CN102321640A - Herbicide resistant gene, and application of herbicide resistant gene in genetically modified crops - Google Patents
Herbicide resistant gene, and application of herbicide resistant gene in genetically modified crops Download PDFInfo
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Abstract
The present invention discloses herbicide resistant gene. According to the gene: 1) an amino acid sequence of the protein encoded by the herbicide resistant gene shares at least more than 82% sequence identity with the amino acid sequence represented by the SEQ ID NO:3; 2) the protein encoded by the herbicide resistant gene can provide resistance for at least two herbicides selected from acetolactate synthase (ALS) inhibitor herbicide, anti-protoporphyrinogen oxidase (PPO) inhibitor herbicide, p-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide and photosystem II inhibitory type herbicide. The present invention further discloses the herbicide resistant protein encoded by the herbicide resistant gene. The present invention further discloses a use of the herbicide resistant gene: the herbicide resistant plant is obtained through the herbicide resistant gene transferring, or the herbicide resistant gene is adopted as the screening gene for the plant transgene.
Description
Technical field
The invention belongs to plant genetic engineering field, specifically, the present invention relates to the gene and the encoded protein matter thereof of weedicides such as anti-nicosulfuron.
Background technology
Need controlling weeds in the proportion of crop planting process.If farm crop can obtain the resistance capacity to a kind of broad-spectrum herbicide, the weeds of so this farm crop just can prevent and treat through spraying broad-spectrum herbicide, thereby make the farm crop can normal growth and the killing of being selected property of weeds.This weed control method is simple, efficient, and low-cost.
Farm crop can obtain the resistance to weedicide through genetically engineered.For example, the plant resistance 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS) that can pass through transgene expression Agrobacterium (Agrobacterium tumefaciens sp CP4) obtains the ability of resistance glyphosate.The farm crop of this resistance glyphosate are quoted (USP 453590,4769061,5094945) aborning.
Cytochrome P450 is very large gene family members.P450 gene in the common kind of plant can have more than 200.Part P450 gene comes to light can degrading herbicide.The cytochrome P450 gene P4507A1 of a kind of animal, yeast NADPH-cytochrome P450 gene (Shiota et al. (1994) Plant Physiol.106:17) and some other cytochrome P450 gene (Didierjean for example, L.et al. (2002) Plant Physiol.130:179-189 for example; Morant, M.S.et al. (2003) Opinion in Biotechnology 14:151-162) is used to obtain the transgenic plant of antiweed.Ability (one Chinese patent application, 200610155661 that a kind of P450 gene in the corn comes to light and has weedicides such as anti-nicosulfuron; U.S. Pat 20080052798A1; 2008/006891).The cytochrome P450 gene CYP81A6 gene of paddy rice kind come to light and have anti-bentazone and part sulfonylurea herbicide (Pan et al, Plant Mol Biol, 2006,61:933-943).
But for resistance level that improves transgenic crop and the variety that increases resistant gene, farm crop still need new antiweed ability in the production application.New anti-herbicide gene is significant in the farm crop improvement.
Summary of the invention
The technical problem that the present invention will solve provides the antiweed protein of a kind of anti-herbicide gene, this genes encoding, and the purposes of this gene--the transgenic plant of-acquisition antiweed.
In order to solve the problems of the technologies described above, the present invention provides a kind of anti-herbicide gene: 1) aminoacid sequence of its encoded protein matter and SEQ ID NO:3 have the homogeny more than at least 82%; 2) its encoded protein mass-energy enough causes at least 2 kinds of following weedicides are produced resistances: acetolactate synthestase (ALS) suppressor factor class weedicide, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, photosystem II inhibition type weedicide.Annotate: for satisfying above 2 conditions simultaneously.
Improvement as anti-herbicide gene of the present invention: acetolactate synthestase (ALS) suppressor factor class weedicide is sulfonylurea, imidazolone type, triazolopyrimidine sulfonamides or pyrimidine salicylic acid; Antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide is phenyl ether, fluoroglycofenethyl, oxyfluorfen, fomesafen, flumioxazin or methylarsonic acid; To two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt is nitre sulphur humulone, mesotrione or isoxazolidinone; Photosystem II inhibition type weedicide is atrazine, chlorotoluron (paraquat) or bromoxynil (bromoxynil).
Further improvement as anti-herbicide gene of the present invention: be SEQ ID NO:1 or the described nucleotide sequence of SEQ ID NO:2.
