Application of soybean low-phosphorus-tolerant gene GsMYB7 in improvement of low-phosphorus stress tolerance of plants

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a plant low-phosphorus-resistant important gene GsMYB7 and application thereof. According to the invention, an MYB family GsMYB7 gene is cloned in soybean for the first time. Researches show that the expression of the GsMYB7 gene is increased under the induction of low phosphorus stress, and under different phosphorus concentration treatment conditions, the overexpression GsMYB7 can obviously increase the biomass of transgenic plants and promote the growth of the plants under the low phosphorus condition; meanwhile, overexpression of GsMYB7 can improve the tolerance of plants to low-phosphorus stress, and reduce the inhibition effect of low phosphorus on plant root growth. Therefore, the GsMYB7 plays an important role in adapting to the low-phosphorus stress of the plant, and the adaptive capacity of the plant to the low-phosphorus stress of the acid soil can be improved through a transgenic technology.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of the soybean low-phosphorus tolerance gene GsMYB7 in improving the low-phosphorus stress tolerance of plants. Background Art

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

[0003] Phosphorus, as one of the essential macronutrients in the process of plant growth and development, plays an irreplaceable role in the growth and development of plants. For leguminous crops, phosphorus deficiency will lead to stunted plants and inhibited leaf growth, thus affecting the growth and development of the plants. In addition, phosphorus deficiency will also affect the root morphology of plants: on the one hand, the development of the main root is inhibited, its length and volume are reduced, and the ability of plants to absorb water and nutrients is decreased; on the other hand, the development of root nodules will also be inhibited, resulting in a decrease in their volume and nitrogen fixation efficiency, and further affecting the acquisition of nitrogen by plants. Moreover, phosphorus deficiency will further affect the reproductive growth and stress resistance of plants. However, the total phosphorus content in general soil is relatively small, and the total phosphorus content in natural soil is about 0.02% - 0.25%, and the phosphorus content that can be absorbed and utilized by plants is even less.

[0004] Therefore, solving the problem of phosphorus deficiency is one of the important ways to improve the yield and quality of leguminous crops. Studying the physiological response and adaptation mechanism of soybeans under low-phosphorus stress conditions is of great significance for improving the phosphorus utilization efficiency of soybeans, enhancing their stress resistance, and achieving high-yield and high-quality cultivation. Summary of the Invention

[0005] To solve the problems in the above background art, the first object of the present invention is to provide an application of the soybean low-phosphorus tolerance gene GsMYB7 in improving the low-phosphorus stress tolerance of plants.

[0006] In this application, by overexpressing the gene GsMYB7 in plants, the low-phosphorus stress tolerance of plants is improved.

[0007] Furthermore, the plant is soybean.

[0008] The second object of the present invention is to provide a method for improving the low-phosphorus stress tolerance of plants, and the method is to increase the expression level of the amino acid sequence shown in SEQ ID NO.2 in the recipient plants; the specific steps include: introducing the soybean low-phosphorus tolerance gene GsMYB7 shown in SEQ ID NO.1 into the recipient plants to obtain transgenic plants, and the low-phosphorus stress tolerance of the transgenic plants is higher than that of the recipient plants; or overexpressing the amino acid sequence shown in SEQ ID NO.2 in the recipient plants.

[0009] Furthermore, the improvement of the low-phosphorus stress tolerance of the plants is manifested as an increase in the main root length or the number of lateral roots.

[0010] The third object of the present invention is to provide a preparation for improving the low-phosphorus stress tolerance of plants, and the active ingredient of the preparation contains the soybean low-phosphorus tolerance gene GsMYB7 or the protein encoded by the soybean low-phosphorus tolerance gene GsMYB7.

[0011] The fourth object of the present invention is to provide the application of the recombinant vector of the soybean low-phosphorus tolerance gene GsMYB7 in improving the low-phosphorus stress tolerance of plants.

[0012] The fifth object of the present invention is to provide the application of a soybean low-phosphorus tolerance gene GsMYB7 in cultivating low-phosphorus stress-tolerant soybean plants.

