Application of GmPG39 gene in regulating soybean growth and phosphorus efficiency

Abstract

The application discloses application of a GmPG39 gene in regulating soybean growth and phosphorus efficiency. The application research finds that the GmPG39 gene is involved in soybean root cell wall remodeling, the protein is expressed in the plant cell wall, and is regulated by the root system under the phosphorus, and is up-regulated in the main root section in response to low-phosphorus stress. Overexpression of the GmPG39 can increase polygalacturonase activity, reduce the pectin content in the cell wall of the hair root, aggravate the decomposition of the cell wall under low-phosphorus stress, and promote the relaxation of the cell wall. Meanwhile, the GmPG39 gene positively regulates the soybean growth and the phosphorus efficiency, under normal phosphorus conditions, overexpression of the GmPG39 gene can significantly promote the growth of the in-vitro hair root of the soybean, increase the fresh weight and the total root length of the hair root, increase the total phosphorus content and the soluble phosphorus concentration, and regulate the phosphorus absorption efficiency of the soybean, and has an important role in promoting the growth of the soybean root system and improving the phosphorus absorption efficiency of the soybean root system.

Description

Technical Field

[0001] The present invention belongs to the field of plant breeding and genetic engineering technology, and more specifically relates to the application of the GmPG39 gene in regulating soybean growth and phosphorus efficiency. Background Art

[0002] Soybeans are an important grain and oil crop in my country, widely distributed across many countries and one of the top five crops. Soybeans contain up to 40% protein, including eight essential amino acids that cannot be synthesized by the human body. Soybeans also contain up to 20% fat, making them a key ingredient in edible vegetable oils. Soybeans also contain numerous essential minerals and vitamins, and processed products like tofu, bean sprouts, soy sauce, and dried bean curd sheets are essential foods. Soybeans and their products play a vital role in health products, pharmaceuticals, and chemical products. Soybeans are also excellent soil-fertilizing crops. During their growth, their roots symbiotically fix nitrogen with rhizobia, playing a crucial role in fertilizing and improving soils and increasing yields. However, soybean production in my country is low, primarily due to reliance on imports. Soybean production is affected by a variety of factors, among which low phosphorus availability is a major factor limiting soybean growth and yield. With the continuous development of my country's economy and rising income levels, residents' demand for plant protein and edible vegetable oils, as well as their quality, has increased significantly. However, soybean yields per hectare have remained at around 1,950 kg / hectare in recent years. The low soybean productivity not only makes it difficult to meet the people's growing demand for plant protein and edible vegetable oil, but also poses a certain threat to my country's food supply and industrial structure to a certain extent.

[0003] Phosphorus, a macronutrient, is essential for crop growth and development. However, a lack of available phosphorus in the soil can lead to stunted crop development and even reduced yields. Phosphorus is the second most important nutrient in plants, after carbon, oxygen, and hydrogen. Its content in plants is second only to nitrogen and potassium, accounting for approximately 0.2-1.1% of their dry matter weight. Phosphorus plays an irreplaceable role in all plant life processes. In organisms, phosphorus is not only involved in the synthesis of biomacromolecules such as nucleic acids and phospholipids, but also plays a vital role in energy metabolism, enzymatic reactions, and signal transduction. Adequate phosphorus can enhance crop resilience, including resistance to drought, cold, pests and diseases, and lodging. Insufficient phosphorus can severely impact plant growth and development, resulting in slowed growth, a purple-red discoloration in stems and leaves due to the accumulation of anthocyanins, dwarfed plants, poor root development, premature aging, delayed fruit and seed ripening, and reduced yield and quality.

