Application of GmPG39 gene in regulation and control of soybean growth and phosphorus efficiency

Abstract

The invention discloses application of a GmPG39 gene in regulation and control of soybean growth and phosphorus efficiency. Researches find that the GmPG39 gene participates in soybean root system cell wall remodeling, the protein of the GmPG39 gene is expressed in a plant cell wall, is regulated and controlled by phosphorus in a root system, and responds to low-phosphorus stress to realize up-regulated expression in a main root segment. The over-expression of the GmPG39 can increase the activity of polygalacturonase, reduce the pectin content of cell walls in hair roots, aggravate the decomposition of the cell walls under the stress of low phosphorus, and promote the relaxation of the cell walls. Meanwhile, the GmPG39 gene positively regulates soybean growth and phosphorus efficiency, under the normal phosphorus condition, overexpression of the GmPG39 gene can remarkably promote growth of soybean in-vitro hair roots, increase fresh weight and total root length of the hair roots, improve total phosphorus content and soluble phosphorus concentration and regulate phosphorus absorption efficiency of the soybeans, and the GmPG39 gene has a significant effect on promoting soybean root growth and improving phosphorus absorption efficiency of the soybeans. The method plays an important role in improving soybean root system phosphorus absorption efficiency.

Description

Technical Field

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

[0002] Soybean is an important grain and oil crop in my country. It is widely distributed in many countries and is one of the five major crops. Soybean protein is as high as 40%, and it contains eight essential amino acids that cannot be synthesized by the human body. Soybean fat is as high as 20%, which is one of the important raw materials for edible vegetable oil. Soybean also contains a variety of minerals and vitamins necessary for the human body. Tofu, bean sprouts, soy sauce, and yuba made from it are necessities in life. Soybean and its products play an important role in health products, medicines, and chemical products. At the same time, soybean is also a good crop for land cultivation. During its growth, its root system will symbiotically fix nitrogen with rhizobia, which plays an important role in fertilizing and improving soil and increasing production. However, the output of soybean in my country is not high, and it mainly depends on imports. The growth of soybean is affected by many factors, among which low phosphorus effectiveness is one of the main factors affecting soybean growth and limiting soybean production. With the continuous development of my country's economy and the continuous improvement of people's income level, residents' demand and quality for plant protein and edible vegetable oil are getting higher and higher. However, in recent years, the yield per unit area of ​​soybean has basically remained at 1950 kg / hectare. The low productivity of soybeans not only makes it difficult to meet the nation'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] As a macronutrient, phosphorus is indispensable in the growth and development of crops, but the lack of effective phosphorus in the soil causes poor crop development and even reduces yields. Phosphorus is the second largest nutrient in plants. In addition to carbon, oxygen and hydrogen, the content of phosphorus in plants is second only to nitrogen and potassium, accounting for about 0.2-1.1% of its dry matter weight. Phosphorus plays an irreplaceable role in all life activities of plants. In organisms, phosphorus is not only involved in the synthesis of biological macromolecules such as nucleic acids and phospholipids, but also plays an important role in energy metabolism, enzymatic reactions and signal transduction. Sufficient phosphorus can improve the resistance of crops, including drought resistance, cold resistance, disease and pest resistance, and lodging resistance. Insufficient phosphorus supply will seriously affect the growth and development of plants, resulting in slow growth process, stems and leaves often appear purple-red due to the accumulation of anthocyanins, short plants, poor root development, premature aging, delayed fruit and seed maturity, reduced yield and quality.

[0004] At present, there are still few genetic resources for regulating soybean growth and phosphorus efficiency. For example, the soybean purple acid phosphatase gene GmPAP35E2, the ubiquitin-binding enzyme GmUBC2 gene, and the GmKIN10 gene disclosed in existing studies can promote soybean growth and increase phosphorus content. Polygalacturonase (PG) is a plant cell wall structural protein that plays an important role in changes in cell wall structure. However, there are few studies on the PG gene in soybeans, and its functional role has not been verified. It is also unknown whether it is involved in the regulation of soybean growth and phosphorus efficiency. Therefore, it is urgent to tap more genetic resources, which is of great significance to the regulation of phosphorus absorption and utilization efficiency and growth metabolism of soybeans. Summary of the invention

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

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

[0009] The 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 the remodeling of soybean root cell walls, and its protein is expressed in plant cell walls and regulated by phosphorus in the root system. It is upregulated in the main root segment in response to low phosphorus stress. The cDNA nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2; overexpression of the GmPG39 gene can increase the activity of polygalacturonase, reduce the pectin content of the cell wall in the hairy root, and aggravate the decomposition of the cell wall under low phosphorus stress, and promote 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 in vitro hairy roots, 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 the growth of soybean roots and improving the phosphorus absorption efficiency of soybean roots. It provides important gene resources for breeding phosphorus-efficient legume varieties, which is of great significance to 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, in preparing growth promoters, in increasing soybean phosphorus content, in preparing products for increasing soybean phosphorus content, or in cultivating soybean plants with high phosphorus content.

