淡色库蚊rDNA-ITS2基因的克隆分析

目的克隆并鉴定淡色库蚊核糖体DNA第二内转录间隔区(rDNA-ITS2)基因并应用于现场孽生蚊媒的种属鉴定,从而为孽生现场的蚊媒预警提供分子生物学依据。方法设计淡色库蚊诊断性引物分别对淡色库蚊实验室标准株及从现场采集的蚊虫的rDNA-ITS2序列进行PCR扩增,PCR产物纯化后连接到pGEM-Teasy载体上并转化到DH5α菌中,然后对其进行DNA序列测定及分析。结果成功克隆了淡色库蚊的rDNA-ITS2基因。序列分析结果表明,实验中所克隆到的淡色库蚊的rDNA-ITS2序列与GenBank所登录的淡色库蚊的rDNA-ITS2序列同源性均为98%以上,同时发现孽生现场存在淡色库蚊。结论rDNA-ITS2序列在淡色库蚊中具有种属特异性,可为孽生现场的蚊媒预警提供分子生物学依据。

Cloning and analysis of the ribosomal DNA-ITS2 gene in Culex pipiens pallens

To clone and analyze the ribosomal DNA-ITS2(rDNA-ITS2) gene from Culex pipiens pallens in Shenzhen so as to provide molecular biology background for the precaution of mosquito vectors in breeding spot.By using the diagnostic primers for C.pipiens pallens,the sequence of the rDNA-ITS2 gene in the standard laboratory strains as well as the field-collected strains of mosquitoes were amplified by PCR.The amplified product was ligated to vector pGEM-Teasy,transformed to host bacteria DH5a and then the DNA sequence determination and analysis were performed accordingly.It was found that the rDNA-ITS2 gene was successfully cloned and the results on sequence analysis indicated that the sequences of rDNA-ITS2 gene of C.pipiens pallens both from the laboratory cloned and the breeding spot showed a homploque of more than 98% with that published in GenBank.Moreover,the existence of C.pipiens pallens in the breeding spot was also found.It is evident that the sequence of rDNA-ITS2 gene exhibits species-specificity,thus it can provide a molecular biological basis for the precaution of mosquito vector in the breeding spot.