CN102776161A - Preparation and use of high-glyphosate-resistance EPSP (5-enolpyruvylshikimate-3-phosphate) synthase separated from soil and coding sequence thereof - Google Patents
Preparation and use of high-glyphosate-resistance EPSP (5-enolpyruvylshikimate-3-phosphate) synthase separated from soil and coding sequence thereof Download PDFInfo
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- CN102776161A CN102776161A CN2012102877019A CN201210287701A CN102776161A CN 102776161 A CN102776161 A CN 102776161A CN 2012102877019 A CN2012102877019 A CN 2012102877019A CN 201210287701 A CN201210287701 A CN 201210287701A CN 102776161 A CN102776161 A CN 102776161A
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Abstract
The invention provides preparation and use of an EPSP (5-enolpyruvylshikimate-3-phosphate) synthase capable of improving glyphosate resistance of plants and a coding sequence thereof. The EPSP synthase is obtained through separation by acquiring a soil sample from the environment extremely polluted by glyphosate and then constructing a glyphosate polluted soil microflora level DNA (deoxyribonucleic acid) library; and the EPSP synthase is proved through experiments to have high glyphosate resistance, and further is capable of improving the glyphosate resistance of plants after being translated into the plants.
Description
Technical field
The invention belongs to biological technical field; Relate to preparation and the purposes of separation, be specifically related to isolatingly from Glyphosate 62 IPA Salt contaminated soil microbial population dna library have 5-enol form acetone thick grass acyl-3-phosphate synthase (EPSP synthase) gene of glyphosate tolerant and the preparation and a purposes of protein product thereof from the high-resistance glyphosate EPSP synthase and the encoding sequence thereof of soil.
Background technology
Glyphosate 62 IPA Salt (Glyphosate) is inner sucting conduction type, wide spectrum steriland herbicide; Its mechanism of action is through suppressing 5-enol form acetone thick grass acyl-3-phosphate synthase (EPSPS) activity in the plant materials; The biosynthesizing of die aromatischen Aminosaeuren in the blocking-up plant materials causes plant dead.
It is the encoding sox of Glyphosate 62 IPA Salt action target EPSP synthase that the bacteriogenic aroA two mutants of applied chemistry mutagenesis, the research of resistance mechanism have been proved conclusively the aroA gene.The country that comprises the U.S. and China in recent years, Glyphosate 62 IPA Salt output sharply increases also seed selection with serial transgenic glyphosate resistant crops kind, and popularization has direct relation with big area.Part patent surplus companies such as U.S. Mosanto and Calegene have applied for 100 at aspects such as the encoding sox of EPSP synthase and resistance glyphosate transgenic plant thereof; Obtain serial crop varieties such as transgenic resistance glyphosate soybean, corn, rape, beet and cotton, wherein multiple genetically modified crops such as soybean have got into commercialization production.
The research and development of resistance glyphosate transgenic plant can be played huge prograding to agricultural development; Therefore, the genetically modified crops this area for the new high-resistance glyphosate of seed selection presses for the new 5-enol form acetone thick grass acyl-3-phosphate synthase with resistance glyphosate purposes of exploitation.
Summary of the invention
The purpose of this invention is to provide a kind of preparation and purposes that improves the EPSP synthase and the encoding sequence thereof of plant glyphosate resistance; The present invention is through collection soil sample from the extreme contaminate environment of Glyphosate 62 IPA Salt; Make up the horizontal dna library of Glyphosate 62 IPA Salt contaminated soil microflora, separate having obtained a kind of EPSP synthase, and through experiment confirm it have the high-resistance glyphosate resistance; And after changing plant over to, can cause the raising of plant resistance glyphosate resistance.
The present invention realizes through following technical scheme:
A kind of separation is used to improve the resistance of plant to Glyphosate 62 IPA Salt from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof.
Separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and high-resistance glyphosate EPSP synthase comprises polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQ ID NO:2 aminoacid sequence.
Separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and high-resistance glyphosate EPSP synthase is following polypeptide,
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have resistance glyphosate resistance and EPSP synthase activity by (a) polypeptides derived.
Separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and the enzymic activity of high-resistance glyphosate EPSP synthase is 25.57U/mg.
Separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and the polynucleotide sequence of the said high-resistance glyphosate EPSP synthase of encoding is selected from down group: (a) polynucleotide of the SEQ ID NO:1 of the above-mentioned EPSP synthase polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.
Separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and the polynucleotide sequence of the SEQ ID NO:1 of said coding EPSP synthase polypeptide is selected from down a kind of of group:
(a) has the sequence of 1-1335 position among the SEQ ID NO:1;
(b) has the sequence of 1-1338 position among the SEQ ID NO:1.
A kind ofly be used to improve the carrier of plant, contain aforesaid polynucleotide the resistance of Glyphosate 62 IPA Salt.
A kind ofly be used to improve the host cell of plant, contain aforesaid carrier the resistance of Glyphosate 62 IPA Salt.
Separation (a) is cultivated the host cell that the above-mentioned carrier that contains polynucleotide as claimed in claim 7 transforms or transduces from the preparation method of the high-resistance glyphosate EPSP of soil synthase; (b) from culture, isolate and have active polypeptide.
A kind of can with aforesaid high-resistance glyphosate EPSP synthase specificity bonded antibody.