The present invention also provides a kind of antiweed protein of anti-herbicide gene coding simultaneously: 1) utilize the some or all of sequence of SEQ ID NO:2 to obtain through the protein engineering transformation; 2) can produce resistance to following at least 2 kinds weedicide: acetolactate synthestase (ALS) suppressor factor class weedicide, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, photosystem II inhibition type weedicide.Annotate: for satisfying above 2 conditions simultaneously.
Antiweed protein improvement as anti-herbicide gene coding of the present invention: the proteinic aminoacid sequence of this antiweed is shown in the SEQ ID NO:3.
The present invention also provides a kind of plasmid vector simultaneously: comprise above-mentioned nucleotide sequence.
The present invention also provides a kind of recombinant plasmid vector that is used for Plant Transformation simultaneously: the polynucleotide function property ground that above-mentioned nucleotide sequence and at least one control are expressed is connected.
The present invention also provides the purposes of above-mentioned anti-herbicide gene simultaneously: obtain herbicide resistant plants through transgenic, perhaps as the screening-gene of plant transgene.
Improvement as the purposes of anti-herbicide gene of the present invention: plant is paddy rice, corn, cotton, soybean, tobacco, rape, barley, wheat or Chinese sorghum.
Be specially:
The present invention provides the varient of a kind of cytochrome P450 gene or this gene; Its nucleotide sequence coded protein of expression in plant can cause transgenic plant that following at least 2 kinds of weedicides are produced resistance: A, acetolactate synthestase (ALS) suppressor factor class weedicide, includes but are not limited to sulfonylurea, imidazolone type, triazolopyrimidine sulfonamides and pyrimidine salicylic acid; B, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, include but are not limited to nitre sulphur humulone, mesotrione, isoxazolidinone etc.; C, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide include but are not limited to phenyl ether, fluoroglycofenethyl, oxyfluorfen, fomesafen, flumioxazin, methylarsonic acid; D, photosystem II inhibition type weedicide include but are not limited to atrazine, chlorotoluron (paraquat) and bromoxynil (bromoxynil).Gene of the present invention also can with other anti-herbicide gene, import in the plant jointly like the EPSPS gene, thereby obtain the anti-simultaneously several herbicides that comprises Glyphosate 62 IPA Salt.Further, gene of the present invention also can import transgenic plant with anti insect gene simultaneously, thereby obtains antiweed and pest-resistant transgenic plant simultaneously.
Cytochrome P450 gene provided by the present invention, its nucleotides sequence are classified SEQ ID NO:1 as, and its encoded protein matter sequence is that SEQ ID NO:3 is said.The present invention also provides the plasmid that comprises the protein expression frame of expressing this nucleotide coding simultaneously, thereby and imports plant and in plant, express the method that obtains herbicide resistant plants.
The present invention from jielu grass (formal name used at school: be cloned into a kind of anti-herbicide gene Zoysia japonica), it can cause plant that at least two kinds of weedicides are as follows produced resistances:
A, acetolactate synthestase (ALS) suppressor factor class weedicide include but are not limited to sulfonylurea, imidazolone type, triazolopyrimidine sulfonamides and pyrimidine salicylic acid; B, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, include but are not limited to nitre sulphur humulone, mesotrione, isoxazolidinone etc.; C, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide include but are not limited to phenyl ether, fluoroglycofenethyl, oxyfluorfen, fomesafen, flumioxazin, methylarsonic acid; D, photosystem II inhibition type weedicide include but are not limited to atrazine, chlorotoluron (paraquat) and bromoxynil (bromoxynil).
