CN103834674A - Separated 5-enolpyruvyl shikimate-3-phosphate synthase gene - Google Patents
Separated 5-enolpyruvyl shikimate-3-phosphate synthase gene Download PDFInfo
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- CN103834674A CN103834674A CN201410108595.2A CN201410108595A CN103834674A CN 103834674 A CN103834674 A CN 103834674A CN 201410108595 A CN201410108595 A CN 201410108595A CN 103834674 A CN103834674 A CN 103834674A
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- phosphate synthase
- epsps
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to separation and identification of roundup ready 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene, and belongs to the technical field of agricultural microbial genetic engineering. The 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene is characterized in that the nucleotide sequence is shown as SEQ ID No.: 1; and the sequence of protein coded by the gene is shown as SEQ ID No.: 2. The 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene is prepared by synthesizing aroA gene sequences of actinobacteria Isoptericola Variabilis as reported in Genbank; and recombinant escherichia coli LZD002 containing the gene plasmid is stored in the China Center for Type Culture Collection (CCTCC), with a storage number of CCTCC No.: M2013632. The biological experiment verifies that the gene shows a certain tolerance for glyphosate.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to one and derive from the separation of actinobacteria genomic 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS) gene, its encoding gene is AroA, by the method for gene recombination and genetic transformation, this gene is proceeded in host cell, make host cell obtain the ability of tolerance glyphosate.The present invention relates to nucleotide sequence and the aminoacid sequence of this gene.
Background technology
Glyphosate is most popular wide spectrum translocated herbicide in world wide, and most plants is had to the natural disposition of going out.The physico-chemical property of glyphosate is stable, and to people and livestock low toxicity, in soil, residual quantity is low, is therefore able to large-scale use.
Shikimic acid pathway is the metabolism critical path in bacterium, fungi, algae and higher plant, has important associated with die aromatischen Aminosaeuren in cell synthetic.5-enol pyruvic acid shikimic acid-3-phosphate synthase (EPSPS) is the key enzyme of the 6th step in this approach, and catalytic substrate phosphoenolpyruvic acid (PEP) and 3-phosphoric acid shikimic acid (S3P) generate 5-enol pyruvic acid shikimic acid-3-phosphoric acid (Bentley and Haslam1990).Because the transition state of one of the effect substrate of glyphosate and EPSPS PEP has similar structure, therefore can suppress competitively the effect of EPSPS.Due to the effect of glyphosate, intracellular chorismic acid is synthetic to be hindered, and the die aromatischen Aminosaeurens such as follow-up tryptophane (Trp), tyrosine (Tyr), phenylalanine (Phe) synthetic is also subject to further inhibition.Therefore, in born of the same parents, normal nitrogen metabolism multilated, finally causes necrocytosis (Pollegioni et al.2011).
The AroA gene of research is mainly divided into two types by gene order feature and resistance feature, i.e. I type and II type at present.The AroA gene of I type is mainly derived from intestinal bacteria (E.coli), Salmonellas (Aeromonas salmonella), Gram-negative bacteria and the plants such as petunia (Petunia) and Arabidopis thaliana (Arabidopsis thaliana).I class EPSPS is natural in glyphosate sensitivity, after mutagenesis transformation, can obtain certain glyphosate tolerant, but follow simultaneously, the avidity of substrate PEP is declined; II type AroA gene and I type similarity in sequence is very low, and textural difference is larger, mainly from agrobacterium tumefaciens CP4 (Agrobacterium tumefaciens CP4), the gram-positive microorganisms such as subtilis (Bacillus subtilis) and Rhodopseudomonas PG.2982 (Pesudomonas sp.), PEP is had to high-affinity, and natural insensitive to glyphosate, this genoid has obtained commercial applications (Funke et al.2006).
In one's early years due to Resistant Herbicide Crops popularization worldwide; II class AroA gene has obtained research widely; in recent years novel aroA starts to be concerned, and has successively the glyphosate resistance gene different from II genoid to be found (Heinrichs et al.2012; Sun et al.2005).The AroA gene that glyphosate resistance is higher is excavated and patent protection mostly, caused significant limitation to the practical application of China.For finding the AroA gene with independent intellectual property right, the present invention has separated novel AroA gene order in actinobacteria, has obtained the insensitive AroA gene of glyphosate differing greatly with isoformgene.
