CN108795956B - Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans - Google Patents

Abstract

The invention disclosesGmMDH12The application of the gene in promoting the nodulation and nitrogen fixation of soybean. In particular malate dehydrogenase genesGmMDH12Controlling the synthesis of the malic acid of the root nodule and the application thereof in promoting the root nodulation of leguminous crops. The research of the invention finds that the invention,GmMDH12has the function of regulating the synthesis of malic acid from soybean root nodule and the over-expressionGmMDH12The endogenous malic acid concentration of the soybean transgenic composite plant root nodule is obviously increased, and the number of the root nodules and the biomass of the root nodules are increased, so that the nitrogen content and the biomass of the plant are increased. In accordance with the present invention, there is provided,GmMDH12the gene has the potential of improving the biological nitrogen fixation function of leguminous crops through a transgenic technology, and has important theoretical and practical significance for improving the efficient utilization of crop nutrients and the development of environment-friendly sustainable agriculture.

Description

Application of GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans

Technical Field

The present invention belongs to the field of plant gene engineering technology. More particularly, toGmMDH12The gene is promoting soybeanThe application of the capability of nodulation and nitrogen fixation.

Background

The root system of leguminous plants can form a symbiotic system-root nodule which is mutually beneficial and complementary with rhizobia. The host plant can provide a carbon source and a proper growth environment for the growth of the rhizobia, and the rhizobia can carry out biological nitrogen fixation through the action of the azotobacter and provide a nitrogen source for the growth of the host. The rhizobium inoculation technology is applied to agricultural production to improve the nitrogen nutrition of crops, is an effective measure for saving fertilizer and increasing yield of leguminous crops, and plays an important role in agricultural production (Qin et al, 2012). The growth of legume rhizobia, the formation of nodules, and biological nitrogen fixation are complex physiological and biochemical processes that are performed by the co-participation of many metabolic pathways, including the carbon metabolic pathway (Hern a ndez et al, 2009). Soybean (semen glycines)Glycine max (L.) Merr]Is an important oil and protein crop worldwide, is an important raw material of edible oil and edible protein, and is also the most important leguminous crop in China. The soybean can be symbiotic with rhizobia to form root nodules, so that the nitrogen nutrition of the soybean can be improved, and the nitrogen fertilizer input can be reduced.

Phosphoenolpyruvate (PEP) is an important intermediate metabolite in the root nodule carbon metabolism. In nodules, PEP is assimilated mainly through two pathways, one being the tricarboxylic acid cycle pathway for the synthesis of organic acids into the nodule, and the other being the amino acid metabolic pathway for nitrogen assimilation (White et al, 2007). Studies suggest that the main direction of carbon metabolism in rhizobia is the pathway for the production of Oxaloacetate (OAA) via PEP and finally the synthesis of malate (Le Roux et al, 2008). By passing¹⁴C-marker test, malic acid found in the nodule has a marker signal, suggesting that malic acid may accumulate in the nodule as a product of carbon metabolism, and malic acid is considered as an important carbon source in the respiratory metabolism of the nodule bacteria, and is further used for metabolic consumption of the nodule bacteria (Rosendahl et al, 1990; Le Roux et al, 2006).

The malate dehydrogenase gene MDH (EC.1.1.1.37) can catalyze the reversible conversion of malate and OAA, and is a key enzyme in the tricarboxylic acid cycle pathway. In alfalfa, pea and vegetableIn legumes such as legumes, inoculation with rhizobia has been found to significantly increase MDH enzyme activity, and increased MDH enzyme activity in nodules is thought to act primarily to regulate malate synthesis in the carbon metabolic pathway of nodules (Miller et al, 1998; Colebatch et al, 2002; Le Roux et al, 2008; Hern a ndez et al, 2009). Moreover, numerous studies have also shown that overexpressionMDHThe gene can increase the content of endogenous malic acid in plants such as alfalfa, soybean and tobacco (Tesfaye et al, 2001; Luet al, 2012). However, it is not clear whether the important genes for regulating malic acid synthesis of root nodules and increasing malic acid synthesis can promote root nodulation and increase nitrogen nutrition of leguminous crops.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art, and to homologously clone a gene in soybean root nodule through analysis methods such as proteomics, quantitative PCR and the likeGmMDH12The gene (the sequence is shown as SEQ ID NO.3, and the coding amino acid sequence is shown as SEQ ID NO. 4) is found to be simultaneously subjected to low-phosphorus enhanced expression on the protein and transcription level, and proves thatGmMDH12The gene coded protein has the biological function of promoting the synthesis of the root nodule malic acid, further promotes the nodulation of the root system, and finally improves the nitrogen fixation capacity and biomass of the root nodule of the transgenic material.

The object of the present invention is to provideGmMDH12The application of the gene in promoting the nodulation and nitrogen fixation of soybean.

Another object of the present invention is to provideGmMDH12The application of gene enhancement in malic acid synthesis of root nodules.

The above purpose of the invention is realized by the following technical scheme:

the present invention relates to the function and application of a gene. Specifically, the invention provides a method for utilizing malic acid dehydrogenase geneGmMDH12Method for increasing malic acid synthesis of leguminous root nodule, promoting nodulation and nitrogen fixation and improving nitrogen efficiency of plant by overexpressionGmMDH12The transgenic soybean composite plants have the phenotype of increasing the endogenous malic acid concentration of the nodules, increasing the number and biomass of the nodules, and increasing the nitrogen content of the plants.

The present invention firstly carries out malate dehydrogenaseGmMDH12Cloning of full-Length cDNA of Gene, and the following studies were carried out

(1) Adopting an escherichia coli protein expression and purification system to analyze the enzymatic properties of GmMDH 12; by adopting the method for constructing the soybean transgenic composite plant, the method analyzes the analysis in the soybean hairy rootGmMDH12Tissue localization of genes, overexpression in Soybean rootGmMDH12And relatively studyGmMDH12The transgenic strain and the control strain have root nodulation and other phenotypes.

