CN105969782A

CN105969782A - Method for screening glyphosate-resistant gene, EPSPS mutant gene, defect strain and application thereof

Info

Publication number: CN105969782A
Application number: CN201610325692.6A
Authority: CN
Inventors: 王博雅; 卢远根; 胥南飞
Original assignee: Gevoto LLC
Current assignee: Gevoto LLC
Priority date: 2016-05-17
Filing date: 2016-05-17
Publication date: 2016-09-28
Anticipated expiration: 2036-05-17
Also published as: CN105969782B

Abstract

The invention discloses a method for screening a glyphosate-resistant gene, an EPSPS mutant gene, a defect strain and application thereof. The method can be used for cloning glyphosate feeling related genes, including plant genes for multidirectional accelerated mutation and high-throughput glyphosate-resistant performance screening, so that a high glyphosate-resistant gene can be quickly evolved. By adopting the screening method, plants and other sourced genes can be quickly evolved to become mutant genes with glyphosate resistance by utilizing the characteristics of quick growth and reproduction and easy culture of bacteria.

Description

Selection for Resistance Gene Glyphosate method, EPSPS mutant gene and deficiency bacterium Strain and application

Technical field

The present invention relates to biological technical field, in particular to Selection for Resistance Gene Glyphosate side Method, EPSPS mutant gene and deficient strain and application.

Background technology

Glyphosate is to be researched and developed by Monsanto Chemicals, is a kind of foliage-spray, extensively imitates, Nonselective systematicness glyphosate.The approach that its main component glyphosate plays a role is suppression 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in shikimic acid pathway in plant Activity, the plant that makes to be injured can not continue synth essential amino acid and then affect plant normal growth and be To dead.

Glyphosate is the glyphosate herbicidal of a kind of wide spectrum, kills nearly all plant Wound property, on existing market, the most frequently used Antiglyphosate gene is CP4 gene, is Monsanto Company The gene of isolated resistance strong to glyphosate from Agrobacterium.Plant can be by transgenic Mode obtains this resistance.Because the crops of energy resistance glyphosate bring substantially for agricultural and environment Benefit, the corn and soybean kind containing CP4 gene had obtained spread past 20 years. But production application is still constantly needed to new Antiglyphosate gene and anti-grass based on this Sweet phosphine crop varieties.

It is desirable to be resistant to grass with gene editing technology creation non-transgenic anti-glyphosate plants sweet The plant EPSPS gene of phosphine.Though with CP4 as representative microorganism EPSPS gene can carry For glyphosate resistance, even if the method for this genoid genetic modification to be proceeded to plant, but because From different plant species, still there is the suspicion of transgenic, it is difficult to obtain the approval of society's ordinary populace, public Many with prejudice to the understanding of genetically modified crops here, hinder the development of transgenic technology to a certain degree With universal.Therefore, the plant EPSPS gene making energy high-resistance glyphosate is that non-transgenic resists One key of glyphosate crops.

Theoretically, the EPSPS gene of plant itself can be used chemistry, radiate or other Method carries out mutation, and under certain glyphosate pressure, screening has the plant of resistance.It is true that In the case of using glyphosate the most in a large number, some weeds have been evolved and have resisted glyphosate Property；Wherein majority is the change of EPSPS gene.But these changes mostly are gene copy number Increase, and resistance is not the highest, it is difficult to be used on crops.The EPSPS of crops own Gene also has sudden change to produce the example of glyphosate resistance, but resistance is not as CP4.Can be high for creating The non-transgenic crop of resistance glyphosate, continues mutation, screening crops or other plant EPSPS gene resistant gene is imperative.

But, existing from crops or other plant screening there is the sudden change of glyphosate resistance The method of gene needs first plant to be carried out mutagenic treatment and obtains substantial amounts of mutant plant, more right These mutant plants carry out resistance screening, obtain the mutant plant with glyphosate resistance, logical The genome detection crossing antagonism plant is analyzed, and finally obtains the sudden change base with glyphosate resistance Cause.Owing to the cycle of plant growing is long, plant the time that substantial amounts of mutant plants not only expends Land area that is long and that need is the hugest.

Summary of the invention

It is an object of the invention to provide the screening of a kind of mutant gene with glyphosate resistance Method, this screening technique can screen the sudden change base obtaining the exogenous gene from plant rapidly Cause, the mutant gene that the screening of this screening technique obtains has glyphosate resistance.

Another mesh of the present invention is to provide a kind of mutant gene, and this mutant gene is by above-mentioned screening Method screening obtains, and it has glyphosate resistance.

The another mesh of the present invention is to provide the application of said mutation gene, dashes forward so that converting this The plant becoming gene has glyphosate resistance.

The another mesh of the present invention is to provide a kind of sudden change for screening with glyphosate resistance The model bacterium of gene, this model bacterium can not be expressed EPSPS, the most not had the merit of cracking glyphosate Energy.

The another mesh of the present invention is the EPSPS providing above-mentioned model bacterium to originate in test plants Application in the function of gene.

The another mesh of the present invention is the EPSPS providing above-mentioned model bacterium to originate in test plants Application in the resistance of gene pairs glyphosate.

The another mesh of the present invention is the mutant providing above-mentioned model bacterium to originate in test plants Application in EPSPS gene pairs glyphosate resistance.

The present invention solves it and technical problem is that and realize by the following technical solutions.

A kind of Selection for Resistance Gene Glyphosate method, comprising:

Use gene Knockout to knock out the interference gene of source bacterial strain, obtain deficient strain, source Bacterial strain, from the one in bacillus coli DH 5 alpha, TOP10 and BL21, disturbs gene bag Including EPSPS gene and C-P Lyase gene, deficient strain is EPSPS and C-P Lyase Deficient strain；

First by external source EPSPS channel genes deficient strain, then after mutagenic treatment, To the first mutant bacteria containing external source EPSPS mutant gene, external source EPSPS gene is from mesh Plant；

Or first by mutated for external source EPSPS gene process, obtain external source EPSPS sudden change base Cause, then external source EPSPS mutant gene is imported deficient strain, obtain the second mutant bacteria；

The screening culture medium being placed in the first mutant bacteria or the second mutant bacteria containing glyphosate is enterprising Row filter is cultivated, and obtains the monoclonal resistance bacterium with glyphosate resistance；

Monoclonal resistance bacterium is carried out sequence verification, obtains the EPSPS with glyphosate resistance Mutant gene.

