CN106520721A

CN106520721A - High-glyphosate-resistance EPSP synthase and application of encoding gene thereof

Info

Publication number: CN106520721A
Application number: CN201710009017.7A
Authority: CN
Inventors: 曹高燚; 刘允军; 杜锦; 向春阳; 谢晓东
Original assignee: Tianjin Agricultural University
Current assignee: Tianjin Agricultural University
Priority date: 2017-01-06
Filing date: 2017-01-06
Publication date: 2017-03-22

Abstract

The invention relates to high-glyphosate-resistance EPSP synthase. The EPSP synthase is a protein, or a polypeptide, or a conservative mutation polypeptide of the protein, or an active fragment of the protein, or an active derivative of the protein, which is formed by an amino acid sequence represented by SQE ID NO.2. The invention further relates to an encoding gene of the high-glyphosate-resistance EPSP synthase. The encoding gene has a nucleotide sequence represented by SQE ID NO.1. The invention further relates to application of the high-glyphosate-resistance EPSP synthase. According to the application, a high-glyphosate-resistance plant is produced, and the encoding gene of the EPSP synthase is taken as a screening marker for the transgenic cell culture of microorganisms or plants; and a new way is provided for improving the glyphosate resistance of the plants, the possibility is provided for changing the glyphosate resistances of existing excellent crop varieties, the encoding gene of the EPSP synthase is taken as the screening marker for the transgenic cell culture of the microorganisms or the plants, and the application prospect is huge.

Description

A kind of application of high-resistance glyphosate EPSP synthase and its encoding gene

Technical field

The invention belongs to field of biology, is related to a kind of microbe-derived 5- enol pyruvylshikimate -3- phosphoric acid and closes Enzyme (5-enolpyruvylshikimate-3-phosphate Synthase, EPSP synthase), its encoding gene and its training Educate the application in anti-glyphosate plants.

Background technology

Glyphosate (glyphosate), is phase early 1970s, by the John E Franz of Monsanto Chemicals Synthesis is found at first.Glyphosate have efficiently, low toxicity, fast, wide spectrum of degrading in the environment go out the good characteristic such as raw, and preparation technology Simply.Due to the characteristic of a variety of protrusions of glyphosate so as to which developing rapidly between decades becomes volume of production in current agricultural production And sales volume is maximum, a kind of most wide herbicide of usable floor area.

The mechanism of action of glyphosate mainly competitively suppresses the catalysis PEP and S3P generation EPSP in shikimic acid pathway EPSPS.Glyphosate is the analog of PEP, its inhibitive factor as EPSPS, can take up EPSPS and Binding Capacity Avtive spot cause being obstructed for shikimic acid pathway, and then shikimic acid in plant body is accumulated in a large number, finally make plant produce grass Sweet phosphine poisons (Steinrucken, H.C., and Amrhein, N. (1980) .The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase.Biochem Biophys Res Commun 94,1207-1212)。

The shikimic acid pathway only discovery in plant and microorganism, not yet finds the report that this approach is present in mammal Road, shikimic acid are the precursors of many compound synthesis, therefore become many antibacterial, the active important target transplanted seedlings with herbicide Mark.EPSPS acts on the 6th step enzyme reaction in shikimic acid pathway, is plant, Microbe synthesis aromatic amino acid and correlation The requisite key enzyme of aromatic series metabolite.EPSPS is by aroA gene codes, catalytic substrate phosphoenolpyruvic acid (PEP) and shikimic acid -3- phosphoric acid (S3P) generate 5- enolpyrul-shikimates acid -3- phosphoric acid (EPSP), discharge in the process Go out 1 molecule Phos (Pi).EPSPS during catalytic substrate is combined into binary complex with S3P first, leads to again afterwards Cross cutting C-O keys to combine to form ternary complex with PEP, ultimately generate EPSP, mediate being normally carried out for shikimic acid pathway (Boocock,M.R.,and Coggins,J.R.(1983).Kinetics of 5-enolpyruvylshikimate-3- phosphate synthase inhibition by glyphosate.FEBS Lett 154,127-133)。

EPSPS is the action target of broad spectrum weeding agent glyphosate, and glyphosate can be competitive with the substrate PEP of EPSPS Ground suppresses the activity of EPSPS, causes being obstructed for plants shikimic acid approach, is suppressed plant growing even dead.Source EPSPS encoding genes in plant, typically do not have glyphosate resistance, and the glyphosate of low dosage just seriously can suppress Enzymatic activity (Funke, T., Yang, Y., Han, H., et al. (2009) the .Structural basis of of EPSPS glyphosate resistance resulting from the double mutation Thr97->Ile and Pro101->Ser in5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.J Biol Chem 284,9854-9860)。

In order to increase the glyphosate resistance ability of plant EPSPS, typically by two kinds of approach, itself and substrate can be increased The affinity of PEP and S3P, or increase the inhibition constant to glyphosate.But due to PEP and glyphosate height in structure It is similar, often while EPSPS is increased with PEP affinities, the inhibition constant of glyphosate is also declined；On the other hand, While EPSPS declines to glyphosate binding ability often also along with to PEP affinities decline (Zhou, M., Xu, H., Wei,X.,et al.(2006).Identification of a glyphosate-resistant mutant of rice5- enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy.Plant Physiol 140,184-195)。

Organism is obtained glyphosate resistance and mainly can be carried out by following approach：1. glyphosate occupies EPSPS's Avtive spot, affects being normally carried out for shikimic acid pathway；The content for increasing EPSPS in shikimic acid pathway can be just made up because grass is sweet The competition of phosphine and EPSPS that content declines.2. orthogenesiss EPSPS be during current relevant glyphosate resistance institute is worked most It is deeply widest.Mainly by the strategy of two aspects being evolved, first is rite-directed mutagenesises to orthogenesiss, second be with Machine is mutated.3. organism can also be made to obtain glyphosate resistance by degradation of glyphosate.Glyphosate degradation approach mainly has two, First is that C-P bond fissions generate sarcosine (Sarcosine), and second is that C-N bond fissions generate AminomethylphosphoniAcid Acid (AMPA).This two The mesostate of kind of metabolic pathway is carbon source, nitrogen source and the phosphorus source of many microorganisms, is studying by microbial metabolism The degradation process of glyphosate provides foundation.4. the glyphosate resistance mechanism of non-target tropism refers to glyphosate and is not directly placed on EPSPS in shikimic acid pathway, glyphosate are also detoxified not over degradation pathway, be many factors it is comprehensive and produce it is careless sweet Phosphine resistance.

In biotechnology in modern agricultural production, shared proportion is increasing, and Transgenic Resistant Herbicide Crops development is fast Suddenly, glyphosate because its numerous advantage become cultivate anti-fecundi-t first-selection.Current glyphosate resistance base Because resource is narrow, need to excavate some new Glyphosate resistance genes, this has great to cultivating transgenic anti-glyphosate plants Meaning.