Summary of the invention
The object of this invention is to provide a kind of isolation identification from actinobacteria genomic 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS) gene, pass through genetic transforming method, this gene is proceeded in host cell, make host cell obtain the ability of tolerance glyphosate.
The glyphosate resistance Gene A roA of separating clone of the present invention comes from the aroA gene order (Genbank Accession Number:YP_004542951) of actinobacteria Isoptericola Variabilis.Applicant obtains this gene by chemosynthesis and is cloned in intestinal bacteria aroA deletion mycopremna AB2829.Still growing containing this recombinant bacterial strain in the M9 substratum of 150mM glyphosate.The unnamed gene that applicant obtains this separation is AroA
i.varigene.By AroA
i.varigene transformation is (conversion process is referring to the step transforming in embodiment 1) in bacillus coli DH 5 alpha, obtain recombination bacillus coli LZD002, deliver China on December 6th, 2013. Wuhan. Wuhan University's Chinese Typical Representative culture collection center (CCTCC) preservation, its preserving number is CCTCC NO:M2013632).Enzymatic determination result shows, glyphosate resistance Gene A roA
i.variin the time of pH8.0, activity is the highest, for normally playing a role condition is provided under meta-alkalescence condition in this gene transferred plant.Glyphosate resistance Gene A roA
i.varito the K of its natural substrate phosphoenolpyruvic acid (PEP)
mvalue is 28 μ M, to the K of shikimic acid-3 phosphoric acid (S3P)
mvalue is 115 μ M, partly suppressing constant IC50 value and suppressing constant K glyphosate
ivalue is respectively 5.4mM and 1.2mM.
By above-mentioned glyphosate resistance Gene A roA
i.varirecombinate to pGEX-6P-1 plasmid expression vector (purchased from GE Healthcare company of the U.S.) and be positioned at after GST label, can be at intestinal bacteria (Escherichia coli) BL21(DE3) carry out heterogenous expression in (purchased from Invitrogen company), the size of the fusion rotein (called after GST-EPSPS) containing GST label giving expression to is 73kDa, and target protein (EPSPS) size of removing after GST label is 47kDa.
The glyphosate resistance Gene A roA of separating clone of the present invention
i.varinucleotide sequence sequence total length as shown in sequence table SEQ ID NO:1 be 1374bp, its 457 amino acid of encoding, by its aminoacid sequence and typical I class AroA aminoacid sequence (AroA
e.coli) and typical II class AroA aminoacid sequence (AroA
cP4) relatively, result shows, AroA
i.varigene order and two nucleotide sequence homology maximums that represent type AroA gene order are only 35% and 29%.（http://blast.ncbi.nlm.nih.gov）。According to glyphosate resistance Gene A roA
i.varithe feature applicant of sequence is classified as I type, is the new gene of one of finding.
Brief description of the drawings
Sequence table SEQ ID NO:1: be the synthetic glyphosate resistance Gene A roA of the present invention
ivarinucleotide sequence, sequence total length is 1374bp, 1-1374bp place is coding region.
Sequence table SEQ ID NO:2: the glyphosate resistance Gene A roA that the present invention separates
ivariaminoacid sequence, 457 amino acid of encoding.
Fig. 1: the technology of the present invention schema.
Fig. 2: AroA
ivariclone's process of gene.
Fig. 2 A is with pUC57-aroA
i.varithe AroA going out for template amplification
i.varigene order;
Fig. 2 B is by AroA
i.varigene clone is to the recombinant plasmid obtaining in pGEX-6P-1.
Fig. 3: intestinal bacteria DE3(BL21) express I.vari EPSPS albumen SDS-PAGE analyze.
Swimming lane 1:pGEX-6p-1-AroA in figure
i.variinduced precipitation;
Swimming lane 2:pGEX-6p-1-AroA
i.variinduction supernatant;
Swimming lane 3:proteins Marker;
Swimming lane 4: the I.vari EPSPS albumen of purifying.
Fig. 4: by Nanjing Jin Sirui company synthetic contain AroA
i.varithe plasmid pUC57-AroA of gene
i.varicollection of illustrative plates.
Fig. 5: containing AroA
i.varithe recombinant plasmid pGEX-6p-1-AroA of gene
i.varicollection of illustrative plates.
Fig. 6: determination of protein concentration canonical plotting.
Fig. 7: inorganic phosphorus (KH
2pO
4) canonical plotting.
