CN100420751C - Pesticidal gene and its use - Google Patents
Pesticidal gene and its use Download PDFInfo
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- CN100420751C CN100420751C CNB2006100496106A CN200610049610A CN100420751C CN 100420751 C CN100420751 C CN 100420751C CN B2006100496106 A CNB2006100496106 A CN B2006100496106A CN 200610049610 A CN200610049610 A CN 200610049610A CN 100420751 C CN100420751 C CN 100420751C
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Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention discloses a disinsection gene whose nucleotide sequence is disclosed in SEQ ID NO: 2. The present invention also discloses disinsection protein coded by the disinsection gene, and the amino acid sequence of the protein is SEQ ID NO: 1. The amino acid sequence of the novel disinsection protein of the present invention is different from that of the existing disinsection protein at present, and the novel disinsection protein has high insecticidal activity to main agricultural pests comprising armyworms, corn borers, beet noctuids, striped rice borers, etc.
Description
Technical field
The present invention relates to a kind of killing gene and uses thereof.
Background technology
Pesticidal toxins is a kind of protein with important applying value, and it can be used for transforming the insecticidal microorganism agricultural chemicals, also can be used for the transgenic pest-resistant crop.At present the transgenic pest-resistant technology in a large number commercialization use (James is ISAAA Briefs No.32.Ithaca C.2004, NY:ISAAA.43pp), for the improvement of insect provides a kind of new technique cheaply.Plant transgenic technology is ripe at present, and the key of transgenic pest-resistant is the good insect-killing protein of obtained performance.
Insect-killing protein has multiple at present.Relatively commonly used is the Bt crystal proteins, Cry1Ab for example, Cry1C, Cry2, (Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J.﹠amp such as Cry3; Dean, D.H.1998Microbiol.Mol.Biol.Rev.62,807-813).The transgenic anti-insect plants that commercialization is now promoted all is based on Bt crystal insect-killing protein.But found that various insects can produce the resistance to the crystal insect-killing protein at present.The influence on development of resistance pest-resistant performance and utilizability (Ferre, the J.﹠amp of these crystal toxins; Van Rie, J. (2002) Annu.Rev.Entomol.47,501-533).Do not have the common use of different insect-killing proteins of crossed resistance or use generation (Zhao J-Z, Cao J, Li YX, Collins HL, Roush RT, the Earle ED ﹠amp that can effectively slow down pest resistance in turn; SheltonAM.2003 Nature Biotechnol.21,1493-1497).Therefore, obtain new Pesticidal toxins, to improving killing ability and having significant application value.
Summary of the invention
At the deficiencies in the prior art part, the invention provides a kind of novel killing gene and application thereof.
The present invention is to realize by such technical scheme for reaching above purpose: a kind of killing gene is provided, and the nucleotides sequence of this gene is classified SEQ ID NO:2 as.
The present invention also provides the insect-killing protein of above-mentioned killing gene coding, and this proteinic aminoacid sequence is SEQ ID NO:1.
The present invention also provides a kind of killing gene, and this gene utilizes the part segment of SEQ ID NO:1 synthetic.
The present invention also provides the insect-killing protein of above-mentioned killing gene coding, and this proteinic aminoacid sequence is: compare the homogeny that has more than 91% with SEQ ID NO:1.
The present invention also provides the purposes of above-mentioned killing gene.
New type disinsection protein of the present invention, its aminoacid sequence is different with present known insect-killing protein, and it comprises mythimna separata to main Agricultural pests, Pyrausta nubilalis (Hubern)., beet armyworm and striped rice borer etc. have high insecticidal activity.
Embodiment
Following examples of the present invention use molecular biological method to be known technology.The Current Protocols in MolecularBiology that publishes in the John Wiley and Sons company that Ausubel writes, write the Molecular Cloning:A Labortory Manual that Cold Spring Harbor Laboratory Press (2001) publishes with J.Sambrook etc., documents such as 3rd ED. all have detailed explanation.
The clone of embodiment 1, gene obtains and synthetic gene:
The step that gene clone obtains is as follows:
1, get 300 different Bt bacterial strains and use the TB nutrient solution 28 ℃ of shaking culture 24 hours respectively, nutrient solution all mixes then, according to method (Reddy, A., Battisti, the L. ﹠amp of Reddy etc.; Thorne, C.B. (1987) J. Bacteriol.169,5263-5270) plasmid of extraction Bt.This plasmid is the plasmid mixture of 300 different B t bacterial strains.
