Application of GsMYB10 gene in plant tolerance to low phosphorus stress

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a kind of important gene GsMYB10 of plant tolerance to low phosphorus and application thereof.Research shows that GsMYB10 gene is induced to express up-regulation under low phosphorus stress, and under different phosphorus concentration treatment conditions, overexpression GsMYB10 can significantly increase the biomass of transgenic plants, thereby promoting plant growth under low phosphorus conditions;Meanwhile, overexpression GsMYB10 can improve the tolerance of plant to low phosphorus stress, and reduce the inhibition of low phosphorus on plant root growth.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the GsMYB10 gene in plant tolerance to low phosphorus stress. Background Technology

[0002] Soybeans are a highly nutritious plant, with a protein content 2.5 to 8 times higher than that of grains and tubers. Furthermore, their levels of fat, calcium, phosphorus, iron, and essential vitamins B1 and B2 are also higher than in grains and tubers. Soybeans have a wide range of uses. For food, soybeans can be processed into tofu, soy milk, and dried bean curd sticks. Fermented soy products include fermented bean curd, stinky tofu, broad bean paste, soy sauce, fermented black beans, and natto, while non-fermented soy products include soft tofu, dried tofu, bean sprouts, and braised soy products. For animal feed, soybean stems, leaves, and residue are excellent fertilizers and fodder, suitable for ruminant livestock such as chickens, pigs, and dairy cows.

[0003] Soybeans are phosphorus-loving crops, and phosphorus plays a crucial role throughout their entire life cycle. Insufficient phosphorus intake inhibits root nodule growth, reduces nitrogen fixation, and ultimately severely diminishes soybean yield and quality. China is the world's largest soybean consumer, with an annual demand exceeding 100 million tons. However, China's soybean self-sufficiency rate is only 18%-20%. China's soybean production is mainly concentrated in Northeast China, accounting for approximately 50%-60% of the national total. While the Huang-Huai-Hai Plain and the red soil regions of southern China possess abundant land resources, phosphorus deficiency in the soil restricts soybean plant growth and reduces pod formation, ultimately impacting soybean production and quality.

[0004] Therefore, screening for phosphorus-efficient genes to improve soybean's tolerance to low phosphorus levels is one of the important ways to increase soybean yield and quality. Summary of the Invention

[0005] To address the problems mentioned above in the background art, the first objective of this invention is to provide the application of the gene GsMYB10 in improving the plant's tolerance to low phosphorus stress, thereby increasing the yield and quality of soybeans under low phosphorus conditions. 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.

[0006] This application improves the ability of plants to tolerate low phosphorus stress by overexpressing the gene GsMYB10 in plants.

[0007] Furthermore, the plant in question is soybean.

[0008] The second objective of this invention is to provide a method for improving the tolerance of plants to low phosphorus stress. The method involves increasing 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 a transgenic soybean plant, wherein the transgenic plant has a higher tolerance to low phosphorus stress than the recipient plant; or overexpressing the amino acid sequence shown in SEQ ID NO.2 in the recipient plant.

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

[0010] The third objective of this invention is to provide the application of recombinant plasmids, recombinant vectors, and transgenic plant cell lines of the gene GsMYB10 in improving the plant's tolerance to low phosphorus stress, wherein 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] This application studies a method for promoting plant growth under low phosphorus stress environments using the soybean low phosphorus tolerance gene GsMYB10, including the following specific steps:

[0012] Using soybean as material, the GsMYB10 gene was genetically transformed into soybean using the Agrobacterium-mediated soybean cotyledon node transformation method, resulting in soybean plants overexpressing the GsMYB10 gene. By comparing the growth performance, phosphorus uptake efficiency, and root development of transgenic plants and wild-type plants under low phosphorus stress conditions, the function and application potential of the GsMYB10 gene in improving soybean's tolerance to low phosphorus were verified.

[0013] In summary, the beneficial effects of this invention are as follows: By cloning and analyzing the soybean gene GsMYB10, and using Agrobacterium-mediated transformation of soybean cotyledonary nodes, this invention can successfully integrate the GsMYB10 gene into the soybean genome and stably express it. At the same time, it was found that the low phosphorus tolerance of GsMYB10 overexpression plants is significantly enhanced, which can lay the foundation for molecular breeding of soybeans with low phosphorus tolerance.

[0014] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0015] Figure 1 Cloning of the GsMYB10 gene;

[0016] Figure 2 Tissue expression pattern analysis of the GsMYB10 gene in wild soybean BW69 line;

[0017] Figure 3 Changes in the expression level of the GsMYB10 gene at different time points;

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

[0019] Figure 5 : A diagram illustrating the transcriptional activation activity of the GsMYB10 protein in yeast cells;

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

[0021] Figure 7 Verification of GsMYB10 transgenic soybean seedlings in hydroponics;

[0022] Figure 8 Data on the verification and evaluation of GsMYB10 transgenic soybeans grown in soil in a net-house throughout its entire growth period;

[0023] Figure 9 Data from the verification and evaluation of GsMYB10 transgenic soybean seedlings in soil cultivation in net houses. Detailed Implementation

[0024] To facilitate a clearer understanding of the present invention, the following description, based on specific embodiments and accompanying drawings, further illustrates the invention. In this invention, the equipment and raw materials used are readily available from the market or commonly used in the field; the soybeans used are provided by the Guangdong Branch of the National Soybean Improvement Center. It should be understood that the implementation of this invention is not limited to the following embodiments; unless otherwise specified, all are within the scope of protection of this invention.

