Application of GmAIR12-2 protein in improving the tolerance of legumes to low phosphorus stress

Abstract

The present invention discloses the application of GmAIR12-2 protein in improving the ability of legumes to tolerate low-phosphorus stress. The present invention finds that under low-phosphorus stress, overexpression of GmAIR12-2 protein in soybean plants can improve the root growth of soybean plants, increase the number of small root nodules and the fresh weight of small root nodules of soybean plants, increase the above-ground parts and root dry weight of soybean plants, increase total root length and root surface area, indicating that the protein can improve the tolerance of soybeans to low-phosphorus stress and reduce the adverse effects of low-phosphorus stress on soybean growth. Thus, the present invention provides the application of GmAIR12-2 protein in improving the ability of legumes to tolerate low-phosphorus stress, and the protein can also be used to cultivate transgenic legume varieties resistant to low-phosphorus stress, so that legumes planted in soils with lower available phosphorus content can also grow normally, thereby increasing the yield of legumes. At the same time, the present invention also provides important gene resources for breeding legume varieties resistant to low-phosphorus stress.

Description

Technical Field

[0001] The present 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] Soybeans (Glycine max) are not only an important crop used as food, oil, and feed, but also a major source of high-quality protein for the human body, playing a crucial role in the human diet. With the continuous development of the global economy, the demand for soybeans is also increasing.

[0003] Nodule nitrogen fixation is very important for soybean production. Studies have shown that after inoculating effective rhizobium strains in the field, it can significantly promote soybean plant growth and nodule development, thereby improving soybean nitrogen fixation efficiency, improving nitrogen and phosphorus nutrient utilization efficiency, and increasing soybean field yields. This 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 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] Large areas of arable land in my country are phosphorus-deficient, particularly in the acidic soil regions of southern China. Acidic soils are rich in ions such as iron, aluminum, and manganese, which can lead to the loss of calcium, magnesium, and phosphate fertilizers. This results in insufficient available phosphorus in the soil during cultivation, hindering soybean production. Identifying genes that confer tolerance to low-phosphorus stress and cultivating soybean varieties tolerant of low-phosphorus stress may effectively mitigate the adverse effects of insufficient soil available phosphorus on soybean production. While genes that confer tolerance to low-phosphorus stress in soybeans have been reported, these genes affect phosphorus metabolism in soybeans, and it is unclear whether they can mitigate the effects of low-phosphorus stress on soybean nodules.

[0005] AIR12 (Auxin Induced in Root Culture) is believed to be a gene that influences lateral root development in Arabidopsis and is induced early by auxin. It exists as a single copy in the Arabidopsis genome. Studies have found that overexpressing the AIR12 gene can improve the freezing tolerance of tobacco, while knocking out the AIR12 gene reduces freezing tolerance in Arabidopsis. Iron toxicity stress increases the abundance of ascorbic acid-reducible cytochrome AIR12 and reduces the abundance of iron-containing proteins. In other words, the AIR12 gene plays a role in regulating plant redox reactions and in plant responses to abiotic and biotic stresses. However, it is unclear whether the AIR12 gene can improve the tolerance of legumes to low-phosphorus stress. Summary of the Invention

[0006] The present invention addresses the problem of soybean growth being severely affected by low-phosphorus stress and provides the use of the GmAIR12-2 protein in improving the tolerance of legumes to low-phosphorus stress. Under low-phosphorus stress, overexpressing the GmAIR12-2 protein in soybean plants can improve the growth of soybean roots and significantly increase the number of small root nodules, thereby improving the soybean's tolerance to low-phosphorus stress.

[0007] The first object of the present invention is to provide a use of the 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 legumes 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 legumes 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 legume varieties resistant to low-phosphorus stress.

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

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

[0013] A seventh object of the present invention is to provide a method for improving the ability of legumes to tolerate low-phosphorus stress or cultivating legume varieties that are 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 the GmAIR12-2 protein in soybean plants and plants them under low-phosphorus stress conditions, and finds that the 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 surface area of ​​the root system, and the number and fresh weight of small nodules, thereby significantly improving the soybean's ability to tolerate low-phosphorus stress. Plants of the same genus have similar genetic backgrounds. For the same type of stress, plants of the same genus may have the same physiological mechanism to adapt to the stress, that is, overexpressing the soybean GmAIR12-2 protein in other leguminous plants may also increase their ability to tolerate low-phosphorus stress. Therefore, the present invention requests protection of the following applications of the GmAIR12-2 protein in leguminous plants:

[0016] The present invention seeks 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 seeks to protect the use of the GmAIR12-2 protein in cultivating legume 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 seeks to protect the use of the gene encoding the GmAIR12-2 protein in improving the ability of legumes to tolerate low-phosphorus stress.

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

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

[0022] The present invention also claims protection for 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 protection for the use of the reagent for promoting the expression of the GmAIR12-2 protein in cultivating legume 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 improvement of the legume plant's ability to tolerate low phosphorus stress is to increase the number and / or fresh weight of its root 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 legume plant's tolerance to low phosphorus stress also includes increasing its 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 legumes to tolerate low-phosphorus stress or cultivating legume varieties tolerant 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 legume plant by an Agrobacterium-mediated transformation method.