Utilize the cytochrome P450 gene of the antiweed that provides in the present invention, other homology anti-herbicide gene just can obtain homologous gene through the method that has had.For example, one of ordinary skill in the art can obtain these homologous genes through Southern hybridizing method or PCR method.Further, those skilled in the art can utilize existing technology to obtain the varient of this gene, for example, from different plant species or same species, can obtain nucleotide sequence and the differentiated gene of aminoacid sequence in the different individuality; Also can introduce amino acid whose special the change, but still keep the performance of antiweed through recombinant gene; Can also be through passing through the gene that artificial gene reorganization methods (for example Gene 271:13-20) such as (gene shuffling) obtains variation with other different cells cytochrome p 450 genes.Therefore, cytochrome P450 gene of the present invention also comprises a kind of gene, and its encoded protein matter is compared with SEQ ID No:3 has at least 82%, 85%, 90% or 95% homogeny and gene that can antiweed.Amino acid whose homogeny can obtain through existing method, for example Karlin and Altschul, 1990, Porc.Natl.Acad.Sci.USA 87:3364; Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877.).The antiweed performance of these genes can obtain checking through the antiweed ability of transgenic plant.The transgenic plant that import these genes can resist at least two kinds of following weedicide: A, acetolactate synthestase (ALS) suppressor factor class weedicide, include but are not limited to sulfonylurea, imidazolone type, triazolopyrimidine sulfonamides and pyrimidine salicylic acid; B, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide include but are not limited to phenyl ether, fluoroglycofenethyl, oxyfluorfen, fomesafen, flumioxazin, methylarsonic acid; C, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, include but are not limited to nitre sulphur humulone, mesotrione, isoxazolidinone etc.; D, photosystem II inhibition type weedicide include but are not limited to atrazine, chlorotoluron (paraquat) and bromoxynil (bromoxynil).
The aminoacid sequence of genes encoding provided by the invention has tangible different with the functional gene that other have been known.The aminoacid sequence of gene provided by the invention reaches 78% with aminoacid sequence (SEQ ID NO:4) homogeny from a kind of cytochrome P450 gene of corn in gene pool.But this corn gene imports to paddy rice does not cause transgenic paddy rice to improve the resistance to weedicides such as nicosulfuron and nitre sulphur humulones later on.Gene of the present invention also has the amino acid homogeny of 65-76% simultaneously with the gene of other several corns and paddy rice; These genes comprise the CYP81A6 gene (SEQ ID NO:6) of paddy rice; Amino acid homogeny 72%, it has function (Pan et al., the Plant Molecular Biology of anti-bentazone and part sulfonylurea herbicide; 2006,61:933-943); The Nsf1 gene of corn (SEQ ID NO:5), amino acid homogeny 77%, it has the function (US patent appl.20070214515) of anti-nicosulfuron, and certain anti-nitre sulphur humulone ability (US patent appl.2008/0006891); A corn P450 gene (SEQ ID NO:7), the sour homogeny 74% that deaminizes, the function of its gene is unclear.
The present invention also provides a kind of plasmid vector that comprises gene provided by the present invention.Anti-herbicide gene of the present invention in this plasmid vector functionally is connected with terminator with promotor and constitutes expression cassette, can in vegetable cell, express.Making up a kind of expression cassette that in plant, can express has been the technology of using always.Thereby gene provided by the invention can import plant and obtain genetically modified herbicide resistant plants, and these plants include but not limited to corn, wheat, barley, Chinese sorghum, paddy rice, soybean, Radix Dauci Sativae, yam, cotton, Sunflower Receptacle, rape, Oak Tree, turfgrass, herbage.
The nucleotide sequence of anti-herbicide gene of the present invention can have multiple different variation, includes but not limited to: 1) utilize same amino acid whose different codons and the different nucleotide sequence that obtains, the identical active protein and peptide of these sequence encodings; The proteinic nucleotide sequence of 2) still still encoding and having the antiweed ability through the variation that imports nucleotide sequence.This variation can be variation at random, also can be to put variation targetedly, can also be to insert or deletion mutation.One of ordinary skill in the art just can produce above-mentioned variation through molecular biological method; 3) through forming the heterozygous genes that the hybrid molecule acquisition still has the antiweed ability with other P450 genes.(Stemmer 1994, Nature 370:389-391 for example to obtain new gene through Domain Swapping and Gene Shuffling method; Crameri et al.1998, Nature 391:288-291).
Utilize nucleotide sequence provided by the invention, also can obtain to have the homologous gene of identical function.A kind of method is to utilize nucleic acid provided by the invention to obtain homologous gene for probe hybridization DNA library, and another kind of method is to clone homologous gene according to nucleotide sequence design primer provided by the invention through PCR.Moreover, those skilled in the art's nucleotide sequence provided by the invention also capable of using and protein sequence are found out the gene of high homology from genomic library through the method for molecular information.As utilize BLAST (
Www.ncbi.nih.gov) method, according to the aminoacid sequence of nucleotide sequence provided by the invention and protein and peptide, find with dna homolog property provided by the invention than higher gene.Usually the aminoacid sequence and the anti-herbicide gene of the present invention of protein and peptide have 80% at least; 85%; 90%; The protein of 95% or 99% homogeny possibly have the antiweed activity, and can confirm through present existent method, as utilizes the method for the embodiment of the invention 3 and 4.