A kind of method that changes plant resistance glyphosate resistance, it comprises step:
(1) Agrobacterium of carrying expression vector is provided, described expression vector contains EPSP synthase dna encoding sequence, and described EPSP synthase is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through a replacement to 20 amino-acid residues, disappearance or interpolation, and have resistance glyphosate resistance and EPSP synthase activity by (a) polypeptides derived;
(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make EPSP synthase dna encoding sequence change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or tissue or the organ that changes EPSP synthase dna encoding sequence over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
In the present invention; Term " EPSPS ", " EPSP synthase ", " epsp synthase " " EPSP polypeptide " or " 5-enol form acetone thick grass acyl-3-phosphate synthase " interchangeable use all refer to have 5-enol form acetone thick grass acyl-albumen or the polypeptide of 3-phosphate synthase (EPSPS) aminoacid sequence (SEQ ID NO:2).They comprise the EPSP synthase that contains or do not contain initial methionine.
As invent usedly, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As invent usedly, " isolating EPSP synthase or polypeptide " is meant that the EPSP polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying EPSP synthase of standard.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises fragment, verivate and the analogue of EPSP synthase.As used herein, term " fragment ", " verivate " are meant biological function or the active polypeptide that keeps natural EPSP synthase of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, verivate and analogue belong to the known scope of those skilled in the art.
In the present invention, term " EPSP polypeptide " refers to have SEQ ID NO. 2 polypeptide of sequence of EPSP synthase activity.This term also comprises having and variant forms EPSP synthase identical function, SEQ ID NO. 2 sequences.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of EPSP synthase.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of EPSPS DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-EPSP polypeptide to obtain.The present invention also provides other polypeptide, as comprises EPSP polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of EPSP polypeptide.Usually, this fragment have the EPSP peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of EPSP synthase or polypeptide.The difference of these analogues and natural EPSP polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology.
In the present invention; " EPSP synthase conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8; More preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala (A) | Val; Leu; Ile | Val |
| Arg (R) | Lys; Gln; Asn | Lys |
| Asn (N) | Gln; His; Lys; Arg | Gln |
| Asp (D) | Glu | Glu |
| Cys (C) | Ser | Ser |
| Gln (Q) | Asn | Asn |
| Glu (E) | Asp | Asp |
| Gly (G) | Pro; Ala | Ala |
| His (H) | Asn; Gln; Lys; Arg | Arg |
| Ile (I) | Leu; Val; Met; Ala; Phe | Leu |
| Leu (L) | Ile; Val; Met; Ala; Phe | Ile |
| Lys (K) | Arg; Gln; Asn | Arg |
| Met (M) | Leu; Phe; Ile | Leu |
| Phe (F) | Leu; Val; Ile; Ala; Tyr | Leu |
| Pro (P) | Ala | Ala |
| Ser (S) | Thr | Thr |
| Thr (T) | Ser | Ser |
| Trp (W) | Tyr; Phe | Tyr |
| Tyr (Y) | Trp; Phe; Thr; Ser | Phe |
| Val (V) | Ile; Leu; Met; Phe; Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, be preferably at least 70%, be more preferred from the polynucleotide of at least 80% homogeny.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 65 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or to separate the polynucleotide of coding epsp synthase.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
EPSPS Nucleotide full length sequence of the present invention or its fragment can use the method for synthetic, pcr amplification method or recombination method to obtain usually.For example at first carrying out complete sequence according to the sequence of SEQ ID NO:1 synthesizes.
For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and with artificial synthetic EPSPS Nucleotide full length sequence or its fragment as template, amplification and must be about sequence.
In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can obtain the dna sequence dna of code book invention albumen (or its fragment and verivate) fully through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or EPSP synthase coding sequence, and produce the method for polypeptide according to the invention through recombinant technology.
Recombinant DNA technology (Science, 1984 through routine; 224:1431), polymerized nucleoside acid sequence of the present invention capable of using can be used to express or produce the EPSP polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding EPSP polypeptide of the present invention, or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, EPSP synthase polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains EPSP synthase DNA sequences encoding and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell; Insect cell etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain the plant that the resistance glyphosate resistance improves.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
On the other hand, the present invention also comprises EPSP synthase DNA or the polypeptide of its segment encoding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Preferably, refer to that those can combine with epsp synthase gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody comprises that those can combine and suppress the molecule of EPSP synthase among the present invention, comprises that also those do not influence the antibody of EPSP synthase function.The present invention also comprise those can with modify or without the epsp synthase gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment or chimeric antibody.
Antibody of the present invention can prepare through the known various technology of those skilled in that art.For example, the epsp synthase gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing EPSP synthase or its has antigenic segmental cell and can be used to immune animal and produce antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize the fragment or the functional zone of epsp synthase gene product, obtains through the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.(for example can use prokaryotic cell prokaryocyte with the unmodified form bonded antibody of epsp synthase gene product
E.Coli) in the gene product of producing come immune animal and produce; With posttranslational modification form bonded antibody (like the albumen or the polypeptide of glycosylation or phosphorylation), can use the gene product that produces in the eukaryotic cell (for example yeast or insect cell) to come immune animal and obtain.The antibody of anti-EPSP synthase can be used for the EPSP synthase in the test sample.