[0025] The full-length cDNA of the GsMYB10 gene in this application was cloned from wild soybean BW69, encoding the same sequence as cultivated soybean Huachun 6. The GsMYB10 gene is located on chromosome 11, named Glyma11g183400, contains 6 exons, has a full length of 3695 bp, a transcribed sequence of 1867 bp, an open reading frame of 1245 bp, and 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 genome 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 primers described above to obtain an amplification product of 1402 bp in length. The full-length CDS sequence of GsMYB10 was amplified according to the reaction system listed in Table 1 below. The reaction program was as follows: 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 at 12°C.

[0030] Table 1 Fragment amplification reaction system

[0031]

[0032] S3. The amplification product was purified by gel extraction using a DNA fragment purification kit (Tiangen Biotech (Beijing) Co., Ltd.) to obtain a high-purity target gene fragment.

[0033] S4. The PCR fragment cloned and purified product was ligated into the pLB vector (zero background rapid cloning kit, Tiangen Biotech Co., Ltd.) to obtain the ligation product (pLB-GsMYB10). The reaction system is shown in Table 2 below. The reaction program is: 20℃, 2min; 70℃, 5min; 22℃, 20min.

[0034] Table 2 Connection Reaction System

[0035]

[0036]

[0037] S5, E. coli transformation: Add 10 μL of ligation product to 100 μL of DH5α E. coli competent cells, gently aspirate and mix, then incubate on ice for 30 min, followed by heat shock at 42°C for 45 s, and immediately transfer to ice for 2 min; then transfer the ligation product to a clean bench, add 500-700 μL of LB liquid medium, and incubate at 37°C and 220 rpm for 1 h to obtain bacterial culture. Spread the bacterial culture on plates containing Amp, incubate overnight at 37°C, pick single clones for detection and sequencing.

[0038] The cloning of the GsMYB10 gene is as follows: Figure 1 As shown, M is the DL2000 DNA marker; 1 indicates that the size of the GsMYB10 gene amplification product is 1402 bp.

[0039] S6. The GsMYB10 gene coding sequence from the correctly sequenced pLB-GsMYB10 plasmid was cloned into the pZY101 vector to construct the GsMYB10-pZY101 overexpression vector.

[0040] Specific steps for carrier construction:

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

[0042] a. Primer design for the insertion fragment. Primers were designed using CE Design V1.03 software from Nanjing Novizan Biotechnology Co., Ltd.

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

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

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

[0046] Table 3 Enzyme digestion reaction system

[0047] reagents volume vector plasmid 3μL 10*KBuffer 2μL 10*BSA 2μL SpeI 1μL NcoI 1μL Sterilized water 11μL Total 20μL

[0048] The added enzyme digestion system was thoroughly mixed and briefly centrifuged, then placed in a PCR instrument. The program was set to 37℃ / 1h, 4℃ / ∽. The digested system was purified and the enzyme-digested vector fragments were recovered using a PCR product purification kit.

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

[0050] d. Reaction system preparation and reaction: The concentrations of the enzyme digestion vector and the insert fragment were determined using a Thermo Fisher NanoDrop 2000 micro UV-Vis spectrophotometer and estimated by electrophoresis before preparing the reaction system. The reaction system was prepared as shown in Table 4 below on an ice box.

[0051] Table 4 Recombinant Linkage Reaction System

[0052]

[0053]

[0054] After the system is prepared, gently pipette up and down to mix all components thoroughly. Then, transfer each component to a centrifuge tube and briefly centrifuge (avoiding the generation of air bubbles). Place the centrifuge tube in a PCR instrument for reaction, with the program set to 37℃ / 30min, 4℃ / ∽. After the reaction is complete, promptly remove the centrifuge tube and place it in an ice-water bath for 5min to cool.

[0055] e. Reaction product conversion and coating.

[0056] f. Cloning identification

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

[0058] S7, the GsMYB10-pZY101 vector was transferred into the soybean cultivar Huachun 6 using Agrobacterium-mediated cotyledon node transformation.

[0059] 1. Preparation of electrocompetent cells of Agrobacterium tumefaciens EHA101

[0060] a. Using a pipette in a clean bench, pick up a single colony and inoculate it into 1 ml of YEP liquid medium containing three antibiotics (Chl). Place the medium on a 28°C constant temperature shaker and incubate at 240 rpm for 6 hours.

[0061] b. At 8 p.m., use a pipette in a clean bench to transfer 1 ml of bacterial culture into 50 ml of YEP liquid culture medium containing the corresponding antibiotics, and continue overnight culture until the OD value is approximately 0.6 the next day.

[0062] c. In a clean bench, transfer the bacterial culture to a 50ml sterile centrifuge tube and place it in an ice box containing ice water for 30 minutes. Then, place it in an ultracentrifuge and centrifuge at 4000 rpm, 4℃, and 10 minutes.

[0063] d. Discard the supernatant in a clean bench, then resuspend the bacterial cells in a small amount of ice-cold sterile water to completely dissolve the bacterial solution, then add ice-cold sterile water to make up to 50 ml, and centrifuge again in the centrifuge set in step c.