[0013] The present application studies the method for the soybean low-phosphorus tolerance gene GsMYB7 to promote the growth of plants in a low-phosphorus stress environment, including the following specific steps:

[0014] Using soybean as the material, by means of the Agrobacterium-mediated cotyledon node transformation method of soybean, the GsMYB7 gene was genetically transformed into soybean to obtain soybean plants with overexpression of the GsMYB7 gene. By comparing the growth performance, phosphorus absorption efficiency and root system development and other indexes of transgenic plants and wild-type plants under low-phosphorus stress conditions, the function and application potential of the GsMYB7 gene in improving the low-phosphorus tolerance of soybean were verified.

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

[0016] The present invention provides the application of a GsMYB7 gene in regulating the low-phosphorus stress tolerance of soybean. The GsMYB7 gene can enhance the low-phosphorus stress tolerance of soybean, and overexpression of the GsMYB7 gene can enhance the low-phosphorus tolerance of plants.

[0017] In addition, the present application constructed a soybean material with overexpression of GsMYB7: by using the Agrobacterium-mediated cotyledon node transformation method of soybean, the GsMYB7 gene can be successfully integrated into the soybean genome and stably expressed.

[0018] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following preferred embodiments are specifically cited and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the domain structure diagram of GsMYB7 protein;

[0020] Figure 2 This is the evolutionary tree analysis diagram of GsMYB7;

[0021] Figure 3 To clone the GsMYB7 gene and identify the GsMYB7 gene overexpression vector by PCR.

[0022] Figure 4 Detection of GsMYB7 transgenic soybean lines, where (A) shows PCR identification using Bar gene primers, (B) shows gene expression profiles of transgenic soybean lines detected by qRT-PCR, and (C) shows the tolerance of transgenic plants to herbicides.

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

[0024] Figure 6 To analyze the expression pattern of GsMYB7 gene for low phosphorus tolerance;

[0025] Figure 7 This is a test of total phosphorus content in GsMYB7 transgenic soybeans;

[0026] Figure 8 To identify the low-phosphorus tolerance phenotype of GsMYB7 transgenic soybean in hydroponic culture at the seedling stage;

[0027] Figure 9 To identify the phenotype of GsMYB7 transgenic soybean throughout its growth period;

[0028] Figure 10 To identify the phenotypic characteristics and analyze the observation indicators of GsMYB7 transgenic soybean seedlings in soil culture. DETAILED DESCRIPTION

[0029] In order to make the contents of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the 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 length of the coding DNA sequence (CDS) is 1002 bp, encoding 333 amino acids.

[0032] S1. Conserved domain analysis: As Figure 1 shown, the GsMYB7 protein in this application contains two conserved DNA-binding domains, which are composed 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 conserved structural pattern of typical R2R3-MYB transcription factors (TFs).

[0033] S2. Phylogenetic tree and homology analysis: As Figure 2 shown, the related homologs of the GsMYB7 protein are mainly distributed in leguminous plants. Although the homology between GsMYB7 and the homologous protein sequence in Arabidopsis thaliana is low, both have a typical MYB DNA-binding domain, indicating that they have retained the key DNA-binding function during evolution.

[0034] S3: Functional speculation and regulatory mechanism: The promoter region of 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 response element), ERE (ethylene response element), TGA (auxin response element), HSE (heat stress response element), TC-rich repeat sequence (adverse stress response element). The presence of these elements suggests that GsMYB7 may play a regulatory role in various environmental signal transduction pathways such as light, ethylene, auxin, heat, and adverse stress.

[0035] In this example, the candidate sequence information of the GsMYB7 gene was retrieved through the National Center for Biotechnology Information (NCBI) database, and its accession number is XP_003527079.1; in addition, this experiment used DNAMAN software to complete the multiple sequence alignment of the soybean GsMYB7 protein and members of the R2R3-MYB family, and used MAGE software to construct a phylogenetic tree by the Neighbor-Joining (NJ) method.

