Application of soybean low-phosphorus-tolerant gene GmAP2 in improvement of low-phosphorus stress tolerance of plants

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses application of a soybean low-phosphorus-tolerant gene GmAP2 to improvement of low-phosphorus stress tolerance of plants. Research finds that under the condition of low phosphorus stress, the expression level of the GmAP2 gene in plants is remarkably increased. The GmAP2 gene is subjected to heterologous overexpression in arabidopsis thaliana, so that stress on plants under a low-phosphorus condition can be effectively relieved, the fresh weight and lateral root length of the arabidopsis thaliana can be remarkably increased, and the growth of the arabidopsis thaliana plants is promoted. In addition, under the low-phosphorus condition, overexpression of the GmAP2 gene can improve the phosphorus deficiency symptom of the soybean, improve the absorption of phosphorus nutrients by plants and promote the growth of plant roots and leaves. Therefore, the GmAP2 gene plays a very important role in dealing with the phosphorus starvation stress environment by the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of a soybean low-phosphorus-tolerant gene GmAP2 in improving the low-phosphorus stress tolerance of a plant. Background Art

[0002] Soybean is an important crop used as both food and oil in the world and plays a very important role in agricultural production. Phosphorus is one of the three elements necessary for plant growth, development and metabolism. It participates in various biochemical processes in plants in many ways. Plants can only obtain phosphorus in the form of inorganic phosphate (Pi). However, most of the soil in South China is acidic red soil with low pH value. Iron (Fe 3+ ) and aluminum (Al 3+ ) ion concentration is high, these ions will react with phosphate (PO4 3- ) to form insoluble iron phosphate (FePO4) and aluminum phosphate (AlPO4) precipitation, making it difficult for plants to absorb and utilize most of the phosphorus in the soil, resulting in low soybean yields. Soil phosphorus deficiency has become an important factor limiting soybean yield and quality. Therefore, analyzing the molecular mechanisms related to soybean tolerance to low phosphorus, discovering and identifying key phosphorus-efficient genes, improving the efficiency of soybean phosphorus absorption and utilization, and cultivating new phosphorus-efficient soybean varieties are of great significance to increasing soybean yields in South China. Summary of the Invention

[0003] In order to solve the problems in the above-mentioned background technology, one of the objects of the present invention is to provide an application of a soybean low-phosphorus tolerance gene GmAP2 in improving the ability of a plant to tolerate low-phosphorus stress, wherein the nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP2 is shown in SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes an amino acid sequence as shown in SEQ ID NO.2.

[0004] The present application improves the plant's ability to tolerate low-phosphorus stress by overexpressing the soybean low-phosphorus tolerance gene GmAP2 in the plant.

[0005] Furthermore, the plant is Arabidopsis thaliana or soybean.

[0006] The second object of the present invention is to provide a method for improving the tolerance of plants to low phosphorus stress, the method comprising increasing the expression level of the amino acid sequence shown in SEQ ID NO.2 in a recipient plant;

[0007] The specific steps include: introducing the soybean low-phosphorus tolerance gene GmAP2 shown in SEQ ID NO.1 into a 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.

[0008] Furthermore, the improvement in the plant's tolerance to low phosphorus stress is reflected in the increase in its root length.

[0009] Furthermore, the improvement in the plant's ability to tolerate low-phosphorus stress is reflected in an increase in the number of its lateral roots.

[0010] Furthermore, the improvement in the plant's tolerance to low phosphorus stress is reflected in an increase in its fresh weight.

[0011] The third object of the present invention is to provide the use of the soybean low-phosphorus tolerance gene GmAP2 in cultivating low-phosphorus stress-tolerant plants, wherein the nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP2 is shown in SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes the amino acid sequence shown in SEQ ID NO.2.

[0012] The fourth object of the present invention is to provide a recombinant plasmid, a recombinant vector or a transgenic plant cell line of the soybean low-phosphorus tolerance gene GmAP2 for use in improving the plant's tolerance to low-phosphorus stress, wherein the nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP2 is shown in SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes an amino acid sequence shown in SEQ ID NO.2.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] S1, extracting soybean root tip RNA and reverse-transcribing it into cDNA, and then cloning the coding DNA sequence (CDS) of the soybean low-phosphorus tolerance gene GmAP2 by PCR amplification;

[0015] S2, using the CDS as a template, PCR amplification was performed to obtain the target fragment;

[0016] S3, sequence the target fragment and compare the sequencing result with the reference sequence of soybean low-phosphorus tolerance gene GmAP2 (known sequence of this gene in NCBI database). The sequence of GmAP2 is consistent with the reference sequence;

[0017] S4, ligating the sequenced and compared target fragments to an expression vector to obtain a recombinant vector, sequencing the recombinant vector to verify the connection, and then transforming the recombinant vector into Agrobacterium;

[0018] S5, using Arabidopsis thaliana as a vector to verify the role of the soybean low-phosphorus tolerance gene GmAP2 in plant tolerance to low-phosphorus stress: construct an overexpression vector for the soybean low-phosphorus tolerance gene GmAP2 and heterologously transform Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana plants. From these T5 generation homozygous lines, fluorescent quantitative identification was performed to screen for lines with high expression levels of the soybean low-phosphorus tolerance gene GmAP2;

[0019] S6, prepared wild-type Arabidopsis thaliana, and performed low-phosphorus tolerance phenotype identification on transgenic and wild-type Arabidopsis plants. The results showed that root elongation and lateral root length of transgenic Arabidopsis were significantly improved compared with wild-type plants, suggesting that the soybean low-phosphorus tolerance gene GmAP2 may be involved in the mechanism of soybean low-phosphorus tolerance.

[0020] S7, gene editing GmAP2 gene (pCas9-GmAP2(CAS9)), overexpression of GmAP2 gene (pTF101-GmAP2(OE)) and empty vector control gene (pTF101(CK)) were constructed simultaneously, pCas9-GmAP2(GmAP2), pTF101-GmAP2(OE), and pTF101(CK) were transformed into K599 Agrobacterium rhizogenes for rooting experiments;

[0021] S8, plants carrying Agrobacterium carrying pCas9-GmAP2 (CAS9), pTF101-GmAP2 (OE) and pTF101 (CK) were identified for low-phosphorus tolerance phenotype. The results showed that the root elongation, lateral roots and dry weight of OE plants were significantly improved compared with CK. Through the above experiments, it is speculated that overexpression of GmAP2 can enhance soybean tolerance to low-phosphorus stress.