Selection for Resistance Gene Glyphosate method, EPSPS mutant gene and the defect that the present invention provides Type bacterial strain and application provide the benefit that: relative to existing from plant screening have grass sweet The screening technique of the mutant gene of phosphine resistance, the screening technique of the present invention is by building EPSPS With C-P Lyase deficient strain, then with this EPSPS and C-P Lyase deficient strain it is Host Strains, in from the external source EPSPS channel genes of purpose plant to this deficient strain, Obtain the mutant bacteria containing external source EPSPS mutant gene i.e. external source EPSPS gene mutation body Storehouse, then filter out from this external source EPSPS gene mutation body storehouse there is glyphosate resistance EPSPS mutant gene.Owing to the reproduction speed of antibacterial is fast, volume is little, therefore, and the present invention's The problem that screening technique overcomes cycle length present in existing screening technique, floor space is big, The screening technique making the present invention goes out to have the EPSPS gene of glyphosate resistance at directed screening Cycle short, the features such as floor space is the least, and cycle short operation is simple.And the sieve of the present invention Choosing method, using the bacterial strain of EPSPS and C-P Lyase deficiency as Host Strains, is effectively discharged out EPSPS gene and the sudden change of C-P Lyase gene of Host Strains itself and produce glyphosate and resist The situation of property so that the result filtered out more can have science, more reliable.

Accompanying drawing explanation

In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be in embodiment The required accompanying drawing used is briefly described, it will be appreciated that the following drawings illustrate only this Some bright embodiment, is therefore not construed as the restriction to scope, common for this area From the point of view of technical staff, on the premise of not paying creative work, it is also possible to according to these accompanying drawings Obtain other relevant accompanying drawings.

Fig. 1 is the pADV5 carrier structure figure of the embodiment of the present invention；

Fig. 2 is the pKD46 carrier structure figure of the embodiment of the present invention；

Fig. 3 is rice EPSP S mutant gene and the wild rice of the embodiment of the present invention 1 The sequence comparing analysis result of EPSPS gene；

Fig. 4 is soy bean EPSPS mutant gene and the Wild-type soy of the embodiment of the present invention 2 The sequence comparing analysis result of EPSPS gene.

Detailed description of the invention

For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below by right Technical scheme in the embodiment of the present invention is clearly and completely described.In embodiment unreceipted Actual conditions person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument Device unreceipted production firm person, being can be by the commercially available conventional products bought and obtain.

Selection for Resistance Gene Glyphosate method, EPSPS mutant gene and defect to the present invention below Type bacterial strain and application are specifically described.

A kind of Selection for Resistance Gene Glyphosate method, comprising:

Step S1: build deficient strain

Use gene Knockout to knock out the interference gene of source bacterial strain, obtain deficient strain, source Bacterial strain, from the one in bacillus coli DH 5 alpha, TOP10 and BL21, disturbs gene bag Including EPSPS gene and C-P Lyase gene, deficient strain is EPSPS and C-P Lyase Deficient strain.

It is to say, EPSPS and C-P Lyase deficient strain be bacillus coli DH 5 alpha, One in TOP10 and BL21 obtains after knocking out EPSPS gene and C-P Lyase gene The deficient strain arrived.The feature of this EPSPS and C-P Lyase deficient strain is: cannot Grow in the basal medium without aminoacid or albumen is also referred to as restricted culture medium, But can grow on the basal medium containing only sugar after importing external source EPSPS gene.

The source bacterial strain of knocking out namely knocks out EPSPS gene and the C-P of wild-type e. coli The effect of Lyase gene is as described below.

Owing to colibacillary endogenous EPSPS gene can express EPSPS5-enolpyruvyl Shikimic acid-3-phosphate synthase (EPSPS), and C-P Lyase gene also be able to express cracking The C-P lyases of C-P key, this C-P lyases can crack glyphosate.Therefore, if with The escherichia coli of wild type are as Host Strains, in follow-up mutagenic treatment step, in Host Strains EPSPS gene and the C-P Lyase gene in source are it can also happen that sudden change, and it is sweet that generation has anti-grass EPSPS mutant gene that phosphine is endogenous and the C-P Lyase mutant gene that cracking ability is strengthened, compose Give Host Strains glyphosate resistance, cause clearly screening and obtain the grass that monoclonal resistance bacterium has Sweet phosphine resistance is the EPSPS sudden change base that external source EPSP mutant gene gives or it is endogenous Cause and C-P Lyase mutant gene give.Therefore, using escherichia coli as Host Strains Time, need to knock out its endogenous EPSPS gene and C-P Lyase gene, it is ensured that it is final To monoclonal resistance bacterium glyphosate resistance by external source EPSPS mutant gene give, The result making screening is more scientific, more reasonable, more reliable.

Certainly, colibacillary EPSPS gene and the gene knockout of C-P Lyase gene are knocked out Technology is a lot, such as such as FRT method, pCas system, utilizes pKD46 system or utilizes homology PCR fragment directly knocks out method etc..Genome of E.coli sequence information clearly in the case of, Use said method all can knock out the EPSPS gene on its genome and C-P relatively easily Lyase gene.

Step S2: build external source EPSPS gene mutation body storehouse

A kind of construction strategy is, using deficient strain as Host Strains, first by external source EPSPS In channel genes deficient strain, then after mutagenic treatment, obtain dashing forward containing external source EPSPS Become the first mutant bacteria of gene.Wherein, it is preferred that mutagenic treatment is chemomorphosis process or spoke Penetrate mutagenic treatment.Chemomorphosis processes for example with chemical mutagens such as EMS or DES the One mutant bacteria carries out mutagenic treatment, so that external source EPSPS gene is sent out along with the propagation of Host Strains Raw sudden change.

Another kind of construction strategy is, first by mutated for external source EPSPS gene process, outside obtaining Source EPSPS mutant gene, then external source EPSPS mutant gene is imported deficient strain, To the second mutant bacteria.Wherein, sudden change processes is to use mistake with external source EPSPS gene for template Join PCR method or DNA Shuffling method carries out the PCR primer that PCR obtains and is external source EPSPS mutant gene.

It should be noted that wherein the first mutant bacteria or the second mutant bacteria all contain external source EPSPS Mutant gene, the first mutant bacteria or the second mutant bacteria are external source EPSPS gene mutation body storehouse.

Wherein, term " first ", " second " are only used for distinguishing the purpose described, and are not understood that For instruction or hint relative importance.