(2) ObtainGmMDH12Promoter driveGUSSoybean transgenic compound plant of reporter gene, analyzedGmMDH12Histochemical localization of the gene.

(3) The GmMDH12-GST fusion protein was obtained.

(4) Obtain excessive expressionGmMDH12The transgenic soybean composite plant of (1).

The research result of the invention shows that:

(1) histochemical localization analysis of the gene of the invention shows that,GmMDH12mainly located in rhizobium tissues such as thallus-like infected cells, vascular bundles, cortex and the like.

(2) The invention is as describedGmMDH12The gene coded protein has the activity of catalyzing oxaloacetate to synthesize malic acid in vitro.

(3) The concentration of the malic acid of the overexpression transgenic composite plant root nodule of the gene is obviously higher than that of a control line, namely, the overexpression of the gene is realizedGmMDH12Increasing the endogenous malic acid concentration of the root nodule of the transgenic composite plant.

(4) Under the conditions of low-nitrogen treatment and rhizobium inoculation, the number and biomass of the rhizobium of the overexpression transgenic composite plant of the gene are obviously higher than those of a control line, and the nitrogen content and dry weight of the plant are obviously higher than those of the control line. I.e. overexpressionGmMDH12Increasing the root nodule number and the root nodule biomass of the transgenic composite plant, and increasing the dry weight and the nitrogen content of the transgenic composite plant.

(5) The invention passes throughGmMDH12The functional analysis of the soybean transgenic composite plant definesGmMDH12In the family of the LeguminosaeFunction of root nodule in growth process, elucidationGmMDH12The participated root nodulation mechanism provides gene resources and theoretical basis for the high-efficiency utilization of nitrogen of leguminous crops including soybeans, has the application prospect of fertilizer saving and high yield, and has important practical significance for developing environment-friendly sustainable agriculture.

Therefore, the following applications should be within the scope of the present invention:

GmMDH12the application of the gene in enhancing malic acid synthesis of leguminous plant nodules.

GmMDH12The application of the gene in promoting nodulation and nitrogen fixation of leguminous plants.

GmMDH12The gene is applied to cultivating leguminous plants with strong root nodulation capacity and/or biological nitrogen fixation capacity.

Preferably, the leguminous plant is soybean.

In addition, based on the invention, the invention also provides a method for constructing the transgenic soybean with strong root nodulation capability and/or biological nitrogen fixation capability, which is to construct the transgenic soybean with strong root nodulation capability and/or biological nitrogen fixation capabilityGmMDH12The gene is overexpressed in soybean.

Specifically, overexpression was constructedGmMDH12The method for transgenic compound soybean comprises the following steps:

s1, constructing an overexpression vector

Using soybean root nodule cDNA as template, using upstream and downstream specific primers shown by SEQ ID NO. 14 and SEQ ID NO. 15 to make PCR amplificationGmMDH12A gene sequence;

after the PCR amplified fragment is recovered and sequenced without errors, the PCR amplified fragment is obtained bySac IAnd XbaI, carrying out double enzyme digestion on the amplified fragment and the target vector PTF101S respectively, and then carrying out double enzyme digestion on the amplified fragment and the target vectorGmMDH12Connecting the gene amplification fragment to a target vector PTF101S to obtain an expression vector;

s2. transformation

Then transferring the constructed expression vector into K599 by a freeze-thaw method, and transforming by utilizing the agrobacterium-mediated soybean transgenic composite plant to obtain a transgenic composite plant;

s3, verifying

Extracting transgenic composite plant root system and root nodule RNA, reverse transcriptionFormed cDNA, detected by quantitative PCRGmMDH12Effect of gene expression.

The invention has the following beneficial effects:

the research of the invention finds that the malic acid dehydrogenase geneGmMDH12Function in soybean root nodule growth metabolism:GmMDH12the gene has expression in the infected cells of mature rhizobium thallus, vascular bundles, cortex and other rhizobium tissues, and the expression amount of the gene is gradually increased along with the growth time of the rhizobium. The function in the aspect of malic acid synthesis of soybean root nodule: GmMDH12 has the function of catalyzing oxaloacetic acid to synthesize malic acid in vitro. And, compared to a control, overexpressionGmMDH12Obviously increases the malic acid concentration of the root nodule of the soybean transgenic composite plant. The function in promoting root nodulation is as follows: overexpression compared to controlGmMDH12Obviously increasing the root nodule number and the root nodule biomass of the soybean transgenic composite plant and improving the nitrogen content and the biomass of the plant.

Thus, a gene is providedGmMDH12The novel application of the gene can be used for increasing the malic acid synthesis of soybean root nodules, promoting the nitrogen fixation of the soybean nodules, cultivating high-efficiency nodulation nitrogen fixation transgenic leguminous plants and improving the nitrogen efficiency of crops, and can provide candidate gene resources for improving the biological nitrogen fixation capacity of leguminous crops including soybeans, and molecular breeding with high nitrogen utilization efficiency and high yield. In accordance with the present invention, there is provided,GmMDH12the gene has the potential of improving the biological nitrogen fixation function of leguminous crops through a transgenic technology, and has important theoretical and practical significance for improving the efficient utilization of crop nutrients and the development of environment-friendly sustainable agriculture.

Drawings

FIG. 1:GmMDH12protein and gene expression analysis.

FIG. 2:GmMDH12promoter driveGUSAnalysis of expression site of (3).

FIG. 3: enzymatic properties of GmMDH 12.

FIG. 4: overexpression ofGmMDH12Influence on nodulation of the transgenic compound plant of the soybean.

