Application of soybean low-phosphorus-tolerant gene GmVQ32 in improvement of low-phosphorus stress tolerance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a soybean low-phosphorus-tolerant gene GmVQ32 in improving low-phosphorus stress tolerance of plants. The soybean low-phosphorus-tolerant gene GmVQ32 disclosed by the invention can be used for enhancing the root growth and phosphorus absorption efficiency of plants by positively regulating the response of plant root systems to low-phosphorus stress, so that the adaptability of the plants to a low-phosphorus environment is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of the soybean low-phosphorus tolerance gene GmVQ32 in improving the low-phosphorus stress tolerance of plants. Background Art

[0002] Soybean is an important protein and oil crop in the world and has a very important position in agricultural production. Phosphorus is one of the three essential elements for plant growth, development and metabolism. It is not only a component in plants, but also a participant in a large number of physiological activities of plants. It participates in various biochemical processes in plants in various ways and plays an important role therein. Phosphorus can not only promote plant growth and development, but also enhance the stress resistance of plants and increase crop yields, which is crucial for crops.

[0003] In China, there are approximately one billion mu of land lacking phosphorus, accounting for two-thirds of the total cultivated land area. Plants can only obtain phosphorus in the form of inorganic phosphate (Pi). In the southern region of China, most of the soil is acidic red soil with a low pH value. Phosphorus is easily oxidized and fixed by metal ions such as iron (Fe) and aluminum (Al) in acidic soil, and is easily precipitated by calcium (Ca) in alkaline soil. Therefore, most of the phosphorus in the soil is difficult to be absorbed and utilized by plants, resulting in low soybean yields and economic benefits that are difficult to meet the production needs of people. Soil phosphorus deficiency has become an important factor restricting soybean yields and quality. At present, there are also relevant studies taking soybean seedlings as the research object to explore methods to help soybeans survive the critical phosphorus-deficient period. There are mainly two compensatory approaches. One is to increase the application amount of phosphate fertilizer to improve the phosphorus supply situation in the soil. However, due to the fact that phosphorus is easily fixed by metal ions in the soil, the phosphorus utilization efficiency of this method is low, and a large amount of fertilization will also cause environmental pollution and other problems, which is not conducive to green agriculture. The other is to improve the genetic characteristics of soybeans for phosphorus absorption and utilization, and to explore gene resources related to low-phosphorus tolerance. Taking phosphorus-efficient soybean varieties as the research object, low-phosphorus-tolerant soybean varieties are obtained through biotechnology. In recent years, the main domestic and foreign research on soybean phosphorus has focused on the mechanism of root traits controlling the efficient absorption and utilization of plant phosphorus, including regulating root morphological architecture to obtain available phosphorus, the physiological and molecular mechanisms of root exudates activating and utilizing insoluble phosphorus, rhizosphere microorganism interaction, and phosphorus signaling networks, etc., and certain progress has been made.

[0004] Although the screening and functional identification of genes related to phosphorus absorption and utilization have provided a large amount of reference and basis for subsequent research. However, we should also see that there are still many gaps in the field of soybean phosphorus, and many key genes and regulatory networks have not been analyzed, restricting the potential of soybean production. Therefore, analyzing the molecular mechanism related to low-phosphorus tolerance in soybeans, excavating and identifying key genes for high phosphorus efficiency, improving the phosphorus absorption and utilization efficiency of soybeans, and cultivating new soybean varieties with high phosphorus efficiency are of great significance for increasing the yield of soybeans in South China. Summary of the Invention

[0005] In order to overcome the problems in the above-mentioned background technology, the first object of the present invention is to provide an application of the soybean low-phosphorus tolerance gene GmVQ32 in improving the low-phosphorus stress tolerance ability of plants. The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

[0006] The soybean low-phosphorus tolerance gene GmVQ32 of the present application positively regulates the response of plant roots to low-phosphorus stress, enhances the root growth and phosphorus absorption efficiency of plants, and thus improves the low-phosphorus stress tolerance ability of plants.

[0007] Preferably, the plant is Arabidopsis thaliana or soybean.

[0008] The second object of the present invention is to provide an expression vector for regulating plant adaptation to low-phosphorus stress. The vector includes the soybean low-phosphorus tolerance gene GmVQ32 and a plasmid carrying the soybean low-phosphorus tolerance gene GmVQ32. The plasmid is a pCas9 or pTF101 vector; the nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

[0009] The third object of the present invention is to provide a host cell for regulating plant adaptation to low-phosphorus stress. The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

[0010] Optionally, the host cell is an Escherichia coli or Agrobacterium cell.

[0011] Objective 4 of the present invention is to provide a method for promoting the growth of Arabidopsis thaliana in a low-phosphorus stress environment. By overexpressing the low-phosphorus tolerance gene GmVQ32 of soybean in Arabidopsis thaliana, the growth of Arabidopsis thaliana is promoted. The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

[0012] Objective 5 of the present invention is to provide a method for promoting the growth of soybean in a low-phosphorus stress environment. By overexpressing the low-phosphorus tolerance gene GmVQ32 of soybean in soybean, the growth of soybean is promoted. The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

[0013] Among them, the method for the soybean low-phosphorus tolerance gene GmVQ32 of the present application to promote the growth of plants in a low-phosphorus stress environment includes the following steps:

[0014] S1, Extract the RNA from the soybean root tip and reverse transcribe it into cDNA, and then clone the coding DNA sequence (Coding DNA Sequence, CDS) of the soybean low-phosphorus tolerance gene GmVQ32 by PCR amplification. The length of this CDS sequence is 666bp;