Fig. 8: optimum temperuture and optimal pH are measured curve.In Fig. 8, A figure is that optimum temperuture is measured curve, and in Fig. 8, B figure is that optimum temperuture is measured curve.
Fig. 9: the impact of ion pair activity.
Figure 10: K
m(PEP) mensuration.
Figure 11: K
m(S3P) mensuration.
Figure 12: partly suppress dosage IC
50the mensuration of value.
Figure 13: K
i(glyphosate) mensuration.
Figure 14: contain AroA
i.varithe growth curve of the glyphosate resistance checking of the intestinal bacteria AroA deficient strain AB2829 of gene.
Embodiment
Synthetic and the clone of embodiment 15-enol acetone shikimic acid-3-phosphate synthase gene
(1) AroA
i.varisynthesizing of gene order
Obtain the AroA of the EPSPS of coding Isoptericola Variabilis bacterial strain at Genbank database
i.varigene order, is synthesized the nucleotide sequence 1374bp of this gene and recombinant plasmid pUC57-AroA is provided by Nanjing Jin Sirui company
i.vari(Fig. 4).The aminoacid sequence of its nucleotide sequence and coding is as shown in sequence table SEQ ID NO:1 and SEQ ID NO:2.
(2) AroA
i.varithe clone of gene order
According to AroA
i.variprimers: add BamHI restriction enzyme site at primer 5 ' end, add restriction enzyme site EcoRI at 3 ' end, the DNA sequence dna of described primer pair is as follows:
Forward primer (5 '-AroA
i.vari-BamHI): 5 '-CGC
gGATCCaTGACGCCAGCGCCCGCCAGC-3 ', wherein underscore part is restriction enzyme site;
Reverse primer (3 '-AroA
i.vari-EcoRI): 5 '-CCG
gAATTCtCAGGCGCCGGCCCCCACGGT-3 ', underscore part is restriction enzyme site.Synthesized by Nanjing Genscript Biotechnology Co., Ltd..
With pUC57-AroA
i.varifor template, pcr amplification EPSPS.PCR reaction system is as follows:
5×TransStart FastPfu buffer 10μL；
dNTP（10mM） 1μL；
pUC57-AroA
I.vari（1μg/μL） 1μL；
Forward primer (5 '-AroA
i.vari-BamHI, 10 μ M) 1 μ L;
Reverse primer (3 '-AroA
i.vari-EcoRI, 10 μ M) 1 μ L;
TransStart FastPfu DNA Polymerase 1μL；
Add distilled water to 50 μ L;
PCR reaction conditions: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C are extended 450s; 30 circulations; 72 DEG C are extended 10min, preserve 5min for 4 DEG C and finish.The result of PCR product electrophoresis is as shown in Fig. 2 A in Fig. 2.
(3) enzymolysis PCR product and plasmid vector pGEX-6p-1 build
By obtain PCR product with restriction enzyme BamHI/EcoRI(purchased from precious biotechnology Dalian company limited) carry out enzymolysis, enzymatic hydrolysis system: in 60 μ L contain 15 μ L PCR products or pGEX-6P-1 plasmid (purchased from GE Healthcare company of the U.S.); 37 μ L ddH
2o, 6 μ L10 × H Buffer; 1 μ L BamHI; 1 μ L EcoRI; Spend the night in 37 DEG C of placements.The recovery that enzyme is cut product is used glue to reclaim test kit (purchased from Beijing Zhuan Meng company), reclaims electrophoresis product according to the specification sheets of this test kit.Detailed process is: under ultraviolet lamp, object band is cut in the centrifuge tube that is placed on 1.5mL, every 100mg adds the sol solutions (this test kit carries) of 300 μ L, bathes 10min, until gel all melts in 55 DEG C of water-bath temperature.Sol solutions is joined to 2mL adsorption tube 12000rpm, 4 DEG C of centrifugal 1min, add the centrifugal wash-out 1min of 500uL elutriant (this test kit carries), add again the centrifugal wash-out 1min of 700uL elutriant, after damping fluid is discarded, centrifugal 1min removes ethanol as far as possible again, adds 30 μ L distilled water wash-outs to collect DNA.
