CN100427600C - Zoophobous fusion protein and use thereof - Google Patents
Zoophobous fusion protein and use thereof Download PDFInfo
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- CN100427600C CN100427600C CNB2006100496110A CN200610049611A CN100427600C CN 100427600 C CN100427600 C CN 100427600C CN B2006100496110 A CNB2006100496110 A CN B2006100496110A CN 200610049611 A CN200610049611 A CN 200610049611A CN 100427600 C CN100427600 C CN 100427600C
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Abstract
The present invention discloses an insect resistant fusion gene which comprises a nucleotide sequence with encoding BT crystal toxin and a nucleotide sequence with encoding Vip 3 toxin from 5' to 3' in sequence, wherein the two nucleotide sequences are positioned in the same open reading frame, and the BT crystal toxin is an active toxin with full length or with a C end cut. The present invention also discloses a fusion protein encoded by the insect resistant fusion gene, which is provided with the BT crystal toxin and the Vip 3 toxin from an N end to a C end in sequence. The present invention also discloses the application of the fusion protein to insect resistant crops of transgenic crops and insecticidal microorganisms of transgenic crops.
Description
Technical field
The present invention relates to the fusion rotein of a kind of pest-resistant fusion gene, the pest-resistant fusion gene coding of this kind, and the concrete application of this fusion rotein.
Background technology
Insect brings annual about 8,000,000,000 dollars loss for global agriculture production.Main at present dependence of the control of insect used chemical pesticide, but pesticide residue can be brought harm to HUMAN HEALTH.Therefore, the selection of people's success to utilize the Pesticidal toxins genetically modified crops to carry out pest-resistant, transgenic insect-resistant corn and cotton plantation in a large number at present.
The pest-resistant gordian technique of genetically modified crops is the good insect-killing protein of obtained performance, and insect-killing protein has multiple.Relatively commonly used is Bt crystal proteins (annotating 1), Cry1Ab for example, and Cry1C etc., they are applied to the transgenic pest-resistant crop in a large number.But found that insect can produce the resistance to crystal toxin at present, therefore influenced pest-resistant performance of these crystal toxins and utilizability (annotating 2).Another kind of insect-killing protein is Vip3 (annotating 3), and it is different with the insecticidal mechanism of crystal toxin, but not high to the killing ability of some primary pest.In addition, no matter single Pesticidal toxins is crystal toxin or Vip3 toxin, and its insecticidal spectrum is all narrow; Therefore influenced the effect of pest control, and a large amount of the use also causes the generation resistance easily.
Therefore, obtain the new type disinsection toxin, to the significant application value that has that improves killing ability and slow down pest resistance.
Reference is as follows:
(1)、
Crickmore，N.，
Zeigler，D.R.，
Feitelson，J.，
Schnepf，E.，
Van Rie， J.，
Lereclus，D.，
Baum，J.&
Dean，D.H.(1998)Microbiol.Mol.Biol.Rev.62，807-813.
(2)、Ferre，J.& Van Rie，J.(2002)Annu.Rev.Entomol.47，501-533.
(3)、
Estruch，J.J.，
Warren，G.W.，
Mullins，M.A.，
Nye，G.J.，
Craig，J.A.&
Koziel，M.G.(1996)Proc.Natl.Acad.Sci.USA 93，5389-5394.
Summary of the invention
At the deficiencies in the prior art part, the invention provides and a kind ofly can improve killing ability and slow down the pest-resistant fusion gene of pest resistance, the fusion rotein of this genes encoding and the purposes of this kind fusion rotein.
The present invention is to realize by such technical scheme for reaching above purpose: a kind of pest-resistant fusion gene is provided, and this gene comprises from 5 '-3 ' and is followed successively by the nucleotide sequence that contains coding BT crystal toxin and the nucleotide sequence of coding Vip3 toxin; And above-mentioned 2 nucleotide sequences are positioned at same open reading frame; The BT crystal toxin is the active toxin of total length or C tip cut-off.
A kind of improvement as pest-resistant fusion gene of the present invention: this gene comprises from 5 '-3 ' and is followed successively by the nucleotide sequence that contains coding BT crystal toxin, any amino acid polypeptide segment and the nucleotide sequence of coding Vip3 toxin.
Further improvement as pest-resistant fusion gene of the present invention: the BT crystal toxin is Cry1Ab, Cry1Ac, Cry2 or Cry3, and the Vip3 toxin is Vip3A, Vip3B, Vip3C, Vip3D or Vip3H.
Further improvement as pest-resistant fusion gene of the present invention: when the BT crystal toxin be Cry1Ab, when the Vip3 toxin is Vip3H, the nucleotides sequence of gene is classified SEQ ID NO:1 as; When the BT crystal toxin be Cry1Ab, when the Vip3 toxin is Vip3A, the nucleotides sequence of gene is classified SEQ ID NO:2 as.
The present invention also provides the fusion rotein of above-mentioned pest-resistant fusion gene coding, and this fusion rotein is held to the C end from N and is followed successively by BT crystal toxin and Vip3 toxin.
A kind of improvement as fusion rotein of the present invention: when the BT crystal toxin be Cry1Ab, when the Vip3 toxin is Vip3H, the aminoacid sequence of fusion rotein is SEQ ID NO:3; When the BT crystal toxin be Cry1Ab, when the Vip3 toxin is Vip3A, the aminoacid sequence of fusion rotein is SEQ ID NO:4.
The present invention also provides the application of above-mentioned fusion rotein aspect genetically modified crops anti-pest crop and genetically modified crops insecticidal microorganism.
Bt crystal toxin of usefulness that the present invention is designed and the artificial proteins molecule that the Vip3 toxin is fused into, compare with Vip3 protein with original crystal proteins, have following advantage: insecticidal spectrum is wide, can kill insects such as mythimna separata, beet armyworm, Pyrausta nubilalis (Hubern)., striped rice borer, killing rate is up to 100%; And the generation that can effectively slow down pest resistance.
Description of drawings
Fig. 1 is the structure iron of fusion insecticidal proteins of the present invention;
Fig. 2 is the preparation process figure of fusion toxin gene.
Embodiment
With reference to above-mentioned accompanying drawing, the specific embodiment of the present invention is elaborated.
The preparation of embodiment 1, Cry1Ab-Vip3H fusion toxin gene:
It is synthetic that the gene of coding Cry1Ab and Vip3H insecticidal proteins is given birth to the worker by Shanghai, and its dna sequence dna is respectively SEQ ID NO:5 and SEQ ID NO:6, and be cloned between the site of the restriction enzyme BamHI of PET28a expression vector and SacI.
