Application of the soybean low phosphorus tolerance gene GsMYB7 in improving plant tolerance to low phosphorus stress

Abstract

This invention relates to the field of plant genetic engineering technology, and discloses an important low-phosphorus tolerance gene, GsMYB7, and its applications. This invention is the first to clone a MYB family gene, GsMYB7, from soybean. Studies show that the expression of the GsMYB7 gene is upregulated under low-phosphorus stress. Under different phosphorus concentration treatments, overexpression of GsMYB7 significantly increases the biomass of transgenic plants and promotes plant growth under low-phosphorus conditions. Simultaneously, overexpression of GsMYB7 enhances the plant's tolerance to low-phosphorus stress and reduces the inhibitory effect of low phosphorus on root growth. This indicates that GsMYB7 plays an important role in plant adaptation to low-phosphorus stress and can improve the plant's adaptability to low-phosphorus stress in acidic soils through transgenic technology.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the soybean low phosphorus tolerance gene GsMYB7 in improving the plant's tolerance to low phosphorus stress. Background Technology

[0002] Soybean (Glycine max (L.) Merr) is an annual herbaceous plant belonging to the legume family. Native to China, it is an important agricultural product and industrial raw material. Due to its rich protein and fat content, soybeans are widely used as a high-quality raw material in food processing, animal feed production, and other industries. Therefore, strengthening soybean cultivation and improving its yield and quality are of great significance for ensuring a stable supply of soybeans, meeting domestic market demand, and reducing import dependence.

[0003] Phosphorus, as one of the essential macronutrients for plant growth and development, plays an irreplaceable role. For legumes, phosphorus deficiency leads to stunted growth and stunted leaf development, thus affecting overall plant growth. Furthermore, phosphorus deficiency also affects root morphology: on the one hand, taproot development is inhibited, with reduced length and volume, decreasing the plant's ability to absorb water and nutrients; on the other hand, root nodule development is also inhibited, resulting in smaller nodules and reduced nitrogen fixation efficiency, further impacting nitrogen acquisition. Moreover, phosphorus deficiency further affects reproductive growth and stress resistance. However, the total phosphorus content in soil is generally low, ranging from approximately 0.02% to 0.25% under natural conditions, with even less readily available for plant absorption and utilization.

[0004] Therefore, addressing phosphorus deficiency is one of the important ways to improve the yield and quality of legume crops. Research on the physiological response and adaptation mechanism of soybeans under low phosphorus stress is of great significance for improving the phosphorus use efficiency of soybeans, enhancing their stress resistance, and achieving high-yield and high-quality cultivation. Summary of the Invention

[0005] To address the problems mentioned above in the background art, the first objective of this invention is to provide an application of the soybean low phosphorus tolerance gene GsMYB7 in improving the plant's tolerance to low phosphorus stress.

[0006] This application improves the plant's tolerance to low phosphorus stress by overexpressing the gene GsMYB7 in the plant.

[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 soybean low phosphorus tolerance gene GsMYB7 shown in SEQ ID NO.1 into the recipient plant to obtain a transgenic 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 the length of its taproot or the number of its lateral roots.

[0010] The third objective of this invention is to provide a formulation for improving the plant's tolerance to low phosphorus stress, wherein the active ingredient of the formulation comprises the soybean low phosphorus tolerance gene GsMYB7 or the protein encoded by the soybean low phosphorus tolerance gene GsMYB7.

[0011] The fourth objective of this invention is to provide the application of a recombinant vector of the soybean low phosphorus tolerance gene GsMYB7 in improving the plant's tolerance to low phosphorus stress.

[0012] The fifth objective of this invention is to provide an application of the soybean low-phosphorus tolerance gene GsMYB7 in the cultivation of soybean plants resistant to low-phosphorus stress.

[0013] This application studies a method for promoting plant growth under low phosphorus stress environments using the soybean low phosphorus tolerance gene GsMYB7, including the following specific steps:

[0014] Using soybean as material, the GsMYB7 gene was genetically transformed into soybean using Agrobacterium-mediated transformation of soybean cotyledon nodes, resulting in soybean plants overexpressing the GsMYB7 gene. By comparing the growth performance, phosphorus uptake efficiency, and root development of transgenic plants and wild-type plants under low phosphorus stress, the function and application potential of the GsMYB7 gene in improving soybean's tolerance to low phosphorus were verified.