Above-mentioned enzyme is cut to the AroA of rear recovery
i.
varigene is connected with pGEX-6P-1 plasmid vector.In 10 μ L linked systems, contain: the pGEX-6P-1 plasmid vector 0.03nmol containing BamHI/EcoRI double enzyme site building through said process; Through the goal gene 0.21nmol of BamHI/EcoRI double digestion; T4DNA ligase(is purchased from Transgene company) 1 μ L; 5 × T4DNA ligase buffer2 μ L; Add distilled water and mend to 10 μ L, connect and spend the night in 4 DEG C of enzymes.Get enzyme and connect product 1 μ L and mix with 50 μ L competent escherichia coli cell DH5 α (purchased from Invitrogen company), join in the 1mm electricity revolving cup (purchased from BioRad company) of precooling, by 2.1KV voltage electric shock; Then add rapidly 600 μ L liquid LB substratum, in 37 DEG C of recovery 40min; Be coated on the LB flat board that contains 100 μ g/mL penbritins (Ampicillin, purchased from Beijing Transgene company), 37 DEG C are spent the night.Picking transformant, is inoculated into liquid LB and cultivates based on 37 DEG C of concussion cultivations, extracts plasmid and also carries out electrophoresis checking.
(4) extraction of plasmid
Picking list bacterium colony from streak plate, with 5mL being housed containing the LB liquid nutrient medium of 100 μ g/mL Ampicillin in vitro, 37 DEG C of concussion overnight incubation.With 1.5mL centrifuge tube collect thalline, the centrifugal 1min of 12000rpm, abandons supernatant, add 200 μ L solution I (formula: 50mM glucose, 25mM TrisHCl, supplies distilled water to 1L, adjust pH to 8.0, for subsequent use, 10mM EDAT, adjust pH to 8.0) resuspended thalline; Mix, add 400mL solution II (formula: 0.2M NaOH, l%SDS, supplies distilled water to 1L, for subsequent use), put upside down and mix gently, be no more than 5min storage period.Add 300mL solution III (formula: 3M KAc, be adjusted to pH4.8 with glacial acetic acid, for subsequent use), put upside down and mix.In the centrifugal 10min of 12000rpm, get supernatant, add the Virahol of supernatant 2/3 volume, in 12000rpm4 DEG C of low-temperature centrifugation 10min; Abandon supernatant, precipitation is with after 70% washing with alcohol, in 12000rpm4 DEG C of low-temperature centrifugation 10min.Abandon supernatant, 30 μ L deionized water dissolvings for precipitation.Get 5 μ L electrophoresis detection, positive colony obtaining contains AroA
i.varithe recombinant plasmid pGEX-6p-1-AroA of gene
i.vari.Electrophoresis result is as shown in Fig. 2 B in Fig. 2, and Fig. 5 is shown in by recombinant plasmid collection of illustrative plates.
Expression, purifying and the analysis of embodiment 2EPSPS albumen
(1) expression of target protein EPSPS
By recombinant plasmid pGEX-6p-1-AroA
i.varibe transformed into expression host cell intestinal bacteria E.coli BL21(DE3).By the activation of spending the night of qualification positive colony, be forwarded to 1L containing in the LB liquid nutrient medium of 100 μ g/mL Ampicillin with the inoculum size of volume ratio 1%, cultivate 2-3h in 37 DEG C, until OD
600reach 0.6 left and right, add inductor isopropyl-β-D-thiogalactoside(IPTG) (IPTG) final concentration to 0.5mM, in 22 DEG C, under 180rpm, cultivate 25h.Centrifugal collection thalline, then use Hepes damping fluid (formula: 50mM4-hydroxyethyl piperazine ethanesulfonic acid (Hepes), 2mM dithiothreitol (DTT) (DTT), adjust pH to 7.0, supply distilled water to 1L, for subsequent use) by thalline washing one time, then use high pressure cell cracker (purchased from GEA Niro Soari company model: NSI0012K) smudge cells after suspending with 50mL Hepes damping fluid.By cytoclasis liquid centrifugal 30min under 4 DEG C, 12000rpm, collect supernatant.Detect by SDS-PAGE method the supernatant obtaining and whether contain target protein.
SDS-PAGE formula is as follows:
Concentrated glue (5% polyacrylamide), fill a prescription as follows:
Separation gel (12% polyacrylamide), fill a prescription as follows:
Get 80 μ L cytoclasis liquid, add 20 μ L5 × albumen sample-loading buffers (6mL1mol/L Tris-HCl, pH8.8), 20mL10%SDS, 50mL50% glycerine, 5mL beta-mercaptoethanol, 1mL1% tetrabromophenol sulfonphthalein, adds distilled water to 100mL, for subsequent use), boiling water bath 5min, then point sample 10 μ L electrophoresis detection, SDS-PAGE electrophoresis detection the results are shown in Figure 3.Fig. 3 shows, target protein EPSPS obtains correction in intestinal bacteria, containing the fusion rotein of GST label and target protein, applicant is by this fusion rotein called after GST-EPSPS, its size is about 73kDa, and the target protein EPSPS removing after GST label is 46kDa, meets and predicts the outcome.