The Cry1Ab-Vip3H building-up process as shown in Figure 2, concrete steps are as follows:
1, obtains Vip3H gene segment: V3HN 5 ' CTCCCC CGG GCC AGG TGG AAG TAA GCT CTC CAC CCG CGC CCT C and V3HC 5 ' TCT TGA GCT CCT ACT TGA TGC TCA CGT CGT AGA AGT GCA by following two primers through PCR.These two primers comprise the site of restriction enzyme XmaI and SacI respectively.The template of PCR is Vip3H (SEQ ID NO:6).
2, handle the PCR product with XmaI and SacI, and be connected with the pET28a-Cry1Ab carrier of handling with same enzyme.
3, change intestinal bacteria over to, the acquisition plasmid is pET28a-Cry1Ab-Vip3H.Fusion toxin gene C ry1Ab-Vip3H (dna sequence dna 3) is cloned among the expression vector pET28a.The open reading frame of the Cry1Ab-Vip3H fusion toxin gene of this synthetic can be expressed a molecular weight and be approximately the 150kD fused protein.
The preparation of embodiment 2, Cry1Ab-Vip3A fusion toxin gene:
It is synthetic that the gene of coding Cry1Ab and Vip3A insecticidal proteins is given birth to the worker by Shanghai, and its dna sequence dna is respectively SEQ ID NO:5 and SEQ ID NO:7, and be cloned between the site of the restriction enzyme BamHI of PET28a expression vector and SacI.
The Cry1Ab-Vip3A building-up process as shown in Figure 2, concrete steps are as follows:
1, obtains Vip3H gene segment: V3AN 5 ' GGCCCC GGG ACC AAG CTG AGC ACC AGG GCC and V3AC 5 ' TCT TGA GCTCCT ACT TGA TGC TCA CGT CGT AGA ACT TCA CG by following two primers through PCR.These two primers comprise the site of restriction enzyme XmaI and SacI respectively.The template of PCR is Vip3H (SEQ IDNO:7).
2, handle the PCR product with XmaI and SacI, and be connected with the pET28a-Cry1Ab carrier of handling with same enzyme.
3, change intestinal bacteria over to, the acquisition plasmid is pET28a-Cry1Ab-Vip3A.Fusion toxin gene C ry1Ab-Vip3H (dna sequence dna 3) is cloned among the expression vector pET28a.The open reading frame of the Cry1Ab-Vip3A fusion toxin gene of this synthetic can be expressed the fused protein that a molecular weight is approximately 150kD.
The preparation of embodiment 3, Cry1Ab-Vip3H fusion rotein:
The expression vector pET28a-Cry1Ab-Vip3H that utilizes universal standard method will contain antigen-4 fusion protein gene is changed over to the single colony inoculation to 100 of e. coli bl21 star. milliliter LB inoculum, and 37 ℃ of vibrations are cultured to OD
600=0.6, add IPTG to 0.5mM, continue under same condition, to cultivate 4 hours.Centrifugal 10 minutes precipitations of nutrient solution 5000g Bacillus coli cells is abandoned the supernatant collecting precipitation then.Add 30 milliliters of 20mM Tris-HCl damping fluids in the precipitation, promptly can be used for the mensuration of insecticidal activity after the ultrasonication.
The preparation of embodiment 4, Cry1Ab-Vip3A fusion rotein:
The expression vector pET28a-Cry1Ab-Vip3A that utilizes universal standard method will contain antigen-4 fusion protein gene is changed over to the single colony inoculation to 100 of e. coli bl21 star. milliliter LB inoculum, and 37 ℃ of vibrations are cultured to OD
600=0.6, add IPTG to 0.5mM, continue under same condition, to cultivate 4 hours.Centrifugal 10 minutes precipitations of nutrient solution 5000g Bacillus coli cells is abandoned the supernatant collecting precipitation then.Add 30 milliliters of 20mM Tris-HCl damping fluids in the precipitation, promptly can be used for the mensuration of insecticidal activity after the ultrasonication.
Embodiment 5, multiple lepidopterous insects there is lethal effect at the Cry1Ab-Vip3H of expression in escherichia coli fusion toxin:
The surface that Cry1Ab-Vip3H that embodiment 3 and embodiment 4 are obtained and Cry1Ab-Vip3A fusion rotein are coated in the insect artificial diet, newborn first-instar young are used for carrying out insecticidal activity assay.The preparation method of negative control is identical with embodiment 3, but plasmid is not for containing the pET28a carrier of any insertion DNA itself.Insecticidal activity result is as shown in table 1:
The killing rate of table 1, Cry1 Ab-Vip3 H
| Mythimna separata | Pyrausta nubilalis (Hubern). | Beet armyworm | |
| Cry1Ab | 0％ | 100％ | 60％ |
| Vip3H | 100％ | 0％ | 100％ |
| Vip3A | 100％ | 0％ | 100％ |
| Cry1Ab-Vip3H | 100％ | 100％ | 100％ |
| Cry1Ab-Vip3A | 100％ | 100％ | 100％ |
| Negative control | 0％ | 0％ | 0％ |
Embodiment 6, transgenic paddy rice
The acquisition step of Cry1Ab-VipH transgenic paddy rice is as follows:
1, the Cry1Ab-Vip3H gene utilizes BamHI and SacI to cut out from pET28a-Cry1Ab-Vip3H (embodiment 1).Corn Ubiqutin promotor obtains (its 5 ' be provided with the HindIII site, 3 ' is provided with the BamHI site) with PCR method from the corn gene group, and with HindIII and BamHI processing.Carrier pCAM1300 handles through HindIII and SacI.More than 3 dna fragmentation linked enzymes and obtain pCAMUbi-Cry1Ab-Vip3H plant transgene plasmid.
2, applying gene small of the stock pCAMUbi-Cry1Ab-Vip3H squeezes into the rice callus tissue.Use present mature technology, carry out thought and cultivation, obtain transgenic paddy rice with 50mg/L hygromycin.