FIG. 5: overexpression ofGmMDH12Influence on the growth and nitrogen/phosphorus content of the soybean transgenic compound plant.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1GmMDH12Gene cloning and expression analysis

1. GmMDH12 protein expression analysis

Root nodule proteomics analysis experimental design: after 5 days of seed germination for soybean variety "HN 66", seedlings were inoculated with Rhizobium BXYD 31 hours and then transferred to low phosphorus (LP, 5. mu.M KH)₂PO₄) Or high phosphorus (HP, 250. mu.M KH)₂PO₄) Low nitrogen nutrient solution (50 μ M NH)₄NO₃) After 25 days of culture, aerial, root and nodule samples were harvested and stored at-80 ℃ for extraction of nodule proteins and RNA. Design of gene expression analysis experiments at different time points: after 5 days of soybean HN66 seed germination, seedlings were inoculated with Rhizobium BXYD 31 hours and then transferred to low phosphorus (LP, 25. mu.M KH)₂PO₄) Or high phosphorus (HP, 500. mu.M KH)₂PO₄) Low nitrogen nutrient solution (250 mu M NH)₄NO₃) Culturing, collecting root and root nodule samples at 14, 21, 30 and 40 days, respectively, and storing at-80 deg.C for malic acid and RNA extraction.

Two-dimensional electrophoresis analysis: adding 8.0 mL of extraction buffer (50% phenol, 0.45M sucrose, 25 mM EDTA, 2% beta-mercaptoethanol, 250 mM Tris-HCl pH 8.8, 2 mM PMSF, 2% PVPP, 0.3-1.4M NaCl, 10-40% ethanol) to 3 g of nodule sample, grinding, mixing, centrifuging at 12000 rpm at 4 ℃ for 15 minutes, sucking the supernatant phenol solution, adding 1/10 times volume of 5M NaCl, adding 5 times volume of precooled methanol (containing 0.1M ammonium acetate and 1% beta-mercaptoethanol), mixing, standing at-20 ℃ for 2 hours, centrifuging at 12000 rpm at 4 ℃ for 15 minutes, discarding the supernatant, washing the precipitate with precooled methanol (containing 0.1M ammonium acetate) for 2 times, washing with 80% acetone for 2 times, and finally washing with 70% alcohol for 1 time, the precipitate was collected, dried under vacuum and stored at-80 ℃ for further use. The protein sample was mixed with 600. mu.L of lysis Buffer (7M urea, 2M thiourea, 4% CHAPS, 80 mM DTT, 1% IPG Buffer) thoroughly, vortexed and shaken for 15 minutes, centrifuged at 12000 rpm at 4 ℃ for 15 minutes, and the supernatant was the protein sample solution. Protein samples were subjected to concentration determination using a protein quantification Kit (2-D Quant-Kit, GE-Healthcare, USA), a bovine serum albumin BSA (GE-Healthcare, USA) standard curve was prepared, the absorbance value of the samples was determined, and the protein concentration of the samples was calculated.

Adding 430 μ L of protein sample solution into the gel strip tank, adding IPG gel strip (24 cm), dripping covering oil, covering the gel strip tank, placing on IPGphor (GE-Healthcare, USA), and isoelectric focusing (IEF) procedure at 25 deg.C and 50 μ A gel^-1The voltage and time parameters are: 30V (rapid 12 hours) → 50V (rapid 2 hours) → 100V (linear 2 hours) → 200V (linear 2 hours) → 500V (linear 2 hours) → 1000V (linear 2 hours) → 8000V (linear 4 hours) → 8000V (rapid 90000 volt hours) → 500V (rapid 10 hours). The focused IPG strip was equilibrated. The rubber strip balance is carried out in two steps: in the first step, a 1% DTT-containing equilibrium solution [ 50 mM Tris-HCl (pH 8.8), 6M urea, 30% (V/V) glycerol, 2% (W/V) SDS, and a trace amount of bromophenol blue]Equilibrating for 15 minutes; the second step was equilibrated with an equilibration solution containing 2.5% iodoacetamide for 15 minutes.

The well-balanced IPG gel strip was transferred to the upper end of the gel and electrophoresed in Ettan DALTsix electrophoresis apparatus (GE-Healthcare, USA) at 10 mA.^-1After electrophoresis for 1 hour, 40 mA glue^-1And (5) electrophoresis is finished. After electrophoresis, the gel is washed by deionized water, and the fixing solution [ 40% (V/V) absolute ethyl alcohol and 10% (V/V) glacial acetic acid]Fixing for more than 1 hr, staining with Coomassie brilliant blue G-250 staining solution [ 0.08% (W/V) Coomassie brilliant blue G-250, 25% (V/V) methanol, 1% (V/V) phosphoric acid]Staining overnight, then washing several times with deionized water until the gel background is clear, scanning with an EPSON 1640' L-type scanner. The 2-DE glue is analyzed by adopting PD-Quest 7.0 (Bio-Rad, USA) software, and a table of significant differences under low-phosphorus and high-phosphorus treatment is screened outThe obtained nodule protein spot was subjected to in-gel trypsin (Trysin) enzymatic analysis, and then subjected to MALDI-TOF/TOF MS mass spectrometry using an ABI 4700 TOF-TOF mass spectrometer (Applied Biosystems, USA).

2、GmMDH12Gene cloning and expression analysis

Performing homologous comparison on soybean genome database Phytozome, predicting 16 members of soybean malate dehydrogenase family, and determining the regulated and controlled rhizobium by phosphorus in combination with proteomics analysisGmMDH12Gene and protein sequence information, designGmMDH12The full-length gene primer, the upstream specific primer 5'-ATGATGAAGCCATCGATGCTCAGAT-3' (SEQ ID NO:1), the downstream specific primer 5'-TTACTGGTTGGCAAATTTGATTCCCTT-3' (SEQ ID NO:2), the soybean root nodule cDNA as the template, PCR amplificationGmMDH12The full-length sequence of the gene Open Reading Frame (ORF) is sequenced and compared to obtainGmMDH12The whole sequence of the gene ORF is 1038 bp (SEQ ID NO:3), 345 amino acid residues are coded (SEQ ID NO:4), and the protein molecular weight is 36 kD.

