CN1384192A - Rice 5-enolpyruvate shikimate-3-phosphorylase and gene encoding this phosphrylase - Google Patents

Abstract

The present invention relates to nucleic acid molecular of rice 5-enolpyruvate shikimate-3-phosphorylase. The present invention also relates to plant cell after being converted and the cell regenerated plant. They express the features of resisting herbicide glyphosate.

Description

The gene of paddy rice 5-enol acetone shikimic acid-3-phosphate synthase and this enzyme of encoding

The present invention relates to paddy rice 5-enol acetone shikimic acid-3-phosphate synthase, the nucleic acid molecule of this enzyme of encoding uses this nucleic acid molecule to transform method of plant and the plant that is obtained by this method.

The present invention relates to carrier and the bacterium that contains these nucleic acid molecule in addition, and by described nucleic acid molecule plant transformed cell and plant, the transgenic plant of generation are significantly increased on the ability of antiweed glyphosate.

Die aromatischen Aminosaeuren comprises that phenylalanine, tyrosine, the synthetic of tryptophane are by shikimic acid pathway, via one section multistep enzymatic reaction process of branched acid.5-enol acetone shikimic acid-3-phosphate synthase (5-enolpyruvylshikimate 3-phosphate synthase, EPSPS, hereinafter to be referred as epsp synthase) be an important enzyme in the die aromatischen Aminosaeuren route of synthesis, also be wherein to study one of maximum enzyme.It is catalysis 3-phosphoric acid shikimic acid (Shikimate-3-phosphate reversibly, S3P) and phosphoenolpyruvic acid (phosphoenolpyrurate, PEP) be condensed into 5-enol acetone shikimic acid-3-phosphoric acid, and release inorganic phosphorus molecule (Gollub, E.1967, J.Biol.Chem.242,5323-5328).

From bacterium (Duncan, 1984, FEBS Lett.170,59-63), fungi (Charles, 1986, Nucleic Acids Res.14,2201-2213) and plant (Gasser, 1988 J.Bio.Chem.263 (9), 4280-4289; Klee, 1987, Mol.Gen.Gent.210,437-442) middle clone has obtained corresponding epsp synthase gene.The epsp synthase of plant and bacterium is a kind of monomeric enzyme, and molecular weight is 44, and 000-48 is between 000.And the epsp synthase of fungi is the part of arom prozyme.Find that by the aminoacid sequence comparative analysis to the epsp synthase of each kind of plant of having cloned and microorganism the aminoacid sequence of their mature peptides is very conservative, homology is higher, infers that they originate from same gene.

Utilize the method research epsp synthase of chemically modified, the result shows that Methionin, arginine and Histidine are necessary to the activity of keeping enzyme.23 Methionin and 28 important component parts that arginine is the active centre of enzyme in the petunia epsp synthase.If 23 lysine mutation arginine, enzyme active unaffected.But if 23 lysine mutation is L-glutamic acid or L-Ala, the activity of enzyme will completely lose.This shows that lysine side-chain positively charged side-chain radical has vital role in the activity of keeping epsp synthase.Can infer like this that the side-chain radical of 23 Methionin and 28 s' arginic positively charged can be discerned the negatively charged ion residue of substrate.Contrast the aminoacid sequence of acquired epsp synthase, near the aminoacid sequence above-mentioned two amino acid sites is very conservative.In addition, higher Histidine consistence in the aminoacid sequence of the epsp synthase of plant and microorganism of 385.

The epsp synthase of plant contains chloroplast(id) leads peptide, about about 72 amino acid of length, and no conservative property, the chloroplast(id) of different plant species is led peptide and is differed greatly.

Important feature of epsp synthase is that it can be suppressed by the glyphosate specificity.Glyphosate is a kind of interior nonselective herbicide of inhaling conduction, is applicable to multiple annual, the perennial Gramineae of control, and Cyperaceae and broadleaf weeds.The glyphosate specificity suppresses epsp synthase, makes the epsp synthase loss of activity, will directly disturb the biosynthesizing of phenylalanine and tyrosine, make not normal (the Steinr ü cken H.C. of endocellular chromosome, 1980, Biochem.Biophys.Res.Commum.94,1207-1212).After glyphosate was administered to plant, they transported and run up to position, particularly stem and the tip of a root of quick growth in plant, cause the stem and the root of the root of plant to be poisoned to death.At present, glyphosate is most widely used nonselective herbicide, can control 76 kinds in 78 kinds of the most serious weeds of whole world harm effectively.It has animal nontoxic, and the soil longevity of residure is short, is easy to advantages such as degraded.Major ingredient among the commercially available commodity Roundup is exactly a glyphosate.

Glyphosate is competitive inhibitor for the inhibition of epsp synthase to PEP, is noncompetitive inhibitor for S3P.It structurally is similar to PEP, combines with epsp synthase competitively, and the overslaugh epsp synthase combines with normal substrate PEP's, thereby causes the epsp synthase vigor to descend and even forfeiture.

Glyphosate has a remarkable shortcoming as weedicide, and it can't distinguish crops and weeds, and this can not use weedicide safely and effectively in the farmland.

For the genetically engineered of plant resistance glyphosate, two kinds of comparatively common strategies are arranged: the one, adopt the epsp synthase gene transformation of sudden change; The 2nd, the excessive generation of epsp synthase.

