Application of GmAIR12-2 protein in improving low-phosphorus stress tolerance of leguminous plants

Abstract

The invention discloses an application of a GmAIR12-2 protein in improving the low-phosphorus stress resistance of leguminous plants. According to the application disclosed by the invention, under low-phosphorus stress, GmAIR12-2 protein is excessively expressed in soybean plants, so that the root growth vigor of the soybean plants can be improved, the number and fresh weight of small root nodules of the soybean plants can be increased, the dry weight of overground parts and root systems of the soybean plants can be increased, and the total root length and the root surface area can be increased; it is shown that the protein can improve the tolerance of soybeans to low-phosphorus stress and relieve adverse effects of low-phosphorus stress on soybean growth and the like. Therefore, the invention provides the application of the GmAIR12-2 protein in improving the low-phosphorus stress resistance of the leguminous plants, and the protein can also be used for cultivating low-phosphorus stress resistant transgenic leguminous plant varieties, so that the leguminous plants planted in soil with relatively low available phosphorus content can also grow normally, and the yield of the leguminous plants is improved. Meanwhile, the invention also provides an important gene resource for breeding leguminous plant varieties resistant to low phosphorus stress.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of GmAIR12-2 protein in improving the low-phosphorus stress tolerance of leguminous plants. Background Art

[0002] Soybean (Glycine max) is not only an important crop for food, oil and feed, but also a major source of high-quality protein for the human body, and plays an important role in the human diet. With the continuous development of the global economy, the demand for soybean is also increasing.

[0003] Nodule nitrogen fixation is very important for soybean production. Studies have shown that after inoculating effective rhizobia strains in the field, it can significantly promote the growth of soybean plants and the development of nodules, thereby improving the efficiency of soybean nitrogen fixation, improving the efficiency of nitrogen and phosphorus nutrient utilization, and increasing soybean field yields, which is of great significance for achieving weight loss and efficiency improvement and developing green agriculture. Compared with non-symbiotic crops, the biological nitrogen fixation process of leguminous crops requires the participation of a large amount of phosphorus, and within a certain range, the ability to maintain nodule phosphorus balance is positively correlated with the nitrogen fixation ability. Phosphorus deficiency will seriously affect the growth and development of leguminous crops and the efficiency of nitrogen fixation. Under low phosphorus stress, soybean plants grow short, nodule growth and development are inhibited, and the final soybean yield will be seriously affected.

[0004] There are large areas of phosphorus-deficient arable land in my country, especially in the acidic soil areas in the south. Acidic soils are rich in acid ions such as iron ions, aluminum ions, and manganese ions, which easily cause the loss of calcium, magnesium, and phosphorus fertilizers, resulting in insufficient supply of effective phosphorus in the soil during the planting process, which is not conducive to soybean production. Exploring plant genes that tolerate low-phosphorus stress and cultivating soybean varieties that tolerate low-phosphorus stress may effectively reduce the adverse effects of insufficient soil effective phosphorus supply on soybean production. Although there are reports of genes that can make soybeans resistant to low-phosphorus stress, they affect the metabolism of soybean phosphorus, and it is unclear whether they can reduce the impact of low-phosphorus stress on soybean nodules.

[0005] AIR12 (Auxin Induced in Root culture) is considered to be a gene that affects the development of Arabidopsis lateral roots and is induced early by auxin. It exists in the Arabidopsis genome in a single copy. Studies have found that overexpression of the AIR12 gene can improve the frost resistance of tobacco, while knocking out the AIR12 gene reduces the frost resistance of Arabidopsis; iron toxicity stress increases the abundance of ascorbic acid-reducible cytochrome AIR12 and reduces the abundance of iron-containing proteins. That is, the AIR12 gene plays a role in regulating plant redox reactions and in the response of plants to abiotic and biotic influences. However, it is not clear whether the AIR12 gene can improve the tolerance of legumes to low-phosphorus stress. Summary of the invention

[0006] The present invention aims at the problem that soybean growth is seriously affected under low phosphorus stress, and provides the application of GmAIR12-2 protein in improving the ability of legumes to tolerate low phosphorus stress. Under low phosphorus stress, overexpressing GmAIR12-2 protein in soybean plants can improve the root growth of soybeans and significantly increase the number of small root nodules, thereby improving the ability of soybeans to tolerate low phosphorus stress.

