gene GsMYB10 encodes an amino acid sequence shown in SEQ ID NO.2.

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