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

[0065] f. In a clean bench, discard the supernatant repeatedly, then resuspend the bacteria in a small amount of ice-cold sterile water to completely dissolve the bacterial solution, then add ice-cold sterile water to make up to 5 ml, and centrifuge in the centrifuge set in step c.

[0066] g. Finally, discard the supernatant in a clean bench and suspend the precipitate with 10% glycerol to completely dissolve the bacterial solution. Dispense 100 μl per tube and store at -80°C for later use.

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

[0068] a. Remove the electrocution cup stored in 70% ethanol from the laminar flow hood, dry it in the laminar flow hood, then sterilize it with ultraviolet light for 20 minutes and use it after air drying.

[0069] b. Take the prepared Agrobacterium EHA101 competent cells from the -80℃ ultra-low temperature freezer and thaw them in an ice box. At the same time, take the plasmid to be transformed from the -20℃ freezer and thaw it at room temperature. Use a pipette to add the completely thawed plasmid to the thawed competent cells. In a clean bench, use a pipette to gently mix the cells and then transfer them to a sterilized and air-dried beaker.

[0070] c. Then place the shock cup into the preheated shock device, set the parameters to: voltage 1800V, time 5ms, and perform the shock.

[0071] d. After electroporation, place the container in a clean bench and use a pipette to slowly add 1 ml of antibiotic-free YEP liquid culture medium into the electroporation cup. Aspirate and mix the bacteria several times to suspend them.

[0072] e. Aspirate the suspended and mixed bacterial solution from the electroporation cup and place it in a centrifuge tube. Then, place the tube on a constant temperature shaker at 28°C, 240 rpm, and 3 hours for incubation.

[0073] f. In a sterile laminar flow hood, use a pipette to draw 50 μl of bacterial suspension and spread it onto a YEP solid plate containing three antibiotics: STR, SPE, and CHL using an alcohol-sterilized spreader. Incubate the plate at 28°C for about 36 hours until colonies grow.

[0074] 3. Genetic transformation of soybean cotyledonary nodes

[0075] a. Seed disinfection: Soybean seeds were disinfected using a dry surface disinfection method. First, the selected and stored Huachun 6 seeds were removed from the -20℃ freezer and arranged in a single layer in a petri dish. Then, the dish was placed in a desiccator within a fume hood, along with a beaker containing 100mL of sodium hypochlorite (250mL). Using a 5ml pipette, 4.2mL of concentrated hydrochloric acid was slowly added along the beaker wall, and the desiccator was immediately covered. After chlorine sterilization for 13.5 hours, the fume hood was opened, the petri dishes in the desiccator were covered, and the dish was quickly placed in a UV-sterilized laminar flow hood with ventilation for approximately 40 minutes before proceeding to the next step.

[0076] b. Germination: Place the pre-prepared germination medium (GM) in the laminar flow hood, and then use tweezers sterilized by alcohol flame to place approximately 12 sterilized soybean seeds, hilum-side down, into the germination medium. After inoculation, seal the plates with medical tape and place the culture dishes in the tissue culture room. The tissue culture room environment is set as follows: 16h light / 8h darkness, temperature 24℃, and light intensity 140μmoles / m² / sec, and culture for 5 days.

[0077] c. Bacterial suspension preparation: Take 1 ml of Agrobacterium tumefaciens strain EHA101 containing GsMYB10-pZY101 (which has already undergone bacterial suspension PCR identification), place it in a sterile laminar flow hood, and pipette it into a 250 ml sterile Erlenmeyer flask containing 100 mg / L SPECIAL, 25 mg / L CHIL, and 100 mg / L STR in YEP liquid medium. Incubate overnight in a shaker at 240 rpm and 28°C. The next day, measure the absorbance (OD600) of the bacterial suspension until it is between 1.0 and 1.2. Then, add it to a sterile 50 ml centrifuge tube and centrifuge at 5000 rpm, 24°C, and 10 min. After centrifugation, remove the supernatant in a laminar flow hood and collect the bacterial precipitate. Resuspend the precipitate in liquid co-culture medium and adjust the absorbance until the OD600 is between 0.6 and 0.8. Store at 4℃ for the next experiment (note that it should not be stored for too long and should be prepared and used immediately).

[0078] d. Explant preparation and transformation: Germinating seedlings grown in the culture room for 5 days were carefully inspected for contamination in the culture dishes. If contamination was found, the dishes were discarded immediately. Uncontaminated germination dishes were placed in a sterilized laminar flow hood. Using forceps and a scalpel, the soybean cotyledons and 0.5 cm hypocotyl were removed from the seedlings. The seeds were then vertically split along the cotyledons, and the hypocotyl was also separated vertically. The buds and epicotyl on the cotyledons were removed. Using a scalpel, approximately 10 shallow incisions were made parallel to the hypocotyl in the cotyledonary node area, each incision approximately 0.5 mm deep and 3-4 mm long.