[0022] The present invention has the following beneficial effects: the expression of the GmAP2 gene is upregulated by low phosphorus stress, and its expression level increases significantly as the phosphorus treatment time is prolonged;

[0023] Under different phosphorus concentration treatment conditions, overexpression of GmAP2 significantly increased the biomass of transgenic plants, indicating that GmAP2 positively regulates the ability of plant roots to adapt to low phosphorus stress;

[0024] Therefore, GmAP2 plays an important role in plant adaptation to low-phosphorus stress, and regulating its expression through transgenic technology can significantly enhance the plant's adaptability to low-phosphorus stress in acidic soil.

[0025] 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

[0026] Figure 1 This is the evolutionary tree analysis diagram of some AP2 / ERF family genes in Arabidopsis and soybean;

[0027] Figure 2 The quantitative PCR results of GmAP2 gene under different phosphorus conditions (NP indicates normal phosphorus treatment, LP indicates low phosphorus treatment);

[0028] Figure 3 The figure shows the cloning of GmAP2 gene and the identification of GmAP2 gene and pLB vector bacterial solution;

[0029] Figure 4 The results of transient expression of super1300-GFP and super1300-GmAP2-GFP in tobacco leaf cells, respectively (GFP is the GFP channel (excitation wavelength 488 nm), DAPI is the UV channel (excitation wavelength 360 nm), Merge indicates the fusion of GFP and DAPI, and Bright field is bright field);

[0030] Figure 5 This is the validation diagram of GmAP2 yeast self-activation;

[0031] Figure 6 To determine the autoactivation region of GmAP2 protein;

[0032] Figure 7 This is a validation diagram of the interaction between GmAP2 protein and candidate genes;

[0033] Figure 8 This is the verification picture of the genetic transformation positive plant;

[0034] Figure 9 The results show the differences in phenotype, main root length, lateral root length, fresh weight and total phosphorus content between transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (Col-0) after 14 days of treatment under normal phosphorus and low phosphorus conditions.

[0035] Figure 10 The relative expression levels in the roots of soybean chimeras expressing GmAP2 gene (OE), gene-edited GmAP2 gene, and empty vector (CK) after 14 days of treatment under normal and low phosphorus conditions;

[0036] Figure 11 The root phenotypes of soybean chimeras overexpressing GmAP2 gene (OE), gene-edited GmAP2 gene, and empty vector (CK) after treatment under normal phosphorus and low phosphorus conditions for 14 days. DETAILED DESCRIPTION

[0037] 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.

[0038] To further illustrate the technical means adopted in the present invention, the following is a further detailed description in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment adopted in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0040] The GmAP2 gene cDNA nucleotide sequence used in the present invention is shown in SEQ ID NO.1:

[0041] ATGTGTGGCGGTGCCATCATCGCTGACTTCATACCCCGCCGTGGAGGCCGCCGCCTCACGGCCTCCGAGCTCTGGCCAAACTCCTTCGCCAAAGACGATGACTTTGACTTGGATTACTCCCACATCGCTACCCAACAACCCTCCACTCTCAAAAGGTCCCAACCTCCCAAAGTTAGCGAGCAGGTTGAGAATAAGCCGGTGAAGAGGCAGAGGAAGAATCTCTACAGAGGGATTCGGCAGCGTCCGTGGGGCAAATGGGCCGCGGAGATTCGCGATCCTAGAAAAGGGGTTCGTGTCTGGTTGGGCACCTTTAACACCGCAGAAGAAGCCGCGAGAGCCTACGATCGTGAAGCTCGAAAAATCCGAGGCAAAAAGGCTAAGGTGAATTTCCCCAACGAGGACGACGAATATTCCATTCAAGCTCGTAATCCAATTCCACCCCTTCCTTTCGCTCCACAACACCCTCCCCTGTACCAGCAACAGTACCGTTGCGATCTCAACAATGCCCCTAAAAATCTCAACTTTGAGTTCGGTTACGACCTGAACCACGCGGAGGCGTTCCCATCCCGCGTGGACGCCGTCAACGCTGACTCGGTGGTTGTCTCCGTTGATGAAAATTCGGGGTCAGCGTCGGGTTCAGAGGGTGCTTATTCGACAACGGAGTTCATGGGGTCCGTTCAGAACGGGAACGGTTATTTGGGTGGCACGGTAATGGAGAAGAAGGAGAAAGAGACAGAGGTTATTGAAGCTGAAGAAGAAAAGAACAAAGTGCTGGAGCTTTCTGAGGAGCTGATGGCGTACGAAAATTACATGAAGTTTTATCAGATTCCGTACTATGATGGACAATCCACGACGAATAATGTTCAGGAAAGCTTGGTTGGGGATCTCTGGAGTTTCGATTAG

[0042] The amino acid sequence of the GmAP2-encoded protein is shown in SEQ ID NO.2:

[0043] MCGGAIIADFIPRRGGRRLTASELWPNSFAKDDDFDLDYSHIATQQPSTLKRSQPPKVSEQVENKPVKRQRKNLYRGIRQRPWGKWAAEIRDPRKGVRVWLGTFNTAEEAARAYDREARKIRGKKAKVNFPNEDDEYSIQARNPIPPLPF APQHPPLYQQQYRCDLNNAPKNLNFEFFGYDLNHAEAFPSRVDAVNADSVVVSVDENSGSASGSEGAYSTTEFMGSVQNGNGYLGGTVMEKKEKETEVIEAEEEKNKVLELSEELMAYENYMKFYQIPYYDGQSTTNNVQESLVGDLWSFD*

[0044] The GmAP2 gene of the present application belongs to the AP2 / ERF gene family. The phylogenetic tree analysis of some AP2 / ERF family genes in Arabidopsis and soybean is as follows: Figure 1 shown.

[0045] Example 1: Expression pattern of GmAP2 gene at different phosphorus levels

[0046] 1. Soybean material processing

[0047] The soybean variety Brazil No. 13 (BRSMG68) of the same period and full growth was selected as plant material, germinated in vermiculite at a germination temperature of 26℃ and a light duration of 16 / 8h. It was cultured for 4 to 5 days (day, d). After the cotyledons were slightly opened, the soybeans were grouped and transferred to culture medium treated with normal phosphorus nutrition NP (Normal Phosphorus, NP, 500μM KH2PO4) and low phosphorus treatment LP (Low Phosphorus, LP, 5μMKH2PO4) for hydroponic treatment. Samples were collected after 0h, 3h, 6h, 12h, 24h, 36h, 2d, 5d, 7d, and 14d, and the collected samples were wrapped in tin foil and stored at -80℃.

[0048] 2. RNA Extraction and Reverse Transcription

[0049] RNA was extracted from soybean root samples obtained at different phosphorus concentrations and after hydroponic treatment, and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was then performed using the cDNA as a template. The specific steps are as follows:

[0050] (1) RNA extraction

[0051] ① Prepare a mortar, grinding rod, spoon, 1.5mL centrifuge tube, and pipette tip, and sterilize them with high temperature and high pressure in advance;

[0052] ② Cool the mortar and grinding rod with liquid nitrogen, then put the soybean root sample in, add liquid nitrogen, and quickly grind it into powder. Use a spoon to scoop the powder into the EP tube. The mass of the powder should be 50-100 mg.