(2) purifying of target protein EPSPS
The present invention utilizes reduced glutathion (GST) affinity purification system purification of recombinant proteins (Nilsson et al.1997), concrete operation step (all operations all carries out at 4 DEG C) is as follows: get 1ml column material GSH Sepharose4B(purchased from Amersham-Pharmacia company) in chromatography column, by 50mL Hepes buffer solution elution balance; The supernatant of cytoclasis liquid is crossed to post with the flow velocity of 1.0ml/min; The foreign protein that there is no combination by 500mL Hepes buffer solution elution, flow rate control is below 1.0mL/min.The HRV 3CP (purchased from Merck company) of getting 5 μ L concentration again and be 10unit/ μ L mixes with 1mL Hepes damping fluid, puts 4 DEG C of enzymolysis 12 hours after adding chromatography column to mix.Collect the Hepes damping fluid that contains target protein EPSPS in previous step after enzymolysis to the centrifuge tube of 1.5ml, then add the rear packing of isopyknic glycerine mixing, be placed in-80 DEG C of preservations (Rippert and Matringe2002).
(3) concentration quantitative of target protein EPSPS is measured
Adopt quantitative Bradford method (Bradford1976) to measure the concentration of the target protein EPSPS of purifying.Concrete steps are as follows: measure the concentration of target protein EPSPS with Bradford protein quantification test kit (purchased from Shanghai Sheng Gong biotechnology company limited), according to the specification sheets operation of test kit.Concrete steps are: get 20 μ L1mg/mL protein standard substance bovine serum albumins (purchased from Shanghai Sheng Gong biotechnology limited liability company) and add Hepes damping fluid and be diluted to 100 μ L, making final concentration is 0.2mg/mL.Get standard substance after dilution by 0,5,10,15,20,25,30 μ L are added to respectively in 96 orifice plates, add Hepes damping fluid and supply 1mL, and every porin content is respectively 0,5,10,15,20,25,30 μ g/mL.3 parallel tests of every group of design.Every hole adds 200 μ L Bradford Reagent(Bradford protein quantification test kits to carry), mix, room temperature uses the microplate reader (Thermo Scientific company) of preheating to measure OD after placing 5min
595value, drawing standard curve, obtains determination of protein concentration typical curve as shown in Figure 6, the protein concentration of calculation sample.
(4) determination of activity of target protein EPSPS
The measuring method of the inorganic phosphorus of target protein EPSPS determination of activity reference literature (Lanzetta et al.1979) report.Concrete steps are as follows:
The making of inorganic phosphorus typical curve: the inorganic phosphorus standardized solution of preparation 10mM (takes 0.2282gK
2hPO
43H
2o, dissolve and be settled to 100mL with Hepes damping fluid), get respectively 0-20 μ L in 20 1.5mL centrifuge tubes, add appropriate Hepes damping fluid to complement to 1mL, make in 20 centrifuge tubes inorganic phosphorus final concentration be respectively the every pipe of 0-0.2mM. and get 100 μ L, after 28 DEG C of standing 3min, add 0.8mL MAT solution (now with the current: 0.045% malachite green 75mL, 4.2% ammonium molybdate is settled to 25mL after dissolving with 10mL concentrated hydrochloric acid, after mixing, filter, add 200 μ L Tritonx-100, for subsequent use), room temperature adds 0.1mL34% sodium citrate solution after placing 1min, after placement 30min, get 200 μ L and measure OD in 96 orifice plates
660value.Design three experimental group (Jin et al.2007).Taking inorganic phosphorus concentration as X-coordinate, with OD
660for ordinate zou mapping obtains inorganic phosphorus canonical plotting (see figure 7).