Embodiment 7, Cry1Ab-VipH express in plant can desinsection:
Cry1Ab-Vip3H is cloned under the control of corn Ubiqutin promotor, changes paddy rice over to particle gun, and the extract of the blade of paddy rice can kill insects such as Cnaphalocrocis medinali(rice leaf roller), striped rice borer, mythimna separata and Pyrausta nubilalis (Hubern)., and insecticidal effect is as shown in table 2:
Table 2
| Insect | Killing rate |
| Mythimna separata | 100％ |
| Beet armyworm | 100％ |
| Pyrausta nubilalis (Hubern). | 100％ |
| Striped rice borer | 100％ |
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
SEQ ID NO：1
1 ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CAACCCCGAG
61 GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CATCAGCCTG
121 AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GCTGGGCCTG
181 GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GGTGCAGATC
241 GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CAGCCGCCTG
301 GAGGGCCTGA GCAACCTGTA CCAAATCTAC GCCGAGAGCT TCCGCGAGTG GGAGGCCGAC
361 CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GAACAGCGCC
421 CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GCTGAGCGTG
481 TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTCAGCGT GTTCGGCCAG
541 CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CCGCCTGATC
601 GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CGTGTGGGGT
661 CCCGACAGCC GCGACTGGAT CAGGTACAAC CAGTTCCGCC GCGAGCTGAC CCTGACCGTG
721 CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CCGCACCGTG
781 AGCCAGCTGA CCCGCGAGAT TTACACCAAC CCCGTGCTGG AGAACTTCGA CGGCAGCTTC
841 CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GGACATCCTG
901 AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CGGCCACCAG
961 ATCATGGCCA GCCCCGTCGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GTACGGCACC
1021 ATGGGCAACG CTGCACCTCA GCAGCGCATA GTGGCACAGC TGGGCCAGGG AGTGTACCGC
1081 ACCCTGAGCA GCACCCTGTA CCGTCGACCT TTCAACATCG GCATCAACAA CCAGCAGCTG
1141 AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CAGCGCCGTG
1201 TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCTCAGAA CAACAACGTG
1261 CCACCTCGAC AGGGCTTCAG CCACCGTCTG AGCCACGTGA GCATGTTCCG CAGTGGCTTC
1321 AGCAACAGCA GCGTGAGCAT CATCCGTGCA CCTATGTTCA GCTGGATTCA CCGCAGTGCC
1381 GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CAAGAGCACC
1441 AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CGACATCCTG
1501 CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CCCCCTGAGC
1561 CAGCGCTACC GCGTCCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CCACACCAGC
1621 ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CGGCAGCAAC
1681 CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CAGCAACGGC
1741 AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GTACATCGAC
1801 CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GGAGAGGGCT
1861 CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GACCGACGTG
1921 ACCGACTACC ACATCGATCA GGTGCGAGGC CCCGGGCCAG GTGGAAGTAA GCTCTCCACC
1981 CGCGCCCTCC CGTCCTTCAT CGACTACTTC AACGGCATCT ACGGCTTCGC CACCGGCATC
2041 AAGGACATCA TGAACATGAT CTTCAAGACC GACACCGGCG GCAACGTCAC CCTCGACGAG
2101 ATCCTCAAGA ACCAGCAGCT CCTCAACGAG ATCAGCGGCA AGCTCGACGG CGTGAACGGC
2161 TCCCTCAACG AGCTCATCGC CCAGGTCAAC CTCAACACCG AGCTGTCCAA GGAGATCCTC
2221 AAGATCTCCA ACGAGCAGAA CCAGGTGCTC AACGACGTGA ACAACAAGCT GGACGCCATC
2281 AACACCATGC TGCACATCTA CCTCCCGAAG ATCACCTCCA TGCTCTCCGA CGTGATGAAG
2341 CAGAACTACG CCCTCTCCCT CCAGATCGAG TACCTCTCCA AGCAGCTCCA GGAGATCAGC
2401 GACAAGCTCG ACATCATCAA CGTGAACGTG CTCATCAACT CCACCCTCAC CGAGATCACC
2461 CCGGCCTACC AGCGCATCAA GTACGTGAAC GAGAAGTTCG AGGAGCTGAC CTTCGCCACC
2521 GAGACCACCC TCAAGGTGAA GAAGGACTCC TCCCCGGCCG ACATCCTCGA CGAGCTGACC
2581 GAGCTGACCG AGCTGGCCAA GTCCGTGACC AAGAACGACG TGGACGGCTT CGAGTTCTAC
2641 CTCAACACCT TCCACGACGT GATGGTGGGC AACAACCTCT TCGGCCGCTC CGCCCTCAAG
2701 ACCGCCTCCG AGCTGATCGC CAAGGAGAAC GTGAAGACCT CCGGCTCCGA GGTGGGCAAC
2761 GTGTACAACT TCCTCATCGT GCTCACCGCC CTGCAGGCCA AGGCCTTCCT CACCCTCACC
2821 ACCTGCCGCA AGCTCCTCGG CCTCGCCGGC ATCGACTACA CCTCCATCAT GAACGAGCAC
2881 CTCAACAAGG AGAAGGAGGA GTTCCGCGTG AACATCCTCC CGACCCTCTC CAACACCTTC
2941 TCCAACCCGA ACTACGCCAA GGTGAAGGGC TCCGACGAGG ACGCCAAGAT GATCGTGGAG
3001 GCCAAGCCGG GCCACGCCCT CGTGGGCTTC GAGATGTCCA ACGACTCCAT CACCGTGCTC
3061 AAGGTGTACG AGGCCAAGCT CAAGCAGAAC TACCAGGTGG ACAAGGACTC CCTCTCCGAG
3121 GTGATCTACG GCGACACCGA CAAGCTCTTC TGCCCGGACC AGTCCGAGCA GATATACTAC
3181 ACCAACAACA TCGTGTTCCC GAACGAGTAC GTGATCACCA AGATCGACTT CACCAAGAAG
3241 ATGAAGACCC TCCGCTACGA GGTGACCGCC AACTTCTACG ACTCCTCCAC CGGCGAGATC
3301 GACCTCAACA AGAAGAAGGT GGAGTCCTCC GAGGCCGAGT ACCGCACCCT CTCCGCCAAC
3361 GACGACGGCG TGTACATGCC GCTCGGCGTG ATCTCCGAAA CCTTCCTCAC CCCGATCAAC
3421 GGCTTCGGCC TCCAGGCCGA CGAGAACTCC CGCCTCATCA CCCTCACCTG CAAGTCCTAC