[0015] S2, Sequence the cloned CDS and compare the sequencing results with the reference sequence of the soybean low-phosphorus tolerance gene GmVQ32 (the known sequence of this gene in the NCBI database). The sequence of the CDS is consistent with the reference sequence;

[0016] S3, Connect the sequenced and compared CDS to an expression vector to obtain a recombinant vector. Sequence the recombinant vector for verification. After ensuring that the connection is correct, transform the recombinant vector into Agrobacterium;

[0017] S4, Use Arabidopsis thaliana as a vector to verify the role of the soybean low-phosphorus tolerance gene GmVQ32 in plant low-phosphorus stress tolerance: Using the Agrobacterium-mediated genetic transformation method, introduce the GmVQ32 gene into Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana plants. Take the T5 generation homozygous lines, and screen the lines with high expression levels of the soybean low-phosphorus tolerance gene GmVQ32 through fluorescence quantification;

[0018] S5, Prepare wild-type Arabidopsis thaliana and conduct the low-phosphorus tolerance phenotype identification of transgenic Arabidopsis thaliana plants and wild-type Arabidopsis thaliana. The results show that the root elongation and lateral roots of transgenic Arabidopsis thaliana are significantly improved compared with the wild type. It is speculated that the soybean low-phosphorus tolerance gene GmVQ32 may be involved in the soybean low-phosphorus tolerance mechanism;

[0019] S6. Meanwhile, construct the gene-edited GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), the overexpressed GmVQ32 gene (pTF101-GmVQ32 (OE)), and the empty vector control gene (pTF101 (CK)). Transform pCas9-GmVQ32 (GmVQ32), pTF101-GmVQ32 (OE), and pTF101 (CK) into Agrobacterium rhizogenes K599, and then use the Agrobacterium-mediated genetic transformation method to introduce the GmVQ32 gene into the target plants;

[0020] S7. Conduct low-phosphorus tolerance phenotype identification on the above target plants carrying Agrobacterium with pCas9-GmVQ32 (GmVQ32), pTF101-GmVQ32 (OE), and pTF101 (CK). The results show that the root elongation, lateral roots, and dry weight of the OE (Overexpression) plants are significantly improved compared to the CK (Control Check). Through the above experiments, it is shown that overexpressing the GmVQ32 gene can enhance the tolerance of soybeans to low-phosphorus stress.

[0021] The present invention has the following beneficial effects: The expression of the GmVQ32 gene is upregulated under low-phosphorus stress, and its expression level increases significantly with the prolongation of phosphorus treatment time;

[0022] Under different phosphorus concentration treatment conditions, overexpressing GmVQ32 can significantly increase the biomass of transgenic plants. GmVQ32 positively regulates the response of plant roots to low-phosphorus stress, enhances root growth and phosphorus absorption efficiency of plants, and thus improves the adaptability of plants to low-phosphorus environments;

[0023] Therefore, GmVQ32 plays an important role in plant adaptation to low-phosphorus stress, and regulating its expression through transgenic technology can significantly enhance the adaptability of plants to low-phosphorus stress in acidic soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0025] Figure 1 For the phylogenetic tree analysis of VQ family genes in Arabidopsis thaliana, rice, and soybeans;

[0026] Figure 2 For the quantitative PCR results of the GmVQ32 gene under different phosphorus concentration treatment conditions;

[0027] Figure 3 For the transient expression results of Super1300-GFP and Super1300-GmVQ32-GFP in tobacco leaf cells respectively;

[0028] Figure 4 is the self-activation verification result of GmVQ32 yeast;

[0029] Figure 5 is the interaction verification result of GmVQ32 protein and candidate genes;

[0030] Figure 6 is the quantitative PCR result of Glyma.12G206400 and Glyma.13G294500 genes under different phosphorus concentration treatments;

[0031] Figure 7 is the difference result of the whole plant phenotype, primary root length, lateral root length, and fresh weight of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana (Col-0) after 14 days of treatment under normal phosphorus and low phosphorus conditions;

[0032] Figure 8 Phenotype of the whole plant, shoot dry weight, root dry weight, and relative root expression results of gene-edited GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), overexpressed GmVQ32 gene (pTF101-GmVQ32 (OE)), and empty vector control gene (pTF101 (CK)) soybeans after 14 days of treatment under normal phosphorus and low phosphorus conditions. Detailed implementation mode

[0033] To further elaborate on the technical means adopted by the present invention, the following provides a more detailed description in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0034] The cDNA nucleotide sequence of the GmVQ32 gene adopted by the present invention is shown in SEQ ID NO.1:

[0035] ATGACTGCCACAGCTGATCAATGCATGCATGAGTTTTATCAACAACCTCTCATGGATGGCATGGCTATGGATCCATCCA

[0036] TAGAAGGACTCATGGATGCATCCATGTCATCATCATCAGAGGGCATGATCATGTTGAGCCCAAGCAATTCACACAACAA

[0037] CACAACAATTGGCCACCACAATTTAACTCCAAAGGGTTGTGCATTCAAACAAATCCGAAGGAGATCTAGAGCTTCTAAG

[0038] AGCACTCCAATTACCCTTCTCAAGGCCAACACCTCCAATTTCAGGGCATTGGTACAACAATTCACTGGGTGTCCCACCA

[0039] CAACAGCCATGTCACTTGCAATCCATAAGGGTCCCGTTACCTTAAATTTCCAACAAGGTGGTAGCAAACAACATATTCA

[0040] TCATCATCACCACACAAAAATAACAACTAGAGGAGCAATGCCACCGTTTATTGGCACCATAAGTTCTAACCAAAACCAA

[0041] GTTTCTGTGCCACTTCCAAAGCAACACTTGATGCAAGAGCAGCAAAGTGGACACTTTCTTCCAACTTCGTCGGGTAACT

[0042] CTTATAGGCCAATTAATTGCATGGATGATGGATTGATCTTTGATAATGATTTTAGTTTACATGAGCTAACCGTGAATGCCATCTCCAATGATATCGATGATTTATTTATGTGA。

