CN103194465B - Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene - Google Patents
Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene Download PDFInfo
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- CN103194465B CN103194465B CN201210006891.2A CN201210006891A CN103194465B CN 103194465 B CN103194465 B CN 103194465B CN 201210006891 A CN201210006891 A CN 201210006891A CN 103194465 B CN103194465 B CN 103194465B
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Abstract
本发明属于基因工程技术领域，具体涉及一种抗草甘膦基因5-烯醇丙酮莽草酸-3-磷酸合酶(EPSPS)基因的分离鉴定，所述的5-烯醇丙酮莽草酸-3-磷酸合酶(EPSPS)基因的核苷酸序列如SEQ ID NO：1所述，该基因编码的氨基酸序列如SEQ ID NO：2所述。5-烯醇丙酮莽草酸-3-磷酸合酶(EPSPS)基因是从深海细菌Janibacter limosus菌株的基因组文库中克隆得到，包含该基因质粒的重组大肠杆菌LZD001保藏在中国典型培养物保藏中心，其保藏编号为CCTCC No：M2011493。经过生物学实验验证，证明该基因对草甘膦具有一定的耐受性。The invention belongs to the technical field of genetic engineering, and specifically relates to the isolation and identification of a glyphosate-resistant gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. The 5-enolpyruvylshikimate-3-phosphate - The nucleotide sequence of the phosphate synthase (EPSPS) gene is set forth in SEQ ID NO:1, and the amino acid sequence encoded by the gene is set forth in SEQ ID NO:2. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was cloned from the genome library of the deep-sea bacterium Janibacter limosus strain, and the recombinant Escherichia coli LZD001 containing the gene plasmid was preserved in the China Type Culture Collection Center. The deposit number is CCTCC No: M2011493. After biological experiment verification, it is proved that the gene has a certain tolerance to glyphosate.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to one and derive from the isolation identification of deep-sea marine microorganism genomic 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS) gene, its encoding gene is AroA, by the method for 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.
Technical background
N-phosphonomethylglycine, be glyphosate (glyphosate), it is the wide spectrum steriland herbicide of everybody a kind of inner sucting conduction type of knowing, have people and livestock low toxicity, in soil, residual quantity is low, make weeds be difficult to produce the features such as resistance, based on these features, glyphosate is widely used in worldwide.
Glyphosate can suppress the activity of a kind of important enzyme in die aromatischen Aminosaeuren building-up process in plant or microbe; it is 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS); the penultimate stride of this kind of enzyme catalysis shikimic acid pathway (shikimate pathway), the 5th hydroxyl the acyl moiety of phosphoenolpyruvic acid (PEP) being transferred to shikimic acid triphosphoric acid (shikimate-3-phophate) forms 5-enol form shikimic acid-3-phosphoric acid (EPSP) and inorganic phosphorus.Because glyphosate and PEP have similar structure, can form epsp synthase S-3-P glyphosate mixture, check the combination of PEP and enzyme, the route of synthesis of blocking-up die aromatischen Aminosaeuren, affected the eubolism of plant, thereby killed most plants, weeds and farm crop are all unexceptional.Therefore,, in order optionally to remove weeds in the process of crop growth, farm crop are the essential ability (Dill 2005 that obtains resistance glyphosate just; Sun, Li et al.2006).
The glyphosate resistance Gene A roA of research is mainly divided into two classes (He, Yang et al.2001) at present.I class AroA gene is mainly derived from plant or microorganism, natural in glyphosate sensitivity, can produce tolerance to certain density glyphosate, but can be affected to the avidity of substrate PEP after transformation; II class AroA gene is mainly derived from agrobacterium tumefaciens CP4 (Agrobacterium tumefaciens CP4), subtilis (Bacillus subtilis) and Rhodopseudomonas PG.2982 (Pesudomonas sp.) etc., to substrate, PEP has high-affinity, and natural insensitive to glyphosate, this genoid is applied (referring to United States Patent (USP) 453590 in production practice, 4769061,5094945).The amino acid homogeny of this two genoids sequence is lower than 30%.