(1) imports the epsp synthase that suddenlys change

Researchists such as Stalker were separated to the bacterial strain to glyphosate performance resistance in 1985 from Salmonella typhimurium and E.coli, studies show that this resistance occurs on the AroA gene.Dna sequence analysis shows that two types sudden change has taken place the AroA gene: a class is the promoter region mutation of AroA gene, causes the raising of gene expression dose, but this sudden change is little to the patience raising of glyphosate; Another kind of is the structure gene origination point sudden change of AroA, causes the proline(Pro) of the 101st of enzyme to be replaced by Serine, and the epsp synthase that is produced has reduced the affinity with glyphosate herbicidal thus, thereby has improved the resistance to glyphosate.Change the AroA gene of this sudden change over to E.coli, the latter grows under the situation that the 2000ug/ml glyphosate exists, and the E.coli of wild-type promptly reaches 40% to growth-inhibiting being added with 100ug/ml.Comai etc. (Comai, L., 1985, Nature317 741-744) has made up the fusion gene of being transcribed signal by aroA and ocs.Then, these two kinds of genes are cloned into respectively on the plant expression vector, change Agrobacterium rhizogenes over to.Use the T-DNA transfer techniques of Ri plasmid media, the said gene structure is imported in tobacco, petunia, the tomato.In transfer-gen plant, can detect the mRNA and the activated zymoprotein of exogenous gene expression.Transfer-gen plant shines the resistance comparison of glyphosate and has improved 2 to 3 times.Particularly the epsp synthase of the isolating sudden change of E.coli to the susceptibility of glyphosate than normal enzyme low 99.98%.But, aspect growth conditions, transfer-gen plant contrast is produced slowly.Epsp synthase from the aroA genes encoding of bacterial origin does not contain chloroplast(id) and leads peptide, transform plant after, the epsp synthase of external source is present in the kytoplasm of vegetable cell.

(2) import excessive epsp synthase, lower the function influence of glyphosate

The activity that improves epsp synthase is an another kind of method comparatively commonly used in the present anti-herbicide gene engineering.The proposition of this method is based on following discovery.People such as Amrhein strengthen the selective pressure of glyphosate by increasing the concentration of glyphosate gradually, and the petunia clone of a glyphosate tolerant of separation acquisition (Amrhein N, et al., 1983, FEBS Lett., 157:191-196).Analyze the epsp synthase in this clone, the result shows that the copy number of the epsp synthase gene in the nuclear of this clone has increased 20 times, produces enzyme in a large number.The researchist utilizes above-mentioned clone to isolate the cDNA clone of the epsp synthase of petunia.Petunia epsp synthase gene is the nuclear gene of an about 9Kb, and 7 introns are arranged, and the zymoprotein molecular weight is 48KD.Be used to come from the promotor of cauliflower mosaic virus (CaMV) 35S and the epsp synthase gene cDNA of petunia has made up chimeric epsp synthase gene.The promotor of cauliflower mosaic virus (CaMV) 35S can make some encoding sequences show high-caliber constructive expression in all plant tissues.The activity of epsp synthase has increased by 20 times in transfer-gen plant, transfer-gen plant to the tolerance degree of glyphosate amount of application be kill more than 4 times of non-transgenic plant consumption (Shah, D.M., 1986, Science 233,478-481).

In the disclosed content of this paper, a lot of terms have been used.In this article, " gene " is meant all or part of nucleic acid fragment of a kind of specific protein of coding, and comprises the regulating and controlling sequence of this front, coding region (5 ' non-coding region) and back (3 ' non-coding region)." foreign gene " is meant the gene that does not have in the host living beings under the normal circumstances, import by transgenosis; Also making a comment or criticism often is present in the host living beings, but is imported the gene of another locus outside its natural gene seat in its genome again, and this variation can cause a kind of appearance of one or several additional copy of encoding sequence of native gene." mosaic gene " is meant the gene that comprises heterologous regulatory sequence and encoding sequence.

" promotor " is meant that one section can combine and initial accurately and effectively dna sequence dna of transcribing with the trans factor that RNA polymerase and some other influences are transcribed." constitutive promoter " is to express in each etap of organism and different sites by the driving purposes gene, and expression level does not have a class promotor of notable difference." tissue-specific promoter " is meant the class promotor that the driving purposes gene is expressed in organism particular organization." organ specific promoters " is meant the class promotor that the driving purposes gene is expressed in the organism certain organs." inducible promoter " is meant that (as light, temperature, chemical substance etc.) induce a class promotor of driving purposes genetic expression down under certain ambient conditions.

" combined promoter " is between the different promoters or promotor and the common formation of regulating and controlling sequence.Controlling element comprises the dna sequence dna that can strengthen exogenous gene expression or regulatory gene various sources of expressive site in plant.The sequence that is intended to strengthen exogenous gene expression is paddy rice Actin1 introne 1, corn Ubiquitin introne 1, corn sucrose synthase gene Sh1 introne 1, maize alcohol dehydrogenase 1-S gene intron 1, corn sucrose synthase Sh1 exon and rice EPSP synthase gene first intron.

" enhanser " is to strengthen the active one section cis regulating and controlling sequence of promoter transcription." intron " is one section non transcribed dna sequence dna, and indivedual introns of some gene can strengthen the transcriptional activity of promotor.

" plant expression vector " is meant the carrier of energy driving purposes gene at plant interior expression." conversion of integral level " is meant with the organism to be the method for transformation of transformation receptor, and it comprises gene transformation, sexual cell infusion method, blastular or the ovary injection etc. of pollen tube passage method mediation.

" genomic library " is the cell carrier that a cover comprises genomic DNA fragment.Carrier commonly used has lambda particles phage, cos plasmid, BAC (Bacteria Artificial Chromosome) carrier, YAC (Yeast Artificial Chromosome) and TAC (Transformation-competentArtificial Chromosome) carrier." cDNA library " is to become the cDNA strand from the mRNA reverse transcription, becomes double-stranded cDNA molecule through the role transformation of archaeal dna polymerase, is inserted in the appropriate carriers then and cloning and the set that forms.

