Application of soybean low phosphorus tolerance gene GmAP2 in improving plant tolerance to low phosphorus stress

Abstract

The application relates to the technical field of plant genetic engineering, and discloses application of a soybean low-phosphorus-tolerant gene GmAP2 in improving the low-phosphorus-stress tolerance of plants. The application finds that, under low-phosphorus stress, the expression level of the GmAP2 gene in plants is significantly up-regulated. By heterologously overexpressing the GmAP2 gene in Arabidopsis, the stress suffered by plants under low-phosphorus conditions can be effectively relieved, and the fresh weight and lateral root length of the Arabidopsis can be significantly increased, so that the growth of the Arabidopsis plant is promoted. In addition, under low-phosphorus conditions, overexpression of the GmAP2 gene can improve the phosphorus deficiency symptoms of soybean, improve the absorption of phosphorus nutrients by plants, and promote the growth of the roots and leaves of the plants. Therefore, the GmAP2 gene plays a very important role in the response of plants to a phosphorus-starvation stress environment.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of the soybean low phosphorus tolerance gene GmAP2 in improving the plant's tolerance to low phosphorus stress. Background Technology

[0002] Soybeans are an important dual-purpose crop for both food and oil production, holding a vital position in agricultural production. Phosphorus is one of the three essential elements for plant growth, development, and metabolism, participating in various biochemical processes within plants. Plants can only obtain phosphorus in the form of inorganic phosphate (Pi), but the soils in southern China are mostly acidic red soils with low pH values, resulting in low iron (Fe) content. 3+ ) and aluminum (Al) 3+ The concentration of ions is high, and these ions will react with phosphate ions (PO4). 3- Phosphorus combines with phosphorus to form insoluble precipitates of iron phosphate (FePO4) and aluminum phosphate (AlPO4), making it difficult for plants to absorb and utilize most of the phosphorus in the soil, resulting in low soybean yield. Soil phosphorus deficiency has become a major factor limiting soybean yield and quality. Therefore, elucidating the molecular mechanisms related to low phosphorus tolerance in soybeans, identifying and characterizing key phosphorus-efficient genes, improving the efficiency of phosphorus absorption and utilization in soybeans, and breeding new phosphorus-efficient soybean varieties are of great significance for increasing soybean yield in South China. Summary of the Invention

[0003] To address the problems mentioned above in the background art, one of the objectives of this invention is to provide an application of the soybean low-phosphorus tolerance gene GmAP2 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.

[0004] This application improves the ability of soybeans to tolerate low phosphorus stress by overexpressing the soybean low phosphorus tolerance gene GmAP2 in plants.

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

[0006] The second objective of this invention is to provide a method for improving the tolerance of plants to low phosphorus stress, wherein the method involves increasing the expression level of the amino acid sequence shown in SEQ ID NO.2 in the 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 increased tolerance of the plant to low phosphorus stress is reflected in the increase in its root length.

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

[0010] Furthermore, the increased tolerance of the plant to low phosphorus stress is reflected in its increased fresh weight.

[0011] The third objective of this invention is to provide the application of the soybean low-phosphorus tolerance gene GmAP2 in the cultivation of plants resistant 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.

[0012] The fourth objective of this invention is to provide the application of recombinant plasmids, recombinant vectors, or transgenic plant cell lines of the soybean low-phosphorus tolerance gene GmAP2 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 the amino acid sequence shown in SEQ ID NO.2.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] S1. Soybean root tip RNA was extracted and reverse transcribed into cDNA, and then the coding DNA sequence (CDS) of soybean low phosphorus tolerance gene GmAP2 was cloned by PCR amplification.

[0015] S2, using CDS as a template, is used for PCR amplification to obtain the target fragment;

[0016] S3. Sequencing of the target fragment and comparison of the sequencing results with the reference sequence of the soybean low phosphorus tolerance gene GmAP2 (the known sequence of this gene in the NCBI database) showed that the GmAP2 sequence was consistent with the reference sequence.

[0017] S4. The sequenced and compared target fragments are ligated into the expression vector to obtain a recombinant vector. The recombinant vector is then sequenced to verify the ligation. After ensuring that the ligation is correct, the recombinant vector is transformed into Agrobacterium.

[0018] S5. Using Arabidopsis thaliana as a vector, we verified the role of the soybean low phosphorus tolerance gene GmAP2 in plant tolerance to low phosphorus stress: We constructed an overexpression vector of the soybean low phosphorus tolerance gene GmAP2 and heterologously transformed Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana plants. We then selected T5 generation homozygous lines and screened lines with high expression levels of the soybean low phosphorus tolerance gene GmAP2 by quantitative fluorescence identification.

[0019] S6. Wild-type Arabidopsis thaliana was prepared, and the low phosphorus tolerance phenotype of transgenic Arabidopsis thaliana plants and wild-type Arabidopsis thaliana was identified. The results showed that the root elongation and lateral root of transgenic Arabidopsis thaliana were significantly improved compared with wild-type. It is speculated that the soybean low phosphorus tolerance gene GmAP2 may be involved in the mechanism of soybean low phosphorus tolerance.