[0015] In summary, the beneficial effects of the present invention are as follows:

[0016] This invention provides an application of the GsMYB7 gene in regulating the tolerance of soybean to low phosphorus stress. The GsMYB7 gene can enhance the tolerance of soybean to low phosphorus stress, and overexpression of the GsMYB7 gene can enhance the plant's tolerance to low phosphorus.

[0017] In addition, this application constructed soybean material overexpressing GsMYB7: using Agrobacterium-mediated transformation of soybean cotyledonary nodes, the GsMYB7 gene was successfully integrated into the soybean genome and stably expressed.

[0018] 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

[0019] Figure 1 Diagram of the GsMYB7 protein domains;

[0020] Figure 2 Phylogenetic analysis diagram of GsMYB7;

[0021] Figure 3 Cloning of the GsMYB7 gene and identification of the GsMYB7 gene overexpression vector by bacterial culture using PCR;

[0022] Figure 4 The detection of the GsMYB7 transgenic soybean line includes (A) PCR identification using Bar gene primers, (B) qRT-PCR detection of gene expression in the transgenic soybean line, and (C) detection of herbicide tolerance in the transgenic plant.

[0023] Figure 5 PCR identification diagram of primers designed for the 35S sequence;

[0024] Figure 6 Analysis of the low-phosphorus-tolerant expression pattern of the GsMYB7 gene;

[0025] Figure 7 Total phosphorus content was tested in GsMYB7 genetically modified soybeans;

[0026] Figure 8 Identification of low phosphorus tolerance phenotype in GsMYB7 transgenic soybean seedlings during hydroponic culture;

[0027] Figure 9 Phenotypic identification of GsMYB7 transgenic soybean throughout its entire growth period;

[0028] Figure 10 Phenotypic identification and observation index analysis of GsMYB7 transgenic soybean seedlings in soil culture. Detailed Implementation

[0029] To make the content of this invention easier to understand, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0030] Experiment 1: Structural and functional analysis of GsMYB7 protein

[0031] The full-length coding sequence of the GsMYB7 gene in this application is 2179 bp, and the coding DNA sequence (CDS) is 1002 bp long, encoding 333 amino acids.

[0032] S1, Conservative Domain Analysis: (e.g.) Figure 1 As shown, the GsMYB7 protein of this application contains two conserved DNA-binding domains, consisting of 46 amino acids (located at amino acid residues 10-60) and 44 amino acids (located at amino acid residues 70-120), respectively. This structural feature conforms to the typical conserved structural pattern of R2R3-MYB transcription factors (TFs).

[0033] S2, Phylogenetic Tree and Homology Analysis: such as Figure 2 As shown, related homologs of the GsMYB7 protein are mainly distributed in legumes. Although GsMYB7 has low sequence homology with homologous proteins in Arabidopsis thaliana, both possess a typical MYB DNA-binding domain, indicating that they have retained their crucial DNA-binding function during evolution.

[0034] S3: Functional Speculation and Regulatory Mechanism: The promoter region 1500 bp upstream of the start codon of the GsMYB7 gene contains various cis-acting elements related to stress response, including but not limited to: AE-box (light-responsive element), ERE (ethylene-responsive element), TGA (auxin-responsive element), HSE (heat-stress-responsive element), and TC-rich repeat sequence (abiotic stress-responsive element). The presence of these elements suggests that GsMYB7 may play a regulatory role in multiple environmental signal transduction pathways, including those related to light, ethylene, auxin, heat, and abiotic stress.

[0035] In this embodiment, candidate sequence information for the GsMYB7 gene was retrieved from the National Center for Biotechnology Information (NCBI) database, with accession number XP_003527079.1. Furthermore, DNAMAN software was used to perform multiple sequence alignment of soybean GsMYB7 protein with members of the R2R3-MYB family, and MAGE software was used to construct a phylogenetic tree using the Neighbor-Joining (NJ) method.