[0079] e. Infection and Co-cultivation: In a laminar flow hood, separately pour the prepared bacterial solution into other sterile 150ml Erlenmeyer flasks. Immerse the nicked explants in the Erlenmeyer flasks until the solution just covers the explants. Infect for 30 minutes, shaking the flasks frequently to ensure the explants come into contact with fresh bacterial solution. Then pour out the bacterial solution and use forceps to transfer the explants from the Erlenmeyer flasks to clean, sterile culture dishes. Place the wound-side down on a solid culture medium (with a layer of sterilized filter paper on top). Place approximately 18 explants on each culture dish. Carefully seal the dishes with medical tape and transfer them to a constant temperature incubator at 22°C for 3 days in the dark.

[0080] f. Shoot Induction: Place the prepared shoot induction medium and the dark-cultured explants together on a sterile laminar flow hood. Use forceps to obliquely insert the explants into the shoot induction medium, ensuring the cut surface of the explant is completely embedded in the medium to guarantee full contact between the wound and the medium. Place 10 explants in each culture dish. Seal the dish with medical tape and place it in the tissue culture room. Culture for 14 days. After 14 days, carefully inspect the medium and discard any contaminated plates. Place the sterile plates back into the laminar flow hood, replace the medium with fresh shoot induction medium, and discard any explants that have not produced clustered shoots. Cut off any plates with only one large shoot using a scalpel. Place 7 explants in each fresh medium dish and seal the dish with medical tape. Place it in the tissue culture room and continue induction for another 14 days.

[0081] g. Shoot growth: Carefully examine the shoot induction medium, discard any contaminated medium, and place the sterile medium into a sterilized laminar flow hood. Simultaneously, place the shoot elongation medium into the laminar flow hood as well. Discard any undifferentiated material. Remove the cotyledons from explants that have developed clusters of shoots, and use a scalpel to make a new incision at the base of each node. Transfer the explants to the shoot elongation medium, placing 9 explants per dish. Culture in a tissue culture room for 2-8 weeks, carefully checking for contaminated dishes every 2 weeks. Discard any contaminated dishes and replace the uncontaminated dishes with fresh shoot elongation medium.

[0082] h. Rooting: During the change of the bud elongation medium, place seedlings with buds at least 3cm long in a clean bench. Use a scalpel to cut the bud from the root of the tissue, soak it in auxin for 2 minutes, and then transfer it to an Erlenmeyer flask containing rooting medium. Cover with a sealing film and place in the tissue culture room to continue growing until several taproots and lateral roots begin to emerge. Remove the plant from the tissue culture room, gently remove it from the rooting medium, and carefully rinse the roots with tap water to remove the medium. Transplant the seedling into a pot containing a 1:2 mixture of autoclaved vermiculite and genetically modified (GM) potting soil. Cover with plastic film to reduce water evaporation. Remove the film once the plant is fully viable. Continue culturing with nutrient solution until pods form.

[0083] S8. Transgenic positive plants were identified using 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] Experimental results are as follows Figure 4 As shown in section A, M represents DL2000 with DNA labeling; - represents ddH2O; + represents pZY101-GsMYB10 plasmid; WT represents wild-type Huachun 6 soybean; and L1-L3 represent GsMYB10 transgenic lines.

[0087] S9, gene expression in transgenic soybean lines was detected by qRT-PCR, and the experimental results are as follows: Figure 4 As shown in Part B, the expression level of the GsMYB10 gene is higher in transgenic soybeans compared to wild-type soybeans. The data in the figure are the mean ± standard deviation of three biological replicates (t-test P<0.05).

[0088] S10, to test the tolerance of transgenic plants to glyphosate; the experimental results are as follows: Figure 4 As shown in section C, genetically modified soybeans are more tolerant to glyphosate than wild-type soybeans.

[0089] Experiment 1: Tissue expression pattern analysis of the GsMYB10 gene in wild soybean BW69:

[0090] Roots, stems, leaves, flowers, pods, and apical meristems were collected from wild soybean BW69 (flowering stage) for total RNA extraction. The RNA was reverse transcribed into cDNA using TRIzol, a plant total RNA extraction reagent. The expression level of the GsMYB10 gene in the above different tissues was then determined using qRT-PCR technology, with cDNA as a template.

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

[0092] a. Take approximately 100 mg of the sample and grind it rapidly and thoroughly in liquid nitrogen. Transfer the mixture to a 2 ml centrifuge tube containing RNase-free gel, and quickly add 1 ml of TRNzol Universal reagent using a pipette. Then, place the tube on a multifunctional tissue cell homogenizer and vortex thoroughly for 5 minutes. Allow the well-mixed sample to stand at room temperature for 5 minutes.

[0093] b. Place the sample in a centrifuge for centrifugation. Set the centrifuge temperature to 4℃, the speed to 12000rpm, and the time to 10min. After centrifugation, take about 1ml of 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, cap the tube, shake vigorously for 30 seconds, and then let it stand at room temperature for 3 minutes.

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

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

[0097] f. Centrifuge under the same conditions as step b, including temperature, rotation speed, and time. Discard the supernatant. RNA will precipitate on the sides 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 minutes. After centrifugation, pour out the liquid in the clean bench and slowly remove the remaining small amount of liquid with a pipette.

[0100] i. Place the RNA on a clean bench at room temperature for 5 minutes to air dry, add 100 μL of RNase-Free ddH2O, and vortex to fully dissolve the RNA.

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

[0102] a. Genomic DNA removal reaction

[0103] Equipped with the following system:

[0104] Table 5 DNA Removal Reaction System

[0105]

[0106]

[0107] Incubate at 42℃ for 2 minutes, then place in an ice box for later use.

