CN112094857B - Rape EPSPS gene containing three mutation sites and cloning method and application thereof - Google Patents

Abstract

The invention discloses a rape EPSPS gene containing three mutation sites and a cloning method and application thereof, belonging to the technical field of biology. The invention creates the rape EPSPS gene (BnmEPSPS) with stronger herbicide tolerance by changing the 518 th basic group of the rape EPSPS gene from C to T, changing the 521 th basic group from C to T and changing the 529 th basic group from C to T, and successfully expresses the rape EPSPS gene in tobacco by adding a chloroplast signal peptide sequence at the 5' end of the created gene and utilizing leaf disc transfection. The rape EPSPS gene created by the invention has the characteristic of high glyphosate herbicide resistance, has a high utilization value, and is expected to be widely applied to cultivation of transgenic glyphosate herbicide-resistant crops.

Description

Rape EPSPS gene containing three mutation sites and cloning method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a rape EPSPS gene containing three mutation sites and a cloning method and application thereof.

Background

Chemical herbicides have been widely used in modern agriculture as a highly effective method of weeding. Glyphosate (also known as Glyphosate) is a non-selective broad-spectrum herbicide, belonging to amino methyl phosphonic acid compounds containing carboxyl groups. Glyphosate is currently introduced and registered by more than 100 countries, and is the herbicide with the widest use area and the largest yield all over the world.

As a systemic herbicide, glyphosate can kill both the above-ground green parts and the underground root tissues. The weeding mechanism is clear, the glyphosate and phosphoenolpyruvate (PEP) have similar molecular structures, compete with PEP for binding sites with EPSPS, and block EPSP generation. EPSPS is a 5-enolpyruvylshikimate-3-phosphate synthase. The EPSPS enzyme is responsible for catalyzing the binding of shikimate-3-phosphate (S3P) to enolpyruvate (PEP) to produce 5-enolpyruvylshikimate-3-phosphate (EPSP), which is an important synthetic precursor for aromatic amino acids. Inhibition of EPSPS enzyme activity by the herbicide glyphosate results in deficiency of aromatic amino acids in plants, accumulation of shikimic acid and ultimately cell death.

According to the mechanism of action of glyphosate, there are theoretically 3 fundamental ways to make plants glyphosate resistant: 1) overexpression of a plant endogenous EPSPS synthase; 2) the inhibition effect of the glyphosate is relieved by degrading or modifying the glyphosate; 3) the plant expresses an exogenous type II EPSPS synthase, i.e. a non-sensitive EPSPS synthase. In practice, resistance to glyphosate in plants is generally obtained by a third pathway, the expression of a non-sensitive EPSPS synthase.

At present, resistance genes of glyphosate-resistant crops are mostly from microbial bacteria, such as agrobacterium CP4-EPSPS, I.variabilis-EPSPS and the like, and EPSPS genes derived from plant mutation are rarely reported, and the main reason is the lack of EPSPS genes with high glyphosate resistance. The introduction of the mutation site by using the in vitro artificial synthesis gene technology to create the glyphosate-tolerant EPSPS gene from plants, and the cultivation of transgenic herbicide-resistant crops through genetic engineering is a way worth exploring.

Disclosure of Invention

The invention aims to provide a rape EPSPS gene containing three mutation sites, a cloning method and application thereof, which are used for solving the problems in the prior art and utilizing the gene as a means for culturing glyphosate-resistant plants.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a rape BnmEPSPS gene containing three mutation sites, wherein the three mutation sites are three mutation sites, namely a 518 th base is changed from C to T, a 521 th base is changed from C to T, and a 529 th base is changed from C to T, and the mutated gene sequence is shown as SEQ ID NO. 3.

The invention also provides the protein coded by the rape BnmEPSPS gene containing the three mutation sites, and the amino acid sequence of the protein is shown as SEQ ID NO. 4.

The invention also provides a construction method of the transgenic plant containing the rape BnmEPSPS gene containing the three mutation sites, wherein a chloroplast signal peptide sequence is added at the 5' end of the transgenic plant containing the three mutation sites, and then the chloroplast signal peptide sequence is cloned to a vector and then is transferred into a target plant.

Preferably, the chloroplast signal peptide sequence is shown in SEQ ID NO. 5.

Preferably, the vector is pCAMBIA 1304.

