棉铃虫中肠P450 CYP6B6基因5′上游序列的克隆及序列分析

Cloning and Sequence Analysis of 5′-Upstream Region of Cytochrome P450 CYP6B6 Gene in the Midgut from Cotton Bollworm,Helicoverpa armigera

已证实2-十三烷酮等植物次生物质的胁迫会诱导棉铃虫(Helicoverpa armigera)CYP6B6基因的过量表达[1]。为探明棉铃虫P450 CYP6B6基因的表达调控机制,根据棉铃虫中肠P450 CYP6B6基因cDNA序列(GenBank登录号:AY950636),运用基因组步移的方法获得其5′上游区的序列,用启动子区的分析软件进行比对分析,结果得到棉铃虫P450 CYP6B6基因5′上游区域1 333 bp的基因片段。分别运用NNPP v.2.2和TRANSFAC v.6.0预测转录起始位点及分析了转录因子结合位点,结果显示,该序列不仅包括TATA盒等这一类核心结构序列,还含有多个转录因子的结合位点,如GATA-1、Dfd、C/EBP、HSF、cytR和AhR(芳香烃受体化合物应答元件)等。该结果为深入研究棉铃虫P450 CYP6B6的表达调控机制及其参与杀虫剂抗性的研究奠定分子基础。

Over-expression of cytochrome P450 CYP6B6 gene has been proved to be associated with natural plant compounds,such as 2-tridecanone in the cotton bollworm,Helicoverpa armigera.Based on the 5′-end nucleotide sequence of the full length cDNA of CYP6B6（AY950636）,the 5′-upstream region of CYP6B6 gene was obtained by genome walking technique,in order to investigate the regulatory mechanism of CYP6B6 gene expression.The 5′-upstream fragment promoter 1 333 bp was successfully cloned and sequenced.The transcription initiation site of CYP6B6 gene was predicted by the NNPP v.2.2 software.Besides we used TRANSFAC v.6.0 software to predict several cis-acting element binding sites in the 5′-upstream region of CYP6B6,the results showed that there are not only core promoter sequences（TATA-box）,but also several transcriptional elements（such as GATA-1,Dfd,C/EBP,AhR）.The current study will provide an important basis for further investigating in insecticide resistance of H.armigera in future.