Available EPSP synthase of the production of polyclonal antibody or polypeptide immune animal, like rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
The invention still further relates to the testing method of quantitative and detection and localization EPSP synthase level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.
A kind of method that whether has the EPSP synthase in the test sample that detects is to utilize the specific antibody of EPSP synthase to detect, and it comprises: sample is contacted with EPSP synthase specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample EPSP synthase.
Polynucleotide of the present invention a part or all can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), be used for the expression of gene analysis.Carry out the transcription product that RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect the EPSP synthase with the special primer of EPSP synthase.
In an instance of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are to exempt from culture technique from employing to make up the horizontal dna library of group of Glyphosate 62 IPA Salt contaminated soil mikrobe isolating and artificial complete synthesis.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1338 bases, and its ORF is positioned at the 1-1335 position, and the coding total length is 445 amino acid whose EPSP synthase (SEQ ID NO:2).This EPSP synthase of the present invention is called " XA3-EPSPS " for short.
EPSP synthase of the present invention has following principal feature:
A) first clearly this EPSP synthase have the resistance glyphosate ability;
B) glyphosate resistance definite functions, and shown higher relatively resistance glyphosate ability.
The EPSP synthase of the present invention for a change resistance glyphosate resistance of plant provides new approach, thereby has great application prospect.Can import through encoding sox, change the resistance glyphosate resistance of existing good variety of crops, can obtain wheat, paddy rice or other variety of crops of resistance glyphosate, solve the practical problems that exists in the agriculture prodn the EPSP synthase.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the comparison result according to EPSPS of the present invention and II class EPSPS.Wherein identical amino acid represented in asterisk; The amino acid that the fullstop mark is conservative; Emit the very conservative amino acid of labelled notation.
Fig. 2 has shown the phylogeny comparison diagram according to XA3 EPSPS of the present invention and part I class and II class EPSPS.
Fig. 3 has shown the glyphosate tolerance experimental result of XA3 EPSPS.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the dna fragmentation clone of high-resistance glyphosate
1, the collection of pedotheque in the extreme contaminate environment of Glyphosate 62 IPA Salt
Particularly in the geographic soil of weedicide such as life-time service Glyphosate 62 IPA Salt, there is miscellaneous bacterial isolates that can tolerate Glyphosate 62 IPA Salt or other weedicide in the physical environment.From by (new Anhua, Zhejiang worker group produce Glyphosate 62 IPA Salt plant area) collected specimens the Glyphosate 62 IPA Salt Contaminated soil.
, adopt and to exempt from cultural method isolates from the extreme contaminated soil sample of Glyphosate 62 IPA Salt flat total DNA that falls into water
Take by weighing Glyphosate 62 IPA Salt contaminated soil sample 2 grams, adding 0.6g micro glass beads (d 0.11mm), 4000 rev/mins vibrate 2 times.Added 300 μ l 2%SDS+12% phenol Tris damping fluid (pH8.0) solution 1 hour on ice, and added equivalent phenol Tris damping fluid, pH8.0 (about 700ml), abundant mixing, 4 ℃ on warp, 13, centrifugal 5 minutes of 000rpm.Upper solution adds the 3M NaAc pH5.2 of 0.1 times of volume, adds 0.6 times of volume Virahol mixing behind the mixing.The DNA deposition is dissolved in 200 μ l 1xTE (thick DNA).Claim that the 100mg cesium chloride places a new 1.5ml Epp. centrifuge tube, add the thick DNA of 100 μ l mixing gently, left standstill under the room temperature dark condition 1-3 hour.Room temperature, 13,000rpm, centrifugal 20 minutes.Add 400 μ l aseptic deionized waters and 300 μ l Virahols in the supernatant, room temperature left standstill 30 minutes.Room temperature, 13,000rpm, centrifugal 20 minutes.Deposition is dissolved in 100 μ l 1xTE and 40 μ l 8M Potassium ethanoates (KAc), and room temperature left standstill 15 minutes.4 ℃, 13, centrifugal 15 minutes of 000rpm.Supernatant adds 0.6 times of volume Virahol mixing.Room temperature left standstill 30 minutes.Room temperature, 15, centrifugal 20 minutes of 000rpm.The DNA deposition is dissolved in 100 μ l 1xTE.
Adopt Wizard spin column clean-up separating kit purify DNA sample.Purify DNA is dissolved in 10mM Tris-EDTA (pH8.0) damping fluid that TV is 100 μ l.
, the total DNA cosmid library of group's level structure
Soil bacteria DNA uses
Sau3AI carries out partially digested trial cut in 10 μ l reaction systems,
SauThe 3AI enzyme is pressed the 1:100 dilution, and 37 ℃, enzyme is cut 10min respectively, 20min, and 30min, 40min, 50min adds 10 * loading buffer, 1 μ l termination reaction, the righttest endonuclease reaction time of electrophoresis detection behind the 60min.Then selecting identical system enzyme to cut 30 min carries out a large amount of enzymes and cuts.It is subsequent use behind agarose gel electrophoresis, to cut glue recovery 2～6kb dna fragmentation.Plasmid vector pACYC184 (available from NEB company) uses
BamCarry out terminal dephosphorylation with SAP alkaline phosphatase lipase behind the HI complete degestion, to reduce carrier from connecting.Soil bacteria DNA (200ng) after the above-mentioned recovery and terminal dephosphorylized plasmid vector pACYC184 (150ng) are connected 16h with the T4 ligase of 2U under 4 ℃.