Preferably, the gene is cloned into a vector using recombinant methods.

Preferably, the step of transferring into the target plant comprises leaf disc explant transfection, tissue culture and pot transplantation.

Preferably, the leaf disc transfection method is an agrobacterium transformation method.

The invention also provides the application of the rape BnmEPSPS gene containing the three mutation sites or the protein or any construction method in culturing glyphosate-resistant plants.

Preferably, the plant is a crop plant.

The invention discloses the following technical effects:

the rape BnmEPSPS gene prepared by the invention has stronger herbicide tolerance. Through a glyphosate herbicide spraying test, a batch of transgenic tobacco herbicide-tolerant is discovered; among the strains with better herbicide tolerance are: a2(T1-2), A4(T1-4), A7(T1-7), B1(T1-8), B2(T1-9), B7(T1-14), C1(T1-15), C3(T1-17), C4(T1-18) and C5 (T1-19). And further identifying by using a high-concentration glyphosate solution, wherein part of transgenic tobacco can tolerate glyphosate with higher concentration, such as T1-2, T1-4, T1-7, T1-8 and the like with the tolerance concentration of 12000 mg/L. And the concentration of T1-2 which can be tolerated is up to 36000mg/L, which is equivalent to 12 times of the concentration used in the field. The rape BnmEPSPS gene created by the invention has the characteristics of high glyphosate tolerance herbicide, has high utilization value and is expected to be applied in cultivating glyphosate-resistant crops in large quantity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic diagram of the mutation site of the EPSPS gene of Brassica campestris;

FIG. 2 is a schematic diagram of vector pCAMBIA 1304;

FIG. 3 is a schematic diagram of a p1304BnmEPSPS recombinant vector;

FIG. 4 is the restriction enzyme map of the recombinant vector p1304 BnmEPSPS;

FIG. 5 shows T1 generation seedlings after 15 days of glyphosate treatment, in which C7 is a non-transgenic control and the rest are T1 generation plants of different transgenic tobacco lines;

FIG. 6 shows PCR identification of transgenic tobacco plants, in which lane M shows DNA molecular weight marker DL 2000; lanes 1-20 are the obtained transgenic regenerated plants, respectively; lanes CK-, CK + are non-transgenic control and plasmid DNA control, respectively;

FIG. 7 is a graph showing the effect of 4-fold glyphosate solution (12000mg/L) on a portion of transgenic tobacco lines after 14 days of treatment;

FIG. 8 shows the effect of 4-16 times (12000L-48000mg/L) glyphosate solution on transgenic tobacco T1-2 seedlings and controls after 14 days;

FIG. 9 shows the relative expression level of the exogenous gene (BnmEPSPS gene) of a part of the transgenic tobacco strains.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

First, extraction and analysis of EPSPS gene of cabbage type rape

The EPSPS gene sequence of the cabbage type rape is searched from the gene library, the gene sequence number is LK033547.1, the gene comprises a plurality of intron sequences, the sequence of a coding region is artificially extracted, and 1548bp (SEQ ID NO.1) is calculated. Through rape genome sequence alignment analysis, the gene is located on BnaC04 chromosome. The encoded protein product consists of 515aa, as shown in SEQ ID NO.2, analyzed by biological software.

Secondly, introduction of mutation sites and complete gene sequence synthesis

3 single base mutation sites are introduced in the sequence 1, namely the 518 th base is changed from C to T, the 521 is changed from C to T, and the 529 is changed from C to T, as shown in figure 1. The mutated sequence is shown in SEQ ID NO.3 and is named as BnmEPSPS. The amino acid sequence coded by The sequence 3 is correspondingly changed, namely 173 site is changed from The position to Ile, 174 site is changed from Ala to Val, 177 site is changed from Pro to Ser; the amino acid sequence coded by the mutated gene is shown in SEQ ID NO. 4.

Third, construction of plant expression vector

In order to allow the expression product of the mutant gene to function in the plant chlorophyll, a chloroplast signal peptide sequence was added to the 5' end of the mutant gene. The sequence is selected from the gene sequence with the number as EU477376.1, wherein the sequence of 1622-1786 (SEQ ID NO.5) is the signal peptide gene sequence of the brassica napus ribulose 1, 5-bisphosphate carboxylase/oxygenase small subunit protein, and the coded amino acid sequence is shown in SEQ ID NO. 6. It is intended to be used to direct the mutated EPSPS gene translation product of Brassica napus into chloroplast for function.