At present; the glyphosate resistance gene of applying in production practice is mainly above-mentioned II class AroA gene; research to I class AroA gene is relatively less; and the I class AroA gene of finding is at present mostly natural in glyphosate sensitivity; this type of studies main selection in land microorganism; and the higher AroA gene of glyphosate resistance is excavated and patent protection mostly, cause significant limitation to the practical application of China.For solving this limitation, the present invention chooses new microorganism resource, i.e. marine microorganism, and the difference that this type of microbial genome and the existence of land microbial genome are very large, obtains AroA gene and other isoformgenes exist very big-difference.
Summary of the invention
The object of this invention is to provide a kind of isolation identification from deep-sea marine microorganism 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 3rd institute screening of China national ocean office and obtains the natural deep-sea bacterium Janibacter of strain limosus 1C00094 (original strain 1C00094 is from Chinese Sea microbial strains preservation administrative center, the 3rd institute of China national ocean office, Fujian, Xiamen, bacterium source address: www.mccc.org.cn), this bacterial strain can be grown on the flat board that contains 150mM glyphosate.Applicant builds above-mentioned bacterium Janibacter limosus genomic library by shotgun (Osoegawa, Woon et al.1998), at (the formula: Na of the M9 substratum containing 20mM glyphosate
2hPO
46.8g/L, KH
2pO
43.0g/L, NaCl 0.5g/L, NH
4cl 1.0g/L, MgSO
47H
2o 0.4929g/L, CaCl
20.111g/L, glucose 4.0g/L, supplements distilled water to 1L, adjust pH to 7.4 before sterilizing, at 115 ℃, high pressure steam sterilization 10min, for subsequent use) top sieve obtains a strain glyphosate resistance bacterial strain, further improve glyphosate concentration to 100mM, this bacterial strain still can be grown.Sequencing result demonstration, the gene of separating clone of the present invention is AroA gene, applicant is AroA by this unnamed gene
j.limo.By AroA
j.limogene transformation is to intestinal bacteria AroA gene defection type bacterial strain (conversion process is referring to the step transforming in embodiment 1), the recombinant bacterial strain obtaining, applicant is by high bacterial strain called after recombination bacillus coli (Escherichia coli) LZD001, this bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Luojiashan, Wuchang, Wuhan City, Hubei Province on December 31st, 2011, and its preserving number is CCTCCNO:M2011493.Through function complementation experiment checking, this recombinant bacterial strain LZD001 can be growing containing in the M9 liquid nutrient medium of 100mM glyphosate.Enzymatic determination result shows, glyphosate resistance Gene A roA
j.limoin the time of pH 8.0, 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
j.limobeing 90.7 μ M to the Km value of its natural substrate phosphoenolpyruvic acid (PEP), is 138 μ M to the Km value of shikimic acid-3 phosphoric acid (S3P), and glyphosate partly suppressed to constant IC
50value is 3.115mM, and maximum reaction velocity (Vmax) is 0.226mmol min
-1mg
-1.
By above-mentioned glyphosate resistance Gene A roA
j.limorecombinate to pGEX-6P-1 plasmid expression vector (purchased from GE Healthcare company of the U.S.) and be positioned at after GST label, can in intestinal bacteria (Escherichia coli) BL21 (DE3) (purchased from Invitrogen company), carry out heterogenous expression, the size of the fusion rotein (called after GST-EPSPS) containing GST label giving expression to is 71.1kDa, and target protein (EPSPS) size of removing after GST label is 45.1kDa.
The glyphosate resistance Gene A roA of separating clone of the present invention
j.limonucleotide sequence as shown in sequence table SEQ ID NO:1.The sequencing results shows, glyphosate resistance Gene A roA
j.limosequence total length is 1299bp, its 432 amino acid of coding (1-1296bp of coding region bit sequence table SEQ ID NO:1), and by its aminoacid sequence and the upper various AroA aminoacid sequence comparison of GeneBank, result shows, AroA
j.limogene order is only 67% (http://blast.ncbi.nlm.nih.gov) with the nucleotide sequence homology maximum of the AroA gene order predicting from Janibacter spp.HTCC2649 bacterial strain (GenBank:EAP99947.1) genome sequence.According to glyphosate resistance Gene A roA
j.limothe feature applicant of sequence is classified as I type, is the new gene of one of finding.This gene transformation is arrived to intestinal bacteria AroA gene defection type bacterial strain (conversion process is referring to the step transforming in embodiment 1), recombinant bacterial strain called after recombination bacillus coli (Escherichia coli) LZD001 obtaining, this bacterial strain is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Luojiashan, Wuchang, Wuhan City, Hubei Province on December 31st, 2011, and its preserving number is CCTCC NO:M2011493.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the glyphosate resistance Gene A roA that the present invention separates
j.limonucleotide sequence, sequence total length is 1299bp, 1-1299bp place is coding region.