Above-mentioned connection product changes over to
E.coliER2799 (available from the NEB company) competent cell that shocks by electricity, coating LB+Cm
r+ Km
rThen the clone who grows on the LB plate is xeroxed M9+Cm
r+ Km
rThe flat board of+50mm Glyphosate 62 IPA Salt is cultivated 48h for 37 ℃.The bacterium that grows on the flat board behind the LB plate loop method, is inoculated these bacterium colonies (100, the 150mm Glyphosate 62 IPA Salt) on the M9 flat board that contains different Glyphosate 62 IPA Salt concentration.With being coated with M9+Cm behind the Transformed E R2799 again after the plasmid extraction in these reorganization bacterium
r+ Km
rDull and stereotyped checking (ER2799+pACYC184 is contrast) is carried out the recombinant plasmid enzyme simultaneously and is cut checking.
, screening glyphosate resistance transformant
Transfection bacterium coating is contained on the LB flat board of Cm (paraxin), Km (kantlex), 37 ℃ cultivate 20h after, 5000 colony growths are arranged approximately, with these bacterial colony photographic reprintings to containing Cm
r, Km
rAfter cultivating 48h on the M9 flat board of 50mM Glyphosate 62 IPA Salt, three colony growths are arranged.These three colony inoculations are cultivated to the M9 flat board of the Glyphosate 62 IPA Salt that contains 100mM, 150mM, found to have only 1 clone on the M9 of the Glyphosate 62 IPA Salt that contains 150mM flat board, to grow, its contained plasmid is named as pACYCXA3.Cloning extractive plasmid pACYCXA3 from this changes over to
E.coliIn ER2799 or the e. coli jm109; Transformant put on the MOPS solid medium that contains the 20mM Glyphosate 62 IPA Salt with aseptic toothpick check resistance; The result proves that the transformant that this clone is produced all has the resistance glyphosate characteristic, shows that the resistance glyphosate characteristic causes owing to changing pACYCXA3 over to really.
, glyphosate tolerance experiment
Intestinal bacteria ER2799 (containing the pACYCXA3 plasmid that carries new clone) is inoculated into the M9 liquid nutrient medium (Cm that contains 0～200mm Glyphosate 62 IPA Salt
r+ Km
r) in, after cultivating through 37 ℃, the shaking table of 72h, measure the OD of culture
600Do not have the negative contrast of intestinal bacteria ER2799 of inserting segmental plasmid to contain simultaneously, insert known glyphosate resistance gene HTG7 to contain
AroAIntestinal bacteria ER2799 (contain pACYCHTG7, this plasmid carries known glyphosate resistance gene HTG7
AroA) as positive control.
The result:
ER2799 (carrying the pACYCXA3 plasmid) is inoculated into the M9 liquid nutrient medium (Cm that contains 0～200mm Glyphosate 62 IPA Salt
r+ Km
r) in, after cultivating through 37 ℃, the shaking table of 72h, find that negative control almost can not grow in M9; Positive ER2799 (pACYCHGT7) can be in only containing the M9 liquid nutrient medium of 100mm Glyphosate 62 IPA Salt well-grown; ER2799 (pACYCXA3) then can also grow in containing the M9 liquid nutrient medium of 150mm Glyphosate 62 IPA Salt.The result shows that the exogenous segment of carrying on the pACYCXA3 has the active (see figure 3) of very strong glyphosate tolerance.
Embodiment 2: the sequential analysis of the dna fragmentation of high-resistance glyphosate and epsp synthase functional verification thereof
1, the sequential analysis of the dna fragmentation of high-resistance glyphosate
High-resistance glyphosate dna fragmentation to institute's subclone among the embodiment 1 carries out full nucleotide sequencing.Analytical results shows; The clip size of inserting is 2643bp; The reading frame that has wherein comprised a 1338bp, its sequence are shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1338 bases; Its ORF is positioned at the 1-1335 position, and the coding total length is 445 amino acid whose epsp synthases (SEQ ID NO:2).
The amino acid sequence homology analytical results shows, EPSP synthase of the present invention with
MagnetococcusThe amino acid identity of the EPSPS of MC-1 is 100%.The polygene sequence alignment shows that it is one type (Fig. 2) that XA3-DEPSPS EPSPS obvious and the II type gathers.This presentation of results XA3 EPSPS belongs to II class EPSPS.The phylogeny comparative result of XA3 EPSPS and part I class and II class EPSPS is as shown in Figure 2.
, high-resistance glyphosate the EPSP synthase functional verification of dna fragmentation
The sequencing results shows the ORF that contains the EPSP synthase in the resistance clone, has the EPSP synthase activity in order to confirm it, and this experiment is a F-strain with the intestinal bacteria EPSP synthase deficient strain ER2799 of routine, adopts CaCl
2Method changes glyphosate resistance subclone pACYCXA3 over to; With aseptic toothpick the transformant bacterium colony is put on the MOPS solid medium; F-strain ER2799, contain the negative contrast of ER2799 of empty carrier plasmid, contain effable epsp synthase gene, the complementary defective of recipient bacterium like the resistance glyphosate subclone; Then can utilize the inorganics synthesizing amino acid and the growth of restricted substratum, otherwise just can not grow.