Artificially synthesized to form a target gene sequence which consists of SEQ ID NO.5 and SEQ ID NO.3 and is shown as SEQ ID NO. 7. To verify the function of the synthesized in vitro mutant gene, the pseudo-target gene sequence (SEQ ID NO. 7): 1713bp (the leader peptide is 165bp, and the rest is the mutant rape BnmEPSPS coding sequence 1548 bp). The recombinant vector was cloned into the pCAMBIA1304(AF234300) vector at Nco I/BstE II, replacing 2-2574bp of CD1 carried by the original vector. The schematic diagram of the vector pCAMBIA1304 is shown in FIG. 2, and the vector obtained after recombination (p1304BnmEPSPS) is shown in FIG. 3. The vector was digested with restriction enzymes to generate 2 specific bands, one of which was about 2570bp in size, as shown in FIG. 4.

Fourth, function verification of mutant gene

To verify the function of the mutant gene, we performed verification using transgenic tobacco.

1) Tobacco was transfected using leaf disc transformation.

A single colony of Agrobacterium EHA105 carrying the vector p1304BnmEPSPS was picked, inoculated into 5ml of LB liquid medium containing Rif20mg/L and Kan 50mg/L, and cultured overnight at 28 ℃ with shaking. Overnight activated agrobacterium was taken and mixed as 1: 50 to YEB liquid culture medium containing Rif20mg/L and Kan 50mg/L, and continuously culturing to OD₆₀₀The value is approximately 0.6-0.8. Centrifuging at 6000rpm for 5min, collecting thallus, washing thallus once with 1/2MS0 liquid culture medium, and diluting to OD₆₀₀The value is 0.3-0.35.

2) Genetic transformation of tobacco leaf discs

The transgenic recipient tobacco material was Nicotiana benthamiana (Nicotiana benthamiana). Selecting a tobacco sterile seedling with the seedling age of about 30 days, taking tobacco leaves with a scalpel under the sterile condition, taking the leaves with the size of about 0.8cm square as explants, putting the explants into prepared agrobacterium liquid, carrying out bacteria staining for 3-5 minutes, taking out, sucking residual liquid attached to the surfaces of the leaves with filter paper, then putting the leaves on a co-culture medium (1 × MS, 3% of sucrose, 1% of agar powder and pH 5.8) in a dark place, and carrying out co-culture for two days at the temperature of 25 ℃.

3) Regeneration of transgenic tobacco seedlings

Transferring to bud induction culture medium (1 × MS, sucrose 3%, BA2.0 mg/L, IAA0.5 mg/L, carbenicillin 500mg/L, hygromycin 20mg/L, agar powder 1%, pH 5.8) for bud induction culture, and subculturing once every 2-3 weeks, wherein the subculturing medium is the same as the bud induction culture medium. After the adventitious bud grows out and the bud grows to 1-1.5cm, the bud is cut off and is switched to a rooting culture medium (1 × MS, 3% of sucrose, 0.5mg/L of IAA, 500mg/L of carbenicillin, 20mg/L of hygromycin, 1% of agar powder, and pH 5.8) to induce rooting.

4) And (4) potting the transgenic tobacco seedlings, and transplanting the rooted tobacco seedlings into a small pot. The culture medium is common gardening culture medium, and is used after autoclaving, and after transplanting tobacco seedlings, attention is paid to soil moisture conservation, and compound fertilizer is properly added. Until the flower and the seed are harvested.

5) The invention obtains 20 transgenic tobacco seedlings in total, and all the transgenic tobacco seedlings receive seeds.

6) Seeds received by the T0 generation tobacco seedlings are sowed in small pots to obtain T1 generation plant seedlings. When the tobacco seedlings grow to 1-2 true leaves, spraying leaves with glyphosate (750mg/L) with lower concentration, wherein each pot is about 0.9ml, the growth condition after 15 days is shown in figure 5, and the non-transgenic control tends to die. The strains with better herbicide tolerance are as follows: a2(T1-2), A4(T1-4), A7(T1-7), B1(T1-8), B2(T1-9), B7(T1-14), C1(T1-15), C3(T1-17), C4(T1-18) and C5 (T1-19). The strains showing poor herbicide tolerance are A1(T1-1), A5(T1-5), B4(T1-11), B5(T1-12), B6(T1-13), C2(T1-16) and C6 (T1-20).

