淡色库蚊细胞色素P450基因的克隆、序列分析及表达差异的鉴定

目的克隆淡色库蚊细胞色素P450（CYP6F1）基因并进行表达差异的鉴定。方法采用RT—PCR技术和RACE策略，从淡色库蚊对溴氰菊酯抗性品系4龄幼虫中克隆细胞色素P450基因（CYP6F1），用相应的软件进行生物信息学分析，并用实时定量PCR和RT—PCR分析敏感、抗性品系蚊的CYP6F1表达水平及对蚊各期CYP6F1进行表达鉴定。结果成功克隆出CYP6F1基因，基因全长1639bp，开放阅读框为1527bp，编码508个氨基酸（GenBank登录号：AY662654）。序列分析显示该基因与已克隆的日本致倦库蚊细胞色素P450基因（AB001324）有99％的同源性，并具有所有的细胞色素P450基因的保守性特征，1个膜锚定信号、2个还原酶结合位点、1个典型的血红素蛋白结合位点和ETLR基序等。实时定量PCR和半定量RT—PCR结果表明，CYP6F1在淡色库蚊对溴氰菊酯抗性品系中表达水平高于敏感品系，且CYP6F1基因在淡色库蚊各个发育阶段有不同的表达。结论CYP6F1基因可能与杀虫剂抗药性相关，并且在淡色库蚊各个发育阶段有不同的表达。

Molecular cloning and differential expression of a cytochrome P450(CYP6F1) in the mosquito, Culex pipiens pallens, in China

Objective To clone cytochrome P450(CYP6F1) gene and determine differential expression at the deltamethrin-resistant and susceptible 4th instar larvae, and all stages of Culex pipiens pallens. Methods CYP6F1 was isolated from deltamethrin-resistant 4th instar larvae of Cx. pipiens pallens using RT-PCR and RACE techniques, made bioinformatics analysis using software, and difference of expression was identified by RT-PCR and Real time PCR. Results A new gene, CYP6F1(GenBank/EMBL AY662654) with complete coding sequence in the cytochrome P450 family 6 was cloned and sequenced. The cDNA sequence of CYP6F1 has an open reading frame of 1 527 bp coding a putative protein of 508 amino acid residues. Sequence analysis showed that CYP6F1 putative protein contains important conserved domains of a putative membrane-anchoring signal, putative reductase binding sites, a typical heme-binding site, ETLR motif and substrate recognition sites in P450s. The amino acid sequence deduced from the CYP6F1 nucleotide sequence showed 99% identity to a cytochrome P450 gene from Cx. quinquesfasciatus Say. Semi-quantitative RT-PCR and Real time PCR analysis indicated that the CYP6F1 gene in the deltamethrin-resistant strain appeared to be expressed more strongly than in the susceptible strain, and there was different expression in the deltamethrin-resistant 1st, 2nd, 3rd, 4th larvae and the adult female mosquitoes, but the expression level was relative higher in the 4th, 3rd larvae. Conclusion CYP6F1 may be a deltamethrin-resistant associated gene of Cx. pipiens pallens and confirm to have different expression in the deltamethrin-resistant 1st, 2nd, 3rd, 4th larvae and the adult female mosquitoes