大劣按蚊和斯氏按蚊核糖体基因内转录第二间隔区序列分析

Sequence analysis of the second internal transcription spacer of ribosomal DNA from Anopheles dirus and Anopheles stephensi

目的分析对约氏疟原虫不易感的大劣按蚊和易感的斯氏按蚊核糖体基因内转录第二间隔区(rDNA ITS2)的基因特征。方法对大劣按蚊和斯氏按蚊ITS2基因进行PCR扩增,PCR产物进行限制性片断长度多态性分析(RFLP)和基因测序,并采用相关在线程序及软件进行序列分析。测序结果与GenBank数据库中其它传疟按蚊的ITS2序列进行多序列比对,构建分子系统树。结果大劣按蚊和斯氏按蚊的ITS2基因PCR产物经限制性内切酶XhoI消化,产生不同的酶切图谱;测序结果表明大劣按蚊和斯氏按蚊ITS2序列分别为715bp和466bp,且两种按蚊ITS2序列的GC含量和简单重复序列等特征存在明显差异,两者碱基差异水平为53.5%;分子进化树显示,大劣按蚊和斯氏按蚊形成单独的支系。结论大劣按蚊和斯氏按蚊ITS2基因具有不同的基因特征,其遗传多态性可能与产生对约氏疟原虫的易感性不同有关,为进一步研究按蚊与疟原虫之间的相互作用奠定了基础。

To analyse the genetic characteristics of the second internal transcription spacer of ribosomal DNA （rDNA ITS2） from Anophleles dirus and Anopheles stephensi, the rDNA ITS2 regions of both species of anopheles were amplified with PCR, cloned and sequenced , and the amplified products were analysed by restriction endonuclease digestion （RFLP）. Then, the corresponding sequences of both species were searched from GenBank in which all these sequences were aligned and compared using Clustal W. program, and the phylogenetic tree was constructed by MEGA program. It was demonstrated that there were significant differences in RFLP pattern of both species, i.e. the length of the ITS2 region was 715 bp and 466 bp in A. dirus and A. stephensi respectively; there were also remarked differences in GC contents of ITS2 and simple sequence repeats between both species, and the level of base pair difference was 53.5 %. The phylogenetic tree constructed by MEGA program showed that these two species of anopheles existed as an independent taxon to each other. From the above observations, it is evident that gene variance is indistin-guished between A. dirus and A. stephensi, and the basis for compatibility between Plasmodium yoelii and mosquito host appears to be, at least in part, determined by the difference in genetic background of A. dirus A. stiphensi. These findings provide basis for further studies on the interactions between anopheline mosquitoe host and plasmodium genotypes.