[0020] S7. Simultaneously, the gene-edited GmAP2 gene (pCas9-GmAP2(CAS9)) was constructed, and the GmAP2 gene (pTF101-GmAP2(OE)) and the empty vector control gene (pTF101(CK)) were overexpressed. pCas9-GmAP2(GmAP2), pTF101-GmAP2(OE), and pTF101(CK) were transformed into K599 Agrobacterium rhizogenes for rooting experiments.

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

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

[0023] Under different phosphorus concentration treatments, 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 ability to adapt to low phosphorus stress in acidic soils.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] Figure 1 Phylogenetic analysis of some AP2 / ERF family genes in Arabidopsis thaliana and soybean;

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

[0028] Figure 3 Image showing the cloning of the GmAP2 gene and the identification of bacterial culture containing the GmAP2 gene linked to the pLB vector;

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

[0030] Figure 5 This is a graph validating the self-activation of GmAP2 yeast.

[0031] Figure 6 Determination of the self-activation region of the GmAP2 protein;

[0032] Figure 7 A diagram validating the interaction between the GmAP2 protein and candidate genes;

[0033] Figure 8 This is a verification image of genetically transformed positive plants;

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

[0035] Figure 10 The relative expression levels of the whole root of soybean chimera overexpressing GmAP2 gene (OE), gene-edited GmAP2 gene, and empty vector (CK) were determined after 14 days of treatment under normal and low phosphorus conditions.

[0036] Figure 11 To investigate the root phenotypes of soybean chimeras overexpressing the GmAP2 gene (OE), gene-edited GmAP2, and empty vector (CK) after 14 days of treatment under normal and low phosphorus conditions. Detailed Implementation

[0037] To make the content of this invention easier to understand, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0038] To further illustrate the technical means employed in this invention, a more detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. However, these embodiments do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

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

[0040] The nucleotide sequence of the GmAP2 gene cDNA used in this 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 in this application belongs to the AP2 / ERF gene family. Phylogenetic analysis of some AP2 / ERF family genes in Arabidopsis thaliana and soybean is as follows: Figure 1 As shown.

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

[0046] 1. Soybean material processing

[0047] Brazilian soybean variety BRSMG68, which was plump and from the same growth stage, was selected as plant material. Germination was carried out in vermiculite at a germination temperature of 26℃ and a photoperiod of 16 / 8h for 4–5 days (days). After the cotyledons began to open slightly, the plants were transferred in groups to nutrient solutions treated with normal phosphorus NP (500 μM KH2PO4) and low phosphorus LP (5 μM KH2PO4) for hydroponic cultivation. Samples were collected at 0h, 3h, 6h, 12h, 24h, 36h, 2d, 5d, 7d, and 14d, and the collected samples were wrapped in aluminum foil and stored at -80℃.

[0048] 2. RNA extraction and reverse transcription

[0049] RNA was extracted from soybean root samples obtained after different phosphorus concentrations and hydroponic treatments, and reverse transcribed into cDNA. The cDNA was then used as a template for real-time quantitative PCR. The specific steps are as follows:

[0050] (1) RNA extraction

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

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

[0053] ③ Add 500 μL of lysis buffer and 700 μL of diluent to the EP tube in sequence, and mix them by vortexing until there are no obvious lumps. Then place the EP tube in a metal bath at 70°C and heat for 3 min. After that, transfer the EP tube to a centrifuge at 4°C and centrifuge at 12000 r / min for 10 min.

[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 vortex the mixture 20-25 times until the liquid in the centrifuge tube is turbid and has white foam, so that the reaction is complete and the reaction solution is obtained.

[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 into the centrifuge for centrifugation. The centrifuge temperature is 4℃, the rotation speed is 12000r / min, and the centrifugation time is 1min.

[0056] ⑥ Remove the centrifuge column and collection tube, discard the filtrate, add 600 μL of RNA washing buffer, and then place the centrifuge column and collection tube into a centrifuge for centrifugation. The centrifuge temperature is 4℃, the speed is 12000 r / min, and the centrifugation time is 45s.

[0057] ⑦ Remove the centrifuge column and collection tube, discard the filtrate, and prepare the 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 adsorption membrane of the centrifuge column and let it stand for 15 min.

[0058] ⑧ Add 600 μL of RNA washing buffer to the centrifuge column and wash the precipitate twice. Then place the centrifuge column and collection tube into the centrifuge and centrifuge at 4℃, 12000 r / min for 45 s. Discard the filtrate.

[0059] ⑨ Transfer the centrifuge column to the elution tube, then add 50-200 μL of nuclease-free water to the centrifuge column, let it stand for 2 minutes, and then place the centrifuge column and elution tube into the centrifuge for centrifugation. The centrifuge temperature is 4℃, the speed is 12000 r / min, and the centrifugation time is 1 minute.