3481 CTCCGCGAGC TGCTCCTCGC CACCGACCTC TCCAACAAGG AGACCAAGCT CATCGTGCCG
3541 CCGTCCGGCT TCATCTCCAA CATCGTGGAG AACGGCGCCA TCGAGGAGGA CAACCTCGAG
3601 CCGTGGAAGG CCAACAACAA GAACGCCTAC GTGGACCACA CCGGCGGCGT GAACGGCACC
3661 AAGGCCCTCT ACGTGCACAA GGACGGCGGC TTCTCCCAGT TCATCGGCGA CAAGCTCAAG
3721 CCGAAGACCG AGTACGTGAT CCAGTACACC GTGAAGGGCA AGGCCAGCAT CTACCTGAAG
3781 GACGAGAAGA ACAACGAGGG CATCTACGAG GAGATCAACA ACGACCTGGA GGACTTCCAG
3841 ACCGTGACCA AGAGGTTCAT CACCGGCACC GACAGCAGCG GCGTGCACCT GATCTTCACC
3901 AGCCAGAACG GCGACGAGGC CTTCGGCGGC AACTTCATCA TCAGCGAGAT CAGGAGCAGC
3961 GAGGAGCTGC TGAGCCCGGA GCTGATCAAG AGCGACGCCT GGGTGGGCAG CCAGGGCACC
4021 TGGATCAGCG GCAACAGCCT GACCATCAAC AGCAACGCCA ACGGCACCTT CAGGCAGAAC
4081 CTGCCGCTGG AGAGCTACAG CACCTACAGC ATGAACTTCA ACGTGAACGG CTTCGCCAAG
4141 GTGACCGTGA GGAACAGCAG GGAGGTGCTG TTCGAGAAGA ACTTCAGCCA GCTGAGCCCG
4201 AAGGACTACA GCGAGAAGTT CACCACCGCC GCCAACAACA CCGGCTTCTA CGTGGAGCTG
4261 AGCAGGGGCA CCCAGGGCGG CAACATCACC TTCAGGGACT TCAGCATCAA GTAA
SEQ ID NO：2
1 ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CAACCCCGAG
61 GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CATCAGCCTG
121 AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GCTGGGCCTG
181 GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GGTGCAGATC
241 GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CAGCCGCCTG
301 GAGGGCCTGA GCAACCTGTA CCAAATCTAC GCCGAGAGCT TCCGCGAGTG GGAGGCCGAC
361 CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GAACAGCGCC
421 CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GCTGAGCGTG
481 TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTCAGCGT GTTCGGCCAG
541 CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CCGCCTGATC
601 GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CGTGTGGGGT
661 CCCGACAGCC GCGACTGGAT CAGGTACAAC CAGTTCCGCC GCGAGCTGAC CCTGACCGTG
721 CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CCGCACCGTG
781 AGCCAGCTGA CCCGCGAGAT TTACACCAAC CCCGTGCTGG AGAACTTCGA CGGCAGCTTC
841 CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GGACATCCTG
901 AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CGGCCACCAG
961 ATCATGGCCA GCCCCGTCGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GTACGGCACC
1021 ATGGGCAACG CTGCACCTCA GCAGCGCATA GTGGCACAGC TGGGCCAGGG AGTGTACCGC
1081 ACCCTGAGCA GCACCCTGTA CCGTCGACCT TTCAACATCG GCATCAACAA CCAGCAGCTG
1141 AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CAGCGCCGTG
1201 TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCTCAGAA CAACAACGTG
1261 CCACCTCGAC AGGGCTTCAG CCACCGTCTG AGCCACGTGA GCATGTTCCG CAGTGGCTTC
1321 AGCAACAGCA GCGTGAGCAT CATCCGTGCA CCTATGTTCA GCTGGATTCA CCGCAGTGCC
1381 GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CAAGAGCACC
1441 AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CGACATCCTG
1501 CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CCCCCTGAGC
1561 CAGCGCTACC GCGTCCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CCACACCAGC
1621 ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CGGCAGCAAC
1681 CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CAGCAACGGC
1741 AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GTACATCGAC
1801 CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GGAGAGGGCT
1861 CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GACCGACGTG
1921 ACCGACTACC ACATCGATCA GGTGCGAGGC CCCGGGACCA AGCTGAGCAC CAGGGCCCTG
1981 CCGAGCTTCA TCGACTACTT CAACGGCATC TACGGCTTCG CCACCGGCAT CAAGGACATC
2041 ATGAACATGA TCTTCAAGAC CGACACCGGC GGCGACCTGA CCCTGGACGA GATCCTGAAG
2101 AACCAGCAGC TGCTGAACGA CATCAGCGGC AAGCTGGACG GCGTGAACGG CAGCCTGAAC
2161 GACCTGATCG CCCAGGGCAA CCTGAACACC GAGCTGAGCA AGGAGATCCT GAAGATCGCC
2221 AACGAGCAGA ACCAGGTGCT GAACGACGTG AACAACAAGC TGGACGCCAT CAACACCATG
2281 CTGAGGGTGT ACCTGCCGAA GATCACCAGC ATGCTGAGCG ACGTGATGAA GCAGAACTAC
2341 GCCCTGAGCC TGCAGATCGA GTACCTGAGC AAGCAGCTGC AGGAGATCAG CGACAAGCTG
2401 GACATCATCA ACGTGAACGT GCTGATCAAC AGCACCCTGA CCGAGATCAC CCCGGCCTAC
2461 CAGAGGATCA AGTACGTGAA CGAGAAGTTC GAGGAGCTGA CCTTCGCCAC CGAGACCAGC
2521 AGCAAGGTGA AGAAGGACGG CAGCCCGGCC GACATCCTGG ACGAGCTGAC CGAGCTGACC
2581 GGCCTGGCCA AGAGCGTGCC GAAGAACGAC GTGGACGGCT TCGAGTTCTA CCTGAACACC
2641 TTCCACGACG TGATGGTGGG CAACAACCTG TTCGGCAGGA GCGCCCTGAA GACCGCCAGC
2701 GAGCTGATCA CCAAGGAGAA CGTGAAGACC AGCGGCAGCG AGGTGGGCAA CGTGTACAAC
2761 AGCCTGATCG TGCTGACCCT GCTGCAGGCC AAGGCCTTCC TGACCCTGAC CACCTGCAGG
2821 AAGCTGCTGG GCCTGGCCGA CATCGACTAC ACCAGCATCA TGAACGAGCA CCTGAACAAG
2881 GAGAAGGAGG AGTTCAGGGT GAACATCCCG CCGACCCTGA GCAACACCTT CAGCAACCCG
2941 AACTACGCCA AGGTGAAGGG CAGCGACGAG GACGCCAAGA TGATCGTGGA GGCCAAGCCG
3001 GGCCACGCCC TGGTGGGCTT CGAGATCAGC AACGACAGCA TCACCGTGCT GAAGGTGTAC
3061 GAGGCCAAGC TGAAGCAGAA CTACCAGGTG GACAAGGACA GCCTGAGCGA GGTGATCTAC
