CN1358858A - Method for optimizing gene and antiroundup gene obtained by said method and expression carrier - Google Patents

Abstract

In present invention, characteristics of homologous recombination of DNA sequence and thermal cycling reaction of traditional PCR amplification method are used to design two or more than two gene sequences whose homology is greater than 50% as source template and adopts simple, convenient and high-effective gene optimization method of two-step PCR amplification reaction, and utilizes said method to obtain two EPSP synzyme genes which can produce multipoint mutation and possess stronger glyphosate resistance, and its coded enzyme possesses K[PEP] lower than that of wild type, and its K[glyp hosate] can be obviously raised. Said invention not only can raise catalytic efficiency of enzyme, but also reduces affinity of enzyme and glyphosate so as to greatly raise the enzyme capability for resisting glyphosate.

Description

A kind of method of gene optimization and the Antiglyphosate gene and the expression vector thereof that obtain with this method

The invention belongs to technical field of bioengineering, be specifically related to a kind of method of gene optimization, the epsp synthase gene of the resistance glyphosate that obtains with this method contains the plasmid and the transformant of this gene.

Glyphosate is to use broad-spectrum herbicide the most widely, has people and animals nontoxicly substantially, and weeds are difficult to it is produced resistance, characteristics such as low soil residual quantity, and market potential is huge.For adapting to mass mechanized production and using the needs of glyphosate, since middle 1980s, people have done a large amount of work for cultivating the resistance glyphosate farm crop.At present, obtained many glyphosate resistant crops by agriculture genetic engineering technique, they have following characteristics: (1) has changed the agricultural tradition tillage method, and cultivation management is more prone to; (2) herbicidal effect is more obvious; (3) reduce the weedicide consumption, help protecting environment; (4) increasing both production and income.The plantation of glyphosate resistant crops will significantly reduce the usage quantity of weedicide, the soybean of U.S.'s plantation resistance glyphosate in 1996, and per hectare weedicide consumption reduces 9% to 39%; The crop of Canada's plantation resistance glyphosate in 1996 makes the weedicide amount ratio routine of average per hectare reduce 1.0 kilograms.The glyphosate resistant crops of U.S. Monsanto Company exploitation, also promptly anti-farming reaches series, by establishing in large scale, and has produced huge economic benefit.

Epsp synthase by the aroA genes encoding exists only in microorganism and the plant, in the die aromatischen Aminosaeuren pathways metabolism, work, catalysis shikimic acid-3-phosphoric acid (S-3-P) and phosphoenolpyruvic acid (PEP) effect generate 5-enol pyruvoyl shikimic acid-3-phosphoric acid (EPSP).The epsp synthase of having identified can be divided into two classes: the first kind can be by generations such as intestinal bacteria, salmonella typhimurium (calling the first kind epsp synthase or first fermentoid in the following text); Second class is found (calling the second class epsp synthase or second fermentoid in the following text) in agrobacterium tumefaciens CP4, achromobacteria LBAA and pseudomonas PG2982.The homology of this two classes epsp synthase is not high, amino acid whose similarity be less than 50%, the second fermentoid can not with the first zymoid monoclonal antibody generation cross reaction, and both zymologic properties are also had any different.

Because glyphosate has the structure similar to PEP, can be used as competitive inhibitor, form epsp synthase S-3-P glyphosate mixture, check combining of PEP and enzyme, thereby the blocking-up die aromatischen Aminosaeuren synthetic, kill most plants, the neither exception of weeds and farm crop.

In the past, the approach that the research of acquisition resistance glyphosate transgenic crop is followed mainly contains: one, import the epsp synthase gene of overexpression, resist the competitive inhibition of glyphosate with this; Two, by rite-directed mutagenesis, acquisition is hanged down affinity to glyphosate or is not had the epsp synthase of affinity, thereby eliminates the restraining effect of glyphosate.Because about resistance enzyme high two orders of magnitude than plant origin of the epsp synthase of bacterium to glyphosate, so, use engineered method, in the epsp synthase gene transfered plant of bacterium, can make plant obtain certain resistance.For improving resistance level, can adopt and improve external source epsp synthase expression of gene amount or reduce this enzyme to measures such as glyphosate avidity by rite-directed mutagenesis.Comai, people such as L. make rite-directed mutagenesis with the aroA gene of salmonella typhimurium, make the 101st proline(Pro) of epsp synthase become Serine, and the gene transformation tobacco with after the sudden change can make tobacco that glyphosate is shown stronger resistance; People such as Padgette SR are made proteic the 96th glycine of colibacillary aroA genetic expression into L-Ala, no matter epsp synthase after the sudden change is present in the intestinal bacteria or in pea, all shows the glyphosate resistance of higher level because of the reduction to glyphosate avidity.In addition, the epsp synthase gene of the resistance glyphosate that derives from Agrobacterium mutation CP4 is introduced in beet, cotton and the soybean, all successfully obtained to have the genetically modified crops of glyphosate resistance.

But the foreign gene overexpression will inevitably influence the growth of plant self.And because near the effect of the amino-acid residue enzyme active center is not understood, so the effect of rite-directed mutagenesis is very limited.At present, the difficulty that runs into is, the epsp synthase behind the rite-directed mutagenesis also is accompanied by decline to former substrate avidity to the decline of glyphosate avidity, also promptly influenced the catalytic activity of enzyme.

The method that the purpose of this invention is to provide a kind of gene optimization can artificially strengthen the probability of producer sudden change with this method; And pass through the epsp synthase gene that this gene optimization method obtains to take place to modify the highly active resistance glyphosate that suddenlys change, and the plasmid and the transformant that contain this gene.The epsp synthase of this genes encoding has the level of higher resistance glyphosate, can be used as the excellent material of cultivating the resistance glyphosate new crop varieties.

Gene optimization method of the present invention be use simultaneously two or more homologys greater than 50% gene order as From Template, design and the corresponding two ends of From Template sequence primer, and adopt two to go on foot the pcr amplification method of amplified reactions, thereby obtain a series of gene fragments with multipoint mutation.

