CN101182521A - Applications of corn cytochrome P450 gene - Google Patents
Applications of corn cytochrome P450 gene Download PDFInfo
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- CN101182521A CN101182521A CNA2007101563954A CN200710156395A CN101182521A CN 101182521 A CN101182521 A CN 101182521A CN A2007101563954 A CNA2007101563954 A CN A2007101563954A CN 200710156395 A CN200710156395 A CN 200710156395A CN 101182521 A CN101182521 A CN 101182521A
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- bentazone
- leu
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Abstract
The invention discloses a maize cytochrome P450 gene, which has the nucleic acid sequence showed by SEQ ID NO: 1. The transgenic crop obtained by the gene has the capability of increasing the high resistance for bentazon or sulfonylurea herbicides. The invention also discloses a DNA plasmid, which contains an inhibiting expression frame of antisense RNA or RNAi constructed by the cytochrome P450 gene or the gene fragment of the cytochrome P450 gene. The invention also discloses a transgenic maize cell and a preparation method of transgenic maize by using the cell. The transgenic crop obtained by the DNA plasmid of the invention can decrease the resistance ability for bentazon or sulfonylurea herbicides, so that the bentazon or sulfonylurea herbicides can be applied to control the transgenic crop selectively.
Description
Technical field
The invention belongs to the genetically engineered field.Specifically, the present invention relates to utilize corn cytochrome P 450 gene can produce the farm crop of antiweed; And utilize this gene constructed sense-rna that obtains or RNAi, can obtain the method for the transgenic crop that available herbicide selective kills.
Background technology
Transgenic crop is planted in the world many countries large-scale popularization at present.For example in the North America large-scale popularization, transgene cotton is also planted in China in a large number for transgenic pest-resistant, antiweed corn.The antiweed ability of improvement farm crop is the importance of farm crop improvement.Therefore find and utilize anti-herbicide gene to have important use to be worth.The research of corn resistance glyphosate is very many, but the genetically engineered of anti-other weedicides is fewer.For example, corn itself has certain anti-bentazone and sulfonylurea herbicide ability, but the resistance deficiency; Therefore further improve the resistance of corn antagonism bentazone and sulfonylurea herbicide, can further improve the controlling weeds ability of corn.
On the other hand, one of important process of transgenic crop application is their propagation of control, prevents that they are blended among the non-transgenic farm crop.Prevent that particularly it is very important may having the transformed variety of undesirable action to sneak in grain and the feed articles for use to humans and animals as bio-reactor production pharmaceutical protein, industrial enzyme and other.Therefore, developing a kind of easy method controls transgenic crop and has great value.
Bentazone and sulfonylurea herbicide are the agriculture weedicides of using always, and it has the good careless ability of killing to many width leaf weeds; Therefore the toxenzyme of separating that has these weedicides in the gramineae farm crop has certain resistance to this weedicide.If increase the expression of detoxifying gene, just can further improve the resistance of farm crop, thereby can utilize these weedicides control weeds better by transgenosis.In addition, in transgenic plant, suppress the expression of separating toxenzyme of anti-bentazone and sulfonylurea herbicide and the transgenic corns that obtains to bentazone and sulfonylurea herbicide sensitivity, where necessary just can the elimination of above-mentioned herbicide selective ground; Thereby play the diffusion that prevents, limits transgenic corns, remove the effect that is blended into the non-transgenic corn, the Biosafety of favourable transgenic corns.
Summary of the invention
The technical problem to be solved in the present invention provides the purposes of corn cytochrome P 450 gene.Utilize this gene to create antagonism farm crop that bentazone and sulfonylurea herbicide have higher resistance also can utilize this gene to obtain the transgenic corns that available herbicide selective is killed.
In order to solve the problems of the technologies described above, the invention provides a kind of corn cytochrome P 450 gene, it has the nucleotide sequence shown in the SEQ ID NO:1; Its purposes is: use the transgenic crop of this gene gained to have enhancing to bentazone and the higher resistance capacity of sulfonylurea herbicide.
The present invention also provides a kind of DNA plasmid, and this DNA plasmid contains the inhibition expression cassette of above-mentioned cytochrome P450 gene or constructed sense-rna that obtains of gene segment wherein or RNAi.
Improvement as DNA plasmid of the present invention: the nucleotides sequence of sense-rna i is classified SEQ ID NO:6 as.
The present invention also provides a kind of transgenic corns cell: it contains above-mentioned plasmid.
The present invention also provides a kind of preparation method of transgenic corns: above-mentioned transgenic corns cell culture is become plant.
The present invention also provides the purposes of above-mentioned DNA plasmid: utilize the transgenic crop of this DNA plasmid gained that bentazone and sulfonylurea herbicide resistance capacity are reduced significantly, thereby utilize bentazone and sulfonylurea herbicide can optionally control (killing) this transgenic crop.
The present invention at first provides a kind of corn cytochrome P 450 gene, and the cDNA nucleotides sequence of this gene is classified SEQ ID NO:1 as, and the aminoacid sequence of encoded protein matter is SEQ ID NO:2; This gene is named as ZM-P450A in the present invention.
ZM-P450A can import corn or other farm crop strengthen their the anti-bentazone and the ability of sulfonylurea herbicide by transgenosis.The antiweed ability that strengthens farm crop helps the control of weeds.The ZM-P450A gene can functionally be connected with a terminator with a promotor and form expression cassette.Promotor can be paddy rice or corn ubiquitin (Ubiqutin) promotor or paddy rice Actin promotor or cauliflower mosaic virus 35S promoter (CaMV35S) or other promoters active; Terminator can utilize the natural terminator of functional gene in the biology, for example NOS terminator.The ZM-P450A gene expression frame can import the farm crop genome by particle gun method and method such as agriculture bacillus mediated.Particle gun method and agriculture bacillus mediated method all are known and sophisticated method (Hiei et al.1994 Effieient transformation of rice (Oryza sativa L.) mediated byagrobacterium and sequence analysis of the boundaries of the T-DNA.The Plant J.6:271-282).The transgenic crop that contains the ZM-P450A gene is owing to strengthened the ability of anti-bentazone and sulfonylurea herbicide; In the operation of therefore actual field, can improve the concentration of employed bentazone and sulfonylurea herbicide, kill careless effect thereby improved, and greatly reduce the poisoning possibility that this kind transgenic crop is caused.
Because any transgenic corns all can by pollen diffusion or directly by the place of seed dispersal beyond the plan plantation, the propagation of this transgenic corns be to use and promote an existing safety issue in the transgenic corns.Particularly express the transgenic corns of pharmaceutical protein and industrial enzyme, their propagation may be deleterious.The present invention utilizes sense-rna or RNAi to suppress the expression of this cytochrome P450 gene in the corn, the transgenic corns that can make gained is to bentazone and sulfonylurea herbicide sensitivity, thereby can reach the purpose that can utilize bentazone and sulfonylurea herbicide optionally to kill transgenic corns.The present invention produces susceptibility by the render transgenic corn to bentazone and sulfonylurea and is implemented in case of necessity and can optionally kills transgenic corns, thereby has guaranteed that goal gene can Be Controlled.For example, need only and use bentazone and sulfonylurea herbicide controlling weeds just can eliminate the transformed variety that to sneak into the big Tanaka of plantation non-transgenic kind.The above-mentioned purpose gene comprises a kind of Antiglyphosate gene, thereby makes the transgenic crop of acquisition have high resistance to glyphosate and to bentazone and sulfonylurea herbicide sensitivity.The above-mentioned purpose gene can also comprise pest-resistant adversity gene, industrial enzyme gene, pharmaceutical protein plasmagene and other the valuable genes that suits to express of waiting in plant; Can be one of them, two or a plurality of gene.
In the present invention, the detoxification genes ZM-P450A of bentazone and sulfonylurea suppresses expression cassette and can be made up of two parts: one is promotor, and it can be paddy rice or corn ubiquitin (Ubiqutin) promotor or paddy rice Actin promotor or cauliflower mosaic virus 35S promoter (CaMV35S) or other promoters active in gramineae farm crop; That another part and promoter function link is sense-rna or the RNAi of cytochrome P450 gene ZM-P450A of the present invention (SEQ ID NO:1).Mechanism and the technology of utilizing sense-rna and RNAi to suppress to express have had complete description, be technology (the Kusaba 2004 RNA Interference in crop plants that had, Current Opinionin Biotechnology, 15:139-143).The dna profiling that produces sense-rna can be a full-length gene, perhaps only is one of them or several segment.Producing RNA interferential sequence (RNAi) can design according to SEQID NO:1.
