Application of soybean low phosphorus tolerance gene GmVQ32 in improving plant tolerance to low phosphorus stress

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a soybean low-phosphorus-tolerant gene GmVQ32 in improving plant low-phosphorus-stress tolerance. The soybean low-phosphorus-tolerant gene GmVQ32 of the application positively regulates the response of a plant root system to low-phosphorus stress, enhances the root system growth and phosphorus absorption efficiency of the plant, and thus improves the adaptability of the plant to a low-phosphorus environment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a soybean low-phosphorus-tolerant gene GmVQ32 in improving plant low-phosphorus-tolerance. BACKGROUND

[0002] Soybean is an important protein and oil crop in the world, and plays a very important role in agricultural production. Phosphorus is one of the three essential elements for plant growth and metabolism, it is not only a component of plant body, 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. Phosphorus can not only promote plant growth and development, but also enhance plant stress resistance and improve crop yield, which is crucial for crops.

[0003] There are about one billion mu of land in China in a phosphorus-deficient state, accounting for two-thirds of the total cultivated land area. Plants can only obtain phosphorus in the form of inorganic phosphate (Pi), and the pH value of acid red soil in South China is low, and phosphorus is easily oxidized and fixed by metal ions such as iron (Fe) and aluminum (Al) in acid soil, and 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 yield and economic benefits that are difficult to meet people's production needs, and soil phosphorus deficiency has become an important factor limiting soybean yield and quality. At present, there are also related researches taking soybean seedlings as the research object to explore the method to help soybean survive the critical phosphorus deficiency period. There are two main ways to make up for it, one is to increase the application amount of phosphorus fertilizer to improve the phosphorus supply of 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 second is to improve the genetic characteristics of soybean for phosphorus absorption and utilization, and to explore the genes related to low-phosphorus tolerance, and to take phosphorus-efficient soybean varieties as the research object to obtain low-phosphorus-tolerant soybean varieties through biotechnology. In recent years, the main research on soybean phosphorus at home and abroad has focused on the mechanism of root system traits controlling plant phosphorus efficient absorption and utilization, including regulation of root system architecture to obtain available phosphorus, physiological and molecular mechanisms of root exudates to activate and utilize insoluble phosphorus, rhizosphere microbial interactions, and phosphorus signal networks, and some progress has been made.

[0004] Although the screening and functional identification of the genes related to phosphorus absorption and utilization provide a large number of references and basis for subsequent research, we should also see that there are still a large number of blanks in the field of soybean phosphorus, and many key genes and regulatory networks have not been analyzed, which restricts the potential of soybean production. Therefore, it is of great significance to analyze the molecular mechanism related to low phosphorus tolerance of soybean, to mine and identify the key genes of high phosphorus efficiency, to improve the phosphorus absorption and utilization efficiency of soybean, and to cultivate new varieties of soybean with high phosphorus efficiency, so as to improve the single yield of soybean in South China. SUMMARY

[0005] In order to overcome the problems in the background art, the purpose of the present application is to provide an application of a soybean low phosphorus tolerance gene GmVQ32 in improving the low phosphorus stress tolerance of plants, wherein the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the amino acid sequence encoded by the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 2.

[0006] The soybean low phosphorus tolerance gene GmVQ32 of the present application can positively regulate the response of plant root system to low phosphorus stress, enhance the root growth and phosphorus absorption efficiency of plants, and thus improve the low phosphorus stress tolerance of plants.

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

[0008] The purpose of the present application is to provide an expression vector for regulating the adaptation of plants to low phosphorus stress, wherein the vector comprises a soybean low phosphorus tolerance gene GmVQ32, and a plasmid carrying the soybean low phosphorus tolerance gene GmVQ32, and the plasmid is pCas9 or pTF101 vector; the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the amino acid sequence encoded by the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 2.

[0009] The purpose of the present application is to provide a host cell for regulating the adaptation of plants to low phosphorus stress, wherein the nucleotide gene sequence of the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the amino acid sequence encoded by the soybean low phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 2.

