热带病与寄生虫学

热带病与寄生虫学 ›› 2020, Vol. 18 ›› Issue (1): 12-16.

• 论著 • 上一篇    下一篇

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

郭秀霞, 程鹏, 刘丽娟, 王海防, 王怀位, 张崇星∗
  

  1. 山东第一医科大学(山东省医学科学院)、山东省寄生虫病防治研究所,济宁 272033
  • 收稿日期:2020-01-20 出版日期:2020-03-10 发布日期:2020-03-10
  • 通信作者: 张崇星,E-mail:chongxingzhang@aliyun.com
  • 作者简介:郭秀霞,女,硕士,助理研究员,研究方向:病原生物学。 E-mail:guoguoxiua@163.com
  • 基金资助:
    山东省自然科学基金项目 (ZR2017YL004),山东省医药卫生科技发展计划项目(2018WS302),山东省医学科学院医药卫生科技创新工程

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

GUO Xiu-xia, CHENG Peng, LIU Li-juan, WANG Hai-fang, WANG Huai-wei, ZHANG Chong-xing∗   

  1. Shandong Institute of Parasitic Diseases, Shandong First Medical University, Shandong Academy of Medical Sciences, Jining 272033, China
  • Received:2020-01-20 Online:2020-03-10 Published:2020-03-10
  • Contact: ∗Corresponding author,E-mail:chongxingzhang@aliyun.com

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摘要： 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。

关键词: 蚊虫, 核糖体, DNA 第二内转录间隔区, 分子鉴定

Abstract: 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 align- ment 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 tradi- tional 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 iden- tify the species of different mosquitoes by genus.

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