At first, select two or more homologys greater than 50% gene order as From Template, and design and the corresponding two ends of From Template sequence primer.The purpose of the first step amplification is the random incorporation by an end primer and From Template, staggered extension, the dna single chain of the reorganization that synthetic a series of sequences are different.The consumption of primer is less, approximately is that 1pmol-5pmol gets final product.The time of DNA chain extension is also shorter, and desirable 1-5 second, promptly each primer at most only extends tens bp with after template annealing combines, and just enters the circulation of next denaturation renaturation-extension.Because DNA has the characteristic of homologous recombination, so, when annealing each time, equating between primer and each template in conjunction with chance.Primer and template bonded randomness have just caused the unhomogeneity of the DNA chain that extends.For increasing DNA chain and the template bonded chance in extending, the present invention has designed two cycling programs, and first circulation is only carried out 5-10 time, and annealing temperature is lower, and about 40-50 ℃, to guarantee the annealed combination of initial primers and template; Second circulation carried out 30-60 time, and annealing temperature also wants corresponding and bring up to 50-60 ℃, makes that synthetic short dna chain has superiority with combining more of template with the binding ratio initial primers of template in the above-mentioned circulation.In circulation each time, extended chain is random incorporation between each template, reads each template sequence information at random, instructs the synthetic of self.Finally, carried the new dna fragmentation of each From Template sequence information with synthesizing a series of heterogeneities.

The product that is obtained with the first step amplification adds the two ends primer according to the From Template design as template, carries out the amplification of second step.Each required temperature-time parameter of thermal cycling requires to get final product routinely.After 30 circulations, just can obtain the complete gene fragment of a series of generation multipoint mutations.

The actual conditions and the operation steps of gene optimization method of the present invention are as follows:

The first step amplified reaction:

Reactive system: 1 * taqDNA polymerase buffer, 4 kinds of each 200umol/L of dNTP are according to each 1pmol-5pmol of primer of each template 5 '-end design, each From Template DNA about 10 ²-10 ⁴Copy, the about 0.5-5U of taqDNA polysaccharase.

Response procedures and parameter variation range:

[1] pre-sex change: 94 ℃, 5-10 minute;

[2] circulation 1:94 ℃ 60 seconds, 40-50 ℃ 60 seconds, 72 ℃ of 1-5 seconds, circulation 5-10 time;

[3] circulation 2:94 ℃ 60 seconds, 50-60 ℃ 60 seconds, 72 ℃ 5 seconds, circulation 30-60 time;

[4] 72 ℃ 10 minutes.

The second step amplified reaction:

Reactive system: 1 * taqDNA polymerase buffer, 4 kinds of each 200umol/L of dNTP, according to each 10pmol-50pmol of primer of each template 5 '-end and 3 '-end design, the reaction product 1ul that gets above-mentioned the first step amplified reaction is as template, the about 0.5-5U of taqDNA polysaccharase.

Response procedures and parameter variation range:

[1] 94 ℃ 5 minutes;

[2] circulation: 94 ℃ 90 seconds, 45-55 ℃ of 30-90 second, 72 ℃ of 60-240 seconds, circulate 30 times;

[3] 72 ℃ 10 minutes.

According to the gene optimization method of the invention described above, selecting the epsp synthase gene order of intestinal bacteria and salmonella typhimurium is From Template, and designs following three and these two the corresponding two ends of template primers:

Primer 1:5 '-CATGCCATGGAATCCCTGACGTTACAA-3 ',

Primer 2: 5 '-CGCGGATCCTCAGGCTGCCTGGCTAATCC-3 ',

Primer 3:5 '-CGCGGATCCTTAGGCAGGCGTACTCATTC-3 ';

The epsp synthase gene that can synthesize sudden change.Product comprises the dna sequence dna of called after aroAM12 and the dna sequence dna of called after aroAM13, and they are respectively shown in sequence table 1 and sequence table 2.

The above-mentioned aroAM12 dna sequence dna that obtains is inserted between the NcoI and BamHI restriction enzyme site of plasmid pET11d, can obtains containing the plasmid pET-aroAM12 of aroAM12 dna sequence dna, its structure as shown in Figure 1; The above-mentioned aroAM13 dna sequence dna that obtains is inserted between the NcoI and BamHI restriction enzyme site of plasmid pET11d, can obtains containing the plasmid pET-aroAM13 of aroAM13 dna sequence dna, its structure as shown in Figure 2.

With the above-mentioned plasmid pET-aroAM12 transformed into escherichia coli BL-21 (DE3) that obtains, can obtain the transformant of called after BL21 (aroAM12) with glyphosate resistance; With the above-mentioned plasmid pET-aroAM13 transformed into escherichia coli BL-21 (DE3) that obtains, can obtain the transformant of called after BL21 (aroAM13) with glyphosate resistance.

Below by embodiment and accompanying drawing the present invention is described in further detail.

Fig. 1 is a pET-aroAM12 plasmid structure iron;

Fig. 2 is a pET-aroAM13 plasmid structure iron;

Fig. 3 is the growth curve of transformant in containing the M9 liquid nutrient medium of 20mM glyphosate;

Fig. 4 is the growth curve of transformant in containing the M9 liquid nutrient medium of 30mM glyphosate;

Fig. 5 is the growth curve of transformant in containing the M9 liquid nutrient medium of 60mM glyphosate;

Fig. 6 is the protein electrophoresis figure of each transformant;

Fig. 7 is the design of graphics of epsp synthase expression vector.

Synthesizing of embodiment 1, sudden change epsp synthase gene

Dna sequence dna according to the epsp synthase gene of intestinal bacteria that from GENE BANK, detect (Escherichia coli) and salmonella typhimurium (Salmonellatyphimurium), and by relatively finding, the homology of these two sequences is 79%, meet the requirement of gene optimization method, can be used as From Template.Because 5 '-terminal sequence of these two sequences is identical, so 5 '-end primer also can be identical.According to the requirement of gene optimization method, design three primers:

Primer 1:5 '-CATGCCATGGAATCCCTGACGTTACAA-3 ',

Primer 2: 5 '-CGCGGATCCTCAGGCTGCCTGGCTAATCC-3 ',

Primer 3:5 '-CGCGGATCCTTAGGCAGGCGTACTCATTC-3 ';

Above-mentioned band underscore place represents corresponding restriction enzyme site.Wherein, primer 1 is the identical homing sequence that is added with the epsp synthase gene of the intestinal bacteria of a Nco I restriction enzyme site and salmonella typhimurium in front.Primer 2 and primer 3 are respectively the restriction enzyme sites that the preceding sequence of epsp synthase gene 1284bp of intestinal bacteria and salmonella typhimurium adds BamHI.