[0043] The amino acid sequence of the protein encoded by GmVQ32 is shown in SEQ ID NO.2:

[0044] MTATADQCMHEFYQQPLMDGMAMDPSIEGLMDASMSSSSEGMIMLSPSNSHNNTTIGHHNLTPKGCAFKQIRRRSRASK

[0045] STPITLLKANTSNFRALVQQFTGCPTTTAMSLAIHKGPVTLNFQQGGSKQHIHHHHHTKITTRGAMPPFIGTISSNQNQ VSVPLPKQHLMQEQQSGHFLPTSSGNSYRPINCMDDGLIFDNDFSLHELTVNAISNDIDDLFM*。

[0046] By comparing the cloned CDS sequence of the GmVQ32 gene, the sequence of the CDS is consistent with the known sequence of this gene in the NCBI database. Figure 1 It is a phylogenetic tree analysis diagram of VQ family genes in Arabidopsis thaliana, rice, and soybean.

[0047] Example 1: Expression pattern of the GmVQ32 gene under different phosphorus levels

[0048] 1. Treatment of soybean materials

[0049] Select the same period and plump soybean variety BRSMG68 as the plant material, germinate it in vermiculite, with a germination temperature of 26 °C, a light cycle of 16 h light and 8 h darkness, and culture for 4 - 5 days (d). After the cotyledons slightly unfold, transfer them to nutrient solutions with normal phosphorus nutrition (NP, 500 μM KH2PO4) and low phosphorus nutrition (LP, 5 μM KH2PO4) in groups for hydroponic treatment, and collect root samples at 0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 2 d, 5 d, 7 d, and 14 d respectively. The collected samples are wrapped with tin foil and stored in a -80 °C refrigerator.

[0050] 2. RNA extraction and reverse transcription

[0051] Extract the RNA of soybean root samples treated with different phosphorus concentrations and hydroponic treatment and stored respectively, and reverse transcribe it into cDNA through a reverse transcription kit (Vazyme, Nanjing), and then use the cDNA as a template for real-time fluorescence quantitative PCR. The specific steps are as follows:

[0052] (1) RNA extraction

[0053] ① Prepare a mortar, pestle, spoon, 1.5 mL centrifuge tube, and pipette tips, and sterilize them in advance by high-temperature and high-pressure sterilization;

[0054] ② Cool the mortar and pestle with liquid nitrogen, then put in the soybean root sample, add liquid nitrogen again, and quickly grind it into powder. Scoop 50 - 100 mg of the powder into an EP tube with a spoon;

[0055] ③ Add 500 μL of lysis buffer and 700 μL of dilution buffer to the EP tube in sequence, then use a vortex mixer to blow and mix until there is no obvious lumpy tissue, then place the EP tube in a 70 °C metal bath and heat for 3 min, and then transfer the EP tube to a 4 °C centrifuge and centrifuge at 12000 r / min for 10 min;

[0056] ④ Take 600 μL of the supernatant from the EP tube and transfer it to a new 1.5 mL centrifuge tube. Subsequently, add 300 μL of absolute ethanol to the centrifuge tube, and then use a vortex mixer to pipette 20 - 25 times until the liquid in the centrifuge tube becomes turbid and has white foam, allowing it to react fully to obtain the reaction solution.

[0057] ⑤ Prepare a centrifugal column and a collection tube. Place the centrifugal column inside the collection tube, and then transfer the reaction solution to the centrifugal column. Subsequently, place the centrifugal column with the reaction solution and the collection tube into a centrifuge for centrifugation. The temperature of the centrifuge is 4°C, the rotation speed is 12000 r / min, and the centrifugation time is 1 min.

[0058] ⑥ Take out the centrifugal column and the collection tube, discard the filtrate therein, and then add 600 μL of RNA wash solution. Subsequently, place the centrifugal column and the collection tube into a centrifuge for centrifugation. The temperature of the centrifuge is 4°C, the rotation speed is 12000 r / min, and the centrifugation time is 45 s.

[0059] ⑦ Take out the centrifugal column and the collection tube, discard the filtrate therein. Prepare the incubation solution in advance (5 μL of 10×DNase I buffer; 5 μL of DNase I; 40 μL of nuclease-free water). Pour 50 μL of the incubation solution onto the center of the adsorption membrane of the centrifugal column and let it stand for 15 min.

[0060] ⑧ Add 600 μL of RNA wash solution to the centrifugal column and wash the precipitate twice. Subsequently, place the centrifugal column and the collection tube into a centrifuge for centrifugation. The temperature of the centrifuge is 4°C, the rotation speed is 12000 r / min, and centrifuge for 45 s, then discard the filtrate.

[0061] ⑨ Transfer the centrifugal column to an elution tube. Subsequently, add 50 - 200 μL of nuclease-free water to the centrifugal column, let it stand for 2 min, and then place the centrifugal column and the elution tube into a centrifuge for centrifugation. The temperature of the centrifuge is 4°C, the rotation speed is 12000 r / min, and the centrifugation time is 1 min.

[0062] ⑩ Measure the concentration value of the RNA and store it in a -80°C refrigerator for later use.