" degeneracy " is the phenomenon of the corresponding a kind of coded amino acid of a plurality of codons that causes of the slewability owing to the 3rd of genetic codon.

In this description, " plant " means any multi-cell organism that can carry out photosynthetic differentiation, and " vegetable cell " means and derive from plant and can form for example callus or differentiated tissues any cell of embryo or plant part or seed for example of indifferent tissue.

In existing plant epsp synthase gene of having cloned, mainly at dicotyledons; And, study lessly for monocotyledons, the proteic separation of epsp synthase (Forlanni, G., 1994.Plant Physiol.105, the 1107-1114 of Chinese sorghum and corn are only arranged; Ream, J.E, 1988 Plant Physiol.87 232-238), still do not derive from the nucleotide sequence report in the literature of monocotyledons epsp synthase gene at present.

An object of the present invention is to provide 5-enol acetone shikimic acid-3-phosphate synthase of paddy rice, fragment or functional deriv that this enzyme has aminoacid sequence shown in the SEQ ID NO:2 or has this enzymic activity.Wherein, functional deriv be meant by one or more amino acid whose replacements, disappearance, increase or modification obtain and have the derivative of 5-enol acetone shikimic acid-3-phosphate synthetase activity simultaneously.

Another object of the present invention provides a kind of 5-enol acetone shikimic acid-3-phosphate synthase of coding or has the fragment of this enzymic activity or the nucleic acid molecule of functional deriv.This molecule can be the nucleic acid molecule that (1) contains the nucleotide sequence shown in the SEQ ID NO:1; (2) coding contains the nucleic acid sequences to proteins of aminoacid sequence shown in the SEQ ID NO:2; (3) contain the nucleic acid molecule of the nucleotide sequence shown in the SEQ IDNO:3; (4) nucleotide sequence that causes because of the degeneracy of genetic code is different but still coding contains the proteinic nucleic acid molecule of aminoacid sequence shown in the SEQ ID NO:2 with the sequence of (1), (2) or (3) described nucleic acid molecule.Described nucleic acid molecule can be a dna molecular.Described dna molecular can be cDNA molecule or genomic dna molecule.The aminoacid sequence of described nucleic acid molecule encoding can have homology more than 85% with SEQ ID NO:2.

A further object of the present invention provides a kind of paddy rice 5-enol acetone shikimic acid-cell of 3-phosphate synthase gene production antiweed and method of plant utilized, this method may further comprise the steps: (1) with the upstream extremity of paddy rice 5-enol acetone shikimic acid-3-phosphate synthase gene along on the nucleic acid fragment that is connected in promotor on the sense orientation, its downstream end is connected in a kind of nucleic acid fragment that is used to control the suitable regulating and controlling sequence of Transcription Termination of coding, and then makes up plant expression vector; (2) plant expression vector that builds is imported vegetable cell; (3) vegetable cell after will transforming under the suitable culture condition is regenerated, and obtains expressing the plant of goal gene.

In aforesaid method, described promotor comprises constitutive promoter, organ specific promoters, tissue-specific promoter, inducible promoter and the combined promoter of being made up of promotor and controlling element.Used method for transformation comprises the method for transformation of method for transformation, electric shocking method and the integral level of agriculture bacillus mediated method for transformation, particle bombardment, protoplastis mediation.Wherein, described weedicide is N-(phosphine methylol) glycine isopropyl amine salt (N-(phosphonomethyl) glycine, a glyphosate).Perhaps, described weedicide comprises glyphosate precursor, glyphosate derivative and is the mixture of main component with the glyphosate.

Of the present inventionly provide plant and the filial generation thereof that obtains by aforesaid method by a purpose.This plant can be a monocotyledons, also can be dicotyledons.For example paddy rice, corn, wheat, barley, Chinese sorghum; Perhaps tobacco, cotton, willow, soybean, sweet potato, potato, Chinese cabbage, wild cabbage or green pepper.

Use a kind of mosaic gene of herbicide glyphosate resistance in the method for the present invention, on the transcriptional orientation of this mosaic gene, comprise: a promoter region, chloroplast(id) is led peptide, the sequence of the enzyme gene of coding glyphosate tolerant and the polyadenylic acid signaling zone of 3 ' terminal untranslated.

Acquisition to the rice EPSP synthetic enzyme among the present invention is by the screening by hybridization to the rice genome library.After obtaining positive colony, carry out subclone then, and finally obtain the genom sequence of rice EPSP synthetic enzyme.These are known the researchist in this area, can be referring to people such as Sambrook, and molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989).

The clone of rice EPSP synthase gene full length cDNA sequence adopts the RT-PCR method, is template with reverse transcription cDNA one chain of the total RNA of bright extensive 63 blades of paddy rice, carries out pcr amplification.These all are that researchist in this area is known, can be referring to people such as Sambrook, and molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).

The plasmid that contains the rice EPSP synthase gene generally contain controlling elements such as suitable promotor, enhanser, and suitable selection resistant gene is (as ampicillin resistance gene; Kalamycin resistance gene etc.), the regrouping process of plasmid comprise that the enzyme of extraction, the plasmid of plasmid is cut, being connected of the segmental recovery of purpose and purpose fragment and related vector.These all are that researchist in this area is known, can be referring to people such as Sambrook, and the molecule gram falls: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).