3121 GGCGACATGG ACAAGCTGCT GGGCCCGGAC CAGAGCGGCC CGATCTACTA CCCGAACAAC
3181 ATCGTGTTCC CGAACGAGTA CGTGATCACC AAGATCGACT TCACCAAGAA GATGAAGACC
3241 CTGAGGTACG AGGTGACCGC CAACTTCTAC GACAGCAGCA CCGGCGAGAT CGACCTGAAC
3301 AAGAAGAAGG TGGAGAGCAG CGAGGCCGAG TACAGGACCC TGAGCGCCAA CGACGACGGC
3361 GTGTACATGC CGCTGGGCGT GATCAGCGAG ACCTTCCTGA CCCCGATCAA CGGCTTCGGC
3421 CTGCAGGCCG ACGAGAACAG CAGGCTGATC ACCCTGACCT GCAAGAGCTA CCTGAGGGAG
3481 CTGCTGCTGG CCACCGACCT GAGCAACAAG GAGACCAAGC TGATCGTGCC GCCGAGCGGC
3541 TTCATCAAGA ACATCGTGGA GAACGGCAGC ATCGAGGAGG ACAACCTGGA GCCGTGGAAG
3601 GCCAACAACA AGAACGCCTA CGTGGACCAC ACCGGCGGCG TGAACGGCAC CAAGGCCCTG
3661 TACGTGCACA AGGACGGCGG CATCAGCCAG TTCATCGGCG ACAAGCTGAA GCCGAAGACC
3721 GAGTACGTGA TCCAGTACAC CGTGAAGGGC AAGCCGAGCA TCCACCTGAA GGACGAGAAC
3781 ACCGGCTACA TCCACTACGA GGACACCAAC AACAACCTGG AGGACTACCA GACCATCACC
3841 AAGAGGTTCA CCACCGGCAC CGACCTGAAG GGCGTGTACC TGATCCTGAA GAGCCAGAAC
3901 GGCGACGAGG CCTGGGGCGA CAACTTCATC ATCCTGGAGA TCAGCCCGAG CGAGAAGCTG
3961 CTGAGCCCGG AGCTGATCAA CACCAACAAC TGGACCAGCA CCGGCAGCAC CAACATCAGC
4021 GGCAACACCC TGACCCTGTA CCAGGGCGGC AGGGGCATCC TGAAGCAGAA CCTGCAGCTG
4081 GACAGCTTCA GCACCTACAG GGTGTACTTC AGCGTGAGCG GCGACGCCAA CGTGAGGATC
4141 AGGAACAGCA GGGAGGTGCT GTTCGAGAAG AGGTACATGA GCGGCGCCAA GGACGTGAGC
4201 GAGATCTTCA CCACCAAGCT GGGCAAGGAC AACTTCTACA TCGAGCTGAG CCAGGGCAAC
4261 AACCTGAACG GCGGCCCGAT CGTGAAGTTC TACGACGTGA GCATCAAGTA A
SEQ ID NO：3
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu
Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu
Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala
Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp GLy Ser Phe
Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val
Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe
Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser
Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly
Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val Arg Gly Pro Gly Pro Gly Gly Ser Lys Leu Ser Thr
Lys Asp Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asn Val Thr Leu Asp Glu
Lys Ile Ser Asn Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile
Asn Thr Met Leu His Ile Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met Lys
Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys Gln Leu Gln Glu Ile Ser
Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr
Pro Ala Tyr Gln Arg Ile Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr
Glu Thr Thr Leu Lys Val Lys Lys Asp Ser Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr
Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val Asp Gly Phe Glu Phe Tyr
Leu Asn Thr Phe His Asp Val Met Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys
Thr Ala Ser Glu Leu Ile Ala Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn
Val Tyr Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr Leu Thr
Thr Cys Arg Lys Leu Leu Gly Leu Ala Gly Ile Asp Tyr Thr Ser Ile Met Asn Glu His
Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe
Ser Asn Pro Asn Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu
Ala Lys Pro Gly His Ala Leu Val Gly Phe Glu Met Ser Asn Asp Ser Ile Thr Val Leu
Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp Lys Asp Ser Leu Ser Glu
Val Ile Tyr Gly Asp Thr Asp Lys Leu Phe Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr
Thr Asn Asn Ile Val Phe Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys
Met Lys Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile
Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr Arg Thr Leu Ser Ala Asn
Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn
Gly Phe Gly Leu Gln Ala Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr
Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile Val Pro
Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ala Ile Glu Glu Asp Asn Leu Glu
Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr
Lys Ala Leu Tyr Val His Lys Asp Gly Gly Phe Ser Gln Phe Ile Gly Asp Lys Leu Lys
Pro Lys Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Ala Ser Ile Tyr Leu Lys
Asp Glu Lys Asn Asn Glu Gly Ile Tyr Glu Glu Ile Asn Asn Asp Leu Glu Asp Phe Gln
Thr Val Thr Lys Arg Phe Ile Thr Gly Thr Asp Ser Ser Gly Val His Leu Ile Phe Thr
Ser Gln Asn Gly Asp Glu Ala Phe Gly Gly Asn Phe Ile Ile Ser Glu Ile Arg Ser Ser
Glu Glu Leu Leu Ser Pro Glu Leu Ile Lys Ser Asp Ala Trp Val Gly Ser Gln Gly Thr
Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn Ser Asn Ala Asn Gly Thr Phe Arg Gln Asn
Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser Met Asn Phe Asn Val Asn Gly Phe Ala Lys
Val Thr Val Arg Asn Ser Arg Glu Val Leu Phe Glu Lys Asn Phe Ser Gln Leu Ser Pro
Lys Asp Tyr Ser Glu Lys Phe Thr Thr Ala Ala Asn Asn Thr Gly Phe Tyr Val Glu Leu