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

[0037] S1. Construction of the intermediate vector pUC18-GsMYB7: Amplify the CDS sequence of GsMYB7 by PCR, and the amplification primers are:

[0038] Forward Primer (SEQ ID NO.3): 5’-GGACCACTATCTCCGACCTG-3’

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

[0040] It was inserted between the BamHI and KpnI restriction sites of the modified intermediate vector pUC18 to construct the intermediate vector pUC18-GsMYB7.

[0041] pUC18-GsMYB7_F (SEQ ID NO.5): 5’-ATACTAGTGGCGCGCCGGGTGGTAGT 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: 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; stored at 12°C.

[0043] Table 1 Fragment amplification reaction system

[0044]

[0045] Recovery of the S2 target fragment: Digest pUC18-GsMYB7 with HindIII restriction endonuclease and recover the target fragment containing 35S-GsMYB7-NOS terminator.

[0046] Construction and transformation of the expression vector pZY101-GsMYB7: Insert the above target fragment into the HindIII site of the pZY101 vector to construct the expression vector pZY101-GsMYB7; then transform the constructed expression vector into the competent cells of Escherichia coli DH5α. The steps for E. coli transformation were: Pipette 10 μL of the ligation product into 100 μL of DH5α E. coli competent cells, gently pipette and mix well, then incubate on ice for 30 min, and then perform heat shock at 42°C for 45 s and immediately transfer to ice for 2 min; then transfer the ligation product to the laminar flow bench, add 500 - 700 μL of LB liquid medium to the ligation product, and then culture at 37°C on a shaker at 220 rpm for 1 h to obtain the bacterial solution. Spread the bacterial solution on a plate containing Amp and culture overnight at 37°C. Pick single colonies and shake the bacteria for detection and sequencing.

[0047] Identification of S4 positive clones: The pZY101-GsMYB7 positive clones were identified by colony PCR, restriction 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 by the genetic transformation method of soybean cotyledon nodes.

[0051] a Preparation of electrocompetent Agrobacterium

[0052] 1) Pick a milky and plump single colony and inoculate it into 5 mL of YEP liquid medium containing 5 μL of spectinomycin, 5 μL of streptomycin, and 5 μL of chloramphenicol, and culture it overnight in a shaker at 28 °C and 250 rpm;

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

[0054] 3) Transfer the bacterial solution to a sterile centrifuge tube, place it on ice for 30 min, and then centrifuge it in a centrifuge at 4 °C, 4000 rpm for 10 min;

[0055] 4) Discard the supernatant, resuspend the cells with a small amount of pre-cooled sterile water, add pre-cooled sterile water to 50 mL, and then centrifuge it in a centrifuge at 4 °C, 4000 rpm for 10 min;

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

[0057] b Transformation by electroporation

[0058] 1) Take out the electroporation cuvette from 70% ethanol, place it in a laminar flow hood to dry, wrap the electroporation cuvette with clean gloves after drying, and place it on ice for pre-cooling for later use;

[0059] 2) Take out the Agrobacterium competent cells from the -80°C refrigerator and thaw them on ice. Then add the plasmid to the thawed competent cells, gently pipette and mix well, and transfer them to the cuvette.

[0060] 3) Place the cuvette in the electroporator for electroporation, and set the parameters as: voltage 1800V, time 5ms.

[0061] 4) After electroporation, slowly add 1 mL of YEP liquid medium without antibiotics to the cuvette, gently pipette a few times to suspend and mix the bacteria.

[0062] 5) Aspirate the bacterial solution from the cuvette and place it in a centrifuge tube, and then culture it in a shaker at 28°C and 250 rpm for 3 h.

[0063] 6) Aspirate 50 μL of the bacterial solution and spread it on a YEP solid plate containing spectinomycin, streptomycin, and chloramphenicol, and culture it in a shaker at 28°C and 250 rpm for about 36 h until single colonies grow.

