Zhao Guo-Dong, Zhang Yi-Ling, Liu Yun-Lei, Li Bing, Chen Yu-Hua, Xu Ya-Xiang, Xia Qing-You, Shen Wei-De, Wei Zheng-Guo
School of Biology and Basic Medical Sciences, Soochow University, No. 199 Ren'ai Road, Dushu Lake Higher Education Town, Suzhou Industrial Park, Suzhou, 215123, People's Republic of China.
National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, 215123, Jiangsu, China.
Mol Genet Genomics. 2015 Oct;290(5):1943-53. doi: 10.1007/s00438-015-1050-6. Epub 2015 Apr 29.
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.
在昆虫中，细胞色素P450单加氧酶（P450s）参与类固醇激素和脂质等内源性化合物的代谢。在本研究中，我们采用双掺入策略，通过逆转录定量实时聚合酶链反应（RT-qPCR）测量了20-羟基蜕皮酮（20E）诱导的CYP6ab4基因转录水平。然后，我们在家蚕中使用RNA干扰实验探究了可能的生理功能。通过萤火虫荧光素酶活性测量了CYP6ab4启动子在各种家蚕组织中的活性，并用海肾荧光素酶活性进行了标准化。我们的结果表明，CYP6ab4启动子的活性在马氏管中最高，其次是脂肪体、丝腺、中肠、表皮和血细胞。基础转录活性和20E诱导转录活性的关键区域位于转录起始位点上游-908至-456 bp之间。通过在家蚕卵巢细胞（BmN）中使用双荧光素酶报告基因检测进行启动子截短分析，我们发现-827至-722 bp之间的区域对于基础转录活性和20E诱导转录活性至关重要。对该区域的序列分析揭示了几个潜在的转录调控元件，如驼背蛋白（Hb）和BR-C Z。BR-C Z结合位点核心碱基的突变表明，BR-C Z诱导20E介导的CYP6ab4转录。进一步鉴定顺式和反式元件及其在CYP6ab4上调中的作用，可能有助于阐明P450对20E反应机制的贡献。