7) PCR identification of transgenic tobacco plants

Taking leaves of transgenic tobacco T1 generation seedlings, extracting genome DNA by a CTAB method, and taking leaf DNA of non-transgenic tobacco seedlings as negative control. PCR identification was performed with specific primer pairs. The primer sequence is as follows:

F：5-ATGACTCTAGCCGTTGTTGCTC-3(SEQ ID NO.8)；

R：5-AAGACCGGCAACAGGATTCAATCTT-3(SEQ ID NO.9)；

amplification reaction 20. mu.l system: 2 × Tag master mix 10 μ l, DNA template 1 μ l, primer F1 μ l, primer R1 μ l, H2O 7 μ l; reaction procedure: after 35 cycles of 94 ℃ for 4min, 94 ℃ for 40sec,58 ℃ for 40sec,72 ℃ for 40min, and 35 cycles of 72 ℃ for 5min, the amplification product was checked by agarose gel electrophoresis. Most transgenic tobacco seedlings could be amplified to a specific PCR product (419 bp). FIG. 6 shows that the specific PCR amplification products can be detected from 1, 2, 4, 6, 7, 8, 9, 11, 12, 14, 15, 16, 17, 18, 19, 20 seedlings.

8) Results of 4-fold concentration glyphosate treatment on transgenic tobacco strains T1-2, T1-4, T1-7, T1-8 and the like

Transgenic tobacco seedlings of part of the strains are treated by applying a glyphosate solution with the concentration 4 times that of the field, and after 10 days, the non-transgenic tobacco grows and tends to die in contrast. The transgenic tobacco still grows vigorously without adverse effects. As shown in fig. 7.

9) Results of treatment of transgenic tobacco T1-2 and non-transgenic controls with 4-16 times the concentration of glyphosate

When the tobacco seedlings are in the 4-5 leaf stage, glyphosate solutions with different concentration gradients (0, 1500mg/L, 12000mg/L, 24000mg/L, 30000mg/L, 36000mg/L and 48000mg/L) are used, which are equivalent to the using concentration of glyphosate in the field of 0, 1/2 times, 4 times, 8 times, 10 times, 12 times and 16 times, transgenic tobacco T1-2 strains are sprayed on leaf surfaces, and the spraying amount is 1.8 ml. After 14 days, the effect was checked. The non-transgenic control tobacco sprayed with 1/2 concentration only tends to die and shows high sensitivity to glyphosate herbicide, while the transgenic tobacco sprayed with 4-12 times of glyphosate solution (12000-36000mg/L) T1-2 still can grow normally, which indicates that the transgenic tobacco has stronger tolerance to glyphosate. The transgenic tobacco seedling leaves sprayed with 16 times (48000mg/L) of the herbicide at high concentration showed the symptoms of shrinkage, yellowing and damage, and part of the seedlings were growth-arrested and tended to die, as shown in FIG. 8.

10) Analysis of Gene expression

Partial transgenic strains (8 samples in total of transgenic T1-2, T1-4, T1-7, T1-8, T1-9, T1-15, T1-19 and non-transgenic CK controls) with better glyphosate resistance are selected to extract total RNA, and the relative expression quantity of the EPSPS gene of the transgenic rape is analyzed by utilizing fluorescent quantitative PCR.

The RNA extraction adopts a polysaccharide polyphenol plant total RNA extraction kit produced by Shanghai Pudy Biotech limited. Taking about 100mg of leaves, grinding the leaves into powder under the condition of liquid nitrogen, and carrying out the steps according to a method provided by a kit. The total RNA was further treated with DNaseI enzyme from SIGMA-ALDRICH to remove residual DNA molecules. Thereafter, cDNA synthesis was carried out using EasyQuick RT-Master Mix kit available from Kangji corporation as a century. RT-PCR was performed using the 2X T5 FastqPCRMix (SYBR Green I) kit from Onychidaceae.

qPCR reaction System: 2X FastqPCR mix 10. mu.L, primer F1. mu.l, primer R1. mu.l, 50X ROX Reference Dye I, 0.4. mu.l, cDNA template 1. mu.l, and complement dd H2O to 20. mu.l.