Ser Arg Gly Thr Gln Gly Gly Asn Ile Thr Phe Arg Asp Phe Ser Ile Lys
SEQ ID NO：4
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu
Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu
Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala
Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val
Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe
Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser
Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly
Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val Arg Gly Pro Gly Thr Lys Leu Ser Thr Arg Ala Leu
Pro Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile
Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu Ile Leu Ly
Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn
Asp Leu Ile Ala Gln Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala
Asn Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met
Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met Lys Gln Asn Tyr
Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys Gln Leu Gln Glu Ile Ser Asp Lys Leu
Asp Ile Ile Asn Val Asn Val Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr
Gln Arg Ile Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser
Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr Glu Leu Thr
Gly Leu Ala Lys Ser Val Pro Lys Asn Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr
Phe His Asp Val Met Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser
Glu Leu Ile Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn
Ser Leu Ile Val Leu Thr Leu Leu Gln Ala Lys Ala Phe Leu Thr Leu Thr Thr Cys Arg
Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile Met Asn Glu His Leu Asn Lys
Glu Lys Glu Glu Phe Arg Val Asn Ile Pro Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro
Asn Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro
Gly His Ala Leu Val Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr Val Leu Lys Val Tyr
Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp Lys Asp Ser Leu Ser Glu Val Ile Tyr
Gly Asp Met Asp Lys Leu Leu Gly Pro Asp Gln Ser Gly Pro Ile Tyr Tyr Pro Asn Asn
Ile Val Phe Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys Thr
Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile Asp Leu Asn
Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly
Val Tyr Met Pro Leu Gly Val Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly
Leu Gln Ala Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu
Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile Val Pro Pro Ser Gly
Phe Ile Lys Asn Ile Val Glu Asn Gly Ser Ile Glu Glu Asp Asn Leu Glu Pro Trp Lys
Ala Asn Asn Lys Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu
Tyr Val His Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys Thr
Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His Leu Lys Asp Glu Asn
Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu Glu Asp Tyr Gln Thr Ile Thr
Lys Arg Phe Thr Thr Gly Thr Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn
Gly Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys Leu
Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly Ser Thr Asn Ile Ser
Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg Gly Ile Leu Lys Gln Asn Leu Gln Leu
Asp Ser Phe Ser Thr Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile
Arg Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val Ser
Glu Ile Phe Thr Thr Lys Leu Gly Lys Asp Asn Phe Tyr Ile Glu Leu Ser Gln Gly Asn
Asn Leu Asn Gly Gly Pro Ile Val Lys Phe Tyr Asp Val Ser Ile Lys
SEQ ID NO：5
1 ATGGACAACA ACCCCAACAT CAACGAGTGC ATCCCCTACA ACTGCCTGAG CAACCCCGAG
61 GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ACCGGCTACA CCCCCATCGA CATCAGCCTG
121 AGCCTGACCC AGTTCCTGCT GAGCGAGTTC GTGCCCGGCG CCGGCTTCGT GCTGGGCCTG
181 GTGGACATCA TCTGGGGCAT CTTCGGCCCC AGCCAGTGGG ACGCCTTCCT GGTGCAGATC
241 GAGCAGCTGA TCAACCAGCG CATCGAGGAG TTCGCCCGCA ACCAGGCCAT CAGCCGCCTG
301 GAGGGCCTGA GCAACCTGTA CCAAATCTAC GCCGAGAGCT TCCGCGAGTG GGAGGCCGAC
361 CCCACCAACC CCGCCCTGCG CGAGGAGATG CGCATCCAGT TCAACGACAT GAACAGCGCC
421 CTGACCACCG CCATCCCCCT GTTCGCCGTG CAGAACTACC AGGTGCCCCT GCTGAGCGTG
481 TACGTGCAGG CCGCCAACCT GCACCTGAGC GTGCTGCGCG ACGTCAGCGT GTTCGGCCAG
541 CGCTGGGGCT TCGACGCCGC CACCATCAAC AGCCGCTACA ACGACCTGAC CCGCCTGATC
601 GGCAACTACA CCGACCACGC CGTGCGCTGG TACAACACCG GCCTGGAGCG CGTGTGGGGT
661 CCCGACAGCC GCGACTGGAT CAGGTACAAC CAGTTCCGCC GCGAGCTGAC CCTGACCGTG
721 CTGGACATCG TGAGCCTGTT CCCCAACTAC GACAGCCGCA CCTACCCCAT CCGCACCGTG
781 AGCCAGCTGA CCCGCGAGAT TTACACCAAC CCCGTGCTGG AGAACTTCGA CGGCAGCTTC
841 CGCGGCAGCG CCCAGGGCAT CGAGGGCAGC ATCCGCAGCC CCCACCTGAT GGACATCCTG
901 AACAGCATCA CCATCTACAC CGACGCCCAC CGCGGCGAGT ACTACTGGAG CGGCCACCAG
961 ATCATGGCCA GCCCCGTCGG CTTCAGCGGC CCCGAGTTCA CCTTCCCCCT GTACGGCACC
1021 ATGGGCAACG CTGCACCTCA GCAGCGCATA GTGGCACAGC TGGGCCAGGG AGTGTACCGC
