广东国境口岸不同蚊种COI序列分析和分子鉴定方法

COI Sequences Analysis and Molecular Identification for Five Species of Mosquitoes from Guangdong Port

〔目的〕建立国境口岸不同蚊种的细胞色素C氧化酶亚基I(COI)分子和氨基酸鉴定方法并分析其系统进化关系。〔方法〕设计1对扩增COI部分编码区的PCR引物,对广州机场、江门和湛江等国境口岸采集的致倦库蚊、三带喙库蚊、白纹伊蚊、中华按蚊、骚扰阿蚊等成蚊和实验室喂养的蚊幼虫进行PCR扩增和序列测定,分析COI核苷酸和氨基酸的系统进化关系。〔结果〕5种蚊种的COI基因扩增片断长度均为415bp,A+T含量为68.77%～70.6%。同源性比较表明,不同蚊种间COI片断碱基变异颠换数都明显高于转化数,核苷酸序列及其编码的氨基酸序列同源性分别为85.1%～93.7%和92.0%～99.3%。COI核苷酸系统进化关系显示,所有蚊虫的COI分子鉴定与其形态学结果吻合,但骚扰阿蚊位于一单独的分支上,其亲缘关系与其它蚊种最远。COI基因编码的氨基酸系统进化与蚊虫形态学亲缘关系一致,库蚊属、伊蚊属、阿蚊属聚类为库蚊亚科,中华按蚊与其它蚊种的亲缘关系最远。〔结论〕建立的COI核苷酸和氨基酸鉴别技术可成功地应用于国境口岸范围内成蚊和幼蚊的属和种的区分,后者更能区分高级分类阶元亚科和正确反映蚊虫的系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点,为广东口岸和其它国境口岸范围内外来的或新发现的蚊种的鉴别提供了分子水平的技术依据。

Objective To establish the molecular and amino acid identification of cytochrome c oxidase subunit I （COI） of mitoehondrial DNA and to analyse the corresponding phylogenetie relationship of five species of mosquitoes from Guangdong ports. Method The specific PCR primers were disigned to amplify the partial COI coding regions. Five species of mosquito （including Culex pipiens quinquefasciatus, Culex. tritaeniorhynchus, Aedes albopictus, Anopheles sinensis and Armigeres subalbatus） from Baiyun Airport, Jiangmen and Zhanjiang frontier ports and the reared larvae in the lab were analyzed by PCR and the amplified fragments were also sequenced. The phylogenetic relationship of COI nucleotide sequences and the corresponding amino acids were tested. Result The amplified COI fragments of five species of mosquitoes were 415bp in size, whose A＋T contents were 68.77% to 70.6%. Nucleotide transversion substitution among different mosqutio species was higher than transition substitution. The homology in nucleotide analysis and amino acid analysis showed 85.1%-93.7% and 92.0% -99.3% respectively. COI molecular phylogenetic study showed that Ar. subalbatus was distantly related to the others. However, the phylogenetic relationship of COI amino acid was consistent with the morphological characteristics of mosquitoes. Genera Aedes and genera Armigeres were grouped together, which was then clustered to subfamily Culicinae with genera Cluex. A n. sinensis was existed on a separate subfamily branch. Conclusion COI molecular and amino acid identificaton could be used to classify genera and species of mosquitoes and larvae from frontier ports. The classification of subfamily and phylogenetuc relationships among mosquitoes could also be solved by COI amino acid identificaton. This method can remedy the shortcoming of the traditional systematic classification such as deficient shape characteristic and provide molecular basis for identifying the foreign mosquito species at Guangdong ports or other frontier ports.