白纹伊蚊溴氰菊酯抗性株 CYP6 cDNA片段克隆、鉴定

Cloning and Identification of cDNA fragments from the mosquito Aedes albopictus

根据家蝇CYP6A1、CYP6D1以及致倦库蚊CYP6E1的保守区氨基酸序列设计一组简并引物,采用RT-PCR的方法,从白纹伊蚊四龄期活体幼虫总RNA中扩增出与设计相符的基因片段。利用T-A克隆法,将获得的基因片段克隆人pGEM-T easy载体。经限制性内切酶酶切和PCR鉴定证明重组成功。将筛选的阳性克隆经序列测定及同源性分析,表明共获得CYP6家族中9个的新cDNA序列。为进一步研究细胞色素P450的多样性及其与抗药性形成的分子机制打下基础。

In This study, a set of degenerate primers were designed according to the conserved amino acid sequences of CYP6A1, CYP6DI in Musca domestia and CYP6E1 in Culex quinquefasciatus. It obtained cDNA fragments by degenerate RT-PCR method from the total RNA of the mosquito, Aedes albopictus. The fragments were cloned into pGEM-T Easy Vector. The recombinant plasmid was transformed into E. coli cell(DH5a). Positive clones were screened out and identified by restriction analysis and PCR. After the recombinat plasmids were sequenced, the obtained sequences were deduced into amino acid sequences by PCGENE program. Through homologus analysis and comparison with the sequences from Genbank, 9 new sequences of P450 were obtained. These may provide the theoretical basis and data for the further study on the diversity of cytochrome P450 and insecticide resistance.