Building-up reactions comprises the two-step pcr amplification, can carry out with condition as follows:

The first step amplified reaction:

Reactive system: in reaction solution, have two templates (the nuclear DNA of intestinal bacteria and salmonella typhimurium) each about 10 ²-10 ⁴Copy and 1.5pmol primer 1, various dNTP 200umol/L, 1 * taqDNA polymerase buffer, the about 0.5-5U of taqDNA polysaccharase.

Reaction conditions:

[1] pre-sex change: 94 ℃, 10 minutes;

[2] circulation 1:94 ℃ 60 seconds, 45 ℃ 60 seconds, 72 ℃ 5 seconds; Circulate 5 times;

[3] circulation 2:94 ℃ 60 seconds, 55 ℃ 60 seconds, 72 ℃ 5 seconds; Circulate 30 times;

[4] 72 ℃ 10 minutes.

The amplification of second step:

The first step amplified reaction finishes, and extracts reaction solution 1 microlitre as second template of taking turns reaction, and with 1 * taqDNA polymerase buffer, 4 kinds of each 200umol/L of dNTP, each 15pmol of primer 1-3, the about 0.5-5U of taqDNA polysaccharase constitutes reactive system.

Reaction conditions:

[1] 94 ℃ 5 minutes;

[2] circulation: 94 ℃ 90 seconds, 55 ℃ 90 seconds, 72 ℃ 120 seconds; Circulate 30 times;

[3] 72 ℃ 10 minutes.

After two steps, amplified reaction finished, agarose electrophoresis was identified the pcr amplification result.Discovery is through pcr amplification, obtains to be about the dna fragmentation about 1.3kb, matches with the size of the epsp synthase gene of expection.Owing to adopted the special genes optimization method, so, the dna fragmentation that is obtained not is the intestinal bacteria of wild-type of sequence homogeneous or the epsp synthase gene of salmonella typhimurium, but carrying two From Template sequence informations to some extent, the epsp synthase gene of one group of reorganization of sudden change has in various degree also promptly taken place.

The structure of embodiment 2, epsp synthase expression vector and the acquisition of transformant

The sequence heterogeneity dna fragmentation that the foregoing description 1 is obtained behind pcr amplification is walked agarose gel electrophoresis, and cut off and contain the gel band that size is about the DNA of 1.3kb, with " the QIAquick Gel Extration Kit " of QIAGEN company, these dna fragmentations of separation and purification.Separation finishes, and cuts with restriction enzyme NcoI and BamHI enzyme respectively simultaneously with plasmid pET 11d, again both is pressed carrier: insert the mixed of fragment=1: 3, connect 12 hours with the T4 ligase enzyme at 16 ℃.

Get part and connect liquid, with standard calcium chloride transformation transformed into escherichia coli BL21 (DE3), the transformant turning point of acquisition to the M9 solid medium that contains 30mM glyphosate and 1mM IPTG, the transformant of the tool glyphosate resistance that screening can be grown on this substratum.The resistance transformant turning point of these preliminary screening acquisitions to the M9 solid medium that contains 60mM glyphosate and 1mM IPTG, has been obtained two and had the strong active transformant of resistance glyphosate, called after BL21 (aroAM12) and BL21 (aroAm13) respectively.

" QIAprep Spin Miniprep Kit " with QIAGEN company extracts plasmid from transformant BL21 (aroAM12) and BL21 (aroAM13), respectively called after pET-aroAM12, pET-aroAM13 (its structure respectively as shown in Figure 1 and Figure 2).With NcoI and these two recombinant plasmids of BamHI double digestion, agarose electrophoretic analysis obtains the fragment that size is about 5.7kb and 1.3kb respectively.Electrophoresis result explanation, between the NcoI and BamHI two restriction enzyme sites of these two plasmids, the 1.3Kb fragment that contains pcr amplification respectively and obtained.Measure to insert segmental dna sequence dna with the ABI377 sequenator, further proof is cloned between the NcoI of carrier pET 11d and BamHI two restriction enzyme sites, is the epsp synthase gene of undergoing mutation.We distinguish called after aroAM12 and aroAM13 to the gene fragment of these two sudden changes.The aminoacid sequence of the dna sequence dna of aroAM12 and aroAM13 and the coded epsp synthase with glyphosate resistance thereof is seen sequence table 1 and sequence table 2 respectively.

Simultaneously, primer 1-3 with the foregoing description 1, nuclear DNA with intestinal bacteria and salmonella typhimurium is a template, the pcr amplification method of employing standard, obtain the intestinal bacteria of wild-type and the epsp synthase gene of salmonella typhimurium, called after EcaroA and StaroA are cloned into respectively between the NcoI and BamHI restriction enzyme site of plasmid pET-11d respectively.Recombinant plasmid is called after pET-EcaroA and pET-StaroA respectively, and with this recombinant plasmid transformed e. coli bl21 (DE3), the gained transformant is called after BL21 (EcaroA) and BL21 (StaroA) respectively, as the above-mentioned contrast that contains the transformant of mutator gene.

The building process of above-described plasmid pET-aroAM12, pET-aroAM13, pET-EcaroA and pET-StaroA as shown in Figure 7." aroA " among Fig. 7 represents epsp synthase gene aroAM12, aroAM13, EcaroA or StaroA; " pET-aroA " represents plasmid pET-aroAM12, pET-aroAM13, pET-EcaroA or pET-StaroA.