The carrier that has the detoxification genes inhibition expression cassette of destination gene expression frame and bentazone and sulfonylurea simultaneously can be by the stable corn that imports of transgenic method.This method comprises particle gun method and agriculture bacillus mediated method, and these two kinds of methods all are sophisticated known methods.
In the present invention, be connected on the same DNA importing segment because the detoxifying gene ZM-P450A of destination gene expression frame and bentazone and sulfonylurea herbicide suppresses expression cassette, thereby reach the inhibition expressing gene close linkage of the detoxifying gene of goal gene and bentazone, sulfonylurea herbicide in transgenic corns.This kind transgenic corns can be killed by bentazone or sulfonylurea antiweed where necessary, thereby prevents their propagation.
The invention provides and utilize different weedicide (bentazone and sulfonylurea herbicide) can just select and bear the technology of selecting transgenic plant.When goal gene was Antiglyphosate gene, it was structured on the same plasmid with the dna segment that suppresses corn cytochrome P 450 gene; Like this, to the resistance of glyphosate and chain at the transgenic corns camber to the susceptibility of bentazone and sulfonylurea.The transgenic plant of Huo Deing can utilize glyphosate to screen and control of weeds in this way, also can utilize the control of bentazone and sulfonylurea herbicide simultaneously and eliminate transgenic corns.
In sum, advantage of the present invention is as follows:
1, gene of the present invention is the gene of corn oneself, therefore utilizes gene of the present invention further to cultivate the higher transgenosis antiweed farm crop of resistance, and its security is higher.When some farm crop does not have resistance to bentazone or sulfonylurea herbicide, use method of the present invention can make it have resistance; When some farm crop possesses resistance to bentazone or sulfonylurea herbicide, use method of the present invention can further strengthen its resistance.
2, in transgenic corns, express in the goal gene, utilize the method for sense-rna or RNAi to suppress the expression of this cytochrome P450 gene in the paddy rice, thereby make this kind transgenic crop to bentazone and sulfonylurea herbicide sensitivity, and then can optionally be killed.
Be connected on the same DNA importing segment because the detoxifying gene of destination gene expression frame and bentazone, sulfonylurea herbicide suppresses expression cassette, thereby reach the inhibition expressing gene close linkage of the detoxifying gene of goal gene and bentazone, sulfonylurea herbicide in transgenic corns.Therefore in case of necessity can by use bentazone commonly used and sulfonylurea herbicide prevent transgenic corns propagation, eliminate the transgenic corns of sneaking in the non-transgenic corn.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is the structure synoptic diagram of the T-DNA conversion carrier of pCAMB1300Rice-GlyR-450i-ZM450A.Among the figure: G6 is the EPSPS gene (gb:EU169459) of artificial synthetic Pseudomonas putida; Carrier pCAMB1300Rice-GlyR-450i has in detail in patent 200610155661.7 and states; ZM-P450A is a gene of the present invention; ZmUbi-1 is the promotor of corn ubiquitin-1; 35S is cauliflower mosaic virus (CaMV35S) 35S promoter; It between LB and RB the T-DNA fragment that can insert Plant Genome.
Fig. 2 is the T-DNA conversion carrier synoptic diagram of pCAMBIA1300-G6-ZM450i.G6 is the EPSPS gene (gb:EU169459) of artificial synthetic Pseudomonas putida; ZmUbi-1, the promotor of corn ubiquitin-1; It between LB and RB the T-DNA fragment that can insert Plant Genome.
Embodiment
The clone of embodiment 1, relevant corn cytochrome P 450 gene:
According to the sequence of corn gene group database, designed and to have obtained to comprise a dna segment of intact cell cytochrome p 450 gene respectively by genomic dna being carried out pcr amplification.This cytochrome P450 gene is ZM-450A.Its cDNA sequence is SEQ ID NO:1, and coded protein amino acid sequence is SEQ ID NO:2.The primer of the PCR of amplification ZM-450A is zm450aF:GGATCCACCATGGATAAGGCCTACGTGGCCGTG (SEQ ID NO:3) and zm450aR:CAGTATACAGGACAACCTGCAGAAGACCACAATTT (SEQ IDNO:4).The condition of pcr amplification is: 95 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 2 minutes, carry out 30 circulations.These cytochrome P450 gene groups PCR product is cloned in the T carrier (worker is given birth in Shanghai), obtains the pT-zm450A plasmid, and the gene that order-checking explanation clone obtains is identical with the sequence of database.
The structure of the T-DNA carrier of embodiment 2, Agrobacterium-mediated Transformation ZM-450A gene:
Agrobacterium-mediated Transformation T-DNA carrier pCAMB1300Rice-GlyR-450i is used as the T-DNA carrier (application number 200610155661.7 " the optionally method of destroying gramineous transgenic plant ") that is structured in expression corn cytochrome P 450 gene ZM-450A in the paddy rice.T-DNA among the pCAMB1300Rice-GlyR-450i comprises one and suppresses degraded bentazone and the RNAi segment of sulfonylurea herbicide gene P450 expression and the gene of a glyphosate tolerant weedicide in the paddy rice.Therefore pCAMB1300Rice-GlyR-450i will cause the sensitivity of transgenic paddy rice to bentazone and sulfonylurea herbicide.If but in pCAMB1300Rice-GlyR-450i, importing the foreign gene that can improve bentazone and sulfonylurea herbicide resistance simultaneously, the transgenic paddy rice that is obtained still can show as the high resistance to bentazone and sulfonylurea herbicide.
The Agrobacterium-mediated Transformation T-DNA carrier pCAMB1300Rice-GlyR-450i-ZM450A that contains the expression cassette of ZM-450A makes up as shown in Figure 1.Carrier pCAMB1300Rice-GlyR-450i is behind BamHI and KpnI double digestion, be connected with corn ubiquitin-1 promotor ZmUbi-1 (HindIII cut pCAMB1300Rice-GlyR-450i and obtain) and 3 segments of ZM-450A gene (BamH1 obtains with KpnI digested plasmid pT-zm450A), obtain Agrobacterium-mediated Transformation T-DNA carrier pCAMB1300Rice-GlyR-450i-ZM450A (SEQID NO:5) with the BamH1 enzyme.
The acquisition of embodiment 3, transgenic paddy rice:
PCAMB1300Rice-GlyR-450i-ZM450A and two conversion carriers of pCAMB1300Rice-GlyR-450i are imported Agrobacterium LBA4404 respectively by electric-shocking method.The preparation method of the transgenic plant of 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 110 seeds of choosing mature and plump shell, and induce to produce callus as converting material.Get the Agrobacterium that contains pCAMB1300Rice-GlyR-450i-ZM450A and pCAMB1300Rice-GlyR-450i and draw plate, choose single colony inoculation preparation conversion respectively and use Agrobacterium.Callus to be transformed is put into the agrobacterium liquid (containing Syringylethanone) of proper concn, allow Agrobacterium be attached to the callus surface, then callus is transferred in the common substratum, cultivated altogether 2～3 days.With the callus after the aseptic water washing conversion, transfer on the screening culture medium that contains the 1-4mM glyphosate screening and culturing two months (middle subculture once).After screening, the callus that growth vigor is good is transferred on the pre-differentiation substratum and was cultivated about 20 days, will break up good callus then in advance and move on to division culture medium, and illumination in 14 hours differentiation is germinateed.2-3 transfers to strengthening seedling and rooting on the root media to the resistance regeneration plant after week, at last regeneration plant flush away agar is transplanted in the greenhouse, as expert evidence.Each conversion carrier has obtained the rice strain of independently regenerating more than 15.
The insertion of T-DNA and expression of gene thereof can detect by PCR and western engram analysis.Genomic dna can extract from transformation tissue culture plant and non-rice transformation according to existing method.