For a person skilled in the art, the multiple eukaryotic method that a dna sequence dna is imported higher plant is arranged.These methods comprise particle bombardment (referring to (1987) Nature327:70-73 such as Klein); The plasmid-mediated method for transformation of Ti of Agrobacterium and Ri is (referring to (1985) Bio/Technology 3:241 such as Pacciotti; Potrykus etc. (1985) Mol.Gen.Genet.199:183-188; Hiei etc. (1994) The Plant J, 6:271-282).Also has other method for transformation in addition, as foreign DNA direct guiding method and electric shocking method etc.Cell is in case after transforming, can be regenerated by those skilled in the art.Behind the transgenosis individual plant that has obtained to transform with one of aforesaid method, in the present invention, can screen resistant plant by kantlex, and the method for binding molecule detection, identify genetically modified integration situation, identify the transformed plant that obtains with herbicide glyphosate, obtained the transgenic plant of resistance glyphosate.

Brief Description Of Drawings

Fig. 1: plasmid pBEF restriction enzyme mapping.Contain rice EPSP synthase gene cDNA complete sequence, the plasmid skeleton is from pBlueSK.

Fig. 2: plasmid pSPROK restriction enzyme mapping.P-35S is the 35S promoter of CaMV, belongs to constitutive promoter.

Fig. 3: plasmid pSPEF restriction enzyme mapping.P-35S is the 35S promoter of CaMV, belongs to constitutive promoter, and the rice EPSP synthase gene is connected under the promotor.The plasmid skeleton is from plasmid pSPROK.

Fig. 4: plasmid pNPT-II restriction enzyme mapping.This plasmid can be used to transform dicotyledons, contains left and right sides border sequence, and the plant selectable marker gene is that neomycin phosphoric acid is translated the enzyme gene.The bacterium selective marker is a kantlex.

Fig. 5: plasmid pCSPEF-NptII restriction enzyme mapping.This plasmid can be used to transform dicotyledons.P-35S is the 35S promoter adjusting and controlling rice epsp synthase gene of CaMV.NptII is that neomycin phosphotransferase gene is as selection markers.The plasmid skeleton is from pC2300.

Further illustrate the present invention below in conjunction with embodiment.In this application, used term and abbreviation are general term of those skilled in the art and abbreviation.Embodiment 1: the segmental acquisition of rice EPSP synthase gene Partial cDNA

According to paddy rice 5-enol acetone shikimic acid-3-phosphate synthase part mRNA sequence information that GeneBank sequence number AB016765 provides, synthetic a pair of specific primer utilizes the RT-PCR method to obtain the cDNA sequence of the EPSP of part.The primer is among the PCR:

Upstream primer EP1:ggctctctgtggaagcag

Downstream primer EP2:gtgatcatcgtaggtgtc

Pcr amplification obtains a specificity band, is about 1000bp, called after EPSP _(p),, obtain recombinant plasmid, and check order the EcoRV site of this fragment cloning to pBluescript KS (+).The result of order-checking shows that the part paddy rice epsp synthase gene order consistence that AB016765 provides among 1000bp fragment that the clone obtains and the GeneBank is 100%, the sudden change that does not produce base.Embodiment 2: the screening of bright extensive 63 genomic libraries of rice varieties

Genomic library is by bright extensive 63 the TAC library that Liu Yaoguang researcher provides, is stored in 8 96 orifice plates, and 100 clones of every Kong Hanyue, the segmental mean length of each clone's insertion is 40Kb.Use EPSP _(p)For probe screens the library.Use the random priming label probe, adopt following labeled reactant: get 20ng EPSP (p) the dna fragmentation template that serves as a mark, after adding 2 μ l (0.5 μ g/ μ l) random primer and adding TE to 10 μ l, 95 ℃ of sex change 5 minutes, took out immediately ice bath 5 minutes, add then 11 μ l mark damping fluids (contain dATP, dGTP, dTTP), big fragment of dna polymerase i and the 10-20 μ Ci a-of 2U ³²P-dCTP, 37 ℃ were reacted 2-3 hour.Nylon membrane is put into hybridization bag, add an amount of (4-6ml/100cm ²) hybridization solution (5 * SSC; 0.1%, SDS; 0.1%, Sarkosyl; 0.6%, Blocking Reagent; 5%, Dextran Sulfate), 65 ℃ of prehybridizations are 1 hour.Add the probe after the sex change then, under 65 ℃, hybridized liquid (16-20 hour).Take out nylon membrane, (0.2 * SSC, 0.1 * SDS) washes 2-3 time, each about 60 minutes with washing lotion down at 65 ℃.After washing film, wrap compressing tablet with preservative film.-70 ℃ of following radioautograph.

First round screening has obtained two positives, 5-10C and 5-11C.Draw the bacterium liquid in a small amount of positive hole, at certain extent of dilution coated plate, to obtain the single bacterium colony that dispersion is opened.The single bacterium colony of picking, (every hole contains 250 μ l LB to place every hole of 384 orifice plates respectively; IPTG10 mg/L; Km 50mg/L).384 orifice plates after 16 hours, change film 37 ℃ of cultivations, are used for hybridization.Second takes turns hybridization has obtained 20 positive colonies altogether, is named as E5-E25 respectively.Extract the plasmid of positive colony, cut, change film behind the electrophoresis with the HindIII enzyme.Results of hybridization shows that the positive colony of determining has 16, is respectively E5, E7, E8, E9, E11, E12, E13, E15, E16, E17, E18, E20, E21, E23, E24, E25.The Hind III restriction enzyme mapping of analyzing positive colony as can be seen, it is identical that these 16 clones insert clip size, about 40kb, the banding pattern unanimity, we judge that they are with a kind of clone.Embodiment 3: the subclone of positive colony