[0053] ③ Add 500 μL of lysis buffer and 700 μL of diluent to the EP tube in sequence, and mix thoroughly using a vortex mixer until no obvious lumps remain. Then, heat the EP tube in a 70°C metal bath for 3 minutes. Then, transfer the EP tube to a centrifuge at 4°C and centrifuge at 12,000 rpm for 10 minutes.

[0054] ④ Take about 600 μL of supernatant from the EP tube and transfer it to another new 1.5 mL centrifuge tube. Then add 300 μL of anhydrous ethanol to the centrifuge tube and use a vortex mixer to pipette 20-25 times until the liquid in the centrifuge tube becomes turbid and white foam appears, allowing it to fully react to obtain a reaction solution;

[0055] ⑤ Prepare the centrifuge column and collection tube, place the centrifuge column in the collection tube, then transfer the thoroughly mixed reaction solution to the centrifuge column. Place the centrifuge column and collection tube containing the reaction solution in a centrifuge and centrifuge at 4°C, 12,000 rpm, and 1 minute.

[0056] ⑥ Remove the centrifuge column and collection tube, discard the filtrate, add 600 μL RNA wash solution, and then place the centrifuge column and collection tube in a centrifuge for centrifugation at 4°C, 12,000 rpm, and 45 seconds.

[0057] ⑦ Remove the centrifuge column and collection tube, discard the filtrate, and prepare incubation solution (5 μL of 10× DNase I buffer; 5 μL of DNase I; 40 μL of nuclease-free water). Then, pour 50 μL of the prepared incubation solution into the center of the centrifuge column adsorption membrane and let it stand for 15 minutes.

[0058] ⑧ Add 600 μL RNA wash solution to the spin column and wash the precipitate twice. Then place the spin column and collection tube in a centrifuge and centrifuge at 4°C, 12,000 rpm, for 45 seconds. Discard the filtrate.

[0059] ⑨ Move the spin column to the elution tube, then add 50-200 μL of nuclease-free water to the spin column, let it stand for 2 minutes, then place the spin column and elution tube in a centrifuge and centrifuge at 4°C, 12,000 rpm, and centrifuge for 1 minute;

[0060] ⑩ Measure the RNA concentration and store at -80°C until use.

[0061] (2) Reverse transcription

[0062] Reverse transcription was performed using a reverse transcription kit (Vazyme, Nanjing). The amount of RNA used (0.1 ng / μg) was calculated based on the OD value of the extracted RNA. The reaction system is shown in Table 1 below. The reaction procedure was as follows: incubation at 50°C for 15 min; followed by heating at 85°C for 5 seconds. All operations must be completed on ice. After the reaction, the cDNA was stored in a refrigerator at -20°C.

[0063] Table 1 Reverse transcription reaction system

[0064]

[0065] 3. Quantitative PCR primer design

[0066] The gene sequence was searched from the Phytozome database. The GmAP2 genome CDS sequence was downloaded from the website as shown in SEQ ID NO.1, and the GmAP2 protein sequence was downloaded as shown in SEQ ID NO.2. Primers were designed and synthesized based on the genome sequence on the NCBI website for specific quantitative amplification:

[0067] GmAP2_qF(SEQ ID NO.3):5'-TACTCCCACATCGCTACCCA-3';

[0068] GmAP2_qR (SEQ ID NO. 4): 5'-GCTGCCGAATCCCTCTGTAG-3'.

[0069] 4. Real-time fluorescence quantitative PCR

[0070] ①Actin3 was used as the internal reference gene;

[0071] ② Dilute the cDNA of all samples 1-fold with ddH2O and use it as a template for quantitative PCR reaction;

[0072] ③ Fluorescence quantitative PCR was performed using the Applied Biosystems StepOnePlus real-time PCR system according to the reaction system in Table 2 below. The reaction program was as follows: 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: 95°C for 10 s; 54.3°C for 5 s; 94.3°C for 5 s;

[0073] Table 2 Real-time fluorescence quantitative PCR reaction system

[0074] Reagents volume 2×ChamQ Blue Universal SYBR qPCR Master Mix 10 μL Primer F (10 μM) 0.4μL Primer R (10 μM) 0.4μL cDNA to 20 μL

[0075] ④ Data were processed and relative expression analysis was performed using the CT method (2^-△△CT )

[0076] 5. Results Analysis

[0077] The expression results of GmAP2 gene under normal phosphorus and low phosphorus levels are as follows Figure 2 As shown, the expression of GmAP2 gene was induced in soybean roots under low phosphorus stress.

[0078] Example 2: Cloning of GmAP2 gene and construction of vector

[0079] 1. Construction of pLB-GmAP2 zero-background expression vector

[0080] ① Amplify the target fragment: extract RNA from the root sample of soybean Huachun No. 6, reverse transcribe it into cDNA, and then use the cDNA of soybean Huachun No. 6 as a template to design and synthesize primers:

[0081] GmAP2_pLB_F(SEQ ID NO.5):5'-ACTCTACTCATTCCACACCCA-3';

[0082] GmAP2_pLB_R(SEQ ID NO.6):5'-CTGTGCATCCCTGCCAAAAC-3',

[0083] The full-length CDS sequence of GmAP2 was amplified using the reaction system in Table 3 below. The reaction procedure was as follows: initial denaturation at 95°C for 3 min; 34 cycles (denaturation at 95°C for 15 s; annealing at 55°C for 15 s; extension at 72°C for 1 min / kb); final extension at 72°C for 5 min; and storage at 12°C.

[0084] Table 3 Fragment amplification reaction system

[0085]

[0086] ②Purification of PCR products:

[0087] S1. Run the PCR product on agarose gel electrophoresis and purify the PCR product with the correct band using a purification kit. Then, centrifuge the PCR product and measure the volume. If the volume is less than 100 μL, add ddH2O to 100 μL. Add 5 volumes of Buffer GDP to the PCR product and mix thoroughly using a vortex mixer to obtain a mixture. Place an adsorption column in a collection tube and transfer the mixture to the adsorption column. Then, place the adsorption column and collection tube in a centrifuge and centrifuge at 12,000 rpm for 1 minute. Discard the filtrate.

[0088] S2. Prepare Buffer GW stock solution by adding 80 mL of anhydrous ethanol to every 100 mL of Buffer GW stock solution and mix well to obtain Buffer GW working solution. Add 700 μL of Buffer GW working solution to the adsorption column. Then, place the adsorption column and collection tube in a centrifuge and centrifuge at 12,000 rpm for 1 min. Discard the filtrate.