[0036] Example 2: Construction of GsMYB7 expression vector and genetic transformation of soybean

[0037] Construction of the S1 intermediate vector pUC18-GsMYB7: The CDS sequence of GsMYB7 was amplified by PCR. Amplification primers:

[0038] Forward Primer(SEQ ID NO.3):5'-GGACCACTATCCCGACCTG-3'

[0039] Reverse Primer(SEQ ID NO.4):5'-GCCCATCTGTTACCCAAA-3'

[0040] The intermediate vector pUC18-GsMYB7 was constructed by inserting it between the BamHI and KpnI restriction sites of the modified intermediate vector pUC18.

[0041] pUC18-GsMYB7_F (SEQ ID NO.5): 5'-ATACTAGTGGCGCCGCCGGGTGGTAGT GATG-3'; pUC18-GsMYB7_R (SEQ ID NO.6): 5'-CCGAGCTCGCCTAGGACTGTGGCTT GCTC-3',

[0042] The full-length CDS sequence of GsMYB7 was amplified according to the reaction system in Table 1 below. The reaction program was as follows: pre-denaturation 95℃, 3 min; 34 cycles (denaturation 95℃, 15 s; annealing 55℃, 15 s; extension 72℃, 1 min / kb); final extension 72℃, 5 min; storage at 12℃.

[0043] Table 1 Fragment amplification reaction system

[0044]

[0045] Recovery of the S2 target fragment: pUC18-GsMYB7 was digested with HindI II restriction endonuclease to recover the target fragment containing the 35S-GsMYB7-NOS terminator.

[0046] Construction and transformation of S3 expression vector pZY101-GsMYB7: The target fragment was inserted into the HindII site of the pZY101 vector to construct the expression vector pZY101-GsMYB7; subsequently, the constructed expression vector was transformed into competent Escherichia coli DH5α cells. The E. coli transformation steps were as follows: 10 μL of the ligation product was added to 100 μL of competent DH5α E. coli cells, gently aspirated and mixed, then incubated on ice for 30 min, followed by heat shock at 42℃ for 45 s, and immediately transferred to ice for 2 min; then the ligation product was transferred to a clean bench, and 500-700 μL of LB liquid medium was added to the ligation product, and the mixture was incubated at 37℃ and shaken at 220 rpm for 1 h to obtain bacterial culture. The bacterial culture was plated on plates containing Amp and incubated overnight at 37℃. Single clones were picked and shaken for detection and sequencing.

[0047] Identification of S4 positive clones: pZY101-GsMYB7 positive clones were identified by bacterial PCR, restriction enzyme digestion and DNA sequencing to ensure the correctness of vector construction.

[0048] pZY101-GsMYB7_F (SEQ ID NO.7): 5'-GGCAGAGGCATCTTCAACG-3';

[0049] pZY101-GsMYB7_R (SEQ ID NO.8): 5'-GCTCACTCATTAGGCACCC-3',

[0050] S5 Agrobacterium tumefaciens transformation and soybean genetic transformation: The pZY101-GsMYB7 expression plasmid was transformed into Agrobacterium tumefaciens EHA101, and the GsMYB7 gene was introduced into the soybean recipient variety Huachun 6 using the soybean cotyledon node genetic transformation method.

[0051] Preparation of Agrobacterium electrocompetents

[0052] 1) Pick a milky white and plump single colony and inoculate it into 5mLYEP liquid medium containing 5μL spectinomycin, 5μL streptomycin and 5μL chloramphenicol. Incubate overnight at 28°C and 250rpm in a shaker.

[0053] 2) Pipette 2 mL of the culture into 50 mL of LYEP liquid medium and incubate at 28 °C and 250 rpm in a shaker until the OD value is approximately 0.6;

[0054] 3) Transfer the bacterial culture to a sterile centrifuge tube, place it on ice for 30 minutes, and then centrifuge it in a centrifuge. Set the centrifuge temperature to 4℃, the speed to 4000 rpm, and the centrifugation time to 10 minutes.

[0055] 4) Discard the supernatant, resuspend the bacterial cells in a small amount of pre-cooled sterile water, add pre-cooled sterile water to 50 mL, and then put it into a centrifuge for centrifugation. The centrifuge temperature is set to 4℃, the speed is 4000 rpm, and the centrifugation time is 10 min.