Utilize anti-herbicide gene nucleotide sequence provided by the invention, can make up the artificial gene that in plant, to express.Equally, according to antiweed protein and peptide sequence provided by the invention, also can artificial sequence synthetic nucleic acid (according to Campbell and Gowri, Plant Physiol.92:1-11), and the further artificial gene that can in plant, express of structure.The artificial gene member that can in plant, express comprises promotor, anti-herbicide gene and terminator.In plant, expressing and produce the needed promotor of antiweed ability, chloroplast(id) signal peptide and terminator is the technology that has had.For example, promotor can be corn Ubiqutin-1 promotor, perhaps paddy rice Actin promotor when transforming monocots; And terminator can be agrobacterium tumefaciens terminator (Nos) or other terminators; This express member can through Agrobacterium (like Agrobacterium bacterial strain LAB4404) or, particle gun method additive method is incorporated into acquisition antiweed transfer-gen plant in the genome of plant.The technology of Plant Transformation and method are known and sophisticated.The method and the step of the conversion of different plants are different.But, pass through immature embryo, mature embryo, undifferentiated callus or the protoplastis that Agrobacterium or particle gun import plant usually.Use corresponding screening culture medium screening and culturing then.Break up again and obtain to transform bud, cultivate through root media and just can obtain the transgenic seedling that to plant.Further, the antiweed transgenic plant can for example spray nicosulfuron and can kill not genetically modified paddy rice through the herbicide spraying screening.The plant that the present invention relates to includes but not limited to paddy rice, corn, cotton, wheat, soybean, turfgrass or herbage.The transgenic technology of these plants is existing technology.
The present invention also provides the nucleotide sequence that utilizes above-mentioned anti-herbicide gene molecule, in the plant transgene cell cultures as selection markers.The above-mentioned antiweed artificial gene that can in vegetable cell, express can transform on the fragment with the same DNA of destination gene expression frame construction at same Plant Transformation plasmid.Goal gene can be any valuable gene.This Plant Transformation plasmid can pass through particle gun, or Agrobacterium method additive method imports plant tissue; Weedicide (like the nicosulfuron) substratum that contains suitable concn then can optionally kill and not import DNA and transform segmental vegetable cell, thereby has selected to contain the vegetable cell of goal gene.
The present invention is applicable to all plants, comprises dicotyledonous and monocotyledons.
In sum, gene of the present invention can be used in plant expressing and make plant obtain at least the resistance capacity to 2 kinds of weedicides, thereby kills weeds with can utilizing herbicide selective.The present invention also can be used in the breeding of crop, the screening of culture plant cell.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the sequence alignment synoptic diagram;
Fig. 2 is the T-DNA synoptic diagram of carrier of expressing the Agrobacterium transduction of ZJ3-1 anti-herbicide gene.The border, the left and right sides of LB and RB:T-DNA; Act-P, a kind of constitutive promoter; Ubi-P, a kind of constitutive promoter; ZJ3-1, anti-herbicide gene provided by the invention, EPSPS, Antiglyphosate gene.3 ' of anti-herbicide gene ZJ3-1 and EPSPS is terminator.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Employed molecular biology of following examples of the present invention and biochemical method are known technology.The Current Protocols in Molecular Biology that publishes in the John Wiley and Sons company that Ausubel writes; Write the Molecular Cloning:ALabortory Manual that Cold Spring Harbor Laboratory Press (2001) publishes with J.Sambrook etc., documents such as 3rd ED. all have detailed explanation.
The resistant determination of embodiment 1, anti-nicosulfuron jielu grass
(the Latin formal name used at school: be the plant of Gramineae Zoysia Zoysia japonica), be that a kind of common weed also is a kind of turfgrass to jielu grass.Whether have anti-nicosulfuron and anti-nitre sulphur ketone ability in order to measure it, spray nicosulfuron (400mg/L) or nitre sulphur ketone (500mg/L), every mu consumption is 20L; Observed later in 10 days; The jielu grass that discovery has sprayed these 2 kinds of weedicides does not have death; And other weeds comprise that Herba Setariae Viridis, ild avena sativa, Amaranthus retroflexus, rule grass, purslane, Sheathed Monochoria, piemarker and nutgrass flatsedge etc. all are killed, and explain that jielu grass can have the gene of anti-nicosulfuron and nitre sulphur ketone.