[0108] b. Reverse transcription reaction

[0109] The reaction solution was prepared on ice.

[0110] Table 6 Reverse Transcription Reaction Solution System

[0111] reagents Usage The reaction solution in step 1 10.0μL PrimeScriptRTEnzymeMixI 1.0μL RTPrimerMix 1.0μ 5×PrimeScriptBuffer2 4.0μL <![CDATA[RNaseFreedH2O]]> 4.0μL Total 20μL

[0112] Place the reaction system in a PCR instrument, set the temperature to 37℃ for 15 min and 85℃ for 5 sec, and store the obtained cDNA in a refrigerator at 4℃ for later use.

[0113] Step 3: Design of Quantitative PCR Primers

[0114] Gene sequences were retrieved from the Phytozome database, and the GsMYB7 genome CDS sequence (as shown in SEQ ID NO. 1) and GsMYB7 protein sequence (as shown in SEQ ID NO. 2) were downloaded from the website. Primers were then designed and synthesized specifically for quantitative amplification based on the genome sequences from 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 quantitative PCR

[0118] ① Actin3 was used as an internal reference gene;

[0119] ② Dilute all cDNA samples by 1-fold with ddH2O to serve as templates for quantitative PCR reactions;

[0120] ③ The real-time quantitative PCR system was run according to the reaction system in Table 7 below. The reaction program was as follows: pre-denaturation 95℃, 30s; 39 cycles (denaturation 95℃, 5s; annealing 60℃, 30s); melting curve 95℃, 10s; 54.3℃, 5s; 94.3℃, 5s;

[0121] Table 7 Real-time Quantitative PCR Reaction System

[0122]

[0123] ④ Data processing: The relative expression level analysis was performed using the 2^-△△CT method.

[0124] Experimental results are as follows Figure 2 As shown, in soybean, the GsMYB10 gene is expressed at higher levels in roots, flowers, and pods compared to stems, leaves, and apical meristems.

[0125] Experiment 2: Analysis of GsMYB10 gene expression patterns in soybean roots under low phosphorus stress duration

[0126] Specific experimental steps:

[0127] S1. Prepare BX13 soybean seeds and disinfect their surface with alcohol. Sow the disinfected seeds in moist vermiculite and place them in a culture room (temperature: 26℃, light: 16h / dark: 8h) for germination. Then, after the cotyledons unfold, seedlings of similar growth are grouped and transferred to normal phosphorus nutrient (Normal Phosphorus, NP, 500μM KH2PO4) and low phosphorus nutrient (Low Phosphorus, LP, 5μM KH2PO4) nutrient solutions. Root samples are collected at 0h, 3h, 6h, 12h, 24h, 36h, 48h, and 7d. RNA is extracted and reverse transcribed into cDNA. Real-time quantitative PCR is performed using the cDNA as a template. Primers for real-time quantitative PCR:

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

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

[0130] The changes in reletive expression level of this gene were observed during the above time periods. The experimental results are as follows: Figure 3 As shown, low phosphorus stress induces the expression of the GsMYB10 gene.

[0131] Experiment 3: Determination of GsMYB10 protein transcriptional activity

[0132] S1, Amplifying the Target Fragment: The GsMYB10 gene is cloned using a pLB vector plasmid (cloning reaction as above). The full-length coding region of GsMYB10 is amplified using special primers to obtain the amplification product. The special primer sequences are as follows:

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

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

[0135] S2, the amplification product is inserted 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, plasmid pGBKT7 (control) and plasmid pGBKT7-GsMYB10 were transformed into yeast Y2HGold, respectively. Yeast cells were grown on control medium plates (SD / -Trp) or selective medium plates (SD / -Trp / -X-α-Gal).

[0137] Transformation and transcriptional autoactivation effects of Y2H yeast cells were detected (referencing the product manual for Y2HGold Chemically Competent Cell from Shanghai Weidi Biotechnology Co., Ltd.).

[0138] a. Take 100 μL tubes of Y2HGold competent cells from a -80°C ultra-low temperature freezer and quickly place them into an ice box containing an ice-water mixture for thawing. Pre-cool the PEG / LiAc in the ice box as well. Place the carrier DNA in a boiling water bath for 5 min, then quickly place it in an ice bath for 3 min, repeating this process once before placing it in the ice box for later use. Then, using a pipette, add 2 μg of pre-cooled target plasmid (with its concentration determined and volume calculated), 10 μL of the pre-treated carrier DNA, and 500 μL of pre-cooled PEG / LiAc, mixing thoroughly with a few pipettes.

[0139] b. Place the well-mixed competent cells into a pre-prepared 30°C constant temperature water bath and incubate for 30 minutes. At the 15-minute mark, remove the cells by hand and invert them 6-8 times to mix thoroughly.

[0140] c. Place the competent cells that have been bathed in water at 30°C for half an hour into another pool in a constant temperature water bath set at 42°C for 15 minutes, and then invert them 6-8 times at the 7.5-minute mark to mix them thoroughly.

[0141] d. Remove the tubes that have undergone water bath and place them in a microcentrifuge. Set the speed to 5000 rpm and the time to 1 min. After centrifugation, discard the supernatant in a clean bench and resuspend the tubes in 400 μL of sterile ddH2O at room temperature. Centrifuge again and discard the supernatant.