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

[0011] The fourth object of the present application is to provide a method for promoting the growth of Arabidopsis thaliana in a low-phosphorus stress environment, by overexpressing the soybean low-phosphorus tolerance gene GmVQ32 in Arabidopsis thaliana to promote the growth of Arabidopsis thaliana, wherein the nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0012] The fifth object of the present application is to provide a method for promoting the growth of soybean in a low-phosphorus stress environment, by overexpressing the soybean low-phosphorus tolerance gene GmVQ32 in soybean to promote the growth of soybean, wherein the nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown as SEQ ID NO. 2.

[0013] In the method for promoting the growth of plants in a low-phosphorus stress environment by using the soybean low-phosphorus tolerance gene GmVQ32 of the present application, the method comprises the following steps:

[0014] S1, extracting soybean root tip RNA and reverse transcribing it into cDNA, and then amplifying and cloning the coding DNA sequence (CDS) of the soybean low-phosphorus tolerance gene GmVQ32 by PCR, wherein the length of the CDS sequence is 666 bp;

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

[0016] S3, connecting the CDS after sequencing and comparison to an expression vector to obtain a recombinant vector, sequencing the recombinant vector to verify the connection, and transforming the recombinant vector into Agrobacterium after ensuring that the connection is correct;

[0017] S4, using Arabidopsis thaliana as a carrier to verify the role of the soybean low-phosphorus tolerance gene GmVQ32 in the low-phosphorus stress tolerance of plants: introducing the GmVQ32 gene into Arabidopsis thaliana by using an Agrobacterium-mediated genetic transformation method to obtain transgenic Arabidopsis thaliana plants, taking a T5 generation homozygous strain thereof, and screening a strain with a high expression amount of the soybean low-phosphorus tolerance gene GmVQ32 by fluorescence quantification;

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

[0019] S6, while constructing gene editing GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), overexpression GmVQ32 gene (pTF101-GmVQ32 (OE)) and empty vector control gene (pTF101 (CK)), transforming pCas9-GmVQ32 (GmVQ32), pTF101-GmVQ32 (OE), pTF101 (CK) into K599 Agrobacterium, and then using the agrobacterium-mediated genetic transformation method to introduce GmVQ32 gene into the target plant;

[0020] S7, the above-mentioned target plants carrying pCas9-GmVQ32 (GmVQ32), pTF101-GmVQ32 (OE) and pTF101 (CK) Agrobacterium are subjected to low phosphorus tolerance phenotype identification, and the results show that the root elongation, lateral root and dry weight of the OE (overexpression) plant are significantly improved compared with the CK (control check), and through the above-mentioned experiment, it is shown that overexpression of GmVQ32 gene can enhance the tolerance of soybean to low phosphorus stress.

[0021] The application has the following beneficial effects: the GmVQ32 gene is up-regulated under low phosphorus stress, and its expression amount is obviously increased with the extension of the phosphorus treatment time;

[0022] Under different phosphorus concentration treatment conditions, overexpression of GmVQ32 can obviously increase the biomass of the transgenic plant, GmVQ32 regulates the response of the plant root system to low phosphorus stress in a positive way, enhances the root growth and phosphorus absorption efficiency of the plant, and thus improves the adaptability of the plant to the low phosphorus environment;

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

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

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

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

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

[0028] Figure 4 GmVQ32 yeast self-activation verification results;

[0029] Figure 5 GmVQ32 protein and candidate gene interaction verification results;

[0030] Figure 6 Glyma.12G206400 and Glyma.13G294500 gene quantitative PCR results under different phosphorus concentration treatment conditions;

[0031] Figure 7 Transgenic Arabidopsis and wild-type Arabidopsis (Col-0) whole plant phenotype, main root length, lateral root length and fresh weight after 14d under normal phosphorus and low phosphorus conditions;

[0032] Figure 8 Gene editing GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), overexpression GmVQ32 gene (pTF101-GmVQ32 (OE)) and empty vector control gene (pTF101 (CK)) soybean chimera whole plant phenotype, aboveground dry weight, underground dry weight, and root relative expression level after 14d under normal phosphorus and low phosphorus conditions. DETAILED DESCRIPTION

[0033] In order to further illustrate the technical means adopted by the present application, the following will be further described in detail in combination with the drawings and specific examples in the specification, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] The cDNA nucleotide sequence of the GmVQ32 gene used in the present application 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 GmVQ32 encoded protein is shown as SEQ ID NO. 2:

[0044] MTATADQCMHEFYQQPLMDGMAMDPSIEGLMDASMSSSSEGMIMLSPSNSHNNTTIGHHNLTPKGCAFKQIRRRSRASK

[0045] STPITLLKANTSNFRALVQQFTGCPTTTAMSLAIHKGPVTLNFQQGGSKQHIHHHHHTKITTRGAMPPFIGTISSNQNQ VSVPLPKQHLMQEQQSGHFLPTSSGNSYRPINCMDDGLIFDNDFSLHELTVNAISNDIDDLFM*.

[0046] The CDS sequence of the GmVQ32 gene clone was compared with the known sequence of the gene in the NCBI database. Figure 1 Phylogenetic analysis of the VQ family genes in Arabidopsis thaliana, rice, and soybean.

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

[0048] 1. Soybean material processing

[0049] 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 temperature of 26℃, with a photoperiod of 16 hours of light followed by 8 hours of darkness. The plants were cultured for 4-5 days (days) until the cotyledons began to open slightly. Then, the plants were transferred to hydroponic culture media containing normal phosphorus (NP, 500 μM KH2PO4) and low phosphorus (LP, 5 μM KH2PO4). Root samples were collected at 0h, 3h, 6h, 12h, 24h, 36h, 2d, 5d, 7d, and 14d. The collected samples were wrapped in aluminum foil and stored at -80℃.

[0050] 2. RNA extraction and reverse transcription

[0051] RNA was extracted from soybean root samples treated with different phosphorus concentrations and those preserved after hydroponics, and then reverse transcribed into cDNA using a reverse transcription kit (Vazyme, Nanjing). The cDNA was then used as a template for real-time quantitative PCR. The specific steps are as follows:

[0052] (1) RNA extraction

[0053] 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;

[0054] 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 50-100mg of powder into the EP tube.

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

[0056] (4) Take 600 μL of supernatant from the EP tube and transfer it to a new 1.5 mL centrifuge tube, then add 300 μL of anhydrous ethanol to the centrifuge tube, and then use a vortex mixer to blow 20-25 times until the liquid in the centrifuge tube is turbid and has white foam, so as to fully react to obtain a reaction solution;

[0057] (5) Prepare a centrifugal column and a collection tube, place the centrifugal column in the collection tube, and then transfer the reaction solution to the centrifugal column, and then place the centrifugal column containing the reaction solution and the collection tube into a centrifuge, and the temperature of the centrifuge is 4°C, the 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 washing solution, and then place the centrifugal column and the collection tube into a centrifuge, and the temperature of the centrifuge is 4°C, the 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, and prepare an incubation solution (10×DNAse I buffer 5 μL; DNAse I 5 μL; nuclease-free water 40 μL) in advance, pour 50 μL of the incubation solution into the center of the centrifugal column adsorption membrane, and stand for 15 min;

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

[0061] (9) Move the centrifugal column to an elution tube, and then add 50-200 μL of nuclease-free water to the centrifugal column, stand for 2 min, and then place the centrifugal column and the elution tube into a centrifuge, and the temperature of the centrifuge is 4°C, the 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 refrigerator at -80°C for use.

[0063] (2) Reverse transcription

[0064] The OD value (Optical Density) of the extracted RNA is measured by ultraviolet spectrophotometry or microplate spectrophotometry, and the amount of RNA (0.1 ng-1 μg) is calculated according to the OD value of the extracted RNA, and the specific calculation formula is: RNA amount=1000 / OD value. Reverse transcription is carried out by a reverse transcription kit (Vazyme, Nanjing), and the reaction system is shown in Table 1, and the reaction procedure is: incubation at 50°C for 15 min; then heated at 85°C for 5 seconds; all operations must be completed on ice, and after the reaction is completed, the cDNA is stored in a refrigerator at -20°C.

[0065] Table 1 Reverse transcription reaction system

[0066]

[0067] 3. Quantitative PCR primer design

[0068] The gene sequence was retrieved from the Phytozome database, and the GmVQ32 genome CDS sequence (as shown in SEQ ID NO. 1) and GmVQ32 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 sequence using the NCBI website.