The content of the invention

In order to solve the above problems, it is an object of the invention to provide a kind of new 5- enols third with glyphosate resistance Keto acyl shikimic acid -3- phosphate synthases (5-enolpyruvylshikimate-3-phosphate Synthase), abbreviation EPSP are closed Enzyme, and its fragment, analogs and derivatives, which has higher glyphosate resistance, can be used to cultivate anti-glyphosate plants, Its encoding gene also can be used as the selection markers in microorganism and plant cell cultivation.

It is a further object of the present invention to provide the gene of the above-mentioned EPSP synthase of coding.

Another object of the present invention is to provide the application of above-mentioned EPSP synthase and its encoding gene, is especially improving plant Application in terms of resistance glyphosate ability.

The present invention solves its technical problem and takes technical scheme below to realize：

A kind of high-resistance glyphosate EPSP synthase, the egg that the EPSP synthase is made up of SEQ ID NO.2 aminoacid sequences In vain, or polypeptide or its conservative variation's polypeptide or its active fragment or its reactive derivative；

SEQ ID NO.2

MKVTFSGRLTQNPTVIEVPGDKSISHRAVMLGSLAKGVTTVRKCLLAADVVSTIGMMRAMGVSIEIANGQVTITGAG ESGLTRPNAELDAGNAGTAMRLMAGILAGQPFESVLVGDHSLSKRPMGRVATPLGQMGAQIQLSASGTAPMRITGTP KLRGITYDLPVASAQVKSAVLLAGLYADGATTVIERKPTRNYTETMVAAFGGNVSVNGQEIQVERSSLKGRDVVVPG DISSAAFFMVAAAINPGASITIRNVGINPTRTGIVDALRLMGARVELSDRRSSGGEEVADITVTGAELSGIELDADI APRMIDEFPVFFIAAAFANGTTVVRGAEELRVKESDRIETMLVGLRQLGVVVESKPDGAVISGVGSRMLKGGVSIET AYDHRIGMAFAIASSRCEDGITVVDAEAIGTSFPGFGDLCAGCGLASSQ-

And, the albumen being made up of SEQ ID NO.2 aminoacid sequences or polypeptide or its conservative variation's polypeptide, Or its active fragment or its reactive derivative be further on the basis of SEQ ID NO.2 aminoacid sequences C-terminal and/ Or N-terminal is substituted, lacks or adds the variant form sequence composition of one or several aminoacid sequences and has glyphosate The derived protein or polypeptide or its conservative variation's polypeptide or its active fragment or its activity of resistance and EPSP synthase activities Derivant.

And, the EPSP synthase is recombinant polypeptide, natural polypeptidess, synthesis polypeptide；EPSP synthase is the product for isolating and purifying Thing, or the product of chemosynthesis, or produced from protokaryon or eucaryon host using recombinant technique.

And, the EPSP synthase includes conservative variation's polypeptide form of EPSP synthase, and these variant forms are：It is homologous Sequence, conservative variant, allelic variant, natural mutation, induced mutants, can be with the conditions of high or low stringency Albumen coded by the DNA of EPSPS DNA hybridizations and the polypeptide that obtains of antiserum or albumen using anti-EPSP synthase； EPSP synthase conservative variation's polypeptides are that have at most 10 aminoacid on the basis of SEQ ID No.2 aminoacid sequences by property phase Like or close aminoacid is replaced and the polypeptide that formed.

A kind of gene of coding high-resistance glyphosate EPSP synthase, the nucleotides sequence of the encoding gene are classified as：SEQ ID NO.1。

SEQ ID NO.1

ATGAAGGTTACGTTTTCCGGCCGTCTGACCCAAAACCCGACAGTGATTGAGGTTCCTGGCGACAAGTCC ATCTCTCACCGCGCGGTGATGCTCGGCTCTCTGGCTAAAGGGGTTACCACGGTTCGGAAGTGCTTGCTGGCAGCAGA CGTGGTCAGCACCATCGGAATGATGCGAGCCATGGGAGTAAGCATTGAGATTGCGAACGGGCAGGTAACCATCACTG GGGCCGGCGAGAGTGGGCTCACGCGGCCAAATGCGGAACTGGACGCGGGCAACGCGGGTACTGCGATGAGGCTTATG GCCGGAATCTTGGCCGGGCAGCCCTTCGAAAGCGTGCTGGTTGGAGACCATTCTTTGTCGAAACGGCCGATGGGGCG TGTGGCGACCCCCCTTGGCCAAATGGGGGCGCAAATTCAGCTGTCAGCTTCCGGGACCGCTCCGATGAGGATCACCG GCACGCCAAAGCTTCGGGGCATCACTTACGATTTGCCCGTGGCCAGCGCCCAGGTGAAGTCGGCGGTACTGTTGGCC GGCCTCTATGCGGATGGAGCCACCACCGTGATAGAAAGGAAACCCACCCGCAACTACACGGAAACCATGGTGGCCGC CTTCGGAGGCAACGTCTCCGTCAACGGGCAAGAAATCCAAGTGGAGCGAAGCAGCCTGAAGGGCCGGGACGTTGTTG TTCCCGGCGACATATCGTCCGCAGCCTTTTTTATGGTGGCGGCTGCTATCAATCCTGGCGCCTCAATTACCATCAGG AACGTGGGAATAAATCCGACACGCACCGGCATTGTGGACGCACTGCGGCTCATGGGCGCGCGGGTAGAGCTTTCGGA CCGCCGCAGCTCTGGTGGAGAGGAAGTCGCTGATATAACGGTCACCGGTGCCGAGCTGTCCGGAATCGAGCTCGACG CGGACATCGCGCCCCGGATGATCGACGAGTTCCCCGTGTTCTTTATTGCCGCGGCGTTTGCCAACGGGACCACTGTA GTGCGCGGAGCGGAGGAGCTCAGAGTTAAGGAAAGTGACCGTATTGAGACCATGCTCGTCGGGTTGCGCCAGCTTGG CGTAGTCGTCGAATCCAAGCCCGACGGCGCCGTGATATCGGGCGTGGGCAGTCGAATGCTCAAGGGCGGTGTCTCAA TTGAGACGGCGTACGACCACCGAATCGGGATGGCTTTCGCCATCGCCAGCTCCCGCTGCGAAGACGGGATAACAGTC GTGGATGCGGAGGCGATCGGGACGTCGTTTCCGGGCTTTGGAGATCTGTGCGCCGGCTGCGGTCTGGCGAGCTCGCA ATAA

And, the recombinant expression carrier of the SEQ ID NO.1 nucleotide sequences is p3301-121-sp-aroA818-2.

A kind of application process of above-mentioned high-resistance glyphosate EPSP synthase, it is as follows that the method comprising the steps of：

(1) build the recombinant expression carrier p3301-121-sp- for carrying the SEQ ID NO.1 nucleotide sequences The Agrobacterium of aroA818-2；

(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), turns EPSP synthasee code genes Enter cell, and be incorporated on the chromosome of plant cell；

(3) select the plant cell or tissue or organ for proceeding to EPSP synthasee code genes；

(4) by the plant cell or tissue in step (3) or neomorph into high-resistance glyphosate plant, and by EPSP synthase Encoding gene is used as microorganism or the selection markers of plant transgene cell culture.

