School of Biology and Basic Medical Sciences, Soochow University, No. 199 Ren'ai Road, Dushu Lake Higher Education Town, Suzhou Industrial Park, Suzhou, 215123, Jiangsu, People's Republic of China.
Mol Biol Rep. 2014 Mar;41(3):1607-16. doi: 10.1007/s11033-013-3007-3. Epub 2014 Jan 11.
Carboxylesterase (CarE) is a multifunctional superfamily, and it plays important roles in xenobiotic detoxification, pheromone degradation, neurogenesis and regulating development. In this research, firstly, we measured the rutin-induced transcriptional level of BmCarE-10 gene by using real-time quantitative RT-PCR method, and dual spike-in strategy. Several possible physiological functions were certified preliminarily by RNAi experiments in silkworm. Promoter truncation analysis using a dual-luciferase reporter assay in Bombyx mori ovary cells (BmN) showed that the region -705 to -625 for BmCarE-10 gene was essential for basal and rutin-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Croc and Dfd. The activities of the BmCarE-10 promoter in various tissues of silkworm were also measured by firefly luciferase activity and normalized by the Renilla luciferase activity. Results showed that the activity of the BmCarE-10 promoter were highest in the Malpighian tubule, followed by fat body, silk gland, midgut, epidermis, and hemocyte. The essential region for basal and rutin-induced transcriptional activity was also -894 to -502 in Malpighian tubule and fat body of silkworm. The potential core promoters of BmCarE-10 gene in B. mori are reported for the first time in this research. Further identification of cis- and trans-elements and their role in upregulation of BmCarE-10 gene may be useful for elucidating the contribution of CarE protein to the response mechanism to rutin.
羧酸酯酶（CarE）是一个多功能超家族，在异生物质解毒、信息素降解、神经发生和调节发育中起着重要作用。在这项研究中，我们首先通过实时定量 RT-PCR 方法和双内参策略测量了芸香苷诱导的 BmCarE-10 基因的转录水平。通过在蚕中的 RNAi 实验初步验证了几个可能的生理功能。双荧光素酶报告基因检测在 Bm 卵巢细胞（BmN）中的启动子截断分析表明，BmCarE-10 基因的-705 至-625 区域对于基础和芸香苷诱导的转录活性是必需的。该区域的序列分析揭示了几个潜在的转录调控元件，如 Croc 和 Dfd。通过萤火虫荧光素酶活性和海肾荧光素酶活性归一化测量，还测量了 BmCarE-10 启动子在蚕各种组织中的活性。结果表明，BmCarE-10 启动子在马氏管中的活性最高，其次是脂肪体、丝腺、中肠、表皮和血细胞。在马氏管和脂肪体中，基础和芸香苷诱导转录活性的必需区域也是-894 至-502。本研究首次报道了 B. mori 中 BmCarE-10 基因的潜在核心启动子。进一步鉴定顺式和反式元件及其在 BmCarE-10 基因上调中的作用，可能有助于阐明 CarE 蛋白对芸香苷反应机制的贡献。