[0089] S3, repeat S2; then place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 30 μL of Solution Buffer to the center of the adsorption column, let it stand for 2 minutes, place the adsorption column and collection tube in a centrifuge and centrifuge at 12,000 rpm for 1 minute; discard the adsorption column and store the purified PCR product at -20°C;

[0090] ③ Connect the target gene to the pLB zero background expression vector:

[0091] The target fragments were ligated according to the reaction system in Table 4 below to obtain pLB-GmAP2. The reaction procedure was as follows: denaturation at 20°C for 2 min; annealing at 70°C for 5 min; and extension at 22°C for 20 min.

[0092] Table 4 Ligation reaction system

[0093]

[0094] ④ E. coli transformation: Pipette 10 μL of pLB-GmAP2 into 100 μL of DH5α competent E. coli, gently pipette to mix, place on ice for 30 minutes, then heat shock in a 42°C water bath for 45 seconds, and immediately transfer to ice to cool for 2 minutes. Then, transfer the ligation product to a clean bench, add 500-700 μL of LB liquid medium to the pLB-GmAP2 ligation product, and incubate on a shaker at 37°C for 1 hour at 220 rpm to obtain a bacterial suspension. The bacterial suspension was spread on a plate containing ampicillin (Amp) antibiotics and incubated overnight at 37°C. Single clones were picked for detection and sequencing.

[0095] Figure 3 This is a diagram of the cloning of the GmAP2 gene and the identification of the GmAP2 gene connected to the pLB vector bacterial solution.

[0096] 2. Construction of pTF101-GmAP2 overexpression vector

[0097] ① Amplify the target fragment: Using the cDNA of soybean Huachun No. 6 as a template, design and synthesize primers:

[0098] GmAP2_pTF101_F (SEQ ID NO.7):

[0099] 5'-gagaacacgggggactctagaATGTGTGGCGGTGCCATC-3';

[0100] GmAP2_pTF101_R (SEQ ID NO.8):

[0101] 5'-cgatcggggaaattcgagctcCTAATCGAAACTCCAGAGACCCC-3',

[0102] The full-length CDS sequence of GmAP2 was amplified according to the reaction system in Table 3. The reaction procedure was as follows: initial denaturation at 95°C for 3 min; 34 cycles (denaturation at 95°C for 15 s; annealing at 55°C for 15 s; extension at 72°C for 1 min / kb); final extension at 72°C for 5 min; storage at 12°C;

[0103] ② Linearized vector: Double digest the pTF101 plasmid DNA with restriction endonucleases SacI and XbaI;

[0104] ③ Ligation: Ligate the target fragment with pTF101 according to the reaction system in Table 5 below. Reaction procedure: Incubate at 37°C for 30 min to obtain pTF101-GmAP2;

[0105] Table 5 Ligation reaction system

[0106]

[0107] ④ Agrobacterium transformation: After sequencing and comparing single clones of pTF101-GmAP2, the plasmid was extracted using a plasmid extraction kit (Vazyme, Nanjing). 4 μL of plasmid was added to 100 μL of GV3101 competent Agrobacterium, incubated on ice for 5 minutes, then frozen in liquid nitrogen for 5 minutes, heat-shocked at 37°C for 5 minutes, and then cooled on ice for 5 minutes. The plasmid was transferred to a clean bench, and 500–700 μL of YEP liquid medium was added. The culture was then shaken at 220 rpm at 28°C for 2–3 hours to obtain a bacterial suspension. The suspension was then plated on plates containing rifampicin (Rif) and spectinomycin (Spe) at a working concentration of 30–50 μg / mL and incubated at 28°C for 2–3 days. Single colonies were selected and shaken for testing. An equal volume of 50% glycerol was added to the successful suspension and stored at −80°C.

[0108] 3. Construction of super1300-GmAP2 subcellular localization vector

[0109] ① Amplify the target fragment: Using the pLB-GmAP2 plasmid as a template, use CE Design to design recombination cloning primers:

[0110] super1300-GmAP2-F(SEQ ID NO.9):

[0111] 5'-acgggggactcttgaccatggCTATGTGTGGCGGTGCCATC-3';

[0112] super1300-GmAP2-R(SEQ ID NO.10):

[0113] 5'-aagttcttctcctttactagtATCGAAACTCCAGAGATCCCCA-3',

[0114] The full-length CDS sequence of the GmAP2 gene was amplified according to the reaction system in Table 3 to obtain an amplified product. The amplification system and PCR amplification method were the same as the amplification procedure in Table 3. The amplified product was subjected to gel electrophoresis, and then the target band was recovered and purified using an agarose gel kit.

[0115] ② Linearized vector: Double-digest the super1300 empty plasmid with XbaI and SpeI restriction endonucleases.

[0116] ③ Ligate the target fragment, GFP fragment, and Super1300, and transform the ligation product into E. coli and Agrobacterium tumefaciens. The methods and conditions for ligation, E. coli transformation, and Agrobacterium transformation are the same as those for the construction of the pTF101-GmAP2 overexpression vector and will not be repeated here. The super1300-GmAP2-GFP vector was obtained for subsequent experiments.

[0117] 4. Construction of pGBKT7-GmAP2 recombinant protein vector

[0118] ① Amplify the target fragment: Using the pLB-GmAP2 plasmid as a template, use CE Design to design recombination cloning primers:

[0119] pGBKT7-GmAP2-F (SEQ ID NO.11):

[0120] 5'atggccatggaggccgaattcATGTGTGGCGGTGCCATC-3';

[0121] pGBKT7-GmAP2-R (SEQ ID NO.12):

[0122] 5'ccgctgcaggtcgacggatccCATCGAAAACTCCAGAGATCCCC-3',

[0123] The full-length CDS sequence of GmAP2 was amplified according to the reaction system in Table 3 to obtain an amplified product. The amplification system and conditions were the same as those in Table 3. The amplified product was subjected to gel electrophoresis and the target band was recovered and purified using an agarose gel kit (Vazyme, Nanjing).

[0124] ② Linearized vector: Double-digest the pGBKT7 plasmid with restriction endonucleases BamHI and EcoRI.

[0125] ③ Ligate the target fragment with the pGBKT7 plasmid and transform the ligation product into E. coli. The ligation and E. coli transformation methods and conditions are identical to those used in the construction of the pTF101-GmAP2 overexpression vector and are not repeated here. After sequencing and alignment, extract the plasmid and store it at -20°C until use. This pGBKT7-GmAP2 vector was used for subsequent experiments.