And, the high-resistance glyphosate plant includes：Semen Maydiss, Oryza sativa L., Semen Tritici aestivi, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Cotton Gossypii, Semen sojae atricolor, Nicotiana tabacum L., Willow, Rhizoma Dioscoreae esculentae, Rhizoma Solani tuber osi, Chinese cabbage, Caulis et Folium Brassicae capitatae and Capsicum annuum L...

Advantages of the present invention and good effect are：

The invention provides a kind of EPSP synthase of new high-resistance glyphosate, the glyphosate resistance for changing plant is provided New approach, with huge application prospect.EPSP synthasee code genes of the present invention are imported into plant and is expressed wherein Afterwards, the transgenic plant for being obtained has enhanced toleration to glyphosate, therefore, it can by importing this in crops The bright EPSP synthasee code genes, change the glyphosate resistance of existing excellent variety of crops, can obtain the jade of resistance glyphosate Rice, Oryza sativa L., Semen Tritici aestivi, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Cotton Gossypii, Semen sojae atricolor, Nicotiana tabacum L., willow, Rhizoma Dioscoreae esculentae, Rhizoma Solani tuber osi, Chinese cabbage, Caulis et Folium Brassicae capitatae, Capsicum annuum L. or other agricultures Crop varieties, solve practical problem present in agricultural production.

Description of the drawings

Screening and Identification figures of the Fig. 1 for glyphosate resistance bacterial strain；

Fig. 2 is the schematic diagram of AroA818-2 and related EPSPS sequence constructs systematic evolution tree；

Fig. 3 is growth curve qualification figure of the recombinant bacterial strain containing AroA818-2 under glyphosate stress；

Fig. 4 plant expression vector construction schematic diagrams.

Specific embodiment

Present invention enforcement is further described below in conjunction with accompanying drawing, following examples are descriptive, is not to limit Property, it is impossible to protection scope of the present invention is limited with this.

A kind of EPSP synthase of new glyphosate highly-tolerant, the EPSP synthase is：

(1) protein of the aminoacid sequence composition shown in SEQ ID NO.2；

(2) aminoacid sequence shown in SEQ ID NO.2 is substituted, lacks or adds one or several aminoacid (preferably 1-20, ground) and the protein by derived from (1) with glyphosate resistance and EPSP synthase activities.

SEQ ID NO.2：

Jing Phylogenetic analysis, the EPSP synthase belong to II type EPSP synthase.

In the present invention, term " EPSP synthase ", " EPSPS polypeptides ", " EPSPS " or " 5- enol pyruvylshikimate -3- phosphorus Acid synthase " is used interchangeably, and all refers to the albumen with the aminoacid sequence shown in SEQ ID No.2 or polypeptide or its conservative Variant polypeptides or its active fragment or its reactive derivative.They include that the EPSP for containing or not contain initial methionine is closed Enzyme.

In the present invention, it is (if crude, original that " detached " refers to that material is separated from its primal environment Environment is natural surroundingses).As the polynucleotide and polypeptide under the native state in active somatic cell is not isolated and purified, But same polynucleotide or polypeptide with separating in other materials for existing, are then isolated and purified such as from native state.

In the present invention, " detached EPSP synthase or EPSPS polypeptides " refers to that EPSPS polypeptides are substantially free of other natural Albumen, lipid, saccharide or other materials.Those skilled in the art can with the purified technology of protein of standard purify EPSP conjunction Enzyme.

The EPSPS polypeptides of the present invention can be recombinant polypeptide, natural polypeptidess, synthesis polypeptide, preferably recombinant polypeptide.This Bright EPSPS polypeptides can be the product for isolating and purifying, or the product of chemosynthesis, or using recombinant technique from protokaryon or true Produce in core host (for example, antibacterial, yeast, higher plant, insecticide and mammalian cell).According to used by recombinant production scheme Host, the EPSPS polypeptides of the present invention can be glycosylated, or can be nonglycosylated.The EPSPS polypeptides of the present invention are also May include or not include the methionine residues of starting.

Present invention additionally comprises the fragment of EPSP synthase, derivant and analog.As used herein, term " fragment ", " derivative Thing " and " analog " refer to the polypeptide for being kept substantially the natural EPSP synthase identical biological function or activity of the present invention. The polypeptide fragment of the present invention, derivant or the like can be that (i) has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by Genetic code encoding, or (ii) polypeptide with substituted radical in one or more amino acid residues, or (iiii) is ripe Polypeptide and another compound merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and shape Into polypeptide (such as targeting sequencing or secretion sequence or the sequence for this polypeptide of purification).Record of the invention, these pieces Section, derivant and analog belong to scope known to those skilled in the art.

In the present invention, term " EPSPS polypeptides " also include with EPSP synthase identical functions, SEQ ID No.2 The variant form of shown aminoacid sequence.These variant forms include (but being not limited to)：Several (usually 1-50, Preferably 1-30, more preferably 1-20, most preferably 1-10) disappearance of aminoacid, insertion and/or replacement, and in C-terminal And/or N-terminal adds one or several (usually 20 within, within preferably 10, more preferably within 5) amino Acid.For example, in the art, when being replaced with similar nature or similar aminoacid, the work(of protein will not generally be changed Energy.Again such as, add the function that or several aminoacid will not generally also change protein in C-terminal and/or N-terminal.Should Active fragment and reactive derivative of the term also including EPSP synthase.

The variant form of " the EPSPS polypeptides " includes：It is homologous sequence, conservative variant, allelic variant, natural prominent Variant, induced mutants, under the conditions of high or low stringency can with the albumen coded by the DNA of EPSPS DNA hybridizations and The polypeptide obtained using the antiserum of anti-EPSPS polypeptides or albumen.Present invention also offers other polypeptides, such as more comprising EPSPS The fusion protein of peptide or its fragment.In addition to the almost polypeptide of total length, present invention includes the solubility piece of EPSPS polypeptides Section.Generally, the fragment has at least about 10 continuous amino acids of EPSPS peptide sequences, typically at least about 30 continuous amino Acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about 100 companies Continuous aminoacid.

Present invention also offers the analog of EPSP synthase or EPSPS polypeptides.These analog and natural EPSPS polypeptides Difference can be the difference on aminoacid sequence, or do not affect sequence modified forms on difference, or and and have It.These polypeptides include natural or induction genetic variant.Induction variant can be obtained by various technologies.

In the present invention, " EPSP synthase conservative variation's polypeptides " refers to and the aminoacid sequence phase shown in SEQ ID No.2 Than, there are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 aminoacid are similar or close by property Aminoacid it is replaced and form polypeptide.These conservative variation's polypeptides are preferably and carry out amino acid substitution according to table 1 and produce.

1 amino acid substitution table of table

Present invention also offers the gene of the coding EPSP synthase.The nucleotides sequence of the gene is classified as：On (a) coding State the polynucleotide of EPSP synthase；(b) polynucleotide complementary with polynucleotide (a).

Preferably, the gene code has the polypeptide of aminoacid sequence shown in SEQ ID NO.2.It is highly preferred that described Gene has the nucleotide sequence shown in SEQ ID NO.1 or with 1-1302 in the nucleotide sequence shown in SEQ ID NO.1 The sequence of position.