The mensuration of embodiment 4, transgenic paddy rice resistance capacity:
10 independent transformation plant rice plants of difference of each conversion carrier acquisition and the rice plant individual plant in the greenhouse that does not have to transform are cultivated.These regeneration plants are divided into 4 processing, handle 1: spray 20mM glyphosate; Handle 2: spray 1000mg/L bentazone; Handle the 3:4mg/L benbbensulfuronmethyl; Handle 4: water spray.Handle the surviving rate of back 7 days record plant, the result is as shown in table 1.
Table 1, the surviving rate of different 10 independent transformation plants under different weedicides
| Glyphosate | Bentazone | Benbbensulfuronmethyl | Water | |
| Non-transformed plant | 0％ | 100％ | 100％ | 100％ |
| pCAMB1300Rice-GlyR-450i-ZM450A | 100％ | 90％ | 80％ | 100％ |
| pCAMB1300Rice-GlyR-450i | 100％ | 10％ | 30％ | 100％ |
The expression of ZM450A makes and should obtain resistance again to the paddy rice of bentazone and sulfonylurea herbicide sensitivity.Therefore illustrate that ZM450A is the gene of anti-bentazone and sulfonylurea herbicide.
The structure of embodiment 5, pCAMB1300-G6-ZM450i corn conversion carrier:
Design and synthesized the dna segment (SEQ ID NO:6) that can produce RNAi according to the cytochrome P450 gene (comprising ZM-P450A) of corn.Be connected to after this dna segment is cut with the XhoI enzyme with XhoI in advance enzyme remove the pCambia1300 carrier (gb:AF234296) of moisture resistance mycin (hyg) gene earnestly, the novel vector that obtains is further cut with EcoRI and HindIII enzyme, the dna segment of cutting with the same enzyme enzyme of usefulness that contains Antiglyphosate gene G6 then and obtaining connects, and obtains carrier pCAMB1300-GlyR-ZM450i (Fig. 2).This carrier includes an inhibition expression cassette that can produce RNAi under the control of CaMVS35 promotor.
The generation of the transgenic corns of embodiment 6, bentazone and sulfonylurea herbicide sensitivity:
Get 8-10 days the Hi-II mealie in pollination back.Collect all immature embryos (size is 1.0-1.5mm).The Agrobacterium and the immature embryo that will contain T-DNA carrier pCAMB1300-GlyR-ZM450i are cultivated common cultivation 2-3 days (22 ℃) altogether.Shift immature embryo (M3, the Timentin that contains 200mg/L kills Agrobacterium) to the callus of induce substratum, 28 ℃ of dark cultivations 10-14 days.All callus are forwarded (M4) on the screening culture medium that has the 2mM glyphosate to 28 ℃ of dark cultivations 2-3 week.
Shift on all screening culture medium that is organized into fresh glyphosate (M4) 28 ℃ of dark cultivations 2-3 week.Then, shift the embryonal connective tissue (M5) to regeneration culture medium that survives after all screenings, 28 ℃ of dark cultivations 10-14 days, strain system of every ware.Shift embryonal connective tissue (M5) to fresh regeneration culture medium, 26 ℃ illumination cultivation 10-14 days.Shift all full-grown plants (M6) to root media, 26 ℃ of illumination cultivation are complete up to root development, and be transplanted in the greenhouse individual plant then and cultivate, and calm their antiweed ability.
The mensuration of embodiment 7, transgenic corns susceptibility:
Conversion pCAMB1300-G6-ZM450i milpa that obtains and the milpa individual plant in the greenhouse that does not have to transform are cultivated.These regeneration plants are divided into 3 processing, and each processing comprises pCAMB1300-G6-ZM450i transformed plant and each 20 strain of non-transformed plant.Handle 1: spray 10mM glyphosate; Handle 2: spray 2000mg/L bentazone; Handle 3: water spray.Handle the surviving rate of back 10 days record plant, the result is as shown in table 2.
Table 2, the surviving rate of maize transformation plant under different weedicides
| Glyphosate | Bentazone | Water | |
| The pCAMB1300-GlyR-ZM450i transformed plant | 100％ | 15％ | 100％ |
| Non-transformed plant | 0％ | 100％ | 100％ |
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, 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
SEQ ID NO 1：
1 atggataagg cctacgtggc cgtgctctcc ttcgccttcc tcttcgtgat ceactacctc
61 gtgggacgtg ctggccgcaa gggcaatggc aagggcaagg gaacgcagcg gttgcctccc
121 agccctccgg ccgtcccgtt cctcggccac ctccacctcg tcaagacgcc gttccacgag
181 gcgctggccg gcctcgccgc gcgccacggg ccggtgttct ccatgcgcat ggggtcccgc
241 cgcgcgctgg tcgtgtcctc gcccgagtgc gccaaggagt gcttcacgga gcacgacgtg
301 gtgttcgcca accggccgag gttcgccacg caggacctcg tgtccttcgg gggcgccgcg
361 ctcgccgcgg ccagctacgg gccctactgg cgcaacctcc ggcgcgtggc cacegtgcag
421 ctcctctccg cgcaccgcgt cgcctgcatg tccgccgtcg tcgccgccga ggtgcgcgcc
481 atggcgcgcc gcatgggccg cgcggccgcg gcggcgcccg gcggcgccgc gcgcgtccag
541 ctcaagcgga ggctgttcga ggtctccctc agcgtgctca tggagaccat cgcgcgcacc
601 aagacgtccc gcgccgaggc ggacgccgac tcggacatgt cgcccgaggc gcacgaattc
661 aagcagatcg tcgacgagat cgtgccgcac ctcggcacgg ccaacctgtg ggactacctg
721 ccggtgctgc ggtggctcga cgtgttcggc gtgaggaaca agatcacggc cgctgtgggc
781 aggagggacg cgttcctgcg gcggctcatc gacgcggagc ggcggaggct ggacgacggc
841 ggcggcgaca gcgacagcga caagaagagc atgatagccg tgctgctctc gtgcagaag
901 tcagagccgg aggtgtacac ggataccatg atcatggcac tttgcgggaa cttatttggc
961 gccggaacgg agaccacgtc gaccacgaca gaatgggcca tgtcgctcct cctgaaccac
1021 ccggaggcgc tgaagaaggc acaggcagag atcgacgcag tcgtgggcac ctcccgccta
1081 ctcgccgcag aggacgtgcc ccgactcggc tacctgcacc gcgtcatcag cgagaccctg
1141 cgcatgtacc cggccgcccc gctgctcctg ccgcacgagt cgtcggcgga ctgcaaggtc
1201 ggcggctacg acgtggcccg cggcacgctg ctcatcgtga acgcgtacgc catccacagg
1261 gacccgctgg tgtgggagga cccggacgag ttcaggccgg agcggttcga ggacggcaag
