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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、Clustal X 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1.482×10~6a～4.117×10~6a。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 analyze the mitochondrial cytochrome C oxidase subunit Ⅰ (mtDNA-COⅠ) gene sequences of nine common mosquitoes and to understand their molecular phylogenetic relationship. Methods In 2016, the human bait tent trap method was used to collect mosquitoes in Beihu district, Jining City. After identification of laboratory species, Culex pipiens pallens, Culex tritaeniorhynchus, Culex tritaeniorhynchus, Aedes albopictus, Aedes albopictus , Anopheles sinensis (Euphorbiaceae), Arbuscular Anoxia mosquitoes (Arborvitae), Manchurian mosquitoes and Aedes albopictus. The mtDNA-COⅠ gene was amplified by PCR and sequenced. Homology comparison was carried out in GenBank, and the sequencing results were compared using Bioedit 7.0 software and DNAman software. The sequence features, transversion rate, differentiation age, and phylogenetic tree were analyzed by DNAStar, Clustal X 1.81 and Mega 6.0 software , To explore the relationship between the system. Results The mtDNA-COⅠ gene of 9 species of mosquitoes was successfully amplified and sequenced. The 528 bp gene fragments were obtained. The (A + T) contents ranged from 65.72% to 69.32%. There were 375 conserved sites and 153 mutated sites , 104 parsimony informative sites and 49 singleton loci. The transversion rates of different mosquito species ranged from 3.409% to 9.470%, and the age of differentiation was 1.482 × 10 ~ 6a ~ 4.117 × 10 ~ 6a. The sequence homology between different mosquitoes ranged from 84.28% to 92.94%. Phylogenetic relationships show that all the mosquitoes COⅠ gene molecular identification and morphological identification of the same genus aggregation, different mosquito species as an independent branch. Conclusion The mtDNA-COⅠ gene can be used for the distinction between mosquitoes and species, providing a scientific basis for the molecular identification of mosquitoes.