SEQ ID NO.1：

In the present invention, the polynucleotide for encoding EPSP synthase can be DNA form or rna form.DNA form includes The DNA of cDNA, genomic DNA or synthetic.DNA can be single-stranded or double-strand.DNA can be coding strand or non-volume Code chain.The coding region sequence of encoding mature polypeptide can or degeneracy identical with the coding region sequence shown in SEQ ID NO.1 Variant.As used herein, " variant of degeneracy " refers to coding with the amino shown in SEQ ID NO.2 in the present invention The protein of acid sequence, but with the differentiated nucleotide sequence of coding region sequence shown in SEQ ID NO.1.

The polynucleotide of the mature polypeptide of the aminoacid sequence shown in coding SEQ ID NO.2 include：Encoding mature is more The coded sequence of peptide；The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence of mature polypeptide is (and optional Additional coding sequence) and non-coding sequence.

The term polynucleotide of EPSPS polypeptides " coding " can be include encoding such peptides polynucleotide, or Also include the polynucleotide of additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotide, its coding and the present invention have many of identical aminoacid sequence The fragment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotide can be the allelic variant of natural generation or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this Known to field, allelic variant is the alternative forms of polynucleotide, it be probably one or more nucleotide replacement, Disappearance is inserted, but will not be from the function of the polypeptide for substantially changing its coding.

The invention further relates to have at least 50% with above-mentioned sequence hybridization and between two sequences, preferably at least 70%, more preferably at least polynucleotide of 80% homogeny.The present invention be more particularly directed to many with of the present invention under strict conditions The interfertile polynucleotide of nucleotide.In the present invention, " stringent condition " is referred to:(1) compared with low ionic strength and higher temperature Under hybridization and eluting, such as 0.2 × SSC, 0.1%SDS, 600 DEG C；Or added with denaturant during (2) hybridization, such as 50% (v/v) first Amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or the homogeny of (3) only between two sequences is at least 90% More than, just there is hybridization when more preferably more than 95%.Also, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO.2 Shown mature polypeptide has identical biological function and activity.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " is extremely Contain 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably at least 100 nucleoside less It is more than acid.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or separate many poly-nuclears of coding EPSP synthase Thuja acid.

Polypeptide and polynucleotide in the present invention is preferably provided with detached form, is more preferably purified to homogenizing.

The full length sequence of the coding epsp synthase gene of the present invention or its fragment can generally use synthetic, PCR The method of TRAP or recombination method is obtained.Complete sequence synthesis is carried out according to the sequence of SEQ ID NO.1 first for example.

For PCR TRAP, can be according to relevant nucleotide sequence, especially open reading frame sequence disclosed in this invention To design primer, and the EPSP synthasee code genes full length sequence with synthetic or its fragment are expanded and must be had as template Close sequence.

Once obtain relevant sequence, it is possible to relevant sequence is obtained in large quantity with recombination method.This is typically by which Carrier is cloned into, then proceeds to cell, then isolated relevant sequence in the host cell by conventional method from after propagation.

Additionally, relevant sequence can also be synthesized with the method for synthetic, when especially fragment length is shorter.Generally, lead to After multiple small fragments are first synthesized, then it is attached again and can obtains the very long fragment of sequence.

At present, it is already possible to obtain encoding completely by chemosynthesis EPSP synthase proteins of the present invention (or its fragment and Derivant) gene.Then the gene can be introduced various existing DNA molecular (or such as carriers) as known in the art and thin In born of the same parents.Additionally, be able to also will be mutated by chemosynthesis being introduced in protein sequence of the present invention.

, through extensively in-depth study, from glyphosate serious pollution soil, to obtain glyphosate resistance micro- for screening for inventor Biological P818, obtains EPSP conjunctions by the method (obtaining aroA818) and sequence measurement (obtaining aroA818-2) of Tail-PCR (wherein aroA818 has been applied for a patent two copy genes of enzyme, the patent No.:ZL201310376641.2；Certificate number：The No. 1695432), it is labeled as and it has high-resistance glyphosate by experimental verification ability, and after proceeding to plant, can leads Glyphosate resistance of plant ability is caused to improve.

Glyphosate resistance microorganism P818 of the present invention：Pseudomonass (Pseudomonas sp.) P818, preservation In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Number, Institute of Microorganism, Academia Sinica, postcode 100101, preservation date August in 2013 12 days, preserving number CGMCC No.7989。

Present invention also offers the carrier containing above-mentioned coding epsp synthase gene.Preferably, the carrier is restructuring table Up to carrier.

In the present invention, the encoding gene of EPSP synthase is can be plugged in recombinant expression carrier.Term " recombinant expression carrier " Refer to bacterial plasmid well known in the art, phage, yeast plasmid, plant cell virus, mammalian cell virus or other loads Body.In a word, as long as can replicate in host's body and stable, any plasmid and carrier can be used.One of recombinant expression carrier Key character is to usually contain replication orgin, promoter, marker gene and translation control element.

Method well-known to those having ordinary skill in the art can be used for building synthasee code gene containing EPSP and suitably transcribe/turn over Translate the expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc.. Described gene can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression vector also includes The ribosome binding site and transcription terminator of translation initiation.

Additionally, expression vector preferably includes one or more selected markers, conversion is selected to provide The dihydrofolate reductase of the phenotypic character of host cell, such as eukaryotic culture, neomycin resistance and green fluorescence egg In vain (GFP), or for colibacillary tetracycline or ammonia section penicillin resistance.

It is further preferred that the recombinant expression carrier is p3301-121-sp-aroA818-2, the recombinant expressed load The construction method of body p3301-121-sp-aroA818-2 comprises the steps：

(1) forward primer 5'-AGGATCCATGAAGGTTACGTTTTCC-3' is designed, 5' ends introduce BamHI restriction enzyme sites (GGATCC)；Downstream primer 5'-GAGCTCTTATACCCATACGATGTTCCAGATTACGCTTTGCGAGCTCGCCAGA- 3', 5' ends introduce SacI restriction enzyme sites (GAGCTC)；

(2) extract the genome DNA of glyphosate resistance microorganism P818, the forward primer designed with step (1) and under Trip primer expands epsp synthase gene for being directed into performing PCR reaction；

(3) PCR primer that amplification is obtained is cloned on pEASY T1 simple carriers, is sequenced, obtains carrier T-p818- 2-3301；

(4) HindIII and EcoRI digested plasmid pBI121 are used, reclaims 35S-GUS-NOS fragments；With HindIII and EcoRI enzyme action carrier pCAMBIA3301, reclaim carrier segments, and connection obtains plasmid pCAMBIA3301-121；

(5) with carrier T-p818-2-3301 of the BamHI and SacI enzyme action containing epsp synthase gene, electrophoresis simultaneously reclaims EPSP Synthase gene fragment；SacI and BamH1 enzyme action pCAMBIA3301-121 carriers, electrophoresis is equally used simultaneously to reclaim carrier segments；Even Connect, obtain carrier p3301-121-aroA818-2；