1321 gccgaggggc ggctcctgat gccgttcggg atggggcggc gcaagtgccc tggggagacc
1381 ctcgcgctgc ggaccateag cctggtgctc ggcacgctga tccagtgctt cgactgggac
1441 cgagtcgacg gcctcgagat cgacatggcc gcgggcggcg ggctcactct gcceagggcc
1501 gtcccgttgg aggccacgtg caagcctcgt gcagctgtgc gccatcttct tctggagctc
1561 tgaatctgat gc
SEQ ID NO 2：
1 Met Asp Lys Ala Tyr Val Ala Val Leu Ser Phe Ala Phe Leu Phe Val Ile His Tyr Leu
21 Val Gly Arg Ala Gly Arg Lys Gly Asn Gly Lys Gly Lys Gly Thr Gln Arg Leu Pro Pro
41 Ser Pro Pro Ala Val Pro Phe Leu Gly His Leu His Leu Val Lys Thr Pro Phe His Glu
61 Ala Leu Ala Gly Leu Ala Ala Arg His Gly Pro Val Phe Ser Met Arg Met Gly Ser Arg
81 Arg Ala Leu Val Val Ser Ser Pro Glu Cys Ala Lys Glu Cys Phe Thr Glu His Asp Val
101 Val Phe Ala Asn Arg Pro Arg Phe Ala Thr Gln Asp Leu Val Ser Phe Gly Gly Ala Ala
121 Leu Ala Ala Ala Ser Tyr Gly Pro Tyr Trp Arg Asn Leu Arg Arg Val Ala Thr Val Gln
141 Leu Leu Ser Ala His Arg Val Ala Cys Met Ser Ala Val Val Ala Ala Glu Val Arg Ala
161 Met Ala Arg Arg Met Gly Ala Ala Ala Ala Ala Ala Pro Gly Gly Ala Ala Arg Val Gln
181 Leu Lys Arg Arg Leu Phe Glu Val Ser Leu Ser Val Leu Met Glu Thr Ile Ala Arg Thr
201 Lys Thr Ser Arg Ala Glu Ala Asp Ala Asp Ser Asp Met Ser Pro Glu Ala His Glu Phe
221 Lys Gln Ile Val Asp Glu Ile Val Pro His Leu Gly Thr Ala Asn Leu Trp Asp Tyr Leu
241 Pro Val Leu Arg Trp Leu Asp Val Phe Gly Val Arg Asn Lys Ile Thr Ala Ala Val Gly
261 Arg Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp Ala Glu Arg Arg Arg Leu Asp Asp Gly
281 Gly Gly Asp Ser Asp Ser Asp Lys Lys Ser Met Ile Ala Val Leu Leu Ser Leu Gln Lys
301 Ser Glu Pro Glu Val Tyr Thr Asp Thr Met Ile Met Ala Leu Cys Gly Asn Leu Phe Gly
321 Ala Gly Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu Asn His
341 Pro Glu Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala Val Val Gly Thr Ser Arg Leu
361 Leu Ala Ala Glu Asp Val Pro Arg Leu Gly Tyr Leu His Arg Val Ile Ser Glu Thr Leu
381 Arg Met Tyr Pro Ala Ala Pro Leu Leu Leu Pro His Glu Ser Ser Ala Asp Cys Lys Val
401 Gly Gly Tyr Asp Val Ala Arg Gly Thr Leu Leu Ile Val Asn Ala Tyr Ala Ile His Arg
421 Asp Pro Leu Val Trp Glu Asp Pro Asp Glu Phe Arg Pro Glu Arg Phe Glu Asp Gly Lys
441 Ala Glu Gly Arg Leu Leu Met Pro Phe Gly Met Gly Arg Arg Lys Cys Pro Gly Glu Thr
461 Leu Ala Leu Arg Thr Ile Ser Leu Val Leu Gly Thr Leu Ile Gln Cys Phe Asp Trp Asp
481 Arg Val Asp Gly Leu Glu Ile Asp Met Ala Ala Gly Gly Gly Leu Thr Leu Pro Arg Ala
501 Val Pro Leu Glu Ala Thr Cys Lys Pro Arg Ala Ala Val Arg His Leu Leu Leu Glu Leu
SEQ ID NO：3
1 Ggatccacca tggataaggc ctacgtggcc gtg
SEQ ID NO：4
1 cagtatacag gacaacctgc agaagaccac aattt
SEQ ID NO：5
1 cgtaat acgact cactat agggcg acatat gatcga tgatat cccatg ggcggc cgcctg
61 cagacc aggtct acagta tacagg acaacc tgcaga agacca caattt gctata aaccat
121 actgtg aaccaa ctactg attgat tgttca cgcaaa cgactg tacaac acattt tacaga
181 agataa cacgca cgaggg tattac gcactc aagctt atttag tttagc acacac catcag
241 gcaaag catact agggat catatg catgcc aagatg ttacta cagcat cagatt cagagc
301 tccaga agaaga tggcgc acagct gcacga ggcttg cacgtg gcctcc aacggg acggcc
361 ctgggc agagtg agcccg ccgccc gcggcc atgtcg atctcg aggccg tcgact cggtcc
421 cagtcg aagcac tggatc agcgtg ccgagc accagg ctgatg gtccgc agcgcg agggtc
481 tcccca gggcac ttgcgc cgcccc atcccg aacggc atcagg agccgc ccctcg gccttg
541 ccgtcc tcgaac cgctcc ggcctg aactcg tccggg tcctcc cacacc agcggg tccctg
601 tggatg gcgtac gcgttc acgatg agcagc gtgccg cgggcc acgtcg tagccg ccgacc
661 ttgcag tccgcc gacgac tcgtgc ggcagg agcagc ggggcg gccggg tacatg cgcagg
721 gtctcg ctgatg acgcgg tgcagg tagccg agtcgg ggcacg tcctct gcggcg agtagg
781 cgggag gtgccc acgact gcgtcg atctct gcctgt gccttc ttcagc gcctcc gggtgg
841 ttcagg aggagc gacatg gcccat tctgtc gtggtc gacgtg gtctcc gttccg gcgcca
901 aataag ttcttc atcaca gagaaa aaacag tcagtg tctctg tttgta ttggga aaaaca
961 aacaaa cacgac agcgac gctgtt gttatg aagatg tcacat cgctag gtttgt caaagc
1021 ttcgct cagaga aagaag tactgg aaggaa aaaaac tcaccc cgcaaa gtgcca tgatca
1081 tggtat ccgtgt acacct ccggct ctgact tctgca acgaga gcagca cggcta tcatgc
1141 tcttct tgtcgc tgtcgc tgtcgc cgccgc cgtcgt ccagcc tccgcc gctccg cgtcga
1201 tgagcc gccgca ggaacg cgtccc tcctgc ccacag cggccg tgatct tgttcc tcacgc
1261 cgaaca cgtcga gccacc gcagca ccggca ggtagt cccaca ggttgg ccgtgc cgaggt
1321 gcggca cgatct cgtcga cgatct gcttga attcgt gcgcct cgggcg acatgt ccgagt
1381 cggcgt ccgcct cggcgc gggacg tcttgg tgcgcg cgatgg tctcca tgagca cgctga
1441 gggaga cctcga acagcc tccgct tgagct ggacgc gcgcgg cgccgc cgggcg ccgccg
1501 cggccg cgcggc ccatgc ggcgcg ccatgg cgcgca cctcgg cggcga cgacgg cggaca
1561 tgcagg cgacgc ggtgcg cggaga ggagct gcacgg tggcca cgcgcc ggaggt tgcgcc
1621 agtagg gcccgt agctgg ccgcgg cgagcg cggcgc ccccga aggaca cgaggt cctgcg
1681 tggcga acctcg gccggt tggcga acacca cgtcgt gctccg tgaagc actcct tggcgc
1741 actcgg gcgagg acacga ccagcg cgcggc gggacc ccatgc gcatgg agaaca ccggcc
1801 cgtggc gcgcgg cgaggc cggcca gcgcct cgtgga acggcg tcttga cgaggt ggaggt
1861 ggccga ggaacg ggacgg ccggag ggctgg gaggca accgct gcgttc ccttgc ccttgc
1921 cattgc ccttgc ggccag cacgtc ccacga ggtagt ggatca cgaaga ggaagg cgaagg
1981 agagca cggcca cgtagg ccttat ccatgg tggatc ctctag agtcga cctgca gaagta
2041 acacca aacaac agggtg agcatc gacaaa agaaac agtacc aagcaa ataaat agcgta
2101 tgaagg cagggc taaaaa aatcca catata gctgct gcatat gccatc atccaa gtatat
2161 caagat caaaat aattat aaaaca tacttg tttatt ataata gatagg tactca aggtta
2221 gagcat atgaat agatgc tgcata tgccat catgta tatgca tcagta aaaccc acatca
2281 acatgt atacct atccta gatcga tatttc catcca tcttaa actcgt aactat gaagat
2341 gtatga cacaca cataca gttcca aaatta ataaat acacca ggtagt ttgaaa cagtat
2401 tctact ccgatc tagaac gaatga acgacc gcccaa ccacac cacatc atcaca accaag
2461 cgaaca aaaagc atctct gtatat gcatca gtaaaa cccgca tcaaca tgtata cctatc
