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. 2013 Feb;40(2):1701-10. doi: 10.1007/s11033-012-2221-8. Epub 2012 Oct 23.
Cytochrome P450s (CYPs) are widespread proteins that interact with exogenous chemicals from the diet or the environment. CYP9A subfamily genes are important in the silkworm Bombyx mori. We previously reported transcriptional levels of two CYP9A genes in different tissues and their responses to sodium fluoride (NaF). In this study, promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN) showed that the regions -1,496 to -1,102 bp for CYP9A19, and -1,630 to -1,210 bp for CYP9A22 were essential for basal transcriptional activity. Sequence analysis of these regions revealed several transcriptional regulatory elements but no typical promoter elements. Promoter activities were regulated after NaF induction and with an obvious dose effect. Although the dual-luciferase assay has been widely used to determine the activity of a given promoter in cell lines, problems with it still exist. Our results indicate that both plasmid size and construct protocols affect the experimental results.
细胞色素 P450 酶（CYPs）是广泛存在的蛋白质，可与饮食或环境中的外源化学物质相互作用。CYP9A 亚家族基因在桑蚕 Bombyx mori 中很重要。我们之前报道了两种 CYP9A 基因在不同组织中的转录水平及其对氟化钠（NaF）的反应。在这项研究中，使用双荧光素酶报告基因检测在桑蚕卵巢细胞（BmN）中的启动子截断分析表明，CYP9A19 的-1,496 至-1,102 bp 区域和 CYP9A22 的-1,630 至-1,210 bp 区域对于基础转录活性是必需的。这些区域的序列分析揭示了几个转录调控元件，但没有典型的启动子元件。NaF 诱导后和具有明显剂量效应时，启动子活性受到调节。尽管双荧光素酶检测已广泛用于确定细胞系中特定启动子的活性，但该检测仍存在问题。我们的结果表明，质粒大小和构建方案都会影响实验结果。