[0004] Currently, there are relatively few genetic resources for regulating soybean growth and phosphorus efficiency. Existing studies have revealed that the soybean purple acid phosphatase gene GmPAP35E2, the ubiquitin-binding enzyme gene GmUBC2, and the GmKIN10 gene can promote soybean growth and increase phosphorus content. Polygalacturonase (PG) is a structural protein in plant cell walls and plays an important role in changes in cell wall structure. However, there are few studies on PG genes in soybeans, and their functional roles have not been verified. Whether they are involved in regulating soybean growth and phosphorus efficiency is also unknown. Therefore, there is an urgent need to explore more genetic resources, which is of great significance for regulating phosphorus absorption and utilization efficiency and growth metabolism in soybeans. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortage of existing gene resources for regulating soybean growth and phosphorus efficiency, and to provide the application of GmPG39 gene in regulating soybean growth and phosphorus efficiency.

[0006] The first object of the present invention is to provide a new application of the GmPG39 gene.

[0007] The second object of the present invention is to provide the use of a preparation for promoting GmPG39 gene expression.

[0008] A third object of the present invention is to provide a product for promoting soybean growth and / or increasing the phosphorus content of soybean.

[0009] A fourth object of the present invention is to provide a method for promoting soybean growth and / or increasing the phosphorus content of soybean.

[0010] A fifth object of the present invention is to provide a method for cultivating soybean plants with high phosphorus content.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention found that the soybean polygalacturonase GmPG39 gene is involved in soybean root cell wall remodeling. Its protein is expressed in the plant cell wall and is regulated by phosphorus in the root system. In response to low phosphorus stress, its expression is upregulated in the main root segment. The cDNA nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. Overexpression of the GmPG39 gene can increase polygalacturonase activity, reduce the pectin content of the cell wall in the hairy root, and aggravate the decomposition of the cell wall under low phosphorus stress, promoting cell wall relaxation. At the same time, the GmPG39 gene positively regulates soybean growth. Overexpression of the GmPG39 gene can significantly promote the growth of soybean detached hairy roots, increase the fresh weight and total root length of the hairy roots, increase the total phosphorus content and soluble phosphorus concentration, regulate the phosphorus absorption efficiency of soybeans, play an important role in promoting soybean root growth and improving the phosphorus absorption efficiency of soybean roots, provide important genetic resources for breeding phosphorus-efficient legume varieties, and have important significance for the development of green agriculture.

[0013] Therefore, the present invention provides the following applications of the GmPG39 gene:

[0014] Application in positive regulation of soybean growth and / or phosphorus efficiency.

[0015] Application in promoting soybean growth.

[0016] Application in increasing phosphorus content in soybeans.

[0017] At the same time, the present invention provides the use of a preparation for promoting GmPG39 gene expression in promoting soybean growth, preparing a growth promoter, increasing soybean phosphorus content, preparing a product with increased soybean phosphorus content, or cultivating soybean plants with high phosphorus content.

[0018] Preferably, the preparation is a plasmid, vector, or recombinant bacterium that overexpresses the GmPG39 gene.

[0019] More preferably, the preparation is a vector that overexpresses the GmPG39 gene.

[0020] Furthermore, the overexpression vector includes an Agrobacterium binary vector, which is prepared according to conventional methods in the art, and the Agrobacterium binary vector is pEGAD, pTF101s or a plant expression vector pTF101s-flag derived therefrom.

[0021] The present invention provides a product for promoting soybean growth and / or increasing soybean phosphorus content, comprising a plasmid, a vector or a recombinant bacterium for overexpressing the GmPG39 gene.

[0022] The present invention provides a method for promoting soybean growth and / or increasing soybean phosphorus content, which comprises over-expressing the GmPG39 gene in soybean or treating the plant with a preparation promoting the expression of the GmPG39 gene.

[0023] The present invention provides a method for cultivating soybean plants with high phosphorus content, wherein a recombinant expression vector for promoting the expression of the GmPG39 gene is transferred into the plant to obtain the plant with high phosphorus content.

[0024] Preferably, a recombinant overexpression vector containing a gene encoding the GmPG39 protein is constructed, and the recombinant vector is transformed into the plant by Agrobacterium-mediated transformation.