2521 ctagat cgatat ttccat ccatca tcttca attcgt aactat gaatat gtatgg cacaca
2581 cataca gatcca aaatta ataaat ccacca ggtagt ttgaaa cagaat tctact ccgatc
2641 tagaac gaccgc ccaacc agacca catcat cacaac caagac aaaaaa aagcat gaaaag
2701 atgacc cgacaa acaagt gcacgg catata ttgaaa taaagg aaaagg gcaaac caaacc
2761 ctatgc aacgaa acaaaa aaaatc atgaaa tcgatc ccgtct gcggaa cggcta gagcca
2821 tcccag gattcc ccaaag agaaac actggc aagtta gcaatc agaacg tgtctg acgtac
2881 aggtcg catccg tgtacg aacgct agcagc acggat ctaaca caaaca cggatc taacac
2941 aaacat gaacag aagtag aactac cgggcc ctaacc atggac cggaac gccgat ctagag
3001 aaggta gagagg gggggg ggggga ggacga gcggcg tacctt gaagcg gaggtg ccgacg
3061 ggtgga tttggg ggagat ctggtt gtgtgt gtgtgc gctccg aacaac acgagg ttgggg
3121 aaagag ggtgtg gagggg gtgtct atttat tacggc gggcga ggaagg gaaagc gaagga
3181 gcggtg ggaaag gaatcc cccgta gctgcc gtgccg tgagag gaggag gaggcc gcctgc
3241 cgtgcc ggctca cgtctg ccgctc cgccac gcaatt tctgga tgccga cagcgg agcaag
3301 tccaac ggtgga gcggaa ctctcg agaggg gtccag aggcag cgacag agatgc cgtgcc
3361 gtctgc ttcgct tggccc gacgcg acgctg ctggtt cgctgg ttggtg tccgtt agactc
3421 gtcgac ggcgtt taacag gctggc attatc tactcg aaacaa gaaaaa tgtttc cttagt
3481 tttttt aatttc ttaaag ggtatt tgttta attttt agtcac tttatt ttattc tatttt
3541 atatct aaatta ttaaat aaaaaa actaaa atagag ttttag ttttct taattt agaggc
3601 taaaat agaata aaatag atgtac taaaaa aattag tctata aaaacc attaac cctaaa
3661 ccctaa atggat gtacta ataaaa tggatg aagtat tatata ggtgaa gctatt tgcaaa
3721 aaaaaa ggagaa cacatg cacact aaaaag ataaaa ctgtag agtcct gttgtc aaaata
3781 ctcaat tgtcct ttagac catgtc taactg ttcatt tatatg attctc taaaac actgat
3841 attatt gtagta ctatag attata ttattc gtagag taaagt ttaaat atatgt ataaag
3901 atagat aaactg cacttc aaacaa gtgtga caaaaa aaatat gtggta attttt tataac
3961 ttagac atgcaa tgctca ttatct ctagag aggggc acgacc gggtca cgctgc actgta
4021 ggcatg caagct tggcac tggccg tcgttt tacaac gtcgtg actggg aaaacc ctggcg
4081 ttaccc aactta atcgcc ttgcag cacatc cccctt tcgcca gctggc gtaata gcgaag
4141 aggccc gcaccg atcgcc cttccc aacagt tgcgca gcctga atggcg aatgct agagca
4201 gcttga gcttgg atcaga ttgtcg tttccc gccttc agttta aactat cagtgt ttgaca
4261 ggatat attggc gggtaa acctaa gagaaa agagcg tttatt agaata acggat atttaa
4321 aagggc gtgaaa aggttt atccgt tcgtcc atttgt atgtgc atgcca accaca gggttc
4381 ccctcg ggatca aagtac tttgat ccaacc cctccg ctgcta tagtgc agtcgg cttctg
4441 acgttc agtgca gccgtc ttctga aaacga catgtc gcacaa gtccta agttac gcgaca
4501 ggctgc cgccct gccctt ttcctg gcgttt tcttgt cgcgtg ttttag tcgcat aaagta
4561 gaatac ttgcga ctagaa ccggag acatta cgccat gaacaa gagcgc cgccgc tggcct
4621 gctggg ctatgc ccgcgt cagcac cgacga ccagga cttgac caacca acgggc cgaact
4681 gcacgc ggccgg ctgcac caagct gttttc cgagaa gatcac cggcac caggcg cgaccg
4741 cccgga gctggc caggat gcttga ccacct acgccc tggcga cgttgt gacagt gaccag
4801 gctaga ccgcct ggcccg cagcac ccgcga cctact ggacat tgccga gcgcat ccagga
4861 ggccgg cgcggg cctgcg tagcct ggcaga gccgtg ggccga caccac cacgcc ggccgg
4921 ccgcat ggtgtt gaccgt gttcgc cggcat tgccga gttcga gcgttc cctaat catcga
4981 ccgcac ccggag cgggcg cgaggc cgccaa ggcccg aggcgt gaagtt tggccc ccgccc
5041 taccct cacccc ggcaca gatcgc gcacgc ccgcga gctgat cgacca ggaagg ccgcac
5101 cgtgaa agaggc ggctgc actgct tggcgt gcatcg ctcgac cctgta ccgcgc acttga
5161 gcgcag cgagga agtgac gcccac cgaggc caggcg gcgcgg tgcctt ccgtga ggacgc
5221 attgac cgaggc cgacgc cctggc ggccgc cgagaa tgaacg ccaaga ggaaca agcatg
5281 aaaccg caccag gacggc caggac gaaccg tttttc attacc gaagag atcgag gcggag
5341 atgatc gcggcc gggtac gtgttc gagccg cccgcg cacgtc tcaacc gtgcgg ctgcat
5401 gaaatc ctggcc ggtttg tctgat gccaag ctggcg gcctgg ccggcc agcttg gccgct
5461 gaagaa accgag cgccgc cgtcta aaaagg tgatgt gtattt gagtaa aacagc ttgcgt
5521 catgcg gtcgct gcgtat atgatg cgatga gtaaat aaacaa atacgc aagggg aacgca
5581 tgaagg ttatcg ctgtac ttaacc agaaag gcgggt caggca agacga ccatcg caaccc
5641 atctag cccgcg ccctgc aactcg ccgggg ccgatg ttctgt tagtcg attccg atcccc
5701 agggca gtgccc gcgatt gggcgg ccgtgc gggaag atcaac cgctaa ccgttg tcggca
5761 tcgacc gcccga cgattg accgcg acgtga aggcca tcggcc ggcgcg acttcg tagtga
5821 tcgacg gagcgc cccagg cggcgg acttgg ctgtgt ccgcga tcaagg cagccg acttcg
5881 tgctga ttccgg tgcagc caagcc cttacg acatat gggcca ccgccg acctgg tggagc
5941 tggtta agcagc gcattg aggtca cggatg gaaggc tacaag cggcct ttgtcg tgtcgc
6001 gggcga tcaaag gcacgc gcatcg gcggtg aggttg ccgagg cgctgg ccgggt acgagc
6061 tgccca ttcttg agtccc gtatca cgcagc gcgtga gctacc caggca ctgccg ccgccg
6121 gcacaa ccgttc ttgaat cagaac ccgagg gcgacg ctgccc gcgagg tccagg cgctgg
6181 ccgctg aaatta aatcaa aactca tttgag ttaatg aggtaa agagaa aatgag caaaag
6241 cacaaa cacgct aagtgc cggccg tccgag cgcacg cagcag caaggc tgcaac gttggc
6301 cagcct ggcaga cacgcc agccat gaagcg ggtcaa ctttca gttgcc ggcgga ggatca
6361 caccaa gctgaa gatgta cgcggt acgcca aggcaa gaccat taccga gctgct atctga
6421 atacat cgcgca gctacc agagta aatgag caaatg aataaa tgagta gatgaa ttttag
6481 cggcta aaggag gcggca tggaaa atcaag aacaac caggca ccgacg ccgtgg aatgcc
6541 ccatgt gtggag gaacgg gcggtt ggccag gcgtaa gcggct gggttg tctgcc ggccct
6601 gcaatg gcactg gaaccc ccaagc ccgagg aatcgg cgtgac ggtcgc aaacca tccggc
6661 ccggta caaatc ggcgcg gcgctg ggtgat gacctg gtggag aagttg aaggcc gcgcag
6721 gccgcc cagcgg caacgc atcgag gcagaa gcacgc cccggt gaatcg tggcaa gcggcc
6781 gctgat cgaatc cgcaaa gaatcc cggcaa ccgccg gcagcc ggtgcg ccgtcg attagg
6841 aagccg cccaag ggcgac gagcaa ccagat tttttc gttccg atgctc tatgac gtgggc
6901 acccgc gatagt cgcagc atcatg gacgtg gccgtt ttccgt ctgtcg aagcgt gaccga
6961 cgagct ggcgag gtgatc cgctac gagctt ccagac gggcac gtagag gtttcc gcaggg