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

[0034] The present invention is to construct a transgenic soybean composite plant that overexpresses GmAIR12-2 protein by injecting Agrobacterium containing an overexpression vector of GmAIR12-2 gene into hypocotyl through Agrobacterium-mediated. Under low-phosphorus stress conditions, compared to wild-type soybean plants (injected with Agrobacterium not containing an overexpression vector), overexpressing GmAIR12-2 protein in soybean plants can improve the root growth of soybean plants, increase the number of small root nodules and the fresh weight of small root nodules of soybean plants under low-phosphorus stress conditions, increase the above-ground part and root dry weight of soybean plants, increase total root length and root surface area, show that GmAIR12-2 protein can improve the tolerance of soybean to low-phosphorus stress, and reduce the adverse effects of low-phosphorus stress conditions on soybean growth, etc. The present invention not only enriches the method for improving the low-phosphorus stress resistance of legumes or cultivating legume varieties resistant to low-phosphorus stress, but also provides gene resources for cultivating low-phosphorus stress-resistant legumes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Response 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; the pictures from left to right are the contents observed and taken 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 overlapping pictures (fusion).

[0037] Figure 3The expression levels of the GmAIR12-2 gene in the roots of different strains were 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 hybrid plants overexpressing GmAIR12-2 and wild-type soybean plants. Figure A shows the phenotypes of the hybrid and wild-type soybean plants and their respective nodules under different phosphorus concentrations. The scale bars in the first row of figures are 10 cm, the scale bars in the second row of figures are 5 cm, the scale bars in the third row of figures are 1 cm, and the scale bars in the fourth row of figures are 1 cm. Figure B shows the aboveground dry weight of the hybrid and wild-type soybean plants. Figure C shows the root dry weight of the hybrid and wild-type soybean plants. Figure D shows the total root length of the hybrid and wild-type soybean plants. Figure E shows the root surface area of ​​the hybrid and wild-type soybean plants. Figure F shows the number of nodules in the hybrid and wild-type soybean plants. Figure G shows the fresh weight of nodules in the hybrid and wild-type soybean plants. Data in the figures are the means and standard errors of 8 replicates. “*” indicates significant differences compared with WT (Student's t-test, P < 0.05). DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. 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 were commercially available.

[0041] Example 1 Analysis of the expression pattern of the 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. Four replicates were set for each treatment.

[0046] Seedling cultivation: Sand seedlings were grown. Uniform soybean seeds (Yuechun 03-3, soybean variety, Yuechun 03-3) with intact seed coats were selected. Sterilize the seeds with 10% sodium hypochlorite solution for 5 minutes, wash three times with pure water, and place them in sand for germination. Incubate in the dark at 28°C for 2 days and in the light for 2-3 days.

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

[0048] Sample treatment: After the roots of the germinated soybean seedlings were soaked in rhizobium solution for 1 hour, the soybean seedlings were moved to 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 applied 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 quickly frozen with liquid nitrogen and stored in a -80°C refrigerator.

[0050] Sample Preparation: Total RNA from each of the treated plant samples was extracted using the TRIzol kit (Invitrogen, USA). RNA, after DNase I treatment, was reverse transcribed into cDNA using the MMLV reverse transcription kit (Promega, USA). qRT-PCR analysis was performed using the SYBR® kit (Promega, USA). After reverse transcription, the samples were diluted 10-fold 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 10 μL, forward and reverse primers 0.4 μL each (primer concentration is 10 μmol / L), CXR Reference Dye 0.2 μL, cDNA template 2 μL, Nuclease-free water 7 μL;

[0064] The reaction procedure was as follows: pre-denaturation at 95°C for 10 min, and 40 cycles of 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 as follows 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. 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 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 soybean GmAIR12-2 gene ORF (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, and 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 was detected by gel electrophoresis and recovered and purified to obtain the PCR product.

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

[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 the recombinant vector: The recombinant vector was transformed into competent E. coli DH5α. 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 stored for future use after testing.

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

[0083] Tobacco leaves were infected with Agrobacterium tumefaciens (GV3101) and injected with the 35S::GmAIR12-2-GFP vector and the empty pEGAD vector. Transient expression of the 35S::GmAIR12-2-GFP vector and the empty pEGAD vector was achieved in tobacco epidermal cells. GFP fluorescence signals in the epidermal cells were observed using a laser confocal microscope.

[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 digested with the SmaI restriction endonuclease, and the digested product was recovered and purified.

[0096] (3) Insert the purified target fragment into the enzyme-digested pTF101s vector.

[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. After the samples were sequenced and confirmed to be correct, 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 testing.

[0100] 2. Obtaining transgenic soybean composite plant materials

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

[0102] (1) Seed germination. Select soybean seeds of uniform size and intact seed coat. Soybean variety Yuechun 03-3 (YC03-3) was sterilized with 10% sodium hypochlorite solution for 5 minutes, washed three times with pure water, and placed in sand for germination. Incubate in the dark at 28°C for 2 days and in the light for 2–3 days.