Test-results shows, in the glyphosate resistance subclone contained dna fragmentation all can be on function complementary EPSP synthase deficient strain, therefore, can confirm to contain among the subclone pACYCXA3 epsp synthase gene of complete function.
Embodiment 3: the synthetic of the epsp synthase gene of high-resistance glyphosate
According to the completed nucleotide sequence that contains the 1338bp coding region, at first divide 8 sections respectively according to normal chain and secondary chain-ordering, synthesize the about 150-200bp of length respectively, have the single stranded oligonucleotide fragment of sticky end.With normal chain and secondary chain each one to one 8 complementary single stranded oligonucleotide fragments anneal respectively, form 8 double chain oligonucleotide fragments that have sticky end.Mix the double chain oligonucleotide fragment, be assembled into a complete epsp synthase gene through the catalysis of T4 dna ligase.This synthetic dna fragmentation contains the nucleotide sequence of 1-1335 position among the SEQ ID NO:1, and the upstream and downstream two ends of synthetic gene contain NdeI and HindIII site.
With the 5' and the 3' end restriction enzyme site of above-mentioned synthetic is NdeI and HindIII site EPSPS gene, is used to express the EPSP synthase of high-resistance glyphosate and makes up corresponding gene plant expression vector.
Embodiment 4: the EPSPS of high-resistance glyphosate expresses
The 5' of above-mentioned synthetic and 3' end restriction enzyme site are NdeI and HindIII site EPSPS gene; After cutting with NdeI and HindIII enzyme, be connected into that carrier pET28a (available from NOVAGEN company) that same enzyme cuts obtains recombinant plasmid pETXA3 and with its transformed into escherichia coli BL21 (DE3).Transformant is first at LB+Kan
rIn the substratum 37 ℃, 200rpm is cultured to OD
600Be worth approximately 0.5, add and change 37 ℃ of inducible proteins over to behind the IPTG (final concentration is 0.75mmol/L) and express the SDS-PAGE electrophoresis detection.
Through the SDS-PAGE electrophoresis detection, contain pETXA3's
E.coliBL21 (DE3) 37 ℃ after IPTG induces 4h expression amount promptly reach mxm..Target protein is a soluble proteins; The about 47kD of size conforms to predictor.
The enzyme activity determination of embodiment 5:EPSPS and the mensuration of kinetic parameter
(a) measuring method
The inorganic phosphorus typical curve: 10mM inorganic phosphorus reference liquid is pressed the 1:10 dilution; Get 0,1,2,3 respectively ... 20 μ l are in 1.5ml Eppendorf centrifuge tube; Add milli-Q pure water to 100 μ l mixing; Add MAT solution 0.8ml mixing, timing adds the rapid mixing of 34% SC solution, 100 μ l after three minutes, and room temperature is measured OD after leaving standstill 20min
660Value.Triplicate.With the inorganic phosphorus concentration is X-coordinate, OD
660Value obtains the inorganic phosphorus typical curve for the ordinate zou mapping.
Enzyme activity determination: enzyme crude extract protein quantification adopts Xylene Brilliant Cyanine G G-250 staining (Bradford, 1976).In 1.5ml Eppendorf centrifuge tube, add following solution on ice: 10mM PEP solution 2 μ l, 10mM S3P solution 2 μ l, 0.5M HEPES solution 2 μ l, 1mM (NH4) 6MO
7O
244H
2O solution 2 μ l and milli-Q pure water 12 μ l mixings; After 28 ℃ of temperature are bathed 5min, respectively manage sample room and add 1 μ l crude enzyme liquid and timing at a distance from 2S; Behind the 2min more at interval 2S add 200 μ l MAT solution successively; Behind the colour developing 3min more at interval 2S add the rapid mixing of 20 μ l, 34% SC solution successively, measure OD behind the color development at room temperature 20min
660Value.Contrast except that not enzyme-added liquid, all the other same sample hoses.The OD of sample hose and control tube
660After value was subtracted each other, contrast inorganic phosphorus typical curve can be tried to achieve the inorganic phosphorus molar weight that reaction discharges, and just obtained the enzyme activity (U/mg) of this enzyme again divided by reaction times and zymoprotein amount.
Partly suppressing dosage (IC50) measures: add 0,10 in the above-mentioned reaction solution
-3, 10
-2, 10
-1, 1,10,100, the 500mM Glyphosate 62 IPA Salt, gained specific activity of enzyme data are the X axle with Glyphosate 62 IPA Salt concentration, adopt logarithmic coordinate, be that the Y axle is mapped with speed of response V (U/mg).
Km (PEP) measures: the S3P strength of solution is constant at 1mM, measures enzyme reaction rate by above-mentioned reaction system down in different PEP concentration (0.05,0.067,0.1,0.2,0.5,1.0mM), the numerical value of surveying press V-v/ [S] (Eadic-Hofstee) method map.
Ki (glyphosate) measures: the enzyme reaction rate of EPSPS when mensuration PEP concentration is 66.7,100,200,500 μ M under different Glyphosate 62 IPA Salt concentration (0,10,50,100 μ M).Employing double-log mapping obtains 1/V-1/ [S] straight line, again with each collinear slope as ordinate zou, Glyphosate 62 IPA Salt concentration obtains a new straight line as X-coordinate, the intersection point of this straight line and X axle is Ki (glyphosate) value.