[0064] Soybean genetic transformation

[0065] 1) Germination: a. Seed disinfection: Take out the screened and stored Huachun 6 seeds from the -20°C refrigerator, soak them in 75% alcohol for 1 min, then dry them, arrange them in a single layer in a petri dish, and place them in a drying oven in the fume hood; Mix 100 mL of sodium hypochlorite and 5 mL of concentrated hydrochloric acid in the fume hood, quickly transfer them to a 250 mL beaker, place it in the drying oven, seal and sterilize for 13.5 hours, then take it out, blow it in the laminar flow hood, and store it in the 4°C refrigerator for later use. b. Sowing: Gently press the seeds with the hilum facing down on the germination medium, and conduct the infection experiment after the cotyledons turn green.

[0066] 2) Soak the seeds: Soak the soybeans in sterilized ddH2O, and place them in a dark environment at 24°C for 10 - 16 h.

[0067] 3) Bacterial solution preparation: Take out the EHA101 bacterial solution transformed with the target plasmid, and add it to the YEP liquid medium containing 100 mg / L Spe and 30 mg / L Rif; Set the constant temperature shaker at 28°C and 240 rpm, culture overnight, and measure the absorbance OD600 of the bacterial solution the next day to be 0.8 - 1.0; Pour the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min; Pour out the supernatant, resuspend the bacteria with CCM liquid medium to make OD600 = 0.6 - 0.8, and store at 4°C for the next experiment.

[0068] 4) Preparation and transformation of explants: In the laminar flow hood, use forceps and a scalpel to cut open the hypocotyl of the soaked seeds, vertically cut the seeds along the cotyledons, and remove the young buds, epicotyls, and seed coats on the cotyledons.

[0069] 5) Infection and co-culture: Immerse the explants in the prepared CCM resuspension, apply ultrasound for 3 min, vacuum for 10 min, shake on a shaker at 120 - 150 rpm, 28 °C, and oscillate for 40 min; after the infection, pour out the bacterial solution, lay the explants flat on the CCM solid medium covered with filter paper using forceps; seal with medical tape, place in the tissue culture room, set the temperature at 22 °C, and incubate in the dark for 48 h, then culture under light for 3 - 5 days until the cotyledons turn completely green.

[0070] 6) Induction of young shoots: Use a scalpel to cut off the overgrown hypocotyl, insert the explants obliquely onto the shoot induction medium, seal with medical tape, place in the tissue culture room, set the temperature at 22 °C, the light time at 16 / 8 h, and culture for 14 days; then transfer the sterile plate to a new shoot induction medium, discard the explants that have not produced clustered shoots, place 6 - 7 explants on each medium, seal with medical tape, and place in the tissue culture room for induction for 14 days.

[0071] 7) Growth of young shoots: Four weeks after the induction of young shoots, discard the undifferentiated materials, cut off the cotyledons of the explants with clustered shoots, transfer them to the shoot growth medium, make the cut surface fit the medium, place 6 - 7 explants in each petri dish, place in the tissue culture room, and change the medium every 14 days.

[0072] 8) After rooting, use a scalpel to cut the seedlings with shoots longer than 3 cm from the roots of the tissue, soak them in auxin for 1 min, transfer them to the rooting medium, place in the tissue culture room to continue growing. When 5 - 6 main roots and lateral roots grow out, gently take the plants out of the rooting medium, and carefully rinse the medium on the roots with tap water; transplant the seedlings into the autoclaved nutrient soil (vermiculite:substrate soil = 1:2), place them in a heat and humidity - retaining seedling - raising tray, and place the seedling - raising tray in an incubator, set the temperature at 26 °C, the light time at 12 / 12 h. Water with nutrient solution for cultivation until pod formation.

[0073] Identification results

[0074] As Figure 3 shown, where M in part a is DL2000 DNA Marker, lane 1 is the full - length cloning fragment of GsMYB7, and the band indicated by ☆ is the target band, approximately 1002 bp;

[0075] M in part b is DL5000 DNA Marker, - is the H2O control, lanes 1, 2, 3, and 4 are PCR detections of pZY101 - GsMYB7 Escherichia coli bacterial solution, and the band indicated by ☆ is the target band, approximately 2000 bp;

[0076] M in part c is DL15000 DNA Marker. Lane 1 is the recombinant plasmid pZY101-GsMYB7. Lane 2 is the double-digested fragment of the full-length GsMYB7 with BamHΙ and KpnΙ. Lane 3 is the double digestion of the recombinant plasmid pZY101-GsMYB7 with BamHΙ and KpnΙ. The ☆ indicates the target band, which is approximately 1002 bp.