Sequence table SEQ ID NO:2 is the glyphosate resistance Gene A roA that the present invention separates
j.limoaminoacid sequence, 432 amino acid of encoding.
Fig. 1: the technology of the present invention schema.
Fig. 2: the enzymolysis process of Janibacter limosus genomic dna.
1.Janibacter limosus genomic dna in figure; 2-7. is respectively 5min, 10min, 15min, 20min, 25min, the enzymolysis product of 30min; 8.DNA Marker; 9. the needed fragment (4-9kb) obtaining after enzymolysis.
Fig. 3: the SDS-PAGE of the EPSPS albumen of expressing with intestinal bacteria DE3 (BL21) analyzes.
1.proteins Marker in figure; 2. empty carrier pGEX-6P-1 does not induce supernatant; 3. empty carrier pGEX-6P-1 induction supernatant;
4.pGEX-6p-1-AroA
j.limodo not induce supernatant; 5.pGEX-6p-1-AroA
j.limonot induced precipitation;
6.pGEX-6p-1-AroA
j.limoinduction supernatant; 7.pGEX-6p-1-AroA
j.limonot induced precipitation.
Fig. 4: positive recombinant clone containing goal gene obtaining from the genomic library screening building: pUC118-insert fraction collection of illustrative plates.
Fig. 5: containing AroA
j.limothe recombinant plasmid pGEX-6p-1-AroA of gene
j.limocollection of illustrative plates.
Fig. 6: containing the recombinant plasmid pGEX-6p-1-AroA of intestinal bacteria AroA gene
e.colicollection of illustrative plates.
Fig. 7: determination of protein concentration canonical plotting.
Fig. 8: inorganic phosphorus (KH
2pO
4) canonical plotting.
Fig. 9: enzyme reaction optimum temperuture is measured curve.
Figure 10: enzyme reaction optimal pH is measured curve.
The mensuration of Figure 11: Km (PEP).
The mensuration of Figure 12: Km (S3P).
Figure 13: partly suppress dosage IC
50the mensuration of value.
Figure 14: growth curve, contains AroA
j.limothe glyphosate resistance checking of the intestinal bacteria AroA deficient strain of gene.
Embodiment
Separation, the clone of embodiment 1 5-enol acetone shikimic acid-3-phosphate synthase gene
(1) structure of genomic library and order-checking, obtains resistant gene
(original strain is from Chinese Sea microbial strains preservation administrative center to screen the natural deep-sea of the strain marine bacteria obtaining the selected original Janibacter limosus 1C00094 bacterial strain that is 150mM to glyphosate resistance (claiming again mud Janus bacterium 1C00094) from the 3rd institute of Chinese National Bureau of Oceanography, it is the 3rd institute of China national ocean office, Fujian China Xiamen, www.mccc.org.cn), build the genomic library of this bacterial strain according to following step and method: extracting deep-sea marine bacteria Janibacter limosus is mud Janus bacterium 1C00094 strain gene group DNA (concrete grammar sees below), Partial digestion (restriction enzyme site is Sau3A I) obtains needed fragment (4-9kb), again with pUC118 carrier (purchased from precious biotechnology Dalian company limited, this carrier carries restriction enzyme site BamH I) connect, then proceed to the efficient competent cell of intestinal bacteria (E.coli) DH5 α.Referring to detailed process below.
Obtain by being added with the M9 Screening of Media of 20mM glyphosate the clone that can grow, be sent to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, to after sequencing result and NCBI BLAST database (http://blast.ncbi.nlm.nih.gov) comparison, obtain the sequence of goal gene AroA, the aminoacid sequence of its coding is as shown in sequence table SEQ ID NO:2.