[0126] 5. Construction of GmAP2 gene editing vector

[0127] Design gRNAs targeting GmAP2 for gene editing on the CRISPR-GE website (http: / / skl.scau.edu.cn / targetdesign / ). Select four editing targets within the exonic region of GmAP2, designated GmAP2-guide13, GmAP2-guide15, GmAP2-guide20, and GmAP2-guide21. Design and synthesize four corresponding primer pairs using the CRISPR / Cas9 vector pUC19 cloning method for legumes.

[0128] pCas9-13-F: GGATTGGAGCGTTCCCATCCCGCG

[0129] pCas9-13-R: AAACCGCGGGATGGGAACGCCTCCA

[0130] pCas9-15-F: GGATTGTAACCGTTCCCGTTCTGAA

[0131] pCas9-15-R: AAACTTCAGAACGGGAACGGTTACA

[0132] pCas9-20-F:GGATTGAAAGGTCCCAACCTCCCAA

[0133] pCas9-20-R: AAACTTGGGAGGTTGGGACCTTTCA

[0134] pCas9-21-F:GGATTGGAAAATTCGGGGTCAGCGT

[0135] pCas9-21-R:AAACACGCTGAACCCGAATTTTCCA

[0136] After annealing the oligonucleotide into a double-stranded structure, it was ligated with the BsaI-digested vector pUC19 plasmid and transformed into DH5α Escherichia coli. After successful bacterial liquid detection, the plasmid was extracted and transformed into Agrobacterium K599 competent cells. An equal volume of 50% glycerol was added to the successfully detected bacterial liquid, and the cells were stored at -80°C to obtain the GmAP2 gene editing vector.

[0137] Example 3: Arabidopsis genetic transformation

[0138] 1. Cultivation of wild-type Arabidopsis

[0139] Wild-type Arabidopsis seeds (Col-0) were soaked in pure water and vernalized at 4°C for three days. After vernalization, the seeds were evenly sown on nutrient soil and sprayed with water. Covered with plastic wrap to retain moisture and placed in the dark for two to three days to break dormancy. Thinning was performed after the Arabidopsis had grown four to six leaves, leaving four to five plants per pot. At the first bolting, the main stem was cut back to increase branching. Transformation was performed using Agrobacterium solution when the plants were next in full bloom.

[0140] 2. Transformation of Arabidopsis inflorescence with Agrobacterium solution

[0141] Mix the Agrobacterium GV3101 stock solution carrying the target vector with 100 mL of YEP (gen+spe+rif) liquid to obtain a bacterial solution. Place the bacterial solution in a 28°C incubator at 200 rpm and incubate overnight until the OD600 value of the bacterial solution reaches 0.6-0.8. The bacterial solution is then centrifuged at 5000 rpm for 10 minutes, and the supernatant is discarded. The Agrobacterium is then resuspended in one volume of fresh infiltration solution (5% sucrose + 1 / 2 MS + Silwet L-77 0.02%) and transferred to a 500 mL beaker. The aerial part of the flowering Arabidopsis plant is immersed in the Agrobacterium solution for 1-2 minutes. The immersed plant is then removed and the transformed Arabidopsis plant is wrapped with plastic film to ensure high humidity. The plant is kept in the dark for 12-16 hours. The plastic film is removed the next day and the plant is placed in an incubator for cultivation and growth. When mature, the T0 generation seeds are collected.

[0142] 3. Identification of transgenic Arabidopsis offspring

[0143] After drying the mature T0 generation seeds, vernalization was carried out and multiplication was carried out. After the two true leaves of Arabidopsis thaliana were fully grown, glyphosate herbicide was sprayed regularly and several times. The results were as follows: Figure 8 As shown in part b of the figure, surviving transformed seedlings were screened and false positives were removed. After the plants grew robust, fresh leaves were selected and DNA was extracted using TPS extraction solution: young leaves were placed in a 1.5 mL sterile centrifuge tube, 200 μL of TPS extraction solution was added, and the leaves were ground using a grinder. The leaves were then placed in a metal bath at 80°C for 10 minutes. The tubes were then centrifuged at 12,000 rpm for 10 minutes. 20 μL of the supernatant was collected and 50 μL of double-distilled water (ddH2O) was added to the supernatant. Mix thoroughly by pipetting, and 2 μL was used for the PCR reaction.

[0144] DNA molecular level identification was performed according to the reaction system in Table 6 below. The reaction procedure was: pre-denaturation at 95°C for 3 min; 28 cycles (denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min / kb); and final extension at 72°C for 5 min.

[0145] The universal primers used in the PCR reaction were pTF101-F (SEQ ID NO. 13): 5'-CCTTCGCAAGACCCTTCCTC-3'; pTF101-R (SEQ ID NO. 14): 5'-TCATCGCAAGACCGGCAAC-3'.

[0146] After the PCR reaction was completed, 1% agarose gel electrophoresis was used for detection. Figure 8 As shown in part a of the figure, where M represents a 2000 DNA marker, and plants 1-12 are all positive. If the band lengths are correct, it can be preliminarily determined to be transgenic material. T2 generation Arabidopsis seeds are harvested upon maturity. This method is then used to identify the T4 generation. RNA is extracted from different Arabidopsis strains and identified by fluorescence quantitative analysis. Strain with high expression levels is selected for large-scale seed propagation for subsequent experiments.

[0147] Table 6 DNA molecular level identification reaction system

[0148]

[0149] 4. Transient Transformation of Nicotiana benthamiana

[0150] Agrobacterium (P19) carrying super1300-GFP and super1300-GmAP2-GFP were inoculated into 50 mL of YEP, and antibiotics (Kan) were added to obtain bacterial liquid. The bacterial liquid was then cultured in a shaker at 28°C for 16 hours until the OD600 reached 0.6-0.8.

[0151] The cultured bacterial solution was placed in a centrifuge for centrifugation at a speed of 4000 rpm for 10 min. The supernatant was then discarded, and the bacteria were resuspended in 15 mL of infiltration solution and cultured at room temperature in the dark for 3 h.

[0152] Then, use a small syringe with the needle removed to carefully inject the resuspended bacterial solution into the back of the tobacco leaves and culture for about 2 days. Then, observe the GFP fluorescence signal of the tobacco epidermal cells under a laser confocal microscope at a wavelength of 488nm, then use DAPI staining for 3-5 minutes, and then observe the blue fluorescence signal at an excitation wavelength of 360nm and an emission wavelength of 460nm.

[0153] Experimental results:

[0154] like Figure 4 As shown, eGFP corresponds to the GFP channel; DAPI corresponds to the UV channel. DAPI dye binds to DNA and emits blue fluorescence; Merge indicates the fusion image of GFP and DAPI; Bright field indicates bright field. Super1300-GFP (corresponding to GFP in the figure) and Super1300-GmAP2-GFP (corresponding to GmAP2-GFP in the figure) were transiently expressed in tobacco epidermal cells. Tobacco epidermal cells transformed with the super1300-GFP empty vector exhibited green fluorescence in the cell membrane, nucleus, and cytoplasm, while the Super1300-GmAP2-GFP expression vector in tobacco leaves mostly emitted green fluorescence only in the nucleus, which overlapped with the blue fluorescence of DAPI, indicating that the GmAP2 protein is localized in the nucleus.