[0025] The present invention has the following beneficial effects:

[0026] The present invention discovered that the soybean polygalacturonase GmPG39 gene is involved in soybean root cell wall remodeling. Its protein is expressed in plant cell walls and is regulated by phosphorus in the root system. In response to low phosphorus stress, its expression is upregulated in the main root segment. Studies have found that the GmPG39 gene has the function of regulating soybean root growth and phosphorus efficiency. The GmPG39 gene positively regulates soybean growth. Overexpression of the GmPG39 gene can significantly promote the growth of soybean hairy roots in vitro, increase the fresh weight of hairy roots and total root length, increase the total phosphorus content and soluble phosphorus concentration, and regulate the phosphorus absorption efficiency of soybeans. It plays an important role in promoting soybean root growth and improving the phosphorus absorption efficiency of soybean roots, and provides an important genetic resource for breeding phosphorus-efficient legume varieties. It is of great significance to the development of green agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 2 shows the expression pattern analysis results of GmPG39 in soybean roots under different phosphorus concentrations (in the figure, (+P) indicates normal phosphorus treatment, (-P) indicates low phosphorus treatment, data are the mean and standard error of 3 replicates, asterisks indicate significant differences between the control (+P) and treatment (-P) (Student's t-test), *: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0028] Figure 2 The results of tobacco subcellular localization analysis of GmPG39 protein (the first row in the figure is the subcellular localization map of tobacco transformed with an empty vector (35S:GFP), and the second row is the subcellular localization map of GmPG39 fused with GFP protein in tobacco leaves (35S:GmPG39-GFP); the pictures from left to right are the contents observed and photographed under a laser confocal microscope using the green fluorescence channel (GFP), the overlapped picture (fusion), and the light microscope channel (bright field); scale bar 50μm).

[0029] Figure 3These are the results of subcellular localization analysis of GmPG39 protein after separation of the onion inner epidermal wall (the first row in the figure shows the subcellular localization map of the onion inner epidermal wall after separation transformed with an empty vector (35S:GFP), and the second row shows the subcellular localization map of GmPG39 fused to GFP protein after separation of the onion inner epidermal wall (35S:GmPG39-GFP); from left to right, the pictures are the contents observed under a laser confocal microscope using the green fluorescence channel (GFP), the overlapped picture (fusion), and the light microscope channel (bright field); scale bar 50 μm).

[0030] Figure 4 This is the tissue localization analysis of GmPG39 in isolated soybean hairy roots (histochemical localization analysis of GmPG39 in isolated soybean hairy roots, the scale of the first column of whole hairy root images is 1 cm, and the scale of the other root system images is 0.5 mm; arrows of different colors correspond to different magnified positions of the root system).

[0031] Figure 5 The graph shows the relative expression levels of the detached hairy roots of GmPG39 beans under different phosphorus treatments (+P in the figure indicates normal phosphorus treatment, -P indicates low phosphorus treatment, the data are the mean and standard error of 3 replicates, asterisks indicate significant differences between the control (+P) and treatment (-P) (Student's t-test), *: P < 0.05, **: P < 0.01, ***: P < 0.001).

[0032] Figure 6 Figure 3 Effects of overexpression of GmPG39 on the growth and development of transgenic soybean hairy roots in vitro (A is the phenotype of the empty control (CK) and transgenic hairy roots overexpressing GmPG39 (OX) under normal and low phosphorus conditions; B is the fresh weight of hairy roots; C is the total root length of hairy roots; D is the total phosphorus content; E is the polygalacturonase activity; F is the pectin content; G is the soluble phosphorus concentration; (+P) indicates normal phosphorus, and (-P) indicates low phosphorus; OX indicates the overexpression line; CK indicates the transgenic line transformed with the empty vector; scale bar is 1 cm; data are the means and standard errors of 8 replicates; asterisks (*) indicate significant differences between the overexpression line and the control line (Student's t-test; *: P < 0.05, **: 0.001) <P<0.01,***:P<0.001)。 DETAILED DESCRIPTION

[0033] 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.

[0034] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0035] The soybean (Glycine max (L.) Merr.) material used in the following examples is Yuechun 03-3 (YC03-3), which is from the Root Biology Research Center of South China Agricultural University.