Reaction conditions are as follows: 95 ℃, 4min, 95 ℃,5 seconds, 64 ℃ for 3 seconds, 40 cycles.

Wherein the primers are respectively as follows:

Bn-mepspsF2:5'－TGTCCTCCTGTTCGTGTC－3'(SEQ ID NO.10)；

Bn-mepspsR2:5'－TATTTCTGACCGCCCTTG－3'(SEQ ID NO.11)；

tobacco ActinF15 '-GTATGGGTCAGAAAGATGC-3' (SEQ ID NO. 12);

tobacco ActinR15 '-AGGACAGCCTGAATAGCA-3' (SEQ ID NO. 13);

RT-PCR detection results show that the expression of rape BnmEPSPS gene can be detected in the detected part of high-glyphosate-resistant transgenic tobacco leaves, and the relative expression quantity of different transformation single plants has larger difference. A single T1-2 plant detected glyphosate solution that was able to tolerate a concentration of 36000mg/L, which was almost 12 times higher than the normal concentration used. The expression levels of the transgenes T1-7, T1-8, T1-9, T1-15 and T1-19 glyphosate-resistant genes are relatively high, some genes are even dozens of times of T1-2, but the maximum glyphosate-resistant concentration of the genes is still evaluated, and the expression of the transgenes cannot be detected by non-transgenic controls. See fig. 9.

Fifth, experimental results

Preliminary experiment results show that the created rape BnmEPSPS gene has stronger herbicide tolerance. Through a glyphosate herbicide spraying test, a batch of transgenic tobacco herbicide-tolerant is discovered; among the strains with better herbicide tolerance are: a2(T1-2), A4(T1-4), A7(T1-7), B1(T1-8), B2(T1-9), B7(T1-14), C1(T1-15), C3(T1-17), C4(T1-18) and C5 (T1-19). The strains showing poor herbicide tolerance comprise A1(T1-1), A5(T1-5), B4(T1-11), B5(T1-12), B6(T1-13), C2(T1-16) and C6 (T1-20).

Further identification is carried out by using a high-concentration glyphosate solution, and part of transgenic tobacco can tolerate the glyphosate solution with higher concentration, such as the transgenic tobacco strain T1-2 can tolerate 36000 mg/L. The rape BnmEPSPS gene created by the invention has the characteristic of high glyphosate herbicide resistance, has high utilization value and is expected to be applied to crops such as transgenic rape and the like.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> agricultural science and academy of Jiangsu province

<120> rape EPSPS gene containing three mutation sites, and cloning method and application thereof

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gaaggtgatg cttctagtgc tagctacttc ttggctggtg ctgccattac tggtgaaact 1020

gtcactgttg aaggttgtgg aacaactagc ctgcagggag atgtgaaatt cgcagaggtt 1080

cttgagaaaa tgggatgtaa agtgtcatgg acagagaaca gtgtgactgt gactggacca 1140

tctagagatg cttttggaat gagacacttg cgtgctgttg atgtgaacat gaacaaaatg 1200

cctgatgtag ccatgactct agccgttgtt gctctctttg ccgatggtcc aacaaccatc 1260

agagatgtgg ctagctggag agttaaggag acagagagga tgattgccat ttgcacagag 1320

cttagaaagc ttggagctac agtggaagaa ggttcagatt attgtgtgat aactccacca 1380

gcaaaggtga aaccggcgga gattgatacg tatgatgatc atagaatggc gatggcgttc 1440

tcgcttgcag cttgtgctga tgttccagtc accatcaagg atcctggctg caccaggaag 1500

actttccctg actacttcca agtccttgaa agtatcacaa agcattaa 1548

<210> 4

<211> 515

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Gln Ser Ser Arg Ile Cys His Gly Val Gln Asn Pro Cys Val