The clone of embodiment 2, resistant gene
Plant has bigger P450 gene family members usually; For example in Arabidopsis thaliana genome, finding has 300 P450 gene (Werck-Reichhart et al. (2000) Trends in Plant Science 5 (3): 116-123) of surpassing.Paddy rice and corn have cytochrome P450 gene (Pan et al., Plant Molecular Biology, 2006, the 61:933-943 of weedicides such as anti-nicosulfuron, bentazone; Chinese invention patent, 200610155661).And the genome sequence of paddy rice and corn is known.Therefore according to the nucleotide sequence of P450 gene family in paddy rice and the corn (like gb:BT043141; Gb:EU957735; Gb:EU955667; Gb:EU955910) conservative region that exists in; Following 2 PCR primers have been designed: 450-470F:CTC TCC GCG CAC CGC GTC and 450-1500R:TCC CAG TCG AAG CAC TGG; And be template with the genomic dna that extract to obtain in the jielu grass, utilize the Pfu enzyme to carry out pcr amplification.The condition of PCR is: 94 ℃ 1 minute, 52 ° 1 minute, 72 ° 2 minutes, totally 32 circulations.PCR has obtained the gene fragment about a 1200bp.This fragment is cloned in the pMD-18T carrier, and has selected 10 clones to carry out sequencing.Result's discovery, but have 4 kinds of similar different sequences in this PCR product at least, be respectively 3ZJ-1,3ZJ-2,3ZJ-3, and 3ZJ-4.Wherein, the sequence of PCR product 3ZJ-1 is used to the genomic DNA fragment and the cDNA fragment of further clones coding entire reading frame.
Confirming of 5 ' terminal sequence of 3ZJ-1 gene is to obtain through genomic PCR.At first be special primer 450-3-1R2: (5 ' CTC CTG GGC CTC TGG CGA CAT GTC CGT) and non-specific primer N-1 (5 ' CGG ACC AAC AAT GGA TAA GGC CTA) with 3ZJ-1; The jielu grass genome is carried out pcr amplification; Then; The dilution of PCR product is worked as template for 200 times; Utilize the further pcr amplification of special primer 450-3-1R1 (5 ' CTC CTG GGC CTC TGG CGA CAT GTC CGT) of non-specific primer N-1 (sequence is the same) and 3ZJ-1, the PCR product of acquisition is cloned in the pMD-18T carrier, and carries out sequencing.
The definite of 3 ' terminal sequence of 3ZJ-1 gene obtains through PCR equally.After extracting jielu grass mRNA, utilize oligo-dT primer DTANCHOR:5 ' GAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TTV reverse transcription to become cDNA.Then, be template with cDNA, carry out the outer end amplification with primer ANCHOR:5 ' GAC CAC GCG TAT CGA TGT CGA C and 3-1C0:5 ' GAG GAG CCG ACG CAG TTC CGG.This PCR product obtains 3 ' end fragment as template with primer ANCHOR (sequence is the same) and 3-1C2:5 ' GGA TGG GCC GGC TGA AGT GCC C amplification after 200 times of dilutions.After this fragment cloning is in the pMD-18T carrier, carries out sequencing, thereby obtained the sequence of 3 ' end fragment.
The acquisition of 3ZJ-1 genome and cDNA full length fragment.With 3-1BAMH:5 ' GGA TCC AAC AAT GGA TAA GGC CTA CGT AGC CAT ACT CTC TTT TG and 3-1KPNI:5 ' GGT ACC GAG ATT GAC TAT TTG TGA GTA TAC TAC C is that primer is a template amplification with cDNA and genome respectively, obtains a complete cDNA and the genomic DNA fragment of reading frame of coding.The cDNA of ZJ3-1 gene is cloned in the pMD-18T carrier, and has carried out sequencing, and the cDNA sequence is SEQ ID NO:1 (that is, sequence table is the 1st).Genomic dna is cloned into the pMD-18T carrier equally, and the sequence of mensuration is SEQ ID NO:2 (that is, sequence table is the 2nd).Relatively ZJ3-1 genomic dna and cDNA sequence are found, have comprised an intron in the genomic dna.The aminoacid sequence of the cDNA of ZJ3-1 (SEQ ID NO:1) encoded protein matter is such as SEQ ID NO:3 (that is, sequence table is the 3rd) shown in.
