棉铃虫P450基因HarmCYP9A33的克隆、 序列分析及原核表达

夜间活动昆虫如夜蛾类主要通过嗅觉来寻找配偶、 寄主植物和产卵场所， 是研究昆虫嗅觉分子机制的理想材料。P450为多功能单加氧酶， 在昆虫对各种内源与外源物质的代谢中起重要作用。为研究P450在昆虫嗅觉中的作用， 本研究采用RT-PCR和RACE技术， 从夜蛾科昆虫棉铃虫Helicoverpa armigera (Hübner)雄蛾触角中扩增得到一条全长1 772 bp的P450基因， 命名为HarmCYP9A33 (GenBank登录号为JX486677)。序列分析表明， HarmCYP9A33开放阅读框全长1 590 bp， 编码529个氨基酸残基， 预测蛋白质分子量和等电点分别为61. 62 kD和7. 97； HarmCYP9A33与甘蓝夜蛾Mamestra brassicae触角毛形感器中高表达的MbraCYP9A13蛋白的氨基酸序列一致性最高， 达75%， 蛋白二级结构相似， 6个底物识别位点(substrate recognition sites， SRSs)序列一致性达61%， 其中底物与酶结合通道开关Ⅰ螺旋中SRS4序列完全相同， 与棉铃虫CYP9A亚家族蛋白有一定的结构相似性。Real-time PCR检测表明, HarmCYP9A33在雌、 雄蛾各组织中均有表达， 以腹部表达量最高， 其次为头部； 在卵至成虫各个时期也均表达， 以蛹中表达量最高； 在触角中的表达量随羽化时间而变化， 且多高于卵和幼虫中的表达量。SDS-PAGE检测和Western blot鉴定表明HarmCYP9A33体外融合表达成功。本研究为深入探讨该基因在棉铃虫触角感器细胞中的定位及其生物学功能奠定了基础。

Cloning, sequence analysis and prokaryotic expression of a P450 gene HarmCYP9A33 from Helicoverpa armigera (Lepidoptera: Noctuidae)

Nocturnal insects such as Noctuidae moths can precisely find mating partners, host plants and oviposition sites by olfaction system, and therefore are used as ideal models to study the molecular basis of olfaction. P450s are multifunctional monooxygenases which play dominant roles in the metabolism of a wide variety of both endogenous and xenobiotic substances. In order to investigate the roles of P450s in the olfaction, a new P450 gene from the male antennae of Helicoverpa armigera (Hübner), named HarmCYP9A33, with a full-length of 1 772 bp, was amplified by using RT-PCR and RACE methods. Sequence analysis showed that the full-length open reading frame of HarmCYP9A33 is 1 590 bp in size, encoding 529 amino acid residues, with the predicted molecular weight and isoelectric point of 61. 62 kD and 7. 97, respectively. HarmCYP9A33 shares a high amino acid sequence identity (75%) and the similar secondary protein structure with the homolog of Mamestra brassicae MbraCYP9A13, which is strongly expressed in sensilla trichodea. Moreover, six SRSs (substrate recognition sites) have 61% amino acid sequence identity, of which SRS4 responsible for the binding switch between substrate and enzyme is completely identical in both species. HarmCYP9A33 also shares certain similarities in the secondary protein structure with CYP9A subfamily proteins in H. armigera. Real-time PCR detection showed that HarmCYP9A33 could be detected in all test tissues of adult body, and the highest expression level of HarmCYP9A33 was in abdomen and then in head. The temporal expression profile analysis revealed that HarmCYP9A33 was expressed at all developmental stages with the highest expression level in the pupa. The expression level of HarmCYP9A33 in adult antennae varied with the time after emergence, which was higher than that in larvae and eggs. SDS-PAGE and Western blot results indicated the fused-protein was successfully expressed. These results provide a foundation to further investigate the cellular localization and biological functions of HarmCYP9A33 in cotton bollworm antennae.