基于核糖体DNA第二内转录间隔区基因的9种蚊虫分子鉴定

目的分析常见9种蚊虫核糖体DNA第二内转录间隔区(rDNA-ITS2)基因序列特征,为蚊虫种类分子鉴定方法探讨提供依据。方法在山东省济宁市采集淡色库蚊、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、常型曼蚊和黄色柯蚊成蚊,形态学种类鉴定后,提取上述9种蚊虫DNA,PCR扩增rDNA-ITS2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Mega6软件分析列特征并构建系统进化树探讨系统发生关系。结果9种蚊虫的rDNA-ITS2长度在343 bp与577 bp之间,共有59个保守位点、449个变异位点、235个简约信息位点和191个单态位点,9种蚊虫种间序列同源性为28.21%~53.76%;所有蚊虫的rDNA-ITS2基因分子鉴定与形态学鉴定吻合率100%,库蚊属的淡色库蚊、三带喙库蚊和二带喙库蚊聚成一类,伊纹属的白纹伊蚊和刺扰伊蚊聚成一类,不同种蚊虫为独立分支。结论核糖体DNA第二内转录间隔(rDNA-ITS2)基因可用于蚊虫属和种的鉴定。

Molecular identification of nine species of mosquitoes based on ribosomal internal transcribed spacer 2 gene

Objective To analyze the gene characteristics of ribosomal internal transcribed spacer 2 sequence(rDNA-ITS2)in the 9 species of common mosquitoes for the basis in molecular identification of the mosquito species.Methods Nine species of mosquitoes,including Culex pipiens pallens,Culex tritaeniorhynchus,Culex bitaeniorhynchus,Aedes albopictus,Aedes vexans,Anopheles sinensis,Armigeres obturbans,Mansonia uniformis,Coquillettidia ochracea,were collected from Jining area in Shandong Province,and morphologically identified.Single mosquito genomic DNA was extracted from each species and rDNA-ITS2 was specifically amplified by PCR and sequenced.Then the gene sequences were subjected to homology alignment in GenBank,compared and analyzed by Bioedit 7.0 and DNAMAN.The gene traits were analyzed by DNASTAR,ClustalX 1.81 and Mega6,and the phylogenetic tree was constructed.Results The amplified rDNA-ITS2 fragments for the nine species of mosquitoes varied from 343 bp to 577 bp,with 59 conserved sites,449 variable sites,235 parsimony informative sites and 191 singleton sites in the sequences.The nucleotide sequence homology among the different mosquito species was 28.21%~53.76%,and the molecular identification of the species based on rDNA-ITS2 was consistent with the traditional morphological findings.The results indicated that Culex pipiens pallens,Culex tritaeniorhynchus and Culex bitaeniorhynchus were clustered to Culex genera,and Aedes albopictus and Aedes vexans to Aedes genera,The different species was independent at its own taxonomy branch.Conclusion rDNA-ITS2 can serve as an useful classification for and molecular marker to identify the species of different mosquitoes by genus.