[0060] ⑩ Measure the RNA concentration and store at -80℃ for later 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 program was as follows: incubation at 50℃ for 15 min; followed by heating at 85℃ for 5 seconds. All operations had to be performed on ice. After the reaction, the cDNA was stored in a freezer at -20℃.

[0063] Table 1 Reverse transcription reaction system

[0064]

[0065] 3. Primer design for quantitative PCR

[0066] Gene sequences were retrieved from the Phytozome database, and the GmAP2 genome CDS sequence (as shown in SEQ ID NO.1) and GmAP2 protein sequence (as shown in SEQ ID NO.2) were downloaded from the website. Primers were then designed and synthesized specifically for quantitative amplification based on the genome sequences on the NCBI website.

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

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

[0069] 4. Real-time quantitative PCR

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

[0071] ② Dilute all cDNA samples by 1-fold with ddH2O to serve as templates for quantitative PCR reactions;

[0072] ③ The Applied Biosystems StepOnePlus real-time PCR system was used to perform real-time PCR according to the reaction system in Table 2 below. The reaction program was as follows: 95℃ pre-denaturation for 30s; 39 cycles (95℃ denaturation for 5s; 60℃ annealing for 30s); melting curve: 95℃, 10s; 54.3℃, 5s; 94.3℃, 5s.

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

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

[0075] ④ Data processing: Relative expression level analysis was performed using the CT method (2^-△△CT )

[0076] 5. Results Analysis

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

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

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

[0080] ① Amplifying the target fragment: RNA was extracted from the root samples of soybean Huachun 6 and reverse transcribed into cDNA. Primers were then designed and synthesized using the cDNA from soybean Huachun 6 as a template.

[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 according to the reaction system in Table 3 below. The reaction program was as follows: pre-denaturation at 95℃ for 3 min; 34 cycles (denaturation at 95℃ for 15 s; annealing at 55℃ for 15 s; extension at 72℃ for 1 min / kb); final extension at 72℃ for 5 min; storage at 12℃.

[0084] Table 3 Fragment amplification reaction system

[0085]

[0086] ②Purify PCR products:

[0087] S1. Perform agarose gel electrophoresis on the PCR products. Purify the PCR products with correct bands using a purification kit. Then, centrifuge the PCR products and measure the volume. If the volume is less than 100 μL, add ddH2O to make up to 100 μL. Add 5 times the volume of Buffer GDP to the PCR products and mix thoroughly using a vortex mixer to obtain a mixture. Place the adsorption column in the collection tube and transfer the mixture into the adsorption column. Then, place the adsorption column and collection tube in a centrifuge and centrifuge at 12000 rpm for 1 min. Discard the filtrate.

[0088] S2, Prepare Buffer GW stock solution. Add 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 put the adsorption column and collection tube into a centrifuge and centrifuge at 12000 rpm for 1 min. Discard the filtrate.

[0089] S3, repeat S2; then place the adsorption column in a 1.5 mL sterile centrifuge tube, add 30 μL of solution buffer to the center of the adsorption column, let stand for 2 min, then place the adsorption column and collection tube in a centrifuge and centrifuge at 12000 rpm for 1 min; discard the adsorption column, and then store the purified PCR product at -20℃.

[0090] ③ Target gene ligated into pLB zero-background expression vector:

[0091] The target fragments were linked according to the reaction system in Table 4 below to obtain pLB-GmAP2. The reaction program was: denaturation at 20℃ for 2 min; annealing at 70℃ for 5 min; extension at 22℃ for 20 min.

[0092] Table 4 Connection Reaction System

[0093]

[0094] ④ E. coli transformation: Add 10 μL of pLB-GmAP2 to 100 μL of DH5α E. coli competent cells, gently aspirate and mix, incubate on ice for 30 min, then heat shock in a water bath at 42℃ for 45 s, and immediately transfer to ice to cool for 2 min. Then transfer the ligation product to a clean bench, add 500–700 μL of liquid medium to the pLB-GmAP2 ligation product, and incubate on a shaker at 37℃ for 1 h at 220 rpm to obtain bacterial culture. Spread the bacterial culture on a plate containing ampicillin (Amp) antibiotic, incubate overnight at 37℃, pick single colonies for detection and sequencing.

[0095] Figure 3 Image showing the cloning of the GmAP2 gene and identification of bacterial culture containing the GmAP2 gene linked to the pLB vector.

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

[0097] ① Amplification of the target fragment: Using cDNA from soybean Huachun 6 as a template, primers were designed and synthesized:

[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 program was as follows: 95℃ pre-denaturation for 3 min; 34 cycles (95℃ denaturation for 15 s; 55℃ annealing for 15 s; 72℃ extension for 1 min / kb); 72℃ final extension for 5 min; and storage at 12℃.

[0103] ② Linearization vector: pTF101 plasmid DNA was double-digested with restriction endonucleases SacI and XbaI;

[0104] ③ Ligation: The target fragment was ligated to pTF101 according to the reaction system in Table 5 below. The reaction procedure was: incubation at 37°C for 30 min to obtain pTF101-GmAP2.