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

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

[0071] 4. Real-time quantitative PCR

[0072] ① Actin3 was used as an internal reference gene;

[0073] ② Dilute all cDNA samples by 1-fold with ddH2O to serve as templates for quantitative PCR reactions;

[0074] ③ The real-time PCR system was used for real-time quantitative 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); the melting curve was 95℃, 10s; 54.3℃, 5s; 94.3℃, 5s.

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

[0076]

[0077] ④ Data processing: Relative expression level analysis was performed using 2... -△△CT Law.

[0078] 5. Results Analysis

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

[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 Amplification of target fragment: RNA was extracted from the root samples of Huachun soybean, and reverse transcribed into cDNA by reverse transcription kit (Vazyme, Nanjing). The cDNA of Huachun 6 soybean variety was used as a template, and primers were designed and synthesized:

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

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

[0085] The CDS full-length sequence of GmVQ32 was amplified according to the reaction system in Table 3 below, and the reaction procedure was as follows: 95°C pre-denaturation for 3 min; 34 cycles of 95°C denaturation for 15 s, 55°C annealing for 15 s, and 72°C extension for 1 min / kb; 72°C final extension for 5 min; and 12°C storage;

[0086] Table 3 Fragment amplification reaction system

[0087]

[0088] ② Purification of PCR product:

[0089] S1, the PCR product was subjected to agarose gel electrophoresis, and the correct PCR product band was purified with a purification kit. Then the PCR product was centrifuged, the volume was measured, and if the volume was less than 100 μL, ddH2O was added to 100 μL. 5 times the volume of Buffer GDP was added, and the vortex mixer was used to mix evenly to obtain a mixture. The adsorption column was fitted in the collection tube, and the mixture was transferred to the adsorption column. Then the adsorption column and the collection tube were placed in the centrifuge, the speed of the centrifuge was set to 12000 rpm, and the centrifugation time was 1 min, and the filtrate was discarded;

[0090] S2, prepare Buffer GW mother liquor, add 80 ml of anhydrous ethanol to 100 mL of Buffer GW mother liquor, mix evenly to obtain Buffer GW working solution, add 700 μL of Buffer GW working solution to the adsorption column, then place the adsorption column and the collection tube in the centrifuge, set the speed of the centrifuge to 12000 rpm, and centrifuge for 1 min, discard the filtrate;

[0091] S3, repeat S2 operation; 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, stand for 2 min, place the adsorption column and the collection tube in a centrifuge, set the speed of the centrifuge to 12000 rpm, and centrifuge for 1 min; discard the adsorption column, and then store the purified PCR product at -20°C;

[0092] ③Ligation of the target gene to the pLB zero background expression vector:

[0093] The target fragment is ligated according to the reaction system in Table 4 below to obtain pLB-GmVQ32, and the reaction procedure is as follows: denaturation at 20°C for 2 min; annealing at 70°C for 5 min; extension at 22°C for 20 min;

[0094] Table 4 Ligation reaction system

[0095]

[0096] ④E. coli transformation: 10 μL pLB-GmVQ32 is added to 100 μL DH5α E. coli competent cells, mixed gently, then placed in ice water for 30 min, and then heated at 42°C for 45 s, immediately transferred to ice for cooling for 2 min; then transferred to a clean bench, 500-700 μL LB liquid medium is added to the pLB-GmVQ32 ligation product, and then placed in a 37°C shaker for 1 h, the speed of the shaker is 220 rpm, a bacterial solution is obtained, the bacterial solution is inoculated on an Amp-containing plate, and then cultured at 37°C overnight, a single colony is shaken, and detection and sequencing are performed.