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

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

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

[0080] The PCR identification results of the Bar gene primers are as shown in Figure 4 Part A. The transgenic lines overexpressing GsMYB7 can amplify a specific Bar gene band with a size of approximately 488 bp.

[0081] The results of qRT-PCR detection of gene expression in transgenic soybean lines are as shown in Figure 4 Part B. The relative expression level (Reletive expression) of GsMYB7 in the transgenic lines is higher than that in the wild type (WT) plants.

[0082] The results of herbicide tolerance identification are as shown in Figure 4 Part C. The leaves with a tick are the side where the herbicide was applied, and the herbicide was not applied on the left side. The experiment shows that the transgenic lines have stronger herbicide tolerance than the WT plants.

[0083] PCR identification was performed using primers designed with the sequences of 35S and the target gene. The results are as shown in Figure 5 Parts a and b. The experiment shows that the transgenic lines overexpressing GsMYB7 can amplify a band with a size of approximately 1110 bp, proving that the GsMYB7 gene sequence has been integrated into the genome of the soybean transformant plants.

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

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

[0086] After disinfecting BX13 soybean seeds with 75% ethanol, they were placed in vermiculite at 22°C with a 16 / 8 h light cycle. After 4 days of germination, when the cotyledons were fully expanded, seedlings with consistent growth were collected and transferred to 1 / 2 Hoagland solution for cultivation. They were divided into two treatment groups: the experimental group and the control group. In the experimental group, the plants were cultivated with a low phosphorus concentration (5 μmol / L K2HPO4), and in the control group, they were cultivated with a normal phosphorus concentration (500 μmol / L K2HPO4). Subsequently, root samples were taken at 0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, 7 d, and 14 d after treatment. RNA was extracted and reverse transcribed into cDNA, and the changes in the expression level of this gene during the above periods were observed by real-time fluorescence quantitative PCR (qRT-PCR).

[0087] Experimental Procedures

[0088] Quantitative PCR Primer Design: The gene sequence was retrieved from the Phytozome database. The genomic CDS sequence of GsMYB7, shown as SEQ ID NO.1, and the protein sequence of GsMYB7, shown as SEQ ID NO.2, were downloaded from the website. Specific quantitative amplification primers were designed and synthesized based on the genomic sequence on the NCBI website:

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

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

[0091] Real-Time Fluorescence Quantitative PCR:

[0092] ① Using Actin3 as the internal reference gene,

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

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

[0095] ②Dilute the cDNA of all samples 1-fold with ddH2O as the template for the quantitative PCR reaction;

[0096] ③The real-time fluorescence quantitative PCR system was carried out according to the reaction system in Table 2 below. The reaction program was: pre-denaturation at 95°C for 30 s; 39 cycles (denaturation at 95°C for 5 s; annealing at 60°C for 30 s); melting curve at 95°C for 10 s; 54.3°C for 5 s; 94.3°C for 5 s;

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

[0098]

[0099] ④Process the data, and the relative expression analysis was performed using the 2^ -△△CT method.

[0100] The experimental results are as Figure 6 shown. The low-phosphorus environment can promote the expression of GsMYB7.

[0101] After disinfecting the GsMYB7 transgenic seeds with 75% ethanol, they were cultured in vermiculite at 22°C with a light time of 16 / 8 h. After 4 days of germination, when the cotyledons were fully unfolded, the seedlings with consistent growth were collected and transferred to 1 / 2 Hoagland solution for culture, and were divided into two treatment groups: the experimental group and the control group. Among them, the experimental group was cultured with a low phosphorus concentration (5 μmol / L K2HPO4), and the control group was cultured with a normal phosphorus concentration (500 μmol / L K2HPO4). Among them, each group included the wild type (WT) and transgenic lines. WT was used as the within-group control (CK). The transgenic lines specifically included three lines: Gm7A, Gm7C, and Gm7H. Each group was set with 3 replicates. The hydroponic nutrient solution was renewed every two days, and the hydroponic phenotypes were observed after 14 days, and the seeds were examined and the total phosphorus content was measured (the total phosphorus content was the total phosphorus content multiplied by the dry weight of the above- and below-ground parts).