External source EPSPS gene used by above-mentioned steps is from purpose plant, and purpose plant is water Rice, Semen sojae atricolor, Semen Tritici aestivi, Semen Maydis, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Nicotiana tabacum L., Cotton Gossypii, Rhizoma Dioscoreae esculentae, willow, horse Bell potato, Chinese cabbage, Caulis et Folium Brassicae capitatae or Capsicum annuum L..In actual screening process, can select according to the actual requirements Select.

Step S3: resistance screening

External source EPSPS gene mutation body storehouse the first mutant bacteria or second will be obtained in step s 2 Mutant bacteria is placed in the enterprising row filter of the screening culture medium containing glyphosate and cultivates, and obtains having grass sweet The monoclonal resistance bacterium of phosphine resistance.It should be noted that this monoclonal resistance bacterium is namely at sieve Select the bacterium colony grown in culture medium, it is also possible to referred to as positive transformant.Certainly, positive transformants The situation of the quantity of son has multiple, such as, have a positive transformant or multiple positive transformant.

Wherein, screening culture medium is the M9 basal medium containing different glyphosate concentration.

Step S4: sequence verification

Take the monoclonal resistance bacterium obtained in step s3 and carry out sequence verification, obtain that there is grass The EPSPS mutant gene of sweet phosphine resistance.

Below in conjunction with embodiment, inventive feature and performance are described in further detail.

Embodiment 1

With purpose plant as Oryza sativa L., (Oryza sativa), external source EPSPS gene are the present embodiment Rice EPSP S gene (its nucleotide sequence is as shown in SEQ ID NO.1), to use homology PCR fragment directly knocks out method and knocks out the EPSPS gene in the bacillus coli DH 5 alpha of wild type EPSPS and the C-P Lyase deficient strain obtained with C-P Lyase gene is that Host Strains is Example, is described in detail the screening technique of the present invention, the present embodiment the primer name Claim and nucleotide sequence is shown in Table 1.

The first step, uses homology PCR fragment directly to knock out the EPSPS in bacillus coli DH 5 alpha Gene and and C-P Lyase gene.

1. knock out the C-P Lyase gene of bacillus coli DH 5 alpha

(1) homology PCR fragment amplification

With forward primer CPF2, reverse primer CP5HA3 (being shown in Table 1), big with wild type Enterobacteria DH5 α is that template carries out PCR, and glue reclaims, it is thus achieved that PCR primer, named CP5HA fragment, a length of 525bp, its nucleotide sequence is as shown in SEQ ID NO.13.

Table 1. the present embodiment the primer and nucleotide sequence thereof

With forward primer with CP3HA5 be, reverse primer CPR2, with bacillus coli DH 5 alpha Carrying out PCR for template, glue reclaims, it is thus achieved that PCR primer, names CP3HA fragment, long Degree is 503bp, and its nucleotide sequence is as shown in SEQ ID NO.14.

Use forward primer SPEC5, reverse primer SPEC3, with containing such as SEQ ID NO.2 The carrier (the named pCPSG7 of this carrier) of shown nucleotide sequence is that template carries out PCR, Glue reclaims, it is thus achieved that PCR fragment, named SPEC fragment, a length of 900bp, its nucleoside Acid sequence is as shown in SEQ ID NO.15.

It is primer with CPF2 and CPR2, with CP5HA fragment, SPEC fragment and CP3HA Fragment is that template carries out PCR (carrying out in same reaction system), and glue reclaims, it is thus achieved that PCR Product, named CP5HA-SPEC-CP3HA fragment, a length of 1849bp, its nucleoside Acid sequence is as shown in SEQ ID NO.16.Wherein, the 1st～the 525th is escherichia coli PhnA gene 5 end and upstream sequence thereof, the 526th～the nucleotide sequence of the 1346th For Spectinomycin resistant gene and promoter thereof, the 1347th～the 1849th is large intestine Bacillus PhnH gene 3 end and downstream sequence thereof.

(2) thermal shock converts

Prepare bacillus coli DH 5 alpha competent cell according to a conventional method.By big for 100 μ L Enterobacteria DH5 α competent cell and 5 μ L CP5HA-SPEC-CP3HA fragments are softly mixed Close, be placed in 10min on ice, 42 DEG C of thermal shock 90s, proceed to 2min on ice immediately.

It is rapidly added the 1mL LB fluid medium (Spec containing 50 μ g/mL (spectinomycin, spextinomyxin)), coat LB solid after cultivating 1hr at 37 DEG C In culture medium (containing the Spec of 50 μ g/mL), 37 DEG C of incubated overnight.

Bacillus coli DH 5 alpha after cultivation utilizes forward primer SPE35 and reverse primer CPR0 After detection, this Strain Designation be EDC, EDC be the large intestine bar knocking out C-P Lyase gene Bacterium DH5 α.

2. knock out the EPSPS of EDC (knocking out the bacillus coli DH 5 alpha of C-P Lyase gene) Gene

(1) homology PCR fragment amplification

With the bacillus coli DH 5 alpha of wild type as template, with forward primer EE5-1K and reversely Primer ES5HA3, carries out PCR, and glue reclaims, it is thus achieved that PCR primer, named ES5HA Fragment, a length of 1194bp, its nucleotide sequence is as shown in SEQ ID NO.17.

With bacillus coli DH 5 alpha as template, with forward primer ES3HA5 and reverse primer EE3-1K carries out PCR, and glue reclaims, it is thus achieved that PCR primer, named ES3HA fragment, A length of 1168bp, its nucleotide sequence is as shown in SEQ ID NO.18.

With forward primer GM5L and reverse GM3L, with containing as shown in SEQ ID NO.3 The carrier (the named pCPSG5 of this carrier) of nucleotide sequence be that template carries out PCR, glue Reclaim, it is thus achieved that PCR primer, named GM fragment, a length of 1050bp, its nucleotide Sequence is as shown in SEQ ID NO.19.

With EE5-1K and EE3-1K as primer, ES5HA fragment, GM fragment and ES3HA Fragment is that template carries out PCR, and glue reclaims, it is thus achieved that PCR primer, named ES5HA-GM-ES3HA fragment, a length of 3322bp, its nucleotide sequence such as SEQ ID Shown in NO.20.Wherein, the 1st～the 1194th is escherichia coli EPSPS upstream region of gene Sequence, the nucleotides sequence of the 1195th～the 2154th be classified as gentamicin resistance gene and Promoter, the 2155th～the 3322nd is escherichia coli EPSPS downstream of gene sequences.