[0056] 5) After repeating step 4) three times, remove the supernatant, resuspend the bacterial cells, suspend the precipitate with 10% glycerol, and finally aliquot and store in a refrigerator at -80℃ for later use.

[0057] b. Electroshock conversion

[0058] 1) Remove the electrocution cup from the electrocution cup in 70% ethanol and place it in a clean bench to dry. After drying, wrap the electrocution cup with clean gloves and place it on ice to pre-cool for later use.

[0059] 2) Take out Agrobacterium competent cells from the -80℃ freezer and thaw them on ice. Then add the plasmid to the thawed competent cells, gently mix with a pipette, and transfer to a click cup.

[0060] 3) Place the shock cup in the shock device and administer the shock, setting the parameters as follows: voltage 1800V, time 5ms;

[0061] 4) After electroporation, slowly add 1 mL of antibiotic-free YEP liquid culture medium to the electroporation cup, and gently aspirate and mix the bacterial cells several times with a pipette.

[0062] 5) Aspirate the bacterial culture from the electroporation cup and place it in a centrifuge tube, then incubate it in a shaker at 28°C and 250 rpm for 3 hours;

[0063] 6) Take 50 μl of bacterial suspension and spread it on a YEP solid plate containing spectinomycin, streptomycin, and chloramphenicol. Incubate at 28°C and 250 rpm for about 36 hours until a single colony grows.

[0064] Soybean genetic transformation

[0065] 1) Germination: a. Seed disinfection: Remove the screened and stored Huachun No. 6 seeds from the -20℃ freezer, soak them in 75% alcohol for 1 minute, wipe them dry, arrange them in a single layer in a petri dish, and place them in a fume hood desiccator; mix 100mL of sodium hypochlorite and 5mL of concentrated hydrochloric acid in the fume hood, quickly transfer the mixture to a 250mL beaker, place it in the desiccator, seal and sterilize for 13.5 hours, then remove it, air it in a laminar flow hood, and store it in a 4℃ freezer for later use. b. Sowing: Gently press the seeds onto the germination medium with the hilum facing down. After the cotyledons turn green, conduct an infection experiment.

[0066] 2) Soaking seeds: Soak soybeans in sterilized ddH2O and place them in a dark environment at 24℃ for 10-16 hours.

[0067] 3) Preparation of bacterial culture: Take the EHA101 bacterial culture that has been transformed with the target plasmid and add it to YEP liquid medium containing 100 mg / L spe and 30 mg / L LRif; set the constant temperature shaker to 28℃ and 240 rpm and incubate overnight. The absorbance of the bacterial culture was measured the next day, OD600 = 0.8-1.0; pour the bacterial culture into a sterile 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min; discard the supernatant and resuspend the bacterial cells in CCM liquid medium to make OD600 = 0.6-0.8, and store at 4℃ for the next experiment.

[0068] 4) Preparation and transformation of explants: In a clean bench, the hypocotyl of the soaked seeds is cut open with tweezers and a scalpel, and the seeds are cut open vertically along the cotyledons to remove the buds, epicotyl and seed coat on the cotyledons.

[0069] 5) Infection and co-culture: Immerse the explants in the prepared CCM resuspension, sonicate for 3 min, vacuum for 10 min, shake on a shaker at 120-150 rpm and 28℃ for 40 min; after infection, pour out the bacterial solution, and use tweezers to spread the explants evenly on the CCM solid culture medium lined with filter paper; seal with medical tape, place in the tissue culture room, set the temperature to 22℃, and incubate in the dark for 48 h, and incubate in the light for 3-5 days until the cotyledons turn completely green.

[0070] 6) For the induction of young shoots, the excessively long hypocotyl is removed with a scalpel. The explants are then inserted obliquely into the shoot induction medium, sealed with medical tape, and placed in the tissue culture room. The temperature is set at 22℃, the light duration is 16 / 8h, and the culture is carried out for 14 days. Subsequently, the sterile plates are replaced with new shoot induction medium. Explants that have not grown clusters of shoots are discarded. 6-7 explants are placed in each medium, sealed with medical tape, and placed in the tissue culture room for induction for 14 days.

