CN100420752C - 一种杀虫基因及其用途 - Google Patents
一种杀虫基因及其用途 Download PDFInfo
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- CN100420752C CN100420752C CNB2006100497804A CN200610049780A CN100420752C CN 100420752 C CN100420752 C CN 100420752C CN B2006100497804 A CNB2006100497804 A CN B2006100497804A CN 200610049780 A CN200610049780 A CN 200610049780A CN 100420752 C CN100420752 C CN 100420752C
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Abstract
本发明公开了一种杀虫基因,该基因的核苷酸序列为SEQ ID NO:1。本发明还公开了上述杀虫基因编码的杀虫蛋白质,该蛋白质的氨基酸序列为SEQID NO:2。本发明的新型杀虫蛋白质,其氨基酸序列与目前已知的杀虫蛋白质不同,它对主要农业害虫,包括粘虫,玉米螟,甜菜夜蛾和二化螟等,具有高杀虫活性。
Description
技术领域
本发明涉及一种杀虫基因及其用途。
背景技术
杀虫毒素是一种具有重要运用价值的蛋白质,它可以用来改造杀虫微生物农药,也可以用于转基因抗虫作物。目前转基因抗虫技术已经大量商业化运用(James C.2004 ISAAA Briefs No.32.Ithaca,NY:ISAAA.43 pp),为害虫的治理提供了一种低成本的新型技术。目前植物转基因技术已经成熟,转基因抗虫的关键是获得性能优良的杀虫蛋白质。
目前杀虫蛋白质有多种。比较常用的是Bt晶体蛋白质,例如Cry1Ab,Cry1C,Cry2,Cry3等(Crickmore,N.,Zeigler,D.R.,Feitelson,J.,Schnepf,E.,Van Rie,J.,Lereclus,D.,Baum,J.& Dean,D.H.1998Microbiol.Mol.Biol.Rev.62,807-813)。现在已经商业化推广的转基因抗虫植物都是基于Bt晶体杀虫蛋白质。但是目前已发现多种昆虫可以产生对晶体杀虫蛋白质的抗性。抗性的发展影响了这些晶体毒素的抗虫性能和可利用性(Ferre,J.& Van Rie,J.(2002)Annu.Rev.Entomol.47,501-533)。没有交叉抗性的不同杀虫蛋白质共同使用或轮流使用可以有效减缓害虫抗性的发生(Zhao J-Z,Cao J,Li YX,Collins HL,Roush RT,Earle ED & SheltonAM.2003 Nature Biotechnol.21,1493-1497)。因此,获得新杀虫毒素,对提高杀虫能力和具有重要应用价值。
发明内容
针对现有技术中存在的不足之处,本发明提供一种新型的杀虫基因及其应用。
本发明为达到以上目的,是通过这样的技术方案来实现的:提供一种杀虫基因,该基因的核苷酸序列为SEQ ID NO:1。
本发明还提供了上述杀虫基因编码的杀虫蛋白质,该蛋白质的氨基酸序列为SEQ ID NO:2。
本发明还提供了一种杀虫基因,其氨基酸序列由SEQ ID NO:2的部分片断合成。
本发明还提供了上述杀虫基因编码的杀虫蛋白质,该蛋白质的氨基酸序列为:与SEQ ID NO:2相比具有90%以上的相同性。
本发明还提供了上述杀虫基因的用途。
本发明的新型杀虫蛋白质,其氨基酸序列与目前已知的杀虫蛋白质不同,根据植物基因的密码子使用频率,上述氨基酸序列被逆翻译为高GC含量的人工基因,用以植物转基因抗虫。它对主要农业害虫,包括粘虫,玉米螟,甜菜夜蛾等具有高杀虫活性。
具体实施方式
本发明的以下实施例所使用分子生物学的方法均为已知的技术。在Ausubel编写的John Wiley and Sons公司出版的Current Protocols in MolecularBiology,和J.Sambrook等编写Cold Spring Harbor Laboratory Press(2001)出版的Molecular Cloning:A Labortory Manual,3rd ED.等文献均有详细的说明。
实施例1、基因的克隆获得:
基因克隆获得的步骤如下:
1、苏云金芽胞杆菌分离方法:
取土壤样品0.5克,置于40毫升左右的BPA(0.5%牛肉膏,1%蛋白胨,3.4%醋酸钠,PH7.2-7.4)培养基中,充分震荡;再置于30℃摇床中培养4.0小时,取出置80℃水浴中处理30分钟,或者培养箱中1.0小时;取0.5毫升转移到BP培养基(0.3%牛肉膏,0.5%蛋白胨,0.5%氯化钠,1.8%琼脂,青霉素G1000u/ml,多粘菌素B3ppm,PH7.2-7.4)上,涂布均匀,置于28℃培养24小时。
2、新基因的筛选:
将候选的菌株在加有50微克/毫升氨苄青霉素的LB固体培养基的上画线,挑取单克隆,转接于5毫升加有50微克/毫升氨苄青霉素的LB液体培养基中,于28℃以190r/min振荡培养到对数生长中期;12000rpm离心一分钟收集菌体,提取质粒。以上述质粒为模板DNA,用两对筛选引物进行PCR扩增(引物序列见表1),扩增条件为95℃预变性5min,再按照95℃30sec,52℃30sec,72℃30sec程序扩增35循环,在1.0%琼脂糖凝胶电泳上检测结果,凡泳道中仅出现一条带或不出现条带者为候选菌株。实验结果表明12号Bt菌株仅出现了一条600bp左右条带,回收测序,分析表明该序列与已报道的营养期杀虫蛋白基因存在较大变异,为一个新型的Vip3杀虫蛋白基因。
表1:筛选引物的序列和片段长度
3、新基因全长序列的获得:
测序结果表明该片断长度为580bp,序列分析表明该片断内含HindIII限制性消化酶位点(211),由此在该位点5‘端和3‘端方向设计正向引物和反向引物,利用反向PCR的方法克隆到该酶切位点上游211bp片段和下游641bp片段;分析641bp片段序列表明内含PstI限制性位点,在该位点两侧设计正向和反向引物,利用反向PCR获得了下游1.766kb长度序列。由此获得了该基因的全长序列,其编码的蛋白质氨基酸序列见SEQ ID NO:2。本步骤中使用的反向PCR引物见表2。
表2:反向PCR引物
根据玉米基因的密码子使用频率,上述氨基酸序列被逆翻译为高GC含量的人工基因,经上海生工公司人工合成(如SEQ ID NO:1所述)并克隆到表达载体PET28a的BamH1和Sac位点间(质粒pET28a-LG01)。
实施例2、蛋白的制备:
利用通用的标准方法将含上述基因的表达载体pET28a-LG01转入大肠杆菌BL21star并获得质粒含有pET28a-LG01的菌落。单个菌落接种到100毫升LB细菌培养液,在37℃下震动培养至OD600=0.6,加IPTG到0.5mM,并继续在同样的条件下培养4小时。培养液经过5000g离心10分钟沉淀大肠杆菌细胞,然后弃上清收集沉淀。沉淀中加30毫升20mM Tris-HCl缓冲液,超声粉碎后即可用于杀虫活性的测定。
实施例3、在大肠杆菌中表达的蛋白对多种鳞翅目昆虫有杀伤作用:
将实施例2所获得的蛋白涂在昆虫人工饲料的表面,放置2小时后,接上新生一龄幼虫进行杀虫活性测定。阴性对照的准备方法与实施例2相同,但质粒为不含任何插入DNA的pET28a载体本身。在25℃条件下,7天后记录杀虫数目。杀虫活性结果如下表:
粘虫 | 玉米螟 | 甜菜夜蛾 | |
本发明 | 100% | 80% | 100% |
阴性对照 | 0% | 0% | 0% |
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
SEQ ID NO:1
1 ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT
61 AATGGCATTT ATGGATTTGC CACTGGTATC AAGGACATTA TGAACATGAT TTTTAAAACG
121 GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGAA
181 ATTTCTGGTA AATTGGATGG GGTAAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAAC
241 TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTTA
301 AATGATGTTA ATAACAAACT TGATGCGATA AATACGATGC TGCATATATA TCTACCTAAA
361 ATTACCTCTA TGTTAAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGAA
421 TACCTAAGTA AACAGTTGCA AGAAATTTCT GATAAATTGG ATATTATTAA CGTAAATGTA
481 CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AACGGATTAA ATATGTGAAC
541 GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAATT TAAAAGTAAA AAAGGATGGC
601 TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAACA
661 AAAAATGATG TGGATGGTTT TGAATTTTAT CTTAATACAT TCCACGATGT AATGGTAGGG
721 AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAAT
781 GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGCT
841 CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT
901 ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA
961 AACATCCTTC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATATAAA AACTAAAGGA
1021 AGCGATGAGG ATGCGGAAGT GATTATTCAA GCTGAACCAG GACATGCTTT GGTGGGATTT
1081 GAAATGATTA ATGATCCAAG TCCAGCGTTA AAAGTGTATC AGGCTAAACT AAAACCAAAT
1141 TATCAAGTTG ATAAGCAGTC ATTATCTGAA ACTGTGTATG GTGATATGGA TAAAATATTA
1201 TGTCCAGATA AATCTCAACA AATGTATTAT CTGCATAATA TAACATTCCC AAATGAATAT
1261 GTGATTACAG AAATTATTTT TACAAAGAAA AAGAACAGTT TAAGGTATGA GGTGATAGCA
1321 AATTATTATG AGTTCTCTTC AGGAGATATC GACTTAAATA AGAAGTTAGT AAAATCAAGT
1381 GAAGCAGAGT ATAGTACGTT AAGTGTTAGT AACGATGCTA TTTATATGCC GCTAGGTGTT
1441 ATCAGTGAAA CATTTTTGAC CCCAATTAAA GGATTCGGAC TAACAGTTGA TGAAAGTTCA
1501 AGACTGGTAA CTTTAACGTG TAAATCATAT TTAAGGGAAA TTCTGTTAGC AACAGATTTA
1561 AGCAATAAAG CAACAAAATT AATTGTCCCA CCTAATGGTT TTATTAGTAA TTTGGTGGAA
1621 AATGGGGATA TTGAGGCTGA CAACATAGAA CCATGGAAGG GAAATAATAA AAATGCGTAT
1681 GTTGATCATA CAGGTGGGGT GAATGGAACT AAAGCTCTGT ACACTCAAGA TGATGGGGAA
1741 TTTTCACAAT TTATTGGAGA TAAGTTGAAA TCGAAAACTG AGTATATAAT CCAATATACT
1801 GTAAAAGGAA ATACTTCTAT TTATTTGAAA GATAAAAAAA ATGAAAATGT TATTTATGAA
1861 GATAAAAATA ATAATTTAGA GGCTTTTCAA ACTATTACTA AAAGGTTTAC TACAGAATTG
1921 GATTCTTCAG ATGTTTACTT AGTGTTTAAA TGCAAAAATG GCTATAAAGC TTGGGGAGAC
1981 AACTTTCTAA TTACAGAAAT TAGGCCTAAG GAAGTGGTAA GCCCAGAATT GATAAAAGTA
2041 GAAAATTGGA TTGGAATGGG TGGTAGTAAT CATGTAAACC CTGATTCACT TTTGCTTTTT
2101 ACAGGTGGGA GGTCAATTTT AAAACAAAAT CTCCAATTAG ATAGTTATTC AACCTATAGA
2161 GTAAGATTTT CTTTAATGGT AATTGGTAAG GCTAAGGTTA TTATAAGGAA TTCAAGTGAA
2221 GTACTGTTTG AAAAAAGTTA TGTGAATGAT TCTGAAGGTG TTTTAGAAGG TGTTTCTGAA
2281 ACTTTTACTA CAAAATCGAT TCAAGATAAC TTCTATGTAG AACTTTCTAA TGAGGGCACT
2341 TTTGGAAGTA AAGATGTTGC ATACTTTTAT AATTTTTCTA TTAAATAA
SEQ ID NO:2
Met Asn Lys Asn Asn 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 Lys Asn Gln Gln Leu Leu Asn Glu
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 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 Asn Leu Lys Val Lys Lys Asp Gly
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 Thr 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 Asp
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 Ile Lys Thr Lys Gly
Ser Asp Glu Asp Ala Glu Val Ile Ile Gln Ala Glu Pro Gly His Ala Leu Val Gly Phe