The structure of embodiment 3, ZJ3-1 expression cassette
The genomic DNA fragment (SEQ ID NO:2) of coding ZJ3-1 obtains (with 3-1BAMH:5 ' GGA TCC AAC AAT GGA TAA GGC CTA CGT AGC CAT ACT CTC TTT TG, and 3-1KPNI:5 ' GGT ACC GAG ATT GAC TAT TTG TGA GTA TAC TAC C is a primer) through PCR.The ubiquitin-1 promotor ZmUbi-1 of corn obtains through PCR from the corn gene group.The PCR primer is respectively ZmUbiF (5 ' GCG
AAGCTTGCAT GCC TAC AGTGC AGCGTGACCCGGTCGTGC has added the HindIII site, and underscore is represented), and ZmUbiR (5 ' GTG
GGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAG has added the BamHI site, and underscore is represented).Be connected with the ubiquitin-1 promotor (ZmUbi-1) of corn through general molecular biology method; Be connected with the 35S terminator of a CaMV at 3 ' end then; The ORFs that formation can be expressed in as plant (its 5 ' end has the HindIII site, and 3 ' end has the KpnI site).This expression cassette is cloned between the HindIII and KpnI site of pCambia 1300 (for conventional carrier) then, has obtained T-DNA carrier pCam1300-ZJ3-1.
The conversion of embodiment 4, paddy rice
The preparation method of transgenic paddy rice is to adopt prior art (the refined Gong ancestral of Lu Xiong an ancient egg-shaped, holed wind instrument 1998 life science 10:125-131; Liu Fan etc., 2003 Molecular Plant Breeding 1:108-115)." elegant water 134 " seed of choosing mature and plump shells, and induces to produce callus as converting material.Get the Agrobacterium that contains goal gene carrier pCam1300-ZJ3-1 and draw plate, choose single colony inoculation preparation conversion and use Agrobacterium.Callus to be transformed is put into the agrobacterium liquid (containing Syringylethanone) of proper concn, let Agrobacterium be attached to the callus surface, transfer to callus in the common substratum then, cultivated altogether 2～3 days.With the callus after the aseptic water washing conversion, transfer on the screening culture medium that contains Totomycin screening and culturing two months (middle subculture once).After screening, the callus that growth vigor is good is transferred on the presorting substratum and was cultivated about 20 days, then the good callus of presorting is moved on to division culture medium, and illumination in 14 hours differentiation is germinateed.2-3 transfers to the resistance regeneration plant strengthening seedling and rooting on the root media that contains nicosulfuron (0.1mg/L) after week, at last regeneration plant flush away agar is transplanted in the greenhouse, as expert evidence.
Embodiment 5: utilize the paddy rice transgenic method of nicosulfuron as selective agent
The preparation method of transgenic paddy rice is to adopt prior art (the refined Gong ancestral of Lu Xiong an ancient egg-shaped, holed wind instrument 1998 life science 10:125-131; Liu Fan etc., 2003 Molecular Plant Breeding 1:108-115)." elegant water 134 " seed of choosing mature and plump shells, and induces to produce callus as converting material.Get the Agrobacterium that contains goal gene carrier pCam1300-ZJ3-1 and draw plate, choose single colony inoculation preparation conversion and use Agrobacterium.Callus to be transformed is put into the agrobacterium liquid (containing Syringylethanone) of proper concn, let Agrobacterium be attached to the callus surface, transfer to callus in the common substratum then, cultivated altogether 2～3 days.With the callus after the aseptic water washing conversion, transfer on the screening culture medium that contains nicosulfuron (0.1mg/L) screening and culturing two months (middle subculture once).After screening, the callus that growth vigor is good is transferred on the presorting substratum and was cultivated about 20 days, then the good callus of presorting is moved on to division culture medium, and illumination in 14 hours differentiation is germinateed.2-3 transfers to the resistance regeneration plant strengthening seedling and rooting on the root media that contains nicosulfuron (0.1mg/L) after week, at last regeneration plant flush away agar is transplanted in the greenhouse, as expert evidence.9 show normal growth on the root media of nicosulfuron (0.1mg/L) independently in the transformant at 15 that obtain, and it is positive that PCR detects the ZJ3-1 gene.
