关键词: 棉铃虫, P450, 时空表达, 蛋白结构分析, 原核表达, 蛋白免疫印迹

Abstract: 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.

Key words: Helicoverpa armigera, P450, temporal-spatial distribution, protein structure analysis, prokaryotic expression, Western blot