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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1772bp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1590bp,编码529个氨基酸残基,预测蛋白质分子量和等电点分别为61.62kD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。
Nighttime activities such as insect moths to find their spiders, host plants and spawning sites mainly through their sense of smell are the ideal materials for studying the molecular mechanism of insect olfactory. P450 is a multifunctional monooxygenase that plays an important role in the metabolism of various endogenous and exogenous substances by insects. In order to study the role of P450 in insect olfactory, a P472 gene of 1772bp in length was amplified from the antenna of Helicoverpa armigera (Hübner) by RT-PCR and RACE techniques and named as HarmCYP9A33 (GenBank accession number JX486677). Sequence analysis showed that the full length of the open reading frame of HarmCYP9A33 was 1590bp and encoded a protein of 529 amino acids. The molecular weight and isoelectric point of the predicted protein were 61.62kD and 7.97, respectively. HarmCYP9A33 and MameCYP9A13 protein highly expressed in the hairy sensor of Mamestra brassicae The sequence identity of the six substrate recognition sites (SRSs) was 61%. Among them, the substrate and enzyme binding channel switch Ⅰ helix SRS4 The sequences were identical and had some structural similarities with the CYP9A subfamily protein of Helicoverpa armigera. Real-time PCR showed that HarmCYP9A33 was expressed in all tissues of female and male moths, with the highest expression in the abdomen, followed by the head, the expression in all stages from egg to adult, and the highest expression in the pupa. The expression level varies with the emergence time and is much higher than the expression level in eggs and larvae. SDS-PAGE and Western blot showed that HarmCYP9A33 fusion protein was successfully expressed in vitro. This study lays the foundation for further exploration of the localization of this gene in the antennae of Helicoverpa armigera and its biological functions.