(2) thermal shock converts

Prepare EDC competent cell according to a conventional method.By thin for 100 μ LEDC competence Born of the same parents softly mix with 5 μ L ES5HA-GM-ES3HA fragments, are placed in 10min on ice, 42 DEG C Thermal shock 90s, proceeds to 2min on ice immediately；It is rapidly added 1mL LB fluid medium, Coat containing Spec's (50 μ g/ml) and Gm (50 μ g/ml) after 37 DEG C of cultivation 1hr On LB solid medium (containing 50 μ g/ml Spec and the Gm of 50 μ g/ml), 37 DEG C of mistakes Night cultivates.

With forward primer EE5-1K and GM3L, reverse primer EE3-1K and ECES35U Bacterial strain after detection cultivation, and be EDCE by this Strain Designation.EDCE is for knocking out EPSPS Bacillus coli DH 5 alpha after gene and C-P Lyase gene, namely EPSPS and C-P Lyase deficient strain.

Certainly, it is possible to the percussion knockout technique of other routines knocks out greatly for example with pCas system EPSPS gene in enterobacteria DH5 α and C-P Lyase gene, or use pKD46 system System knocks out the EPSPS gene in bacillus coli DH 5 alpha and and C-P Lyase gene.

Second step, with EPSPS and the C-P Lyase deficiency bacterium obtained in first step step Strain, as Host Strains, by this Host Strains of EPSPS channel genes of Oryza sativa L., is dashed forward Become bacterium i.e. rice EPSP S gene mutation body storehouse.Concrete operations are as follows.

1. utilize error-prone PCR method to build EPSP gene mutation body storehouse

Use conventional method the mRNA reverse transcription of rice EPSP S gene is become cDNA and gram Grand to pADV5 carrier (its structure is as shown in Figure 1).

With forward primer PV325 and reverse primer PV323, there is rice EPSP S base to connect The pADV5 support template of cause carries out first round error-prone PCR, and this PCR reaction system includes: The H of 25.3 μ L₂O, the fallibility PCR MIX of 4 μ L, the fallibility PCR dNTP of 4 μ L, 4 μ L MnCl₂, the PV325 of 0.8 μ L, the PV323 of 0.8 μ L, the Taq enzyme of 0.1 μ L, 2 μ L Template.This PCR response procedures: 95 DEG C, 30 seconds；60 DEG C, 30 seconds；72 DEG C, 2 points Clock；40 cycle P CR products, through 1% sepharose electrophoresis, are then cut glue and are reclaimed, obtain the One takes turns PCR primer.

With above-mentioned first round PCR primer as template, with forward primer 2M1H, reverse primer 2M1T, carries out second and takes turns PCR.PCR system is: the H of 31.9 μ L₂O, 2.5 μ L DMSO, the 10xPCR buffer of 5 μ L, the dNTP of 5 μ L, the MgCl of 4 μ L₂、0.5μL 2M1H, the 2M1T of 0.5 μ L, the Taq enzyme of 0.1 μ L, the template of 0.5 μ L.This PCR Response procedures: 95 DEG C, 30 seconds；60 DEG C, 30 seconds；72 DEG C, 2 minutes；60 circulations

The PCR primer obtained is carried out 1% sepharose electrophoresis, with purpose stripe size (1.5kb) Consistent band carries out glue and reclaims purification, and product after purification is carried out Pac1 and Sbf1 double digestion, It is then attached on the new pADV5 carrier after same double digestion, it is thus achieved that connect product. It is the pADV5 carrier carrying rice EPSP S mutant gene that this step obtains connecting product.

It is of course also possible to use DNA Shuffling method to obtain carry rice EPSP S sudden change The pADV5 carrier of gene, its concrete operations are as follows.

The acquisition of DNA Shuffling method carries the gene mutation body of rice EPSP S gene PADV5 carrier: 1. carry out PCR amplification, amplified production with rice EPSP S gene order Using 1% sepharose electrophoresis, then glue reclaims purification；2. recovery product DNase enzyme is carried out Digestion, runs 1.2% sepharose electrophoresis, cuts 100bp, 200bp or 300bp big after having digested Little fragment is made glue and is reclaimed purification；3. reclaim product 3 μ L with the glue in the 2. step and do template, Carrying out gene shuffling first round PCR, this takes turns and is not added with any primer in PCR, expands 60 Individual circulation；4. take 10 μ L the 3rd step PCR primer and run electrophoresis, see whether successive range Large fragment, as met expection, remaining PCR primer then carries out next round PCR as template； 5. taking the PCR primer 0.5 μ L 3. walked and do template, carry out next round PCR, this takes turns PCR Primer is to design the primer with restriction enzyme site, expands 60 circulations；6. take and 5. walk PCR Product 1% agarose gel electrophoresis, cut the single band more than 500bp do glue reclaim pure Change；7. the glue 6. walked with restriction enzymes double zyme cutting the reclaims product, with 1% after double digestion Agarose gel electrophoresis, cuts purpose fragment, with plastic squeeze water after liquid nitrogen freezing, then with as PADV5 carrier after double digestion connects.Obtain multiple base carrying rice EPSP S gene PADV5 carrier because of mutant.

(2) Transformed E DCE (knocks out the large intestine after EPSPS gene and C-P Lyase gene Bacillus DH5 α)

Prepare EDCE competent cell according to a conventional method.Above-mentioned connection product (is carried water The pADV5 carrier of rice EPSPS mutant gene) join 50 μ L EDCE competent cells In, fully mixing is placed in 30min on ice；42 DEG C of heat shock 90s, add after ice bath 2min LB fluid medium 500 μ L；37 DEG C of low speed (150r/min) shaken cultivation 90min.

Carry the pADV5 vector of rice EPSP S mutant gene in EDCE, I.e. obtain mutant bacteria, namely rice EPSP S gene mutation body storehouse.This rice EPSP S base Because of mutant library, to contain the quantity of rice EPSP S mutant gene the most.Wherein, each Individual mutant bacteria is equivalent to a rice EPSP S gene mutation body plant.Therefore, if screening The rice EPSP S mutant gene of same order, relative to existing screening technique, this Bright screening technique requires no the cultivation cycle of Oryza sativa L. and shared land area, used by it Time and efficiency and operating process are the most quick, simple, and especially floor space is the least, In culture medium, only just can complete screening operation.

3rd step, is seeded in screening culture medium carry out resistance screening by said mutation bacterium.