The extracting method of genomic dna: single bacterium colony of the above-mentioned deep-sea of picking marine bacteria Janibacter limosus bacterial strain from streak plate, with the 100ml 2216 liquid nutrient mediums (special culture media of cultivation deep-sea bacterium Janibacter limosus (mud Janus bacterium 1C00094): peptone 10g/L is housed, yeast powder 5g/L, beef extract powder 1g/L, sodium acetate 1g/L, ammonium nitrate 0.2g/L, NaCl 19.45g/L, MgCl
2, MgSO
4, CaCl
22～3g/L, KCl 0.55g/L, NaHCO
30.16g/L, supplements distilled water to 1L, adjusts pH to 7.6 before sterilizing, and at 121 ℃, high pressure steam sterilization 30min, for subsequent use) triangular flask in 37 ℃ of incubated overnight; Collect thalline 2 times with 2ml centrifuge tube, discard substratum, add 200 μ L1 × TE damping fluids (formula: 10mM Tris-HCl, 1mM ethylenediamine tetraacetic acid (EDTA) (EDTA), add distilled water to 1L, for subsequent use) resuspended thalline, add 30-50 μ L N,O-Diacetylmuramidase (concentration is 50mg/mL), in 37 ℃ of placement 30min, mixed once every 5 minutes.Add 300 μ L lysates (formula: 50mM Tris-HCl, 2.5%NaAc, 2% sodium ethylene diamine tetracetate (EDTA), 30% sodium laurylsulfonate (SDS), 8% Trisodium Citrate, 20% cetyl trimethylammonium bromide (CTAB), supplies distilled water to 1L, for subsequent use), put upside down and mix; Add 150 μ L 5MNaCl, put into 70 ℃ of water-baths, 10min.Then put into-20 ℃ of refrigerators, 10min.Add phenol, the each 350 μ L of chloroform/primary isoamyl alcohol (volume ratio is 24: 1), on vortex vibrator, shake after 30s, after centrifugal 10min, draw supernatant liquor.Add chloroform/primary isoamyl alcohol (with supernatant equal-volume), after mixing, centrifugal 10min, fully removes phenol, draws supernatant; Add the Virahol of above-mentioned supernatant 2/3 volume, put-20 ℃ of refrigerator 10min after mixing, rear centrifugal 10min, abandons supernatant.Adding concentration is 70% ethanol 700 μ L, and centrifugal 7min, abandons supernatant.After being placed at 37 ℃ and drying, every pipe adds 30 μ L distilled waters to dissolve.
Partial digestion: good extraction quality Janibacter limosus genomic dna is carried out to Partial digestion, reclaim the fragment of 4-9kb size.Described enzymatic hydrolysis system is 30 μ L:3 μ L 10 × Hbuffer, 1 μ L Sau3A I (diluting 160 times) (purchased from precious biotechnology Dalian company limited), 26 μ L genomic dnas.First pre-enzyme is cut: after system adds, within every 5 minutes, get 5 μ L, add 1 μ L loading buffer termination reaction, sampling is until take successively.After point sample electrophoresis detection, determine that best enzymolysis time is 25min.A large amount of enzymolysis again: according to the Best Times of pre-enzymolysis, expand reaction system to 300 μ L, enzymolysis rear electrophoresis, reclaiming size is the fragment of 4-9kb.Result as shown in Figure 2.
Enzyme connects: 10 μ L enzyme disjunctor systems are containing T4 DNA ligase (purchased from Transgene company) 1 μ L; 5 × T4 DNA ligase buffer, 2 μ L; Genomic DNA fragment 0.3pmol after enzyme is cut; PUC118 (BamHI/EcoRI) carrier (purchased from precious biotechnology Dalian company limited) 0.03pmol; Supply distilled water to 10 μ L, the enzyme that spends the night at 4 ℃ connects.
Transform: get enzyme and connect product 1 μ L and mixes 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, use 2.1KV voltage shocks by electricity; Then add rapidly 600 μ L liquid LB substratum, in 37 ℃ of recovery 40min; Be coated on the LB flat board that contains 100 μ g/mL penbritins (Ampicillin, purchased from Beijing Transgene company), 37 ℃ are spent the night.Picking transformant, is inoculated into liquid LB and cultivates based on 37 ℃ of concussion cultivations, extracts plasmid and also carries out electrophoresis checking.