[0155] Example 4: Verification of GmAP2 yeast self-activation and interaction

[0156] 1. Verification of GmAP2 yeast self-activation

[0157] Step 1: Construction of GmAP2-BD fusion expression vector

[0158] S1, gene amplification and purification: similar to the steps in Example 2, amplify the coding sequence of the GmAP2 gene by PCR to obtain an amplified product, and purify the amplified product;

[0159] S2, vector linearization: pGBKT7 vector (BD) was double-digested with restriction endonucleases EcoRI and SacI to linearize it, and the linearized vector was purified by agarose gel electrophoresis;

[0160] S3, recombination ligation: perform ligation according to the ligation reaction system in Table 5, and recombinantly ligate the purified amplified product with the linearized pGBKT7 vector to form the fusion expression vector GmAP2-BD;

[0161] S4, transformation and identification: The fusion expression vector GmAP2-BD was transformed into competent E. coli cells using the same transformation method as above. The cells were plated on LB plates containing kanamycin and cultured overnight at 37°C. Single colonies were picked for PCR identification. Once the amplified band size was correct, the cells were sent to a biological sequencing company for sequencing verification to confirm that the fusion vector GmAP2-BD had been successfully constructed.

[0162] Step 2: Yeast two-hybrid assay

[0163] S1, co-transformation: The constructed GmAP2-BD recombinant plasmid and the pGADT7 (AD) empty plasmid were co-transformed into the competent cells of the yeast strain Y2HGold.

[0164] S2, plate screening: The transformed yeast cells were spread on plates containing SD / -Trp-Leu / X-α-Gal to obtain experimental groups.

[0165] Step 3: Control settings:

[0166] Positive control: BD-53 and AD-T were co-transfected into Y2HGold competent cells.

[0167] Negative control: BD-lam and AD-T were co-transfected into Y2HGold competent cells.

[0168] The self-activation verification result is as follows Figure 5 As shown in the figure, parts a and b show the electrophoresis images of the clone constructed from the GmAP2-BD yeast vector and the bacterial culture for identification, respectively. The experiment shows that the GmAP2-BD protein appears blue. To further verify the self-activation activity of GmAP2-BD, yeast cultures carrying the GmAP2-BD and AD empty plasmids were streaked onto SD / -Leu-Trp-His-Ade / X-α-Gal medium. The results, shown in parts c and d, show that GmAP2-BD and the positive control cells turned blue after normal growth in the culture medium, while the negative control did not appear blue.

[0169] 2. Determination of the autoactivation region of GmAP2 protein

[0170] The full-length CDS of the GmAP2 gene was cut into two segments, S1 and S2, as Figure 6 The GmAP2 gene truncation image in part a is shown. S1 and S2 were ligated to the BD vector and then co-transformed with the AD empty vector into the Y2HGold competent medium and spotted on a plate containing SD / -Trp-Leu / X-α-Gal.

[0171] Figure 6Parts b and c in the figure show the electrophoresis patterns of clones and bacterial cultures containing truncated GmAP2 genes, respectively. In part b, the M (Marker) band is a marker, and bands 1, 2, and 3 are recombinant fragments containing the full-length and partial CDS sequences of the GmAP2 gene, respectively. The electrophoresis bands are 943 bp, 709 bp, and 274 bp long. In part c, the M (Marker) band is a marker, and bands 1 and 2 represent identification of the S1 bacterial culture, 3 and 4 represent identification of the S2 bacterial culture, and 5 and 6 represent identification of the GmAP2-BD bacterial culture.

[0172] The cells carrying S1-BD and AD empty vector plasmids were spotted on SD / -Trp-Leu / X-α-Gal and SD / -Leu-Trp-His-Ade / X-α-Gal culture media respectively. Figure 6 Parts d and e show that on the plate containing SD / -Trp-Leu / X-α-Gal, S1 does not show blue, indicating that the S1 protein has no self-activation. However, the S2 protein shows blue. The strain S1-BD and the negative control do not grow and do not show blue, indicating that S1-BD has no self-activation. S1-BD was used for yeast two-hybrid experiment.

[0173] 3. Verification of the interaction between GmAP2 and candidate proteins

[0174] The SMART website was used to predict 10 genes with high interaction scores. The basic information of these 10 genes was searched in the NCBI and Phytozome databases, as shown below:

[0175] Casein kinase 1-like protein 1: Glyma.13G105900, Glyma.15G171100, Glyma.07G007600, Glyma.09G065200, Glyma.17G053500; Casein kinase 1-like protein 2: Glyma.13G348100, Glyma.08G190300; Casein kinase 1: Glyma.15G025900; scof-1 protein: Glyma.17G236200; transcription factor: Glyma.10G205000.

[0176] Based on their functional annotation, two proteins were further selected for interaction verification. S1-BD was used as bait protein, and Glyma.15G171100 and Glyma.17G053500 proteins were co-transformed into yeast strains as prey proteins, spread on SD / -Leu-Trp plates containing X-α-Gal, and cultured upside down at 30°C. Figure 7As shown, in part a, M1 and M2 are 2000 DNA markers, and 1, 2, 3, 4, 5, 6, 7, 8, and 9 are clones of Glyma.17G236200, Glyma.13G105900, Glyma.15G171100, Glyma.07G007600, Glyma.08G190300, Glyma.09G065200, Glyma.17G05350, Glyma.15G025900, and Glyma.10G205000, respectively; in part b, M is 200 0DNAMarker, 1 and 2 are Glyma.17G236200, 3 and 4 are Glyma.13G105900, 5 and 6 are Glyma.15G171100, 7 and 8 are Glyma.08G190300, 9 and 10 are Glyma.09G065200, 11 and 12 are Glyma.17G053500, 13 and 14 are Glyma.15G025900, 15 and 16 are Glyma.10G205000 for bacterial liquid identification. Figure 7 The experimental results in part c are all blue. Two interacting genes were selected from the SD / -Leu-Trp / X-α-Gal plate, diluted with NaCl to OD600 = 0.6, and spotted on SD / -Leu-Trp-His-Ade / X-α-Gal plates. GmAP2 interacts with both proteins.

[0177] Example 5: Arabidopsis genetic transformation and identification of transgenic Arabidopsis root phenotypes

[0178] ① Preparation of genetic transformation materials

[0179] The Arabidopsis floral dipping method was used for genetic transformation. Wild-type Arabidopsis seeds were vernalized at 4°C for 3 days and sown in small planting pots containing moist nutrient soil and an appropriate amount of vermiculite. The pots were covered with plastic wrap, leaving a certain height. The plastic wrap was removed after 3 days, leaving 4-5 seedlings in each pot. When there were many wild-type Arabidopsis flower buds, infection began, and the blooming flowers were cut off before infection.

