Abstract:

Objective To identify CYP6P5 gene of Anopheles sinensis and to analyze its bioinformatic characteristics for further research on its role in insecticide resistance. Methods We searched the transcriptome dataset of An. sinensis for CYP6P5 cDNA sequence with bidirectional Blast, using the sequence of An. funestus CYP6P5 gene as query. The primary structure, hydrophobic region, transmembrane region, structural domain, and 3D structure of translated protein of the resultant sequence were analyzed using bioinformatic software. The phylogenetic relationship of CYP6P5 sequences of An. sinensis and representative mosquito species was constructed using maximum likelihood methods. Results A complete cDNA sequence of An. sinensis CYP6P5 was obtained by BLAST searching. The whole sequence was 1583 bp long with an open reading frame of 1527 bp, encoding 508 amino acids. The deduced amino acid sequence was found with characteristic conserved regions of superfamily P450 family 6 (CYP6). The gene was designated as CYP6P5 (GenBank No. KF358704). Bioinformatic prediction indicated that the molecular weight of CYP6P5 protein was 58.32×103, and the isoelectric point was 7.17. The sequence contained a predicted hydrophobic region spanning from locus 5 to 21, a transmembrane region from 5 to 22, a P450 domain from 36 to 505, and multiple enzyme active sites. Phylogenetic and similarity analyses of amino acid sequences showed that An. sinensis CYP6P5 had the closest phylogenetic relationship with CYP6P5s of An. funestus and An. gambiae, with similarity values of 89.4% and 89.0%, respectively. They formed a monophyletic group. Conclusion This study laid a foundation for further study on the molecular mechanism of An. sinensis CPY6P5s role in pyrethroid resistance.

Key words: Cytochrome P450, Anopheles sinensis, CYP6P5, Identification, Bioinformatic analysis

摘要：

目的 鉴定中华按蚊CYP6P5基因并分析其序列特征, 为研究中华按蚊CYP6P5基因杀虫剂抗性机制提供理论依据。方法 以催命按蚊基因CYP6P5为询问基因, 基于中华按蚊转录组测序数据, 采用双向Blast方法搜寻中华按蚊CYP6P5 cDNA序列。利用生物信息学相关软件对该基因编码蛋白序列的一级结构、疏水区、跨膜区、结构域、3D结构等进行预测分析, 并采用最大似然法(maximum likelihood, ML)建立代表性蚊种的系统发育关系。结果 搜索获得1条中华按蚊CYP6P5全长cDNA序列。该cDNA序列具备P450超家族和CYP6家族特征性的保守序列, 命名为CYP6P5(GenBank登录号:KF358704)。该cDNA全长为1583 bp, 开放阅读框为1527 bp, 编码508个氨基酸, 推测其相对分子质量为58.32×103, 等电点为7.17。分析表明该基因编码的蛋白序列第5～21位氨基酸是疏水区, 第5～22位氨基酸是跨膜区, 第36～505位氨基酸是P450保守结构域, 并具有多个酶活性位点。系统发育关系和相似性分析表明, 中华按蚊CYP6P5与催命按蚊CYP6P5和冈比亚按蚊CYP6P5亲缘关系最近, 形成一个单系, 相似率分别为89.4%和89.0%。结论 为进一步研究中华按蚊CYP6P5基因溴氰菊酯抗性的分子机制奠定了基础。

关键词: 细胞色素P450, 中华按蚊, CYP6P5, 鉴定, 生物信息学分析