[0142] e. Resuspend the plate in 50 μL ddH2O, spread it onto an SD plate using a spreader that has been flame-treated with alcohol, seal the plate with sealing film, and incubate it in a 29°C incubator for 48-96 h.

[0143] f. Once colonies on the SD plate reach 1 mm in size, pick out single colonies of pGBKT7 and pGBKT7-GsMYB10 and spread them separately on SD / -Trp plates containing Xa-Gal. After sealing with sealing film, incubate upside down at 29°C for 2-3 days and observe whether the colonies on the culture medium turn blue to perform self-activation detection.

[0144] The results are as follows Figure 5 As shown, the Y2H strain containing pGBKT7-GsMYB10 grows and shows a blue color on selective medium, indicating that the GsMYB10 protein has transcriptional activation activity.

[0145] Experiment 4: Verifying the subcellular localization of 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 the GFP reporter gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter, to form the construct 35S::GsMYB10-GFP. For vector construction, please refer to the overexpression vector construction guide. The following are the primers for recombinant cloning:

[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 GV3101 transformation (refer to the GV3101 Chemically Competent Cell instruction manual from Shanghai Weidi Biotechnology Co., Ltd.)

[0151] a. Remove the Agrobacterium GV3101 competent cells stored in the -80℃ ultra-low temperature freezer and place them on a work surface at room temperature. When they partially thaw, transfer them to an ice box.

[0152] b. Take the plasmid to be transformed. First, determine the plasmid concentration. Calculate the required plasmid volume by adding 0.01-1 μg of plasmid DNA per 100 μL of competent cells. Then, use a pipette to add the corresponding volume of plasmid to the competent cells and gently stir the bottom of the tube by hand. Perform the following experimental steps in sequence: stand in an ice-water bath for 5 minutes, place in liquid nitrogen for 5 minutes, then add a float plate and place in a water bath at 37°C for 5 minutes (be careful of the centrifuge tubes spraying out during this time), and finally place in an ice-water bath for 5 minutes.

[0153] c. Using a pipette, add 700 μL of antibiotic-free YEB liquid culture medium to the transformed competent cells in a sterilized laminar flow hood, and then place the cells on a 28°C constant temperature shaker for 3 hours.

[0154] d. Take out the bacterial culture that has been revived and whose resistance plasmid has been expressed, place it in a microcentrifuge, set the speed to 6000 rpm, the time to 1 min, centrifuge, plate, and incubate for 2-3 days.

[0155] e. When a single colony grows to a diameter of 1 mm, use a sterile pipette tip to pick up the single colony in a clean bench and place it into YEP liquid medium containing the corresponding antibiotic. Then, incubate it on a 28°C constant temperature shaker for 6 hours. Then, perform the culture by shaking on a clean bench and PCR in a PCR instrument. Finally, run an electrophoresis for identification.

[0156] 2. Cultivation of Tobacco Benedict

[0157] a. The proportions of each component in the substrate preparation are as follows: potting soil: vermiculite = 2:1, 26℃ for 16h / 8h (light / dark), 70% humidity (cover with a transparent white box to maintain moisture for 2 weeks).

[0158] b. After thoroughly watering the substrate, sow the tobacco seeds in the substrate using a pipette, leaving 4 seedlings in each pot, and water with 1 / 6 Hogland nutrient solution every 5 days.

[0159] 3. Agrobacterium injection into tobacco leaves

[0160] a. Select healthy, vigorous tobacco plants (young, fully expanded, 4-week-old leaves) from tobacco plants that have grown for about one month (4-leave stage) for Agrobacterium infection. Water thoroughly before conversion, and do not water after conversion until the photos are taken.

[0161] b. Agrobacterium preparation: Pre-culture, inoculate a single clone into a 2ml centrifuge tube containing 1Ml YEP containing the corresponding antibiotic. Culture: Inoculate 1mL of pre-culture medium into 50ml YEB liquid medium containing three antibiotics: Rif (20mg / mL), Gen (50mg / mL), and Kan (50mg / mL) to activate Agrobacterium (GV3101). When the bacterial culture reaches the late exponential growth stage (OD600 = 0.6), centrifuge at 4000rpm for 10min at room temperature to harvest the bacterial culture.

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

[0163] d. Resuspend to OD600 using a resuspension solution (10 mM ES-KOH, pH 5.6; 10 mM MgCl2; 100 μM Macetosyringone). The resuspension solution should be prepared fresh each time it is used.

[0164] e. Suspend the same cultured Agrobacterium containing P19 and adjust the concentration of the suspension to OD600 = 1.

[0165] f. According to the experimental design, the two types of Agrobacterium were mixed at a volume ratio of 1:1 and allowed to stand at room temperature for 3-4 hours.

[0166] g. Place the tobacco under a white fluorescent light for 1 hour before injection to allow the pores to fully open.