Multiple mutant bacterias obtained above are inoculated in respectively multiple containing different glyphosate concentration Screening culture medium on (glyphosate concentration that screening culture medium contains each other difference, Containing concentration of glyphosate, it is that 10mM, 20mM, 50mM etc. have Concentraton gradient respectively poor Other glyphosate, the concentration of certain glyphosate can set according to practical situation), in 37 DEG C, Incubated overnight.Wherein, screening culture medium is culture medium based on M9, then adds the denseest Degree antibiotic Spec (Spectinomycin, spextinomyxin), Gen (Gentamycin, Gentamycin), Amp (Ampicillin, ampicillin) and the glyphosate of variable concentrations The culture medium obtained.The composition of M9 culture medium is as follows: Na₂HPO₄13～14g/L, KH₂PO₄ 5.7～6.3g/L, NaCl 0.9～1.1g/L, NH₄Cl 1.8～2.2g/L, glucose 37～43g/L, MgSO₄·7H₂O 48～52g/L, CaCl₂21～23g/L.

4th step, sequence verification.

Select, separate the monoclonal resistance bacterium grown in screening culture medium, detect its glyphosate Resistance, and sequence verification, obtain having the rice EPSP S mutant gene of glyphosate resistance Sequence.Illustrating with one of them rice EPSP S mutant gene, its nucleotide sequence is such as Shown in SEQ ID NO.4, by 1365 base compositions.By this rice EPSP S mutant gene (named OsEM gene), with rice EPSP S gene (the named OsE of wild type Gene) nucleotide sequence (as shown in SEQ ID NO.1) and coding aminoacid sequence Row compare, and result is as shown in Figure 3.This rice EPSP S mutant gene is held to 3 ' from 5 ' 209th bit base of end is sported " G " by " C ", and the 240th bit base is sported " C " by " T ", 346th and the 347th continuous two bases " CT " sport " TC ", the 396th bit base " T " sports " C ", and the 453rd sports " G " for base " A ", the 606th bit base " C " sudden change For " T ", the 831st bit base " A " sports " G "；Wherein, the only the 209th bit base is by " C " Sport " G ", cause its amino acid residue sequence encoded from aminoterminal to c-terminus the 70th Position is sported glycine residue, and the 346th and the 347th continuous two by alanine residue Individual base " CT " sports " TC ", cause its amino acid residue sequence encoded the 116th by Leucine residue sports serine residue, and remaining base mutation does not results in the amino of its coding Acid residue changes.

The glyphosate resistance detection of rice EPSP S mutant gene, has OsEM to convert respectively Escherichia coli (EPSPS and the C-P Lyase of gene (experimental group) and OsE gene (matched group) Deficient strain) inoculate with containing 0mM, 1mM, 5mM, 10mM, 20mM, 50 In the culture medium of mM, 10mM strength glyphosate, observe colibacillary upgrowth situation and (use Growth saturation index represents, saturation index=0, not growth；Saturation index=1, on a small quantity Growth；Saturation index=2, grow to semi-saturation；Saturation index=3, vigorous growth, but also There is growth leeway；Saturation index=4, fast-growth, antibacterial has reached the highest (full the most With) concentration grows in other words and reached capacity).Result is as shown in table 2.

Table 2. converts the escherichia coli of OsEM gene and OsE gene containing in variable concentrations glyphosate culture medium Growth saturation index

As shown in Table 2, in the culture medium containing 0mM glyphosate experimental group (containing OsEM base Cause) and matched group (containing OsE gene) all energy normal growth (saturation index is 4)；? In culture medium containing 1mM, 5mM, 10mM, 20mM, 50mM glyphosate, comparison Group can not grow (saturation index is 0), and experimental group energy normal growth (saturation index is 4)； In the culture medium containing 500mM glyphosate, experimental group and matched group all can not (be satisfied by normal growth It is 0 with index).It is indicated above that the present embodiment screening obtains rice EPSP S mutant gene (its Nucleotide sequence is as shown in SEQ ID NO.4) can give EPSPS and C-P Lyase lack The escherichia coli glyphosate resistance of swaged, makes escherichia coli in the training of up to 50mM glyphosate Support in base and grow.

The Selection for Resistance Gene Glyphosate method using the present embodiment of the present invention to provide is screened to be obtained The such as rice EPSP S mutant gene of the mutant gene with glyphosate resistance, its nucleotide Sequence is as shown in SEQ ID NO 4, and it has the resistance of anti-50mM glyphosate.

The Selection for Resistance Gene Glyphosate method directly using the present embodiment of the present invention to provide is screened The mutant gene with glyphosate resistance obtained such as rice EPSP S mutant gene (its core Nucleotide sequence is as shown in SEQ ID NO 4) rice transformation or Semen sojae atricolor or other plant, so that The plant converted is provided with glyphosate resistance.Certainly, conventional the turning in genetic engineering field can be used Change method such as agrobacterium-mediated transformation, particle bombardment, protoplast mediated method, electric shocking method or whole The rice transformations such as the method for transformation of body level or Semen sojae atricolor or other plant, so that the plant tool converted There is glyphosate resistance.

Embodiment 2

The present embodiment is (Glycine max) with purpose plant as Semen sojae atricolor, and exogenous gene is Semen sojae atricolor EPSPS gene (its nucleotide sequence is as shown in SEQ ID NO.5), to use homology FRT Method directly knocks out method and knocks out the EPSPS gene in the bacillus coli DH 5 alpha of wild type and C-P As a example by EPSPS and the C-P Lyase deficient strain that Lyase gene obtains is Host Strains, come The screening technique of the present invention is illustrated, the present embodiment the primer title and nucleotides sequence thereof Row are shown in Table 3.

The first step, knocks out the C-P Lyase gene of bacillus coli DH 5 alpha.

The method utilizing FRT knocks out the EPSPS gene in e.colistraindh5α and C-P Lyase gene, knocks out in two steps, first knocks out C-P Lyase gene, after knock out EPSPS base Cause.

1. the preparation of the bacillus coli DH 5 alpha competent cell containing pKD46 plasmid

Take the pKD46 plasmid (its structure is as shown in Figure 2) of 0.5 μ L, convert escherichia coli DH5 α competent cell, filters out the positive on LB culture medium flat plate (containing Amp100) Bacterium colony；

Choose positive monoclonal bacterium colony, be inoculated in a small amount of M9-sucrose fluid medium (containing sugarcane Sugar), in rotating speed 180rpm, at 30 DEG C, overnight incubation；

After cultivation terminates, it is inoculated in a large amount of M9-sucrose fluid medium in the ratio of 1:10 In (containing sucrose+100 μ g/mL Amp+10mM L-arabinose), cultivate to bacterium in 30 DEG C The OD600 of liquid to about 0.7；

By above-mentioned bacterium solution in cooled on ice 20min, in 4 DEG C, centrifugal under 4000rpm collect bacterium Body；Resuspended with 10% (v/v) glycerol of 40mL pre-cooling, abandon supernatant after repeated washing 3 times, Glycerol with the 10% of 400 μ L pre-coolings is resuspended and is distributed into 100 μ L/ pipes, obtains having Amp The DH5 α of resistance.