[0036] Example 1 Expression pattern of GmPG39 gene

[0037] 1. Plant samples

[0038] Using the paper roll seedling method, select uniform-sized soybean YC03-3 seeds with intact seed coats. Sterilize with chlorine gas generated by reacting 100 mL of sodium hypochlorite with 4.2 mL of hydrochloric acid for 12 hours, then air-bleach the seeds under a clean bench for 1 hour before use. Cut a 20 × 20 cm square of filter paper and prepare a 1 / 4 soybean complete nutrient solution with a pH of 5.8 and sterile water. Sterilize the mixture until ready for use.

[0039] To perform paper roll culture, place plastic wrap on a test bench and soak filter paper with 1 / 4 complete soybean nutrient solution. Place seven sterilized beans approximately 1 cm from one side of the filter paper, with the hilum facing down, and roll the paper from the first bean to the end. Place the rolled filter paper, with the end without beans facing down, in a 500 mL beaker filled with 1 / 4 complete soybean nutrient solution. Wrap the top of the rolled filter paper with plastic wrap. Place the beaker in an incubator at 24-26°C. Incubate in the dark for 1 day, then cycle light / dark (12h / 12h) for 3-4 days until the radicle is 5-6 cm.

[0040] Seedlings of uniform growth were selected and transferred to complete soybean nutrient solution treated with different phosphorus concentrations: normal phosphorus (+P: 1250μM KH2PO4) and low phosphorus (-P: 12.5μM KH2PO4). Each treatment consisted of eight replicates, with eight seedlings per replicate. The pH of the nutrient solution was adjusted to approximately 5.8 every two days and replaced weekly. Soybean tissue samples were harvested at 7 and 14 days post-fertilization, frozen in liquid nitrogen, and stored at -80°C until further use.

[0041] 2. Real-time fluorescence quantitative PCR analysis

[0042] Total RNA was extracted from plant samples treated with different phosphate concentrations (normal phosphate treatment (+P: 1250 μM KH2PO4) and low phosphate treatment (-P: 12.5 μM KH2PO4)) 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 using the Applied Biosystems StepOnePlus Real-Time PCR system.

[0043] The soybean polygalacturonase gene GmPG39 was selected as a candidate gene. Its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. Quantitative PCR primers for the GmPG39 gene were designed and synthesized. The internal reference gene was the soybean housekeeping gene EF1-α (Glyma17g23900). The specific primers are shown below:

[0044] GmPG39-RT-F (SEQ ID NO:3): 5'-CCAAAGAATTCAGGTCATGGATG T-3';

[0045] GmPG39-RT-R (SEQ ID NO:4): 5'-TGAGAACCACCACTTGCTGC-3';

[0046] EF1-α-F (SEQ ID NO:5): 5'-TGCAAAGGAGGCTGCTAACT-3';

[0047] EF1-α-R (SEQ ID NO:6): 5'-CAGCATCACCGTTCTTCAAA-3'.

[0048] Prepare the reaction system: Calculate the required reaction volumes, mix all reagents except the cDNA, and aliquot 18 μL into each tube. Add 2 μL of cDNA template to a final reaction volume of 20 μL. Each 20 μL reaction system contains: 0.5 μL forward / reverse primers, 10 μL SYBR Premix Ex Taq (2×), 2 μL template, and 7 μL ddH2O.

[0049] Preparation of standard curve: 1-2 μL of sample was taken from the original cDNA solution of each sample into a new PCR centrifuge tube, and then the reaction system was gradient diluted, with each dilution of 10 times as a standard sample, and 5 concentration gradient standards were obtained by dilution in this way.

[0050] Quantitative PCR reaction program: 95°C pre-denaturation for 30 s, PCR reaction (95°C denaturation for 30 s, 60°C recombination for 15 s, 72°C extension for 30 s) for 40 cycles.