[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 supplement the corresponding K.

[0103] 1. Method for determination of total phosphorus in plants by molybdenum-antimony anti-colorimetry:

[0104] All reagents used in this experiment were of analytical grade, and the water was deionized water or 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 Antimonyl Tartrate Solution: Weigh 0.5 g of chemically pure potassium antimonyl tartrate and dissolve it in 100 mL of water;

[0110] (6) Molybdenum Antimony Sulfate Stock Solution: Measure 126 mL of concentrated sulfuric acid and slowly add it to 100 mL of water, stirring continuously and cooling. Weigh another 10 g of finely ground ammonium molybdate and dissolve it in about 300 mL of water at 60 °C, then cool. Then slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimonyl tartrate solution, cool, and dilute to 1000 mL with water, 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 Ascorbic Acid Color Reagent: Weigh 1.5 g of ascorbic acid and dissolve it in 100 mL of molybdenum antimony stock solution. This solution has a short shelf life and should be prepared freshly when used;

[0112] (8) Phosphorus Standard Stock Solution: Accurately weigh 0.4390 g of potassium dihydrogen phosphate dried at 105 °C for 2 h, dissolve it in water, add 5 mL of concentrated sulfuric acid, and then dilute to 1000 mL with water. This solution contains 100 mg / L of phosphorus and can be stored in the 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 and dilute to the mark with water. This solution should be prepared freshly when used.

[0114] 2. Experimental Procedures

[0115] (1) Preparation of Sample Solution

[0116] Weigh an appropriate amount of the sample, accurate to 0.001 g, and place it in a 500 mL digestion tube (do not let the sample adhere to the neck of the tube). First, drip a little water to moisten the sample, then add 6 mL of sulfuric acid, shake gently and let stand overnight. Place a bent-neck funnel at the mouth of the tube and digest at 250 °C on a digestion furnace (start timing after the temperature stabilizes, for about 30 min). After the H2SO4 decomposes and emits a large amount of white smoke, raise the temperature to 400 °C. When the solution becomes uniformly dark brown, remove it from the furnace.

[0117] After cooling slightly, add 10 drops of H2O2, shake well, and heat to just boiling, digest for about 5 min. After removing it from the furnace and cooling slightly, repeat adding 5 - 10 drops of H2O2 and digest again. Repeat this 3 - 5 times. The amount of H2O2 added each time should be gradually reduced. Digest until the solution becomes colorless or clear and the bottom is grayish white, then heat for about 5 - 10 min to remove the remaining H2O2.

[0118] Remove the digestion tube and let it cool. Rinse the bent-neck funnel with a small amount of water and let the washing solution flow into the digestion tube. Transfer the digestion solution into a 100 mL volumetric flask without loss, make up the volume with water, and shake well. Filter or let it stand for clarification for the determination of phosphorus.

[0119] (2) Preparation of blank solution

[0120] Except for not adding the test sample, use the same reagents and operating procedures as above.

[0121] (3) Plot the standard curve

[0122] Respectively pipette 0, 2, 4, 6, 8, 10, 14, 20 mL of 5 mg / L phosphorus standard solution into 50 mL volumetric flasks. At the same time, add the same volume of blank solution as the sample solution used for color development determination and 2 - 3 drops of dinitrophenol indicator. Adjust the solution to just turn slightly yellow with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony anti-color reagent, shake well, make up the volume with water, and obtain a series of standard solutions with phosphorus contents of 0.0, 0.2, 0.4, 0.8, 1.0, 1.4, 2.0 mg / L respectively. Shake well, after standing at a temperature above 15 °C for 30 min, measure the absorbance at a wavelength of 880 nm. On the rectangular coordinate paper, use the absorbance as the ordinate and the phosphorus concentration (mg / L) as the abscissa to plot the calibration curve.