7021 ccggcc ggcatg gccagt gtgtgg gattac gacctg gtactg atggcg gtttcc catcta
7081 accgaa tccatg aaccga taccgg gaaggg aaggga gacaag cccggc cgcgtg ttccgt
7141 ccacac gttgcg gacgta ctcaag ttctgc cggcga gccgat ggcgga aagcag aaagac
7201 gacctg gtagaa acctgc attcgg ttaaac accacg cacgtt gccatg cagcgt acgaag
7261 aaggcc aagaac ggccgc ctggtg acggta tccgag ggtgaa gccttg attagc cgctac
7321 aagatc gtaaag agcgaa accggg cggccg gagtac atcgag atcgag ctagct gattgg
7381 atgtac cgcgag atcaca gaaggc aagaac ccggac gtgctg acggtt cacccc gattac
7441 tttttg atcgat cccggc atcggc cgtttt ctctac cgcctg gcacgc cgcgcc gcaggc
7501 aaggca gaagcc agatgg ttgttc aagacg atctac gaacgc agtggc agcgcc ggagag
7561 ttcaag aagttc tgtttc accgtg cgcaag ctgatc gggtca aatgac ctgccg gagtac
7621 gatttg aaggag gaggcg gggcag gctggc ccgatc ctagtc atgcgc taccgc aacctg
7681 atcgag ggcgaa gcatcc gccggt tcctaa tgtacg gagcag atgcta gggcaa attgcc
7741 ctagca ggggaa aaaggt cgaaaa ggtctc tttcct gtggat agcacg tacatt gggaac
7801 ccaaag ccgtac attggg aaccgg aacccg tacatt gggaac ccaaag ccgtac attggg
7861 aaccgg tcacac atgtaa gtgact gatata aaagag aaaaaa ggcgat ttttcc gcctaa
7921 aactct ttaaaa cttatt aaaact cttaaa acccgc ctggcc tgtgca taactg tctggc
7981 cagcgc acagcc gaagag ctgcaa aaagcg cctacc cttcgg tcgctg cgctcc ctacgc
8041 cccgcc gcttcg cgtcgg cctatc gcggcc gctggc cgctca aaaatg gctggc ctacgg
8101 ccaggc aatcta ccaggg cgcgga caagcc gcgccg tcgcca ctcgac cgccgg cgccca
8161 catcaa ggcacc ctgcct cgcgcg tttcgg tgatga cggtga aaacct ctgaca catgca
8221 gctccc ggagac ggtcac agcttg tctgta agcgga tgccgg gagcag acaagc ccgtca
8281 gggcgc gtcagc gggtgt tggcgg gtgtcg gggcgc agccat gaccca gtcacg tagcga
8341 tagcgg agtgta tactgg cttaac tatgcg gcatca gagcag attgta ctgaga gtgcac
8401 catatg cggtgt gaaata ccgcac agatgc gtaagg agaaaa taccgc atcagg cgctct
8461 tccgct tcctcg ctcact gactcg ctgcgc tcggtc gttcgg ctgcgg cgagcg gtatca
8521 gctcac tcaaag gcggta atacgg ttatcc acagaa tcaggg gataac gcagga aagaac
8581 atgtga gcaaaa ggccag caaaag gccagg aaccgt aaaaag gccgcg ttgcgt gcgttt
8641 ttccat aggctc cgcccc cctgac gagcat cacaaa aatcga cgctca agtcag aggtgg
8701 cgaaac ccgaca ggacta taaaga taccag gcgttt ccccct ggaagc tccctc gtgcgc
8761 tctcct gttccg accctg ccgctt accgga tacctg tccgcc tttctc ccttcg ggaagc
8821 gtggcg ctttct catagc tcacgc tgtagg tatctc agttcg gtgtag gtcgtt cgctcc
8881 aagctg ggctgt gtgcac gaaccc cccgtt cagccc gaccgc tgcgcc ttatcc ggtaac
8941 tatcgt cttgag tccaac ccggta agacac gactta tcgcca ctggca gcagcc actggt
9001 aacagg attagc agagcg aggtat gtaggc ggtgct acagag ttcttg aagtgg tggcct
9061 aactac ggctac actaga aggaca gtattt ggtatc tgcgct ctgctg aagcca gttacc
9121 ttcgga aaaaga gttggt agctct tgatcc ggcaaa caaacc accgct ggtagc ggtggt
9181 tttttt gtttgc aagcag cagatt acgcgc agaaaa aaagga tctcaa gaagat cctttg
9241 atcttt tctacg gggtct gacgct cagtgg aacgaa aactca cgttaa gggatt ttggtc
9301 atgcat tctagg tactaa aacaat tcatcc agtaaa atataa tatttt attttc tcccaa
9361 tcaggc ttgatc cccagt aagtca aaaaat agctcg acatac tgttct tccccg atatcc
9421 tccctg atcgac cggacg cagaag gcaatg tcatac cacttg tccgcc ctgccg cttctc
9481 ccaaga tcaata aagcca cttact ttgcca tctttc acaaag atgttg ctgtct cccagg
9541 tcgccg tgggaa aagaca agttcc tcttcg ggcttt tccgtc tttaaa aaatca tacagc
9601 tcgcgc ggatct ttaaat ggagtg tcttct tcccag ttttcg caatcc acatcg gccaga
9661 tcgtta ttcagt aagtaa tccaat tcggct aagcgg ctgtct aagcta ttcgta taggga
9721 caatcc gatatg tcgatg gagtga aagagc ctgatg cactcc gcatac agctcg ataatc
9781 ttttca gggctt tgttca tcttca tactct tccgag caaagg acgcca tcggcc tcactc
9841 atgagc agattg ctccag ccatca tgccgt tcaaag tgcagg accttt ggaaca ggcagc
9901 tttcct tccagc catagc atcatg tccttt tcccgt tccaca tcatag gtggtc ccttta
9961 taccgg ctgtcc gtcatt tttaaa tatagg ttttca ttttct cccacc agctta tatacc
10021 ttagca ggagac attcct tccgta tctttt acgcag cggtat ttttcg atcagt tttttc
10081 aattcc ggtgat attctc atttta gccatt tattat ttcctt cctctt ttctac agtatt
10141 taaaga tacccc aagaag ctaatt ataaca agacga actcca attcac tgttcc ttgcat
10201 tctaaa acctta aatacc agaaaa cagctt tttcaa agttgt tttcaa agttgg cgtata
10261 acatag tatcga cggagc cgattt tgaaac cgcggt gatcac aggcag caacgc tctgtc
10321 atcgtt acaatc aacatg ctaccc tccgcg agatca tccgtg tttcaa acccgg cagctt
10381 agttgc cgttct tccgaa tagcat cggtaa catgag caaagt ctgccg ccttac aacggc
10441 tctccc gctgac gccgtc ccggac tgatgg gctgcc tgtatc gagtgg tgattt tgtgcc
10501 gagctg ccggtc ggggag ctgttg gctggc tggtgg caggat atattg tggtgt aaacaa
10561 attgac gcttag acaact taataa cacatt gcggac gttttt aatgta ctgaat taacgc
10621 cgaatt aattcg ggggat ctggat tttagt actgga ttttgg ttttag gaatta gaaatt
10681 ttattg atagaa gtattt tacaaa tacaaa tacata ctaagg gtttct tatatg ctcaac
10741 acatga gcgaaa ccctat aggaac cctaat tccctt atctgg gaacta ctcaca cattat
10801 tatgga gaaact cgagca gtgcac caggag tcacag aaacac atcaca cattcg tgagct
10861 cagctt agccat ggataa cgccta cattat tgccat tctctc tgtagc tatcct cttctt
10921 gctcca ctacta cctcct cggccg cggcaa tggcgg ggcggc gcggct gccgcc gggtcc
10981 accggc cgtccc gatcct gggaca cctcca cctcgt caagaa gcagat ctcggt gaagca
11041 ctccct ggcgca ccccgg cgacga caccac cacggc gcgccg cgaccc gaggcg cagcga
11101 gaacac cggccc gtaccg ctcggc gaggcg ggacat ggtggc gtgcat cggctt cttgac
11161 gaggtg gaggtg tcccag gatcgg gacggc cggtgg acccgg cggcag ccgcgc cgcccc
11221 gccatt gccgcg gccgag gaggta gtagtg gagcaa gaagag gatagc tacaga gagaat
11281 ggcaat aatgta ggcgtt atccat ggctaa gctgag ctcacg aatgtg tgatgt gtttct
11341 gtgact ctggtg cactgc tcgaga gagata gatttg tagaga gagact ggtgat ttcagc
11401 gtgtcc tctcca aatgaa atgaac ttcctt atatag aggaag gtcttg cgaagg atagtg
11461 ggattg tgcgtc atccct tacgtc agtgga gatatc acatca atccac ttgctt tgaaga
11521 cgtggt tggaac gtcttc tttttc cacgat gctcct cgtggg tggggg tccatc tttggg