(b) result
The enzymic activity of XA3 EPSPS is 25.57U/mg.
The kinetic parameter of XA3 EPSPS is measured as shown in the table:
| Kinetic parameter | XA3 EPSPS |
| IC50 (glyphosate; mM) | 25.035±0.022 |
| K m (PEP; mM) | 0.0443±0.012 |
| K i (glyphosate; mM) | 0.746±0.007 |
Kinetic parameter according to XA3 EPSPS can know that XA3 EPSPS not only has higher glyphosate resistance, but also is keeping the affinity stronger with PEP, and these characteristics will provide possibility for the cultivation that XA3 EPSPS is used for genetically modified crops.
Embodiment 6: the structure of the epsp synthase gene plant expression vector of high-resistance glyphosate
The concrete grammar of the epsp synthase gene plant expression vector construction of high-resistance glyphosate is following:
A. pBI121 (available from ClonTech company) and pCAMBIA2300 (available from ClonTech company) are with HindIII and EcoRI double digestion, and the fragment that pBI121 is had p35S-GUS-Nos-ter is connected into pCAMBIA2300, form intermediate carrier p35S-2300-GUS;
B. with XbaI and the two EPSPS genes of cutting p35S-2300-GUS and above-mentioned synthetic of SacI, replace the GUS of the corresponding restriction enzyme site of p35S-2300-GUS, thereby obtain the epsp synthase gene plant expression vector of high-resistance glyphosate with EPSPS.Again it is changed in the Agrobacterium, be used for the transformation mode plant tobacco.
Embodiment 7: utilize leaf dish method to transform the transgene tobacco that makes up resistance glyphosate
(1) select the positive colony of preparation among the embodiment 5 on the flat board with aseptic toothpick picking YPE, be inoculated in 2MLYPE liquid (Sm+, Kan+), 28 ℃, 200rpm shaking culture 24-36 hour;
(2) under the room temperature 4, centrifugal 10 minutes of 000g;
(3) abandon supernatant, thalline suspends with the 1/2MS liquid nutrient medium, is diluted to 5-20 times of original volume, makes the OD of thalline
600About 0.5;
(4) get the aseptic blade of the tobacco of growth about two weeks, remove its main lobe arteries and veins, it is cut into about 1cm
2Square vanelets;
(5) blade is put into the bacterium liquid for preparing, soaked 2-5 minute, on aseptic filter paper, blot bacterium liquid; Be put on the MS substratum 28 ℃ of dark cultivations 48 hours through the blade of contaminating;
(6) blade is forwarded on the callus substratum (MS+6-BA 1.0mg/l+NAA 0.1mg/l+Kan 50mg/l+ Pyocianil 250mg/l), 25-28 ℃ of illumination is cultivated down, the formation of 7-15 days visible callus;
(7) visible differentiation bud grows after about 20 days, treat that bud is grown up after, downcut, place on the root media (1/2MS+NAA 0.5mg/l+Kan 25mg/l) and carry out root culture, take root about 2-7 days;
(8) treat well developed root system after, plant is taken out, clean the solid medium that is adhering to sterilized water; Move in the soil; Just begin several days with lens cover several days, treated to take off lens again behind the robust plant, be transferred to the plant of screening glyphosate resistance in the solid medium of the Glyphosate 62 IPA Salt that contains 10mM.
(9) resistant plant is verified as genetically modified resistant plant through Southern, Northern hybridization and Westhern blot.
(10) in the greenhouse, experiment showed, that through the glyphosate resistance gradient transgenic plant can tolerate the processing of 30mM Glyphosate 62 IPA Salt and growth is normal.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.Also need to prove, comprise this specification sheets each several part and arbitrary combination thereof from the category of the technical scheme of the purposes of the high-resistance glyphosate EPSP synthase of soil and encoding sequence thereof according to separation of the present invention.