[0007] The first object of the present invention is to provide a use of a GmAIR12-2 protein having an amino acid sequence as shown in SEQ ID NO. 2 in improving the ability of leguminous plants to tolerate low phosphorus stress.

[0008] The second object of the present invention is to provide an application of the gene encoding the GmAIR12-2 protein in improving the ability of leguminous plants to tolerate low-phosphorus stress.

[0009] The third object of the present invention is to provide an application of an agent for promoting the expression of the GmAIR12-2 protein in improving the ability of leguminous plants to tolerate low-phosphorus stress.

[0010] The fourth object of the present invention is to provide the use of the GmAIR12-2 protein in cultivating leguminous plant varieties resistant to low-phosphorus stress.

[0011] The fifth object of the present invention is to provide the use of the gene encoding the GmAIR12-2 protein in cultivating leguminous plant varieties resistant to low-phosphorus stress.

[0012] The sixth object of the present invention is to provide an application of an agent for promoting the expression of the GmAIR12-2 protein in cultivating leguminous plant varieties resistant to low-phosphorus stress.

[0013] The seventh object of the present invention is to provide a method for improving the ability of legume plants to tolerate low-phosphorus stress or cultivating legume plant varieties resistant to low-phosphorus stress.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The present invention overexpresses GmAIR12-2 protein in soybean plants and plants them under low-phosphorus stress conditions, and finds that GmAIR12-2 protein can significantly increase the dry weight of the aboveground part of the plant, the dry weight of the root system, the total root length, the root surface area, and the number and fresh weight of small nodules, and significantly improves the ability of soybean to tolerate low-phosphorus stress. Plants of the same family have similar genetic backgrounds. For the same type of stress, plants of the same family may have the same physiological mechanism to adapt to the stress, that is, overexpressing soybean GmAIR12-2 protein in other leguminous plants may also increase their ability to tolerate low-phosphorus stress. Therefore, the present invention requests protection for the following applications of the GmAIR12-2 protein in leguminous plants:

[0016] The present invention claims to protect the use of GmAIR12-2 protein in improving the ability of leguminous plants to tolerate low-phosphorus stress.

[0017] The present invention also claims to protect the use of the GmAIR12-2 protein in cultivating leguminous plant varieties resistant to low-phosphorus stress.

[0018] Specifically, the amino acid sequence of the GmAIR12-2 protein is shown in SEQ ID NO.2.

[0019] The present invention also claims to protect the use of the gene encoding the GmAIR12-2 protein in improving the ability of leguminous plants to tolerate low-phosphorus stress.

[0020] The present invention also claims to protect the use of the gene encoding the GmAIR12-2 protein in cultivating legume plant varieties resistant to low-phosphorus stress.

[0021] As one of the options, the nucleotide sequence of the gene encoding the GmAIR12-2 protein is shown as SEQ ID NO.1.

[0022] The present invention also claims to protect the use of the reagent for promoting the expression of the GmAIR12-2 protein in improving the ability of leguminous plants to tolerate low-phosphorus stress.

[0023] The present invention also claims to protect the use of the reagent for promoting the expression of the GmAIR12-2 protein in cultivating leguminous plant varieties resistant to low-phosphorus stress.

[0024] Specifically, the reagent includes an overexpression vector containing a gene encoding the GmAIR12-2 protein.

[0025] Specifically, the overexpression vector includes an Agrobacterium binary vector.

[0026] Optionally, the Agrobacterium binary vector is pEGAD or pTF101s.

[0027] Specifically, the method of improving the low-phosphorus stress tolerance of leguminous plants is to increase the number of nodules and / or the fresh weight of nodules under low-phosphorus stress conditions.

[0028] Specifically, the method of improving the legume plant's tolerance to low phosphorus stress also means increasing its dry weight under low phosphorus stress conditions; the dry weight includes the dry weight of the aboveground part and the dry weight of the root system.