[0103] (2) Preparation of bacterial solution. Two days before soybean seed germination, K599 bacterial solution containing the overexpression vector OX-GmAIR12-2-pTF was streaked and activated. On the third day, a single clone was picked 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 180 rpm and 28 °C for 15 h. 200 μL of bacterial solution was then transferred to 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 of 50 mg / μL) for plating. 100 μL of K599 competent cells that were not transformed with any vector were plated on YEP solid medium containing str antibiotics (antibiotic concentration of 50 mg / μL) for plating as a blank control. Pipette 1 μL of bacterial solution for PCR detection. After confirming that the strain is correct, continue to culture 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 with unexpanded cotyledons and use a 1 mL syringe needle to dip the bacterial solution (K599 bacterial solution containing the overexpression vector plasmid OX-GmAIR12-2-pTF or K599 bacterial solution without any vector) into the hypocotyl of the seedling near the cotyledon node. Inject the solution at three fixed points back and forth. Cover the wound with K599 bacteria and moist sand to continue culturing the seedling. The seedling needs 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 rooting. After about 2 weeks, the callus begins to grow hairy roots. Check the injection site and cut off the roots outside the injection site, retaining the hairy roots growing from 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] Total RNA was extracted from the hairy roots of the transgenic plants and reverse transcribed into cDNA. The expression level of the GmAIR12-2 gene was then detected by fluorescent quantitative PCR (the primer sequences for the detection genes are shown in SEQ ID NOs. 5 and 6). The soybean housekeeping gene EF1-a (the primer sequences for the detection gene are shown in SEQ ID NOs. 3 and 4) was used as an internal reference. The relative expression level was calculated as 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 the 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] Transgenic soybean composites overexpressing GmAIR12-2 and wild-type soybean plants were inoculated with rhizobia. After inoculation, the composites and wild-type soybean plants were transplanted into sand culture and irrigated every three days with either a 5 μmol / L KH2PO4 nitrogen-containing nutrient solution or a 250 μmol / L KH2PO4 nitrogen-containing nutrient solution, for a total nitrogen level of 1036 μmol / L. After 35 days of treatment, samples were collected for determination of nodule number, nodule fresh weight, and plant biomass.

[0111] The results of the effects of different phosphorus concentration treatments 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 represents the phenotypes of composite lines and wild-type soybean plants and their respective nodules under different phosphorus concentration treatments; Figure 4Figures B to G represent the aboveground dry weight, root dry weight, total root length, root surface area, number of nodules, and fresh weight of nodules of the composite and wild-type soybean plants, respectively. As shown in the figure, under low phosphorus treatment (-P), overexpression of the GmAIR12-2 gene significantly promoted the growth of the aboveground parts and nodules of the composite soybean plants compared to wild-type 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), total root length and root surface area increased by 114% and more than 98% respectively ( Figure 4 D, E), the number of small nodules and the fresh weight of small nodules increased by more than 245% and 260%, respectively ( Figure 4 (F, G). Under normal phosphorus treatment, overexpression of the GmAIR12-2 gene had no significant effect on root and nodule growth and development in transgenic soybean hybrids compared to wild-type soybean plants. These results suggest that GmAIR12-2 can enhance soybean tolerance to low-phosphorus stress, regulate the growth of the aboveground parts, roots, and nodules of soybean hybrids, 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of the GmAIR12-2 protein having an amino acid sequence as shown in SEQ ID NO. 2 in improving the tolerance of legumes to low phosphorus stress, characterized in that: The legume is soybean.

2. Use of the gene encoding the GmAIR12-2 protein according to claim 1 in improving the tolerance of legumes to low phosphorus stress, characterized in that: The legume is soybean.

3. Use of the agent for promoting the expression of the GmAIR12-2 protein according to claim 1 in improving the ability of leguminous plants to tolerate low-phosphorus stress, characterized in that: The legume is soybean.

4. Use of the GmAIR12-2 protein according to claim 1 in cultivating legume varieties resistant to low phosphorus stress, characterized in that: The legume is soybean.

5. Use of the gene encoding the GmAIR12-2 protein according to claim 1 in cultivating legume varieties resistant to low-phosphorus stress, characterized in that: The legume is soybean.

6. Use of the reagent for promoting the expression of the GmAIR12-2 protein according to claim 1 in cultivating leguminous plant varieties resistant to low-phosphorus stress, characterized in that: The legume is soybean.

7. The use according to any one of claims 1 to 3, characterized in that: The method of improving the low phosphorus stress tolerance of legumes 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, 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, characterized in that: The reagent comprises a recombinant overexpression vector containing a gene encoding the GmAIR12-2 protein according to claim 1.

10. A method for improving the ability of legumes to tolerate low-phosphorus stress or cultivating legume varieties tolerant to low-phosphorus stress, characterized in that: The GmAIR12-2 protein of claim 1 is overexpressed in a legume plant; the legume plant is soybean.

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