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

[0098] ①Amplification of target fragment: the cDNA of Huachun No. 6 soybean variety is used as a template, and primers are designed and synthesized:

[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 CDS full-length sequence of GmVQ32 was amplified according to the reaction system of Table 3, and the reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 34 cycles of 95℃ denaturation for 15 s, 55℃ annealing for 15 s, and 72℃ extension for 1 min / kb; 72℃ final extension for 5 min; and 12℃ storage;

[0104] ②Linearized vector: the pTF101 plasmid DNA was subjected to double enzyme digestion reaction using restriction endonucleases SacI and XbaI to obtain the intermediate vector pTF101;

[0105] ③Ligation: the target fragment was ligated with the intermediate vector pTF101 according to the reaction system in Table 5 below, and the reaction procedure was as follows: incubation at 37℃ for 30 min to obtain pTF101-GmVQ32;

[0106] Table 5 Ligation reaction system

[0107]

[0108] ④Escherichia coli transformation: the method and conditions were the same as those in the step of Escherichia coli transformation in the construction of pLB-GmVQ32 zero background expression vector;

[0109] ⑤Agrobacterium transformation: after monoclone sequencing of pTF101-GmVQ32 and comparison, the plasmid was extracted using a plasmid extraction kit. 4 μL of the plasmid was added to 100 μL of GV3101 Agrobacterium competent cells, and then subjected to ice bath for 5 min, liquid nitrogen freezing for 5 min, and heat shock at 37℃ for 5 min. Subsequently, the plasmid was transferred to an ultraclean workbench, and then 500-700 μL of YEP liquid medium was added. The mixture was placed in a 28℃ shaker and cultured at a speed of 220 rpm for 2-3 h to obtain a bacterial solution. The bacterial solution was inoculated on a plate containing Spe, and then cultured at 28℃ for 2-3 d. A single colony was picked and detected. An equal volume of 50% glycerol was added to the bacterial solution which passed the detection, and then the mixture was stored in a refrigerator at -80℃.

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

[0111] ①Amplification of gene fragment: the pLB-GmVQ32 plasmid was used as a template cDNA, and the recombinant cloning primer Super1300-GmVQ32-F (SEQ ID NO. 9) was designed by CE Design:

[0112] 5'-ccaaatcgactctagtctagaATGACTGCCACAGCTGATCAATG-3';

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

[0114] 5'-catggtaccggatccactagtCATAAATAAATCATCGATATCATTGGAG-3',

[0115] The CDS full-length sequence of GmVQ32 was amplified according to the reaction system in Table 3 to obtain an amplified gene, wherein the amplification method and conditions were the same as those in Table 3. After gel electrophoresis, the amplified gene was recovered and purified using an agarose gel kit.

[0116] 2) Linearized vector: the Super1300 empty vector was double-digested with XbaI and Spel restriction endonucleases to obtain the intermediate vector Super1300.

[0117] 3) Linking the amplified gene, GFP fragment, and intermediate vector Super1300, and E. coli transformation and Agrobacterium transformation of the linking product, wherein the linking, E. coli transformation, and Agrobacterium transformation were the same as those in the linking, E. coli transformation, and Agrobacterium transformation steps in the construction of the pTF101-GmVQ32 overexpression vector, and will not be described here again, to obtain the Super1300-GmVQ32-GFP vector for subsequent experiments.

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

[0119] 1) Amplified gene fragment: using the pLB-GmVQ32 plasmid as a template, a recombinant cloning primer was designed using 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] The CDS full-length sequence of GmVQ32 was amplified according to the reaction system in Table 3 to obtain an amplified gene, wherein the amplification method and conditions were the same as those in Table 3. After gel electrophoresis, the amplified gene was recovered and purified using an agarose gel kit.

[0125] 2. Linearization of the vector: The pGBKT7 plasmid was double-digested with restriction enzymes BamHI and EcoRI to obtain the intermediate vector pGBKT7.

[0126] 3. Ligation: The amplified gene was ligated with the intermediate vector pGBKT7, and E. coli transformation of the ligation product was performed. The method and conditions of ligation and E. coli transformation were the same as those in the ligation and E. coli transformation step in the construction of the pTF101-GmVQ32 overexpression vector, which will not be repeated here. After sequencing and comparison, the plasmid was extracted and stored at -20°C for standby, and the pGBKT7-GmVQ32 vector was obtained for subsequent experiments.