(6) by chloroplast localisation signal peptide (signal peptide, the Accession number from Semen Pisi sativi： X00806.1), XbaI and BamHI restriction enzyme sites are added respectively at 5' ends and 3' ends, by enzyme action method of attachment, sub-clone is to load The BamHI of body p3301-121-aroA818-2 and XbaI enzyme cutting site, obtain recombinant expression carrier p3301-121-sp- aroA818-2。

Wherein, in step (2), PCR reaction systems：

PCR response procedures：98 DEG C 5 minutes；98 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 45 seconds, 35 circulation；72 DEG C 10 minutes； 4 DEG C of insulations；

Present invention also offers containing above-mentioned EPSP synthasee code genes or containing the recombinant expressed of EPSP synthasee code genes The host cell of carrier.The host cell be by the EPSP synthasee code genes directly conversion or the host cell transduceed or By the conversion of the recombinant expression carrier containing EPSP synthasee code genes or the host cell transduceed.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryotic cell, such as yeast cells；Or it is high Deng eukaryotic cell, such as plant cell.Representative example has：Escherichia coli, streptomyces, Agrobacterium；Fungal cell's such as yeast；Plant Thing cell；Insect cell etc..

When the EPSP synthasee code genes of the present invention are expressed in higher eucaryotic cells, if inserting enhancer in the carrier When sequence transcription will be strengthened.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 base pairs, Promoter is acted on the transcription of enhancing gene.

Persons skilled in the art are aware that how to select appropriate carrier, promoter, enhancer and host cell.

Can be carried out with routine techniquess well known to those skilled in the art with recombinant DNA transformed host cell.When host is original When core biology is such as escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, use CaCl₂Method process, institute With the step of it is generally well-known in the art.Another kind of method is to use MgCl₂.If desired, conversion also can be with the side of electroporation Method is carried out.When host is eukaryote, following DNA transfection methods are can select：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..Conversion plant can also use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, example Such as leaf disk method.Plant cell, tissue or organ for conversion can regenerate plant with conventional method, sweet so as to obtain anti-grass The plant that phosphine resistance is improved.

Carrier comprising the EPSP synthasee code genes and appropriate promoter or control sequence, can be used for conversion Appropriate host cell, allows it to marking protein.

Present invention also offers a kind of method for preparing the polypeptide with glyphosate resistance and EPSP synthase activities, the method Including：A () under conditions suitable for the expression, cultivates the above-mentioned host cell for being converted or transduceing；B () is isolated from culture Polypeptide with the activity.

By conventional recombinant DNA technology, can be used to express or produce weight using the EPSP synthasee code genes of the present invention The EPSPS polypeptides of group.In general there are following steps:

(1) by the EPSP synthasee code genes (or variant) of the present invention, or will be containing the EPSP synthasee code genes Recombinant expression carrier conversion or suitable host cell of transduceing；

(2) host cell is cultivated in suitable culture medium；

(3) separation, EPSPS polypeptides described in purification from culture medium or cell.

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, in culture, culture medium used may be selected from various conventional mediums.Under conditions of host cell growth is suitable to Cultivated.After host cell growth is to appropriate cell density, with suitable method (such as temperature transition or chemical induction) The promoter of selection is induced, cell is further cultured for into a period of time.

Recombinant polypeptide in the above methods can in the cell, or on cell membrane expression, or be secreted into extracellular.Such as Fruit need, can using its physics, chemistry separated by various separation methods with other characteristics and purification of Recombinant albumen.This A little methods are well-known to those skilled in the art.The example of these methods is included but is not limited to：The renaturation process of routine, use Protein precipitant process (salting-out method), centrifugation, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), suction Attached chromatography, ion-exchange chromatography, the combination of high performance liquid chroma- tography (HPLC) and other various liquid chromatography (LC) technologies and these methods.

Present invention also offers a kind of method for improving plant glyphosate resistance, methods described comprises the steps：

(1) build the Agrobacterium for carrying the recombinant expression carrier containing above-mentioned EPSP synthasee code genes；

(4) by the plant cell or tissue in step (3) or neomorph into plant.

Preferably, the plant is Semen Maydiss, Oryza sativa L., Semen Tritici aestivi, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Cotton Gossypii, Semen sojae atricolor, Nicotiana tabacum L., willow, Rhizoma Dioscoreae esculentae, horse Bell potato, Chinese cabbage, Caulis et Folium Brassicae capitatae, Capsicum annuum L. or other varieties of crops.

Present invention also offers the antibody that can be specifically bound with above-mentioned EPSP synthase, including polyclonal antibody and Dan Ke Grand antibody.That is, present invention also offers having to above-mentioned EPSP synthasee code genes or the polypeptide of its fragment coding The polyclonal antibody of specificity and monoclonal antibody, especially monoclonal antibody.It is preferred that referring to that those can be encoded with EPSP synthase Gene outcome or fragment are combined but nonrecognition and are incorporated into the antibody of other non related antigen molecules.In the present invention, antibody includes that A bit can with reference to and suppress the molecule of EPSP synthase, also have no effect on the antibody of EPSP synthase functions including those.The present invention is also Including the antibody that those can be combined with modification or without the EPSP synthasee code gene products of modified forms.

The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetent antibody piece Section, or chimeric antibody.

The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.Such as purification EPSP synthasee code genes product or its there is antigenic fragment, animal can be applied to induce the product of polyclonal antibody It is raw.Similar, express EPSP synthase or there is the cell of antigenic fragment can be used to immune animal to produce antibody for which. Such monoclonal antibody can be prepared using hybridoma technology.Each antibody-like of the present invention can encode base using EPSP synthase Because of fragment or the functional areas of product, obtained by common immunological techniques.These fragments or functional areas can utilize recombination method system It is standby or using Peptide synthesizer synthesis.The antibody combined with the unmodified form of EPSP synthasee code gene products can use protokaryon In cell (such as E.coli), the gene outcome of production is carried out immune animal and is produced；The antibody combined with post translational modification form (such as glycosylation or the albumen or polypeptide of phosphorylation), can be with the gene produced in eukaryotic cell (such as yeast or insect cell) Product carrys out immune animal and obtains.The antibody of anti-EPSP synthase can be used for the EPSP synthase in detection sample.

The available EPSP synthase of the production of polyclonal antibody or polypeptide immune animal, such as rabbit, mice, rat etc..Various assistants Agent can be used to strengthen immunoreation, including but not limited to Freund adjuvant etc..

The invention further relates to the method for testing of quantitative and detection and localization EPSP synthase levels.These tests are that this area institute is ripe Know, and determine including FISH and radioimmunoassay.

A kind of method in detection sample with the presence or absence of EPSP synthase is examined using the specific antibody of EPSP synthase Survey, it includes：Sample is contacted with EPSP synthase specific antibodies；See whether to form antibody complex, define antibody multiple There is EPSP synthase in meaning that sample in compound.

Present invention also offers EPSP synthasee code genes conduct in microorganism or plant transgene cell culture The application of selection markers molecule.