[0051] The quantitative PCR detection result was calculated by Rotor-Gene Real-Time Analysis Software to obtain the expression amount of each sample.

[0052] The expression results of GmPG39 gene in soybean roots under different phosphorus concentrations are shown in Table 1. Figure 1 As shown in Table 1, it is shown that GmPG39 gene is expressed in the roots of soybeans under different days and different phosphorus concentrations. Compared with normal phosphorus treatment, the expression amount of GmPG39 gene in soybean roots is significantly up-regulated under low phosphorus condition.

[0053] Example 2 Construction of expression vector

[0054] 1. Construction of overexpression vector pTF101s-GmPG39-OX

[0055] According to the full-length CDS sequence of GmPG39 gene, specific primers were designed, and the cDNA of soybean YC03-3 was used as a template to amplify the gene fragment by using the kit Phanta Max Super Fidelity DNA Polymerase (Novozyme, China). The specific primers are as follows:

[0056] GmPG39-OX-F (SEQ ID NO: 7): 5'-CTCTCGAGCTTTCGCGAGCTCATGTGTCCCTATGAAACCATACCA-3';

[0057] GmPG39-OX-R (SEQ ID NO: 8): 5'-GGATCCCCGGGTACCGAGCTCTAAGTTCCTTCTGTAAGTGCACGTTTG-3'.

[0058] Reaction system (50 μL): 2 x phata max buffer 25 μL, forward and reverse primers (10 mM) 2 μL each, 2 mM dNTPs 1 μL, Phanta Max Super Fidelity DNA Polymerase 1 μL, cDNA template 2 μL, ddH2O 18 μL.

[0059] PCR program: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 sec, annealing at 58°C for 30 s, annealing at 72°C for 1 min, and final extension at 72°C for 5 min, with denaturation and annealing repeated 30 times.

[0060] After gel electrophoresis, the PCR product was purified and recovered using an agarose gel kit (Megi Biotech, China) according to the manufacturer's instructions. The fragment was then inserted into the Sam I restriction site of the linearized pTF101s plasmid using the Clone Express II One Step Cloning Kit 29 (Novozymes, China). The fragment was then transformed into E. coli DH5α cells and cultured at 37°C for 12 hours. Positive clones were isolated and sent to the company for sequencing. After successful plasmid extraction, the target gene fragment was ligated into the pTF101s vector using a one-step cloning method, successfully generating the overexpression vector pTF101s-GmPG39-OX.

[0061] 2. Construction of subcellular localization expression vector 35s::GmPG39-GFP

[0062] Using soybean YC03-3 cDNA as a template, the forward and reverse specific primers of the GmPG39-GFP gene were used to amplify the full-length CDS sequence of the gene. The specific primers are as follows:

[0063] GmPG39-GFP forward specific primer (SEQ ID NO: 9): 5′-TCTAGCGCTACCGGTATGTGTCCCTATGAAACCATACCA-3′;

[0064] GmPG39-GFP reverse specific primer (SEQ ID NO: 10): 5′-ATGGTGGCGACCGGTCCTAAGTTCCTTCTGTAAGTGCACGTTTG-3′.

[0065] The PCR amplification system was as follows: 25 μL 2× Vazyme phata buffer, 1 μL Vazyme phata enzyme, 1 μL dNTP, 1 μL each of forward and reverse primers, 3 μL cDNA template, and finally 16 μL ddH2O was added to make up to 50 μL.

[0066] The PCR amplification program was as follows: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 30 seconds, 30 cycles from denaturation to extension, and storage of the PCR product at 16°C.

[0067] The amplified product was then detected by gel electrophoresis. If the band size was correct, the target fragment was further recovered and purified using an agarose gel DNA gel recovery kit (Meiji Bio, China). Age Ι was selected as the single restriction enzyme site of the pEGAD vector, and the target gene fragment was connected to the pEGAD vector using a one-step cloning method. The ligation reaction system was the same as above, and the ligation product was stored in -20 ° C refrigerators for standby use. Finally, the ligation product was transferred to the Escherichia coli DH5α competence, and the positive clone was shaken after being cultivated at 37 ° C for 12 hours. After successful sequencing, the subcellular localization expression vector 35s::GmPG39-GFP was obtained, and the positive strain was stored in -80 ° C refrigerators and the plasmid was extracted and transformed into Agrobacterium GV3101. The positive GV3101 strain was retained by PCR detection again, and the subcellular localization expression vector 35s::GmPG39-GFP was successfully constructed.