SEQUENCE LISTING
< 110>Biological Technology institute, Chinese Academy of Agricultural Sciences
< 120>separation is from the preparation and the purposes of the high-resistance glyphosate EPSP synthase and the encoding sequence thereof of soil
<130> ZFSZZ110123
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1338
<212> DNA
< 213>artificial sequence
<400> 1
atgtccagca cccatcccgg acgcaccatc cgtagcggcg ccacgcaaaa cctctccggc 60
accatccgcc ccgccgccga taaatccatc tcccaccgct ccgtgatctt tggcgccctg 120
gccgaaggcg aaacccacgt taaaggcatg ctggaaggcg aagatgtgct gcgtaccatc 180
accgcctttc gtaccatggg tatctctatc gaacgctgca acgaaggtga ataccgcatc 240
caaggccaag gactcgacgg cctaaaagaa cccgatgacg tgctggatat gggtaactcc 300
ggtaccgcca tgcgcctgct gtgcggcctg ctggccagcc aaccctttca ctctatcctc 360
accggcgatc actccctacg cagccgcccc atgggccgcg tagtgcaacc cctaaccaaa 420
atgggcgctc gcatccgtgg ccgcgacggt ggccgcctgg cccccctcgc catcgaaggc 480
actgaactgg tacccattac ctacaatagc cccatcgcct cggcccaagt gaagtccgcc 540
attatcctgg ccggactcaa taccgccggc gaaaccacca tcattgaacc cgccgtcagc 600
cgcgaccaca ccgaacgtat gctcatcgcc ttcggtgccg aagtgacccg cgatggcaac 660
caagtgacca tcgaaggctg gcccaacctg caaggccaag agatcgaagt gcccgccgat 720
atctccgccg ccgccttccc catggtggcc gcccttatca ccccaggatc tgatattatc 780
ctggaaaatg tgggtatgaa cccaacccgt accggtattc tcgacctgct cctggctatg 840
ggcggcaata tccaacgcct caacgaacgg gaagttggcg gcgaacccgt ggccgaccta 900
caggtgcgct actcccaact ccaaggcatc gagatagacc ccaccgtggt gccccgtgcc 960
attgatgagt tccccgtgtt ttttgtagcc gccgccctcg cccaaggcca aaccctggtg 1020
caaggcgccg aagagctgcg cgttaaagag agcgaccgca tcaccgccat ggccaacggt 1080
cttaaagccc taggtgccat catagaagaa cgccccgatg gcgcacttat taccggaaat 1140
cccgacggtc tggccggtgg ggccagcgta gactccttta ccgaccaccg tatcgccatg 1200
agcctgctgg tggccggcct gcgctgtaaa gagtccgtat tggtgcaacg ctgcgataat 1260
atcaatacct cctttcccag cttttcccaa ttaatgaaca gtcttggttt tcaattggag 1320
gatgtcagcc atggctga 1338
<210> 2
<211> 445
<212> PRT
< 213>artificial sequence
<400> 2
Met Ser Ser Thr His Pro Gly Arg Thr Ile Arg Ser Gly Ala Thr Gln
1 5 10 15
Asn Leu Ser Gly Thr Ile Arg Pro Ala Ala Asp Lys Ser Ile Ser His
20 25 30
Arg Ser Val Ile Phe Gly Ala Leu Ala Glu Gly Glu Thr His Val Lys
35 40 45
Gly Met Leu Glu Gly Glu Asp Val Leu Arg Thr Ile Thr Ala Phe Arg
50 55 60
Thr Met Gly Ile Ser Ile Glu Arg Cys Asn Glu Gly Glu Tyr Arg Ile
65 70 75 80
Gln Gly Gln Gly Leu Asp Gly Leu Lys Glu Pro Asp Asp Val Leu Asp
85 90 95
Met Gly Asn Ser Gly Thr Ala Met Arg Leu Leu Cys Gly Leu Leu Ala
100 105 110
Ser Gln Pro Phe His Ser Ile Leu Thr Gly Asp His Ser Leu Arg Ser
115 120 125
Arg Pro Met Gly Arg Val Val Gln Pro Leu Thr Lys Met Gly Ala Arg
130 135 140
Ile Arg Gly Arg Asp Gly Gly Arg Leu Ala Pro Leu Ala Ile Glu Gly
145 150 155 160
Thr Glu Leu Val Pro Ile Thr Tyr Asn Ser Pro Ile Ala Ser Ala Gln
165 170 175
Val Lys Ser Ala Ile Ile Leu Ala Gly Leu Asn Thr Ala Gly Glu Thr
180 185 190
Thr Ile Ile Glu Pro Ala Val Ser Arg Asp His Thr Glu Arg Met Leu
195 200 205
Ile Ala Phe Gly Ala Glu Val Thr Arg Asp Gly Asn Gln Val Thr Ile
210 215 220
Glu Gly Trp Pro Asn Leu Gln Gly Gln Glu Ile Glu Val Pro Ala Asp
225 230 235 240
Ile Ser Ala Ala Ala Phe Pro Met Val Ala Ala Leu Ile Thr Pro Gly
245 250 255
Ser Asp Ile Ile Leu Glu Asn Val Gly Met Asn Pro Thr Arg Thr Gly
260 265 270
Ile Leu Asp Leu Leu Leu Ala Met Gly Gly Asn Ile Gln Arg Leu Asn
275 280 285
Glu Arg Glu Val Gly Gly Glu Pro Val Ala Asp Leu Gln Val Arg Tyr
290 295 300
Ser Gln Leu Gln Gly Ile Glu Ile Asp Pro Thr Val Val Pro Arg Ala
305 310 315 320
Ile Asp Glu Phe Pro Val Phe Phe Val Ala Ala Ala Leu Ala Gln Gly
325 330 335
Gln Thr Leu Val Gln Gly Ala Glu Glu Leu Arg Val Lys Glu Ser Asp
340 345 350
Arg Ile Thr Ala Met Ala Asn Gly Leu Lys Ala Leu Gly Ala Ile Ile
355 360 365
Glu Glu Arg Pro Asp Gly Ala Leu Ile Thr Gly Asn Pro Asp Gly Leu
370 375 380
Ala Gly Gly Ala Ser Val Asp Ser Phe Thr Asp His Arg Ile Ala Met
385 390 395 400
Ser Leu Leu Val Ala Gly Leu Arg Cys Lys Glu Ser Val Leu Val Gln
405 410 415
Arg Cys Asp Asn Ile Asn Thr Ser Phe Pro Ser Phe Ser Gln Leu Met
420 425 430
Asn Ser Leu Gly Phe Gln Leu Glu Asp Val Ser His Gly
435 440 445
Claims (10)
1. a separation is from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof, and said encoding sequence comprises SEQ ID NO:1, it is characterized in that: be used to improve the resistance of plant to Glyphosate 62 IPA Salt.