1081 ACCCTGAGCA GCACCCTGTA CCGTCGACCT TTCAACATCG GCATCAACAA CCAGCAGCTG
1141 AGCGTGCTGG ACGGCACCGA GTTCGCCTAC GGCACCAGCA GCAACCTGCC CAGCGCCGTG
1201 TACCGCAAGA GCGGCACCGT GGACAGCCTG GACGAGATCC CCCCTCAGAA CAACAACGTG
1261 CCACCTCGAC AGGGCTTCAG CCACCGTCTG AGCCACGTGA GCATGTTCCG CAGTGGCTTC
1321 AGCAACAGCA GCGTGAGCAT CATCCGTGCA CCTATGTTCA GCTGGATTCA CCGCAGTGCC
1381 GAGTTCAACA ACATCATCCC CAGCAGCCAG ATCACCCAGA TCCCCCTGAC CAAGAGCACC
1441 AACCTGGGCA GCGGCACCAG CGTGGTGAAG GGCCCCGGCT TCACCGGCGG CGACATCCTG
1501 CGCCGCACCA GCCCCGGCCA GATCAGCACC CTGCGCGTGA ACATCACCGC CCCCCTGAGC
1561 CAGCGCTACC GCGTCCGCAT CCGCTACGCC AGCACCACCA ACCTGCAGTT CCACACCAGC
1621 ATCGACGGCC GCCCCATCAA CCAGGGCAAC TTCAGCGCCA CCATGAGCAG CGGCAGCAAC
1681 CTGCAGAGCG GCAGCTTCCG CACCGTGGGC TTCACCACCC CCTTCAACTT CAGCAACGGC
1741 AGCAGCGTGT TCACCCTGAG CGCCCACGTG TTCAACAGCG GCAACGAGGT GTACATCGAC
1801 CGCATCGAGT TCGTGCCCGC CGAGGTGACC TTCGAGGCCG AGTACGACCT GGAGAGGGCT
1861 CAGAAGGCCG TGAACGAGCT GTTCACCAGC AGCAACCAGA TCGGCCTGAA GACCGACGTG
1921 ACCGACTACC ACATCGATCA GGTGCGAGGC CCCGGG
SEQ ID NO：6
1 ATGAACAAGA ACAACAGTAA GCTCTCCACC CGCGCCCTCC CGTCCTTCAT CGACTACTTC
61 AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC
121 GACACCGGCG GCAACGTCAC CCTCGACGAG ATCCTCAAGA ACCAGCAGCT CCTCAACGAG
181 ATCAGCGGCA AGCTCGACGG CGTGAACGGC TCCCTCAACG AGCTCATCGC CCAGGTCAAC
241 CTCAACACCG AGCTGTCCAA GGAGATCCTC AAGATCTCCA ACGAGCAGAA CCAGGTGCTC
301 AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGCACATCTA CCTCCCGAAG
361 ATCACCTCCA TGCTCTCCGA CGTGATGAAG CAGAACTACG CCCTCTCCCT CCAGATCGAG
421 TACCTCTCCA AGCAGCTCCA GGAGATCAGC GACAAGCTCG ACATCATCAA CGTGAACGTG
481 CTCATCAACT CCACCCTCAC CGAGATCACC CCGGCCTACC AGCGCATCAA GTACGTGAAC
541 GAGAAGTTCG AGGAGCTGAC CTTCGCCACC GAGACCACCC TCAAGGTGAA GAAGGACTCC
601 TCCCCGGCCG ACATCCTCGA CGAGCTGACC GAGCTGACCG AGCTGGCCAA GTCCGTGACC
661 AAGAACGACG TGGACGGCTT CGAGTTCTAC CTCAACACCT TCCACGACGT GATGGTGGGC
721 AACAACCTCT TCGGCCGCTC CGCCCTCAAG ACCGCCTCCG AGCTGATCGC CAAGGAGAAC
781 GTGAAGACCT CCGGCTCCGA GGTGGGCAAC GTGTACAACT TCCTCATCGT GCTCACCGCC
841 CTGCAGGCCA AGGCCTTCCT CACCCTCACC ACCTGCCGCA AGCTCCTCGG CCTCGCCGGC
901 ATCGACTACA CCTCCATCAT GAACGAGCAC CTCAACAAGG AGAAGGAGGA GTTCCGCGTG
961 AACATCCTCC CGACCCTCTC CAACACCTTC TCCAACCCGA ACTACGCCAA GGTGAAGGGC
1021 TCCGACGAGG ACGCCAAGAT GATCGTGGAG GCCAAGCCGG GCCACGCCCT CGTGGGCTTC
1081 GAGATGTCCA ACGACTCCAT CACCGTGCTC AAGGTGTACG AGGCCAAGCT CAAGCAGAAC
1141 TACCAGGTGG ACAAGGACTC CCTCTCCGAG GTGATCTACG GCGACACCGA CAAGCTCTTC
1201 TGCCCGGACC AGTCCGAGCA GATATACTAC ACCAACAACA TCGTGTTCCC GAACGAGTAC
1261 GTGATCACCA AGATCGACTT CACCAAGAAG ATGAAGACCC TCCGCTACGA GGTGACCGCC
1321 AACTTCTACG ACTCCTCCAC CGGCGAGATC GACCTCAACA AGAAGAAGGT GGAGTCCTCC
1381 GAGGCCGAGT ACCGCACCCT CTCCGCCAAC GACGACGGCG TGTACATGCC GCTCGGCGTG
1441 ATCTCCGAAA CCTTCCTCAC CCCGATCAAC GGCTTCGGCC TCCAGGCCGA CGAGAACTCC
1501 CGCCTCATCA CCCTCACCTG CAAGTCCTAC CTCCGCGAGC TGCTCCTCGC CACCGACCTC
1561 TCCAACAAGG AGACCAAGCT CATCGTGCCG CCGTCCGGCT TCATCTCCAA CATCGTGGAG
1621 AACGGCGCCA TCGAGGAGGA CAACCTCGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC
1681 GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTCT ACGTGCACAA GGACGGCGGC
1741 TTCTCCCAGT TCATCGGCGA CAAGCTCAAG CCGAAGACCG AGTACGTGAT CCAGTACACC
1801 GTGAAGGGCA AGGCCAGCAT CTACCTGAAG GACGAGAAGA ACAACGAGGG CATCTACGAG
1861 GAGATCAACA ACGACCTGGA GGACTTCCAG ACCGTGACCA AGAGGTTCAT CACCGGCACC
1921 GACAGCAGCG GCGTGCACCT GATCTTCACC AGCCAGAACG GCGACGAGGC CTTCGGCGGC
1981 AACTTCATCA TCAGCGAGAT CAGGAGCAGC GAGGAGCTGC TGAGCCCGGA GCTGATCAAG
2041 AGCGACGCCT GGGTGGGCAG CCAGGGCACC TGGATCAGCG GCAACAGCCT GACCATCAAC
2101 AGCAACGCCA ACGGCACCTT CAGGCAGAAC CTGCCGCTGG AGAGCTACAG CACCTACAGC
2161 ATGAACTTCA ACGTGAACGG CTTCGCCAAG GTGACCGTGA GGAACAGCAG GGAGGTGCTG
2221 TTCGAGAAGA ACTTCAGCCA GCTGAGCCCG AAGGACTACA GCGAGAAGTT CACCACCGCC
2281 GCCAACAACA CCGGCTTCTA CGTGGAGCTG AGCAGGGGCA CCCAGGGCGG CAACATCACC
2341 TTCAGGGACT TCAGCATCAA GTAA
SEQ ID NO：7
1 ATGAACAAGA ACAACACCAA GCTGAGCACC AGGGCCCTGC CGAGCTTCAT CGACTACTTC
61 AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC
121 GACACCGGCG GCGACCTGAC CCTGGACGAG ATCCTGAAGA ACCAGCAGCT GCTGAACGAC
181 ATCAGCGGCA AGCTGGACGG CGTGAACGGC AGCCTGAACG ACCTGATCGC CCAGGGCAAC
241 CTGAACACCG AGCTGAGCAA GGAGATCCTG AAGATCGCCA ACGAGCAGAA CCAGGTGCTG
301 AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGAGGGTGTA CCTGCCGAAG
361 ATCACCAGCA TGCTGAGCGA CGTGATGAAG CAGAACTACG CCCTGAGCCT GCAGATCGAG
421 TACCTGAGCA AGCAGCTGCA GGAGATCAGC GACAAGCTGG ACATCATCAA CGTGAACGTG
481 CTGATCAACA GCACCCTGAC CGAGATCACC CCGGCCTACC AGAGGATCAA GTACGTGAAC
541 GAGAAGTTCG AGGAGCTGAC CTTCGCCACC GAGACCAGCA GCAAGGTGAA GAAGGACGGC
601 AGCCCGGCCG ACATCCTGGA CGAGCTGACC GAGCTGACCG GCCTGGCCAA GAGCGTGCCG
661 AAGAACGACG TGGACGGCTT CGAGTTCTAC CTGAACACCT TCCACGACGT GATGGTGGGC