Optimum temperuture and optimal pH are measured:: 50 μ L reaction systems: 1mM shikimic acid triphosphoric acid, 1mM phosphoenolpyruvic acid (PEP), adds the EPSPS albumen of 1uL purifying, adds Hepes damping fluid and supplies 50 μ L.28 DEG C of reaction 4min, add 800 μ L MAT solution, add 100 μ L34% Trisodium Citrate (SC) solution after 1min, and room temperature is placed after 30min, gets 200 μ L in 96 orifice plates, with the microplate reader mensuration OD of preheating
660value.Control group does not add shikimic acid triphosphoric acid, designs 3 experimental group.After the lower reaction of differing temps (0,10,20,30,40,50,60 DEG C), measure OD with the reaction system identical with above-mentioned 50 μ L systems
660value is also calculated speed of response.Taking temperature as X-coordinate, map as ordinate zou taking the mean value of relative reactivity, obtain optimum temperuture and measure curve (seeing Fig. 8 A).With different pH(4,5,6,7,8,9,10,11) Hepes damping fluid configure above-mentioned 50uL reaction system, after reaction, measure OD600 value and calculate speed of response, taking pH value as X-coordinate, the mapping taking relative reactivity as ordinate zou, the optimal pH shown in obtaining is measured curve (seeing Fig. 8 B).
The impact of positively charged ion on enzymic activity: 50 μ L reaction systems: 1mM shikimic acid triphosphoric acid, 1mM phosphoenolpyruvic acid (PEP), adds the EPSPS albumen of 1uL purifying, adds respectively KCl, KBr, NacCl, MgSO
4, NH4Cl, K2SO4, CaCl2, MgCl2 salts solution to final concentration be to measure enzyme reaction rate after 100mM.Fig. 9 is shown in the impact of ions enzyme activity.
Km (PEP) measures: the concentration of shikimic acid triphosphoric acid (S3P) in system is fixed as to 1mM, measure enzyme reaction rates by above-mentioned 50 μ L reaction systems different PEP concentration (0.05,0.067,0.1,0.2,0.5,1.0mM) is lower, taking PEP concentration as X-coordinate, taking speed of response V(U/mg) be ordinate zou mapping, obtain the measurement result of Km (PEP) as shown in figure 10.
Km (S3P) measures: the concentration of phosphoenolpyruvic acid in system (PEP) is fixed as to 1mM, measure enzyme reaction rates by above-mentioned 50 μ L reaction systems different shikimic acid triphosphoric acids (S3P) concentration (0,0.02,0.05,0.1,0.2,0.4,0.8,1.0mM) is lower, taking S3P concentration as X-coordinate, taking speed of response V(U/mg) be ordinate zou mapping, obtain Km(S3P as shown in figure 11) measurement result.
Partly suppress dosage (IC
50) measure: in above-mentioned reaction system, add different concns (10
-5, 10
-4, 10
-3, 10
-2, 10
-1, 10
0, 10
1, 100,1000mM) glyphosate, the data obtained is taking glyphosate concentration as X-coordinate, adopts logarithmic coordinates, taking speed of response V(U/mg) map for ordinate zou, obtain as shown in figure 12 partly suppress dosage IC
50the measurement result of value.
Suppress constant K
i(glyphosate) measure: the concentration of shikimic acid triphosphoric acid (S3P) in system is fixed as to 1mM, adopt different PEP concentration (0.067,0.1,0.2,0.5,1.0mM) preparation 50ul reaction system, in the system of different PEP concentration, add respectively the glyphosate of 0.5mM, 1mM, 2mM, 5mM, measure enzyme reaction rate, taking the concentration of glyphosate as X-coordinate, taking speed of response V(U/mg) inverse be ordinate zou mapping, obtain K as shown in figure 13
i(PEP) measurement result.
Embodiment 3: genetic transformation experiment (checking has complementary functions)
By recombinant plasmid pGEX-6p-1-AroA
j.limo(conversion process is referring to conversion process in above-described embodiment 1 to be transformed into AroA gene defection type competent escherichia coli cell.This bacterial strain is not containing AroA gene, can get rid of in general intestinal bacteria with the interference of AroA gene) (Vaithanomsat and Brown2007), be transferred to respectively containing 0mM, 50mM, 100mM, M9 liquid nutrient medium (the formula: Na2HPO46.8g/L of 150mM glyphosate, KH2PO43.0g/L, NaCl0.5g/L, NH4Cl1.0g/L, MgSO47H2O0.4929g/L, CaCl20.111g/L, glucose 4.0g/L, supplement distilled water to 1L, before sterilizing, adjust pH to 7.4, high pressure steam sterilization 10min at 115 DEG C) in, growth curve measured.