[0071] 7) Growth of young shoots: After 4 weeks of induction of young shoots, discard the undifferentiated material, cut off the cotyledons of the explants that have grown clustered shoots, transfer them to the shoot growth medium, and make sure the cut is in contact with the medium. Place 6-7 explants in each culture dish and place them in the tissue culture room. Change the medium every 14 days.

[0072] 8) After rooting, use a scalpel to cut the buds of seedlings with buds longer than 3cm from the root of the tissue. Soak them in auxin for 1 minute, then transfer them to rooting medium and place them in a tissue culture room to continue growing. When 5-6 roots have grown and lateral roots have also emerged, gently remove the plant from the rooting medium and carefully rinse the roots with tap water to remove the medium. Transplant the seedlings into high-temperature and high-pressure sterilized nutrient soil (vermiculite: substrate soil = 1:2), and place them in a heat-insulating and moisture-retaining seedling tray. Place the seedling tray in an incubator with a temperature of 26℃ and a light duration of 12 / 12h. Water with nutrient solution and continue culturing until pods form.

[0073] Test results

[0074] like Figure 3 As shown, M in part a is the DL2000 DNA Marker, lane 1 is the full-length clone of GsMYB7, and ☆ indicates the target band, which is approximately 1002bp.

[0075] In part b, M represents the DL5000 DNA Marker, - represents the H2O control, lanes 1, 2, 3, and 4 are for pZY101-GsMYB7 Escherichia coli culture PCR detection, and ☆ indicates the target band, which is approximately 2000bp.

[0076] In section c, M stands for DL15000 DNA Marker, lane 1 is the pZY101-GsMYB7 recombinant plasmid, lane 2 is the full-length GsMYB7 fragment digested with BamHI and KpnI, lane 3 is the pZY101-GsMYB7 recombinant plasmid digested with BamHI and KpnI, and ☆ indicates the target band, which is approximately 1002 bp.

[0077] 9) The obtained transgenic plants were identified through herbicide tolerance, DNA, and RNA analysis. Total genomic DNA was extracted from the leaves of the T4 generation transgenic plants overexpressing GsMYB7, and PCR identification was performed using Bar gene primers. The Bar gene primers are shown below:

[0078] Forward Primer(SEQ ID NO.9):5'-AAGTCCAGCTGCCAGAAACC-3'

[0079] Reverse Primer (SEQ ID NO.10): 5'-AAGCACGGTCAACTTCCGTA-3'

[0080] PCR identification results of Bar gene primers are as follows: Figure 4 As shown in Part A, transgenic lines overexpressing GsMYB7 can amplify a Bar gene-specific band of approximately 488 bp.

[0081] The results of qRT-PCR detection of gene expression in transgenic soybean lines are as follows: Figure 4 As shown in Part B, the transgenic lines have a higher relative expression of GsMYB7 than the wild-type (WT) plants.

[0082] Herbicide tolerance test results as follows Figure 4 As shown in section C, the leaves marked with a checkmark are the side where herbicide was applied, while the left side was not treated with herbicide. The experiment shows that the transgenic line is more tolerant to herbicide than the WT plant.

[0083] Primers were designed using the 35S and target gene sequences for PCR identification, and the results were as follows: Figure 5 As shown in sections a and b, the experiment demonstrated that the transgenic lines overexpressing GsMYB7 could amplify a band of approximately 1110 bp, proving that the GsMYB7 gene sequence had been integrated into the genome of the soybean transformant plants.

[0084] In the three independent biological experiments above, the relative gene expression levels of Huachun 6 WT and GsMYB7 transgenic soybean lines were calculated using the 2^-△△CT method. L1, L2, L3, L4, and L5 are the T4 generation lines of GsMYB7 transgenic soybean.

[0085] Example 3: Hydroponic Validation and Expression Pattern Analysis of GsMYB7

[0086] After disinfecting BX13 soybean seeds with 75% ethanol, they were exposed to light for 16 / 8 hours in vermiculite at 22℃. Four days after germination, when the cotyledons were fully expanded, seedlings with uniform growth were harvested and transferred to 1 / 2 Hoagland solution for cultivation. They were 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). Root samples were then collected at 0h, 3h, 6h, 12h, 24h, 36h, 48h, 7d, and 14d after treatment. RNA was extracted and reverse transcribed into cDNA. The expression level of the gene was observed at these time points using real-time quantitative PCR (qRT-PCR).