2. knock out the C-P Lyase gene of bacillus coli DH 5 alpha

With forward primer C-P Lyase_P15, reverse primer C-P Lyase_P13 (being shown in Table 3), Carry out PCR amplification with bacillus coli DH 5 alpha genome for template, obtain P1 fragment, P1 The nucleotide sequence of fragment is as shown in SEQ ID NO.6；

With forward primer C-P Lyase_P25, reverse primer C-P Lyase_P23, escherichia coli DH5 α genome is that template carries out PCR amplification, obtains P2 fragment, the nucleotide of P2 fragment Sequence is as shown in SEQ ID NO.7；

Purify P1 and P2 by the sepharose electrophoresis of 1%, obtain the PCR primer of purification, by than The plasmid that example adds containing Gen resistance fragments makes mixing pit, as template, uses forward Primer C-P Lyase_P15 and reverse primer C-P Lyase_P23, for carrying out PCR amplification, obtains To PRC fragment, a length of 1586bp, its nucleotide sequence is as shown in SEQ ID NO.21.

By 50 μ L bacillus coli DH 5 alpha competent cells (there is the DH5 α of Amp resistance) Softly mix with 30 μ L PRC fragment after purification, be placed in the electric shock cup of 0.1cm pre-cooling, Shock by electricity at 1.8kV with Bio-Rad electroporation；

It is rapidly added the 1mL M9-sucrose fluid medium containing 10mM arabinose, 30 DEG C cultivate coat after 1h LB solid medium (Amp containing 100 μ g/mL and The Gen of 30 μ g/mL) on filter out the recombinant bacterial strain of the most anti-Amp and Gen, at 30 DEG C Incubated overnight；

After cultivation terminates, with forward primer C-P Lyase_5UTR and reverse primer C-P The Lyase_Gen3 screening positive colony containing Gen gene, the existence of checking Gen gene；

Positive colony is inoculated in LB+Amp fluid medium, 30 DEG C of incubated overnight (12hr), Then it is forwarded to fresh LB fluid medium, continues 30 DEG C and cultivate 12hr；

Culture fluid is diluted to debita spissitudo, is coated with LB flat board, uses forward primer C-P Lyase_5UTR and reverse primer Lyase_3DSR primer screening are without the clone of Gen gene；

Choose monoclonal order-checking, preserve strain, named DH46 △ C-P Lyase.DH46△C-P Lyase is the bacillus coli DH 5 knocking out C-P Lyase gene.

Table 3. the present embodiment the primer title and nucleotide sequence thereof

3. knock out DH46 △ C-P Lyase (knocks out the escherichia coli of C-P Lyase gene DH5 α) EPSPS gene

(1) preparation of DH46 △ C-P Lyase competent cell

Go bail for the DH46 △ C-P Lyase deposited, and at the flat lining out of LB+Amp, 30 DEG C overnight Cultivate；Choose positive monoclonal bacterium colony, be inoculated in M9-sucrose fluid medium in a small amount, in Rotating speed 180rpm, 30 DEG C, overnight incubation；

Cultivate and be inoculated in a large amount of M9 fluid medium (containing sucrose in the ratio of 1:10 after terminating + 100 μ g/mL Amp+10mM L-arabinose) in, cultivate to bacterium solution in 30 DEG C OD600 is 0.7；

By above-mentioned bacterium solution (containing DH46 △ C-P Lyase) cooled on ice 20min, in 4 DEG C, Centrifugal collection thalline under 4000rpm；It is resuspended with 10% (v/v) glycerol of 40mL pre-cooling, Abandoning supernatant after repeated washing 3 times, the glycerol with the 10% of 400 μ L pre-coolings is resuspended and is distributed into 100 μ L/ pipes.

(2) homology PCR fragment amplification

With forward primer EcEPSPS_P35, reverse primer EcEPSPS_P33 with DH46 △ C-P Lyase strain gene group DNA is template amplification, obtains product P3 fragment, The nucleotide sequence of P3 fragment is as shown in SEQ ID NO.8；

With forward primer EcEPSPS_P45, reverse primer EcEPSPS_P43, with DH46 △ C-P Lyase genomic DNA is template amplification, obtains product P4 fragment, P4 The nucleotide sequence of fragment is as shown in SEQ ID NO.9；

Purify P3 fragment and P4 fragment by the sepharose electrophoresis of 1%, be proportionally added into containing The plasmid of Gen resistance fragments makes mixing pit, as template, with EcEPSPS_P35 and EcEPSPS_P43 is that primer expands, and obtains product PRE fragment, a length of 1607bp, Its nucleotide sequence is as shown in SEQ ID NO.22.

(3) thermal shock converts

By 50 μ L DH46 △ C-P Lyase competent cell and 35 μ L PRE sheets after purification The soft mixing of section, is placed in the electric shock cup of 0.1cm pre-cooling, with Bio-Rad electroporation 1.8 KV shocks by electricity；

It is rapidly added the 1mL M9-sucrose fluid medium containing 10mM arabinose, Screening recombinant bacterial strain on LB solid medium is coated, at 30 DEG C after cultivating 1hr at 37 DEG C Incubated overnight；Next day, with EcEPSPS_P35 and EcEPSPS_P43 primer screening containing Gen The existence of the positive colony checking Gen gene of gene.

Positive colony is inoculated in LB fluid medium, 37 DEG C of incubated overnight (12hr), so After be forwarded to fresh LB fluid medium, continue 37 DEG C cultivate 12hr；

Culture fluid is diluted to debita spissitudo, is coated with LB flat board, uses forward primer EcES25 The clone without GM gene is screened with reverse primer forward primer EcES23；

Choose monoclonal to check order, preservation strain, named DH5 α △ PhnFGH △ EPSPS, DH5 α △ PhnFGH △ EPSPS is to knock out C-P Lyase gene and the large intestine of EPSPS gene Bacillus DH5 α, namely EPSPS and C-P Lyase deficient strain.