The 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 ℃ of concussion overnight incubation.Collect thalline with 1.5mL centrifuge tube, the centrifugal 1min of 12000rpm, abandons supernatant, adds 200 μ L solution I (formula: 50mM glucose, 25mM TrisHCl, supplies distilled water to 1L, adjusts pH to 8.0, for subsequent use, 10mM EDAT, adjusts pH to 8.0) resuspended thalline; Mix, add 400mL solution II (formula: 0.2M NaOH, 1%SDS, adds water and complement 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 4 ℃ of low-temperature centrifugation 10min of 12000rpm; Abandon supernatant, precipitation is with after 70% washing with alcohol, in 4 ℃ of low-temperature centrifugation 10min of 12000rpm.Abandon supernatant, 30 μ L deionized water dissolvings for precipitation.Get 5 μ L electrophoresis detection.
Sequential analysis: (Jin Sirui order-checking company provides by Nanjing, forward primer F:5 ' TGTAAAACGACGGCCAGT-3 ' with pUC118 universal primer M13; Reverse primer R:5 ' CAGGAAACAGCTATGACC-3 ') be sequencing primer,, checked order by Nanjing Genscript Biotechnology Co., Ltd. as template take recombinant plasmid (pUC118-insert fraction).
(2) AroA
j.limothe clone of gene
According to sequencing result, obtain AroA through NCBI BLAST comparison
j.limothe sequence of gene, design of amplification primers, adds BamHI restriction enzyme site at primer 5 ' end, adds restriction enzyme site EcoRI at 3 ' end, and the DNA sequence dna of described primer pair is as follows:
Forward primer (5 '-AroA
j.limo-BamHI): 5 ' CGC
gGATCCaTGACCAGTCCTGATTGGCATGC-3 ', underscore part is restriction enzyme site;
Reverse primer (3 '-AroA
j.limo-EcoRI): 5 ' CCG
gAATTCtCAGCCCTCCGACGCCTCG-3 ', underscore part is restriction enzyme site.Synthesized by Nanjing Genscript Biotechnology Co., Ltd..
Take by the above-mentioned deep-sea bacterium of above-mentioned extracting be mud Janus bacterium genomic dna as template, pcr amplification EPSPS.PCR reaction system is as follows:
PCR reaction conditions: 94 ℃ of denaturation 2min; 94 ℃ of sex change 20s, 55 ℃ of annealing 20s, 72 ℃ are extended 50s; 30 circulations; 72 ℃ are extended 10min, preserve 5min for 4 ℃ and finish.
Enzymolysis PCR product and plasmid vector pGEX-6p-1 build: by the PCR product obtaining restriction enzyme BamHI/EcoRI (purchased from precious biotechnology Dalian company limited), enzymatic hydrolysis system: in 100 μ L containing 82 μ L PCR products or pGEX-6P-1 plasmid (conventional plasmid in biological test, the present invention is purchased from GE Healthcare company of the U.S., and this plasmid extraction method is with embodiment 1); 10 μ L 10 × H Buffer; 4 μ L BamHI; 4 μ L EcoRI; Spend the night in 37 ℃ of placements.Recombinant plasmid pGEX-6p-1-AroA
j.limoconcrete building process as shown in Figure 1, the recombinant plasmid building is as shown in Figure 5.
The recovery that enzyme is cut product is used glue to reclaim test kit (purchased from AxyGen 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 DE-A damping fluid (this test kit carries) of 300 μ L, bathes 10min, until gel all melts in 70 ℃ of water-bath temperature.Add again the DE-B damping fluid (this test kit carries) of 1/2 times of DE-A damping fluid (this test kit carries) volume, sol solutions is joined to 2mL adsorption tube 12000rpm, 4 ℃ of centrifugal 1min, add the centrifugal wash-out 1min of 500uL W1 damping fluid (this test kit carries), add again the centrifugal wash-out 1min of 700uL W2 damping fluid (this test kit carries), after damping fluid is discarded, centrifugal 1min removes ethanol as far as possible again, adds 50 μ L distilled water wash-outs to collect DNA.Electrophoresis result as shown in Figure 2.
Clone's enzyme connects: 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; T4 DNA ligase (purchased from Transgene company) 1 μ L; 5 × T4 DNA ligase buffer, 2 μ L; Add distilled water and mend to 10 μ L, connect and spend the night in 4 ℃ of enzymes.