[0105] Table 5 Connection Reaction System

[0106]

[0107] ④ Agrobacterium transformation: After successful single-clone sequencing and comparison of pTF101-GmAP2, plasmids were extracted using a plasmid extraction kit (Vazyme, Nanjing). 4 μL of plasmid was added to 100 μL of GV3101 Agrobacterium competent cells, incubated on ice for 5 min, then frozen in liquid nitrogen for 5 min, followed by heat shock at 37℃ for 5 min, and then cooled on ice for 5 min. The plasmid was then transferred to a clean bench, and 500–700 μL of LYEP liquid medium was added. The mixture was then placed in a shaker at 28℃ and cultured at 220 rpm for 2–3 h to obtain bacterial culture. The bacterial culture was plated on plates containing 30–50 μg / mL rifampicin and spectinomycin, and cultured at 28℃ for 2–3 days. Single clones were picked and cultured for testing. An equal volume of 50% glycerol was added to the successfully tested bacterial culture, and the culture was stored at -80℃.

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

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

[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 listed in Table 3 to obtain the amplification product. The amplification system and PCR amplification method were the same as the amplification procedure in Table 3. The amplification product was subjected to gel electrophoresis, and then the target band was recovered and purified using an agarose gel electrophoresis kit.

[0115] ② Linearization vector: The super1300 empty vector plasmid was digested with XbaI and SpeI restriction endonucleases.

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

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

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

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

[0120] 5'atggccatggaggccgaattcATGTGTGGCGGTGCCATC-3';

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

[0122] 5'ccgctgcaggtcgacggatccCATCGAAACTCCAGAGATCCCC-3',

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

[0124] ② Linearization vector: pGBKT7 plasmid was double-digested with restriction endonucleases BamHI and EcoRI.

[0125] ③ Ligation of the target fragment with the pGBKT7 plasmid and transformation of the ligation product into E. coli were performed. The methods and conditions for ligation and E. coli transformation were the same as those for the ligation and E. coli transformation steps in the construction of the pTF101-GmAP2 overexpression vector, and will not be repeated here. After sequencing and alignment confirmed to be correct, the plasmid was extracted and stored at -20℃ for later use. The pGBKT7-GmAP2 vector was obtained for subsequent experiments.

[0126] 5. Construction of GmAP2 gene editing vector

[0127] gRNAs targeting GmAP2 for gene editing were designed using the CRISPR-GE website (http: / / skl.scau.edu.cn / targetdesign / ). Four editing target sites were selected from the exon regions of GmAP2 and named GmAP2-guide13, GmAP2-guide15, GmAP2-guide20, and GmAP2-guide21, respectively. Four pairs of primers were designed and synthesized following the cloning method of the legume CRISPR / Cas9 vector pUC19.

[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] The oligonucleotides were annealed to double strands and ligated with the BsaI-digested vector pUC19 plasmid, which was then transformed into DH5α Escherichia coli. After successful bacterial culture 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 culture, and the culture was stored at -80℃ to obtain the GmAP2 gene editing vector.

[0137] Example 3: Genetic transformation in Arabidopsis thaliana

[0138] 1. Cultivation of wild-type Arabidopsis thaliana

[0139] Soak wild-type Arabidopsis seeds (Col-0) in pure water and vernalize them at 4℃ for 3 days. After vernalization, sow them evenly on nutrient soil, spray with an appropriate amount of water, cover with plastic film to retain moisture, and place in the dark for 2-3 days to break seed dormancy. When the Arabidopsis has grown 4-6 leaves, thin the seedlings to 4-5 plants per pot. When the first flower stalk is 1cm long, cut off the main stem to encourage branching. When the Arabidopsis flowers again, transform the seeds with Agrobacterium-methyl solution.

[0140] 2. Transformation of Arabidopsis inflorescences by Agrobacterium tumefaciens liquid

[0141] The Agrobacterium GV3101 preservation solution carrying the target vector was mixed with 100 mL of YEP (gen+spe+rif) liquid to obtain a bacterial suspension. The suspension was placed in a constant temperature shaker at 28℃ and shaken at 200 rpm overnight until the OD600 value of the suspension reached 0.6-0.8. The suspension was then centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the Agrobacterium was resuspended in an equal volume of fresh infusion buffer (5% sucrose + 1 / 2 MS + Silwet L-77 0.02%), then transferred to a 500 mL beaker. The aerial parts of flowering Arabidopsis thaliana were immersed in the Agrobacterium suspension for 1-2 minutes. The plants were then removed, and the transformed Arabidopsis were wrapped in plastic film to ensure high humidity. They were then treated in the dark for 12-16 hours. The next day, the plastic film was removed, and the plants were placed in an incubator for further growth. T0 generation seeds were harvested upon maturity.