2. separation as claimed in claim 1 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: high-resistance glyphosate EPSP synthase comprises polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQ ID NO:2 aminoacid sequence.
3. separation as claimed in claim 2 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: high-resistance glyphosate EPSP synthase is following polypeptide,
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have resistance glyphosate resistance and EPSP synthase activity by (a) polypeptides derived.
4. separation as claimed in claim 3 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: the enzymic activity of high-resistance glyphosate EPSP synthase is 25.57U/mg.
5. separation as claimed in claim 4 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: high-resistance glyphosate EPSP synthase
K mValue is 0.0443 ± 0.012.
6. separation as claimed in claim 5 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: high-resistance glyphosate EPSP synthase
K iValue is 0.746 ± 0.007.
7. separation as claimed in claim 6 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: the polynucleotide sequence of the said high-resistance glyphosate EPSP synthase of encoding is selected from down group: (a) polynucleotide of the SEQ ID NO:1 of the above-mentioned EPSP synthase polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.
8. separation as claimed in claim 7 is characterized in that from the high-resistance glyphosate EPSP synthase of soil and the purposes of encoding sequence thereof: the polynucleotide sequence of the SEQ ID NO:1 of said coding EPSP synthase polypeptide is selected from down a kind of of group:
(a) has the sequence of 1-1335 position among the SEQ ID NO:1;
(b) has the sequence of 1-1338 position among the SEQ ID NO:1.
One kind contain polynucleotide as claimed in claim 8 be used to improve the carrier of plant to the resistance of Glyphosate 62 IPA Salt.
10. one kind contains carrier as claimed in claim 9 and is used to improve the host cell of plant to the resistance of Glyphosate 62 IPA Salt.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158475B (en) * | 2015-09-18 | 2016-10-05 | 中国农业科学院生物技术研究所 | For detect the monoclonal antibody of transgenic crop to and Double-antibody sandwich enzymelinked immunosorbent detection kit |
| CN108291236A (en) * | 2015-09-30 | 2018-07-17 | 先锋国际良种公司 | Plant EPSP synthase and application method |
| CN110592039A (en) * | 2019-08-30 | 2019-12-20 | 浙江新安化工集团股份有限公司 | Application of hybridoma cell and monoclonal antibody generated by hybridoma cell in detection of AM79 EPSPS protein |
| CN112725365A (en) * | 2021-01-13 | 2021-04-30 | 浙江新安化工集团股份有限公司 | BNAM79EPSPS glyphosate-resistant gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973035A (en) * | 2004-06-24 | 2007-05-30 | 孟山都技术有限公司 | Microbial glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthases |
| CN101570744A (en) * | 2008-04-28 | 2009-11-04 | 中国农业科学院生物技术研究所 | EPSP synthase of high-resistance glyphosate as well as coded sequence and application thereof |
| US20110262995A1 (en) * | 2006-06-27 | 2011-10-27 | Cheryl Peters | GRG32: A Novel EPSP Synthase Gene Conferring Herbicide Resistance |
-
2012
- 2012-08-14 CN CN2012102877019A patent/CN102776161A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973035A (en) * | 2004-06-24 | 2007-05-30 | 孟山都技术有限公司 | Microbial glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthases |
| US20110262995A1 (en) * | 2006-06-27 | 2011-10-27 | Cheryl Peters | GRG32: A Novel EPSP Synthase Gene Conferring Herbicide Resistance |
| CN101570744A (en) * | 2008-04-28 | 2009-11-04 | 中国农业科学院生物技术研究所 | EPSP synthase of high-resistance glyphosate as well as coded sequence and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| BAZYLINSKI,D.A.等: "Magnetococcus marinus MC-1, complete genome", 《DATABASE GENBANK [ONLINE]》 * |
| JAMES A. SIKORSKI等: "EPSP Synthase: The Design and Synthesis of", 《PHOSPHORUS, SULFUR, AND SILICON》 * |
| 向文胜等: "EPSP合成酶的纯化与制备", 《农药学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105158475B (en) * | 2015-09-18 | 2016-10-05 | 中国农业科学院生物技术研究所 | For detect the monoclonal antibody of transgenic crop to and Double-antibody sandwich enzymelinked immunosorbent detection kit |
| CN108291236A (en) * | 2015-09-30 | 2018-07-17 | 先锋国际良种公司 | Plant EPSP synthase and application method |
| CN108291236B (en) * | 2015-09-30 | 2022-07-26 | 先锋国际良种公司 | Plant EPSP synthases and methods of use |
| CN110592039A (en) * | 2019-08-30 | 2019-12-20 | 浙江新安化工集团股份有限公司 | Application of hybridoma cell and monoclonal antibody generated by hybridoma cell in detection of AM79 EPSPS protein |
| CN112725365A (en) * | 2021-01-13 | 2021-04-30 | 浙江新安化工集团股份有限公司 | BNAM79EPSPS glyphosate-resistant gene and application thereof |
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