[0018] Preferably, the preparation is a plasmid, a vector, or a 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 a conventional method in the art, and the Agrobacterium binary vector is pEGAD, pTF101s or a plant expression vector pTF101s-flag derived therefrom.

[0021] The 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 a GmPG39 gene.

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

[0023] The 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 plants to obtain the plants 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 a plant by an Agrobacterium-mediated transformation method.

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

[0026] The present invention found that the soybean polygalacturonase GmPG39 gene is involved in the remodeling of soybean root cell walls, and its protein is expressed in plant cell walls and regulated by phosphorus in the root system. It is upregulated in the main root segment in response to low phosphorus stress. 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 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 soybean root phosphorus absorption efficiency, and provides important gene resources 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 The results of the expression pattern analysis of GmPG39 in soybean roots under different phosphorus concentrations (in the figure, (+P) represents normal phosphorus treatment, (-P) represents low phosphorus treatment, the data are the mean and standard error of 3 replicates, and the asterisk indicates a significant difference between the control (+P) and the 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 to 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 3The results of subcellular localization analysis of GmPG39 protein after separation of the onion inner epicortical wall (the first row in the figure is the subcellular localization map of the onion inner epicortical wall after separation transformed with an empty vector (35S:GFP), and the second row is the subcellular localization map of GmPG39 fused to GFP protein after separation of the onion inner epicortical wall (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).

[0030] Figure 4 This is the tissue localization analysis of GmPG39 in soybean ex vivo hairy roots (histochemical localization analysis of GmPG39 in soybean ex vivo 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 positions of the enlarged root system).

[0031] Figure 5 The relative expression results of the in vitro hairy roots of overexpressed GmPG39 beans under different phosphorus treatments (+P in the figure represents normal phosphorus treatment, -P represents low phosphorus treatment, the data are the mean and standard error of 3 replicates, and the asterisk indicates a significant difference between the control (+P) and the treatment (-P) (Student's t-test), *: P < 0.05, **: P < 0.01, ***: P < 0.001).

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

[0033] 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.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are 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 soybean YC03-3 seeds with intact seed coat and uniform size, sterilize with chlorine gas produced by the reaction of 100mL sodium hypochlorite and 4.2mL hydrochloric acid for 12 hours, and then blow for 1 hour on a clean bench for later use. Cut a 20×20cm square filter paper, prepare 1 / 4 soybean complete nutrient solution with a pH of 5.8 and sterile water, and sterilize for later use.

[0039] When rolling paper culture, spread the plastic wrap on the test table, soak the filter paper with the prepared 1 / 4 soybean complete nutrient solution, place 7 sterilized beans about 1 cm away from one side of the filter paper, with the hilum of the beans facing down, and roll from the first bean to the end. Put the rolled filter paper with the end without beans facing down into a 500mL beaker filled with 1 / 4 soybean complete nutrient solution, and wrap the upper filter paper with beans with plastic wrap. Put the beaker in the incubator at 24-26℃, first culture in the dark for 1 day, and then culture in light / darkness (12h / 12h) for 3-4 days until the radicle is 5-6cm.

[0040] Seedlings with consistent growth were selected and transferred to soybean complete nutrient solution with different phosphorus concentrations: normal phosphorus treatment (+P: 1250μM KH2PO4) and low phosphorus treatment (-P: 12.5μM KH2PO4), with 8 replicates for each treatment and 8 seedlings for each replicate. The pH of the nutrient solution was adjusted to about 5.8 every two days, and the nutrient solution was changed once a week. Samples of different soybean tissues were harvested at 7d and 14d, frozen in liquid nitrogen, and stored in a -80℃ refrigerator for later use.

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

[0042] The total RNA of the plant samples treated with different phosphorus concentrations (normal phosphorus treatment (+P: 1250μM KH2PO4) and low phosphorus treatment (-P: 12.5μM KH2PO4)) was extracted using the TRizol kit (Invitrogen, USA). The 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 sample was diluted 10 times and then analyzed by real-time fluorescence quantitative PCR using the Applied Biosystems StepOnePlus Real-Time PCR system.

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

[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: first calculate the required reaction volume, mix all reagents except cDNA, dispense 18μL into each tube, and then add 2μL cDNA template to make the final reaction volume 20μL. Each 20μL reaction system contains: 0.5μL forward / reverse primer, 10μL SYBR Premix Ex Taq (2×), 2μL template, 7μL ddH2O.

[0049] Preparation of standard curve: First, pipette 1-2 μL of the cDNA stock solution of each sample into a new PCR centrifuge tube, and then dilute the reaction system in sequence, with each 10-fold dilution as a standard sample, and so on to obtain 5 concentration gradient standards.

