抗氰戊菊酯棉铃虫细胞色素P450 CYP6B7基因的克隆及分析

Cloning and Characterization of Cytochrome P450 CYP6B7 from Fenvalerate Resistant Cotton Bollworm,Helicoverpa armigera

以抗氰戊菊酯棉铃虫六龄幼虫中肠组织总RNA为模板,采用特异性引物,通过对反转录-聚合酶链式反应(RT-PCR)的条件进行不断探索和优化,成功克隆出全长为1 557bp的基因片段(GenBank登录号DQ497428)。该片段包括一个完整的开放阅读框架(1 515bp)及5′端的42个碱基,编码504个氨基酸残基。与国外报道的细胞色素P450CYP6B7基因(GenBank登录号AF031468)的核苷酸、氨基酸同源性分别为97.75%和98.81%,为CYP6B7的等位基因。Northern杂交分析表明,抗性品系棉铃虫中肠组织中CYP6B7mRNA的表达量明显高于敏感品系的,初步表明CYP6B7在棉铃虫对氰戊菊酯的抗药性中起着重要作用。

After optimizing the reaction condition and parameters of RT-PCR, a cDNA clone of cytochrome P450s was obtained from total RNA of midgut of 6th instar larvae of fenvalerate resistant strain of Helicoverpa armigera, using specific primers from CYP6B7 gene. The cDNA has an open reading frame of 1515 nucleotides, encoding a protein of 504 amino acids residues （GenBank No. DQ497428 ）. Comparing with previously reported CYP6B7 （GenBank No. AF031468 ）, the similarity of nucleotides and identity of amino acids of sequence were 97.75 % and 98.81%, respectively. The sequence was classified as an allele of CYP6B7. Northern Blotting analysis showed that the expression of CYP6B7 mRNA in HDFR strains of H. armigera was significantly higher than that of the HDS strain, suggesting that CYP6B7 played an important role in the resistance of H. armigera to fenvalerate.