[0127] 5. Construction of GmVQ32 gene editing vector

[0128] The sgRNA for GmVQ32 gene editing was designed on the CRISPR-P 2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2). Two editing target points were selected from the exon region of GmVQ32, and were named as:

[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-guide35-F: 5'-GGATTGTGCATTCAAACAAATCCGA-3';

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

[0136] After annealing the oligonucleotide into double strand, it is connected with Bsal enzyme cut vector pUC19 plasmid and transferred into DH5a E. coli; after successful bacteria liquid detection, the plasmid is extracted and transferred into Agrobacterium K599, equal volume of 50% glycerol is added to the successfully detected bacteria liquid, and it is stored at -80°C to obtain GmVQ32 gene knockout vector.

[0137] Example 3: Genetic transformation of Arabidopsis thaliana

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

[0139] Wild-type Arabidopsis thaliana seeds (Col-0) are soaked with pure water and subjected to vernalization treatment at 4°C for 3 days. After vernalization, they are evenly sown on nutrient soil and sprayed with an appropriate amount of water, then covered with plastic film for moisture retention and placed in the dark for 2-3 days to break seed dormancy. After the Arabidopsis thaliana grows 4-6 leaves, thinning is performed, leaving 4-5 plants per pot. When the main stem is 1 cm tall, it is cut to increase branching. When the next Arabidopsis thaliana is in full bloom, Agrobacterium liquid is used for transformation.

[0140] 2. Agrobacterium liquid transformation of Arabidopsis thaliana inflorescences

[0141] Agrobacterium GV3101 carrying the target vector is mixed with 100 mL YEP (gen+spe+rif) liquid to obtain a bacterial liquid. The bacterial liquid is incubated in a constant temperature shaker at 28°C at a speed of 200 rpm overnight until the OD600 value of the bacterial liquid reaches 0.6-0.8. The bacterial liquid is then centrifuged at 5000 rpm for 10 minutes, and the supernatant is discarded. The Agrobacterium is resuspended in an equal volume of fresh infiltration liquid (5% sucrose + 1 / 2MS + Silwet L-770 0.02%) and transferred to a 500 mL beaker. The aerial part of the flowering Arabidopsis thaliana is immersed in the Agrobacterium liquid for 1-2 min, then taken out and wrapped with plastic film to ensure high humidity. Dark treatment is performed for 12-16 h, and the next day the plastic film is removed and the plants are placed in an incubator for growth. Mature T0 seeds are harvested.

[0142] 3. Identification of transgenic Arabidopsis thaliana offspring

[0143] The mature T0 generation seeds are dried and then vernalized, propagated, and after two fully expanded true leaves, herbicides are sprayed regularly and repeatedly. False positive seedlings are screened out. After the plants grow healthily, fresh leaves are selected and DNA is extracted using TPS extraction solution: plant young leaves are taken in 1.5 mL sterilized centrifuge tubes, 200 μL TPS extraction solution is added, and the leaves are ground using a grinder, then the centrifuge tubes are placed in a metal bath at 80°C for 10 min, and then the centrifuge tubes are placed in a centrifuge for centrifugation at 12000 rpm for 10 min; 20 μL supernatant is taken, and 50 μL double distilled water (ddH2O) is added to the supernatant, which is mixed uniformly by blowing, and 2 μL is taken for PCR reaction.

[0144] DNA molecular level identification is performed according to the reaction system in Table 6 below, and the reaction procedure is as follows: 95°C pre-denaturation for 3 min; 28 cycles of 95°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 1 min / kb; 72°C final extension for 5 min; and 12°C storage.

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

[0146] After the PCR reaction is completed, 1% agarose gel electrophoresis is used for detection, and the correct band length can preliminarily determine that it is a transgenic material. Mature T1 generation Arabidopsis seeds are harvested, and this method is used for identification and propagation to T4 generation. Arabidopsis RNA of different strains is extracted, and fluorescence quantification is used for identification, and strains with high expression are selected for mass propagation for subsequent experiments.

[0147] Table 6 DNA molecular level identification reaction system

[0148]

[0149] 4. Transient transformation of Nicotiana benthamiana

[0150] The Agrobacterium (P19) carrying Super1300-GFP and Super1300-GmVQ32-GFP is inoculated into 50 mL of YEP, and antibiotics (50 μL Kan+25 μL Rif) are added at the same time to obtain a bacterial solution, and then the bacterial solution is placed in a 28°C shaker for 16 h of culture until the OD600 is 0.6-0.8.

