褐飞虱V-ATPased亚基基因的克隆与mRNA表达分析-应用昆虫学报.doc

褐飞虱V-ATPase d亚基基因的克隆与mRNA表达分析 朱友理，王剑，谢志娟，刘亚萍，王建军基金项目：国家科技支撑计划（ 基金项目：国家科技支撑计划（2012BAD19B01） *通讯作者（Author for correspondence）：王建军（1970-），江苏海安人，博士，教授，主要从事昆虫抗药性与转座子研究。（Tel）0514 (E-mail) wangjj@yzu.edu.cn （扬州大学园艺与植物保护学院，江苏 扬州，225009） 摘要：液泡型H+-ATPase (V-ATPase)在昆虫生长发育过程中具有重要作用。本文通过RT-PCR获得褐飞虱V-ATPase d亚基基因NlVHA-d的cDNA序列，并通过实时荧光定量PCR对NlVHA-d基因的表达进行了分析。结果表明，NlVHA-d基因编码349个氨基酸，不同昆虫V-ATPase d亚基高度保守。NlVHA-d基因在褐飞虱 2龄若虫中表达量最高，雌虫表达量显著高于雄虫表达量。LD10和LD30三唑磷处理的羽化三天短翅雄虫NlVHA-d基因相对表达倍数分别是丙酮处理的2.15和2.46倍。亚致死剂量三唑磷处理褐飞虱短翅雄虫NlVHA-d基因的表达上调可能与褐飞虱再猖獗相关。 关键词：褐飞虱；V-ATPase基因；荧光定量PCR；亚致死剂量；三唑磷 Cloning and mRNA expression analysis of Vacuolar H+-ATPase d subunit gene in the brown planthopper, Nilaparvata lugens (St?l) Zhu You-Li Wang Jian Xie Zhi-Juan Liu Ya-Ping Wang Jian-Jun* (College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China) Abstract: The vacuolar H+-ATPase (V-ATPase) plays an important role in growth and development of insects. In this study, a cDNA sequence of V-ATPase d subunit gene was obtained from the brown planthopper, Nilaparvata lugens (St?l), by RT-PCR and named as NlVHA-d, and its expression was analyzed by qRT-PCR. The results showed that the open reading frame of NlVHA-d is 1047bp in length, encoding 349 amino acid residues, and the V-ATPase d genes are highly conserved among different insects. The qRT-PCR analysis revealed that the mRNA expression level of NlVHA-d was the highest in second instar nymphs, and the female adults showed higher levels of expression than the male adults. Furthermore, at third day after eclosion, the relative expressions of NlVHA-d in brachypterous male adults treated with LD10 and LD30 of triazophos were 2.15 and 2.46 times higher than that in control treatment with acetone, respectively. The over-expression of NlVHA-d gene in brachypterous male adults treated with subleathal dose of triazophos might contribute to the resurgence of brown planthopper. Keywords: Nilaparvata lugens; V-ATPase gene; q