The check of embodiment 3, transformant aroAM12, aroAM13 glyphosate resistance

With transformant BL21 (aroAM12) and BL21 (aroAM13) and BL21 (EcaroA) and BL21 (StaroA), be inoculated into respectively contain 1mM IPTG, 50 μ g/ml acillins and 20,30 or the M9 basic medium of 60mM glyphosate in, cultivate with the condition of 200rpm, 37 ℃ of airbaths.From being cultured to 16 hours, measured a nutrient solution OD600 every 2 hours, write down its growing state, measurement result such as Fig. 3 are to shown in Figure 5.From the growth curve of Fig. 3 to Fig. 5 as can be seen, the transformant that carries mutator gene still can be survived containing on the M9 minimum medium of 60mM glyphosate, and the growth of transformant on the M9 of the glyphosate that contains 20mM minimum medium that carries wild type gene has been subjected to serious inhibition.This shows that the resistance glyphosate of muton can the force rate wild-type be significantly improved.

The mensuration of embodiment 4, sudden change epsp synthase gene DNA sequence reaches the comparison with wild type gene

Mutator gene aroAM12 of the present invention and aroAM13 are done the comparison of sequence similarity respectively with the epsp synthase gene (EcaroA and StaroA) of wild-type, find that aroAM12 is similar to contrast StaroA, aroAM13 is similar to contrast EcaroA, undergo mutation in different loci respectively, the result is shown in following sequence.

aroAM12EPSP：StaroA MESLTLQPIARVDGAINLPGSKSVSNRALLLAALPCGKTALTNLLDSDDVRHMLNALSAL 60M12 LAC TVLMN 60StaroA GINYTLSADRTRCDITGNGGALRAPGALELFLGNAGTAMRPLAAALCLGQNEIVLTGEPR 120M12 LHA 120StaroA MKERPIGHLVDSLRQGGANIDYLEQENYPPLRLRGGFTGGDIEVDGSVSSQFLTALLMTA 180M12 180StaroA PLAPKDTIIRVKGELVSKPYIDITLNLMKTFGVEIANHHYQQFVVKGGQQYHSPGRYLVE 240M12 PED YND QKY 240StaroA GDASSASYFLAAGAIKGGTVKVTGIGRKSMQGDIRFADVLEKMGATITWGDDFIACTRGE 300M12 300StaroA LHAIDMDMNHIPDAAMTIATTALFAKGTTTLRNIYNWRVKETDRLFAMATELRKVGAEVE 360M12 360StaroA EGHDYIRITPPAKLQHADIGTYNDHRMAMCFSLVALSDTPVTILDPKCTAKTFPDYFEQL 420M12 420StaroA ARMSTPA 427M12 427aroAM13EPSP：EcaroA MESLTLQPIARVDGTINLPGSKTVSNRALLLAALAHGKTVLTNLLDSDDVRHMLNALTAL 60aroAM13 KSV 60EcaroA GVSYTLSADRTRCEIIGNGGPLHAEGALELFLGNAGTAMRPLAAALCLGSNDIVLTGEPR 120aroAM13 120EcaroA MKERPIGHLVDALRLGGAKITYLEQENYPPLRLQGGFTGGNVDVDGSVSSQFLTALLMTA 180aroAM13 180EcaroA PLAPEDTVIRIKGDLVSKPYIDITLNLMKTFGVEIENQHYQQFVVKGGQSYQSPGTYLVE 240aroAM13 240EcaroA GDASSASYFLAAAAIKGGTVKVTGIGRNSMQGDIRFADVLEKMGATICWGDDYISCTRGE 300aroAM13 300EcaroA LNAIDMDMNHIPDAAMTIATAALFAKGTTRLRNIYNWRVKETDRLFAMATELRKVGAEVE 360aroAM13 TTL 360EcaroA EGHDYIRITPPEKLNFAEIATYNDHRMAMCFSLVALSDTPVTILDPKCTAKTFPDYFEQL 420aroAM13 420EcaroA ARISQAA 427aroAM13 427

From above-mentioned sequence as seen:

The feature of the dna sequence dna of called after aroAM12 is, the coded aminoacid sequence of the aminoacid sequence of the epsp synthase with glyphosate resistance that it is coded and salmonella typhimurium aroA gene (StaroA) is compared, and contains following amino-acid substitution: proline 35 → L-Ala; L-Ala 40 → Xie Ansuan; Threonine 42 → methionine(Met); Arginine 83 → Histidine; Methionin 185 → L-glutamic acid; Different propylhomoserin 201 → l-asparagine; Glutamine 230 → Methionin.

The feature of the dna sequence dna of called after aroAM13 is, the coded aminoacid sequence of the aminoacid sequence of the epsp synthase with glyphosate resistance that it is coded and intestinal bacteria aroA gene (EcaroA) is compared, and contains following amino-acid substitution: Threonine 23 → Serine; Arginine 330 → Threonine.

The analysis of the protein expression situation of embodiment 5, each transformant

The LB liquid nutrient medium that contains 50 μ g/ml acillins with 5ml is cultivated transformant BL21 (aroAM12), BL21 (aroAM13) and BL21 (EcaroA), BL21 (StaroA) respectively, reaches 10 with condition to the cell concn of 200rpm, 37 ℃ of airbaths ⁸/ ml, the inoculum size with 1% is transferred in the fresh LB liquid nutrient medium that contains 50 μ g/ml acillins, when 200rpm, 37 ℃ continue to be cultured to ODG00=0.75, adds IPTG to 1mM to induce the synthetic of recombinant protein.Subject to the foregoing, continue to cultivate three hours.The centrifugal thalline of collecting adopts the described method of Laemmli, earlier with the resuspended thalline of SDS-PAGE sample buffer, 12.5%SDS-PAGE gel electrophoresis isolated protein.Electrophoresis finishes, and the Ma Shi light blue of examining with 20% dyes.Electrophoresis result as shown in Figure 4, first road is not for containing the protein crude extract administration of intestinal bacteria BL-21 of plasmid; Second to the 5th road is respectively to contain plasmid pET-StaroA, pET-EcaroA, the protein crude extract administration of the BL-21 of pET-aroAM12 and pET-aroAM13.From the protein graphical spectrum as seen, after IPTG induces, in containing the protein sample of transformant of plasmid, all have all detected the specific protein leukorrhea (arrow indication position in as figure) of epsp synthase 45KD, and, then do not produce this protein band as the intestinal bacteria BL-21 that does not contain plasmid of blank.Protein electrophoresis is the result show, the epsp synthase gene obtains effective expression.The transformant of aroAM12, aroAM13 and EcaroA, StaroA gene is promptly carried in two mutator genes that detected and contrast thereof respectively, and is under same culture conditions, obviously not different to the proteic expression level of epsp synthase.This results suggest we, to the raising of glyphosate resistance, be not that the raising owing to the epsp synthase expression amount causes.