[0167] h. Agrobacterium injection: Resuspend the bacterial cells, draw up the bacterial suspension using a 1 mL syringe (without the needle), and gently poke a small hole in the middle of the leaf with the syringe needle. Then, using your left hand to hold the upper surface of the leaf, gently press the syringe nozzle vertically against the lower surface of the leaf with your right hand, and slowly and evenly push with your right thumb, observing the slow movement of the bacterial suspension under the leaf epidermis (ideally, the bacterial suspension should be observed to form liquid bubbles in the lower epidermis for optimal injection), until the entire leaf is infected. It is important to select the best-growing leaf on the plant (do not use cotyledons) for injection, and inject no more than half of the leaf per plant. Cover with plastic film and incubate at 24℃ for 2.5-3 days. Afterward, peel off the lower epidermis of the leaf and observe it under a laser confocal microscope.

[0168] like Figure 6As shown in the figure, eGFP corresponds to the GFP channel; RFP corresponds to the RFP channel; Merge represents the GFP-RFP fusion image; Bright field indicates the bright field. Laser confocal microscopy analysis shows that 35S-GFP is distributed throughout the cell, while the 35S-GsMYB10-GFP fusion protein is only localized in the cell nucleus, indicating that GsMYB10 is a nuclear localization protein. Experiment 5: Hydroponic Validation of GsMYB10

[0169] After sterilizing the GsMYB10 transgenic seeds, the seedlings were germinated in vermiculite for 6 days. Seedlings with fully expanded cotyledons and uniform growth were then transferred to a 1 / 2 Hoagland solution for culture and divided into two treatment groups: an experimental group and a control group. The experimental group was cultured at a low phosphorus concentration (5 μmol / L K2HPO4), while the control group was cultured at a normal phosphorus concentration (500 μmol / L K2HPO4). Each group included a wild-type (WT) and a transgenic line. WT served as the intragroup control (CK). The transgenic lines specifically included Gm10A and Gm10B. Each group was replicated three times. After 14 days, the hydroponic phenotype was observed, and the seed quality and total phosphorus content (total phosphorus content is the total phosphorus content multiplied by the dry weight of the above-ground and underground parts) were measured.

[0170] ① Colorimetric determination method for total phosphorus, molybdenum, and antimony resistance in plants:

[0171] 1. All reagents used in this experiment are of analytical grade, and the water is deionized water, 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 0.5g of chemically pure potassium antimony tartrate and dissolve it in 100mL of water;

[0177] (6) Stock solution of antimony molybdate sulfate: Measure 126 mL of concentrated sulfuric acid and slowly add it to 100 mL of water while stirring continuously and cooling. Separately weigh 10 g of finely ground ammonium molybdate and dissolve it in 300 mL of water at approximately 60 °C, then cool. Slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimony tartar solution, cool, 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 development agent: Weigh 1.5g of ascorbic acid and dissolve it in 100mL of molybdenum-antimony stock solution. This solution has a short shelf life and should be prepared fresh when needed.

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

[0180] (9) 5 mg / L phosphorus standard solution: Take 5 mL of phosphorus stock solution and put it into a 100 mL volumetric flask. Add water to make up to volume. Prepare the solution fresh when needed.

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

[0182] III. Experimental Procedure

[0183] (1) Sample solution preparation

[0184] Weigh an appropriate amount of sample, accurate to 0.001 g, and place it in a 500 ml digestion tube (do not allow the sample to adhere to the neck of the tube). First, add a small amount of water to moisten the sample, then add 6 mL of sulfuric acid, gently shake well, and let it stand overnight. Place a small curved-neck funnel at the mouth of the tube, and digest it on a digestion oven at 250 °C (start timing after the temperature stabilizes, the time is about 30 min). After the H2SO4 decomposes and emits a large amount of white fumes, increase the temperature to 400 °C. When the solution turns a uniform brownish-black color, remove it from the oven.

[0185] After cooling slightly, add 10 drops of H2O2, shake well, and heat to a gentle boil. Digest for about 5 minutes, then remove and let cool slightly. Repeat this process, adding 5-10 drops of H2O2 each time, and then continue digesting. Repeat this process 3-5 times, gradually reducing the amount of H2O2 added each time. Digest until the solution is colorless or clear, with a grayish-white bottom. Then heat for about 5-10 minutes to remove any remaining H2O2.

[0186] Remove the digestion tube and allow it to cool. Rinse the bent-neck funnel with a small amount of water, allowing the washings to flow into the digestion tube. Transfer the digestion solution intact into a 100 mL volumetric flask, dilute to volume with water, and mix well. Filter or allow to settle before use for phosphorus determination.

[0187] (2) Preparation of blank solution

[0188] Except for the absence of a sample, the reagents and operating procedures are the same as above.

[0189] (3) Draw the standard curve

[0190] Pipette 0, 2, 4, 6, 8, 10, 14, and 20 mL of 5 mg / L phosphorus standard solution into 50 mL volumetric flasks, respectively. Simultaneously add an equal volume of blank solution (as used for the colorimetric assay) and 2-3 drops of dinitrophenol indicator. Adjust the solution to a slightly yellow hue with 10% sodium carbonate or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony colorimetric reagent, shake well, and dilute to volume with water to obtain a series of standard solutions with phosphorus contents of 0.0, 0.2, 0.4, 0.8, 1.0, 1.4, and 2.0 mg / L. Shake well, incubate at 15°C or higher for 30 min, and then measure the absorbance at 880 nm. Plot a calibration curve on graph paper with absorbance on the ordinate and phosphorus concentration (mg / L) on the abscissa.