[0180] ② Agrobacterium infection

[0181] The GV3101 bacterial solution containing the pTF101-GmAP2 vector was cultured to OD = 0.6, then placed in a centrifuge and centrifuged at 5000rpm for 10 minutes, and the supernatant was poured off. 50mL of fresh resuspension (5% sucrose + 1 / 2MS + Silwet L-770.02%) was added to the bacterial solution. It should be noted that the resuspension needs to be stirred on a magnetic stirrer in advance until foam is produced. After adding the fresh resuspension, use the tip of the pipette to blow and mix well to thoroughly resuspend the bacteria. The wild-type Arabidopsis buds were then completely immersed in the resuspension and infected for 2 minutes. The infected Arabidopsis was covered with a fresh-keeping bag for preservation, and then treated in the dark for 24 hours.

[0182] ③Selective training

[0183] S1: Vernalize all harvested seeds at 4°C for 3 days. Place seeds in trays filled with moistened soil and sow them on the soil surface. Place seeds in an incubator (16h light / 8h dark; 24°C) for incubation. After the two cotyledons of Arabidopsis thaliana are fully expanded, prepare a 20μg / mL glyphosate solution and spray every two days. Keep the soil moist during this period. Approximately 7 days after spraying, non-positive seedlings will fail to grow normally. Positive seedlings that remain unaffected are considered positive and are transplanted individually into small pots. Each individual plant is harvested as a single strain, and positive seedlings are screened and identified until homozygous T4 generations are formed.

[0184] ④Verification of transgenic plants

[0185] The leaves of transgenic plants were selected and DNA was extracted. The wild type WT was used as a negative control and the pTF101-GmAP2 plasmid was used as a positive control. DNA was used to verify whether the vector carrying the target fragment was transferred into the Arabidopsis genome.

[0186] After the T4 generation was cultivated, RNA was extracted and reverse transcribed from the aboveground part and root system, and identified by fluorescence quantitative analysis. The results are as follows: Figure 8 As shown in part c of the figure, three independent strains (OE-3, OE-4, and OE-6) with overexpression of the GmAP2 gene were obtained. The strains with high expression levels were selected for large-scale propagation and used in subsequent experiments.

[0187] ⑤ Pre-culture: Seeds of transgenic Arabidopsis thaliana strains OE-3, OE-4, and OE-6 were sterilized by soaking in a 10% sodium hypochlorite solution with shaking for 10 minutes, then rinsed five times with sterile water. After washing, the seeds were placed in a 4°C refrigerator for vernalization. The spring flowering period was three days. The seeds were then planted on 1 / 2 MS medium and transferred to a vertical culture room. Once the roots reached approximately 1 cm in length, phosphate treatment was performed.

[0188] ⑥ Phosphorus treatment: A modified 1 / 2 Hoagland solution solid medium was prepared with a concentration gradient of 500 μmol / L (NP), 5 μmol / L, and 0 μmol / L KH2PO4, with the pH adjusted to 5.8. Arabidopsis plants of uniform growth were transferred to this medium, maintaining root elongation. After vertical cultivation for approximately 2 weeks, photographs were taken and the taproot length, total lateral root length, fresh weight, and whole-plant phosphorus content of the transgenic lines OE-3, OE-4, and OE-6 were measured (the experimental procedures were consistent with the method for measuring total phosphorus content in hairy roots in Experiment 6, with whole-plant measurement used for total phosphorus content).

[0189] The results of transgenic Arabidopsis root phenotype identification are as follows Figure 9 As shown in Figure 2, it was shown that heterologous overexpression of GmAP2 in Arabidopsis could alleviate the inhibitory effect of low phosphorus stress on the growth and development of Arabidopsis. The statistical results of biomass indicators are shown in Figure 2. Figure 9 As shown in the bar graph, compared with the wild type, heterologous overexpression of GmAP2 in Arabidopsis thaliana enhances its low-phosphorus tolerance by increasing the main root length, lateral root length and fresh weight of the plant, and the phosphorus content of the transgenic line is also significantly increased.

[0190] Example 6: Identification of soybean hairy root tolerance to low phosphorus

[0191] ① Transformation of K599 Agrobacterium rhizogenes: Transform the gene-edited GmAP2 gene (pCas9-GmAP2), the overexpressed GmAP2 gene (pTF101-GmAP2(OE)), and the empty vector control gene (pTF101(CK)) into K599 Agrobacterium rhizogenes. After identifying positive single cells, expand and shake the cells.

[0192] ② Infection: Shake untransformed K599 Agrobacterium and K599 Agrobacterium carrying gene editing and overexpression vectors to an OD600 of 0.6, centrifuge, and resuspend in a resuspension buffer. Select plump, disease-free Brazilian No. 13 soybeans and plant them on water-permeable vermiculite, then cover them with dry vermiculite. After 2-3 days, when the above-ground part of the soybeans reaches 2-3 cm, use a 1 mL syringe to inject the infection 1-2 cm below the soybean cotyledons. Incubate at high humidity for approximately one week. Once young roots have grown from the injection wound, cover with vermiculite, watering frequently to ensure the vermiculite remains moist, and continue incubation for approximately one week.

[0193] ③ Identification of hairy root phenotypes: When hairy roots develop to approximately 5 cm, remove them without damaging them. The primary roots are cut off and hydroponically cultured in a solution of 500 μmol / L KH2PO4 and 5 μmol / L pH 5.8 for 14 days. Lateral roots attached to the taproot are selected, and DNA is extracted from positive roots. RNA is extracted from roots of each treatment and analyzed by fluorescence quantitative analysis. The fresh weight of the aboveground and underground parts of the soybean plants is measured. After the samples are completely dried, the dry weight is weighed. Dry samples are then taken to determine the total phosphorus content in the aboveground and underground parts of the plants. At least three biological replicates and three technical replicates are used for each data set.

[0194] ④ Colorimetric determination method of total phosphorus, molybdenum and antimony in plants:

[0195] 1. All reagents used in this experiment are of analytical grade, and the water used is deionized water, distilled water or water of equivalent purity.

[0196] (1) Sulfuric acid;

[0197] (2) 30% hydrogen peroxide;

[0198] (3) 10% sodium hydroxide;

[0199] (4) 0.2% dinitrophenol indicator;

[0200] (5) 0.5% potassium antimony tartrate solution: weigh 0.5 g of chemically pure potassium antimony tartrate and dissolve it in 100 mL of water;

[0201] (6) Molybdenum antimony sulfate stock solution: Measure 126 mL of concentrated sulfuric acid and slowly add it to 100 mL of water, stirring constantly and cooling. Weigh 10 g of finely ground ammonium molybdate and dissolve it in 300 mL of water at a temperature of about 60°C and cool. Then slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimony sulfate 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.