Choose positive colony E11 and E20 arbitrarily, select BglII for use, DraI, SalI, ScaI, the XbaI enzyme cutting plasmid changes film.Use EPSP _(p)Hybridize as probe.Results of hybridization shows that the DraI enzyme is cut swimming lane, and hybridization is a band.Corresponding this band that reclaims, and be inserted into the EcoRV site of cloning vector pBluescriptKS (+), schedule of operation according to BIO-RAD company electric exciter, adopt electrization Transformed E .coli DH5 α, select to select white colony on the substratum at the penbritin that contains X-gal and IPTG, obtain recombinant plasmid pBlueE7.Check order according to Applied Biosystem company Sequenase Dye-Primer Sequencing Kit method, give the order-checking of the white good biotech company in Shanghai.Sequencing result shows, subclone E7 total length 3, and 370bp comprises 7 exons and 6 introns.Have 5 complete ' terminal sequence, but 3 ' end is incomplete.The paddy rice 5-enol acetone shikimic acid-3-phosphate synthase part mRNA sequence information that provides according to E7 sequence and GeneBank sequence number AB016765, design two primers, plasmid with positive colony E11 is a template, add 10 * Taq DNA enzyme buffer liquid, 5 μ l successively, dNTPs (2.5umol/L) 5 μ l, TaqDNA polysaccharase 1U adds water at last and mends to cumulative volume 50 μ l, adds 50 μ l paraffin oils again and is covered in the surface.Carry out the PCR reaction by following condition: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1min carry out 35 circulations altogether; Extend 10min in 72 ℃ at last.Amplified production is electrophoretic separation in 1% sepharose.3 ' end of order-checking amplified fragments is in conjunction with the E7 sequence, with the complete sequence of the epsp synthase gene that obtained total length.The acquisition of embodiment 4:EPSP synthetic enzyme cDNA complete sequence

Complete sequence according to the epsp synthase gene, utilize the ORF (Open Reading Fragment) in the DNA Star software analysis genome sequence, ORF is according to synthetic following primer successively, EP3:5 ' gct cta gag cat ggc ggc gac cat gg 3 ' EP4:5 ' ggg gta ccc caa cag aaccta gac 3 ' gets excellent 63 reverse transcription product of Xian 2 μ l as above-mentioned template, add 10 * TaqDNA enzyme buffer liquid, 5 μ l successively, dNTPs (2.5umol/L) 5 μ l, EP5 primer 10pmol, EP6 primer 10pmol, Taq archaeal dna polymerase 1U, add water at last and mend, add 50 μ l paraffin oils again and be covered in the surface to cumulative volume 50 μ l.Carry out the PCR reaction by following condition: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1min carry out 35 circulations altogether; Extend 10min in 72 ℃ at last.Amplified production is electrophoretic separation in 1% sepharose.In order to narrate conveniently the RT-PCR product called after EPSP (h) in this step.The PCR product reclaims amplified fragments with the low melting-point agarose electrophoresis after XbaI and KpnI enzyme are cut, and is inserted into XbaI and the KpnI site of cloning vector pBluescriptKS (+).According to the schedule of operation of BIO-RAD company electric exciter, adopt electrization Transformed E .coli DH5 α, select to select white colony on the substratum at the penbritin that contains X-gal and IPTG, through detailed enzymolysis analysis, obtain recombinant plasmid pBEPSP (h).Check order according to Applied Biosystem company Sequenase Dye-Primer Sequencing Kit method, give the order-checking of the white good biotech company in Shanghai.Embodiment 5: the structure of dicotyledons conversion carrier pCSPEF-NptII

Select the expression of plants intermediate carrier of pSPROK, have CaMV35S promotor and no terminator on it as the rice EPSP synthase gene; Select the plant expression vector of pCNPT-II, have the T-DNA border sequence RB and the LB that derive from Ti-plasmids on it, between border sequence, have plant selectable marker NptII and MCS sequence as the rice EPSP synthase gene.PSPEF is an interstitial granules in the expression of plants, and pCSPEF-NptII is final plant expression vector.

The electric exciter of BIO-RAD company is used in the conversion of root Agrobacterium LBA4404, competent preparation and the electricity method that swashs is carried out with reference to product description, (Rif25, Km50) dull and stereotyped last 28 ℃ of dark cultivations promptly have the bacterium colony of conversion to grow after 2 days, further extract plasmid and identified from transformed bacteria at YEB.Embodiment 6: tobacco transforms the acquisition of seedling

The root Agrobacterium LBA4404 inoculation 20ml YEB substratum (Km50mg/L that will contain plant expression vector, Ref 50mg/L) 28 ℃ of overnight incubation, cultivated 3 hours by 2% middle continuation of YEB substratum (Syringylethanone 0.2%) that is forwarded to antibiotic-free, bacterium liquid suitably dilutes with the MS substratum and is used for Plant Transformation.

Adopt Ye Panfa (Horsch, 1985, Methods in Enzymology, 118:627-641) transformation of tobacco, the root Agrobacterium LBA4404 difference transformation of tobacco that will contain plasmid pCSPEF-NptII and pCNPT-II, wherein pCNPT-II is not for containing the plant expression vector of paddy rice epsp synthase gene, with in contrast.After transforming for two weeks, containing the enterprising row filter of 75mg/L kantlex screening culture medium.Unconverted contrast flavescence gradually, bleach, the blade periphery that transforms has then grown budlet.Continuation is containing resistance glyphosate (effective ingredient is N-(phosphine methylol) glycine isopropyl amine salt) the enterprising row filter of screening culture medium, and Pignus pignoris grain pCNPT-II tobacco leaf bleaches gradually.And change pCSPEF-NptII plasmid tobacco leaf, the energy normal growth, and differentiate budlet.Downcut budlet, change in the root media, treat the seedling well developed root system, after plant is strong, spray screening, obtained the transgenic tobacco plant of antiweed glyphosate with herbicide glyphosate.