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

[0192] Pipette 2-10 mL of the sample solution (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 a slightly yellow color with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum-antimony colorimetric reagent, shake well, and dilute to volume with water. Incubate at room temperature (above 15°C) for 30 min.

[0193] The colored sample solution was measured on a spectrophotometer using an 880nm, 1cm path length cuvette. The instrument was zeroed using a blank test as a reference, and the absorbance was read. The corresponding phosphorus content was then determined from the standard curve.

[0194] IV. Result Calculation

[0195] All samples

[0196] In the formula:

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

[0198] C0 — Blank value

[0199] V – Volume of colorimetric solution;

[0200] D – Dividing factor: Volume of the digestion solution to the final volume / Volume of the digestion solution removed;

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

[0202] 1000 – Conversion factor for converting mL to L. The result should be rounded to three decimal places.

[0203] Primers for real-time quantitative PCR:

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

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

[0206] The results are as follows Figure 7 As shown, compared with WT, the transgenic plants have increased taproot length and number of lateral roots, significantly increased above-ground and underground fresh weight, and significantly higher total phosphorus content than the control group.

[0207] Experiment 6: Verification of GsMYB10's tolerance to low phosphorus throughout its entire growth period in soil cultivation

[0208] The genetically modified soybean was planted in the experimental greenhouse of South China Agricultural University in August 2024. The soybean variety was Huachun 6.

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

[0210] Processing group processing method

[0211] Low phosphorus treatment group: During the seedling stage, a 1 / 2 Hoagland solution containing 5 μmol / L K₂HPO₄ was sprayed evenly. During the flowering and pod-setting stages, the roots were irrigated with the same solution. Normal phosphorus treatment group: During the seedling stage, a 1 / 2 Hoagland solution containing 500 μmol / L K₂HPO₄ was sprayed evenly. During the flowering and pod-setting stages, the roots were irrigated with the same solution. A total of three treatments were conducted: one spraying and two irrigations. The amount of low-phosphorus and normal-phosphorus 1 / 2 Hoagland solution used for spraying during the seedling stage was approximately 7 L each. The amount used for irrigation during the flowering and pod-setting stages was approximately 15 L each.

[0212] After the soybeans matured, their growth was recorded by taking photos. The experimental results are as follows: Figure 8As shown, in the LP and NP treatment groups, the growth of GsMYB10 transgenic soybeans was superior to that of the control WT within the same group. Yield trait analysis revealed that the number of pods per transgenic plant and the height of the bottom pod were higher in the LP treatment than in the control group. In the LP treatment, the plant height of Gm10A transgenic plants was higher than that of the control group, while other lines showed no significant changes. The 100-seed weight showed no significant change. These results can help demonstrate the application of this gene in plant tolerance to low phosphorus. Experiment 7: Soil culture verification of GsMYB10 low phosphorus tolerance in seedlings.

[0213] The genetically modified soybean was planted in the experimental greenhouse of South China Agricultural University. The soybean variety was Huachun 6.

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

[0215] Processing group processing method

[0216] Low phosphorus treatment group: Seedlings were uniformly sprayed with a 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4. Normal phosphorus treatment group: Seedlings were uniformly sprayed with a 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4.

[0217] The growth of soybeans was recorded by photographing during the V2 stage of cultivation. The experimental results are as follows: Figure 9 As shown, under low phosphorus (LP) and normal phosphorus (NP) treatments, the aboveground weight of the transgenic lines was significantly higher than that of the control group (CK).

[0218] This invention provides the application of the GsMYB10 gene in regulating soybean's tolerance to low phosphorus stress. The GsMYB10 gene can enhance soybean's tolerance to low phosphorus stress. Overexpression of the GsMYB10 gene enhances the plant's tolerance to low phosphorus.

[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] MYSTHQQHQGKNIHSSSSSSRMPIPSERHMFLQTGNGSGDSGLVLSTDAKPRLKWTPDLHARFIEAVQQLGGADKATPKTVMKLMGIPGLTLYHLKSHLQKYR LSKSLHGQSNNATHKITINSGSATDERLRENNETHVMNNLNLAPQSINKDLHISEALQMQIEVQRRLNEQLQVQRLLQLRIEAQGKYLQAVLEKAQETLGRQNL GVVGLEAAKLQLSELVSKVSSQCLNSAFSELKEIQGFSPHHQKQTQTNNNQPINANDCSMDSCLTSCEGSSQKDQQEIQNRGMNLIPFNVHTFMEGPNLNNLPN TDLKWCDPVKKNNTFLTRLSMHAERSPSNLSMSIGLLEGETTENRSTIVRTESIKPAVAEKVSQDYGLPSNYFAASKLDQTTEDNKDTKTSCKQLDLNGFSWN*

[0224] The embodiments described above are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. The application of overexpression of gene GsMYB10 in improving the tolerance of soybean to low phosphorus stress, characterized by: The nucleotide sequence of the gene GsMYB10 is shown in SEQ ID NO.1; The specific steps of the application are as follows: the gene GsMYB10 shown in SEQ ID NO.1 is introduced into the recipient soybean to obtain transgenic soybean, wherein the gene GsMYB10 encodes the amino acid sequence shown in SEQ ID NO.2; The improved tolerance to low phosphorus stress is reflected in an increase in root length or the number of lateral roots.

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