And extracting the soybean nodule RNA harvested at different time points, and diluting cDNA obtained by reverse transcription of the RNA sample by 50 times to be used as a template of quantitative PCR reaction. Quantitative PCR analysis was performed using SYBR Green (Takara, Japan) quantitation kit, and reaction was performed using Rotor-Gene 3000 qRT-PCR system (Corbett Research, Australia). 20 μ L reaction system 10 μ L2 XSSYBR Green PCR master mix, 6.4 μ L Mili-Q water, 0.8 μ L10 μ M primer, 2 μ L100-fold diluted cDNA template. The reaction program was 95 ℃ for 1 minute, 94 ℃ for 15 seconds, 60 ℃ for 15 seconds, 72 ℃ for 30 seconds, 40 cycles. With soybeanGmEF1aRelative expression amount = target gene expression amount/housekeeping gene as housekeeping gene(s) (ii)GmEF1a) The expression level.GmMDH12The primers of the gene quantitative PCR are as follows: 5'-CTCACTCACTAATCGCCACTCTCAC-3' (SEQ ID NO:5) and R5'-GCGGAGTGGAGAGATCTGAGCA-3' (SEQ ID NO: 6). Soybean housekeeping geneGmEF1aThe quantitative PCR primers are as follows: 5'-TGCAAAGGAGGCTGCTAACT-3' (SEQ ID NO:7) and R5'-CAGCATCACCGTTCTTCAAA-3' (SEQ ID NO: 8).

3. Extraction and determination of endogenous malic acid in soybean root nodule

A0.2 g sample of soybean root nodule is ground with 0.6 mL of 0.5M HCl, the ground mortar is washed with 0.6 mL of distilled water and then transferred into a centrifuge tube together, the mixture is uniformly mixed for 20 minutes in a water bath at 80 ℃, the mixture is centrifuged for 15 minutes at 12000 rpm at 4 ℃, the supernatant is sucked up, and 1 mL of the supernatant is filtered through a 0.45 mu M microporous membrane. A high performance liquid chromatograph 1260 Infinity LC series (Agilent, USA) is adopted, the column temperature is 35 ℃, the mobile phase is 0.2% metaphosphoric acid, after a sample passes through a C18 column (4.6 mm multiplied by 250 mm), the absorption value of the wavelength of 220 nm is detected, and the concentration of malic acid in the sample is calculated by making a malic acid standard curve.

4. Results of the experiment

FIG. 1 is a drawing ofGmMDH12Protein and gene expression analysis. Wherein A: expression of GmMDH12 protein in nodules; b:GmMDH12expression of genes in roots and nodules; c:GmMDH12expression in nodules at different developmental stages; d: endogenous malic acid concentrations of nodules at different developmental stages. Data in the figure are mean and standard error of 3 biological replicates. The results show that:GmMDH12both proteins and genes were expressed in nodules under the control of phosphorus availability (FIGS. 1A, B). As shown in fig. 1C, under high and low phosphorus treatment, as the growth time of the nodules is prolonged,GmMDH12the gene expression level is gradually increased, the expression level is highest at 30 days, and the expression level is reduced in 40 days of root nodules. AndGmMDH12the gene expression pattern is similar, and the endogenous malic acid concentration of the nodules tends to increase and then decrease along with the increase of the growth time, wherein the malic acid concentration is highest in the nodules at 21 days and is lowest in the aged nodules at 40 days. The results show thatGmMDH12Participates in the growth and metabolism process of the root nodule.

Example 2 GmMDH12Gene promoter cloning, vector construction and tissue localization analysis

1. Experimental methods

GmMDH12Cloning of gene promoter and construction of GUS expression vector: extracting soybean root genome DNA, using soybean genome DNA as template, using upstream specific primer 5'-GGGATCCCCGTGCATGTGTTGA-3' (SEQ ID NO:9) and downstream specific primer 5'-ATGAATTCAGTGGCGATTAGTGAG-3' (SEQ ID NO:10) to make amplificationGmMDH12Promoter 2182 bp (SEQ ID NO:11), PCR fragment recoveryAfter sequencing, byBamH IAnd EcoRI, carrying out double enzyme digestion on the recovered fragment and a target vector pCAMBIA1391, connecting a promoter fragment to the pCAMBIA1391, and then transferring the constructed GUS expression vector into Agrobacterium rhizogenes K599 by a freeze-thaw method (the K599 strain is a gift from the Leguminosae comprehensive research center of university of Queensland, Australia and is stored in the root biology research center laboratory of university of south China, and the specific description is in Kereszt et al, 2007).

Constructing an agrobacterium-mediated soybean transgenic composite plant: after the soybean variety HN66 seed germinates for 5 days in sand culture, the soybean variety HN66 seed is used for induction of hair roots, a1 mL syringe is used for being stained with the cultured K599 thallus, the lower embryonic axis position of a seedling is pricked back and forth, and the thallus is coated on a wound, so that the thallus can fully infect the wound. After the infected seedlings were transferred to normal nutrient solution and cultured, hair roots were seen to grow out of the wound treatment after about 14 days, during which time care was taken to maintain the moisture of the infected wounds. Removing the main root after the hair root grows out, and placing the hair root in a nutrient solution for continuous culture. When the hair root grows to about 10 cm, inoculating rhizobium to the hair root for 1 hour, and culturing in low nitrogen (50 mM NH)₄NO₃) And low phosphorus (5 mu M KH)₂PO₄) After 21 and 30 days of culture in nutrient solution, GUS staining and paraffin section analysis were performed to analyze the tissue localization of the gene in roots and nodules.