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

[0124] Pipette 2 - 10 mL of the sample solution to be tested (containing 0.04 - 1.0 g of phosphorus) into a 50 mL volumetric flask. Add 2 - 3 drops of dinitrophenol indicator, and adjust the solution to just turn slightly yellow with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony anti-color reagent, shake well, make up the volume with water. Place it at room temperature above 15 °C for 30 min.

[0125] For the colored sample solution on the spectrophotometer, use a 1 cm optical path cuvette at 880 nm, adjust the instrument zero with the blank test as the reference solution, conduct colorimetric determination, and read the absorbance. Check the corresponding phosphorus content from the standard curve.

[0126] 3. Result calculation

[0127]

[0128] Where:

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

[0130] C0 —— Blank value

[0131] V —— Volume of the colored solution;

[0132] D——Sub-sampling multiple, volume of digested solution made up to a fixed volume / volume of digested solution taken;

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

[0134] 1000——Conversion factor for converting the unit mL to L. The resulting value should be rounded to three decimal places.

[0135] The test results of total phosphorus content are as Figure 7 shown. Under NP and LP conditions, the total phosphorus content in the above-ground parts and the total phosphorus content in the underground parts of transgenic plants are both higher than those of WT plants.

[0136] The hydroponic phenotypes are as Figure 8 shown. Compared with WT, the root length of transgenic plants increases, and compared with WT, the above-ground fresh weight and underground fresh weight of transgenic plants increase significantly.

[0137] Example 4: Identification of phosphorus-efficient phenotypes during the whole growth period in soil culture

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

[0139] The soybeans were set up into the following treatment groups: low phosphorus (LP) treatment experimental group, normal phosphorus (NP) treatment control group. Among them, each group included wild type (WT) and transgenic lines. WT was used as the within-group control (CK). The transgenic lines specifically included three lines, namely Gm7A, Gm7C, and Gm7H. Each group was set with 3 replicates.

[0140] Treatment methods for treatment groups

[0141] Low phosphorus treatment experimental group: Uniformly spray 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the seedling stage, and irrigate the roots with 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the flowering stage and pod-setting stage respectively. Normal phosphorus treatment group: Uniformly spray 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the seedling stage, and irrigate the roots with 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the flowering stage and pod-setting stage respectively. A total of three treatments were carried out, one spraying and two irrigations. The spraying dosage of low phosphorus and normal phosphorus 1 / 2 Hoagland solution at the seedling stage was about 7 L respectively, and the irrigation dosage of low phosphorus and normal phosphorus 1 / 2 Hoagland solution at each time of the flowering stage and pod-setting stage was 15 L respectively.

[0142] After the soybeans matured, their growth conditions were photographed and recorded. As Figure 9 shown, the analysis of yield traits showed that the number of pods per plant and the height of the bottom pod of the transgenic plants were both higher than those of the control group under the LP treatment; under the low-phosphorus (LP) and normal-phosphorus (NP) treatment conditions, the 100-seed weight of the transgenic line Gm7H was significantly higher than that of the control group (CK), and there were no obvious changes in other lines. There was no obvious change in the number of nodes on the main stem. The above results proved that GsMYB7 could improve the low-phosphorus tolerance of plants.

[0143] Example 5: Identification of high phosphorus efficiency phenotype in the seedling stage by soil culture

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

[0145] The soybeans were set up into the following treatment groups: a low-phosphorus (Low Phosphorus, LP) treatment experimental group and a normal-phosphorus (Normal Phosphorus, NP) treatment control group. Specifically, each group included a wild type (Wild type, WT) and transgenic lines. WT was used as the within-group control (CK). The transgenic lines specifically included three lines, Gm7A, Gm7C and Gm7H. Each group was set with 3 replicates.

[0146] Treatment methods for treatment groups

[0147] Low-phosphorus and normal-phosphorus treatment experimental groups: Uniformly spray a 1 / 2 Hoagland solution containing 5 μmol / L K2HPO4 at the seedling stage. Normal-phosphorus treatment group: Uniformly spray a 1 / 2 Hoagland solution containing 500 μmol / L K2HPO4 at the seedling stage.