1 5 10 15

Ile Ile Ser Asn Leu Ser Lys Ser Asn Gln Asn Lys Ser Pro Ser Ser

20 25 30

Val Ser Leu Lys Thr Gln Pro Arg Ala Ser Ser Trp Gly Leu Lys Lys

35 40 45

Ser Gly Thr Met Leu Asn Gly Ser Val Ile Arg Pro Val Lys Val Thr

50 55 60

Ala Ser Val Ser Thr Ala Glu Lys Ala Ser Glu Ile Val Leu Gln Pro

65 70 75 80

Ile Arg Glu Ile Ser Gly Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu

85 90 95

Ser Asn Arg Ile Leu Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Ala

100 105 110

Val Asp Asn Leu Leu Asn Ser Asp Asp Ile Asn Tyr Met Leu Asp Ala

115 120 125

Leu Lys Lys Leu Gly Leu Asn Val Glu Arg Asp Ser Val Asn Asn Arg

130 135 140

Ala Val Val Glu Gly Cys Gly Gly Ile Phe Pro Ala Ser Leu Asp Ser

145 150 155 160

Lys Ser Asp Ile Glu Leu Tyr Leu Gly Asn Ala Gly Ile Val Met Arg

165 170 175

Ser Leu Thr Ala Ala Val Thr Ala Ala Gly Gly Asn Ala Ser Tyr Val

180 185 190

Leu Asp Gly Val Pro Arg Met Arg Glu Arg Pro Ile Gly Asp Leu Val

195 200 205

Val Gly Leu Lys Gln Leu Gly Ala Asp Val Glu Cys Thr Leu Gly Thr

210 215 220

Asn Cys Pro Pro Val Arg Val Asn Ala Asn Gly Gly Leu Pro Gly Gly

225 230 235 240

Lys Val Lys Leu Ser Gly Ser Ile Ser Ser Gln Tyr Leu Thr Ala Leu

245 250 255

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

260 265 270

Asp Lys Leu Ile Ser Val Pro Tyr Val Glu Met Thr Leu Lys Leu Met

275 280 285

Glu Arg Phe Gly Val Ser Ala Glu His Ser Asp Ser Trp Asp Arg Phe

290 295 300

Phe Val Lys Gly Gly Gln Lys Tyr Lys Ser Pro Gly Asn Ala Tyr Val

305 310 315 320

Glu Gly Asp Ala Ser Ser Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile

325 330 335

Thr Gly Glu Thr Val Thr Val Glu Gly Cys Gly Thr Thr Ser Leu Gln

340 345 350

Gly Asp Val Lys Phe Ala Glu Val Leu Glu Lys Met Gly Cys Lys Val

355 360 365

Ser Trp Thr Glu Asn Ser Val Thr Val Thr Gly Pro Ser Arg Asp Ala

370 375 380

Phe Gly Met Arg His Leu Arg Ala Val Asp Val Asn Met Asn Lys Met

385 390 395 400

Pro Asp Val Ala Met Thr Leu Ala Val Val Ala Leu Phe Ala Asp Gly

405 410 415

Pro Thr Thr Ile Arg Asp Val Ala Ser Trp Arg Val Lys Glu Thr Glu

420 425 430

Arg Met Ile Ala Ile Cys Thr Glu Leu Arg Lys Leu Gly Ala Thr Val

435 440 445

Glu Glu Gly Ser Asp Tyr Cys Val Ile Thr Pro Pro Ala Lys Val Lys

450 455 460

Pro Ala Glu Ile Asp Thr Tyr Asp Asp His Arg Met Ala Met Ala Phe

465 470 475 480

Ser Leu Ala Ala Cys Ala Asp Val Pro Val Thr Ile Lys Asp Pro Gly

485 490 495

Cys Thr Arg Lys Thr Phe Pro Asp Tyr Phe Gln Val Leu Glu Ser Ile

500 505 510

Thr Lys His

515

<210> 5

<211> 165

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggcttact ctatgctctc ctctgccacc gtggttagct caccggctca agcggccatg 60

gttgctccat tcacaggctt gaagtcatcc gctgcattcc cagtcactcg caagaccgac 120

actgacatta cttccatggc aagcaatgga ggaagagtta actcg 165

<210> 6

<211> 55

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ala Tyr Ser Met Leu Ser Ser Ala Thr Val Val Ser Ser Pro Ala

1 5 10 15

Gln Ala Ala Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Val Thr Arg Lys Thr Asp Thr Asp Ile Thr Ser Met Ala Ser