2. Results of the experiment

FIG. 2 isGmMDH12Promoter driveGUSAnalysis of expression site of (3). The results show that, in the root section,GUSthe gene was mainly expressed in the root tip and the middle column (10 mm in each scale, FIGS. 2B-D). FIGS. 2E and 2F are longitudinal and transverse views, respectively, of a 21-day-old nodule, FIG. 2G is a transverse view of a 30-day-old nodule, and FIG. 2H is a partial enlarged view of FIG. 2G, all 200 mm in scale.

The results show that, in the nodule,GUSthe gene is mainly expressed in rhizoid thallus, vascular bundle and cortex, and the expression amount in thallus-like infected cell is gradually increased along with the growth time of the root nodule, which shows thatGmMDH12Participates in the growth process of the root nodule.

Example 3 enzymatic analysis of GmMDH12 Properties

1. Experimental methods

(1) Construction of prokaryotic expression vector: using soybean root nodule cDNA as template, using upstream specific primer 5'-GGATCCATGATGAAGCCATCGA-3' (SEQ ID NO:12) and downstream specific primer 5'-ACATGAATTCTTACTGGTTGGCAAATT-3' (SEQ ID NO:13) to make amplificationGmMDH12The full length of the gene is recovered and sequenced without errors by PCRBamH IAnd EcoRI after double digestion of the fragment and prokaryotic expression vector pGEX6P-3 (GE-Healthcare, USA), the fragment and the prokaryotic expression vector are addedGmMDH12The gene is connected to a target vector pGEX6P-3, and then the constructed expression vector is transferred into BL21 Escherichia coli by a freeze-thaw method.

(2) Taking a proper amount of BL21 bacterial liquid containing the target gene to 200 mL of fresh LB liquid culture medium, culturing at 28 ℃ until OD600 is 0.4, adding 0.2 mM IPTG to continue culturing for 12 hours, centrifuging the bacterial liquid at 12000 rpm for 10 minutes, collecting thalli, and using 8 mL Binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na)₂HPO₄, 1.8 mM KH₂PO₄pH 7.3), and sonicating the cells, centrifuging, collecting the supernatant, reacting the supernatant with 2 mL of Glutathione Sepharose 4B (GE-Healthcare, USA) at 4 ℃ for 3 hours, eluting the hybrid protein 3 times with a Binding buffer, eluting the column 3 times with an Elution buffer (50 mM Tris-HCl, 10 mM Reduced Glutathione, pH 8.0), and collecting the protein of GmMDH 12.

(3) The optimum reaction temperature is determined by placing the reaction mixture in different temperatures (20-90 ℃) for 10 minutes, adding the same amount of enzyme when the reaction is carried out, uniformly mixing, placing in an ultraviolet spectrophotometer, determining the change value of OD340 in 2 minutes, and recording the change condition of data. And (3) determining the optimum reaction pH, preparing 100 mM Tris-HCl in the mixture into different gradients with pH of 7.0-9.0 at the same temperature, preparing the reaction mixture, adding enzyme liquid during activity analysis, uniformly mixing, placing in an ultraviolet spectrophotometer, determining the change value of OD340 within 2 minutes, and recording the change condition of data. Measuring different substrate concentrations, setting different OAA substrate concentrations (0.02-3 mM), adding equal amount of enzyme and reaction mixture during reaction, mixing, and placing in ultraviolet spectrophotometerMeasuring the change value of OD340 in 2 minutes, recording the change condition of data, and obtaining the K of the enzymatic reaction by using a double reciprocal method (Lineweaver-Burk)_mThe value is obtained. Analysis of the influence of different Metal ions on the enzyme Activity, 0.1 mM of different Metal ions (Mn) were added to the same amount of enzyme and reaction mixture, respectively²⁺、Zn²⁺、Mg²⁺、Fe²⁺、Cu²⁺、Al³⁺、Ag²⁺And Hg²⁺) After mixing, the mixture was placed in an ultraviolet spectrophotometer to measure the change in OD340 in 2 minutes. 1. mu. mol of NADH, whose molar extinction coefficient is 6.22L mmol/cm, is defined as 1 unit (U) of enzyme activity per minute of catalytic reaction^-1。

2. Results of the experiment

FIG. 3 shows the analysis of the protease properties of GmMDH 12. Wherein, A: effect of different OAA substrate concentrations on enzyme activity; b: the influence of different pH values on the enzyme activity; c: effect of different temperatures on enzyme activity; d: the effect of different metal ions on the enzyme activity.

The results show that: the optimal substrate concentration of the GmMDH12 protein is 1.5 mM OAA, K of enzymatic reaction_mThe value was 0.434 mM (FIG. 3A), the optimum reaction pH and temperature were pH 8.0 and 50 ℃ respectively (FIGS. 3B, C), and 0.1 mM Mg²⁺、Ag⁺And Hg⁺Ions significantly inhibited the enzymatic activity of GmMDH12 (fig. 3D).

Example 4 overexpressionGmMDH12Vector construction, acquisition and detection of soybean transgenic composite plant

Construction of overexpression vector: using soybean root nodule cDNA as template, using upstream specific primer 5'-ACAAAGAGCTCGAGAGAGAGAAA-3' (SEQ ID NO:14) and downstream specific primer 5'-ATATCTAGAAACCTACCAGACATGTATG-3' (SEQ ID NO:15) to make amplificationGmMDH12Sequence, after recovery and sequencing of the PCR fragment, bySac IAnd XbaI double digestion of the fragment and the target vector PTF101S, and thenGmMDH12The gene is connected to a target vector PTF101S, and then the constructed expression vector is transferred into K599 by a freeze-thaw method, and the agrobacterium-mediated soybean transgenic composite plant is transformed as described above, and a transfer empty vector PTF101S strain is taken as a Control (CK).

Extracting transgenic composite plant root system and root nodule RNA, reverse transcription to cDNA, and further quantitative PCR detectionGmMDH12Effect of Gene expression, detection method and procedure of the sameGmMDH12Gene expression pattern analysis was identical.

