国境口岸常见11种蚊虫DNA条形码分子鉴定研究

DNA-barcode based molecular identification for eleven common mosquito species at frontier ports

目的通过研究国境口岸常见11种蚊虫的细胞色素C氧化酶亚基I（COI）基因序列差异性,并分析其系统进化关系,建立口岸常见蚊种的分子鉴定方法。方法设计l对扩增COI部分编码区的PCR引物,对广东、海南和云南等国境口岸采集的致倦库蚊、白纹伊蚊、中华按蚊等常见11种成蚊进行PCR扩增和序列测定,依据COI核苷酸序列进行系统进化分析。结果 11种蚊虫的COI基因扩增片断长度均为650 bp左右,A＋T含量为66.51%-69.97%。同源性比较表明,同一蚊种间核苷酸序列同源性为94.8%-100.0%;不同蚊种间核苷酸序列同源性为77.7%-91.8%。系统进化关系显示,从种的水平上看,白纹伊蚊、致倦库蚊、中华按蚊等上述所有蚊种均聚集成簇,即同种之间呈明显的聚集,与形态学鉴定结果相一致。在属的水平上,库蚊属、阿蚊属、伊蚊属等均聚集成簇,形成各自的单独一支;各属间,阿蚊属与伊蚊属先聚类,再分别与库蚊属、曼蚊属聚类为库蚊亚科,按蚊属与上述蚊种亲缘关系较远。结论本次研究中的COI基因差异可作为国境口岸常见11种蚊虫分类鉴定的分子标记,为口岸常见蚊种的分子鉴定提供帮助,也可为口岸外来的或新发现的蚊种的鉴别提供分子水平的技术依据。

Objective To establish a molecular identification method for eleven common mosquito species at frontier ports via analysis of their cytochrome C oxidase subunit I（COI） gene and phylogenetic evolution. Methods Specific PCR primers were designed to amplify the COI coding region（Barcode） of eleven common mosquito species,including Culex quinquefasciatus, Aedes albopictus, Anopheles sinensis, from Guangdong, Hainan and Yunnan frontier ports. The amplified fragments were sequenced and then subjected to phylogenetic analysis. Results The amplified COI fragments were 650 bp in length, with A＋T contents between 66.51% to 69.97%. Homologous comparison showed that the nucleotide sequence homology among the same mosquito species were 94.8% -100%, while those among different mosquito species were 77.7%-91.8%. Molecular phylogenetic study showed that different mosquito species were clustered tightly at their own branch at species level, which was consistent with traditional morphological identification.At the generic level, genus Culex, genus Armigeres, genus Aedes, and other genus were clustered separately to form their own tree. Above the generic level, genus Armigeres and genus Aedes were the first cluster, and then clustered with genus Culex and genus Mansonia to form a subfamily, while genus Anopheles were relatively distant from those genus. Conclusion COI gene based barcode in this study can be used as an useful classification and identification molecular markers for eleven common mosquito species. The method established can help to identify not only common mosquito species at frontier ports but also newly discovered or invaded exotic species.