It should be noted that DH5 α △ PhnFGH △ EPSPS is lacking without antibiotic resistance gene Swaged bacterial strain.Most of PhnF of bacillus coli DH 5 alpha, whole PhnG and part PhnH The gene relevant with the phosphonate species that degradation of glyphosate is representative is knocked.FRT DNA The nucleic acid sheet of the downstream sequence that the upstream sequence of 5 ends connections of fragment and its 3 end connect The nucleic acid sequence fragments of section is as shown in SEQ ID 11.Wherein, the 1st～the 318th is big Enterobacteria PhnF gene 5 end and upstream sequence thereof, the 319th～the nucleoside of the 347th Acid sequence is FRT fragment, and the 348th～the 1021st is escherichia coli PhnH gene 3 End and downstream sequence thereof.Additionally, the EPSPS gene in DH5 α △ PhnFGH △ EPSPS Major part replaced by FRT fragment, as shown in SEQ ID12, wherein, the 1st～the 357th Position is escherichia coli EPSPS gene 5 end sequences, and the 358th～the 386th is FRT Fragment, the 387th～the 818th is escherichia coli EPSPS gene 3 end sequences.

Second step, to obtain EPSPS and C-P Lyase in this embodiment first step step Soybean EPSPS gene from Semen sojae atricolor, as Host Strains, is imported this host by deficient strain In bacterium, obtain mutant bacteria i.e. soybean EPSPS gene mutant library.

Use conventional method, soybean EPSPS gene is cloned into pADV5 carrier, then will PADV5 vector DH5 α △ PhnFGH △ EPSPS Host Strains.

DH5 α △ PhnFGH △ EPSPS after conversion is seeded to MA fluid medium (M9 Basal medium+100 μ g/mL Amp) in, 37 DEG C, cultivated under the conditions of 300r/min Night；

The bacterium solution of muddiness will be grown to, use radioinduction to process irradiation 2-5 under such as ultraviolet Min, so that soybean EPSPS gene sudden change obtains corresponding soy bean EPSPS mutant gene, I.e. obtain mutant bacteria i.e. soybean EPSPS gene mutant library.Certainly, this step may be used without Chemomorphosis processes and i.e. adds chemical mutagen such as EMS or DES in MA culture medium Deng, so that soybean EPSPS gene is undergone mutation.

3rd step, screening and culturing.

Take the 5 above-mentioned bacterium solution containing mutant bacteria of μ L and join in screening culture medium, continue 300r/min, overnight incubation at 37 DEG C.

4th step, sequence verification.

Select, separate the monoclonal resistance bacterium grown in screening culture medium, detect its glyphosate Resistance, and sequence verification, obtain having the soy bean EPSPS mutant gene of glyphosate resistance Sequence.

Illustrating with one of them soy bean EPSPS mutant gene, its nucleotide sequence is such as Shown in SEQ ID NO.10, by 1368 base compositions.Suddenly change base by this soy bean EPSPS Because of (named GmEM gene), with the soybean EPSPS gene of wild type (named GmE Gene) nucleotide sequence (as shown in SEQ ID NO.5) and coding aminoacid sequence Row compare, and result is as shown in Figure 4.This soy bean EPSPS mutant gene is held to 3 ' from 5 ' Insert between base " G " and the 45th and the 46th between the 6th to the 8th of end Lack base " A ", cause the base of the 7th to the 44th to there occurs frameshift mutation, phase The amino acid residue sequence coded by this section answered is from the 3rd, aminoacid to c-terminus to 15 also there occurs sudden change (as shown in Figure 4)；Additionally, the 629th bit base is suddenlyd change by " A " For " T ", the 210th amino acids residue of amino acid residue sequence is caused to be dashed forward by glutaminic acid residue Become valine residue；1110th bit base is sported " G " by " A ", the 1125th bit base Being sported " C " by " T ", the base mutation in these two sites does not all result in the amino of its corresponding encoded Acid residue is undergone mutation.

The glyphosate resistance detection of soy bean EPSPS mutant gene, has GmEM to convert respectively Escherichia coli (EPSPS and the C-P Lyase of gene (experimental group) and GmE gene (matched group) Deficient strain) inoculate with containing 0mM, 1mM, 5mM, 10mM, 20mM, 50 In the culture medium of mM, 10mM strength glyphosate, observe colibacillary upgrowth situation.Knot Fruit is as shown in table 4.

Table 4. converts the escherichia coli of GmEM gene and GmE gene containing variable concentrations glyphosate culture medium In growth saturation index

As shown in Table 4, in the culture medium containing 0mM glyphosate, experimental group is (containing GmEM Gene) and matched group (containing GmE gene) all energy normal growth (saturation index is 4)；But Containing 1mM, 5mM, 10mM, 20mM, glyphosate concentration culture medium on, comparison Group can not normal growth, and experimental group energy normal growth (saturation index is 4)；Containing 50mM In the culture medium of glyphosate, matched group can not normal growth, and experimental group can grow (full vigorously It is 3 with index)；In the culture medium containing 100mM glyphosate, experimental group and matched group are the most not Can normal growth (saturation index is all 0).It is indicated above that the present embodiment screening obtains Semen sojae atricolor EPSPS mutant gene (its nucleotide sequence is as shown in SEQ ID NO.10) can give The escherichia coli glyphosate resistance of EPSPS and C-P Lyase deficiency, makes escherichia coli at height Reach in the culture medium of 50mM glyphosate and grow.

Use the screening of the mutant gene with glyphosate resistance that the present embodiment of the present invention provides Method is screened the soy bean EPSPS mutant gene with glyphosate resistance obtained, its nucleoside Acid sequence is as shown in SEQ ID NO 10, and it has the resistance of anti-50mM glyphosate.

The mutant gene with glyphosate resistance of the present embodiment of the present invention offer is directly provided Screening technique screened obtain there is glyphosate resistance soy bean EPSPS mutant gene (its core Nucleotide sequence is as shown in SEQ ID NO 10), soybean transformation or Oryza sativa L. or other plant, with The plant making conversion is provided with glyphosate resistance.

Embodiment 3

Present embodiments providing a kind of deficient strain, specifically, this deficient strain is EPSPS and C-P Lyase deficient strain.This EPSPS and C-P Lyase deficient strain Appointing in bacillus coli DH 5 alpha, TOP10 and BL21 is knocked out by using gene Knockout Anticipate a kind of colibacillary EPSPS gene and C-P Lyase gene obtains.Specifically, this reality Execute gene knockout method used by the gene knockout method such as embodiment 1 used by example or embodiment 2 Identical.