[0068] 3. Construction of the Histochemical Localization Expression Vector pGmPG39::GUS

[0069] Based on the GmPG39 gene sequence, specific primers pGmPG39::GUS-F / R were designed. Root DNA from soybean genotype YC03-3 was used as a template for PCR amplification of a 2000-bp sequence above the GmPG39 start codon. The specific primers are as follows:

[0070] pGmPG39::GUS-F (SEQ ID NO. 11): 5'-CATGATTACGAATTC ACGACCAGGTGTCATGCTTT-3';

[0071] pGmPG39::GUS-R (SEQ ID NO. 12): 5'-CCTACCCGGGGATCC TGTAGGTAAACATATGTGTG-3'.

[0072] The reaction conditions were pre-denaturation at 98°C for 5 min, denaturation at 98°C for 30 s, annealing at 58°C for 30 s, and renaturation at 72°C for 1 to 3 min (depending on the fragment size). This process was cycled 30 times, and extension was performed at 72°C for 10 min.

[0073] The PCR amplified product was purified by gel electrophoresis and recovered using a kit. After obtaining the purified PCR product, a homologous recombination kit was used. II. Recombination ligation of the PCR product with the pTF102 vector digested with restriction enzymes EcoRI and BamHI. A 20 μL reaction system contains 6 μL of PCR product, 8 μL of linearized vector plasmid, 2 μL of Exnase II, and 4 μL of reaction buffer. The reagent mixture is incubated at 37°C for 30 minutes. The recombinant plasmid is transformed into Escherichia coli and sequenced to obtain a plant expression vector for the soybean pGmPG39::GUS fusion gene. Finally, the target vector is transformed with Agrobacterium tumefaciens GV3101, positive clones are detected, and the bacterial suspension is stored at -80°C.

[0074] Example 3 Subcellular localization analysis of GmPG39 protein

[0075] 1. Subcellular localization analysis of GmPG39 protein transiently expressed in tobacco epidermis

[0076] Tobacco leaves were infected with Agrobacterium tumefaciens (GV3101) and injected with the 35S::GmPG39-GFP vector and the empty pEGAD vector. Transient expression of the 35S::GmPG39-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.

[0077] The results of subcellular localization analysis of GmPG39 protein in tobacco leaves are as follows Figure 2 As shown in the figure, the fluorescence of 35S::GmPG39-GFP is distributed in the cell wall membrane of tobacco epidermal cells, indicating that the GmPG39 protein is expressed in the cell wall membrane of the plant.

[0078] 2. Subcellular localization analysis of GmPG39 protein after separation of the inner epidermal wall of onion scales

[0079] Agrobacterium GV3101 containing 35S::GFP and 35S:GmPG39-GFP vectors was inoculated into YEP medium and cultured at 28°C for 24 h. The bacterial liquid was centrifuged and the cells were retained to prepare 10 mmol·L -1 MgCl2, 0.20 g·L -1 MES, 0.029 g·L -1 Resuspend the cells in a mixture of acetosyringone and acetosyringone and adjust the OD value to 0.6. Onion scale inner epidermal cells must be cultured on MS solid medium for 24 hours before transformation. Submerge the cells in the bacterial solution for 4-6 hours. Transformed onions are cultured for 24 hours and then observed for subcellular localization under a laser confocal microscope (Zeiss LSM780, Germany). Plasmolysis is performed using 7% sodium chloride solution. The excitation wavelength for the laser confocal microscope is 488 nm.