Example 5 overexpressionGmMDH12Influence on nodulation of soybean transgenic composite plants

1. Experimental methods

When the soybean transgenic composite plant obtained in example 4 and the control line have the hairy root length of 10 cm, respectively inoculating rhizobium BXYD 31 hours, and then transferring to low-phosphorus (LP, 5 mu M KH)₂PO₄) Or high phosphorus (HP, 250. mu.M KH)₂PO₄) Low nitrogen nutrient solution (50 μ M NH)₄NO₃) In the middle, after 30 days of culture, overground part, root and nodule samples are harvested, and relevant physiological indexes are determined, including: the number of nodules and the fresh weight of nodules, and the malic acid concentration of nodules.

2. Results of the experiment

FIG. 4 is overexpressionGmMDH12Influence on nodulation of the transgenic compound plant of the soybean. Wherein, A: nodule phenotype, scale 1 cm; b: transgenic nodulesGmMDH12Analyzing expression; c: endogenous malic acid concentration of transgenic nodules; d: fresh and heavy root nodules; e: the number of nodules. CK represents an empty vector control strain; OX represents overexpressionGmMDH12Transgenic composite plants. The experimental data are the mean and standard error of 5 biological replicates. Number indicates that the difference between the control CK strain and the OX strain is significant,p<0.05; ns: indicating that the difference was not significant.

The results show that: overexpression relative to the empty vector control strain CKGmMDH12The endogenous malic acid concentration of the transgenic composite plant nodules was significantly increased (FIG. 4C), as well as the number and biomass of nodules (FIG. 4D, E).

Example 6 OverflowGmMDH12Influence on growth and nitrogen/phosphorus content of soybean transgenic composite plants

1. Experimental methods

When the soybean transgenic composite plant and the control line have root length of 10 cm, respectively inoculating rhizobia for 1 hour, and then transferringMove to low phosphorus (LP, 5. mu.M KH)₂PO₄) Or high phosphorus (HP, 250. mu.M KH)₂PO₄) Low nitrogen nutrient solution (50 μ M NH)₄NO₃) In (c), after 30 days of culture, the aerial parts and roots were harvested and relevant physiological indices were determined, including: plant biomass and nitrogen phosphorus content.

And (3) measuring plant biomass: the harvested aerial parts and root samples were de-enzymed in an oven at 105 ℃ for 30 minutes, then dried at 75 ℃ to constant weight, and the dry weight was weighed.

And (3) measuring nitrogen and phosphorus of the plants: respectively weighing about 0.1 g of overground part and root part samples, adding 5 mL of concentrated sulfuric acid for digestion, then diluting with distilled water to 50 mL, diluting the samples by 4 times, and then placing in a continuous flow analyzer (model SAN + +, produced in the Netherlands) for determination. And calculating the nitrogen and phosphorus content of the plant according to the measured nitrogen and phosphorus concentration of the sample and the dry weight of the plant.

2. Results of the experiment

FIG. 5 is overexpressionGmMDH12Influence on the growth and nitrogen/phosphorus content of the soybean transgenic compound plant. Wherein, A: dry weight of the plant; b: the nitrogen content of the plant; c: plant phosphorus content. The transgenic soybean composite plants were inoculated with rhizobia for 1 hour and transferred to low phosphorus (LP, 5. mu.M KH)₂PO₄) Or high phosphorus (HP, 250. mu.M KH)₂PO₄) Low nitrogen nutrient solution (50 μ M NH)₄NO₃) Cultured for 30 days. CK represents an empty vector control strain; OX represents overexpressionGmMDH12Transgenic composite plants. The experimental data are the mean and standard error of 5 biological replicates. Number indicates that the difference between the control CK strain and the OX strain is significant,p<0.05; ns: indicating that the difference was not significant.

The results show that: overexpression relative to the empty vector control strain CKGmMDH12Transgenic composite plants were significantly increased in dry weight (fig. 5A) and nitrogen content (fig. 5B), but had no significant effect on phosphorus content (fig. 5C).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> GmMDH12 gene in promoting nodulation and nitrogen fixation of soybeans

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> amplification of the full-length upstream specific primer (forward primer) of GmMDH12 Gene

<400> 1

atgatgaagc catcgatgct cagat 25

<210> 2

<211> 27

<212> DNA

<213> amplification of the full-Length downstream specific primer (reverse primer) of GmMDH12 Gene

<400> 2

ttactggttg gcaaatttga ttccctt 27

<210> 3

<211> 1038

<212> DNA

<213> Gene GmMDH12 sequence (GmMDH12)

<400> 3

atgatgaagc catcgatgct cagatctctc cactccgccg ccacccgcgg cgcctctcat 60

ctcttccgcc gtgggtacgc ctccgagccg gtgccggagc gcaaggtagc cgttctcggc 120

gccgccggcg ggatcgggca gcctctctcc cttctcatga agctcaatcc cctcgtttcg 180

agcctctccc tttacgatat cgccggaact cccggcgtcg ccgccgatat cagccacata 240

aacaccagat ctgaggtagt ggggtaccaa ggtgatgaag agcttggaaa agctttggag 300

ggtgccgatg ttgttataat tcctgctggt gtgcccagaa agcctggaat gactcgtgat 360

gatcttttta acatcaatgc tggcattgtt aagacgctgt gtactgctat tgctaagtat 420

tgcccccatg cccttgttaa catgataagc aatcctgtga actccactgt tcctattgct 480

gctgaagttt tcaagaaggc aggaacttat gatgagaaga gattgtttgg tgttaccacc 540

cttgatgttg ttagggcaaa aactttctat gctgggaaag ccaatgttcc agttgctggt 600

gttaatgtac ctgttgtggg cggccatgca ggcattacta ttctgccact attttctcaa 660

gccacaccaa aagccaatct tgatgatgat gtcattaagg ctcttacaaa gaggacacaa 720

gatggaggaa cagaagttgt agaagctaag gctggaaagg gttctgcaac tttgtcaatg 780

gcctatgctg gtgccctttt tgctgatgct tgcctcaagg gcctcaatgg agtcccagat 840

gttgtcgagt gctctttcgt gcaatccact gttactgaac ttcccttctt tgcttccaag 900

gtgaggcttg ggacggttgg tgtggaggaa gttctgggct tggggcacct ctcagatttt 960

gagcaacaag gcctcgaaag ccttaagcct gaactcaaat catcaattga gaagggaatc 1020

aaatttgcca accagtaa 1038

<210> 4

<211> 345

<212> PRT

<213> protein sequence encoded by GmMDH12 gene (GmMDH12)