The preparation of embodiment 6, epsp synthase crude extract

The LB liquid nutrient medium that contains 50 μ g/ml acillins with 5ml is cultivated transformant aroAM12, aroAM13 and EcaroA, StaroA respectively, reaches 10 with condition to the cell concn of 200rpm, 37 ℃ of airbaths ⁸/ ml, the inoculum size with 1% is transferred in the fresh LB liquid nutrient medium that contains 50 μ g/ml acillins, when 200rpm, 37 ℃ continue to be cultured to OD600=0.75, adds IPTG to 1mM to induce the synthetic of recombinant protein.Subject to the foregoing, continue to cultivate three hours.The centrifugal thalline of collecting.With buffer A (50mM Tris-Cl, 0.1mM DTT, pH7.2) resuspended thalline.Cell suspension is put-70 ℃ and is freezed, and heavily melts at room temperature again.Handle cell suspension with the mulser of Fluko company then and make its broken wall.With 12000rpm centrifugal 30 minutes, discard cell debris.Supernatant liquor is the epsp synthase crude extract, can be used for the mensuration of every index of relevant this enzyme.

Embodiment 7, preparation S-3-P (shikimate-3-phosphate)

The method of P.F.Knowles etc. is taked in the preparation of S-3-P, extracts from the nutrient solution of Klebsiella pneumoniae (ATCC 25597).Inoculate this bacterium in 12 liters of nutrient broth liquid nutrient mediums (yeast extract 2.8g/L, extractum carnis 7.8g/L, casein 7.8g/L, glucose 5.6g/L, sodium-chlor 5.6g/L, pH7.5) in, 37 ℃ of joltings were cultivated 90 hours.The centrifugal thalline that goes is regulated supernatant liquor pH to 3 with hydrochloric acid, and adds 0.5ml toluene, and last 30 order activated carbon columns are successively with 2 liters of water of regulating pH to 2.5 with hydrochloric acid; 1.5%, 5% and 10% ethanol is washed post.At last, do not have acidifying 5% ethanol to wash pillar once with 500ml.Then, with 2.5 liter of 5% ethanol, 3 liter of 10% ethanol, 2 liter of 25% desired S-3-P of ethanol elution.Merge all elutriants, dewater, obtain melicera residue with the boulton process drying.This syrup is dissolved in the 150ml water, transfers pH to 7 with ammoniacal liquor, add the 100ml barium acetate and react with it, add the concentration of ethanol to 80% (v/v) then, 0 ℃ of placement is spent the night.The centrifugal precipitation of collecting, and wash precipitation twice with 75% ethanol, wash precipitation once with dehydrated alcohol, after the vacuum-drying, just obtain the barium salt of S3P, can be used for the mensuration that the epsp synthase enzyme is lived.

The mensuration of embodiment 8, epsp synthase vigor and Michaelis-Menton constant

The mensuration of epsp synthase vigor adopts the method for the described Victoria Green WPB colour developing of Lanzetta quantitative assay inorganic phosphorus.Reactive system contains 50mM Hepes/NaOH, pH7.2,1mM PEP, 0.5mM S-3-P, 0.1mM (NH ₄) Mo ₇O ₂₄4H ₂O.After each component adds well according to quantity, earlier 30 ℃ of preheatings 5 minutes, add an amount of above-mentioned enzyme liquid of slightly carrying then, enzyme reaction continues 20 minutes, promptly adds the malachite green solution termination reaction.Accurately reaction is 60 seconds, add 34% sodium citrate solution at once, left standstill behind the mixing 15 minutes, measure OD660, the composition of used blank pipe is identical with the initial composition of reaction tubes, and difference is not have time of enzyme effect, promptly add enzyme liquid after, add malachite green solution at once, other each step operation is identical with the enzyme reaction pipe.

The mensuration of Michaelis-Menton constant adopts traditional double-reciprocal plot method.Measurement result as shown in Table 1.

Table one

Michaelis-Menton constant

The ratio of enzyme is lived ^aMichaelis-Menton constant K _M[PEP] ^b

The title of enzyme

(nkat/mg protein) be K (mM) _{I[glyphosate]} ^c

(mM)

StaroA 0.096±0.014 0.801±0.008 0.003±0.001

EcaroA 0.107±0.015 0.464±0.014 0.012±0.001

aroA-M12 1.358±0.21 0.038±0.0004 0.351±0.028

aroA-M13 1.245±0.20 0.025±0.002 0.338±0.017

A. the ratio of enzyme is lived, and is under the excessive condition of substrate, i.e. the ratio vigor of the enzyme of measuring in the reactive system that contains 1.0mMPEP and 0.5mM S-3-P.

B. represent the pairing Michaelis-Menton constant of one of substrate PEP, in the reactive system, the content of S-3-P is fixed on 0.5mM, and PEP then gets a series of changing values by 0.1mM to 0.5 mM.

C. represent the competitive inhibitor glyphosate of PEP and the dissociation constant of enzyme.

D. above each data are two batches of epsp synthase crude extracts, obtain after each measures three times.

Though in existing report, be no lack of the successful example that makes the glyphosate resistance raising of first kind epsp synthase with the means of genetic engineering.But first fermentoid but has typical characteristics: the raising of resistance, be accompanied by the decline of the avidity of one of enzyme-to-substrate PEP, and also be K _[PEP]Increase, thereby cause the decline of the catalytic efficiency of enzyme.Aforesaid successful example, the 101st proline(Pro) after being about to suddenly change becomes the aroA gene transformation tobacco of the salmonella typhimurium of Serine, makes tobacco show stronger resistance to glyphosate; And the 96th glycine after will suddenling change become the colibacillary aroA gene transformation pea of L-Ala, because of epsp synthase makes pea after the conversion show the glyphosate resistance of higher level to the reduction of glyphosate avidity, all belongs to this situation.Because K _[PEP]Increase, reduced the catalytic efficiency of enzyme, so the acquisition of resistance, depend on the overexpression of epsp synthase, and tend to cause the minimizing of crop yield like this.