[0142] 3. Identification of transgenic Arabidopsis progeny

[0143] Mature T0 generation seeds were dried and vernalized, then propagated. After the two true leaves of Arabidopsis thaliana had fully developed, glyphosate herbicide was sprayed regularly and multiple times. The results were as follows: Figure 8 As shown in section b, surviving transformed seedlings were screened, and false positive seedlings were removed. After the plants grew vigorously, fresh and tender leaves were selected, and DNA was rapidly extracted using TPS extraction buffer: tender plant leaves were placed in a 1.5 mL sterile centrifuge tube, 200 μL of TPS extraction buffer was added, and the mixture was ground with a grinder. Then, the mixture was placed in a metal bath at 80 °C for 10 min. Subsequently, the centrifuge tube was placed in a centrifuge and centrifuged at 12000 rpm for 10 min. 20 μL of the supernatant was taken, and 50 μL of double-distilled water (ddH2O) was added to the supernatant. The mixture was mixed thoroughly by pipetting, and 2 μL was used for the PCR reaction.

[0144] DNA molecular-level identification was performed using the reaction system listed in Table 6 below. The reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 28 cycles (95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min / kb); 72℃ final extension for 5 min.

[0145] The universal primers used for 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, the results were detected by 1% agarose gel electrophoresis. Figure 8 As shown in section a, where M is the 2000 DNA Marker, and plants 1-12 are all positive, the correct band length can preliminarily confirm that it is a transgenic material. T2 generation Arabidopsis seeds will be harvested upon maturity. This method will be used to identify plants up to the T4 generation. RNA will be extracted from different Arabidopsis lines and identified by quantitative real-time fluorescence. Lines with high expression levels will be selected for large-scale propagation for subsequent experiments.

[0147] Table 6 DNA Molecular Level Identification Reaction System

[0148]

[0149] 4. Instantaneous conversion of native tobacco

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

[0151] The cultured bacterial culture was centrifuged at 4000 rpm for 10 min. The supernatant was then discarded, and the bacterial cells were resuspended in 15 mL of infiltration solution and cultured in the dark at room temperature for 3 h.

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

[0153] Experimental results:

[0154] like Figure 4 As shown, eGFP corresponds to the GFP channel; DAPI corresponds to the UV channel, and DAPI dye binds to DNA and emits blue fluorescence; Merge represents the fusion image of GFP and DAPI; Bright field represents the bright field. When super1300-GFP (corresponding to GFP in the figure) and Super1300-GmAP2-GFP (corresponding to GmAP2-GFP in the figure) were transiently expressed in tobacco lower epidermal cells, tobacco epidermal cells transformed with the super1300-GFP empty vector showed 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 cell nucleus, and this fluorescence overlapped with the blue fluorescence of DAPI, indicating that the GmAP2 protein is located in the cell nucleus.

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

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

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

[0158] S1, Gene amplification and purification: Following the steps in Example 2, the coding sequence of the GmAP2 gene was amplified by PCR to obtain the amplification product, and the amplification product was purified.

[0159] S2, Vector linearization: The pGBKT7 vector (BD) was linearized by double digestion with restriction endonucleases EcoRI and SacI, and the linearized vector was purified by agarose gel electrophoresis.

[0160] S3, Recombination Ligation: Ligation was carried out according to the ligation reaction system in Table 5. The purified amplification product was recombined 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 agar plates containing kanamycin and incubated overnight at 37°C. Single colonies were picked for colony PCR identification. After confirming that the amplified band size was correct, the cells were sent to a biological sequencing company for sequencing verification, confirming that the fusion vector GmAP2-BD had been successfully constructed.

[0162] Step 2: Yeast Two-Hybrid Experiment

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

[0164] S2, Plate Screening: Transformed yeast cells were plated on plates containing SD / -Trp-Leu / X-α-Gal to obtain the experimental group.

[0165] Step 3: Check the settings:

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

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

[0168] The self-activation verification result is as follows: Figure 5 As shown in the figure, parts a and b are the cloning and bacterial culture identification electrophoresis images of the constructed GmAP2-BD yeast vector, respectively. The experiment shows that the GmAP2-BD protein appears blue. To further verify this, the self-activation activity of GmAP2-BD was verified by streaking yeast culture carrying GmAP2-BD and the empty AD vector plasmid onto SD / -Leu-Trp-His-Ade / X-α-Gal medium. The results are shown in parts c and d. GmAP2-BD and the positive control turned blue after normal growth on the medium, while the negative control did not show blue.

[0169] 2. Determination of the self-activation region of the GmAP2 protein

[0170] The full-length CDS of the GmAP2 gene was cut into two segments, S1 and S2, as follows: Figure 6 The image shows the truncated GmAP2 gene segment a. S1 and S2 were ligated into the BD vector and then co-transformed with the AD empty vector into Y2HGold competent cells, and spotted onto plates containing SD / -Trp-Leu / X-α-Gal.