[0029] Specifically, the method of improving the low-phosphorus stress tolerance of leguminous plants also includes increasing the total root length and / or root surface area under low-phosphorus stress conditions.

[0030] The present application also claims a method for improving the ability of legume plants to tolerate low-phosphorus stress or cultivating legume plant varieties resistant to low-phosphorus stress.

[0031] Specifically, the method is to overexpress the GmAIR12-2 protein in leguminous plants.

[0032] As an optional embodiment, the method for overexpressing the GmAIR12-2 protein includes: constructing a recombinant overexpression vector containing a gene encoding the GmAIR12-2 protein, and transforming the recombinant vector into a leguminous plant by an Agrobacterium-mediated transformation method.

[0033] The present invention has the following beneficial effects:

[0034] The present invention constructs a transgenic soybean composite plant overexpressing GmAIR12-2 protein by injecting Agrobacterium containing an overexpression vector of the GmAIR12-2 gene into the hypocotyl through Agrobacterium-mediated. Under low-phosphorus stress conditions, compared to wild-type soybean plants (injected with Agrobacterium without an overexpression vector), overexpressing GmAIR12-2 protein in soybean plants can improve the root growth of soybean plants, increase the number of small nodules and fresh weight of small nodules of soybean plants under low-phosphorus stress conditions, increase the above-ground part of soybean plants and the dry weight of the root system, increase the total root length and root surface area, indicating that GmAIR12-2 protein can improve the tolerance of soybeans to low-phosphorus stress, and reduce the adverse effects of low-phosphorus stress conditions on soybean growth. The present invention not only enriches the method for improving the ability of legumes to resist low-phosphorus stress or cultivating legume varieties resistant to low-phosphorus stress, but also provides gene resources for cultivating legumes resistant to low-phosphorus stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The responses of GmAIR12-2 gene to low phosphorus stress in soybean roots and nodules; data are the mean and standard error of four replicates; “*” indicates significant difference between low phosphorus treatment (-P) and normal phosphorus treatment (+P) (Student's t-test, P<0.05).

[0036] Figure 2 The results of subcellular localization analysis of GmAIR12-2 protein; the first row in the figure shows the subcellular localization map of tobacco transformed with an empty vector (35S:GFP), with a scale of 40μm; the second row shows the subcellular localization map of GmAIR12-2 fused to GFP protein in tobacco leaves (35S:GFP-GmAIR12-2), with a scale of 50μm; from left to right, the pictures are respectively the contents observed and photographed under a laser confocal microscope using the green fluorescence channel (GFP), the red fluorescence channel (cell membrane marker gene), the light microscope channel (bright field), and the overlapped picture (fusion).

[0037] Figure 3The expression level of GmAIR12-2 gene in the roots of different strains was detected; WT: wild-type soybean plant; OX: transgenic soybean composite plant overexpressing GmAIR12-2; the data in the figure are the mean and standard error of 4 replicates; "*" indicates a significant difference compared with WT (Student's t-test, P<0.05).

[0038] Figure 4 Figure 2 shows the effects of different phosphorus concentrations on the growth of transgenic soybean composite plants overexpressing GmAIR12-2 and wild-type soybean plants; Figure A shows the phenotypes of composite lines and wild-type soybean plants and their respective nodules under different phosphorus concentrations, the scale bars of the first row of figures are 10 cm, the scale bars of the second row of figures are 5 cm, the scale bars of the third row of figures are 1 cm, and the scale bars of the fourth row of figures are 1 cm; Figure B shows the aboveground dry weight of composite lines and wild-type soybean plants; Figure C shows the root dry weight of composite lines and wild-type soybean plants; Figure D shows the total root length of composite lines and wild-type soybean plants; Figure E shows the root surface area of ​​composite lines and wild-type soybean plants; Figure F shows the number of nodules of composite lines and wild-type soybean plants; Figure G shows the fresh weight of nodules of composite lines and wild-type soybean plants; the data in the figure are the mean and standard error of 8 repetitions; "*" indicates significant difference compared with WT (Student's t-test, P<0.05). DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1 Analysis of the expression pattern of GmAIR12-2 gene

[0042] 1. Experimental methods

[0043] Experimental design: Low phosphorus treatment (-P): 5 μmol / L KH2PO4 nitrogen-containing nutrient solution, nitrogen level was 1036 μmol / L total nitrogen;

[0044] Normal phosphorus treatment (+P): 250 μmol / L KH2PO4 nitrogen-containing nutrient solution, nitrogen level was 1036 μmol / L total nitrogen;

[0045] The cultivation device was a 15L blue light-proof bread box, and the culture was carried out in a solar greenhouse, with four replicates for each treatment.