[0063] (2) Reverse transcription

[0064] Measure the OD value (Optical Density) of the extracted RNA using an ultraviolet spectrophotometer or a microplate spectrophotometer. Calculate the RNA dosage (0.1 ng - 1 μg) based on the OD value of the extracted RNA. The specific calculation formula is: RNA dosage = 1000 / OD value. Perform reverse transcription using a reverse transcription kit (Vazyme, Nanjing). The reaction system is shown in Table 1 below. The reaction program is: incubate at 50°C for 15 min; then heat at 85°C for 5 s; all operations must be completed on ice. After the reaction, store the cDNA in a -20°C refrigerator.

[0065] Table 1 Reverse transcription reaction system

[0066]

[0067] 3. Design of quantitative PCR primers

[0068] Search for the gene sequence in the Phytozome database, and download the genomic CDS sequence of GmVQ32 as shown in SEQ ID NO.1 and the protein sequence of GmVQ32 as shown in SEQ ID NO.2 from the website; and design primers on the NCBI website according to the genomic sequence and synthesize its specific quantitative amplification primers:

[0069] GmVQ32_qF (SEQ ID NO.3): 5'-CTCTCATGGATGGCATGGCTA-3';

[0070] GmVQ32_qR (SEQ ID NO.4): 5'-TTGTGGTGGCCAATTGTTGTG-3'.

[0071] 4. Real-time fluorescence quantitative PCR

[0072] ① Use Actin3 as the internal reference gene;

[0073] ② Dilute the cDNA of all samples 1-fold with ddH2O as the template for the quantitative PCR reaction;

[0074] ③ The real-time PCR system performs fluorescence quantitative PCR according to the reaction system in Table 2 below. The reaction program is: pre-denaturation at 95°C for 30 s; 39 cycles (denaturation at 95°C for 5 s; annealing at 60°C for 30 s); the melting curve is 95°C, 10 s; 54.3°C, 5 s; 94.3°C, 5 s;

[0075] Table 2 Real-time fluorescence quantitative PCR reaction system

[0076]

[0077] ④ Process the data, and the relative expression analysis adopts the 2 -△△CT -ΔΔCt method.

[0078] 5. Result analysis

[0079] The expression results of the GmVQ32 gene under normal phosphorus and low phosphorus levels are as Figure 2 shown. The experimental results show that the GmVQ32 gene is induced to express under low phosphorus stress in soybean roots.

[0080] Example 2: Cloning of the GmVQ32 gene and construction of the vector

[0081] 1. Construction of pLB-GmVQ32 Zero-background Expression Vector

[0082] ① Amplify the target fragment: Extract the RNA from the root samples of Huachun soybeans, and reverse transcribe it into cDNA using a reverse transcription kit (Vazyme, Nanjing). Then, using the cDNA of Huachun No. 6 soybean variety as a template, design and synthesize primers:

[0083] GmVQ32_pLB_F (SEQ ID NO.5): 5’-ACCACCCAAATTCCTGAACGA-3’;

[0084] GmVQ32_pLB_R (SEQ ID NO.6): 5’-GGGGAACTCAACATTGAAAAAGC-3’,

[0085] Amplify the full-length CDS sequence of GmVQ32 according to the reaction system in Table 3 below. The reaction program is: pre-denaturation at 95°C for 3 min; 34 cycles (denaturation at 95°C for 15 s; annealing at 55°C for 15 s; extension at 72°C for 1 min / kb); final extension at 72°C for 5 min; obtain the PCR product and store it at 12°C;

[0086] Table 3 Fragment Amplification Reaction System

[0087]

[0088] ② Purify the PCR product:

[0089] S1. Perform agarose gel electrophoresis on the PCR product, and purify the PCR product with the correct band using a purification kit. Subsequently, centrifuge the PCR product, measure the volume, and if the volume is less than 100 μL, supplement it with ddH2O to 100 μL; add 5 times the volume of Buffer GDP, mix well using a vortex mixer to obtain a mixed solution. Place the adsorption column on the collection tube, transfer the mixed solution to the adsorption column, and then place the adsorption column and the collection tube in the centrifuge for centrifugation. Set the centrifuge speed to 12000 rpm and the centrifugation time to 1 min, and discard the filtrate;

[0090] S2. Prepare the Buffer GW stock solution, add 80 ml of absolute ethanol to every 100 mL of the Buffer GW stock solution, mix well to obtain the Buffer GW working solution. Add 700 μL of the Buffer GW working solution to the adsorption column, and then place the adsorption column and the collection tube in the centrifuge for centrifugation. Set the centrifuge speed to 12000 rpm and the centrifugation time to 1 min, and discard the filtrate;

[0091] S3. Repeat the operation of S2; then place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 30 μL Elution Buffer to the center of the adsorption column, let it stand for 2 min, put the adsorption column and the collection tube into the centrifuge for centrifugation, set the centrifuge speed to 12,000 rpm, and the centrifugation time to 1 min; discard the adsorption column, and then store the purified PCR product at -20 °C.

[0092] ③ Ligate the target gene to the pLB zero-background expression vector:

[0093] Ligate the target fragment according to the reaction system in Table 4 below to obtain pLB-GmVQ32. The reaction program is: denature at 20 °C for 2 min; anneal at 70 °C for 5 min; extend at 22 °C for 20 min.