2, be template DNA with above-mentioned Bt plasmid, carry out pcr amplification with primer StartN 5 ' atg aac aag aat aat act aaatta agc aca aga and StopC 5 ' cat gtt act cga gaa ttc tcg ag tta.Pcr amplification carries out 30 circulations altogether.Five circulations at first are 95 ℃, 30 seconds; 50 ℃, 60 seconds; 72 ℃, 2 minutes 30 seconds is 25 circulations then: 95 ℃, and 30 seconds; 60 ℃, 60 seconds; 72 ℃, 2 minutes 30 seconds.
3, to the PCR product by electrophoretic analysis, the about DNA of 2.4kb is reclaimed among rear clone gives birth to the T-carrier that the worker produces to Shanghai.To the cloning and sequencing that obtains, its encoded protein matter aminoacid sequence is seen sequence 1 (SEQ ID NO:1).
Codon usage frequency according to corn gene, above-mentioned aminoacid sequence is the artificial gene of high GC content by reverse translation, gives birth to worker's company's synthetic (as described in SEQ ID NO:2) through Shanghai and is cloned between the BamH1 and Sac site of expression vector PET28a (plasmid pET28a-LG01).
Embodiment 2, proteic preparation:
The expression vector pET28a-LG01 that utilizes general standard method will contain said gene changes e. coli bl21 star over to and obtains the bacterium colony that plasmid contains pET28a-LG01.Single colony inoculation to 100 milliliter LB inoculum, vibrations are cultured to OD under 37 ℃
600=0.6, add IPTG to 0.5mM, and continue under same condition, to cultivate 4 hours.Nutrient solution is abandoned the supernatant collecting precipitation then through centrifugal 10 minutes precipitations of 5000g Bacillus coli cells.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 3, multiple lepidopterous insects there is lethal effect at the albumen of expression in escherichia coli:
The albumen that embodiment 2 is obtained is coated in insect artificial diet's surface, places after 2 hours, connects newborn first-instar young and carries out insecticidal activity assay.The preparation method of negative control is identical with embodiment 2, but plasmid is not for containing the pET28a carrier of any insertion DNA itself.Under 25 ℃ of conditions, record desinsection number after 7 days.Insecticidal activity result such as following table:
| Mythimna separata | Pyrausta nubilalis (Hubern). | Beet armyworm | |
| The present invention | 100％ | 80％ | 100％ |
| Negative control | 0％ | 0％ | 0％ |
Embodiment 4, expressed proteins has insecticidal activity in the rice callus tissue:
In the rice callus tissue, obtained expression as follows, and shown to have insecticidal activity:
1, gene of the present invention utilizes BamHI and SacI to cut out from pET28a-LG01 (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-LG01 plant transgene plasmid.
2, utilize particle gun that pCAMUbi-LG01 is squeezed into the rice callus tissue, and under the hygromycin of 50mg/L, screen and cultivate.After 20 days, the rice callus that screening and culturing obtains is organized in ultrasonication among the 20mM Tris-HCl.Carry out the mensuration of insecticidal activity behind the concussion mixing.
3, the mixing sample is coated in insect artificial diet's surface, and newborn first-instar young is inoculated into above the artificial diet, carries out insecticidal activity assay.Negative control is the rice callus tissue that does not import foreign gene.Determination of activity is added up insect mortality after carrying out 7 days under 125 ℃.
The result shows the transgenic paddy rice callus to Cnaphalocrocis medinali(rice leaf roller), striped rice borer, and the insecticide efficiency of insects such as mythimna separata and Pyrausta nubilalis (Hubern). is 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
Met Asn Lys Asn Asn Ser 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 Asn Val Thr Leu Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Glu
Ile Ser Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Glu Leu Ile Ala Gln Val Asn
Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu 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：2
1 ATGAACAAGA ACAACAGCAA GCTGAGCACC AGGGCCCTGC CGAGCTTCAT CGACTACTTC
61 AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC
121 GACACCGGCG GCAACGTGAC CCTGGACGAG ATCCTGAAGA ACCAGCAGCT GCTGAACGAG