<400> 4

Met Met Lys Pro Ser Met Leu Arg Ser Leu His Ser Ala Ala Thr Arg

1 5 10 15

Gly Ala Ser His Leu Phe Arg Arg Gly Tyr Ala Ser Glu Pro Val Pro

20 25 30

Glu Arg Lys Val Ala Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Pro

35 40 45

Leu Ser Leu Leu Met Lys Leu Asn Pro Leu Val Ser Ser Leu Ser Leu

50 55 60

Tyr Asp Ile Ala Gly Thr Pro Gly Val Ala Ala Asp Ile Ser His Ile

65 70 75 80

Asn Thr Arg Ser Glu Val Val Gly Tyr Gln Gly Asp Glu Glu Leu Gly

85 90 95

Lys Ala Leu Glu Gly Ala Asp Val Val Ile Ile Pro Ala Gly Val Pro

100 105 110

Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Asn Ile Asn Ala Gly

115 120 125

Ile Val Lys Thr Leu Cys Thr Ala Ile Ala Lys Tyr Cys Pro His Ala

130 135 140

Leu Val Asn Met Ile Ser Asn Pro Val Asn Ser Thr Val Pro Ile Ala

145 150 155 160

Ala Glu Val Phe Lys Lys Ala Gly Thr Tyr Asp Glu Lys Arg Leu Phe

165 170 175

Gly Val Thr Thr Leu Asp Val Val Arg Ala Lys Thr Phe Tyr Ala Gly

180 185 190

Lys Ala Asn Val Pro Val Ala Gly Val Asn Val Pro Val Val Gly Gly

195 200 205

His Ala Gly Ile Thr Ile Leu Pro Leu Phe Ser Gln Ala Thr Pro Lys

210 215 220

Ala Asn Leu Asp Asp Asp Val Ile Lys Ala Leu Thr Lys Arg Thr Gln

225 230 235 240

Asp Gly Gly Thr Glu Val Val Glu Ala Lys Ala Gly Lys Gly Ser Ala

245 250 255

Thr Leu Ser Met Ala Tyr Ala Gly Ala Leu Phe Ala Asp Ala Cys Leu

260 265 270

Lys Gly Leu Asn Gly Val Pro Asp Val Val Glu Cys Ser Phe Val Gln

275 280 285

Ser Thr Val Thr Glu Leu Pro Phe Phe Ala Ser Lys Val Arg Leu Gly

290 295 300

Thr Val Gly Val Glu Glu Val Leu Gly Leu Gly His Leu Ser Asp Phe

305 310 315 320

Glu Gln Gln Gly Leu Glu Ser Leu Lys Pro Glu Leu Lys Ser Ser Ile

325 330 335

Glu Lys Gly Ile Lys Phe Ala Asn Gln

340 345

<210> 5

<211> 25

<212> DNA

<213> primer F (forward primer F) for quantitative PCR of GmMDH12 gene

<400> 5

ctcactcact aatcgccact ctcac 25

<210> 6

<211> 22

<212> DNA

<213> primer R (reverse primer R) for quantitative PCR of GmMDH12 Gene

<400> 6

gcggagtgga gagatctgag ca 22

<210> 7

<211> 20

<212> DNA

<213> quantitative PCR primer F (forward primer F) for soybean housekeeping gene GmEF1a

<400> 7

tgcaaaggag gctgctaact 20

<210> 8

<211> 20

<212> DNA

<213> quantitative PCR primer R (reverse primer R) for soybean housekeeping gene GmEF1a

<400> 8

cagcatcacc gttcttcaaa 20

<210> 9

<211> 22

<212> DNA

<213> amplification of a specific primer upstream of the GmMDH12 promoter (forward primer F)

<400> 9

gggatccccg tgcatgtgtt ga 22

<210> 10

<211> 24

<212> DNA

<213> amplification of a primer specific for the downstream of the GmMDH12 promoter (reverse primer R)

<400> 10

atgaattcag tggcgattag tgag 24

<210> 11

<211> 2182

<212> DNA

<213> Promoter sequence of GmMDH12 (GmMDH12 Promoter)