Part or all of the EPSP synthasee code genes of the present invention can be fixed on microarray as probe (microarray) or in DNA chip (being also called " gene chip "), for the expression analysis of gene.It is special with EPSP synthase Primer carries out the transcription product that RNA- polymerase chain reactions (RT-PCR) amplification in vitro also can detect EPSP synthase.

In an example of the present invention, there is provided a kind of detached polynucleotide, it is encoded with SEQ ID NO.2 institutes Show the polypeptide of aminoacid sequence.The polynucleotide of the present invention are divided from the community level DNA library built using Culture-independent method From, it is and artificial complete synthesis.As shown in SEQ ID NO.1, the polynucleotide sequence total length that it includes is its nucleotide sequence 1305 bases, encoding full leng are the EPSP synthase (SEQ ID NO.2) of 434 aminoacid.

The Screening and Identification of 1 resistant strain of embodiment

Glyphosate serious pollution pedotheque carries out gradient dilution with liquid M9 culture medium, is 10 by extension rate⁶Soil Earth suspension is coated on the M9 solid mediums containing 300mM glyphosates, and 37 DEG C are cultivated.As M9 culture medium lacks for nutrition Swaged, the antibacterial that can be survived and grow are glyphosate resistance bacterial strain.The homozygosis bacterial strain of acquisition is shaken after bacterium with without glyphosate The resuspended thalline of liquid M9 culture medium, draw 2 Μ l resuspended bacterium solutions points afterwards in the solid M9 culture medium containing 350mM glyphosates On flat board, 37 DEG C are inverted culture, observe the upgrowth situation of thalline, and final identification is resistant to the bacterium of high-strength glyphosate stress Strain.

The present invention obtains one plant of glyphosate resistance bacterial strain through screening, is named as 818 (Fig. 1), and it can be sweet in grass containing 300mM Grow in the M9 culture medium of phosphine.

The present invention obtains the 16SrDNA sequences of bacterial strain 818 by PCR method.The genome DNA of bacterial strain 818 is extracted, Reacted with following forward primer and reverse primer as performing PCR is directed into.Expanded by PCR and expanded from the genomic DNA of antibacterial 818 Increase its 16SrDNA sequence.

Forward primer sequence：5'-AGAGTTTGATCATGGCTCAG-3', reverse primer sequences：5'- TACGGTTACCTTGTTACGACTT-3'。

PCR reaction systems：

PCR response procedures：98 DEG C 5 minutes；98 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 45 seconds, totally 35 circulation；72 DEG C 10 points Clock；4 DEG C of insulations.

The PCR primer that amplification is obtained is cloned on pEASY T1 simple carriers, with universal primer M13 (F) and M13 (R) it is sequenced, the result of acquisition is carried out into sequence alignment on NCBI, as a result shows which with pseudomonass Pseudomonas Mendocina ymp, Pseudomonas mendocina strain NS2 etc. have 99% or so homogeneity, therefore speculate The bacterial strain is class pseudomonass Pseudomonas sp, is named as P818.

The similarity of 2 P818EPSP synthase of embodiment and other known glyphosate resistance EPSP synthase

On existing Research foundation, by genome sequencing (by Beijing, Joint Genome Institute completes), obtain false single Second copy of the EPSPS encoding genes of born of the same parents bacterium P818, is named as aroA818-2.AroA818-2 total lengths 1305bp, coding 434 aminoacid.The typical part EPSPS sequences that collection has certain Research foundation adopt Neighbor-Joining algorithm structures Build phyletic evolution development tree (MEGA6.0), value of bootstrapping 1000 times.

Phylogenetic analysis show that AroA818-2 belongs to the EPSPS of II types, it and Halomonas HTG7EPSPS and CP4 EPSPS affinities are recently (Fig. 2).

Growth curve identification of recombinant bacterial strain of the embodiment 3 containing AroA818-2 under glyphosate stress

Using BamHI the and SalI restriction enzyme sites of prokaryotic expression carrier PUC18, by aroA818, maroA818 is (based on CP4 The 97th special A of sequence, carries out the point mutation of G/A to AroA818, with higher glyphosate resistance) and aroA818-2 difference Build to BamHI the and SalI restriction enzyme sites of PUC18, convert aroA deficient strains ER2799, recombinant bacterial strain is with containing The liquid M9 culture medium of 200mM glyphosate is cultivated, and determines the growth curve of recombinant bacterial strain.

Carrier construction method is adoptedHD Cloning Kit (clontech), the primer is, positive： 5 '-CGGTACCCGGGGATCCGATGAAGGTTACGTTTTCC-3 ', reversely：5’- ATGCCTGCAGGTCGACTTATTGCGAGCTCGCCAGA-3‘。

PCR reaction systems：

In-fusion reaction systems are：

50 DEG C of reaction 15min, convert DH5 α, screening positive clone, and sequencing identification afterwards, obtain recombiant plasmid.Restructuring Plasmid transformation escherichia coli aroA deficient strains ER2799 carry out glyphosate resistance, with plasmid PUC18, PUC- AroA818, PUC-MaroA818 are control.

As a result show (Fig. 3), there is the recombinant bacterial strain containing aroA818-2 (being labeled as 1305) certain glyphosate to resist Property, aroA818-2 can give recombinant bacterial strain opposing high concentration glyphosate stress, aroA818-2 have cultivate heredity repair The potentiality of decorations plant.

The acquisition of the structure and transgene tobacco and arabidopsiss of 4 plant expression vector of embodiment

1st, gene cloning

According to the sequence of complete aroA818-2 genes, specific primer is designed.Simultaneously before the termination codon of gene End addition HA sequence labels, beneficial to the translation situation of late detection gene.Design forward primer 5'- AGGATCCATGAAGGTTACGTTTTCC-3', 5' end introduces BamHI restriction enzyme sites (GGATCC), downstream primer 5'- GAGCTCTTATACCCATACGATGTTCCAGATTACGCTTTGCGAGCTCGCCAGA-3', 5' end introduces SacI restriction enzyme sites (GAGCTC).Genomic DNA with antibacterial P818 is reacted as performing PCR is directed into as template with forward primer and downstream primer.

PCR reaction systems：

PCR response procedures：98 DEG C 5 minutes；98 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 45 seconds, 35 circulation；72 DEG C 10 minutes； 4 DEG C of insulations.

The PCR primer that obtains of amplification is cloned on pEASY T1 simple carriers, with universal primer M13 (F) 5 '- TGTAAAACGACGGCCAGT-3 ' and M13 (R) 5 '-CAGGAAACAGCTATGACC-3 ' are sequenced, and sequencing result shows gram Grand correct, carrier is named as T-p818-2-3301.