Sequence table (1) general information: (i) applicant:

(A) name:

(B) street: No. 3, Da Tun road first

(C) city: Beijing

(D) country: China

(E) (ii) invention exercise question of postcode (ZIP) .:100101: the nucleic acid molecule of coding paddy rice 5-enol acetone shikimic acid-3-phosphate synthase is sequence number (iii): 4 (iv) computer-reader forms:

(A) media type: floppy disk

(B) computer: but IBM PC compatible

(C) operating system:

(D) software: the information of (2) SEQ ID NO:1

(i) sequence signature:

(A) length: 3661 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: genomic dna sequence

(iii) primary source:

(A) biology: bright extensive 63 genomic libraries

(B) strain: long-grained nonglutinous rice

(C) types of organization: blade

(iv) direct sources:

(A) result of positive colony subclone fragment and PCR product splicing

(B) clone: pBlueE7

(v) sequence description: SEQ ID NO:1

(2) information of SEQ ID NO:2:

(i) sequence signature:

(A) length: 515 amino acid

(B) type: amino acid

(C) topological framework: linearity

(ii) molecule type: protein is sequence description (ii): SEQ ID NO:2Met Ala Ala Thr Met Ala Ser Asn Ala Ala Ala Ala Ala Ala Val Ser Leu Asp Gln Ala Val

5 10 15 20Ala?Ala?Ser?Ala?Ala?Phe?Ser?Ser?Arg?Lys?Gln?Leu?Arg?Leu?Pro?Ala?Ala?Ala?Arg?Gly?Gly

25 30 35 40Met?Arg?Val?Arg?Val?Arg?Ala?Arg?Gly?Arg?Arg?Glu?Ala?Val?Val?Val?Ala?Ser?Ala?Ser?Ser

45 50 55 60Ser?Ser?Val?Ala?Ala?Pro?Ala?Ala?Lys?Ala?Glu?Glu?Ile?Val?Leu?Gln?Pro?Ile?Arg?Glu?Ile 65 70 75 80Ser?Gly?Ala?Val?Gln?Leu?Pro?Gly?Ser?Lys?Ser?Leu?Ser?Asn?Arg?Ile?Leu?Leu?Leu?Ser?Ala85 90 95 100 105Leu?Ser?Glu?Gly?Thr?Thr?Val?Val?Asp?Asn?Leu?Leu?Asn?Ser?Glu?Asp?Val?His?Tyr?Met?Leu

110 115 120 125Glu?Ala?Leu?Lys?Ala?Leu?Gly?Leu?Ser?Val?Glu?Ala?Asp?Lys?Val?Ala?Lys?Arg?Ala?Val?Val

130 135 140 145Val?Gly?Cys?Gly?Gly?Lys?Phe?Pro?Val?Glu?Lys?Asp?Ala?Lys?Glu?Glu?Val?Gln?Leu?Phe?Leu

150 155 160 165Gly?Asn?Ala?Gly?Thr?Ala?Met?Arg?Pro?Leu?Thr?Ala?Ala?Val?Thr?Ala?Ala?Gly?Gly?Asn?Ala 170 175 180 185Thr?Tyr?Val?Leu?Asp?Gly?Val?Pro?Arg?Met?Arg?Glu?Arg?Pro?Ile?Gly?Asp?Leu?Val?Val?Gly190 195 200 205 210Leu?Lys?Gln?Leu?Gly?Ala?Asp?Val?Asp?Cys?Phe?Leu?Gly?Thr?Glu?Cys?Pro?Pro?Val?Arg?Val

215 220 225 230Lys?Gly?Ile?Gly?Gly?Leu?Pro?Gly?Gly?Lys?Val?Lys?Leu?Ser?Gly?Ser?Ile?Ser?Ser?Gln?Tyr

235 240 245 250Leu?Ser?Ala?Leu?Leu?Met?Ala?Ala?Pro?Leu?Ala?Leu?Gly?Asp?Val?Glu?Ile?Glu?Ile?Ile?Asp

255 260 265 270Lys?Leu?Ile?Ser?Ile?Pro?Tyr?Val?Glu?Met?Thr?Leu?Arg?Leu?Met?Glu?Arg?Phe?Gly?Val?Lys 275 280 285 290Ala?Glu?His?Ser?Asp?Ser?Trp?Asp?Arg?Phe?Tyr?Ile?Lys?Gly?Gly?Gln?Lys?Tyr?Lys?Ser?Pro295 300 305 310 315Gly?Asn?Ala?Tyr?Val?Glu?Gly?Asp?Ala?Ser?Ser?Ala?Ser?Tyr?Phe?Leu?Ala?Gly?Ala?Ala?Ile

320 325 330 335Thr?Gly?Gly?Thr?Val?Thr?Val?Gln?Gly?Cys?Gly?Thr?Thr?Ser?Leu?Gln?Gly?Asp?Val?Lys?Phe

340 345 350 355Ala?Glu?Val?Leu?Glu?Met?Met?Gly?Ala?Lys?Val?Thr?Trp?Thr?Asp?Thr?Ser?Val?Thr?Val?Thr

360 365 370 375Gly?Pro?Pro?Arg?Glu?Pro?Tyr?Gly?Lys?Lys?His?Leu?Lys?Ala?Val?Asp?Val?Asn?Met?Asn?Lys 380 385 390 395Met?Pro?Asp?Val?Ala?Met?Thr?Leu?Ala?Val?Val?Ala?Leu?Phe?Ala?Asp?Gly?Pro?Thr?Ala?Ile400 405 410 415 420Arg?Asp?Val?Ala?Ser?Trp?Arg?Val?Lys?Glu?Thr?Glu?Arg?Met?Val?Ala?Ile?Arg?Thr?Glu?Leu