By AroA
j.limogene (its nucleotide sequence is shown in shown in SEQ ID NO:1) is inserted into pGEX-6P-1 carrier, and positive colony correct checking is extracted to recombinant plasmid, called after pGEX-6p-1-AroA
j.limo, its size is 6280bp (seeing Fig. 5).
Expression, purifying and the analysis of embodiment 2 EPSPS albumen
(1) expression of target protein EPSPS
By recombinant plasmid pGEX-6p-1-AroA
j.limobe transformed into expression host cell intestinal bacteria E.coli BL21 (DE3).Checking positive colony, will identify the activation of spending the night of correct positive colony, is forwarded to 1L containing in the LB liquid nutrient medium of 100 μ g/mL Ampicillin with the inoculum size of volume ratio 1%, in 37 ℃ of cultivation 2-3h, until OD
600reach 0.6 left and right, add inductor isopropyl-β-D-thiogalactoside(IPTG) (IPTG) final concentration to 0.5mM, in 22 ℃, under 180rpm, cultivate 25h.Centrifugal collection thalline, then use Hepes damping fluid (formula: 50mM 4-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 ℃, 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 (6mL 1mol/LTris-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 71.1kDa, the target protein EPSPS removing after GST label is 45.1kDa, 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, Stahl et al.1997), concrete operation step (all operations all carries out at 4 ℃) is as follows: get 2ml column material GSH Sepharose 4B (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 300mLHepes buffer solution elution, flow rate control is below 1.0mL/min.The HRV 3CP (purchased from Merck company) of getting 10 μ L concentration again and be 10unit/ μ L mixes with 1mL Hepes damping fluid, puts 4 ℃ 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 ℃ of preservations (Rippert and Matringe 2002).
(3) concentration quantitative of target protein EPSPS is measured
Adopt quantitative Bradford method (Bradford 1976) 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 0,1,2,4,6,8,10,15 μ L are added to respectively in 96 orifice plates, add Hepes damping fluid and supply 20 μ L, and every porin content is respectively 0.0,0.2,0.4,0.8,1.2,1.6,2.0mg/mL.3 parallel tests of every group of design.Every hole adds 200 μ L Bradford Reagent (Bradford protein quantification test kit carries), mixes, and 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 7, 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, Alvarez 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 0.2282g K
2hPO
43H
2o, dissolve and be settled to 100mL with Hepes damping fluid), get 10 times of 30mL standardized solution Hepes damping fluid dilutions, get respectively 0,5,10,20,30,40 (μ L) in 1.5mL centrifuge tube, every pipe inorganic phosphorus concentration is respectively 0,0.1,0.2,0.4,0.8mM; Add respectively appropriate Hepes damping fluid to complement to 50 μ L, after 28 ℃ of reaction 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 0.1mL 34% sodium citrate solution after placing 1min, gets 200 μ L and measure OD after placement 30min in 96 orifice plates
660value.Design three experimental group (Jin, Lu et al.2007).Take inorganic phosphorus concentration as X-coordinate, with OD
660for ordinate zou mapping obtains inorganic phosphorus canonical plotting (seeing Fig. 8).
Optimum temperuture is measured: reaction system is measured system with above-mentioned pH.After adding well, system under differing temps (0,10,20,30,40,50,60 ℃), reacts 3min, add 0.8mL MAT solution, add 0.1mL34% sodium citrate solution after 1min, room temperature is placed after 30min, get 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 (S3P), designs 3 experimental group).Take temperature as X-coordinate, the mapping take relative reactivity as ordinate zou, the optimum temperuture obtaining is as shown in Figure 9 measured curve.
Optimal pH is measured: 50 μ L reaction systems: 1mM shikimic acid triphosphoric acid, 1mM phosphoenolpyruvic acid (PEP), add the EPSPS albumen of 1uL purifying, the Hepes damping fluid that adds different pH (3,4,5,6,7,8,9,10,11) is supplied 50 μ L.28 ℃ 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.Take pH value as X-coordinate, the mapping take relative reactivity as ordinate zou, the optimal pH obtaining is as shown in figure 10 measured curve.