[0080] The results of subcellular localization analysis of GmPG39 protein after separation of the inner cortical wall of onion scales are as follows: Figure 3 As shown in the figure, the fluorescence of 35S::GmPG39-GFP is distributed in the cell walls of epidermal cells in onion scales, indicating that the GmPG39 protein is expressed in the cell walls of plants.

[0081] Example 4 Analysis of Tissue Localization of GmPG39 in Soybean Hairy Roots in Vitro

[0082] The in vitro hairy root transformation method of soybean cotyledonary nodes mediated by Agrobacterium rhizogenes was used. Soybean seeds with uniform size and intact seed coat were selected and surface sterilized in chlorine for 12 to 14 hours. The seeds were then placed in a clean workbench and blown for 30 minutes to remove excess chlorine. The seeds were then sown on germination medium and cultured at 28°C under light conditions for 4 days. Agrobacterium rhizogenes K599 containing the pGmPG39::GUS plasmid was streaked on a plate, and a single clone was picked and cultured at 28°C, 200 rpm / min for 12 hours until the OD 600 The soybean seeds were taken out after culturing for 4 days, and the germinated seeds were cut off at the hypocotyl region about 0.5 cm from the cytoplasm with a scalpel. The seeds were then split vertically along the ridge with a scalpel to remove the seedlings.

[0083] In addition, a scalpel was dipped in the bacterial solution and multiple cuts were made perpendicular to the cotyledonary nodes and hypocotyls. The cut explants were transferred horizontally upward to a culture dish containing moistened filter paper; then, the culture dish was sealed with plastic wrap and transferred to an incubator (24°C) for 5 days under light. The co-cultivated explants were transferred to a medium containing herbicides and carbenicillin and grown for 10 days. Fresh, healthy, and similarly morphologically transgenic hairy roots weighing approximately 0.1 g were then selected and transferred to MS solid medium containing normal phosphorus (+P: 1250 μM KH2PO4) or low phosphorus (-P: 10 μM KH2PO4) and grown for 5 days. After 5 days of exposure to various phosphorus treatments, excised transgenic soybean hairy roots were removed, rinsed three times with secondary water, and placed in GUS staining solution (0.1M Na₂HPO₄ / NaH₂PO₄, pH 7.2, 1mM X-Glu). The solution was vacuum-treated for 40 minutes and then transferred to a 37°C incubator for 24 hours in the dark. After staining, the roots were transferred to 75% (v / v) ethanol for storage. The GUS staining of the roots was observed and photographed under a stereomicroscope (Leica, Germany).

[0084] The results of tissue localization analysis of GmPG39 in soybean hairy roots in vitro are as follows: Figure 4As shown in the figure, under low phosphorus conditions, the expression level of GmPG39 promoter fused with GUS reporter gene in soybean detached hairy roots was significantly higher than that in the control treatment, and was mainly upregulated in the main root segment of soybean hairy roots.

[0085] Example 5 Effect of Overexpression of GmPG39 on the Growth and Development of Transgenic Soybean Hairy Roots in Vitro

[0086] To investigate the effects of overexpressing GmPG39 on the growth and development of transgenic soybean hairy roots in vitro, approximately 0.2 grams of transgenic hairy roots were inoculated in solid MS medium supplemented with either normal (+P: 1250 μM KH2PO4) or low (-P: 12.5 μM KH2PO4) phosphorus for 14 days. Ten independent biological replicates were performed for each treatment. After treatment, GmPG39 expression was measured using real-time fluorescence quantitative PCR, using the same method as in Example 1. The phenotypes of plants treated differently were also observed, and their galacturonase activity and pectin content were measured.

[0087] The expression results are as follows Figure 5 As shown, after 14 days of culture under normal phosphorus and low phosphorus conditions, the expression level of GmPG39 in the detached hairy roots of soybeans overexpressing GmPG39 was significantly higher than that of the empty control (CK), with the expression levels increased by about 3.5 times and 7.3 times, respectively.

