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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。
Objective To identify the CYP6P5 gene of Anopheles sinensis and analyze its sequence characteristics and provide theoretical basis for studying the resistance mechanism of CYP6P5 gene insecticide in Anopheles sinensis. Methods An anopheles stephensi gene CYP6P5 was used as a query gene. Based on the sequencing data of the Anopheles sinensis transcriptome, the two-way Blast method was used to search for the CYP6P5 cDNA sequence of Anopheles sinensis. The primary structure, hydrophobic region, transmembrane region, domain and 3D structure of the protein sequence were predicted by bioinformatics software and the maximum likelihood (ML) was used to establish the representative mosquito Phylogenetic relationships. Results A total length cDNA sequence of Anopheles sinensis CYP6P5 was obtained. The cDNA sequence has the conserved sequence of P450 superfamily and CYP6 family, named CYP6P5 (GenBank accession number: KF358704). The full-length cDNA was 1583 bp in length and 1527 bp in open reading frame, encoding a polypeptide of 508 amino acids. The deduced amino acid sequence was 58.32 × 103 with an isoelectric point of 7.17. The results showed that amino acids 5 to 21 of the protein encoded by this gene were hydrophobic, amino acids 5 to 22 were transmembrane domains, amino acids 36 to 505 were P450 conserved domains and had multiple enzyme active sites. Phylogenetic relationships and similarity analysis showed that Anopheles sinensis CYP6P5 had the closest genetic relationship with CYP6P5 and Anopheles gambiae CYP6P5, forming a single line with similarity rates of 89.4% and 89.0%, respectively. Conclusion This study laid the foundation for the further study on the molecular mechanism of CYP6P5 resistance to deltamethrin in Anopheles sinensis.