Glu Met Ile Asn Asp Pro Ser Pro Ala Leu Lys Val Tyr Gln Ala Lys Leu Lys Pro Asn
Tyr Gln Val Asp Lys Gln Ser Leu Ser Glu Thr Val Tyr Gly Asp Met Asp Lys Ile Leu
Cys Pro Asp Lys Ser Gln Gln Met Tyr Tyr Leu His Asn Ile Thr Phe Pro Asn Glu Tyr
Val Ile Thr Glu Ile Ile Phe Thr Lys Lys Lys Asn Ser Leu Arg Tyr Glu Val Ile Ala
Asn Tyr Tyr Glu Phe Ser Ser Gly Asp Ile Asp Leu Asn Lys Lys Leu Val Lys Ser Ser
Glu Ala Glu Tyr Ser Thr Leu Ser Val Ser Asn Asp Ala Ile Tyr Met Pro Leu Gly Val
Ile Ser Glu Thr Phe Leu Thr Pro Ile Lys Gly Phe Gly Leu Thr Val Asp Glu Ser Ser
Arg Leu Val Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Ile Leu Leu Ala Thr Asp Leu
Ser Asn Lys Ala Thr Lys Leu Ile Val Pro Pro Asn Gly Phe Ile Ser Asn Leu Val Glu
Asn Gly Asp Ile Glu Ala Asp Asn Ile Glu Pro Trp Lys Gly Asn Asn Lys Asn Ala Tyr
Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Thr Gln Asp Asp Gly Glu
Phe Ser Gln Phe Ile Gly Asp Lys Leu Lys Ser Lys Thr Glu Tyr Ile Ile Gln Tyr Thr
Val Lys Gly Asn Thr Ser Ile Tyr Leu Lys Asp Lys Lys Asn Glu Asn Val Ile Tyr Glu
Asp Lys Asn Asn Asn Leu Glu Ala Phe Gln Thr Ile Thr Lys Arg Phe Thr Thr Glu Leu
Asp Ser Ser Asp Val Tyr Leu Val Phe Lys Cys Lys Asn Gly Tyr Lys Ala Trp Gly Asp
Asn Phe Leu Ile Thr Glu Ile Arg Pro Lys Glu Val Val Ser Pro Glu Leu Ile Lys Val
Glu Asn Trp Ile Gly Met Gly Gly Ser Asn His Val Asn Pro Asp Ser Leu Leu Leu Phe
Thr Gly Gly Arg Ser Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Tyr Ser Thr Tyr Arg
Val Arg Phe Ser Leu Met Val Ile Gly Lys Ala Lys Val Ile Ile Arg Asn Ser Ser Glu
Val Leu Phe Glu Lys Ser Tyr Val Asn Asp Ser Glu Gly Val Leu Glu Gly Val Ser Glu
Thr Phe Thr Thr Lys Ser Ile Gln Asp Asn Phe Tyr Val Glu Leu Ser Asn Glu Gly Thr
Phe Gly Ser Lys Asp Val Ala Tyr Phe Tyr Asn Phe Ser Ile Lys
Claims (3)
1. 一种杀虫基因,其特征是:该基因的核苷酸序列为SEQ ID NO:1。
2. 根据权利要求1所述杀虫基因编码的杀虫蛋白质,其特征是:所述蛋白质的氨基酸序列为SEQ ID NO:2。
3. 如权利要求1所述杀虫基因的用途,其特征是:用于杀灭鳞翅目昆虫。
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CN101679491B (zh) * | 2007-03-28 | 2013-11-06 | 先正达参股股份有限公司 | 杀虫的蛋白质 |
CN101173288B (zh) * | 2007-12-03 | 2010-09-08 | 浙江大学 | 人工合成的多核苷酸及获得转基因植物的方法 |
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CN1260397A (zh) * | 1999-08-26 | 2000-07-19 | 中国科学院遗传研究所 | 杀虫蛋白基因及其应用 |
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