[0088] The results of the effects on soybean growth are as follows Figure 6 As shown in the figure, the phenotypes of different hairy roots after 14 days of culture showed that under normal phosphorus (+P) conditions, overexpression of GmPG39 significantly promoted the growth of soybean hairy roots in vitro, while under low phosphorus (-P) conditions, overexpression of GmPG39 inhibited the growth of soybean hairy roots in vitro ( Figure 6 A).

[0089] Under normal phosphorus (+P) conditions, compared with the empty control (CK), the fresh weight of transgenic hairy roots overexpressing GmPG39 increased by 30.2%, the total root length increased by 34.8%, the total phosphorus content increased by 39.1%, and the soluble phosphorus concentration increased by 15.6% ( Figure 6 B, C, D, E).

[0090] The results of the effect on the cell wall remodeling process of soybean showed that under normal phosphorus (+P) conditions, overexpression of GmPG39 increased the polygalacturonase activity of transgenic detached hairy roots. Compared with the empty control (CK), the polygalacturonase activity of transgenic hairy roots overexpressing GmPG39 increased by 3.8%; and reduced the content of pectin, the main component of the cell wall in soybean detached hairy roots, with the pectin content reduced by 10.8%.

[0091] Under low phosphorus (-P) conditions, overexpression of GmPG39 significantly increased the polygalacturonase activity of transgenic detached hairy roots. Compared with the empty control (CK), the polygalacturonase activity of transgenic detached hairy roots overexpressing GmPG39 increased by 16.1% ( Figure 6 F), the pectin content decreased by 27.9% ( Figure 6 G).

[0092] In summary, the study found that the GmPG39 gene is involved in the remodeling of soybean root cell walls. Overexpression of GmPG39 can increase polygalacturonase activity and reduce the pectin content in the cell walls of root hairs. It also responds to low phosphorus stress. Under low phosphorus stress, it will aggravate the decomposition of cell walls and promote cell wall relaxation. At the same time, the GmPG39 gene positively regulates soybean growth. Under normal phosphorus conditions, overexpression of the GmPG39 gene can significantly promote the growth of soybean in vitro root hairs, increase the fresh weight of root hairs and total root length, increase the total phosphorus content and soluble phosphorus concentration, regulate the phosphorus absorption efficiency of soybeans, and play an important role in promoting soybean root growth and improving the phosphorus absorption efficiency of soybean roots.

[0093] 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. GmPG39 The application of the gene in promoting soybean growth is characterized in that: Overexpression in soybean under normal phosphorus conditions GmPG39 gene; GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. GmPG39 The application of the gene in increasing the phosphorus content of soybean is characterized in that: Overexpression in soybean under normal phosphorus conditions GmPG39 gene; GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

3. Promote GmPG39 Use of a gene-expressed preparation for promoting soybean growth under normal phosphorus conditions or in preparing growth-promoting products, characterized in that: described GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the preparation is an overexpression GmPG39 Gene vector or recombinant bacteria.

4. Promote GmPG39 Use of a gene-expressed preparation for increasing the phosphorus content of soybeans under normal phosphorus conditions or in preparing products with increased phosphorus content in soybeans, characterized in that: described GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the preparation is an overexpression GmPG39 Gene vector or recombinant bacteria.

5. Promote GmPG39 The use of a gene-expressed preparation in cultivating soybean plants with high phosphorus content under normal phosphorus conditions is characterized in that: described GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the preparation is an overexpression GmPG39 Gene vector or recombinant bacteria.

6. A method for promoting soybean growth and increasing soybean phosphorus content, characterized in that: Overexpression in soybean under normal phosphorus conditions GmPG39 Genes, or adoption of GmPG39 Plants are treated with a gene-expressing agent; GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the preparation is an overexpression GmPG39 The gene vector or recombinant bacteria, the plant is soybean.

7. A method for cultivating soybean plants with high phosphorus content, characterized in that: Under normal phosphorus conditions, GmPG39 The recombinant expression vector expressing the gene is transferred into the plant to obtain a plant with high phosphorus content; GmPG39 The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2. The plant is soybean.

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