11581 accact gtcggc agaggc atcttg aacgat agcctt tccttt atcgca atgatg gcattt
11641 gtaggt gccacc ttcctt ttctac tgtcct tttgat gaagtg acagat agctgg gcaatg
11701 gaatcc gaggag gtttcc cgatat taccct ttgttg aaaagt ctcaat agccct ttggtc
11761 ttctga gactgt atcttt gatatt cttgga gtagac gagagt gtcgtg ctccac catgtt
11821 atcaca tcaatc cacttg ctttga agacgt ggttgg aacgtc ttcttt ttccac gatgct
11881 cctcgt gggtgg gggtcc atcttt gggacc actgtc ggcaga ggcatc ttgaac gatagc
11941 ctttcc tttatc gcaatg atggca tttgta ggtgcc accttc cttttc tactgt cctttt
12001 gatgaa gtgaca gatagc tgggca atggaa tccgag gaggtt tcccga tattac cctttg
12061 ttgaaa agtctc aatagc cctttg gtcttc tgagac tgtatc tttgat attctt ggagta
12121 gacgag agtgtc gtgctc caccat gttggc aagctg ctctag ccaata cgcaaa ccgcct
12181 ctcccc gcgcgt tggccg attcat taatgc agctgg cacgac aggttt cccgac tggaaa
12241 gcgggc agtgag cgcaac gcaatt aatgtg agttag ctcact cattag gcaccc caggct
12301 ttacac tttatg cttccg gctcgt atgttg tgtgga attgtg agcgga taacaa tttcac
12361 acagga aacagc tatgac catgat tacgaa ttcgcc actgga ttttgg ttttag gaatta
12421 gaaatt ttattg atagaa gtattt tacaaa tacaaa tacata ctaagt tctgca caaagt
12481 ggagta gtcagt catcga tcagga accaga caccag actttt attcat acagtg aagtga
12541 agtgaa gtgcag tgcagt gagttg ctggtt tttgta caacag atcttg agctct cagctc
12601 ttgccc tcctcg gccacc ctgatg cccacc tcggcg cacagg gccagg aagttc gggaag
12661 ctggtg gccacg ttggcg cagtcg tggatt ctgatc ggggcg ctggcc ctcagg ctggcc
12721 acgctg aaggcc atggcg atcctg tggtcg ccgtgg ccgtgc acctcg ccgccg cccagc
12781 tggccg ccgtcg atgatg atgccg tccggg gtcggc tcgcac ttgatg cccagg gtgatc
12841 aggccg tcggcc atcacc tgaatc ctgtcg ctctcc ttcacc ctcagc tcctcg gcgccc
12901 ctcagc acggtc ctgccc tcggcg caggcg gcggcc acgaac agcacc gggaac tcgtcg
12961 atggcc agcggc accagg gcctcc gggatg tcgatg cccttc agctgg gcgccc ctcacc
13021 ctcagg tcggcc accggc tcgccg cccacc tccctc tggttc tccagg gtgatg tcgccg
13081 cccatc agcctc aggatg tcgatc acgccg gtcctg gtcggg ttgatg cccacg tgctcc
13141 agcacc agctcg ctgccc tcggcg atgctg gcggcc accagg aagaag gcggcg ctgctg
13201 atgtcg gccggc acctcg atcctg gtggcg gtcagc ttgccg ccgctc tgcagg ctggcc
13261 accggg ccgttg ctctcc acgctg tagccg aagccc ctcagc atcctc tcggtg tggtcc
13321 ctggtc ggggcc ggctcg gtcacg gtggtc ttgccc tcggcg tacagg ccggcc agcagc
13381 aggcag ctcttc acctgg gcgctg gccatc ggcagg gtgtag gtcagg cccttc agcttg
13441 tggccg cccctg atggtc agcggc ggcctg ccctcc gggccg gtctcc accacg gcgccc
13501 atctcc ctcagc gggttg gccacc ctgttc atcggc ctcttg ctcagg ctggcg tcgccg
13561 gtcatg gtcacg tcgaag ctctgg ccggcc agcagg ccgctc agcagc ctcatg ctggtg
13621 ccgctg ttgccc acgtac agcggg cccggc ggcggc ttcagg ccgtgc aggccc acgccg
13681 tggatg gtcacc ctgccg tggttc gggccc tcgatc accacg cccatg tccctg aaggcc
13741 tgcagg gtggcc agggcg tcctcg ccctcc aggaag ccctcc acctcg gtggtg ccctcg
13801 gccagg ctgccc agcatg atgctc ctgtgg ctgatg ctcttg tcgccc ggcacc ctgatc
13861 ctgccg ttcagc ctgccg cccggc tgggcc aggaag atcagg tcgttg gcccca gatccc
13921 ttgcgg ggatcg gtgggg ccccac ggccgg agcggg gtggcc ggggga gccatc ggcatg
13981 gcgggt gacgcc gctgag cacctg atgggc gcggcg agggcg cggcgg gtggcc aggagg
14041 tgcgcc cggcgc ctcgcc ttgggc gcagcg gtagtg gcgcca gtgagc gcggta gacgcg
14101 gcggcg gcggtg gccatg gtagat cctcta gagtcg acctgc agaagt aacacc aaacaa
14161 cagggt gagcat cgacaa aagaaa cagtac caagca aataaa tagcgt atgaag gcaggg
14221 ctaaaa aaatcc acatat agctgc tgcata tgccat catcca agtata tcaaga tcaaaa
14281 taatta taaaac atactt gtttat tataat agatag gtactc aaggtt agagca tatgaa
14341 tagatg ctgcat atgcca tcatgt atatgc atcagt aaaacc cacatc aacatg tatacc
14401 tatcct agatcg atattt ccatcc atctta aactcg taacta tgaaga tgtatg acacac
14461 acatac agttcc aaaatt aataaa tacacc aggtag tttgaa acagta ttctac tccgat
14521 ctagaa cgaatg aacgac cgccca accaca ccacat catcac aaccaa gcgaac aaaaag
14581 catctc tgtata tgcatc agtaaa acccgc atcaac atgtat acctat cctaga tcgata
14641 tttcca tccatc atcttc aattcg taacta tgaata tgtatg gcacac acatac agatcc
14701 aaaatt aataaa tccacc aggtag tttgaa acagaa ttctac tccgat ctagaa cgaccg
14761 cccaac cagacc acatca tcacaa ccaaga caaaaa aaagca tgaaaa gatgac ccgaca
14821 aacaag tgcacg gcatat attgaa ataaag gaaaag ggcaaa ccaaac cctatg caacga
14881 aacaaa aaaaat catgaa atcgat cccgtc tgcgga acggct agagcc atccca ggattc
14941 cccaaa gagaaa cactgg caagtt agcaat cagaac gtgtct gacgta caggtc gcatcc
15001 gtgtac gaacgc tagcag cacgga tctaac acaaac acggat ctaaca caaaca tgaaca
15061 gaagta gaacta ccgggc cctaac catgga ccggaa cgccga tctaga gaaggt agagag
15121 gggggg gggggg aggacg agcggc gtacct tgaagc ggaggt gccgac gggtgg atttgg
15181 gggaga tctggt tgtgtg tgtgtg cgctcc gaacaa cacgag gttggg gaaaga gggtgt
15241 ggaggg ggtgtc tattta ttacgg cgggcg aggaag ggaaag cgaagg agcggt gggaaa
15301 ggaatc ccccgt agctgc cgtgcc gtgaga ggagga ggaggc cgcctg ccgtgc cggctc
15361 acgtct gccgct ccgcca cgcaat ttctgg atgccg acagcg gagcaa gtccaa cggtgg
15421 agcgga actctc gagagg ggtcca gaggca gcgaca gagatg ccgtgc cgtctg cttcgc
15481 ttggcc cgacgc gacgct gctggt tcgctg gttggt gtccgt tagact cgtcga cggcgt
15541 ttaaca ggctgg cattat ctactc gaaaca agaaaa atgttt ccttag tttttt taattt
15601 cttaaa gggtat ttgttt aatttt tagtca ctttat tttatt ctattt tatatc taaatt
15661 attaaa taaaaa aactaa aataga gtttta gttttc ttaatt tagagg ctaaaa tagaat
15721 aaaata gatgta ctaaaa aaatta gtctat aaaaac cattaa ccctaa acccta aatgga
15781 tgtact aataaa atggat gaagta ttatat aggtga agctat ttgcaa aaaaaa aggaga
15841 acacat gcacac taaaaa gataaa actgta gagtcc tgttgt caaaat actcaa ttgtcc
15901 tttaga ccatgt ctaact gttcat ttatat gattct ctaaaa cactga tattat tgtagt
15961 actata gattat attatt cgtaga gtaaag tttaaa tatatg tataaa gataga taaact
16021 gcactt caaaca agtgtg acaaaa aaaata tgtggt aatttt ttataa cttaga catgca