[0094] Table 4 Ligation reaction system

[0095]

[0096] ④ Transformation of Escherichia coli: Pipette 10 μL of pLB-GmVQ32 and add it to 100 μL of DH5α Escherichia coli competent cells, gently pipette and mix well, then incubate on ice for 30 min, and then perform heat shock at 42 °C for 45 s, immediately transfer it to ice for cooling for 2 min; then transfer it to the laminar flow hood, add 500 - 700 μL of LB liquid medium to the pLB-GmVQ32 ligation product, and then place it on a shaker at 37 °C for 1 h, the shaker speed is 220 rpm, to obtain a bacterial solution. Spread the bacterial solution on a plate containing Amp and culture it overnight at 37 °C. Pick a single clone and shake the bacteria for detection and sequencing.

[0097] 2. Construction of the pTF101-GmVQ32 overexpression vector

[0098] ① Amplify the target fragment: Using the cDNA of the soybean variety Huachun 6 as a template, design and synthesize primers:

[0099] GmVQ32_pTF101_F (SEQ ID NO.7):

[0100] 5’-gagaacacgggggactctagaATGACTGCCACAGCTGATCAATG-3’;

[0101] GmVQ32_pTF101_R (SEQ ID NO.8):

[0102] 5’-cgatcggggaaattcgagctcTCACATAAATAAATCATCGATATCATTGG-3’,

[0103] The full-length CDS sequence of GmVQ32 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;

[0104] ② Linearized vector: Double digest the pTF101 plasmid DNA with restriction endonucleases SacI and XbaI to obtain the intermediate vector pTF101;

[0105] ③ Ligation: Ligate the target fragment with the intermediate vector pTF101 according to the reaction system in Table 5 below. Reaction procedure: Incubate at 37°C for 30 min to obtain pTF101-GmVQ32;

[0106] Table 5 Ligation reaction system

[0107]

[0108] ④ E. coli transformation: The method and conditions are the same as those for E. coli transformation in the construction of the pLB-GmVQ32 zero-background expression vector;

[0109] ⑤ Agrobacterium transformation: After sequencing and comparing single clones of pTF101-GmVQ32, extract the plasmid using a plasmid extraction kit. Pipette 4 μL of plasmid into 100 μL of GV3101 competent Agrobacterium, incubate on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, heat shock at 37°C for 5 minutes, and then cool the plasmid on ice for 5 minutes. Transfer the plasmid to a clean bench and add 500-700 μL of YEP liquid medium. Incubate in a shaker at 28°C and incubate at 220 rpm for 2-3 hours to obtain a bacterial suspension. Spread the suspension onto a plate containing Spe and incubate at 28°C for 2-3 days. Pick a single clone and shake it for testing. Add an equal volume of 50% glycerol to the successful suspension and store it in a -80°C freezer.

[0110] 3. Construction of Super1300-GmVQ32 subcellular localization vector

[0111] ① Amplify gene fragment: Using the pLB-GmVQ32 plasmid as template cDNA, CE Design designed recombination cloning primer Super1300-GmVQ32-F (SEQ ID NO.9):

[0112] 5'-ccaaatcgactctagtctagaATGACTGCCACAGCTGATCAATG-3';

[0113] Super1300 - GmVQ32 - R (SEQ ID NO.10):

[0114] 5’ - catggtaccggatccactagtCATAAATAAATCATCGATATCATTGGAG - 3’,

[0115] Amplify the full - length CDS sequence of GmVQ32 according to the reaction system in Table 3 to obtain the amplified gene, where the amplification method and conditions are the same as those in the amplification system of Table 3. After the amplified gene is subjected to gel electrophoresis, use an agarose gel kit to recover and purify the target band.

[0116] ② Linearize the vector: Double - digest the Super1300 empty plasmid with XbaI and SpeI restriction endonucleases to obtain the intermediate vector Super1300.

[0117] ③ Carry out the ligation of the amplified gene, GFP fragment and the intermediate vector Super1300, as well as the transformation of the ligation product into Escherichia coli and Agrobacterium. The methods and conditions of ligation, Escherichia coli transformation, and Agrobacterium transformation are the same as those in the steps of constructing the pTF101 - GmVQ32 overexpression vector, which will not be elaborated here. Obtain the Super1300 - GmVQ32 - GFP vector for subsequent experiments.

[0118] 4. Construction of the pGBKT7 - GmVQ32 recombinant protein vector

[0119] ① Amplify the gene fragment: Using the pLB - GmVQ32 plasmid as a template, design recombinant cloning primers with CE Design:

[0120] pGBKT7 - GmVQ32 - F (SEQ ID NO.11):

[0121] 5’ - atggccatggaggccgaattcATGACTGCCACAGCTGATCAATG - 3’;

[0122] pGBKT7 - GmVQ32 - R (SEQ ID NO.12):

[0123] 5’ - ccgctgcaggtcgacggatccCCATAAATAAATCATCGATATCATTGG - 3’,

[0124] Amplify the full - length CDS sequence of GmVQ32 according to the reaction system in Table 3 to obtain the amplified gene, where the amplification method and conditions are the same as those in the amplification system of Table 3. After the amplified gene is subjected to gel electrophoresis, use an agarose gel kit to recover and purify the target band.

[0125] ② Linearized vector: The pGBKT7 plasmid was digested with the restriction endonucleases BamHI and EcoRI to obtain the intermediate vector pGBKT7.

[0126] ③ Ligation: Ligation of the amplified gene with the intermediate vector pGBKT7 and transformation of the ligation product into Escherichia coli were carried out. The methods and conditions of ligation and Escherichia coli transformation were the same as those in the ligation and Escherichia coli transformation steps in the construction of the pTF101-GmVQ32 overexpression vector, which will not be elaborated here. After sequencing and correct alignment, the plasmid was extracted and stored at -20 °C for later use to obtain the pGBKT7-GmVQ32 vector for subsequent experiments.