[0050] The quantitative PCR reaction program was as follows: pre-denaturation at 95°C for 30 s, followed by PCR reaction (denaturation at 95°C for 30 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s) for 40 cycles.

[0051] The quantitative PCR detection results were used to calculate the expression level of each sample using Rotor-Gene's Real-Time Analysis Software.

[0052] The expression results of GmPG39 gene in soybean root system under different phosphorus concentrations are shown in Figure 2. Figure 1 As shown, the GmPG39 gene was expressed in the roots of soybeans at different days and different phosphorus concentrations. Compared with the normal phosphorus treatment, the low phosphorus treatment significantly upregulated the expression level of the GmPG39 gene in soybean roots under low phosphorus conditions.

[0053] Example 2 Construction of expression vector

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

[0055] Specific primers were designed based on the full-length CDS sequence of the GmPG39 gene, and the cDNA of soybean YC03-3 was used as a template to amplify the gene fragment 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 (50μL): 2×phata max buffer 25μL, forward and reverse primers (10mM) 2μL each, 2mMdNTPs 1μL, Phanta Max Super Fidelity DNA Polymerase 1μL, cDNA template 2μL, ddH2O 18μL.

[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, renaturation at 72°C for 1 min, final extension at 72°C for 5 min, with denaturation to renaturation repeated 30 times.

[0060] After gel electrophoresis, the PCR product was recovered and purified using an agarose gel kit (Meiji Biotech, China) according to the instructions. Then, the fragment was inserted into the Sam I restriction site of the linearized pTF101s plasmid using the Clone Express II One Step Cloning Kit 29 Recombination Exchange Kit (Novozyme, China), and then transferred into E. coli DH5α. After culturing at 37°C for 12 hours, the positive clone was shaken and sent to the company for sequencing. After successful extraction of the plasmid, the target gene fragment was connected to the pTF101s vector using the one-step cloning method, and the overexpression vector pTF101s-GmPG39-OX was successfully prepared.

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

[0062] Using soybean YC03-3 cDNA as a template, the forward specific primer and reverse specific primer 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: 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 agarose gel DNA gel recovery kit (Meiji Bio, China) was further used to recover and purify the target fragment. AgeΙ was selected as the single restriction 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 a -20 ° C refrigerator for standby use. Finally, the ligation product was transferred to the Escherichia coli DH5α competence, and the positive clone was shaken after culturing at 37 ° C for 12 hours. After the company successfully sequenced, the subcellular localization expression vector 35s::GmPG39-GFP was obtained, and the positive strain was stored in a -80 ° C refrigerator, the plasmid was extracted, and Agrobacterium GV3101 was transformed. 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 histochemical localization expression vector pGmPG39::GUS

[0069] According to the GmPG39 gene sequence, the specific primer pGmPG39::GUS-F / R was designed. The root DNA of soybean genotype YC03-3 was used as a template for PCR amplification to amplify the 2000bp sequence on the start codon of GmPG39. 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 repeated 30 times, and extension was performed at 72°C for 10 min.

[0073] The PCR amplified product was purified by gel electrophoresis using a kit. After obtaining the purified PCR product, a homologous recombination kit was used. II The PCR product was recombined with the pTF102 vector cut by restriction endonucleases EcoR I and BamH I. The reaction system was 20 μL, including 6 μL of PCR product, 8 μL of linearized vector plasmid, 2 μL of recombinant ligase Exnase II, and 4 μL of reaction buffer. The reagent mixture was reacted at 37°C for 30 minutes, the recombinant plasmid was transformed into Escherichia coli and sequenced to obtain the plant expression vector of soybean pGmPG39::GUS fusion gene. Finally, the target vector was transformed with GV3101 Agrobacterium, the positive clones were detected and the bacterial solution was 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] Agrobacterium was used to infect tobacco, and Agrobacterium GV3101 loaded with 35S::GmPG39-GFP vector and pEGAD empty vector was injected into tobacco leaves to transiently express 35S::GmPG39-GFP vector and pEGAD empty vector in tobacco epidermal cells. GFP fluorescence signal of tobacco epidermal cells was observed using laser confocal microscopy.

[0077] The results of subcellular localization analysis of GmPG39 protein in tobacco leaves are as follows Figure 2 As shown in the figure, it can be seen that 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 with YEP medium and cultured at 28°C for 24 h. The bacterial solution 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 A mixed solution of acetosyringone was used to resuspend the bacteria and adjust its OD value to 0.6. The epidermal cells of onion scales should be cultured on MS solid medium for 24 hours in advance. During transformation, the bacterial solution was used to immerse the epidermal cells of onion scales for 4 to 6 hours. After 24 hours of culture, the subcellular localization of the transformed onions was observed under a laser confocal microscope (Zeiss LSM780, Germany). 7% sodium chloride solution was used for plasmolysis. The excitation wavelength of the laser confocal microscope was 488nm.