[0171] Figure 6Parts b and c in the image show electrophoresis images of clones and bacterial cultures identified by GmAP2 gene truncation, respectively. In part b, M (Marker) represents the marker band, and bands 1, 2, and 3 represent amplified recombinant fragments containing the full-length and partial CDS sequences of the GmAP2 gene, with band lengths of 943 bp, 709 bp, and 274 bp, respectively. In part c, M (Marker) represents the marker band, and bands 1 and 2 represent S1 bacterial culture identification, bands 3 and 4 represent S2 bacterial culture identification, and bands 5 and 6 represent GmAP2-BD bacterial culture identification.

[0172] Plasmids carrying the empty vectors S1-BD and AD were spotted onto SD / -Trp-Leu / X-α-Gal and SD / -Leu-Trp-His-Ade / X-α-Gal media, respectively. The results are as follows: Figure 6 Parts d and e show that on plates containing SD / -Trp-Leu / X-α-Gal, S1 does not turn blue, indicating that the S1 protein has no self-activation. However, the S2 protein turns blue. Strain S1-BD and the negative control do not grow or turn blue, indicating that S1-BD has no self-activation. S1-BD was then subjected to a yeast hybridization experiment.

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

[0174] The SMART website was used to predict 10 genes with high interaction scores. Basic information about these 10 genes was retrieved from 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 annotations, two proteins were further selected for interaction verification: S1-BD was selected as the bait protein, and Glyma.15G171100 and Glyma.17G053500 were selected as the prey proteins. These proteins were co-transformed into a yeast strain and plated on SD / -Leu-Trp plates containing X-α-Gal. The plates were then incubated 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 clone diagrams 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 a 200 Identification of bacterial cultures using DNA Marker 0, Glyma.17G236200 for 1 and 2, Glyma.13G105900 for 3 and 4, Glyma.15G171100 for 5 and 6, Glyma.08G190300 for 7 and 8, Glyma.09G065200 for 9 and 10, Glyma.17G053500 for 11 and 12, Glyma.15G025900 for 13 and 14, and Glyma.10G205000 for 15 and 16. Figure 7 The experimental results in section c are all blue. Two interacting genes were picked from SD / -Leu-Trp / X-α-Gal, diluted with NaCl to OD600 = 0.6, and spotted onto SD / -Leu-Trp-His-Ade / X-α-Gal plates. GmAP2 interacts with two of the proteins.

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

[0178] ①Preparation of genetic transformation materials

[0179] Genetic transformation was performed using the Arabidopsis thaliana flower-dipping method. Wild-type Arabidopsis thaliana seeds were vernalized at 4℃ for 3 days and then 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. After 3 days, the plastic wrap was removed, leaving 4-5 seedlings per pot. When there were many wild-type Arabidopsis thaliana flower buds, infection began. Before infection, the fully opened flowers were cut off.

[0180] ② Agrobacterium infection

[0181] GV3101 bacterial culture containing the pTF101-GmAP2 vector was cultured to OD = 0.6, then centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. 50 mL of fresh resuspension (5% sucrose + 1 / 2 MS + Silwet L-77 0.02%) was added to the bacterial culture. Note that the resuspension should be stirred on a magnetic stirrer until foaming occurs beforehand. After adding the fresh resuspension, the mixture was thoroughly resuspended by pipetting. Wild-type Arabidopsis flower buds were then completely immersed in the resuspension for 2 min. The infected Arabidopsis were then placed in a plastic bag for preservation and treated in the dark for 24 h.

[0182] ③Selective cultivation

[0183] S1. After harvesting, all seeds were vernalized at 4℃ for 3 days. Moist nutrient soil was placed in trays, and the seeds were then sown on the surface of the soil. The trays were then placed in an incubator (16h light / 8h dark; 24℃). Once the two cotyledons of Arabidopsis thaliana were fully expanded, a 20μg / mL glyphosate solution was prepared and sprayed every two days, keeping the soil consistently moist. Approximately 7 days after spraying the glyphosate solution, non-positive seedlings failed to grow normally, while those that grew normally were identified as positive seedlings. These positive seedlings were then transplanted individually into small planting pots. Each plant was harvested as a lineage, and positive seedlings were continuously screened and identified until the T4 generation homozygous strain was formed.

[0184] ④ Verification of transgenic plants

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

[0186] After being cultured to the T4 generation, RNA was extracted and reverse transcribed from the aboveground parts and roots, and identified by quantitative fluorescence. The results are as follows: Figure 8 As shown in section c, three independent lines (OE-3, OE-4, and OE-6) with overexpression of the GmAP2 gene were obtained. Lines with high expression levels were selected and propagated in large numbers for subsequent experiments.

[0187] ⑤ Pre-culture: Seeds of different transgenic Arabidopsis lines OE-3, OE-4, and OE-6 were sterilized by soaking and shaking in a 10% sodium hypochlorite solution for 10 minutes, followed by rinsing five times with sterile water. The washed seeds were then vernalized at 4°C for 3 days to induce spring flowering. Afterward, the seeds were planted on 1 / 2 MS medium and transferred to a culture room for vertical cultivation. Once the roots reached approximately 1 cm in length, phosphorus treatment was applied.