[0202] (7) Molybdenum antimony anti-coloring agent: Weigh 1.5g ascorbic acid and dissolve it in 100mL molybdenum antimony stock solution. This solution has a short shelf life and should be prepared freshly when needed.

[0203] (8) Phosphorus standard stock solution: Accurately weigh 0.4390 g of potassium dihydrogen phosphate that has been dried at 105°C for 2 h, dissolve it in water, add 5 mL of concentrated sulfuric acid, and then add water to make up to 1000 mL. The solution contains 100 mg / L of phosphorus and can be stored in a refrigerator for long-term use.

[0204] (9) 5 mg / L phosphorus standard solution: Pipette 5 mL of phosphorus stock solution into a 100 mL volumetric flask and add water to make up to volume. This solution should be prepared when needed.

[0205] 2. 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.

[0206] 3. Experimental steps

[0207] (1) Sample solution preparation

[0208] Weigh an appropriate amount of sample to the nearest 0.001g and place it in a 500mL 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 6mL of sulfuric acid, gently shake, and let it sit overnight. Place a small curved-neck funnel at the end of the tube. Initially, digest at 250°C in a digestion furnace (start timing after the temperature stabilizes, approximately 30 minutes). Wait until the H2SO4 decomposes, emitting a large amount of white smoke, then raise the temperature to 400°C. Remove the solution when it becomes a uniform brown-black color.

[0209] After it cools slightly, add 10 drops of H2O2, shake well, and heat to a slight boil. Cook for about 5 minutes. Remove and cool slightly, then add 5-10 drops of H2O2 and cook again. Repeat this process 3-5 times, gradually reducing the amount of H2O2 added each time. Cook until the solution is colorless or clear, with the lower part being off-white. Then heat for about 5-10 minutes to remove any remaining H2O2.

[0210] Remove the digestion tube and cool it. Rinse the bent-neck funnel with a small amount of water, allowing the rinse to flow into the digestion tube. Pour the digestion solution intact into a 100mL volumetric flask, bring to volume with water, and shake well. Filter or allow the solution to settle before phosphorus determination.

[0211] (2) Preparation of blank solution

[0212] The reagents and operating steps used are the same as above, except that no sample is added.

[0213] (3) Draw a standard curve

[0214] Pipette 0, 2, 4, 6, 8, 10, 14, and 20 mL of a 5 mg / L phosphorus standard solution into a 50 mL volumetric flask. Simultaneously, add a blank solution equal in volume to the sample solution used for the colorimetric assay and 2-3 drops of dinitrophenol indicator. Adjust the solution to a slightly yellowish color with 10% sodium carbonate solution or 5% sulfuric acid solution. Add 5 mL of molybdenum antimony anti-colorimetric reagent, shake well, and bring to volume with water. This yields a series of standard solutions containing phosphorus contents of 0.0, 0.2, 0.4, 0.8, 1.0, 1.4, and 2.0 mg / L, respectively. Shake well, incubate at a temperature above 15°C for 30 minutes, and then measure the absorbance at 880 nm. Plot a calibration curve on grid paper with absorbance as the ordinate and phosphorus concentration (mg / L) as the abscissa.

[0215] (4) Determination of phosphorus in sample solution

[0216] 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 yellowish color with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony colorimetric reagent, shake well, and bring to volume with water. Allow to stand at room temperature above 15°C for 30 minutes.

[0217] The colorimetric determination of the sample solution is performed on a spectrophotometer using a 1 cm light path cuvette at 880 nm. The instrument zero point is adjusted using the blank test solution as the reference solution. The absorbance is then read. The corresponding phosphorus content is then determined from the standard curve.

[0218] 4. Result calculation

[0219]

[0220] Where:

[0221] C——the mass concentration of phosphorus in the colorimetric solution obtained from the calibration curve or regression equation, μg / L;

[0222] C0——Blank value

[0223] V——volume of developing solution;

[0224] D——dispensing multiple, digestion solution fixed volume / absorption digestion solution volume;

[0225] m——weighed sample mass g;

[0226] 1000 – Conversion factor for converting mL to L. The result should be rounded to three decimal places.

[0227] The quantitative results and phenotypic identification results of GmAP2 hairy roots are shown in Figure 2. Figure 10 and Figure 11 As shown, overexpression of pTF101-GmAP2 can enhance soybean tolerance to low-P stress, and gene editing of pCas9-GmAP2 can enhance soybean sensitivity to low-P stress.

[0228] The data in this example are the mean and standard error of three replicates, and "*", "**", "***" and "****" respectively indicate that the difference between the treatment and the control is significant (*P≤0.05), extremely significant (**P≤0.001), extremely significant (***P≤0.001) and extremely extremely significant (****P≤0.0001); the same below.) The above examples are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention fall within the scope of protection of the present invention.

Claims

1. Application of the soybean low-phosphorus tolerance gene GmAP2 in improving the plant's tolerance to low-phosphorus stress, characterized by: The nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP2 is shown as SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes an amino acid sequence shown as SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The plant is Arabidopsis thaliana or soybean.

3. A method for improving plant tolerance to low phosphorus stress, characterized in that: The method is to increase the expression level of the amino acid sequence shown in SEQ ID NO. 2 in the recipient plant; The specific steps include: introducing the soybean low-phosphorus tolerance gene GmAP2 shown in SEQ ID NO.1 into a 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.

4. The method according to claim 3, characterized in that The improvement of the plant's ability to tolerate low phosphorus stress is reflected in the increase of its root length.

5. The method according to claim 3, characterized in that The improvement of the low-phosphorus stress tolerance of the plant is reflected in the increase in the number of its lateral roots.

6. The method according to claim 3, characterized in that The improvement of the plant's ability to tolerate low phosphorus stress is reflected in the increase of its fresh weight.

7. Application of the soybean low-phosphorus tolerance gene GmAP2 in cultivating plants tolerant to low-phosphorus stress, characterized by: The nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP 2 is shown as SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes an amino acid sequence shown as SEQ ID NO.

2.

8. Use of a recombinant plasmid, recombinant vector or transgenic plant cell line of the soybean low-phosphorus tolerance gene GmAP2 in improving plant tolerance to low-phosphorus stress, wherein the nucleotide sequence of the soybean low-phosphorus tolerance gene GmAP2 is shown in SEQ ID NO.1; or the soybean low-phosphorus tolerance gene GmAP2 encodes the amino acid sequence shown in SEQ ID NO.2.

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