Promoter analysis and RNA interference of CYP6ab4 in the silkworm Bombyx mori
2015
Zhao, Guo-Dong | Zhang, Yi-Ling | Liu, Yun-Lei | Li, Bing | Chen, Yu-Hua | Xu, Yaxiang | Xia, Qing-You | Shen, Wei-De | Wei, Zheng-Guo
In insects, cytochrome P450 monooxygenases (P450s) are involved in the metabolism of endogenous compounds such as steroid hormones and lipids. In this study, we measured the 20-hydroxyecdysone (20E)-induced transcriptional level of the CYP6ab4 gene using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) with a dual spike-in strategy. We then probed possible physiological functions using RNAi experiments in the silkworm Bombyx mori. The activity of the CYP6ab4 promoter in various silkworm tissues was measured by firefly luciferase activity and normalized by Renilla luciferase activity. Our results showed that the activity of the CYP6ab4 promoter was highest in the malpighian tubule, followed by the fat body, the silk gland, the midgut, the epidermis, and the hemocyte. The essential region for basal and 20E-induced transcriptional activity was between −908 and −456 bp from the transcription start site. Through promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN), we showed that the region between −827 and −722 bp was essential for basal and 20E-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Hunchback (Hb) and BR-C Z. Mutation of the core bases of the BR-C Z binding site demonstrated that BR-C Z induces 20E-mediated CYP6ab4 transcription. Further identification of cis- and trans-elements and their roles in the upregulation of CYP6ab4 may be useful for elucidating the contribution of P450 to the response mechanism to 20E.
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