From sequencing result also as seen, the sudden change of zymoprotein is because amino acid whose displacement but not owing to amino acid whose increase or disappearance cause.And, metathetical amino acid takes place, be not comprised in the relevant amino acid of forefathers' binding site report and active centre enzyme or enzyme-to-substrate (comprising PEP, S-3-P and glyphosate).Infer that according to former study K22, R27, G96, amino acid such as R100 and P101 are enzyme-to-substrate S-3-P bonded related locus; And H385, C408 is then relevant with the combination of PEP with enzyme with amino acid such as K411.And amino-acid substitution involved in the present invention is not seen relevant its report that plays an important role yet in glyphosate resistance.

The present invention has founded the method for a unique and effective gene optimization, on the basis of conventional P CR TRAP, improved, selected template for use more than one, and design the primer that adapts with it, pass through random incorporation, the pcr amplification process of staggered extension has overcome the limitation of rite-directed mutagenesis, has increased the probability and the amplitude of transgenation.And utilization present method, obtained to take place the epsp synthase gene of multipoint mutation, therefrom screen the muton of two high resistances of forward mutation, compare with wild-type enzyme as template, have a much higher enzyme activity (K of reduction _[PEP]) and the enhanced glyphosate resistance (Ki of rising greatly _[glyphosate]).This is the report that relevant first first kind epsp synthase has this character.The Ki of aroAM12 _[glyphosate]Than the raising of salmonella typhimurium 117 times, and K _[PEP]It but only is 0.047% of salmonella typhimurium.The Ki of aroAM13 _[glyphosate]Improved 28 times than colibacillary, and K _[PEP]But only be colibacillary 0.054%.The Ki of aroAM12 _[glyphosate]/ K _[PEP]Value than the increase of salmonella typhimurium 2600 times; And the Ki of aroAM13 _[glyphosate]/ K _[PEP]Value has increased 510 times than colibacillary.Two mutator genes provided by the invention all are expected to become the excellent material that makes up the resistance glyphosate new crop varieties.

Sequence table 11. sequence signatures: length: 1284bp; Type: nucleic acid and respective egg white matter; Chain number: strand; Geometry: linearity; 2. molecule type: DNA; 3. originate: synthetic; 4. sequence description; SEQ ID NO.1ATG GAA TCC CTG ACG TTA CAA CCC ATC GCG CGG GTC GAT GGC GCC ATT 48 M E S L T L Q P I A R V D G A I 1 5 10 15AAT TTA CCT GGC TCC AAA AGT GTT TCA AAC CGT GCT TTG CTC CTG GCG 96 N L P G S K S V S N R A L L L A

20 25 30GCT TTA GCT TGT TGT AAA ACC GTT CTG ATG AAT CTG CTG GAT AGC GAT 144?A L A C G K T V L M N L L D S D

35 40 45GAC GTC CGC CAT ATG CTC AAT GCC CTG AGC GCG TTG GGG ATC AAT TAC 192?D V R H M L N A L S A L G I N Y

50 55 60ACC CTT TCT GCC GAT CGC ACC CGC TGT GAT ATC ACG GGT AAT GGC GGC 240?T L S A D R T R C D I T G N G G65 70 75 80GCA TTA CAT GCG CCA GGC GCT CTG GAA CTG TTT CTC GGT AAT GCC GGA 288?A L H A P G A L E L F L G N A G

85 90 95ACC GCG ATG CGT CCG TTA GCG GCA GCG CTT TGT CTG GGG CAA AAT GAG 336?T A M R P L A A A L C L G Q N E

100 105 110ATA GTG TTA ACC GGC GAA CCG CGT ATG AAA GAG CGT CCG ATA GGC CAT 384?I V L T G E P R M K E R P I G H

115 120 125CTG GTC GAT TCG CTG CGT CAG GGC GGG GCG AAT ATT GAT TAC CTG GAG 432?L V D S L R Q G G A N I D Y L E

130 135 140CAG GAA AAC TAT CCG CCC CTG CGT CTG CGC GGC GGT TTT ACC GGC GGC 480?Q E N Y P P L R L R G G F T G G145 150 155 160GAC ATT GAG GTT GAT GGT AGC GTT TCC AGC CAG TTC CTG ACC GCT CTG 528?D I E V D G S V S S Q F L T A L

165 170 175CTG ATG ACG GCG CCG CTG GCG CCT GAA GAC ACA ATT ATT CGC GTT AAA 576?L M T A P L A P E D T I I R V K

180 185 190GGC GAA CTG GTA TCA AAA CCT TAC AAC GAT ATC ACG CTA AAT TTA ATG 624?G E L V S K P Y N D I T L N L M

195 200 205AAA ACC TTT GGC GTG GAG ATA GCG AAC CAT CAC TAC CAA CAA TTT GTC 672?K T F G V E I A N H H Y Q Q F V

210 215 220GTG AAG GGC GGT CAA AAG TAT CAC TCT CCA GGT CGC TAT CTG GTC GAG 720?V K G G Q K Y H S P G R Y L V E225 230 235 240GGC GAT GCC TCG TCA GCG TCC TAT TTT CTC GCC GCT GGG GCG ATA AAA 768?G D A S S A S Y F L A A G A I K

245 250 255GGC GGC ACG GTA AAA GTG ACC GGG ATT GGC CGC AAA AGT ATG CAG GGC 816?G G T V K V T G I G R K S M Q G

260 265 270GAT ATT CGT TTT GCC GAT GTG CTG GAG AAA ATG GGC GCG ACC ATT ACC 864?D I R F A D V L E K M G A T I T

275 280 285TGG GGC GAT GAT TTT ATT GCC TGC ACG CGC GGC GAA TTG CAC GCC ATA 912?W G D D F I A C T R G E L H A I

290 295 300GAT ATG GAT ATG AAC CAT ATT CCG GAT GCG GCG ATG ACG ATT GCC ACC 960?D M D M N H I P D A A M T I A T305 310 315 320ACG GCG CTG TTT GCG AAA GGA ACC ACG ACG TTG CGC AAT ATT TAT AAC 1008?T A L F A K G T T T L R N I Y N