2nd, vector construction and the conversion to Agrobacterium

35S-GUS-NOS fragments are reclaimed with HindIII and EcoRI digested plasmids pBI121, be connected to HindIII and On the pCAMBIA3301 of EcoRI enzyme action, plasmid pCAMBIA3301-121 is built.Contain aroA818-2 with BamHI and SacI enzyme action The carrier T-p818-2-3301 of gene, digestion products run agarose gel electrophoresiies, are separately recovered the fragment of about 1.3k.It is same to use SacI and BamH1 enzyme action pCAMBIA3301-121 carriers, electrophoresis simultaneously reclaim 11kb fragments.By the genetic fragment for reclaiming and carrier Fragment is connected with T4DNA ligases, makes aroA818-2 genetic fragments replace the GUS fragments from pBI121 on the carrier, Carrier is named as p3301-121-aroA818-2.For making aroA818-2 genes preferably act in chloroplast, Because of front end, using XbaI and BamHI restriction enzyme sites, sub-clone one derives from the chloroplast localisation signal peptide of Semen Pisi sativi to aroA818-2 (signal peptide, Accession number：X00806.1), pass through at the end of chloroplast localisation signal peptide 5 ' and 3 ' ends The method addition XbaI and BamHI restriction enzyme sites of PCR clones, by enzyme action method of attachment, sub-clone to carrier p3301-121- The BamHI of aroA818-2 and XbaI enzyme cutting site, obtain recombinant vector p3301-121-sp-aroA818-2.Vector construction plan Slightly see (Fig. 4).

3rd, the acquisition of transfer-gen plant

200 μ L Agrobacterium EHA105 competent cells are taken, the DNA of l μ g carrier p3301-121-sp-aroA818-2 is added, Quick-freezing 1 minute in liquid nitrogen, 37 DEG C of water-baths 5 minutes are subsequently adding lmL blank YEB culture medium, 28 DEG C of shaken cultivation 4 hours at a slow speed； L000rpm is centrifuged 30 seconds, abandons supernatant, adds 0.lmLYEB culture medium suspension cell again, coat containing 100 μ g/mL cards that On the YEB flat boards of mycin and 50 μ g/mL rifampicin, 28 DEG C are cultivated about 48 hours.The single bacterium colony grown on picking flat board, is inoculated in In YEB liquid mediums, 28 DEG C of shaken cultivation are overnight；Plasmid DNA is extracted in a small amount, enters performing PCR amplification mirror by template of plasmid DNA It is fixed.

Transformation of tobacco method：By the Agrobacterium inoculation containing plant expression vector p3301-121-sp-aroA818-2 in In YEB liquid mediums (containing 100 μ g/mL kanamycin and 50 μ g/mL rifampicin), 28 DEG C of shaken cultivation to OD₆₀₀For 0.6- 0.8.4000rpm, room temperature are centrifuged 10 minutes, with MS saline solution (PH7.0) again suspension thalline, dilute using MS saline solution during use Release to OD600=0.3 or so.Aseptic tobacco leaf cuts edge and main vein, in above-mentioned Agrobacterium bacterium solution soaks 10 Minute；The bacterium solution of plant material surface is blotted with filter paper, the MS minimal mediums of one metafiltration paper of upper berth, 28 DEG C of light cultures three are proceeded to After it.Material is gone to into (the MS+3mg/L 6-BA+0.2mg/L NAA+20mg/L Basta+ of the division culture medium containing antibiotic 250 μ g/mL fill in p0-357).Whne resistant budses grow to 2-3cm it is high when, cut budlet and proceed in root media that (MS is cultivated substantially Base+20mg/L Basta), further culture obtains transgenic positive Seedling, extracts DNA and enters performing PCR identification.

Transformation of Arabidopsis thaliana method：By the Agrobacterium containing plant expression vector p3301-121-sp-aroA818-2 in solid It is after cultivating 2-3 days on YEB flat boards, with spreader scraping bacterium layer into a little flask, resuspended with 30mL YEB liquid.After resuspended Bacterium solution OD value is 2.0 or so.

The concentration that preparation 120mL contains 0.03%Silwet L-77 in addition is 5% sucrose solution.Both the above is molten Liquid is mixed to get conversion conversional solution used every time.The little holder for being filled with arabidopsiss is inclined, the inflorescence of Arabidopsis plant is enable Enough it is fully immersed in 30 seconds in Agrobacterium-mediated Transformation liquid.Plant is inserted in plastics PE glove, then holder is kept flat, and One lid of lid on young plant, is placed under light after making young plant lucifuge horizontal positioned 16-24 hour and normally cultivates.By the T for collecting₀In generation, turns The seed of gene plant is put in sterilized water 4 DEG C of purification three days, is uniformly laid on containing Vermiculitum with pipettor transfer afterwards：Nutrition Soil (3：1) in small flower, and overlay film moisturizing, 25 DEG C, cultivate under 16hr illumination/8hr dark conditions, treat seedling length to 10 days or so When, spray 1：The Basta of 1000 times of dilutions, the Seedling for surviving are transgenic positive Seedling, extract DNA and enter performing PCR identification.

SEQUENCE LISTING

<110>TanJin Agricultural College

<120>A kind of application of high-resistance glyphosate EPSP synthase and its encoding gene