[0188] ⑥ Phosphorus Treatment: A modified 1 / 2 Hoagland solution solid medium with KH₂PO₄ concentration gradients of 500 μmol / L (NP), 5 μmol / L, and 0 μmol / L was prepared, and the pH was adjusted to 5.8. Arabidopsis thaliana strains with uniform growth were transferred to this medium, maintaining root elongation. After approximately two weeks of vertical culture, 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 procedure was the same as the method for measuring total phosphorus content in hairy roots in Experiment 6; for Arabidopsis thaliana, the whole-plant total phosphorus content was measured).

[0189] Results of transgenic Arabidopsis root phenotypic identification: Figure 9 As shown, heterologous overexpression of GmAP2 in Arabidopsis thaliana can alleviate the inhibitory effect on growth and development of Arabidopsis thaliana under low phosphorus stress. The statistical results of its biomass indicators are as follows: Figure 9 The bar chart shows that, compared to the wild type, heterologous overexpression of GmAP2 in Arabidopsis thaliana enhances its tolerance to low phosphorus by increasing the length of the taproot, lateral roots, and fresh weight of the plant, and the phosphorus content of the transgenic lines is also significantly increased.

[0190] Example 6: Identification of low phosphorus tolerance in soybean hairy roots

[0191] ① Transformation of K599 Agrobacterium rhizogenes: Gene-edited GmAP2 gene (pCas9-GmAP2), GmAP2 gene overexpression (pTF101-GmAP2(OE)), and empty vector control gene (pTF101(CK)) were transformed into K599 Agrobacterium rhizogenes. After identifying positive single bacteria, the bacteria were amplified and preserved by shaking.

[0192] ② Infection: K599 Agrobacterium without any transformation and K599 Agrobacterium carrying gene editing and overexpression vectors were shaken to OD600 = 0.6, centrifuged, and resuspended in a resuspension solution. Plump, disease-free Brazilian 13 soybeans were selected and planted on well-draining vermiculite, covered with dry vermiculite. After 2-3 days, when the above-ground parts of the soybeans reached 2-3 cm in height, a 1 mL syringe was used to inject the soybeans 1-2 cm below the cotyledons for infection. Afterward, the mixture was cultured in high humidity for about one week. Once the injection wound had developed roots, it was covered with vermiculite, and watered frequently to keep the vermiculite moist. Cultured for another week was then continued.

[0193] ③ Hairy root phenotypic identification: When the hairy roots developed to about 5 cm, they were removed without damaging them. The primary roots were cut off, and Hogrange solutions with a KH2PO4 concentration of 500 μmol / L and a pH of 5.8 were prepared and hydroponically cultured for 14 days. Lateral roots on the main root were selected, and DNA was extracted to detect positive roots. RNA was extracted from the roots of each treatment and subjected to quantitative fluorescence analysis. The fresh weight of the aboveground and underground parts of soybean plants was measured. After the samples were completely dried, the dry weight was measured, and the total phosphorus content of the aboveground and underground parts of the plant was determined. Each data point was analyzed with at least three biological replicates and three technical replicates.

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

[0195] 1. All reagents used in this experiment are of analytical grade, and the water 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 0.5g of chemically pure potassium antimony tartrate and dissolve it in 100mL 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 while stirring continuously and cooling. Separately weigh 10 g of finely ground ammonium molybdate and dissolve it in 300 mL of water at approximately 60 °C, then cool. Slowly pour the sulfuric acid solution into the ammonium molybdate solution. Add 100 mL of 0.5% potassium antimony tartar solution, cool, dilute with water to 1000 mL, shake well, and store in a brown reagent bottle. This stock solution contains 1% ammonium molybdate and 2.25 mol / L sulfuric acid.

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

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

[0204] (9) 5 mg / L phosphorus standard solution: Take 5 mL of phosphorus stock solution and put it into a 100 mL volumetric flask. Add water to make up to volume. Prepare the solution fresh when needed.

[0205] II. Experimental instruments: analytical balance (0.0001g), 100mL volumetric flask, 50mL volumetric flask, funnel, several small test tubes, graphite digester (SH220F), spectrophotometer (UV-1800PC), marker pen, etc.

[0206] III. Experimental Procedure

[0207] (1) Sample solution preparation

[0208] Weigh an appropriate amount of sample, accurate to 0.001 g, into a 500 mL digestion tube (do not allow the sample to adhere to the neck of the tube). First, add a small amount of water to moisten the sample, then add 6 mL of sulfuric acid, gently shake well, and let stand overnight. Place a small bent-neck funnel at the mouth of the tube, and digest at 250 °C on a digestion furnace (start timing after the temperature stabilizes, approximately 30 min). After the H2SO4 decomposes and emits a large amount of white fumes, increase the temperature to 400 °C. Remove the tube when the solution turns a uniform brownish-black color.