425 430 435 440Thr?Lys?Leu?Gly?Ala?Ser?Val?Glu?Glu?Gly?Pro?Asp?Tyr?Cys?Ile?Ile?Thr?Pro?Pro?Glu?Lys

445 450 455 460Leu?Asn?Ile?Thr?Ala?Ile?Asp?Thr?Tyr?Asp?Asp?His?Arg?Met?Ala?Met?Ala?Phe?Ser?Leu?Ala

The information of 465 470 475 480Ala Cys Ala Asp Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro Asn, 485 490 495 500Tyr Phe Asp Val Leu Ser Thr Phe Val Arg Asn505,510 515 (3) SEQ ID NO:3:

(iii) sequence signature:

(D) length: 1,604 base pair

(E) type: nucleic acid

(F) topological framework: linearity

(ii) molecule type: cDNA sequence

(iii) sequence description: SEQ ID NO:31 GCAATGGCGGCGACC ATG GCG TCC AAC GCC GCG GCT GCG GCG GCG GTG TCC CTG GAC CAG GCC GTG GCG GCG TCG GCG 78

M A S N A A A A A A V?r S L D Q A V A A S A79 GCG?TTC?TCG?TCG?CGG?AAG?CAG?CTG?CGG?CTG?CCC?GCC?GCG?GCG?CGC?GGG?GGG?ATG?CGG?GTG?CGG?GTG?CGG?GCG?CGG 153

A F S S R K Q L R L P A A A R G G M R V R V R A R154 GGG?CGG?CGG?GAG?GCG?GTG?GTG?GTG?GCG?TCC?GCG?TCG?TCG?TCG?TCG?GTG?GCA?GCG?CCG?GCG?GCG?AAG?GCG?GAG?GAG 228

G R R E A V V V A S A S S S S V A A P A A K A E E229 ATC?GTG?CTC?CAG?CCC?ATC?AGG?GAG?ATC?TCC?GGG?GCG?GTT?CAG?CTG?CCA?GGG?TCC?AAG?TCG?CTC?TCC?AAC?AGG?ATC 303

I V L Q P I R E I S G A V Q L P G S K S L S N R I304 CTC?CTC?CTC?TCC?GCC?CTC?TCC?GAG?GGC?ACA?ACA?GTG?GTG?GAC?AAC?TTG?CTG?AAC?AGT?GAG?GAT?GTT?CAC?TAC?ATG 378

L L L S A L S E G T T V V D N L L N S E D V H Y M379 CTT?GAG?GCC?CTG?AAA?GCC?CTC?GGG?CTC?TCT?GTG?GAA?GCA?GAT?AAA?GTT?GCA?AAA?AGA?GCT?GTA?GTC?GTT?GGC?TGT 453

L E A L K A L G L S V E A D K V A K R A V V V G C454 GGT?GGC?AAG?TTT?CCT?GTT?GAG?AAG?GAT?GCG?AAA?GAG?GAA?GTG?CAA?CTC?TTC?TTG?GGG?AAC?GCT?GGA?ACT?GCA?ATG 528

G G K F P V E K D A K E E V Q L F L G N A G T A M529 CGA?CCA?TTG?ACA?GCA?GCC?GTG?ACT?GCT?GCT?GGT?GGA?AAT?GCA?ACT?TAT?GTG?CTT?GAT?GGA?GTG?CCA?CGA?ATG?AGG 603

R P L T A A V T A A G G N A T Y V L D G V P R M R604 GAG?AGA?CCG?ATT?GGT?GAC?TTG?GTT?GTC?GGG?TTG?AAA?CAA?CTT?GGT?GCG?GAT?GTC?GAC?TGT?TTC?CTT?GGC?ACT?GAA 678

E R P I G D L V V G L K Q L G A D V D C F L G T E679 TGC?CCA?CCT?GTT?CGT?GTC?AAG?GGA?ATT?GGA?GGA?CTT?CCT?GGT?GGC?AAG?GTT?AAG?CTC?TCT?GGT?TCC?ATC?AGC?AGT 753

C P P V R V K G I G G L P G G K V K L S G S I S S754 CAG?TAC?TTG?AGT?GCC?TTG?CTG?ATG?GCT?GCT?CCT?TTG?GCC?CTT?GGG?GAT?GTG?GAG?ATC?GAA?ATC?ATT?GAC?AAA?CTA 828

Q Y L S A L L M A A P L A L G D V E I E I I D K L829 ATC?TCC?ATT?CCT?TAC?GTT?GAA?ATG?ACA?TTG?AGA?TTG?ATG?GAG?CGT?TTT?GGT?GTG?AAG?GCA?GAG?CAT?TCT?GAT?AGT 903

I S I P Y V E M T L R L M E R F G V K A E H S D S904 TGG?GAC?AGA?TTC?TAT?ATT?AAG?GGA?GGG?CAG?AAG?TAC?AAA?TCT?CCT?GGA?AAT?GCC?TAT?GTT?GAA?GGT?GAT?GCC?TCA 978

W D R F Y I K G G Q K Y K S P G N A Y V E G D A S?979 AGC?GCG?AGC?TAT?TTC?TTG?GCT?GGT?GCT?GCA?ATC?ACT?GGA?GGC?ACT?GTG?ACA?GTT?CAA?GGT?TGT?GGT?ACG?ACC?AGT 1053