325 330 335TGG CGA GTG AAA GAA ACC GAT CGC CTG TTC GCG ATG GCG ACC GAG CTA 1056?W R V K E T D R L F A M A T E L

340 345 350CGT AAA GTG GGC GCT GAA GTC GAA GAA GGG CAC GAC TAT ATT CGT ATC 1104?R K V G A E V E E G H D Y I R I

355 360 365ACG CCG CCG GCG AAG CTC CAA CAC GCG GAT ATT GGC ACG TAC AAC GAC 1152?T P P A K L Q H A D I G T Y N D

370 375 380CAC CGT ATG GCG ATG TGT TTC TCA CTG GTC GCA CTG TCC GAT ACG CCA 1200?H R M A M C F S L V A L S D T P385 390 395 400GTC ACG ATC CTG GAC CCT AAA TGT ACC GCA AAA ACG TTC CCT GAT TAT 1248?V T I L D P K C T A K T F P D Y

405 410 415TTC GAA CAA CTG GCG CGA ATG AGT ACG CCT GCC TAA 1284?F E Q L A R M S T P A *

420 425

Sequence table 21. sequence signatures:length:1284bp; Type:nucleic acid and respective egg white matter; Chain number:strand; Geometry; Linear; 2. molecule type:DNA; 3. originate:synthetic; 4. sequence description: SEQ ID NO.2ATG GAA TCC CTG ACG TTA CAA CCC ATC GCT CGT GTC GAT GGC ACT ATT 48 M E S L T L Q P I A R V D G T I 15 10 15AAT CTG CCC GGT TCC AAG AGC GTT TCT AAC CGC GCT TTA TTG CTG GCG 96 N L P G S K S V S N R A L L L A

20 25 30GCA TTA GCA CAC GGC AAA ACA GTA TTA ACC AAT CTG CTG GAT AGC GAT 144?A L A H G K T V L T N L L D S D

35 40 45GAC GTG CGC CAT ATG CTG AAT GCA TTA ACA GCG TTA GGG GTA AGC TAT 192?D V R H M L N A L T A L G V S Y

50 55 60ACG CTT TCA GCC GAT CGT ACG CGT TGC GAA ATT ATC GGT AAC GGC GGT 240?T L S A D R T R C E I I G N G G65 70 75 80CCA TTA CAC GCA GAA GGT GCC CTG GAG TTG TTC CTC GGT AAC GCC GGA 288?P L H A E G A L E L F L G N A G

85 90 95ACG GCA ATG CGT CCG CTG GCG GCA GCT CTT TGT CTG GGT AGC AAT GAT 336?T A M R P L A A A L C L G S N D

100 105 110ATT GTG CTG ACC GGT GAG CCG CGT ATG AAA GAA CGC CCG ATT GGT CAT 384?I V L T G E P R M K E R P I G H

115 120 125CTG GTG GAT GCG CTG CGC CTG GGC GGG GCG AAG ATC ACT TAC CTG GAA 432?L V D A L R L G G A K I T Y L E

130 135 140CAA GAA AAT TAT CCG CCG TTG CGT TTA CAG GGC GGC TTT ACT GGC GGC 480?Q E N Y P P L R L Q G G F T G G145 150 155 160AAC GTT GAC GTT GAT GGC TCC GTT TCC AGC CAA TTC CTC ACC GCA CTG 528?N V D V D G S V S S Q F L T A L

165 170 175TTA ATG ACT GCG CCT CTT GCG CCG GAA GAT ACG GTG ATT CGT ATT AAA 576?L M T A P L A P E D T V I R I K

180 185 190GGC GAT CTG GTT TCT AAA CCT TAT ATC GAC ATC ACA CTC AAT CTG ATG 624?G D L V S K P Y I D I T L N L M

195 200 205AAG ACG TTT GGT GTT GAA ATT GAA AAT CAG CAC TAT CAA CAA TTT GTC 672?K T F G V E I E N Q H Y Q Q F V

210 215 220GTA AAA GGC GGG CAG TCT TAT CAG TCT CCG GGT ACT TAT TTG GTC GAA 720?V K G G Q S Y Q S P G T Y L V E225 230 235 240GGC GAT GCA TCT TCG GCT TCT TAC TTT CTG GCA GCA GCA GCA ATC AAA 768?G D A S S A S Y F L A A A A I K

245 250 255GGC GGC ACT GTA AAA GTG ACC GGT ATT GGA CGT AAC AGT ATG CAG GGT 816?G G T V K V T G I G R N S M Q G

260 265 270GAT ATT CGC TTT GCT GAT GTG CTG GAA AAA ATG GGC GCG ACC ATT TGC 864?D I R F A D V L E K M G A T I C

275 280 285TGG GGC GAT GAT TAT ATT TCC TGC ACG CGT GGT GAA CTG AAC GCT ATT 912?W G D D Y I S C T R G E L N A I

290 295 300GAT ATG GAT ATG AAC CAT ATT CCT GAT GCG GCG ATG ACC ATT GCC ACG 960?D M D M N H I P D A A M T I A T305 310 315 320GCG GCG TTA TTT GCA AAA GGC ACC ACC ACG CTG CGC AAT ATC TAT AAC 1008?A A L F A K G T T T L R N I Y N

325 330 335TGG CGT GTT AAA GAG ACC GAT CGC CTG TTT GCG ATG GCA ACA GAA CTG 1056?W R V K E T D R L F A M A T E L

340 345 350CGT AAA GTC GGC GCG GAA GTG GAA GAG GGG CAC GAT TAC ATT CGT ATC 1104?R K V G A E V E E G H D Y I R I

355 360 365ACA CCT CCG GAA AAA CTG AAC TTT GCC GAG ATC GCG ACA TAC AAT GAT 1152?T P P E K L N F A E I A T Y N D

370 375 380CAC CGG ATG GCG ATG TGT TTC TCG CTG GTG GCG TTG TCA GAT ACA CCA 1200?H R M A M C F S L V A L S D T P385 390 395 400GTG ACG ATT CTT GAT CCC AAA TGC ACG GCC AAA ACA TTT CCG GAT TAT 1248?V T I L D P K C T A K T F P D Y

405 410 415TTC GAG CAG CTG GCG CGG ATT AGC CAG GCA GCC TGA 1284?F E Q L A R I S Q A A *

420 425

Claims (10)

1, a kind of gene optimization method, it is characterized in that using simultaneously two or more homologys greater than 50% gene order as From Template, design and the corresponding two ends of From Template sequence primer, and adopt two the step amplified reactions the pcr amplification method, obtain a series of gene fragments with multipoint mutation.