[0127] 5. Construction of the GmVQ32 gene editing vector

[0128] sgRNAs targeting GmVQ32 for gene editing were designed on the CRISPR-P 2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2). Two editing targets were selected from the exon region of GmVQ32 and named as follows:

[0129] GmVQ32-guide2 (ATAGCCATGCCATCCATGAGAGG);

[0130] GmVQ32-guide35 (GTGCATTCAAACAAATCCGAAGG),

[0131] According to the requirements of the CRISPR / Cas9 system, two pairs of primers were designed and synthesized. The specific primer sequences are as follows:

[0132] GmVQ32-guide2-F: 5’-GGATTGTAGCCATGCCATCCATGAG-3’;

[0133] GmVQ32-guide2-R: 5’-AAACCTCATGGATGGCATGGCTACA-3’;

[0134] GmVQ32-gu ide35-F: 5’-GGATTGTGCATTCAAACAAATCCGA-3’;

[0135] GmVQ32-guide35-R: 5’-AAACTCGGATTTGTTTGAATGCACA-3’,

[0136] The oligonucleotides were annealed into double strands, ligated with the BsaI-digested vector pUC19 plasmid, and then transferred into Escherichia coli DH5α. After successful detection of the bacterial solution, the plasmid was extracted and transferred into Agrobacterium tumefaciens K599. An equal volume of 50% glycerol was added to the successfully detected bacterial solution, and it was stored at -80 °C to obtain the GmVQ32 gene knockout vector.

[0137] Example 3: Arabidopsis thaliana genetic transformation

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

[0139] The wild-type Arabidopsis thaliana seeds (Col-0) were soaked in pure water and vernalized at 4 °C for 3 days. After vernalization, they were evenly sown on nutrient soil, sprayed with an appropriate amount of water, covered with a plastic film to keep moist, and placed in the dark for 2-3 days to break seed dormancy. When Arabidopsis thaliana had 4-6 leaves, thinning was carried out, leaving 4-5 plants per pot. When the main stem grew to 1 cm during the first bolting, the main stem was cut off to increase branching. When Arabidopsis thaliana was in full bloom next time, it was transformed with the Agrobacterium solution.

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

[0141] The preservation solution of Agrobacterium tumefaciens GV3101 carrying the target vector was mixed with 100 mL of YEP (gen+spe+rif) liquid to obtain a bacterial solution. The bacterial solution was placed in a constant temperature shaker at 28 °C and shaken at a speed of 200 rpm overnight until the OD600 value of the bacterial solution reached 0.6-0.8. Subsequently, the bacterial solution was centrifuged at a speed of 5000 rpm for 10 minutes. Then, the supernatant was discarded, and the Agrobacterium was resuspended with one volume of fresh infiltration solution (5% sucrose + 1 / 2 MS + Silwet L-77 0.02%) and transferred to a 500 mL beaker. The above-ground part of the Arabidopsis thaliana in full bloom was immersed in the Agrobacterium solution for 1-2 minutes, then the soaked plants were taken out and the transformed Arabidopsis thaliana was wrapped with a plastic film to ensure high humidity. After dark treatment for 12-16 h, the plastic film was removed the next day, and the plants were placed in an incubator for cultivation and growth. When mature, the T0 generation seeds were harvested.

[0142] 3. Identification of transgenic Arabidopsis thaliana offspring

[0143] Dry the mature T0 generation seeds and then vernalize them for multiplication. After two true leaves have fully grown, spray herbicides regularly multiple times. Screen out false positive seedlings. After the plants grow vigorously, select fresh and tender leaves and quickly extract DNA with TPS extraction solution: Take young and tender plant leaves and put them into a 1.5 mL sterilized centrifuge tube, add 200 μL of TPS extraction solution, grind them with a grinder, then perform a 10-minute metal bath at 80 °C, and then place the centrifuge tube in a centrifuge for centrifugation. The centrifugation speed is 12,000 rpm and the centrifugation time is 10 minutes; Take 20 μL of the supernatant, add 50 μL of double-distilled water (ddH2O) to the supernatant, pipette and mix evenly, and take 2 μL during the PCR reaction.

[0144] Perform DNA molecular level identification according to the reaction system in Table 6 below. The reaction program is: pre-denaturation at 95 °C for 3 minutes; 28 cycles (denaturation at 95 °C for 30 seconds; annealing at 58 °C for 30 seconds; extension at 72 °C for 1 minute / kb); final extension at 72 °C for 5 minutes; preservation at 12 °C.

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

[0146] After the PCR reaction is completed, use 1% agarose gel electrophoresis for detection. If the band length is correct, it can be preliminarily determined that it is transgenic material. Wait for the T1 generation Arabidopsis thaliana seeds to mature and be harvested, and identify the generations up to T4 using this method. Extract the RNA of different Arabidopsis thaliana lines, and through fluorescence quantitative identification, select the lines with high expression levels for large-scale seed multiplication for subsequent experiments.

[0147] Table 6 Reaction system for DNA molecular level identification

[0148]

[0149] 4. Transient transformation of Nicotiana benthamiana

[0150] Respectively inoculate the Agrobacterium (P19) carrying Super1300-GFP and Super1300-GmVQ32-GFP into 50 mL of YEP, and at the same time add antibiotics (50 μL Kan + 25 μL Rif) to obtain the bacterial solution. Then place the bacterial solution in a shaker at 28 °C for 16 hours of culture until the OD600 is 0.6 - 0.8.