181 ATCAGCGGCA AGCTGGACGG CGTGAACGGC AGCCTGAACG AGCTGATCGC CCAGGTGAAC
241 CTGAACACCG AGCTGAGCAA GGAGATCCTG AAGATCAGCA ACGAGCAGAA CCAGGTGCTG
301 AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGCACATCTA 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 GAGACCACCC TGAAGGTGAA GAAGGACAGC
601 AGCCCGGCCG ACATCCTGGA CGAGCTGACC GAGCTGACCG AGCTGGCCAA GAGCGTGACC
661 AAGAACGACG TGGACGGCTT CGAGTTCTAC CTGAACACCT TCCACGACGT GATGGTGGGC
721 AACAACCTGT TCGGCAGGAG CGCCCTGAAG ACCGCCAGCG AGCTGATCGC CAAGGAGAAC
781 GTGAAGACCA GCGGCAGCGA GGTGGGCAAC GTGTACAACT TCCTGATCGT GCTGACCGCC
841 CTGCAGGCCA AGGCCTTCCT GACCCTGACC ACCTGCAGGA AGCTGCTGGG CCTGGCCGGC
901 ATCGACTACA CCAGCATCAT GAACGAGCAC CTGAACAAGG AGAAGGAGGA GTTCAGGGTG
961 AACATCCTGC CGACCCTGAG CAACACCTTC AGCAACCCGA ACTACGCCAA GGTGAAGGGC
1021 AGCGACGAGG ACGCCAAGAT GATCGTGGAG GCCAAGCCGG GCCACGCCCT GGTGGGCTTC
1081 GAGATGAGCA ACGACAGCAT CACCGTGCTG AAGGTGTACG AGGCCAAGCT GAAGCAGAAC
1141 TACCAGGTGG ACAAGGACAG CCTGAGCGAG GTGATCTACG GCGACACCGA CAAGCTGTTC
1201 TGCCCGGACC AGAGCGAGCA GATCTACTAC ACCAACAACA 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 TCATCAGCAA CATCGTGGAG
1621 AACGGCGCCA TCGAGGAGGA CAACCTGGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC
1681 GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTGT ACGTGCACAA GGACGGCGGC
1741 TTCAGCCAGT TCATCGGCGA CAAGCTGAAG 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
Claims (3)
1. killing gene, it is characterized in that: the nucleotides sequence of this gene is classified SEQ ID NO:2 as.
2. according to the insect-killing protein of the described killing gene coding of claim 1, it is characterized in that: described proteinic aminoacid sequence is SEQ ID NO:1.
3. the purposes of killing gene according to claim 1 is characterized in that: be used to kill mythimna separata, Pyrausta nubilalis (Hubern)., beet armyworm or striped rice borer.
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| CNB2006100496106A CN100420751C (en) | 2006-02-27 | 2006-02-27 | Pesticidal gene and its use |
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| CNB2006100496106A CN100420751C (en) | 2006-02-27 | 2006-02-27 | Pesticidal gene and its use |
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| CN100420751C true CN100420751C (en) | 2008-09-24 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421816A (en) * | 2012-12-05 | 2013-12-04 | 北京大北农科技集团股份有限公司 | Insecticidal gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101679491B (en) * | 2007-03-28 | 2013-11-06 | 先正达参股股份有限公司 | Insecticidal proteins |
| CN101173288B (en) * | 2007-12-03 | 2010-09-08 | 浙江大学 | Methods for synthesizing ribonucleotide sequence and acquiring transgenic plant |
| CN102786585B (en) * | 2012-08-02 | 2013-12-18 | 北京大北农科技集团股份有限公司 | Insecticidal protein, coding gene of insecticidal protein and purpose of insecticidal protein |
| CN105624177A (en) * | 2016-02-04 | 2016-06-01 | 浙江大学 | Insect-fusion-resistant gene, coding protein, carrier and application thereof |
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| CN1199321A (en) * | 1995-10-13 | 1998-11-18 | 陶氏农业科学有限公司 | Modified bacillus thuringiensis gene for lepidopteran control in plants |
| US6603063B1 (en) * | 1999-05-07 | 2003-08-05 | Mycogen Corp. | Plants and cells transformed with a nucleic acid from Bacillus thuringiensis strain KB59A4-6 encoding a novel SUP toxin |
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| CN1199321A (en) * | 1995-10-13 | 1998-11-18 | 陶氏农业科学有限公司 | Modified bacillus thuringiensis gene for lepidopteran control in plants |
| US6603063B1 (en) * | 1999-05-07 | 2003-08-05 | Mycogen Corp. | Plants and cells transformed with a nucleic acid from Bacillus thuringiensis strain KB59A4-6 encoding a novel SUP toxin |
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| Bt Cry1Ac毒蛋白对棉铃虫和粘虫作用机理研究. 宋萍.河北农业大学硕士学位论文. 2003 |
| Bt Cry1Ac毒蛋白对棉铃虫和粘虫作用机理研究. 宋萍.河北农业大学硕士学位论文. 2003 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421816A (en) * | 2012-12-05 | 2013-12-04 | 北京大北农科技集团股份有限公司 | Insecticidal gene and application thereof |
| CN103421816B (en) * | 2012-12-05 | 2016-06-08 | 北京大北农科技集团股份有限公司 | Killing gene and purposes thereof |
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| CN1810976A (en) | 2006-08-02 |
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