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

[0152] The resuspended bacteria solution was carefully injected into the back of tobacco leaves with a small syringe (without needle), and cultured for about 2 days, then the GFP fluorescence signal of tobacco epidermal cells was observed under laser confocal microscope at 488 nm wavelength, and then the blue fluorescence signal was observed at excitation wavelength 360 nm and emission wavelength 460 nm after DAPI dye staining for 3-5 min.

[0153] Experimental results:

[0154] As shown in Figure 3 , eGFP corresponds to the GFP channel; DAPI corresponds to the ultraviolet channel, DAPI dye binds to DNA to emit blue fluorescence; Merge represents the fusion 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 lower epidermal cells, and the tobacco epidermal cells transformed by Super1300-GFP empty vector had green fluorescence in the cell membrane, nucleus and cytoplasm, while the Super1300-GmVQ32-GFP expression vector only emitted green fluorescence in the nucleus in most of the tobacco leaves, and it overlapped with the blue fluorescence of DAPI, indicating that the GmVQ32 protein was located in the nucleus.

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

[0156] 1. Yeast self-activation verification of GmVQ32

[0157] ① Set up 3 treatment groups, i.e. experimental group 1, positive control group and negative control group, wherein the plasmids transfected in the experimental group 1 are pGBKT7-GmVQ32 and pGADT7, the plasmids transfected in the positive control group are pGADT7-T and pGBKT7-p53, and the plasmids transfected in the negative control group are pGADT7-T and pGBKT7-lam; each treatment group is treated 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, mix gently, 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 and yeast cell mixture at 42°C for 15 minutes, then centrifuge in a centrifuge at 5000 rpm for 45 seconds, discard the supernatant, and resuspend the bacterial cells with 50 μL of RNase-free ddH2O to obtain 3 types of transformants.

[0158] ② Spread the above 3 types of transformants on -Trp-Leu double deficiency SD medium, and incubate at 28°C for 2-3 days under inversion, for observing whether the recombinant plasmid can be normally expressed.

[0159] ③ Dilute the 3 types of single colonies on the -Trp-Leu double deficiency SD medium with ddH2O, streak on -Trp-Leu double deficiency SD medium coated with X-α-Gal, and incubate at 28°C for 1 day under inversion, for observing whether the colonies turn blue.

[0160] ④ Dilute the 3 types of single colonies on the -Trp-Leu double deficiency SD medium with ddH2O, and spot on -Trp-Leu double deficiency SD medium coated with X-α-Gal and containing 4 different concentrations of ABA, and incubate at 28°C for 1 day under inversion, for observing whether the colonies turn blue.

[0161] The results of self-activation verification are shown in Table 1. Figure 4 As shown in Table 1, the GmVQ32 protein has self-activation activity, and the self-activation is inhibited at 400 ng / mL ABA.

[0162] 2. GmVQ32 protein interaction verification

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

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

[0165] ② The above 7 kinds of transformants are respectively coated on-Trp-Leu double deficiency SD medium, and cultured at 28°C for 2-3d, for observing whether the recombinant plasmid can be normally expressed.

[0166] ③ Pick 7 kinds of single colonies on-Trp-Leu double deficiency SD medium and dilute them with ddH2O by 10 0 , 10 -1 , 10 -2 , 10 -3 , respectively, and then point them to SD / -Trp-Leu / X-α-Gal / ABA(400ng / mL) medium, and culture them at 28°C for 1d, for observing whether the colonies are blue.

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

[0168] 3. Expression analysis of GmVQ32 interaction genes

[0169] The quantitative primer sequences of the two interaction 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 fluorescent quantitative PCR method, specifically, the PCR method and conditions are the same as in Example 1.

[0175] The expression results of the interaction genes under normal phosphorus and low phosphorus levels are shown in Table 1. Figure 6 As shown in Table 1, the interaction genes Glyma.12G206400 and Glyma.13G294500 were induced to express in the roots of soybean under low phosphorus stress.

[0176] Example 5: Phenotype identification of transgenic Arabidopsis

[0177] ①Pre-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 a control group. The above four groups of Arabidopsis were sterilized by soaking in 10% sodium hypochlorite solution for 10 min, and then washed with sterile water for 5 times. The seeds after washing were placed in a refrigerator at 4°C for vernalization, and the vernalization time was 3d. Then the seeds were planted on 1 / 2MS medium and transferred to a culture room for vertical culture. When the roots grew to about 1cm, phosphorus treatment was performed.

