CN100420751C - 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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- 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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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:2。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:1。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.43pp),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:2。
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:1。
本发明还提供了一种杀虫基因,该基因利用SEQ ID NO:1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:1相比具有91%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,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。
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、基因的克隆获得和人工合成基因:
基因克隆获得的步骤如下:
1、取300个不同的Bt菌株分别用TB培养液在28℃振荡培养24小时,然后培养液全部混合,根据Reddy等的方法(Reddy,A.,Battisti,L. & Thorne,C.B.(1987)J. Bacteriol.169,5263-5270)提取Bt的质粒。此质粒为300个不同Bt菌株的质粒混合物。
2、以上述Bt质粒为模板DNA,用引物StartN 5’atg aac aag aat aat act aaatta agc aca aga和StopC 5’cat gtt act cga gaa ttc tcg ag tta进行PCR扩增。PCR扩增共进行30个循环。首先的五个循环为95℃,30秒;50℃,60秒;72℃,2分30秒,然后是25个循环:95℃,30秒;60℃,60秒;72℃,2分30秒。
3、对PCR产物通过电泳分析,把大约2.4kb的DNA回收后克隆到上海生工生产的T-载体之中。对获得的克隆测序,其编码的蛋白质氨基酸序列见序列1(SEQ ID NO:1)。
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:2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***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% |
实施例4、在水稻愈伤组织中表达的蛋白具有杀虫活性:
通过如下步骤在水稻愈伤组织中获得了表达,并表明具有杀虫活性:
1、本发明的基因从pET28a-LG01(实施例1)利用BamHI和SacI切出。玉米Ubiqutin启动子用PCR方法从玉米基因组获得(其5’设有HindIII位点,3’设有BamHI位点),并用HindIII和BamHI处理。载体pCAM1300经过HindIII和SacI处理。以上3个DNA片段经连接酶而获得pCAMUbi-LG01植物转基因质粒。
2、利用基因枪把pCAMUbi-LG01打入水稻愈伤组织,并在50mg/L的hygromycin下进行筛选和培养。20天后,筛选培养获得的水稻愈伤组织在20mM Tris-HCl中超声粉碎。震荡混匀后进行杀虫活性的测定。
3、混匀样品涂在昆虫人工饲料的表面,新生一龄幼虫接种到人工饲料上面,进行杀虫活性测定。阴性对照是没有导入外源基因的水稻愈伤组织。活性测定在125℃下进行7天后统计昆虫死亡率。
结果表明转基因水稻愈伤组织对稻纵卷叶螟,二化螟,粘虫和玉米螟等害虫的杀虫效率均为100%。
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
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. 一种杀虫基因,其特征是:该基因的核苷酸序列为SEQ ID NO:2。
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:1。
3. 如权利要求1所述杀虫基因的用途,其特征是:用于杀灭粘虫、玉米螟、甜菜夜蛾或二化螟。
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CN103421816A (zh) * | 2012-12-05 | 2013-12-04 | 北京大北农科技集团股份有限公司 | 杀虫基因及其用途 |
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US8309516B2 (en) * | 2007-03-28 | 2012-11-13 | Syngenta Participations Ag | Insecticidal proteins |
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 |
CN102786585B (zh) * | 2012-08-02 | 2013-12-18 | 北京大北农科技集团股份有限公司 | 杀虫蛋白质、其编码基因及用途 |
CN105624177A (zh) * | 2016-02-04 | 2016-06-01 | 浙江大学 | 一种抗虫融合基因、编码蛋白、载体及其应用 |
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CN1199321A (zh) * | 1995-10-13 | 1998-11-18 | 陶氏农业科学有限公司 | 用于在植物中防治鳞翅目昆虫的、修饰的苏云金芽胞杆菌基因 |
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Cited By (2)
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CN103421816A (zh) * | 2012-12-05 | 2013-12-04 | 北京大北农科技集团股份有限公司 | 杀虫基因及其用途 |
CN103421816B (zh) * | 2012-12-05 | 2016-06-08 | 北京大北农科技集团股份有限公司 | 杀虫基因及其用途 |
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