棉铃虫α-微管蛋白基因的克隆、序列分析及表达模式检测  

Molecular Cloning, Sequence Analysis and Expression Pattern Detection of α-Tubulin Gene from Helicoverpa armigera (Hübner)

作　　者：闫硕[1] 朱家林[1] 朱威龙[1] 潘李隆[1] 张青文[1] 刘小侠[1] 

机构地区：[1]中国农业大学农学与生物技术学院昆虫学系,北京100193

出　　处：《中国农业科学》2013年第9期1808-1817,共10页Scientia Agricultura Sinica

基　　金：国家"973"项目(2012CB114103)

摘　　要：【目的】克隆及序列分析棉铃虫(Helicoverpa armigera)α-微管蛋白基因的cDNA序列,并检测棉铃虫α、β两种微管蛋白基因的表达情况。【方法】以棉铃虫3龄幼虫为试验材料,采用RT-PCR及RACE技术克隆棉铃虫α-微管蛋白基因(HeTubA),采用荧光定量PCR(QRT-PCR)技术分析棉铃虫α、β-微管蛋白基因在不同生长发育阶段及成虫器官中的表达模式。【结果】克隆得到棉铃虫α-微管蛋白基因(GenBank登录号为JQ069957)。序列分析表明,HeTubA开放阅读框1 353 bp,编码450个氨基酸组成的多肽,氨基酸序列包含多个α-微管蛋白保守区。与其它一些昆虫的一致性分析表明,HeTubA与八字地老虎(Xestia c-nigrum)、柑橘凤蝶(Papilio xuthus)及家蚕(Bombyx mori)的α-微管蛋白氨基酸序列同源性最高,α-微管蛋白基因在长期进化中非常保守。荧光定量PCR表明,棉铃虫α、β-微管蛋白基因不具有生长发育阶段及成虫器官特异性,且二者均在复眼表达高,腹部表达低;HeTubA在末龄幼虫期和蛹期高水平表达,HeTubB在末龄幼虫期和成虫期高水平表达。【结论】成功克隆了棉铃虫α-微管蛋白基因,对2种微管蛋白基因的表达模式进行了检测,进一步进行了蛋白质3D结构的构建,可用于深入研究2种微管蛋白基因的功能及开发新型杀虫剂。[Objective] The objective of this study is to clone and analyze a novel eDNA, named as HeTubA, encoding the α-tubulin from Helicoverpa armigera （Hfibner）, and detect the relative expression levels of HeTubA and HeTubB by real time PCR （QRT-PCR）. [ Method] The total RNA was extracted from H. armigera 3rd instar larvae, and α-tubulin gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA ends （RACE）. The relative expression levels of HeTubA and HeTubB were detected in different adult organs and developmental stages by QRT-PCR. [Result] Sequencing and structural analysis showed that the ORF of HeTubA was 1 353 bp in size, encoding 450 amino acid residues （GenBank accession number JQ069957）. HeTubA contained conserved residues of α-tubulin. The homologue analysis revealed that HeTubA shared high identity with Xestia c-nigrum, Papilio xuthus and Bombyx mori α-tubulin, which indicated that insect α-tubulin was conserved in evolutional process. QRT-PCR revealed that HeTubA and HeTubB mRNA expression was neither adult organ-specific nor developmental-stage-specific. Relative expression levels of HeTubA and HeTubB were the highest in compound eye, the lowest in abdomen. Expression of HeTubA peaked at last larval instar and pupal development, whereas that of HeTubB peaked at last larval instar and adult development.[ ConclusionlHeTubA was cloned and mRNA expression levels of HeTubA and HeTubB were detected. 3D structural of HeTubA was constructed. The study is helpful for further researches on the function of HeTubA and HeTubB and development of a new-type pesticide.

关 键 词：棉铃虫 Α-微管蛋白 Β-微管蛋白 基因克隆 基因表达 3D结构 

分 类 号：S435.622.3[农业科学—农业昆虫与害虫防治]