16081 atgctc attatc tctaga gagggg cacgac cgggtc acgctg cactgt aggcat gcacgg
16141 tac
SEQ ID NO：6
1 ctcgagggtt ttcagagcct cttaagaaca ccacgcatag ctgtacgcgg cctgcacatg
61 gcctccaacg ggacggcccg gggcatggtc agcccgccgc tagccttcat gtcaacctga
121 gctccatcaa ccgtgtccca gtcgaagcac tggagcagcg tggcgagcac cagcccgacg
181 gtccgcagcg cgagcgtctc cccggggcac ttgcgccgcc ccatcccgaa cggcatcagg
241 agccgcccct cggccttgcc gtcctcgaac cgctccggcc tgaactcgtc cgggtcctcc
301 cacaccagcg ggtccctgtg gatggtgtac gcgttcacga tgagcagcgt gccgcgggcc
361 acgtcgtagc cgccgacctt gcagtccgcc gacgactcgt gcggcaggag cagcggggcg
421 gccgggtaca tgcgcagggt ctcgctgatg acgcggtgca ggtagccgag tcggggcacg
481 tcctctgcgg cgagtaggcg ggaggtgccc acgactgcgt cgatctctgc ctgtgccttc
541 ttcagcgcct ccgggtggtt caggaggagc gacatggccc attctgtcgt ggtcgacgtg
601 gtctccgttc cggcgccaaa taagatcccg actcggctac ctgcaccgcg tcatcagcga
661 gaccctgcgc atgtacccgg ccgccccgct gctcctgccg cacgagtcgt cggcggactg
721 caaggtcggc ggctacgacg tggcccgcgg cacgctgctc atcgtgaacg cgtacgccat
781 ccacagggac ccgctggtgt gggaggaccc ggacgagttc aggccggagc ggttcgagga
841 cggcaaggcc gaggggcggc tcctgatgcc gttcgggatg gggcggcgca agtgccccgg
901 ggagacgctc gcgctgcgga ccgtcgggct ggtgctcgcc acgctgctcc agtgcttcga
961 ctgggacacg gttgatggag ctcaggttga catgaaggct agcggcgggc tgaccatgcc
1021 ccgggccgtc ccgttggagg ccatgtgcag gccgcgtaca gctatgcgtg gtgttcttaa
1081 gaggctctga aaacctcctc gagt
Claims (6)
1. a corn cytochrome P 450 gene has the nucleotide sequence shown in the SEQ ID NO:1; Its purposes is: use the transgenic crop of this gene gained to have the high resistance capacity of enhancing to bentazone or sulfonylurea herbicide.
2. a DNA plasmid is characterized in that: the inhibition expression cassette that contains constructed sense-rna that obtains of described cytochrome P450 gene of claim 1 or gene segment wherein or RNAi.
3. DNA plasmid according to claim 2 is characterized in that: the nucleotides sequence of described sense-rna i is classified SEQ ID NO:6 as.
4. a transgenic corns cell is characterized in that: contain claim 2 or 3 described plasmids.
5. the preparation method of a transgenic corns is characterized in that: the described transgenic corns cell culture of claim 4 is become corn.
6. as the purposes of claim 2 or 3 described DNA plasmids: utilize the transgenic crop of this DNA plasmid gained to reduce resistance capacity, thereby utilize bentazone or sulfonylurea herbicide optionally to control described transgenic crop to bentazone and sulfonylurea herbicide.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101563954A CN101182521A (en) | 2007-11-02 | 2007-11-02 | Applications of corn cytochrome P450 gene |
| PCT/CN2007/071373 WO2008083598A1 (en) | 2006-12-30 | 2007-12-28 | Transgenic crop being selectively killed, preparation and utilization thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101563954A CN101182521A (en) | 2007-11-02 | 2007-11-02 | Applications of corn cytochrome P450 gene |
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| Publication Number | Publication Date |
|---|---|
| CN101182521A true CN101182521A (en) | 2008-05-21 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101563954A Pending CN101182521A (en) | 2006-12-30 | 2007-11-02 | Applications of corn cytochrome P450 gene |
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| Country | Link |
|---|---|
| CN (1) | CN101182521A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321640A (en) * | 2011-08-18 | 2012-01-18 | 杭州瑞丰生物科技有限公司 | Herbicide resistant gene, and application of herbicide resistant gene in genetically modified crops |
| WO2012068966A1 (en) * | 2010-11-22 | 2012-05-31 | 杭州瑞丰生物科技有限公司 | Herbicide resistant gene and use thereof |
| CN103421836A (en) * | 2013-07-22 | 2013-12-04 | 浙江大学 | Obtaining method of transgenic plant provided with efficient color development marker and killed selectively |
| WO2014030090A1 (en) * | 2012-08-20 | 2014-02-27 | Basf Se | Herbicide-metabolizing cytochrome p450 monooxygenase |
| CN106148558A (en) * | 2016-09-19 | 2016-11-23 | 山东农业大学 | A kind of corn nicosulfuron anti-sense molecule mark and application thereof |
| CN108179154A (en) * | 2011-12-23 | 2018-06-19 | Kws种子欧洲股份公司 | Develop pathogen-response chimeric promoters novel plant source it is cis--regulating element |
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2007
- 2007-11-02 CN CNA2007101563954A patent/CN101182521A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012068966A1 (en) * | 2010-11-22 | 2012-05-31 | 杭州瑞丰生物科技有限公司 | Herbicide resistant gene and use thereof |
| CN102321640A (en) * | 2011-08-18 | 2012-01-18 | 杭州瑞丰生物科技有限公司 | Herbicide resistant gene, and application of herbicide resistant gene in genetically modified crops |
| CN102321640B (en) * | 2011-08-18 | 2013-08-07 | 杭州瑞丰生物科技有限公司 | Herbicide resistant gene, and application of herbicide resistant gene in genetically modified crops |
| CN108179154A (en) * | 2011-12-23 | 2018-06-19 | Kws种子欧洲股份公司 | Develop pathogen-response chimeric promoters novel plant source it is cis--regulating element |
| WO2014030090A1 (en) * | 2012-08-20 | 2014-02-27 | Basf Se | Herbicide-metabolizing cytochrome p450 monooxygenase |
| CN103421836A (en) * | 2013-07-22 | 2013-12-04 | 浙江大学 | Obtaining method of transgenic plant provided with efficient color development marker and killed selectively |
| CN106148558A (en) * | 2016-09-19 | 2016-11-23 | 山东农业大学 | A kind of corn nicosulfuron anti-sense molecule mark and application thereof |
| CN106148558B (en) * | 2016-09-19 | 2019-08-02 | 山东农业大学 | A kind of anti-sense molecule label of corn nicosulfuron and its application |
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Open date: 20080521 |