<400> 11

cccgtgcatg tgttgatagt agttatgaat ttttaagatt aaaaactaaa acttcattta 60

ggcatttcat caaacacata acttacaacc catatcggga agatgacgaa aaaattagcc 120

cgggaaaata aaaaaggaac caaaaacaga ggctaaaaat cagtaagaaa aaataattat 180

tattaagaag aaaacatgca taacaacaaa caaaattaga accttgcaaa taaaaataaa 240

tagtaatgaa acaaacatgc ataatagcaa aagaggaaaa aacgtgcctg ttggattctt 300

cttcgtcgat agcctggcaa aataaaaaga aaccaaaatc aaaattgaaa aatcaataag 360

aaaaaataac tattattaag aagaaaacat gcataacaac aaataaaatt aaaaccttcc 420

aaatagaagc aaatatcaac gaaacaaaca tgcataacag taaaaaagga aaaaactttc 480

ccgttggatg ctttttcatt gatagcctgg gaaaattaaa aggaacctca tttttggggg 540

aaggctaacc tcttctctac agcctaccag gtccatataa ccatagcaaa ttcttacaac 600

agcaagagat atattacaaa caatattacc tttcttgttt ttggtaacca aaccaaattc 660

ttatagtagc aagcttaatt ttaaaaatga tatattttca agagaggaag caaaaagaag 720

aaagattcat cgcaagctca tatccatcaa ggcataacaa tctcaacata tgcaatgagc 780

ttaaacacat ttagaatgtt tccacataca tatagtcatt ttcttggtaa cacccgtgca 840

tatgttgata gtagttatga atttttaaga ttaaaaacta aaacttcatt taggcatttc 900

atcaaacaca acttacaatc catatgggga agatgacgaa aaaattagcc cgggaaaata 960

aaaaaggaac caaaaacaga gttgaaaaat cagtaagaaa aaataactat tattaagaag 1020

aaaacatgca taacaacaaa tcaaattaaa accttccaaa tagaagcaaa tatcaatgaa 1080

acaaacatgc ataacagtaa aaagggaaaa acctttgcga gaaacaggaa cgtttcggta 1140

ataggaactt gcgtgaatga tcctaacacc gattttcatg ttgggtcgag accaattgta 1200

agcgattgaa cgcgaaatac cactgagcca cagacaccct gatccaaata aaaatcgcaa 1260

aaaacacaaa aagatactaa gaggtgcaga gcttgcatgc aaatccagat aattaagaac 1320

tgaggaaaat gcataaaaac tcatagaatt tcctctaaaa gtatgaacaa aataaaaaat 1380

gagttttagg gaaaaacaac aacgtagcat aaaaaaacac aaaaaatcag acaaaaaacg 1440

atgatagaat aacaattaga gattggcgat aagcaaaagg ggcttacacg atcagtatgt 1500

atatcctttg taacctttga tggcttcacg aatctctttc ctgcagagga aaacgatgac 1560

aaattattga aagagataga aggaaggaaa aaaagagagg aaaaaagaca tgcacaacct 1620

cacattttgt attgcaaaaa gcgggtggct tcagcattct ggggaaattt cagtttccga 1680

aaaaaaaaaa gagaaaagag aaagagataa acagaggatg gtacgtggag ttggcagcaa 1740

aaaaaaaaag ttcataggaa aacacatggc agtgaggtgt cacaaaatca aagcaaagct 1800

gagggctata aaatgaccaa aaaataaaaa taaaaaatgg acaggtgtca agctctcaac 1860

tatgggtatt ttgaccaaaa aaccaaaatt ttggacagct gtcaaccttt cagtcatgga 1920

tatttcagta ttttccattt ccttctgatt tcatatatag atattgattt tcaaatatat 1980

tattcgtttt attttataca ttcttgaata aaaaaatggc accagacatg ttccactttc 2040

tgtttttaca tatatttaat atttaattta atatatataa tatttaatag aatatataat 2100

ttctcatttt ttaaggcatt attatttttc tgtggacccg aaatagatgc aggcgctggc 2160

tcgctcactc actaatcgcc ac 2182

<210> 12

<211> 22

<212> DNA

<213> amplification of the forward specific primer (forward primer F) for the full-length GmMDH12 cDNA

<400> 12

ggatccatga tgaagccatc ga 22

<210> 13

<211> 27

<212> DNA

<213> amplification of the downstream specific primer (reverse primer R) of the full-length GmMDH12 cDNA

<400> 13

acatgaattc ttactggttg gcaaatt 27

<210> 14

<211> 23

<212> DNA

<213> amplification of upstream specific primer (forward primer F) for overexpression fragment of GmMDH12 gene

<400> 14

acaaagagct cgagagagag aaa 23

<210> 15

<211> 28

<212> DNA

<213> amplification of downstream specific primer (reverse primer R) of overexpression fragment of GmMDH12 Gene

<400> 15

atatctagaa acctaccaga catgtatg 28

Claims (7)

1. The nucleotide sequence is shown as SEQ ID NO.3GmMDH12 The application of the gene in enhancing malic acid synthesis of soybean root nodule.

2. As recited in claim 1GmMDH12 The application of the gene in promoting the nodulation and nitrogen fixation of soybean.

3. As recited in claim 1GmMDH12The gene is applied to enhancing the nodulation capacity of soybean plant roots and/or the biological nitrogen fixation capacity.

4. A method for enhancing nodulation ability of soybean plant root system and/or biological nitrogen fixation ability, which comprises the step of adding the compound of claim 1GmMDH12The gene is overexpressed in soybean.

5. The method of claim 4, wherein the method of claim 1 is performedGmMDH12After the gene is connected to a vector PTF101S to obtain an over-expression vector, the over-expression vector is transferred into agrobacterium K599, and the agrobacterium-mediated soybean transgenic composite plant is used for transformation to obtain transgenic soybean.

6. The method according to claim 5, characterized by the steps of:

s1, constructing an overexpression vector

PCR amplification of the DNA of claim 1 using soybean root nodule cDNA as template and upstream and downstream specific primers shown in SEQ ID NO. 14 and SEQ ID NO. 15GmMDH12A gene sequence;

after the PCR amplified fragment is recovered and sequenced without errors, the PCR amplified fragment is obtained bySacI andXbai, carrying out double enzyme digestion on the amplified fragment and a target vector PTF101S respectively, and then connecting the amplified fragment to a target vector PTF101S to obtain an expression vector;

s2, transformation: and then transferring the constructed expression vector into agrobacterium K599 by a freeze-thaw method, and transforming by using the agrobacterium-mediated soybean transgenic composite plant to obtain a transgenic composite plant.

7. The method of claim 6, wherein the resulting transgenic composite plant is verified to be: extracting RNA from transgenic composite plant root and root nodule, reverse transcribing to cDNA, and detecting by quantitative PCR the DNA of claim 1GmMDH12Effect of gene expression.

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