EPSPS and the C-P Lyase deficient strain that the present embodiment provides can be in test from mesh Plant EPSPS gene function in apply.Specifically, with the present embodiment EPSPS and C-P Lyase deficient strain is as Host Strains, by the EPSPS of purpose plant Host Strains, in this Host Strains, is then placed in the basis without aminoacid or albumen by channel genes Culture medium i.e. restricted culture medium are cultivated, if it is observed that just have in restricted culture medium Normal bacterium colony produces and then shows that this has can express from the EPSPS gene of purpose plant The function of EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), and EPSPS has Normal biological activity.

The present embodiment provide EPSPS and C-P Lyase deficient strain also can test from The resistance of the EPSPS gene pairs glyphosate of purpose plant is applied.Specifically, with this EPSPS and the C-P Lyase deficient strain of embodiment is as Host Strains, by purpose plant Host Strains, in this Host Strains, is then placed in containing different glyphosates dense by EPSPS channel genes Cultivate in the M9 culture medium of degree, such as containing 10mM, 20mM, 50mM grass Training growth is carried out, to test this EPSPS from purpose plant in the M9 culture medium of sweet phosphine The glyphosate resistance of gene.

EPSPS and the C-P Lyase deficient strain that the present embodiment provides can have grass in screening The EPSPS mutant gene from purpose plant of sweet phosphine resistance is applied.Concrete makes The screening that can be found in embodiment 1 or embodiment 2 offer by method has glyphosate resistance The method of EPSPS mutant gene.

To sum up, the embodiment of the present invention provide screening technique by build EPSPS and C-P Lyase deficient strain, then with this EPSPS and C-P Lyase deficient strain as Host Strains, In from the external source EPSPS channel genes of purpose plant to this deficient strain, contained There is a mutant bacteria i.e. external source EPSPS gene mutation body storehouse of external source EPSPS mutant gene, then from This external source EPSPS gene mutation body storehouse filters out the EPSPS sudden change with glyphosate resistance Gene.Owing to colibacillary reproduction speed is fast, volume is little, therefore, and the screening side of the present invention The problem that method overcomes cycle length present in existing screening technique, floor space is big so that this The screening technique of invention goes out to have the EPSPS mutant gene of glyphosate resistance at directed screening The features such as having the cycle in operation short, floor space is the least, and cycle short operation is simple.And this The screening technique of invention is using the escherichia coli of EPSPS and C-P Lyase deficiency as host Bacterium, has been effectively discharged out EPSPS gene and the sudden change of C-P Lyase gene of Host Strains itself And producing the situation of glyphosate resistance so that the result filtered out more can have science, more reliable. Screen the gene mutation body of EPSPS gene from plant, it is possible to greatly improve screening Speed and time, the most only need 1～2 time-of-weeks can complete screening operation, obtain that there is grass sweet The mutant gene of phosphine resistance, reduces the cost of screening operation.Additionally, the screening that the present invention provides The mutant gene obtained by method with glyphosate resistance can be used further to convert corresponding plant Kind, overcomes current major part can only transfer in the resistant gene of microorganism to crops Bottleneck, contribute to eliminate the public to transgenic plant understanding on prejudice, and then promote turn The development of gene technology and popularization.Additionally, EPSPS and the C-P Lyase that the present invention provides Deficient strain can be applied, also in the function of the EPSPS gene of test plants Can apply in the resistance of the EPSPS gene pairs glyphosate of test plants, its application Convenient, result is more scientific more reliable.

The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, For a person skilled in the art, the present invention can have various modifications and variations.All at this Within the spirit of invention and principle, any modification, equivalent substitution and improvement etc. made, all should Within being included in protection scope of the present invention.

Claims

1. a Selection for Resistance Gene Glyphosate method, it is characterised in that comprising:

Use gene Knockout to knock out the interference gene of source bacterial strain, obtain deficient strain, institute State source bacterial strain from the one in bacillus coli DH 5 alpha, TOP10 and BL21, described dry Disturbing gene and include EPSPS gene and C-P Lyase gene, described deficient strain is EPSPS With C-P Lyase deficient strain；First by deficient strain described in external source EPSPS channel genes In, then after mutagenic treatment, obtain the first mutant bacteria containing external source EPSPS mutant gene, Described external source EPSPS gene is from purpose plant；

Or first by the described mutated process of external source EPSPS gene, obtain external source EPSPS and dash forward Become gene, more described external source EPSPS mutant gene is imported described deficient strain, obtain Second mutant bacteria；

Described first mutant bacteria or described second mutant bacteria are placed in the screening training containing glyphosate Support the enterprising row filter of base to cultivate, obtain the monoclonal resistance bacterium with glyphosate resistance；

Described monoclonal resistance bacterium is carried out sequence verification, obtains that there is glyphosate resistance EPSPS mutant gene.

Selection for Resistance Gene Glyphosate method the most according to claim 1, it is characterised in that Described mutagenic treatment is chemomorphosis process or radioinduction process.

Selection for Resistance Gene Glyphosate method the most according to claim 1, it is characterised in that Described sudden change process be with described external source EPSPS gene as template, use error-prone PCR method or DNA Shuffling method carries out PCR and obtains described external source EPSPS mutant gene.

Selection for Resistance Gene Glyphosate method the most according to claim 1, it is characterised in that Described purpose plant be Oryza sativa L., Semen sojae atricolor, Semen Tritici aestivi, Semen Maydis, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Nicotiana tabacum L., Cotton Gossypii, Rhizoma Dioscoreae esculentae, willow, Rhizoma Solani tuber osi, Chinese cabbage, Caulis et Folium Brassicae capitatae or Capsicum annuum L..

5. one kind according to the Selection for Resistance Gene Glyphosate method described in any one of Claims 1 to 4 Screened the EPSPS mutant gene with glyphosate resistance obtained.

EPSPS mutant gene the most according to claim 5 is converting plant so that plant There is the application of described glyphosate resistance.

7. a deficient strain, it is characterised in that described deficient strain by source bacterial strain through adopting Obtaining after knocking out interference gene with gene Knockout, described interference gene includes EPSPS base Cause and C-P Lyase gene, described source bacterial strain selected from bacillus coli DH 5 alpha, TOP10 and One in BL21, described deficient strain is EPSPS and C-P Lyase deficient strain.

Deficient strain the most according to claim 7 is being tested from purpose plant Application in the function of EPSPS gene.

Deficient strain the most according to claim 7 is being tested from purpose plant Application in the resistance of EPSPS gene pairs glyphosate.

Deficient strain the most according to claim 7 has glyphosate resistance in screening Application in the EPSPS mutant gene of purpose plant.