[0046] Seedling cultivation: Sand seedling cultivation was adopted. Soybean seeds with uniform size and intact seed coat were selected. The soybean variety was Yuechun 03-3 (YC03-3). After being sterilized with 10% sodium hypochlorite solution for 5 minutes, they were washed 3 times with pure water and placed in sand for germination. They were cultured in the dark at 28°C for 2 days and in the light for 2 to 3 days.

[0047] Preparation of rhizobia: Take a BXYD3 glycerol strain from a -80℃ freezer (the strain is currently stored in the Root Center of South China Agricultural University), streak and activate it, pick a single clone and inoculate it into YMA culture medium (1000mL of secondary water containing 10g of mannitol, 0.2g of MgSO4·7H2O, 0.1g of NaCl, 3g of yeast powder, 0.25g of K2HPO4 and 0.25g of KH2PO4), set the temperature to 28℃, the rotation speed to 180rpm / min, and culture until the OD of the bacterial solution reaches 600 The value (absorbance at a wavelength of 600 nm) is approximately 1.0.

[0048] Sample treatment: After the roots of the germinated soybean seedlings were immersed in the rhizobium solution for 1 hour, the soybean seedlings were moved into sand culture and subjected to low-phosphorus treatment and normal-phosphorus treatment respectively. The seeds were watered with 5μmol / L or 250μmol / L KH2PO4 nitrogen-containing nutrient solution every 3 days, with a nitrogen level of 1036μmol / L total nitrogen. Secondary water was used for 2 days in between, and the treatment lasted for 35 days.

[0049] Sample acquisition: Roots were harvested on days 1, 3, 6, 9, 12, 14, 21, 28, and 35 after treatment, and nodules were harvested on days 21, 28, and 35 after treatment. They were first quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.

[0050] Sample treatment: Total RNA of the plant samples after the above treatments was extracted using the TRIzol kit (Invitrogen, USA). RNA treated with DNase I was reverse transcribed into cDNA using the MMLV reverse transcription kit (Promega, USA). qRT-PCR analysis was then performed using the SYBR (Promega, USA) kit. After reverse transcription, the samples were diluted 10 times and analyzed by real-time fluorescence quantitative PCR using the Applied Biosystems StepOnePlus Real-Time PCR system.

[0051] The nucleotide sequence of the GmAIR12-2 gene is shown below (SEQ ID NO.1):

[0052] SEQ ID NO.1:

[0053] ATGGCATTCCATGATCTACTCTTCACACCCACCATTTCCCTCTTCATAATTCTATTTTCTCTCTTTTCCACCCCTTCCCATTCTGCCCTCACTTGCGCTTCCCAGAAGCTCAACCGCACCTACGCCAACTGCACCAACCTTCCCACCCTCGGCGCCACCCTCCACTTCACCTTCAACGCCACTAACCGCTCCCTCTCCGTCGCCTTCTCCGCCGAGCCTCCCTCCCGCTCCGGCTGGGTCGCCTGGGGCCTCAACCTCGTCGGCGATGGCATGCGCGGCGCCGAAGCCTTCCTCGCCTTCCCCTCCTCCGCCTCCGCCTCCGCCATCACCCTCGGCCGCTACAACCTCACCTCCTACAAAGCCATCGACGAAGTCAAAGCCTTTACCTTCGACTCGTGGGACCTCGCGGCCGAAGAATCCAACGGCGCCGTAACCATCTACGGCTCCGTCAAGATCCCCGATTCGGCGAGGAACGTCAGCCACGTGTGGCAGGTGGGTCCCGTGGCCGCGGGCAAGCCTGGGGTCCACAGCTTCGAAAAGAAGAATACGGACTCCAAGGCCGCGTTCCCTGTCGCGCTGGTGGGGCCCAACACAACTACACCAGCGAGTGGCGAAAACGCGACTGCGCCCGCGAGTGGTGGGGACAAGAAGAACGGTGCTGCTGGGGAGAGCTTTGGGGTTGGGTTTTACTTTTGGTTGGTTTTCGCATTGATGATTGGTGTCGTTGCTATTTGA

