嗜人按蚊rDNA—ITS2基因的克隆鉴定及SNP分析

目的克隆和分析嗜人按蚊核糖体DNA（rDNA）第2内转录间隔区（ITS2）序列，研究嗜人按蚊rDNA-ITS2序列的种属特异性及不同个体rDNA-ITS2序列的单核苷酸多态性（Single Nuclear Polymorphism，SNP）。方法用DNA提取试剂盒从嗜人按蚊中提取DNA摸板，利用蚊虫5．8s和28S序列的保守性设计特异引物进行聚合酶链反应以扩增嗜人按蚊rDNA-ITS2基因，扩增产物经回收纯化后连接到TA载体，经amp^＋LB固体平板筛选的阳性克隆。经amp^＋LB液体培养基培养后进行PCR鉴定、EcoRI酶切鉴定和序列分析。结果经PCR反应扩增出全长556bp的嗜人按蚊rDNA-ITS2基因。纯化后重组克隆经PCB鉴定可重现556bp的特异性条带，其酶切产物亦与目的基因PCR产物位置相同。嗜人按蚊6个克隆的同一性为99．6％，其rDNA-ITS2基因单核苷酸存在颠换和插入，在556的范围内有一个A→T，一个G→T的颠换；一个T的插入。结论嗜人按蚊rDNA-ITS2序列在种间高度保守，但不同个体间也存在一定的SNP多态性。

Cloning and SNP Analysis of rDNA-ITS2 of Anopheles anthropophagus

Objective To clone and analyse rDNA-ITS2 of Anopheles anthropophagus, study the unique feature and Single Nuclear Polymorphism of rDNA-ITS2. Methods DNA was extracted from Anopheles anthropophagus and the rDNA-ITS2 sequences of Anopheles anthropophasus was amplified by PCR. The PCR products were purified and ligated to T-vector. The positive clones were selected by plating on amp＋LB agar plate and identified by PCR, EcoR- Ⅰ digestion and DNA sequencing. Results 556 bp full fragment of rDNA-ITS2 of Anopheles anthropophagus was cloned. The six cloned ITS2 sequences of Anopheles anthropophagus showed 99.6% homology,with transversion and insertion Single Nuclear Polymorphism. There were one A→T transversion,one G→T transversion and one T insertion. Conclusion SNPs were found in the rDNA-ITS2 gene of Anopheles anthropophagus.