35 40 45

Asn Gly Gly Arg Val Asn Ser

50 55

<210> 7

<211> 1713

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggcttact ctatgctctc ctctgccacc gtggttagct caccggctca agcggccatg 60

gttgctccat tcacaggctt gaagtcatcc gctgcattcc cagtcactcg caagaccgac 120

actgacatta cttccatggc aagcaatgga ggaagagtta actcgatggc gcaatctagc 180

agaatctgcc atggcgtgca gaacccatgt gttatcatat ccaatctctc caaatccaac 240

caaaacaaat caccgtcctc tgtctccttg aagactcagc ctcgagcttc ttcgtgggga 300

ttgaagaaga gtggaacgat gctaaacggt tctgttattc gaccggttaa ggtaacagct 360

tctgtttcca ctgccgagaa agcttcagag attgtgcttc aaccaatcag agaaatctcg 420

ggtctcatta agctacccgg atccaaatct ctctccaatc ggatcctcct tcttgccgct 480

ctatctgagg gaactactgc agtggacaac ttgttgaaca gtgatgacat caactacatg 540

cttgatgcgt tgaagaagct ggggcttaac gtggaacgtg acagtgtaaa caaccgtgcg 600

gttgttgaag gatgcggtgg aatattccca gcttccttag attccaagag tgatattgag 660

ttgtaccttg ggaatgcagg aatagtcatg cgttcactca ccgctgcagt tacagctgca 720

ggtggcaacg cgagttatgt gcttgatggg gtgcctagaa tgagggaaag acctatagga 780

gatttggttg ttggtctcaa gcagcttggt gctgatgttg agtgtactct tggcactaac 840

tgtcctcctg ttcgtgtcaa tgctaatggt ggccttcccg gtggaaaggt gaagctttct 900

ggatcgatca gtagtcagta cttgactgcc ctcctcatgg tagctccttt agctcttgga 960

gacgtggaga ttgagatcat tgataaactg atatctgttc catatgttga aatgacattg 1020

aagttgatgg agcgttttgg tgttagtgcc gagcatagtg atagctggga tcgtttcttt 1080

gtcaagggcg gtcagaaata caagtcgcct ggtaatgctt atgtagaagg tgatgcttct 1140

agtgctagct acttcttggc tggtgctgcc attactggtg aaactgtcac tgttgaaggt 1200

tgtggaacaa ctagcctgca gggagatgtg aaattcgcag aggttcttga gaaaatggga 1260

tgtaaagtgt catggacaga gaacagtgtg actgtgactg gaccatctag agatgctttt 1320

ggaatgagac acttgcgtgc tgttgatgtg aacatgaaca aaatgcctga tgtagccatg 1380

actctagccg ttgttgctct ctttgccgat ggtccaacaa ccatcagaga tgtggctagc 1440

tggagagtta aggagacaga gaggatgatt gccatttgca cagagcttag aaagcttgga 1500

gctacagtgg aagaaggttc agattattgt gtgataactc caccagcaaa ggtgaaaccg 1560

gcggagattg atacgtatga tgatcataga atggcgatgg cgttctcgct tgcagcttgt 1620

gctgatgttc cagtcaccat caaggatcct ggctgcacca ggaagacttt ccctgactac 1680

ttccaagtcc ttgaaagtat cacaaagcat taa 1713

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgactctag ccgttgttgc tc 22

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aagaccggca acaggattca atctt 25

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgtcctcctg ttcgtgtc 18

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tatttctgac cgcccttg 18

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtatgggtca gaaagatgc 19

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aggacagcct gaatagca 18

Claims (8)

1. A rape BnmEPSPS gene containing three mutation sites is characterized in that the 518 th base of the rape EPSPS gene with the nucleotide sequence of SEQ ID NO.1 is changed from C to T, the 521 th base is changed from C to T, and the 529 th base is changed from C to T.

2. The rape BnmEPSPS gene containing three mutation sites as claimed in claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 3.

3. A protein encoded by the rape BnmEPSPS gene at the triple mutation site as set forth in claim 1 or 2, wherein the amino acid sequence of said protein is as set forth in SEQ ID No. 4.

4. A recombinant vector comprising the gene according to any one of claims 1 to 2.

5. The recombinant vector according to claim 4, further comprising a signal peptide gene sequence.

6. The recombinant vector according to claim 5, wherein the gene sequence of the signal peptide is shown in SEQ ID No. 5.

7. Use of a rape BnmEPSPS gene containing a triple mutation site as defined in any one of claims 1 to 2 or a protein as defined in claim 3 or a recombinant vector as defined in any one of claims 4 to 6 for breeding glyphosate herbicide tolerant plants.

8. The use of claim 7, wherein the plant is a crop plant.

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