S A S Y F L A G A A I T G G T V T V Q G C G T T S1054 TTG?CAG?GGT?GAT?GTC?AAA?TTT?GCT?GAG?GTA?CTT?GAG?ATG?ATG?GGA?GCA?AAG?GTT?ACA?TGG?ACT?GAC?ACC?AGT?GTA 1128

L Q G D V K F A E V L E M M G A K V T W T D T S V1129 ACC?GTA?ACT?GGT?CCA?CCA?CGT?GAG?CCT?TAT?GGG?AAG?AAA?CAC?CTG?AAA?GCT?GTT?GAT?GTC?AAC?ATG?AAC?AAA?ATG 1203

T V T G P P R E P Y G K K H L K A V D V N M N K M1204 CCT?GAT?GTT?GCC?ATG?ACC?CTT?GCC?GTT?GTT?GCA?CTC?TTC?GCT?GAT?GGT?CCA?ACT?GCT?ATC?AGA?GAT?GTG?GCT?TCC 1278

P D V A M T L A V V A L F A D G P T A I R D V A S1279 TGG?AGA?GTA?AAG?GAA?ACC?GAA?AGG?ATG?GTT?GCA?ATT?CGG?ACC?GAG?CTA?ACA?AAG?CTG?GGA?GCA?TCG?GTT?GAA?GAA 1353

W R V K E T E R M V A I R T E L T K L G A S V E E1354 GGT?CCT?GAC?TAC?TGC?ATC?ATC?ACC?CCA?CCG?GAG?AAG?CTG?AAC?ATC?ACG?GCA?ATC?GAC?ACC?TAC?GAT?GAT?CAC?AGG 1428

G P D Y C I I T P P E K L N I T A I D T Y D D H R1429 ATG?GCC?ATG?GCC?TTC?TCC?CTC?GCT?GCC?TGC?GCC?GAC?GTG?CCC?GTG?ACG?ATC?AGG?GAC?CCT?GGT?TGC?ACC?CGC?AAG 1503

M A M A F S L A A C A D V P V T I R D P G C T R K1504 ACC?TTC?CCC?AAC?TAC?TTC?GAC?GTT?CTA?AGC?ACT?TTC?GTC?AGG?AAC?TGA?ACTGAGCTTTTAAAAGAGTGAGTCTAGGTTCTGTTG 1588

T F P N Y F D V L S T F V R N ***

Claims (16)

1. one kind has the paddy rice 5-enol acetone shikimic acid-3-phosphate synthase of aminoacid sequence shown in the SEQ ID NO:2 or fragment or the functional deriv with this enzymic activity.

2. the described enzyme of the claim 1 of encoding or have the fragment of this enzymic activity or the nucleic acid molecule of functional deriv.

3. by the described nucleic acid molecule of claim 2, this molecule is selected from:

(a) contain the nucleic acid molecule of the nucleotide sequence shown in the SEQ ID NO:1;

(b) coding contains the nucleic acid sequences to proteins of aminoacid sequence shown in the SEQ ID NO:2;

(c) contain the nucleic acid molecule of the nucleotide sequence shown in the SEQ ID NO:3;

(d) nucleotide sequence that causes because of the degeneracy of genetic code and (a) and (b) or (c) sequence of described nucleic acid molecule is different but still coding contains the proteinic nucleic acid molecule of aminoacid sequence shown in the SEQ ID NO:2.

4. by the described nucleic acid molecule of claim 3, it is a dna molecular.

5. by the described nucleic acid molecule of claim 4, wherein, described dna molecular is cDNA molecule or genomic dna molecule.

6. by the described nucleic acid molecule of claim 2, it is characterized in that its amino acid sequence coded and SEQ ID NO:2 have the homology more than 85%.

7. one kind is utilized paddy rice 5-enol acetone shikimic acid-cell of 3-phosphate synthase gene production antiweed and the method for plant, and this method may further comprise the steps:

(a) with the upstream extremity of paddy rice 5-enol acetone shikimic acid-3-phosphate synthase gene along on the nucleic acid fragment that is connected in promotor on the sense orientation, its downstream end is connected in a kind of nucleic acid fragment that is used to control the suitable regulating and controlling sequence of Transcription Termination of coding, and then makes up plant expression vector;

(b) plant expression vector that builds is imported vegetable cell;

(c) vegetable cell after will transforming under the suitable culture condition is regenerated, and obtains expressing the plant of goal gene.

8. by the described method of claim 7, it is characterized in that described promotor comprises constitutive promoter, organ specific promoters, tissue-specific promoter, inducible promoter and the combined promoter of being made up of promotor and controlling element.

9. by the described method of claim 7, it is characterized in that used method for transformation comprises the method for transformation of method for transformation, electric shocking method and the integral level of agriculture bacillus mediated method for transformation, particle bombardment, protoplastis mediation.

10. by the described method of claim 7, wherein, described weedicide is N-(phosphine methylol) glycine isopropyl amine salt (N-(phosphonomethyl) glycine, a glyphosate).

11. by the described method of claim 10, wherein, described weedicide comprises glyphosate precursor, glyphosate derivative and is the mixture of main component with the glyphosate.

12. the plant and the filial generation thereof that obtain by the described method of claim 7.

13. the plant of claim 10 is characterized in that, this plant is a monocotyledons.

14. the described plant of claim 12, it is paddy rice, corn, wheat, barley or Chinese sorghum.

15. the plant of claim 10 is characterized in that, this plant is a dicotyledons.

16. the described plant of claim 13, it is tobacco, cotton, willow, soybean, sweet potato, potato, Chinese cabbage, wild cabbage or green pepper.

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