Embodiment 6: the antiweed ability of transgenic paddy rice is measured
Select transgenic paddy rice strain system through 10 different commentaries on classics pCam1300-ZJ3-1 of embodiment 4 acquisitions; With receptor parent kind " elegant water 134 " is contrast; (temperature 15-25 ℃) cultivated when the about 10cm of height of seedling in the greenhouse, and the spray amount is at 10 milligrams/square metre nicosulfuron.Inspection after 14 days find that the non-transgenic strain is all dead, and transgenic paddy rice strain mortality ratio is 0%, and wherein 8 transgenic lines have no visible growth to suppress, and the growth of 2 strain systems slows down.
Selecting the transgenic paddy rice strain system through the commentaries on classics pCam1300-G6-ZJ3-1 of embodiment 4 acquisitions, is contrast with receptor parent kind " elegant water 134 ", and (temperature 15-25 ℃) cultivated when the about 10cm of height of seedling in the greenhouse; The spray amount is at 15 milligrams/square metre nitre sulphur ketone; Inspection after 14 days finds that the albefaction of non-transgenic strain blade is serious, and part is dead; And in 10 transgenic paddy rice strains system; Have 6 not have tangible poisoning, the blade albefaction that 3 transgenic lines one are more slight has only the albefaction of a strain blade serious.
Mensuration result shows that transgenic paddy rice has obviously improved the resistance of nitre sulphur ketone and nicosulfuron.
Embodiment 7: the anti-mixed herbicide ability of transgenic paddy rice is measured
Select transgenic paddy rice strain system through 10 different commentaries on classics pCam1300-ZJ3-1 of embodiment 4 acquisitions; With receptor parent kind " elegant water 134 " is contrast; (temperature 15-25 ℃) cultivated when the about 10cm of height of seedling in the greenhouse, spray mixed herbicide (nicosulfuron 6 gram/mus+nitre sulphur ketone 10 gram/mus).Inspection after 14 days, the result finds have 6 transgenic lines not have tangible poisoning, other 4 poisoning that have in various degree, and the non-transgenic strain is all dead.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
Claims (9)
1. anti-herbicide gene, it is characterized in that: 1) aminoacid sequence of its encoded protein matter and SEQ ID NO:3 have the homogeny more than at least 82%; 2) its encoded protein mass-energy enough causes at least 2 kinds of following weedicides are produced resistances: acetolactate synthestase (ALS) suppressor factor class weedicide, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, photosystem II inhibition type weedicide.
2. anti-herbicide gene according to claim 1, said acetolactate synthestase (ALS) suppressor factor class weedicide is sulfonylurea, imidazolone type, triazolopyrimidine sulfonamides or pyrimidine salicylic acid; Antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide is phenyl ether, fluoroglycofenethyl, oxyfluorfen, fomesafen, flumioxazin or methylarsonic acid; To two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt is nitre sulphur humulone, mesotrione or isoxazolidinone; Photosystem II inhibition type weedicide is atrazine, chlorotoluron (paraquat) or bromoxynil (bromoxynil).
3. anti-herbicide gene according to claim 1 and 2 is characterized in that: be SEQ ID NO:1 or the described nucleotide sequence of SEQ ID NO:2.
4. the antiweed protein of anti-herbicide gene coding according to claim 1 is characterized in that: 1) utilize the some or all of sequence of SEQ ID NO:2 to obtain through the protein engineering transformation; 2) can produce resistance to following at least 2 kinds weedicide: acetolactate synthestase (ALS) suppressor factor class weedicide, antigen protoporphyrinogen oxidase (PPO) suppressor factor class weedicide, to two oxydase (HPPD) the suppressor factor class weedicides of phenylor pyruvate salt, photosystem II inhibition type weedicide.
5. the antiweed protein of anti-herbicide gene coding as claimed in claim 3, it is characterized in that: the proteinic aminoacid sequence of this antiweed is shown in the SEQ ID NO:3.
6. a plasmid vector is characterized in that: comprise claim 1,2 or 3 said nucleotide sequences.
7. a recombinant plasmid vector that is used for Plant Transformation is characterized in that: the polynucleotide function property ground of claim 1,2 or 3 described nucleotide sequences and at least one control expression is connected.
8. like the purposes of claim 1,2 or 3 described anti-herbicide genes, it is characterized in that: obtain herbicide resistant plants through transgenic, perhaps as the screening-gene of plant transgene.
9. the purposes of anti-herbicide gene according to claim 8, it is characterized in that: said plant is paddy rice, corn, cotton, soybean, tobacco, rape, barley, wheat or jowar.
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