721 AACAACCTGT TCGGCAGGAG CGCCCTGAAG ACCGCCAGCG AGCTGATCAC CAAGGAGAAC
781 GTGAAGACCA GCGGCAGCGA GGTGGGCAAC GTGTACAACA GCCTGATCGT GCTGACCCTG
841 CTGCAGGCCA AGGCCTTCCT GACCCTGACC ACCTGCAGGA AGCTGCTGGG CCTGGCCGAC
901 ATCGACTACA CCAGCATCAT GAACGAGCAC CTGAACAAGG AGAAGGAGGA GTTCAGGGTG
961 AACATCCCGC CGACCCTGAG CAACACCTTC AGCAACCCGA ACTACGCCAA GGTGAAGGGC
1021 AGCGACGAGG ACGCCAAGAT GATCGTGGAG GCCAAGCCGG GCCACGCCCT GGTGGGCTTC
1081 GAGATCAGCA ACGACAGCAT CACCGTGCTG AAGGTGTACG AGGCCAAGCT GAAGCAGAAC
1141 TACCAGGTGG ACAAGGACAG CCTGAGCGAG GTGATCTACG GCGACATGGA CAAGCTGCTG
1201 GGCCCGGACC AGAGCGGCCC GATCTACTAC CCGAACAACA TCGTGTTCCC GAACGAGTAC
1261 GTGATCACCA AGATCGACTT CACCAAGAAG ATGAAGACCC TGAGGTACGA GGTGACCGCC
1321 AACTTCTACG ACAGCAGCAC CGGCGAGATC GACCTGAACA AGAAGAAGGT GGAGAGCAGC
1381 GAGGCCGAGT ACAGGACCCT GAGCGCCAAC GACGACGGCG TGTACATGCC GCTGGGCGTG
1441 ATCAGCGAGA CCTTCCTGAC CCCGATCAAC GGCTTCGGCC TGCAGGCCGA CGAGAACAGC
1501 AGGCTGATCA CCCTGACCTG CAAGAGCTAC CTGAGGGAGC TGCTGCTGGC CACCGACCTG
1561 AGCAACAAGG AGACCAAGCT GATCGTGCCG CCGAGCGGCT TCATCAAGAA CATCGTGGAG
1621 AACGGCAGCA TCGAGGAGGA CAACCTGGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC
1681 GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTGT ACGTGCACAA GGACGGCGGC
1741 ATCAGCCAGT TCATCGGCGA CAAGCTGAAG CCGAAGACCG AGTACGTGAT CCAGTACACC
1801 GTGAAGGGCA AGCCGAGCAT CCACCTGAAG GACGAGAACA CCGGCTACAT CCACTACGAG
1861 GACACCAACA ACAACCTGGA GGACTACCAG ACCATCACCA AGAGGTTCAC CACCGGCACC
1921 GACCTGAAGG GCGTGTACCT GATCCTGAAG AGCCAGAACG GCGACGAGGC CTGGGGCGAC
1981 AACTTCATCA TCCTGGAGAT CAGCCCGAGC GAGAAGCTGC TGAGCCCGGA GCTGATCAAC
2041 ACCAACAACT GGACCAGCAC CGGCAGCACC AACATCAGCG GCAACACCCT GACCCTGTAC
2101 CAGGGCGGCA GGGGCATCCT GAAGCAGAAC CTGCAGCTGG ACAGCTTCAG CACCTACAGG
2161 GTGTACTTCA GCGTGAGCGG CGACGCCAAC GTGAGGATCA GGAACAGCAG GGAGGTGCTG
2221 TTCGAGAAGA GGTACATGAG CGGCGCCAAG GACGTGAGCG AGATCTTCAC CACCAAGCTG
2281 GGCAAGGACA ACTTCTACAT CGAGCTGAGC CAGGGCAACA ACCTGAACGG CGGCCCGATC
2341 GTGAAGTTCT ACGACGTGAG CATCAAGTAA
Claims (9)
1, a kind of pest-resistant fusion gene is characterized in that: described gene comprises from 5 '-3 ' and is followed successively by the nucleotide sequence that contains coding BT crystal toxin and the nucleotide sequence of coding Vip3 toxin; And above-mentioned 2 nucleotide sequences are positioned at same open reading frame; Described BT crystal toxin is the active toxin of total length or C tip cut-off.
2, pest-resistant fusion gene according to claim 1 is characterized in that: described gene comprises from 5 '-3 ' and is followed successively by the nucleotide sequence that contains coding BT crystal toxin, any amino acid polypeptide segment and the nucleotide sequence of coding Vip3 toxin.
3, pest-resistant fusion gene according to claim 1 and 2 is characterized in that: described BT crystal toxin is Cry1Ab, Cry1Ac, Cry2 or Cry3, and described Vip3 toxin is Vip3A, Vip3B, Vip3C, Vip3D or Vip3H.
4, pest-resistant fusion gene according to claim 3 is characterized in that: described BT crystal toxin is that Cry1Ab, Vip3 toxin are Vip3H, and the nucleotides sequence of gained gene is classified SEQ ID NO:1 as.
5, pest-resistant fusion gene according to claim 3 is characterized in that: described BT crystal toxin is that Cry1Ab, Vip3 toxin are Vip3A, and the nucleotides sequence of gained gene is classified SEQ ID NO:2 as.
6, the fusion rotein of pest-resistant fusion gene coding as claimed in claim 1 is characterized in that: described fusion rotein is held to the C end from N and is followed successively by BT crystal toxin and Vip3 toxin.
7, fusion rotein according to claim 6 is characterized in that: described BT crystal toxin is that Cry1Ab, Vip3 toxin are Vip3H, and the aminoacid sequence of gained fusion rotein is SEQ ID NO:3.
8, fusion rotein according to claim 6 is characterized in that: described BT crystal toxin is that Cry1Ab, Vip3 toxin are Vip3A, and the aminoacid sequence of gained fusion rotein is SEQ ID NO:4.
9, as claim 6,7 or 8 described fusion roteins, the application aspect genetically modified crops anti-pest crop and genetically modified crops insecticidal microorganism.
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| CN106834318B (en) * | 2016-12-31 | 2020-05-29 | 浙江大学 | Insect-resistant fusion gene, encoding protein and application thereof |
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Effective date of registration: 20090313 Address after: Room 6, No. 8, ancient Pier Road, Xihu District, Zhejiang, Hangzhou Province, room 1301 Patentee after: Shen Zhicheng Address before: Hangzhou City, Zhejiang Province, Xihu District Ancient Jade Road 20 Patentee before: Zhejiang University |
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