[0087] Experimental steps

[0088] Quantitative PCR primer design: Gene sequences were retrieved from the Phytozome database. 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 designed and synthesized based on the genome sequences on the NCBI website.

[0089] Forward Primer (SEQ ID NO.11): 5'-GGACCAACTATTCCCGACCTG-3';

[0090] Reverse Primer (SEQ ID NO. 12): 5'-GCCCATCTGTTACCCAAA-3'.

[0091] Real-time quantitative PCR:

[0092] ①Actin3 was used as an internal reference gene.

[0093] Forward primer (SEQ ID NO.13): 5'-GCACCACCGGAGAGAAAATA-3';

[0094] Reverse primer (SEQ ID NO.14): 5'-GTGCACAATTGATGGACCAG-3';

[0095] ② Dilute all cDNA samples by 1-fold with ddH2O to serve as templates for quantitative PCR reactions;

[0096] ③ The real-time quantitative PCR system was run according to the reaction system in Table 2 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;

[0097] Table 2 Real-time quantitative PCR reaction system

[0098]

[0099] ④ Data processing: Relative expression level analysis was performed using 2^ -△△CT Law.

[0100] Experimental results are as follows Figure 6 As shown, a low-phosphorus environment can promote the expression of GsMYB7.

[0101] GsMYB7 transgenic seeds were sterilized with 75% ethanol and cultured in vermiculite at 22℃ for 16 / 8 hours of light. After 4 days of germination, when the cotyledons were fully expanded, seedlings with uniform growth were harvested and transferred to 1 / 2 Hoagland solution for further culture. The seedlings were 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 wild-type (WT) and transgenic lines. WT served as the intragroup control (CK). The transgenic lines specifically included Gm7A, Gm7C, and Gm7H. Each group was replicated three times. The hydroponic nutrient solution was changed every two days. After 14 days, the hydroponic phenotype was observed, and the seed quality and total phosphorus content (total phosphorus content is calculated by multiplying the total phosphorus content by the dry weight of the above-ground and below-ground parts) were measured.

[0102] It should be noted that the P concentration should be adjusted according to the corresponding experimental treatment. If KH2PO4 is used for P treatment, K2SO4 or KCl should be used to replenish the corresponding K.

[0103] 1. Colorimetric determination method for total phosphorus, molybdenum, and antimony resistance in plants:

[0104] All reagents used in this experiment were of analytical grade, and the water was deionized water, distilled water, or water of equivalent purity.

[0105] (1) Sulfuric acid;

[0106] (2) 30% hydrogen peroxide;

[0107] (3) 10% sodium hydroxide;

[0108] (4) 0.2% dinitrophenol indicator;

[0109] (5) 0.5% potassium antimony tartrate solution: Weigh 0.5g of chemically pure potassium antimony tartrate and dissolve it in 100mL of water;

[0110] (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.

[0111] (7) Molybdenum-antimony anti-coloring 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.

[0112] (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.

[0113] (9) 5 mg / L phosphorus standard solution: Pipette 5 mL of phosphorus stock solution into a 100 mL volumetric flask, add water to make up to volume. Prepare the solution fresh each time you need it.

[0114] 2. Experimental Procedure

[0115] (1) Sample solution preparation

[0116] Weigh an appropriate amount of sample, accurate to 0.001 g, into 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 stand overnight. Place a small bent-neck funnel at the mouth of the tube, and digest at 250 °C on a digestion furnace (start timing after the temperature stabilizes, approximately 30 min). After the H2SO4 decomposes and emits a large amount of white fumes, increase the temperature to 400 °C. Remove the tube when the solution turns a uniform brownish-black color.

[0117] 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.

[0118] 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.

[0119] (2) Preparation of blank solution

[0120] Except for the absence of a sample, the reagents and operating procedures are the same as above.

[0121] (3) Draw the standard curve

[0122] 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.

[0123] (4) Determination of phosphorus in sample solution

[0124] 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.

[0125] 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.