[0209] After cooling slightly, add 10 drops of H2O2, shake well, and heat to a gentle boil. Digest for about 5 minutes, then remove and let cool slightly. Repeat this process, adding 5-10 drops of H2O2 each time, and then continue digesting. Repeat this process 3-5 times, gradually reducing the amount of H2O2 added each time. Digest until the solution is colorless or clear, with a grayish-white bottom. Then heat for about 5-10 minutes to remove any remaining H2O2.

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

[0211] (2) Preparation of blank solution

[0212] Except for the absence of a sample, the reagents and operating procedures are the same as above.

[0213] (3) Draw the standard curve

[0214] Pipette 0, 2, 4, 6, 8, 10, 14, and 20 mL of 5 mg / L phosphorus standard solution into 50 mL volumetric flasks, respectively. Simultaneously add an equal volume of blank solution (as used for the colorimetric assay) and 2-3 drops of dinitrophenol indicator. Adjust the solution to a slightly yellow hue with 10% sodium carbonate or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony colorimetric reagent, shake well, and dilute to volume with water to obtain a series of standard solutions with phosphorus contents of 0.0, 0.2, 0.4, 0.8, 1.0, 1.4, and 2.0 mg / L. Shake well, incubate at 15°C or higher for 30 min, and then measure the absorbance at 880 nm. Plot a calibration curve on graph paper with absorbance on the ordinate and phosphorus concentration (mg / L) on the abscissa.

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

[0217] The colored sample solution was measured on a spectrophotometer using an 880nm, 1cm path length cuvette. The instrument was zeroed using a blank test as a reference, and the absorbance was read. The corresponding phosphorus content was then determined from the standard curve.

[0218] IV. Result Calculation

[0219]

[0220] In the formula:

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

[0222] C0 — Blank value

[0223] V – Volume of the colorimetric solution;

[0224] D – Dividing factor: Volume of the digestion solution to the final volume / Volume of the digestion solution removed;

[0225] m — Mass of the sample taken (g);

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

[0227] Quantitative results and phenotypic identification results of GmAP2 hairy roots are as follows: Figure 10 and Figure 11 As shown, overexpression of pTF101-GmAP2 can enhance the tolerance of soybean to low phosphorus stress, and gene editing of pCas9-GmAP2 can enhance the sensitivity of soybean to low phosphorus stress.

[0228] The data in this embodiment are the average and standard error of three replicates. "*", "**", "***" and "****" indicate that the difference between the treatment and the control is significant (*P≤0.05), highly significant (**P≤0.001), extremely significant (***P≤0.001), and extremely significant (****P≤0.0001), respectively; the same applies below. The above-described embodiments are only preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. The application of soybean low phosphorus tolerance gene GmAP2 in improving the low phosphorus stress tolerance of plants, characterized in that: The application is overexpression of the GmAP2 gene in plants, the plants are Arabidopsis thaliana or soybean, the nucleotide sequence of the low-phosphorus-tolerant gene GmAP2 of the soybean is shown as SEQ ID NO. 1; or the low-phosphorus-tolerant gene GmAP2 of the soybean encodes an amino acid sequence shown as SEQ ID NO.

2.

2. A method for improving the tolerance of plants to low phosphorus stress, characterized in that, The method is to improve the expression amount of the amino acid sequence shown as SEQ ID NO. 2 in the receptor plant. The specific steps include: introducing the low-phosphorus-tolerant gene GmAP2 of the soybean shown as SEQ ID NO. 1 into a receptor plant to obtain a transgenic plant, and the low-phosphorus-stress tolerance of the transgenic plant is higher than that of the receptor plant; the plants are Arabidopsis thaliana or soybean.

3. The method of claim 2, wherein, The improvement of the low-phosphorus-stress tolerance of the plants is embodied as an increase in root length.

4. The method of claim 2, wherein, The improvement of the low-phosphorus-stress tolerance of the plants is embodied as an increase in the number of lateral roots.

5. The method of claim 2, wherein, The improvement of the low-phosphorus-stress tolerance of the plants is embodied as an increase in fresh weight.

6. The use of soybean low phosphorus tolerance gene GmAP2 in breeding low phosphorus stress tolerant plants, characterized in that: The application is overexpression of the GmAP2 gene in plants, the plants are Arabidopsis thaliana or soybean, the nucleotide sequence of the low-phosphorus-tolerant gene GmAP2 of the soybean is shown as SEQ ID NO. 1; or the low-phosphorus-tolerant gene GmAP2 of the soybean encodes an amino acid sequence shown as SEQ ID NO.

2.

7. The use of the recombinant plasmid, recombinant vector or transgenic plant cell line of the soybean low phosphorus tolerance gene GmAP2 in improving the low phosphorus stress tolerance of plants, characterized in that, The plants are Arabidopsis thaliana or soybean, the nucleotide sequence of the low-phosphorus-tolerant gene GmAP2 of the soybean is shown as SEQ ID NO. 1; or the low-phosphorus-tolerant gene GmAP2 of the soybean encodes an amino acid sequence shown as SEQ ID NO. 2.

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