<130> 2017-01-06

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1305

<212> DNA

<213> SEQ ID NO.1

<400> 1

atgaaggtta cgttttccgg ccgtctgacc caaaacccga cagtgattga ggttcctggc 60

gacaagtcca tctctcaccg cgcggtgatg ctcggctctc tggctaaagg ggttaccacg 120

gttcggaagt gcttgctggc agcagacgtg gtcagcacca tcggaatgat gcgagccatg 180

ggagtaagca ttgagattgc gaacgggcag gtaaccatca ctggggccgg cgagagtggg 240

ctcacgcggc caaatgcgga actggacgcg ggcaacgcgg gtactgcgat gaggcttatg 300

gccggaatct tggccgggca gcccttcgaa agcgtgctgg ttggagacca ttctttgtcg 360

aaacggccga tggggcgtgt ggcgaccccc cttggccaaa tgggggcgca aattcagctg 420

tcagcttccg ggaccgctcc gatgaggatc accggcacgc caaagcttcg gggcatcact 480

tacgatttgc ccgtggccag cgcccaggtg aagtcggcgg tactgttggc cggcctctat 540

gcggatggag ccaccaccgt gatagaaagg aaacccaccc gcaactacac ggaaaccatg 600

gtggccgcct tcggaggcaa cgtctccgtc aacgggcaag aaatccaagt ggagcgaagc 660

agcctgaagg gccgggacgt tgttgttccc ggcgacatat cgtccgcagc cttttttatg 720

gtggcggctg ctatcaatcc tggcgcctca attaccatca ggaacgtggg aataaatccg 780

acacgcaccg gcattgtgga cgcactgcgg ctcatgggcg cgcgggtaga gctttcggac 840

cgccgcagct ctggtggaga ggaagtcgct gatataacgg tcaccggtgc cgagctgtcc 900

ggaatcgagc tcgacgcgga catcgcgccc cggatgatcg acgagttccc cgtgttcttt 960

attgccgcgg cgtttgccaa cgggaccact gtagtgcgcg gagcggagga gctcagagtt 1020

aaggaaagtg accgtattga gaccatgctc gtcgggttgc gccagcttgg cgtagtcgtc 1080

gaatccaagc ccgacggcgc cgtgatatcg ggcgtgggca gtcgaatgct caagggcggt 1140

gtctcaattg agacggcgta cgaccaccga atcgggatgg ctttcgccat cgccagctcc 1200

cgctgcgaag acgggataac agtcgtggat gcggaggcga tcgggacgtc gtttccgggc 1260

tttggagatc tgtgcgccgg ctgcggtctg gcgagctcgc aataa 1305

<210> 2

<211> 434

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<213> SEQ ID NO.2

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

1 5 10 15

Glu Val Pro Gly Asp Lys Ser Ile Ser His Arg Ala Val Met Leu Gly

20 25 30

Ser Leu Ala Lys Gly Val Thr Thr Val Arg Lys Cys Leu Leu Ala Ala

35 40 45

Asp Val Val Ser Thr Ile Gly Met Met Arg Ala Met Gly Val Ser Ile

50 55 60

Glu Ile Ala Asn Gly Gln Val Thr Ile Thr Gly Ala Gly Glu Ser Gly

65 70 75 80

Leu Thr Arg Pro Asn Ala Glu Leu Asp Ala Gly Asn Ala Gly Thr Ala

85 90 95

Met Arg Leu Met Ala Gly Ile Leu Ala Gly Gln Pro Phe Glu Ser Val

100 105 110

Leu Val Gly Asp His Ser Leu Ser Lys Arg Pro Met Gly Arg Val Ala

115 120 125

Thr Pro Leu Gly Gln Met Gly Ala Gln Ile Gln Leu Ser Ala Ser Gly

130 135 140

Thr Ala Pro Met Arg Ile Thr Gly Thr Pro Lys Leu Arg Gly Ile Thr

145 150 155 160

Tyr Asp Leu Pro Val Ala Ser Ala Gln Val Lys Ser Ala Val Leu Leu

165 170 175

Ala Gly Leu Tyr Ala Asp Gly Ala Thr Thr Val Ile Glu Arg Lys Pro

180 185 190

Thr Arg Asn Tyr Thr Glu Thr Met Val Ala Ala Phe Gly Gly Asn Val

195 200 205

Ser Val Asn Gly Gln Glu Ile Gln Val Glu Arg Ser Ser Leu Lys Gly

210 215 220

Arg Asp Val Val Val Pro Gly Asp Ile Ser Ser Ala Ala Phe Phe Met

225 230 235 240

Val Ala Ala Ala Ile Asn Pro Gly Ala Ser Ile Thr Ile Arg Asn Val

245 250 255

Gly Ile Asn Pro Thr Arg Thr Gly Ile Val Asp Ala Leu Arg Leu Met

260 265 270

Gly Ala Arg Val Glu Leu Ser Asp Arg Arg Ser Ser Gly Gly Glu Glu

275 280 285

Val Ala Asp Ile Thr Val Thr Gly Ala Glu Leu Ser Gly Ile Glu Leu

290 295 300

Asp Ala Asp Ile Ala Pro Arg Met Ile Asp Glu Phe Pro Val Phe Phe

305 310 315 320

Ile Ala Ala Ala Phe Ala Asn Gly Thr Thr Val Val Arg Gly Ala Glu

325 330 335

Glu Leu Arg Val Lys Glu Ser Asp Arg Ile Glu Thr Met Leu Val Gly

340 345 350

Leu Arg Gln Leu Gly Val Val Val Glu Ser Lys Pro Asp Gly Ala Val

355 360 365

Ile Ser Gly Val Gly Ser Arg Met Leu Lys Gly Gly Val Ser Ile Glu

370 375 380

Thr Ala Tyr Asp His Arg Ile Gly Met Ala Phe Ala Ile Ala Ser Ser

385 390 395 400

Arg Cys Glu Asp Gly Ile Thr Val Val Asp Ala Glu Ala Ile Gly Thr

405 410 415

Ser Phe Pro Gly Phe Gly Asp Leu Cys Ala Gly Cys Gly Leu Ala Ser

420 425 430

Ser Gln

Claims

1. a kind of high-resistance glyphosate EPSP synthase, it is characterised in that：The EPSP synthase is by SEQ ID NO.2 aminoacid sequences The albumen or polypeptide of composition or its conservative variation's polypeptide or its active fragment or its reactive derivative.

2. high-resistance glyphosate EPSP synthase according to claim 1, it is characterised in that：It is described by SEQ ID NO.2 amino The albumen or polypeptide or its conservative variation's polypeptide of acid sequence composition or its active fragment or its reactive derivative are further It is on the basis of SEQ ID NO.2 aminoacid sequences to be substituted, lack or add one or several in C-terminal and/or N-terminal The variant form sequence composition and derived protein or many with glyphosate resistance and EPSP synthase activities of aminoacid sequence Peptide or its conservative variation's polypeptide or its active fragment or its reactive derivative.

3. high-resistance glyphosate EPSP synthase according to claim 1 and 2, it is characterised in that：The EPSP synthase is restructuring Polypeptide, natural polypeptidess, synthesis polypeptide；EPSP synthase is the product for isolating and purifying, or the product of chemosynthesis, or using restructuring Technology is produced from protokaryon or eucaryon host.

4. high-resistance glyphosate EPSP synthase according to claim 1 and 2, it is characterised in that：The EPSP synthase includes Conservative variation's polypeptide form of EPSP synthase, these variant forms are：Homologous sequence, conservative variant, allelic variant, Natural mutation, induced mutants, can be with the egg coded by the DNA of EPSPS DNA hybridizations under the conditions of high or low stringency In vain, and using anti-EPSP synthase antiserum obtain polypeptide or albumen；EPSP synthase conservative variation's polypeptides are in SEQ There are at most 10 aminoacid to be replaced and formed by the similar or close aminoacid of property on the basis of ID No.2 aminoacid sequences Polypeptide.

5. it is a kind of coding high-resistance glyphosate EPSP synthase gene, it is characterised in that：The nucleotides sequence of the encoding gene is classified as： SEQ ID NO.1。

6. it is according to claim 5 coding high-resistance glyphosate EPSP synthase gene, it is characterised in that：The SEQ ID The recombinant expression carrier of NO.1 nucleotide sequences is p3301-121-sp-aroA818-2.

7. a kind of application process of above-mentioned high-resistance glyphosate EPSP synthase, it is characterised in that：It is as follows that the method comprising the steps of：

(1) build the recombinant expression carrier p3301-121-sp-aroA818-2 for carrying the SEQ ID NO.1 nucleotide sequences Agrobacterium；

(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), EPSP synthasee code genes is proceeded to carefully Born of the same parents, and be incorporated on the chromosome of plant cell；

(4) by the plant cell or tissue in step (3) or neomorph into high-resistance glyphosate plant, and EPSP synthase is encoded Gene is used as microorganism or the selection markers of plant transgene cell culture.

8. the application process of high-resistance glyphosate EPSP synthase according to claim 7, it is characterised in that：The anti-grass of the height is sweet Plant includes phosphine：Semen Maydiss, Oryza sativa L., Semen Tritici aestivi, Fructus Hordei Vulgaris, Sorghum vulgare Pers., Cotton Gossypii, Semen sojae atricolor, Nicotiana tabacum L., willow, Rhizoma Dioscoreae esculentae, Rhizoma Solani tuber osi, Chinese cabbage, Caulis et Folium Brassicae capitatae And Capsicum annuum L..