2,, it is characterized in that the actual conditions and the operation steps of used pcr amplification method is as follows according to the described gene optimization method of claim 1:

The first step amplified reaction:

Reactive system: 1 * taqDNA polymerase buffer, 4 kinds of each 200umol of dNTP, according to each template 5 '-each 1pmol-5pmol of primer of end design, each From Template DNA10 ²-10 ⁴Copy, taqDNA polysaccharase 0.5-5U;

Response procedures and parameter:

[1] pre-sex change: 94 ℃, 5-10 minute;

[2] circulation 1:94 ℃ 60 seconds, 40-50 ℃ 60 seconds, 72 ℃ of 1-5 seconds, circulation 5-10 time;

[3] circulation 2:94 ℃ 60 seconds, 50-60 ℃ 60 seconds, 72 ℃ 5 seconds, circulation 30-60 time;

[4] 72 ℃ 10 minutes;

The second step amplified reaction:

Reactive system: 1 * taqDNA polymerase buffer, 4 kinds of each 200umol of dNTP, according to each template 5 '-end and 3 '-each 10pmol-50pmol of primer that end designs, the reaction product 1ul that gets above-mentioned the first step amplified reaction is as template, taqDNA polysaccharase 0.5-5U;

Response procedures and parameter:

[1] 94 ℃ 5 minutes;

[2] circulation: 94 ℃ of 90-120 seconds, 45-55 ℃ of 30-90 second, 72 ℃ of 60-240 seconds, circulate 30 times;

[3] 72 ℃ 10 minutes.

3, according to claim 1 or 2 described gene optimization methods, it is characterized in that used From Template is the epsp synthase gene order of intestinal bacteria and salmonella typhimurium, designed comprises following three primers with these two the corresponding two ends of template primers:

Primer 1:5 '-CATGCCATGGAATCCCTGACGTTACAA-3 ',

Primer 2: 5 '-CGCGGATCCTCAGGCTGCCTGGCTAATCC-3 ',

Primer 3:5 '-CGCGGATCCTTAGGCAGGCGTACTCATTC-3 ';

Synthetic product is the epsp synthase gene of sudden change.

4,, it is characterized in that the step and the condition of used two-step pcr amplification method is as follows according to the described gene optimization method of claim 3:

The first step amplified reaction:

Reactive system: in reaction solution, have intestinal bacteria and salmonella typhimurium two templates of nuclear DNA each 10 ²-10 ⁴Copy, and 1.5pmol primer 1, various dNTP 200umol/L, 1 * taqDNA polymerase buffer, taqDNA polysaccharase 0.5-5U;

Response procedures and parameter:

[1] pre-sex change: 94 ℃, 10 minutes;

[2] circulation 1:94 ℃ 60 seconds, 45 ℃ 60 seconds, 72 ℃ 5 seconds, circulate 5 times;

[3] circulation 2:94 ℃ 60 seconds, 55 ℃ 60 seconds, 72 ℃ 5 seconds, circulate 30 times;

[4] 72 ℃ 10 minutes;

The amplification of second step:

The first step amplified reaction finishes, extract reaction solution 1 microlitre as second template of taking turns reaction, and with 1 * taqDNA polymerase buffer, 4 kinds of each 200umol/L of dNTP, according to each 10pmol-50pmol of primer of each template 5 '-end and 3 '-end design, taqDNA polysaccharase 0.5-5U constitutes reactive system;

Reaction conditions:

[1] 94 ℃ 5 minutes;

[2] circulation: 94 ℃ 90 seconds, 55 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times;

[3] 72 ℃ 10 minutes.

5, obtain by the described method of claim 3, the dna sequence dna of called after aroAM12, it is characterized in that its coded epsp synthase with glyphosate resistance compares with the aminoacid sequence of salmonella typhimurium aroA coded by said gene, contain following amino-acid substitution: proline 35 → L-Ala; L-Ala 40 → Xie Ansuan; Threonine 42 → methionine(Met); Arginine 83 → Histidine; Methionin 185 → L-glutamic acid; Different propylhomoserin 201 → l-asparagine; Glutamine 230 → Methionin.

6, obtain by the described method of claim 3, the dna sequence dna of called after called after aroAM13, it is characterized in that its coded epsp synthase with glyphosate resistance compares with the aminoacid sequence of intestinal bacteria aroA coded by said gene, contain following amino-acid substitution: Threonine 23 → Serine; Arginine 330 → Threonine.

7, the plasmid pET-aroAM12 that contains the described aroAM12 dna sequence dna of claim 5 is characterized in that the aroAM12 dna sequence dna is inserted between the NcoI and Bam HI restriction enzyme site of plasmid pET11d.

8, the plasmid pET-aroAM13 that contains the described aroAM13 dna sequence dna of claim 6 is characterized in that the aroAM13 dna sequence dna is inserted between the NcoI and Bam HI restriction enzyme site of plasmid pET11d.

9, the transformant with glyphosate resistance of called after BL21 (aroAM12) is obtained by plasmid pET-aroAM12 transformed into escherichia coli BL-21 as claimed in claim 7 (DE3).

10, the transformant with glyphosate resistance of called after BL21 (aroAM13) is obtained by plasmid pET-aroAM13 transformed into escherichia coli BL-21 as claimed in claim 8 (DE3).

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