Km (PEP) measures: the concentration of shikimic acid triphosphoric acid (S3P) in system is fixed as to 1mM, measure enzyme reaction rate (Sun by above-mentioned 50 μ L reaction systems different PEP concentration (0,0.02,0.05,0.1,0.2,0.4,0.8,1.0mM) is lower, Chen et al.2005), take PEP concentration as X-coordinate, map take speed of response V (U/mg) as ordinate zou, obtain the measurement result of Km (PEP) as shown in figure 11.
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, take S3P concentration as X-coordinate, map take speed of response V (U/mg) as ordinate zou, obtain the measurement result of Km (S3P) as shown in figure 12.
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 take glyphosate concentration as X-coordinate, adopts logarithmic coordinates, maps take speed of response V (U/mg) as ordinate zou, obtain as shown in figure 13 partly suppress dosage IC
50the measurement result of value.
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 andBrown 2007), be transferred to respectively in the M9 liquid nutrient medium containing 0mM, 50mM, 100mM glyphosate, measure growth curve.
(1) set contrast
The design of primer is with synthetic.Intestinal bacteria E.coli K-12 AroA gene (the called after AroA announcing according to GENEBANK
e.coli) nucleotide sequence (accession number: NP_415428.1), the synthetic following primer pair of design:
Forward primer (5 '-AroA
e.coli-BamHI):
5 '-CGC
gGATCCaTGGAATCCCTGACGTTACAACCCATCGCTCGTG-3 ', underscore part is BamHI restriction enzyme site;
Reverse primer (3 '-AroA
e.coli-XhoI):
5 '-CCG
cTCGAGtCAGGCTGCCTGGCTAATCCGCGCCAGCT-3 ', underscore part is XhoI restriction enzyme site.This primer pair is synthesized by Nanjing Genscript Biotechnology Co., Ltd..
Build pGEX-6p-1-AroA
e.colirecombinant plasmid, concrete steps and construction process are with pGEX-6p-1-AroA in above-described embodiment 2
j.limothe structure of recombinant plasmid, pGEX-6p-1-AroA
e.colirecombinant plasmid collection of illustrative plates as shown in Figure 6.
(2) mensuration of growth curve
By the recombinant plasmid pGEX-6p-1-AroA building
j.limoand pGEX-6p-1-AroA
e.colibe transformed into respectively competent cell intestinal bacteria AroA deficient strain AB2829, transformant is inoculated into respectively (containing 100 μ g/mL Ampicillin) in the M9 liquid nutrient medium containing containing 0mM, 50mM, 100mM glyphosate, cultivates 40h in 37 ℃ of concussions.Three parallel laboratory tests of every group of design, every 10h gets 200 μ L nutrient solutions and measures cell concn, i.e. OD
600value.Take incubation time as X-coordinate, with OD
600value is ordinate zou mapping, and the growth curve obtaining as shown in figure 14.
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Claims (3)
1. 5-enol acetone shikimic acid-3-phosphate synthase gene, its nucleotide sequence is as described in sequence table SEQ ID NO:1.
2. 5-enol acetone shikimic acid-3-phosphate synthase gene, the sequence of the protein of its coding is as described in sequence table SEQ ID NO:2.
3. a strain comprises intestinal bacteria (Escherichia coli) LZD001 that expresses 5-enol acetone shikimic acid-3-phosphate synthase gene recombinant plasmid, be deposited in Chinese Typical Representative culture collection center (CCTCC), deposit number is CCTCC NO:M2011493, and this bacterial strain contains just like the nucleotide sequence shown in sequence table SEQ ID NO:1.
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| CN1720330A (en) * | 2002-12-12 | 2006-01-11 | 拜尔农科股份有限公司 | Expression cassette encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and herbicide-tolerant plants containing it |
| CN101100676A (en) * | 2007-06-15 | 2008-01-09 | 中国农业科学院生物技术研究所 | Bivalent expression vectors for breeding glyphosate-resistant plants |
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| CN1720330A (en) * | 2002-12-12 | 2006-01-11 | 拜尔农科股份有限公司 | Expression cassette encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and herbicide-tolerant plants containing it |
| CN101100676A (en) * | 2007-06-15 | 2008-01-09 | 中国农业科学院生物技术研究所 | Bivalent expression vectors for breeding glyphosate-resistant plants |
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