[0080] The results of subcellular localization analysis of GmPG39 protein after separation of the inner epidermal wall of onion scales are as follows Figure 3 As shown in the figure, it can be seen that 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. They were then sown on germination medium and cultured at 28°C for 4 days under light conditions. Agrobacterium rhizogenes K599 containing the pGmPG39::GUS plasmid was streaked on a plate, a single clone was picked, and cultured at 28°C, 200 rpm / min for 12 hours until the OD 600 The value is about 1.0. Take out the soybean seeds after 4 days of cultivation, cut the germinated seeds with a scalpel at the hypocotyl area about 0.5 cm from the ion leaf, and then cut the seeds vertically along the ridge with a scalpel to cut off the seed sprouts.

[0083] In addition, a scalpel was used to dip the bacterial solution and cut multiple wounds vertically at the cotyledonary node and hypocotyl. The cut explants were moved 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 light culture for 5 days. The co-cultured explants were transferred to a medium containing herbicides and carbenicillin and grown for 10 days. After that, transgenic hairy roots with fresh growth, good condition, similar root morphology, and a weight of about 0.1g were selected and transferred to MS solid medium containing normal phosphorus (+P: 1250μM KH2PO4) or low phosphorus (-P: 10μM KH2PO4) for 5 days. After the transgenic soybean in vitro hairy roots were treated with different phosphorus for 5 days, the treated hairy roots were first taken out, rinsed with secondary water for 3 times, and placed in GUS staining solution (0.1M Na2HPO4 / NaH2PO4, pH 7.2, 1mM X-Gluc), vacuumed for 40 minutes, and then transferred to a 37°C constant temperature incubator for staining in the dark for 24 hours. After the roots were colored, the hairy roots were transferred to 75% (v / v) ethanol for storage, and the root GUS staining was observed under a stereo microscope (Leica, Germany) and photographed.

[0084] The results of tissue localization analysis of GmPG39 in soybean hairy roots in vitro are as follows Figure 4As shown, under low-phosphorus conditions, the expression level of GmPG39 promoter fused with GUS reporter gene in soybean hairy roots in vitro 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 explore the effect of overexpression of GmPG39 on the growth and development of transgenic soybean hairy roots in vitro, about 0.2 grams of transgenic hairy roots were inoculated in solid MS medium with normal phosphorus (+P: 1250μM KH2PO4) and low phosphorus (-P: 12.5μM KH2PO4) concentrations and cultured for 14 days, with ten independent biological replicates for each treatment. After treatment, the expression of GmPG39 was detected (real-time fluorescence quantitative PCR, the method was the same as in Example 1). At the same time, the phenotypes of plants with different treatments were observed, and their galacturonase activity and pectin content were determined.

[0087] The expression results are as follows Figure 5 As shown, after 14 days of cultivation under normal phosphorus and low phosphorus conditions, the expression level of GmPG39 in the in vitro 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, after 14 days of culture, the phenotypes of different hairy roots 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, and the pectin content decreased 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 the activity of polygalacturonase and reduce the pectin content of cell walls in 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, 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.

[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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The GmPG39 gene is used in positively regulating soybean growth and / or phosphorus efficiency, characterized in that: The nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. The application of GmPG39 gene in promoting soybean growth is characterized in that: The GmPG39 gene is overexpressed in soybean; the nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

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

2.

4. Use of a preparation for promoting GmPG39 gene expression in promoting soybean growth or in preparing a growth-promoting agent, characterized in that: The nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

5. Use of a preparation for promoting GmPG39 gene expression in increasing the phosphorus content of soybeans, or in preparing a product for increasing the phosphorus content of soybeans, characterized in that: The nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

6. Use of a preparation for promoting the expression of the GmPG39 gene in cultivating soybean plants with high phosphorus content, characterized in that: The nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

7. The use according to any one of claims 4 to 6, characterized in that: The preparation is a plasmid, a vector or a recombinant bacterium that overexpresses the GmPG39 gene.

8. A product for promoting soybean growth and / or increasing the phosphorus content of soybean, characterized in that: A plasmid, vector, or recombinant bacterium containing an overexpressed GmPG39 gene; the nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

9. A method for promoting soybean growth and / or increasing the phosphorus content of soybean, characterized in that: The GmPG39 gene is overexpressed in soybeans, or the plants are treated with a preparation that promotes the expression of the GmPG39 gene; the nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

10. A method for cultivating soybean plants with high phosphorus content, characterized in that: The recombinant expression vector that promotes the expression of the GmPG39 gene is transferred into the plant to obtain a plant with a high phosphorus content; the nucleotide sequence of the GmPG39 gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

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