目的 分析常见的9种蚊虫线粒体DNA细胞色素C氧化酶亚基Ⅰ（mtDNA-COⅠ）基因序列特征，了解其分子系统进化关系。方法 2016年采用人饵帐诱捕法在济宁市北湖地区采集蚊虫，经实验室种类鉴定后，提取淡色库蚊、三带喙库蚊、二带喙库蚊、白纹伊蚊、刺扰伊蚊、中华按蚊、骚扰阿蚊、常型曼蚊和黄色轲蚊的DNA，PCR扩增mtDNA-COⅠ基因并测序。在GenBank中进行同源性比对，采用Bioedit 7.0软件及DNAman软件对测序结果进行比对分析，通过DNAStar、ClustalX 1.81和Mega 6.0软件分析序列特征、颠换率、分化年代并构建系统进化树，探讨系统发生关系。结果 9种蚊虫的mtDNA-COⅠ基因成功扩增并测序，获得长度均为528 bp的基因片段，（A＋T）含量为65.72%～69.32%，有375个保守位点，153个变异位点，104个简约信息位点，49个单态位点。不同蚊种间的颠换率为3.409%～9.470%，分化年代为1.482×106a～4.117×106a。不同蚊种间序列同源性为84.28%～92.94%。系统进化关系显示，所有蚊虫的COⅠ基因分子鉴定与形态学鉴定一致，同属间聚集，不同蚊种为独立分支。结论 mtDNA-COⅠ基因可用于蚊虫属和种的区分，为蚊虫的分子鉴定提供科学依据。
Objective To explore gene characteristics of mitochondrial DNA cytochrome C oxidase subunitⅠ (mtDNA-COⅠ) in nine common mosquito species and analyze the corresponding phylogenetic relationship of these mosquitoes. Methods Nine species of mosquitoes (Culex pipiens pallens, Cx. tritaeniorhynchus, Cx. bitaeniorhynchus, Aedes albopictus, Ae. vexans, Anopheles sinensis, Armigeres subalbatus, Mansonia uniformis, Coquillettidia ochracea) were collected with man-bait net trap from Beihu area of Jining district in 2016 and identified in the laboratory. Single mosquito genomic DNA was extracted from each species and mtDNA-COⅠ was specifically amplified by PCR and sequenced. Gene sequences were blasted in GenBank, compared and analyzed by Bioedit 7.0 and DNAman. The gene traits, transversion rates, and divergence times were analyzed by DNAStar、ClustalX 1.81 and Mega 6.0. The phylogenetic tree was constructed. Results The amplified mtDNA-COⅠfragments of nine species of mosquitoes were 528 bp with (A+T)contents 65.72%-69.32%. There were 375 conserved sites, 153 variable sites, 104 parsimony informative sites and 49 singleton sites in the sequences. The transversion rates were from 3.409% to 9.470%, and the divergence times were from 1.482×106a to 4.117×106a. The nucleotide sequence homology among the different mosquito species were 84.28%-92.94%. Molecular phylogenetic study showed that different species were clustered at their own branch at genus level, which was consistent with traditional morphological identification. Conclusion mtDNA-COⅠ can serve as a useful classification and identification molecular marker for different mosquito species and genus, providing a molecular method for the identification and phylogenetic analysis of the mosquitoes.
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