[0054] The amino acid sequence of the GmAIR12-2 protein is as follows (shown in SEQ ID NO.2):

[0055] MAFHDLLFTPTISLFISLFSTPSHSALTCASQKLNRTYANCTNLPTLGATLHFTFNATNRSSLSVAFSAEPPSRSGWVAWGLNLVGDGMRGAEAFLAFPSSASAITLGRYNLTSYKAI DEVKAFTFDSWDLAAEESNGAVTIYGSVKIPDSARNVSHVWQVGPVAAGKPGVHSFEKKNTDSKAAFPVALVGPNTTTPASGENATAPASGGDKKNGAAGESFGVGFYFWLVFALMIGVVAI

[0056] The housekeeping gene EF1-α (Glyma17g23900) was used as an internal reference, and the relative expression level was expressed as the ratio of the expression level of the GmAIR12-2 gene to the expression level of the EF1-α gene.

[0057] The quantitative primer for soybean housekeeping gene EF1-α is EF1-α-F / R, and the nucleotide sequence of the primer is as follows:

[0058] EF1-α-F (SEQ ID NO.3): 5'-TGCAAAGGAGGCTGCTAACT-3'

[0059] EF1-α-R (SEQ ID NO.4): 5'-CAGCATCACCGTTCTTCAAA-3'

[0060] The quantitative primer for GmAIR12-2 is GmAIR12-2-RT-F / R, and the nucleotide sequence of the primer is as follows:

[0061] RT-GmAIR12-2-F(SEQ ID NO.5):5'-GCCCAACACAACTACACCAG-3'

[0062] RT-GmAIR12-2-R(SEQ ID NO.6):5'-AGCAACGACACCAATCATCAA-3'

[0063] Reaction system: 2×Go Taq qPCR Master Mix 10μL, forward and reverse primers 0.4μL each (primer concentration is 10μmol / L), CXR Reference Dye 0.2μL, cDNA template 2μL, Nuclease-free water 7μL;

[0064] Reaction procedure: pre-denaturation at 95°C for 10 min, 40 cycles (denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min).

[0065] 2. Experimental results

[0066] The results of the response of GmAIR12-2 gene to low phosphorus stress in soybean roots and nodules are shown in Figure 2. Figure 1 As shown. Figure 1 It can be seen that compared with normal phosphorus treatment, the expression of GmAIR12-2 gene was extremely significantly upregulated on the 6th, 9th, 12th, 14th, 21st and 28th days after low phosphorus treatment; from the basic formation of nodules on the 21st day after inoculation of rhizobia to the full maturity of nodules on the 28th day, compared with normal phosphorus treatment, the expression of GmAIR12-2 gene was significantly downregulated after low phosphorus treatment.

[0067] Example 2 Subcellular localization analysis of GmAIR12-2 protein

[0068] 1. Construction of expression vector for subcellular localization analysis:

[0069] (1) Primer design: The specific primer for GmAIR12-2 is GFP-GmAIR12-2-F / R, and the nucleotide sequence of the primer is as follows:

[0070] GFP-GmAIR12-2-F (SEQ ID NO.7):

[0071] 5'-CTCTAGCGCTACCGGTCCAATGGCATTCCATGATCT-3'

[0072] GFP-GmAIR12-2-R (SEQ ID NO.8):

[0073] 5'-CATGGTGGCGACCGGTCG AATAGCAACGACACCAATC-3'

[0074] (2) PCR amplification: Using the nodule cDNA of soybean variety YC03-3 grown under normal phosphorus treatment for 28 days as a template (hydroponically cultivated in a nitrogen-containing nutrient solution containing 250 μmol / L KH2PO4 and a total nitrogen level of 1036 μmol / L, with the nutrient solution replaced every 7 days), the full-length ORF of the soybean GmAIR12-2 gene (as shown in SEQ ID NO.1) was amplified using gene-specific primers GFP-GmAIR12-2-F / R.