By the recombinant plasmid pGEX-6p-1-AroA building
i.varibe transformed into competent cell intestinal bacteria AroA deficient strain AB2829, transformant is inoculated into respectively and is contained containing 0mM, 50mM, 100mM, in the M9 liquid nutrient medium of 150mM glyphosate (containing 100 μ g/mL Ampicillin), cultivate 40h in 37 DEG C of concussions.Three parallel laboratory tests of every group of design, every 10h gets 100 μ L nutrient solutions and measures cell concn, i.e. OD
600value.Taking incubation time as X-coordinate, with OD
600value is ordinate zou mapping, and the growth curve obtaining as shown in figure 14.
Reference
1.Bentley R,Haslam E(1990)The Shikimate Pathway-A Metabolic Tree with Many Branche.Critical reviews in biochemistry and molecular biology25(5):307-384
2.Bradford MM(1976)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry72(1):248-254
3.Funke T,Han H,Healy-Fried ML,Fischer M,Schonbrunn E(2006)Molecular basis for the herbicide resistance of Roundup Ready crops.Proceedings of the National Academy of Sciences of the United States of America103(35):13010-5doi:10.1073/pnas.0603638103
4.Heinrichs V,Schouten LC,Berg BV(2012)Directed evolution of GRG31EPSP synthase enzyme.US Patent8,247,535
5.Jin D,Lu W,Ping S,Zhang W,Chen J,Dun B,Ma R,Zhao Z,Sha J,Li L(2007)Identification of a new gene encoding EPSPS with high glyphosate resistance from the metagenomic library.Current microbiology55(4):350-355
6.Lanzetta PA,Alvarez LJ,Reinach PS,Candia OA(1979)An improved assay for nanomole amounts of inorganic phosphate.Analytical Biochemistry100(1):95-97
7.Nilsson J,Stahl S,Lundeberg J,Uhlén M,Nygren PA(1997)Affinity fusion strategies for detection,purification,and immobilization of recombinant proteins.Protein expression and purification11(1):1-16
8.Pollegioni L,Schonbrunn E,Siehl D(2011)Molecular basis of glyphosate resistance-different approaches through protein engineering.The FEBS journal278(16):2753-66doi:10.1111/j.1742-4658.2011.08214.x
9.Rippert P,Matringe M(2002)Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.European Journal of Biochemistry 269(19):4753-4761
10.Sun YC,Chen YC,Tian ZX,Li FM,Wang XY,Zhang J,Xiao ZL,Lin M,Gilmartin N,Dowling DN,Wang YP(2005)Novel AroA with high tolerance to glyphosate,encoded by a gene of Pseudomonas putida4G-1isolated from an extremely polluted environment in China.Applied and environmental microbiology71(8):4771-6doi:10.1128/AEM.71.8.4771-4776.2005
11.Vaithanomsat P,Brown KA(2007)Isolation and mutation of recombinant EPSP synthase from pathogenic bacteria Pseudomonas aeruginosa.Process Biochemistry42(4):592-598。
Claims (4)
1. 5-enol acetone shikimic acid-3-phosphate synthase gene of separation, its nucleotide sequence is as shown in sequence table SEQ ID NO:1.
2. 5-enol acetone shikimic acid-3-phosphate synthase gene of separation, the sequence of the protein of its coding is as shown in sequence table SEQ ID NO:2.
3. a strain comprises the intestinal bacteria LZD002 that expresses 5-enol acetone shikimic acid-3-phosphate synthase gene recombinant plasmid, be deposited in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M2013632, and the nucleotide sequence of described 5-enol acetone shikimic acid-3-phosphate synthase gene is as shown in SEQ ID NO:1.
4. 5-enol acetone shikimic acid-3-phosphate synthase gene claimed in claim 1 turns the application in plant at Antiglyphosate gene.
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| CN104004777A (en) * | 2014-06-06 | 2014-08-27 | 中国农业科学院作物科学研究所 | Glyphosate-resistant gene, special expression vector and application of glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat |
| CN104004777B (en) * | 2014-06-06 | 2016-01-20 | 中国农业科学院作物科学研究所 | Antiglyphosate gene, dedicated expression vector therefor and the application in acquisition resistance glyphosate transgenic wheat thereof |
| CN111197052A (en) * | 2019-07-27 | 2020-05-26 | 华中农业大学 | Cold-adapted I-type 5-enol pyruvoyl shikimic acid-3-phosphate synthase gene |
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