[0148] After the soybeans matured, their growth conditions were photographed and recorded. The experimental results were as Figure 10 shown. Under the low-phosphorus (LP) and normal-phosphorus (NP) treatment conditions, the above-ground weight of the transgenic lines was significantly higher than that of the control group (CK).

[0149] The verification of the above experiments showed that the GsMYB7 gene could enhance the tolerance of soybeans to low-phosphorus stress. Overexpression of the GsMYB7 gene could enhance the low-phosphorus tolerance of plants.

[0150] GsMYB7 gene sequence and amino acid sequence

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

[0152] ATGGGAAGACCACCTTGCTGTGATAAAATTGGGATTAAGAAAGGGCCTTGGACTCCTGAGGAAGACA

[0153] TCATCTTGGTCTCTTACATTCAAGAACATGGACCCGGAAATTGGAGATCGGTTCCCAGTAACACAGG

[0154] TTTGATGAGATGCAGCAAAAGCTGCAGACTCAGATGGACCAACTATCTCCGACCTGGTATCAAACGA

[0155] GGCAATTTCACCGATCATGAAGAGAAAATGATAATCCACCTCCAAGCTCTTTTGGGTAACAGATGGG

[0156] CTGCTATAGCTTCCTACCTTCCACAAAGGACAGACAATGACATAAAGAACTATTGGAACACCCATTT

[0157] GAAGAAGAAGCTGAAGAAGATGCAAATTGGGGGTGGTAGTGATGATGATAATAATGATGACAAATCA

[0158] AACTCTTCTAACAATTCACAAATAAAGGGTCAATGGGAAAGAAGACTTCAAACAGATATCCACATGG

[0159] CCAAACAAGCCTTATGTGAGGCCCTATCTCTTGACAAACCAACCCAAATTTTCCCAGAGACCAAATT

[0160] ACCCTCCACTTCTTCACACCACCACCCCACAACAACAACAACACCAAACCAAACAACATCCTTGTAT

[0161] GCATCAAGCACAGAAAACATAGCCAGATTGTTGGAGAATTGGATGAAGAAATCACCAAATATGACGA

[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 FPETKLPSTSSHHHPTTTTTPNQTTSLYASST

[0171] ENIARLLENWMKKSPNMTTTTTTTMETKPFSNNNMVITTGSSSSEGTQSTITCTQEYALDSLWSFNSERSSQSEENTNLGESKPQYQEPQETQVPLMLLENWLFDDAAPQCNEDLMNMSLEESTEGLF*

[0172] The above-described embodiments are only the preferred embodiments of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantive changes and modifications made by those skilled in the art based on the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of soybean low-phosphorus tolerance gene GsMYB7 in improving plant low-phosphorus stress tolerance, characterized in that: The nucleotide gene sequence of the soybean low phosphorus tolerance gene GsMYB7 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GsMYB7 encodes an amino acid sequence shown in SEQ ID NO.

2.

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

3. A method for improving the low phosphorus stress tolerance of plants, characterized in that, The method is to increase the expression level of the amino acid sequence shown in SEQID NO.2 in the recipient plant; The specific steps include: introducing the soybean low phosphorus tolerance gene GsMYB7 shown in SEQ ID NO.1 into the recipient plant to obtain a transgenic plant, and the low phosphorus stress tolerance of the transgenic plant is higher than that of the recipient plant; or overexpressing the amino acid sequence shown in SEQ ID NO.2 in the recipient plant.

4. The method according to claim 3, wherein The improvement of the low phosphorus stress tolerance of the plant is reflected in the increase of its primary root length or the number of lateral roots.

5. Use of the low phosphorus tolerance gene GsMYB7 of soybean in cultivating plants tolerant to low phosphorus stress, characterized in that: The nucleotide sequence of the soybean low phosphorus tolerance gene GsMYB7 is shown in SEQ ID NO.1; or the amino acid sequence encoded by the soybean low phosphorus tolerance gene GsMYB7 is shown in SEQ ID NO.2.

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