[0075] PCR reaction system: 25 μL 2×Phanta Max Buffer, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL dNTP, 1 μL each of forward and reverse primers (primer concentration is 10 μmol / L), 2 μL cDNA template, add ddH2O to make up to 50 μL.

[0076] PCR reaction conditions: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 58°C for 40 s, extension at 72°C for 30 s, 30 cycles from denaturation to extension, and storage of PCR products at 16°C.

[0077] (3) PCR product purification: The PCR amplified product is detected by gel electrophoresis and the PCR product is obtained after recovery and purification.

[0078] (4) Connecting PCR products to vectors: using homologous recombination kit II. Carry out recombination ligation reaction between the PCR product and the linearized vector pEGAD digested with Age I.

[0079] Reaction system: 6 μL of PCR product, 8 μL of pEGAD linearized vector, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer.

[0080] Reaction conditions: 37°C for 30 min.

[0081] (5) Transformation of recombinant vector: The recombinant vector was transformed into competent E. coli DH5α, and after sequencing, the recombinant vector was extracted to obtain the 35S::GmAIR12-2-GFP vector. The 35S::GmAIR12-2-GFP vector was transformed into Agrobacterium GV3101, and after testing, it was stored for future use.

[0082] 2. Subcellular localization analysis of GmAIR12-2

[0083] Agrobacterium was used to infect tobacco, and Agrobacterium GV3101 loaded with 35S::GmAIR12-2-GFP vector and pEGAD empty vector was injected into tobacco leaves to transiently express 35S::GmAIR12-2-GFP vector and pEGAD empty vector in tobacco epidermal cells. GFP fluorescence signal of tobacco epidermal cells was observed using laser confocal microscopy.

[0084] The results of subcellular localization analysis of GmAIR12-2 protein in tobacco leaves are as follows Figure 2 As shown. Figure 2 It can be seen that the fluorescence of 35S::GFP-GmAIR12-2 is distributed in the nucleus and cell membrane of tobacco epidermal cells, indicating that the GmAIR12-2 protein is expressed in the nucleus and cell membrane of plants.

[0085] Example 3 Obtaining transgenic soybean composite plants overexpressing GmAIR12-2

[0086] 1. Construction of overexpression vector OX-GmAIR12-2-pTF

[0087] (1) Design the GmAIR12-2 gene-specific primer OX-GmAIR12-2-pTF-F / R. The nucleotide sequence of the primer is as follows:

[0088] OX-GmAIR12-2-pTF-F (SEQ ID NO.9):

[0089] 5'-GTACCCGGGGATCCTCTAGACCAATGGCATTCCATGATCT-3'

[0090] OX-GmAIR12-2-pTF-R (SEQ ID NO.10):

[0091] 5'-GCCTGCAGGTCGACTCTAGATCAAATAGCAACGACACCAATC-3'

[0092] (2) PCR amplification: Using the cDNA of Example 2 1 as a template, the full-length ORF of the soybean GmAIR12-2 gene (as shown in SEQ ID NO. 1) was amplified using gene-specific primers OX-GmAIR12-2-pTF-F / R.

[0093] PCR reaction system: Reaction system: 25 μL 2×Phanta Max Buffer, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL dNTP, 1 μL each of forward and reverse primers (primer concentration is 10 μmol / L), 2 μL cDNA template, add ddH2O to make up to 50 μL.

[0094] PCR reaction conditions: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 58°C for 40 s, extension at 72°C for 30 s, 30 cycles from denaturation to extension, and storage of PCR products at 16°C.

[0095] The target band was recovered and purified. The PCR product was detected by gel electrophoresis, and the target fragment was recovered and purified. The pTF101s vector was single-digested with SmaI restriction endonuclease, and the single-digested product was recovered and purified.