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

[0152] The resuspended bacterial solution was carefully injected into the abaxial surface of tobacco leaves using a small syringe (without the needle). After culturing for about 2 days, the GFP fluorescence signal of tobacco epidermal cells was observed at a wavelength of 488 nm under a laser confocal microscope. Then, it was stained with DAPI dye for 3 - 5 minutes, and the blue fluorescence signal was observed at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

[0153] Experimental results:

[0154] As Figure 3 shown, eGFP corresponds to the GFP channel; DAPI corresponds to the ultraviolet channel, and the DAPI dye binds to DNA and emits blue fluorescence; Merge represents the merged image of GFP and DAPI; Bright field is the bright field. Super1300 - GFP (corresponding to GFP in the figure) and Super1300 - GmVQ32 - GFP (corresponding to GmVQ32 - GFP in the figure) were transiently expressed in tobacco abaxial epidermal cells. The tobacco epidermal cells transformed with the Super1300 - GFP empty vector had green fluorescence in the cell membrane, nucleus, and cytoplasm, while the Super1300 - GmVQ32 - GFP expression vector mostly emitted green fluorescence only in the nucleus in tobacco leaves, and its green fluorescence overlapped with the DAPI blue fluorescence, indicating that the GmVQ32 protein is localized in the nucleus.

[0155] Example 4: Verification of GmVQ32 yeast self - activation and interaction verification

[0156] 1. Verification of GmVQ32 yeast self - activation

[0157] ① Three treatment groups, namely experimental group 1, positive control group, and negative control group, were set up. Among them, the plasmids transfected in experimental group 1 were pGBKT7-GmVQ32 and pGADT7, the plasmids transfected in the positive control group were pGADT7-T and pGBKT7-p53, and the plasmids transfected in the negative control group were pGADT7-T and pGBKT7-lam. Each treatment group was processed as follows: Take 4 μL of each of the two plasmids in each treatment group, add 10 μL of denatured salmon sperm DNA and 500 μL of PEG / LiAC to 100 μL of Y2HGold yeast competent cells, gently pipette and mix well, then incubate at 30 °C for 30 minutes to allow the plasmid DNA to fully bind to the yeast cells. Then, heat shock the plasmid DNA-yeast cell mixture in a 42 °C water bath for 15 minutes, and then centrifuge in a centrifuge with a rotation speed set at 5000 rpm for 45 s. Discard the supernatant, add 50 μL of RNase-free ddH2O to resuspend the cells to obtain 3 kinds of transformants.

[0158] ② Spread the above 3 kinds of transformants on -Trp-Leu double-deficient SD medium and incubate them upside down at 28 °C for 2 - 3 d to observe whether the recombinant plasmids can be normally expressed.

[0159] ③ Pick 3 kinds of single colonies on -Trp-Leu double-deficient SD medium, dilute them with ddH2O, and streak them on -Trp-Leu double-deficient SD medium coated with X-α-Gal, and incubate them upside down at 28 °C for 1 d to observe whether the colonies turn blue.

[0160] ④ Pick 3 kinds of single colonies on -Trp-Leu double-deficient SD medium, dilute them with ddH2O, and spot them on -Trp-Leu double-deficient SD medium with 4 different concentrations of ABA and coated with X-α-Gal, and incubate them upside down at 28 °C for 1 d to observe whether the colonies turn blue.

[0161] The results of self-activation verification are as Figure 4 shown. The GmVQ32 protein has self-activation activity, and the self-activation is inhibited in 400 ng / mL ABA.

[0162] 2. Interaction verification of GmVQ32 protein

[0163] ① Predict the candidate genes of the GmVQ32 protein on the SMART online website, search for the gene sequences in the Phytozome database, and design primers on the NCBI website according to the genomic sequences. The methods and conditions for gene cloning and vector construction are the same as in Example 2. Extract the AD plasmid of the interacting gene and store it at -20 °C for standby.

[0164] ② Five experimental groups of BD-GmVQ32+AD-Gmlyma.12G20640, BD-GmVQ32+AD-Gmlyma.13G294500, BD-GmVQ32+AD, AD-Gmlyma.12G206400+BD, and AD-Gmlyma.13G294500+BD, a positive control group of BD-53+AD-T, and a negative control group of BD-lam+AD-T, a total of 7 treatment groups, were each transformed into yeast competent cells to obtain 7 kinds of transformants.

[0165] ② The above 7 kinds of transformants were respectively spread on -Trp-Leu double-deficient SD medium and incubated in an inverted position at 28 °C for 2 - 3 d to observe whether the recombinant plasmid could be normally expressed.

[0166] ③ Seven single colonies on the -Trp-Leu double-deficient SD medium were picked and diluted 4 different times (10 0 , 10 -1 , 10 -2 , 10 -3 ) with ddH2O, and then spotted onto SD / -Trp-Leu / X-α-Gal / ABA (400 ng / mL) medium and incubated in an inverted position at 28 °C for 1 d to observe whether the colonies turned blue.

[0167] The verification results of the interaction of GmVQ32 protein are as Figure 5 , showing that the GmVQ32 protein interacts with the candidate genes Glyma.12G206400 and Glyma.13G294500 proteins.

[0168] 3. Expression analysis of GmVQ32 interacting genes

[0169] The quantitative primer sequences of the two interacting genes are as follows:

[0170] 12G206400-qF (SEQ ID NO.15): 5'-AGGTCCGTGACGAATATCGC-3';

[0171] 12G206400-qR (SEQ ID NO.16): 5'-ACCCAAAGAACCAGTGCCAT-3';

[0172] 13G294500-qF (SEQ ID NO.17): 5'-GGTAGAGGTGGCTACGGGA-3';

[0173] 13G294500-qR (SEQ ID NO.18): 5'-AGAACCCAAAGAACCAGCACC-3',

[0174] Real-time fluorescence quantitative PCR method. Specifically, the PCR method and conditions are the same as those in Example 1.