[0126] 3. Result Calculation

[0127]

[0128] In the formula:

[0129] C—The mass concentration of phosphorus in the colorimetric solution obtained from the calibration curve or regression equation, in μg / L;

[0130] C0 — Blank value

[0131] V – Volume of the colorimetric solution;

[0132] D – Dividing factor: Volume of the digestion solution to the final volume / Volume of the digestion solution removed;

[0133] m — Mass of the sample taken, in g;

[0134] 1000 – Conversion factor for converting mL to L. The result should be rounded to three decimal places.

[0135] The results of the total phosphorus content test are as follows: Figure 7 As shown, under NP and LP conditions, the total phosphorus content in both the aboveground and underground parts of transgenic plants was higher than that in WT plants.

[0136] Hydroponic phenotypes such as Figure 8 As shown, compared to WT, the transgenic plants have longer roots, and the above-ground and underground fresh weights of the transgenic plants are significantly increased.

[0137] Example 4: Identification of high phosphorus efficiency phenotypes in soil culture throughout the entire growth period

[0138] GsMYB7 genetically modified soybeans were planted in the greenhouse of South China Agricultural University in August 2024. The soybean variety was Huachun 6. Experimental conditions: an outdoor environment of high temperature, high humidity and strong light stress with an average daily temperature of 28-32℃, an average daily sunshine duration of 10-12 hours, a relative humidity of 75-90%, and short-term heavy rainfall (50mm / h).

[0139] Soybeans were divided into the following treatment groups: a low phosphorus (LP) treatment experimental group and a 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 three lines: Gm7A, Gm7C, and Gm7H. Each group was set up with 3 replicates.

[0140] Processing group processing method

[0141] 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. During the flowering and pod-setting stages, the amount of low-phosphorus and normal-phosphorus 1 / 2 Hoagland solution used for irrigation was 15 L each.

[0142] After the soybeans mature, take photos to record their growth during this period, such as... Figure 9 As shown, yield trait analysis revealed that the number of pods per plant and the height of the bottom pods were higher in the LP treatment than in the control group. Under low phosphorus (LP) and normal phosphorus (NP) treatments, the 100-seed weight of the transgenic line Gm7H was significantly higher than that of the control group (CK), while other lines showed no significant changes. The number of nodes on the main stem did not change significantly. These results demonstrate that GsMYB7 can improve the plant's tolerance to low phosphorus.

[0143] Example 5: Identification of Phosphorus-Efficient Phenotypes in Soil Culture During Seedling Stage

[0144] GsMYB7 transgenic soybeans were planted in the experimental greenhouse of South China Agricultural University. The soybean variety was Huachun 6. Experimental conditions: an outdoor environment of high temperature, high humidity and strong light stress with an average daily temperature of 28-32℃, an average daily sunshine duration of 10-12 hours, a relative humidity of 75-90%, and short-term heavy rainfall (50mm / h).

[0145] Soybeans were divided into the following treatment groups: a low phosphorus (LP) treatment experimental group and a normal phosphorus (NP) treatment control group. Specifically, each group included wild type (WT) and transgenic lines. WT served as the intragroup control (CK), and the transgenic lines specifically included three lines: Gm7A, Gm7C, and Gm7H. Each group was set up with 3 replicates.

[0146] Processing group processing method

[0147] Low phosphorus and normal phosphorus treatment groups: 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.

[0148] After the soybeans matured, their growth was recorded by taking photos. The experimental results are as follows: Figure 10 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).

[0149] The above experiments demonstrate that the GsMYB7 gene can enhance soybean's tolerance to low phosphorus stress. Overexpression of the GsMYB7 gene can enhance the plant's tolerance to low phosphorus.