[0096] (3) Insert the purified target fragment into the pTF101s vector that has been digested with enzymes.

[0097] Reaction system: 6 μL of target fragment, 8 μL of pTF101s linearized vector, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer.

[0098] Reaction conditions: 37°C for 30 min.

[0099] The ligation product was transformed into competent Escherichia coli DH5α and plated. After culturing at 37°C for 12 h, the positive clones were shaken out and sent for sequencing. After the samples were sequenced and confirmed, the plasmid was extracted to obtain the overexpression vector OX-GmAIR12-2-pTF. The vector was transformed into Agrobacterium rhizogenes K599 and stored for future use after detection.

[0100] 2. Obtaining transgenic soybean composite plant materials

[0101] The main steps of soybean hypocotyl injection transformation mediated by Agrobacterium tumefaciens include:

[0102] (1) Seed germination. Soybean seeds with uniform seed coat and intact seed coat were selected. The soybean variety was Yuechun 03-3 (YC03-3). They were sterilized with 10% sodium hypochlorite solution for 5 min, washed with pure water 3 times, and placed in sand for germination. They were cultured in the dark at 28°C for 2 days and in the light for 2 to 3 days.

[0103] (2) Preparation of bacterial solution. Two days before soybean seeds germinate, K599 bacterial solution containing the overexpression vector OX-GmAIR12-2-pTF was streaked and activated. On the third day, a single clone was selected and cultured in 0.8 mL YEP culture medium (YEP culture medium formula: 500 mL secondary water containing 5 g peptone, 5 g yeast powder, 2.5 g NaCl), at a speed of 180 rpm and 28 °C for 15 h. 200 μL of bacterial solution was then plated on YEP solid medium (YEP solid medium formula: 500 mL secondary water containing 5 g peptone, 5 g yeast powder, 2.5 g NaCl, 5 g agar) containing kanamycin sulfate and streptomycin (kan+str) antibiotics (antibiotic concentrations were both 50 mg / μL) for culture. 100 μL of K599 competent cells that were not transformed with any vector were plated on YEP solid medium containing str antibiotics (antibiotic concentration was 50 mg / μL) as a blank control. Pipette 1 μL of bacterial solution for PCR detection. After confirming that the strain is correct, continue culturing for 2 to 3 days. When the colonies turn pink, hypocotyl injection can be performed.

[0104] (3) Hypocotyl injection transformation. Four days after seed germination, select seedlings whose cotyledons have not unfolded, dip the bacterial solution (K599 bacterial solution containing the overexpression vector plasmid OX-GmAIR12-2-pTF or K599 bacterial solution without any vector) with a 1 mL syringe needle, and inject the bacterial solution into the hypocotyl of the seedling near the cotyledon node. Inject three points back and forth, cover the wound with K599 bacteria, cover with moist sand and continue to cultivate the seedlings. The seedlings need to be covered with a layer of plastic wrap and sprayed with water several times a day to keep the wound moist.

[0105] (4) Induce root growth. After about 2 weeks, the callus begins to grow hairy roots. Check the injection site, cut off the roots that are not at the injection site, and keep the hairy roots that grow at the injection site. After the seedlings have grown for 1 day, the hairy roots of the soybean composite plant can be inoculated with rhizobia.

[0106] 3. Identification of transgenic soybean composite plants

[0107] The total RNA of the transgenic plant hairy roots was extracted and reverse transcribed into cDNA, and then the expression of the GmAIR12-2 gene was detected by fluorescence quantitative PCR (the primer sequences of the detection genes are shown in SEQ ID NO.5-6). The housekeeping gene EF1-a of soybean (the primer sequences of the detection gene are shown in SEQ ID NO.3-4) was used as an internal reference, and the relative expression level was the ratio of the expression level of the target gene GmAIR12-2 to the expression level of the housekeeping gene EF1-a.

[0108] The relative expression levels of the GmAIR12-2 gene in the roots of different strains were detected as follows: Figure 3 As shown, compared with the wild-type soybean plants (injected with K599 bacterial solution without any transformation vector), the expression level of GmAIR12-2 gene in the hairy roots of the GmAIR12-2 overexpressing transgenic soybean composite plants increased by about 1.2 times, and the difference was extremely significant, indicating that the transgenic soybean composite plants overexpressing GmAIR12-2 were successfully constructed.