[0175] The expression results of the interacting genes under normal phosphorus and low phosphorus levels are as Figure 6 shown. The interacting genes Glyma.12G206400 and Glyma.13G294500 are induced to express in the roots of soybeans under low phosphorus stress.

[0176] Example 5 Phenotypic identification of transgenic Arabidopsis

[0177] ① Pre-culture: The transgenic Arabidopsis was randomly divided into three groups: OE-2, OE-6, and OE-8 groups, and wild-type Arabidopsis (WT) was used as the control group. The above four groups of Arabidopsis were soaked and shaken in 10% sodium hypochlorite solution for 10 min for disinfection, and then washed 5 times with sterile water. The washed seeds were placed in a refrigerator at 4 °C for vernalization. The vernalization time was 3 d, and then the seeds were planted on 1 / 2 MS medium and transferred to the culture room for vertical culture. When the roots grew to about 1 cm, phosphorus treatment was carried out.

[0178] ② Phosphorus treatment: MS media with concentration gradients of 500 μmol / L (NP), 0 μmol / L, 5 μmol / L, and 100 μmol / L KH2PO4 were set, and the pH value was adjusted to 5.8. Arabidopsis with consistent growth was transferred to this medium to keep the roots in an elongated state. After vertical culture for about 2 weeks, the phenotypes were photographed and recorded, and the root length, number of lateral roots, fresh weight of the above-ground part, and fresh weight of the underground part were measured.

[0179] The phenotypic identification results of Arabidopsis and the statistical results of biomass indicators are as Figure 7 shown. Heterologous overexpression of GmVQ32 in Arabidopsis can alleviate the inhibitory effect on the growth and development of Arabidopsis under low phosphorus stress. Compared with the wild type, heterologous overexpression of GmVQ32 in Arabidopsis can enhance its low phosphorus tolerance by increasing the main root length, number of lateral roots, fresh weight, etc. of the plant.

[0180] Example 6 Identification of low phosphorus tolerance of soybean hairy roots

[0181] ① Transformation of Agrobacterium rhizogenes K599: The gene-edited GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), the overexpressed GmVQ32 gene (pTF101-GmVQ32 (OE)), and the empty vector control gene (pTF101 (CK)) were transformed into Agrobacterium rhizogenes K599. After identifying positive single bacteria, they were amplified and cultured for preservation.

[0182] ② Infection: Shake the K599 Agrobacterium without any transformation and the K599 Agrobacterium carrying the gene editing vector and the overexpression vector until OD600 = 0.6, centrifuge and resuspend with the resuspension solution. Select plump and disease-free No. 13 Brazilian soybeans, plant them on water-absorbent vermiculite, and cover them with dry vermiculite. After 2 - 3 days, the above-ground part of the soybeans grows to 2 - 3 cm. Use a 1 mL syringe to inject and infect 1 - 2 cm below the cotyledons of the soybeans. After completion, culture in high humidity for about 1 week. After young roots grow from the injection wounds, cover them with vermiculite, water more to ensure that the vermiculite is always in a moist state, and then continue to culture for about 1 week.

[0183] ③ Identification of the hairy root phenotype: Wait until the hairy roots develop to about 5 cm, take them out without damaging the hairy roots, cut off the primary roots, set the Hoagland solution with KH2PO4 concentrations of 500 μmol / L and 5 μmol / L at pH = 5.8, and hydroponically culture for 14 days. Select the lateral roots on the main root, extract DNA to detect positive roots. Extract the RNA from the roots of each treatment for fluorescence quantitative analysis; measure the fresh weights of the above-ground and underground parts of the soybean plants, weigh the dry weights after the samples are completely dried, take the dry samples to determine the total phosphorus content, and take at least three biological replicates for each data. Store the collected samples at -80 °C.

[0184] The identification results of the GmVQ32 hairy root phenotype are as Figure 8 shown. The comparison results show that overexpressing pTF101-GmVQ32 can enhance the tolerance of soybeans to low phosphorus stress, and gene editing of pCas9-GmVQ32 will enhance the sensitivity of soybeans to low phosphorus stress.

Claims

1. Use of the soybean low-phosphorus tolerance gene GmVQ32 in improving the low-phosphorus stress tolerance ability of plants, characterized in that, The nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.

2.

2. According to the application described in claim 1, the plant is Arabidopsis thaliana or soybean.

3. An expression vector for regulating plant adaptation to low phosphorus stress, characterized in that, The expression vector contains the soybean low phosphorus tolerance gene GmVQ32, and a plasmid carrying the soybean low phosphorus tolerance gene GmVQ32, and the plasmid is a pCas9 or pTF101 vector; The nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or soybean.

4. A host cell for regulating plant adaptation to low phosphorus stress, characterized in that, The host cell contains the soybean low phosphorus tolerance gene GmVQ32, the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or soybean.

5. The host cell according to claim 4, characterized in that, The host cell is an Escherichia coli or Agrobacterium cell.

6. A method for promoting the growth of Arabidopsis thaliana in a low-phosphorus stress environment, characterized in that, By overexpressing the soybean low phosphorus tolerance gene GmVQ32 in Arabidopsis thaliana to promote the growth of Arabidopsis thaliana, the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.

2.

7. A method for promoting the growth of soybeans in a low-phosphorus stress environment, characterized in that, By overexpressing the soybean low phosphorus tolerance gene GmVQ32 in soybean to promote the growth of soybean, the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown in SEQ ID NO.1, or the soybean low phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown in SEQ ID NO.2.

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