[0178] ②Phosphorus treatment: MS medium with concentration gradient of 500μmol / L (NP), 0μmol / L, 5μmol / L, 100μmol / L KH2PO4 was set, and the pH value was adjusted to 5.8. The Arabidopsis with consistent growth was transferred to the medium to maintain the elongation of the roots. After vertical culture for about 2 weeks, the phenotype was recorded by taking pictures, and the root length, lateral root number, fresh weight of aboveground part and fresh weight of underground part were measured.

[0179] The phenotype identification results of Arabidopsis and the statistical results of biomass indicators are shown in Table 2. Figure 7 As shown in Table 2, overexpression of GmVQ32 in Arabidopsis can alleviate the inhibitory effect of low phosphorus stress on the growth and development of Arabidopsis. Compared with the wild type, overexpression of GmVQ32 in Arabidopsis can increase the main root length, lateral root number and fresh weight of the plant, thereby enhancing the low phosphorus tolerance.

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

[0181] ①Transformation of K599 hairy root Agrobacterium: The gene edited GmVQ32 gene (pCas9-GmVQ32 (GmVQ32)), overexpressed GmVQ32 gene (pTF101-GmVQ32 (OE)) and empty vector control gene (pTF101 (CK)) were transformed into K599 hairy root Agrobacterium. After identification of positive single bacteria, the bacteria were expanded and preserved.

[0182] ②Infection: K599 Agrobacterium without any transformation and K599 Agrobacterium with gene editing vector and overexpression vector were shaken to OD600 = 0.6, and then resuspended with resuspension solution after centrifugation. Select full and disease-free Brazilian No. 13 soybeans and plant them on water-absorbing vermiculite, covered with dry vermiculite. After 2-3 days, the above-ground part of the soybean grows to 2-3 cm, and then 1 mL syringe is used to inject and infect 1-2 cm below the soybean cotyledon. After completion, high humidity culture for about 1 week, and then cover with vermiculite after the injection wound grows young roots, and water frequently to ensure that the vermiculite is always in a wet state, and continue to culture for about 1 week.

[0183] ③Hairy root phenotype identification: When the hairy roots develop to about 5 cm, they are taken out without damaging the hairy roots, and the original roots are cut off. Set the KH2PO4 concentration of Hoagland solution to 500 μmol / L and 5 μmol / L, pH = 5.8, and hydroponic for 14 days. Select the lateral roots on the main roots, extract DNA to detect positive roots. Extract the root RNA of each treatment, and perform fluorescence quantitative analysis; measure the fresh weight of the above-ground and underground parts of the soybean plants, dry the samples completely, weigh the dry weight, and measure the total phosphorus content. Each data is taken at least three biological replicates, and the samples are stored at -80°C.

[0184] The results of GmVQ32 hairy root phenotype identification are shown in Table 2. Figure 8 The results show that overexpression of pTF101-GmVQ32 can enhance the tolerance of soybean to low phosphorus stress, and gene editing of pCas9-GmVQ32 will enhance the sensitivity of soybean to low phosphorus stress.

Claims

1. Application of a soybean low phosphorus tolerance gene GmVQ32 in improving the low phosphorus stress tolerance of plants, characterized in that, The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown as SEQ ID NO. 2; the application is to enhance the phosphorus absorption efficiency of plants under low-phosphorus stress by overexpressing the GmVQ32 gene in the plants; and the plants are Arabidopsis or soybean.

2. A method of promoting growth of a plant in a low phosphorus stress environment, characterized by, The nucleotide gene sequence of the soybean low-phosphorus tolerance gene GmVQ32 is shown as SEQ ID NO. 1, or the soybean low-phosphorus tolerance gene GmVQ32 encodes an amino acid sequence shown as SEQ ID NO. 2; the application is to enhance the phosphorus absorption efficiency of plants under low-phosphorus stress by overexpressing the GmVQ32 gene in the plants; and the plants are Arabidopsis or soybean.

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