[0109] Example 4 Effect of overexpression of GmAIR12-2 gene on soybean plant and nodule growth

[0110] The transgenic soybean composite plants overexpressing GmAIR12-2 and wild-type soybean plants were inoculated with rhizobia. After inoculation with rhizobia, the composite plants and wild-type soybean plants were moved to sand culture and fertilized with 5 μmol / L KH2PO4 nitrogen-containing nutrient solution or 250 μmol / L KH2PO4 nitrogen-containing nutrient solution every 3 days, with a nitrogen level of 1036 μmol / L total nitrogen. Samples were taken 35 days after treatment to determine the number of nodules, fresh weight of nodules, and plant biomass.

[0111] The results of the effects of different phosphorus concentrations on the growth of transgenic soybean composite plants (OX) overexpressing GmAIR12-2 and wild-type soybean plants (WT) are shown in Figure 2. Figure 4 As shown; Figure 4 A in the figure is the phenotype of composite lines and wild-type soybean plants and their respective nodules under different phosphorus concentration treatments; Figure 4B to G in the figure are the aboveground dry weight, root dry weight, total root length, root surface area, number of nodules and fresh weight of nodules of the composite lines and wild-type soybean plants, respectively. As shown in the figure, under low phosphorus treatment (-P), compared with wild-type soybean plants, overexpression of the GmAIR12-2 gene can significantly promote the growth of the aboveground parts and nodules of the composite soybean plants ( Figure 4 Under low phosphorus treatment (-P), overexpression of the GmAIR12-2 gene increased the aboveground dry weight of soybean composite plants by more than 113% compared with wild-type soybean plants ( Figure 4 B), the root dry weight increased by more than 102% ( Figure 4 C), the total root length and root surface area increased by 114% and more than 98% respectively ( Figure 4 D and E), the number and fresh weight of nodules increased by more than 245% and 260%, respectively ( Figure 4 F, G in the figure). Under normal phosphorus treatment, overexpression of the GmAIR12-2 gene had no significant effect on the root system and nodule growth and development of transgenic soybean composite plants compared with wild-type soybean plants. These results indicate that under low phosphorus stress, GmAIR12-2 can improve the tolerance of soybean to low phosphorus stress, regulate the growth of the aboveground parts, roots and nodules of soybean composite plants, and promote soybean growth and development.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of the GmAIR12-2 protein having an amino acid sequence as shown in SEQ ID NO.2 in improving the tolerance of leguminous plants to low phosphorus stress.

2. Use of the gene encoding the GmAIR12-2 protein described in claim 1 in improving the ability of leguminous plants to tolerate low-phosphorus stress.

3. Use of the agent for promoting the expression of the GmAIR12-2 protein described in claim 1 in improving the ability of leguminous plants to tolerate low-phosphorus stress.

4. Use of the GmAIR12-2 protein described in claim 1 in cultivating leguminous plant varieties resistant to low-phosphorus stress.

5. Use of the gene encoding the GmAIR12-2 protein described in claim 1 in breeding leguminous plant varieties resistant to low-phosphorus stress.

6. Use of the agent for promoting the expression of the GmAIR12-2 protein described in claim 1 in cultivating leguminous plant varieties resistant to low-phosphorus stress.

7. The use according to any one of claims 1 to 3, It is characterized in that The method of improving the low-phosphorus stress tolerance of leguminous plants is to increase the number and / or fresh weight of nodules under low-phosphorus stress conditions.

8. The use according to claim 2 or 5, It is characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

9. The use according to claim 3 or 6, It is characterized in that The reagent comprises a recombinant overexpression vector containing a gene encoding the GmAIR12-2 protein described in claim 1.

10. A method for improving the ability of leguminous plants to tolerate low phosphorus stress or cultivating leguminous plant varieties resistant to low phosphorus stress, It is characterized in that The GmAIR12-2 protein of claim 1 is overexpressed in legumes.

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