[0150] GsMYB7 gene sequence and amino acid sequence

[0151] The gene sequence of GsMYB7 is shown in SEQ ID NO.1:

[0152] ATGGGAAGACCACCTTGCTGTGATAAAATTGGGATTAAGAAAGGGCCTTGGACTCCTGAGGAGAGACA

[0153] TCATCTTGGTCTCTTACATTCAAGAACATGGACCCGGAAATTGGAGATCGGTTCCCAGTAACACAGG

[0154] TTTGATGAGATGCAGCAAAAGCTGCAGACTCAGATGGACCAACTATCTCCGACCTGGTATCAAACGA

[0155] GGCAATTTCACCGATCATGAAGAGAAATGATAATCCACCCTCCAAGCTCTTTTGGGTAACAGATGGG

[0156] CTGCTATAGCTTCCTACCTCCCAAAGGACAGACAATGACATAAAGAACTATTGGAACACCCATTT

[0157] GAAGAAGAAGCTGAAGAAGATGCAAATTGGGGGTGGTAGTGATGATGATAATAATGATGACAAATCA

[0158] AACTCTTCTAACAATTCACAAATAAAGGGTCAATGGGAAGAAGACTTCAAACAGATATCCACATGG

[0159] CCAAACAAGCCTTATGTGAGGCCCTATCTCTTGACAAACCAACCCAAATTTTCCCAGAGACCAAATT

[0160] ACCCTCCACTTCTTCACACCACCACCCACAACAACAACAACACCAAACCAAACAACATCCTTGTAT

[0161] GCATCAAGCACAGAAACATAGCCAGATTGTTGGAGAATTGGATGAAGAAATCACCAAATATGACGA

[0162] CCACGACGACAACAACAATGGAGACAAAACCCTTCAGCAATAATAACATGGTAATAACCACAGGGTC

[0163] TAGTTCTAGTGAGGGAACACAAAGCACAATCACATGCACACAGGAGTATGCCCTTGACTCCTTGTGG

[0164] AGCTTCAACTCTGAACGCTCTTCTCAATCTGAAGAAAACACCAACTTGGGTGAGAGCAAGCCACAGT

[0165] ACCAAGAGCCTCAAGAGACACAAGTCCCTCTCATGTTGCTGGAGAATTGGCTCTTTGATGATGCTGC

[0166] ACCTCAATGCAATGAAGATCTAATGAACATGTCACTCGAGGAAAGTACAGAAGGGTTGTTCTAA

[0167] The amino acid sequence encoded by the GsMYB7 gene is shown in SEQ ID NO.2:

[0168] MGRPPCCDKIGIKKGPWTPEEDI ILVSYIQEHGPGNWRSVPSNTGLMRCSKSCRLRWTNYLRPGIKR

[0169] GNFTDHEEKMIIHLQALLGNRWAAIASYLPQRTDNDIKNYWNTHLKKKLKKMQIGGGSDDDNNDDKSNS

[0170] SNNSQI KGQWERRLQTDI HMAKQALCEALSLDKPTQI FPETKLPSTSSSHHHPTTTTTPNQTTSLYASST

[0171] ENIARLLENWMKKSPNMTTTTTTTMETKPFSNNNMVITTGSSSSEGTQSTITCTQEYALDSLWSFNSERSSQSEENTNLGESKPQYQEPQETQVPLMLLENWLFDDAAPQCNEDLMNMSLEESTEGLF*

[0172] 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. Application of soybean low phosphorus tolerance gene GsMYB7 in improving the low phosphorus stress tolerance of soybean, characterized in that: The nucleotide sequence of the soybean low-phosphorus tolerance gene GsMYB7 is shown as SEQ ID NO. 1, or the amino acid sequence encoded by the soybean low-phosphorus tolerance gene GsMYB7 is shown as SEQ ID NO.

2.

2. A method for improving the low phosphorus stress tolerance of soybean, characterized in that, The method is to improve the expression amount of the amino acid sequence shown as SEQ ID NO. 2 in the recipient soybean; The specific steps include: introducing the soybean low-phosphorus tolerance gene GsMYB7 shown as SEQ ID NO. 1 into the recipient soybean to obtain transgenic soybean, and the low-phosphorus stress tolerance ability of the transgenic soybean is higher than that of the recipient soybean.

3. The application of soybean low phosphorus tolerance gene GsMYB7 in breeding soybean with low phosphorus stress tolerance, characterized in that: The nucleotide sequence of the soybean low-phosphorus tolerance gene GsMYB7 is shown as SEQ ID NO. 1; or the amino acid sequence encoded by the soybean low-phosphorus tolerance gene GsMYB7 is shown as SEQ ID NO. 2.

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