黄鳝胃瘤线虫ITS及5.8S rDNA的克隆及序列分析

本研究旨在阐明黄鳝胃瘤线虫湖南分离株的核糖体DNA(rDNA)内转录间隔区(ITS)及5.8 S rDNA序列的遗传变异情况,并用ITS序列重构胃瘤线虫与其它线虫的种群遗传关系.利用聚合酶链反应(PCR)扩增胃瘤线虫rDNA的ITS-1、5.8S及ITS-2片段,将PCR扩增出的片段纯化后克隆至pGEM-T Easy载体,重组质粒通过菌落PCR鉴定后,对阳性菌落进行序列测定并进行序列分析.结果显示所获得的胃瘤线虫ITS及5.8 S rDNA序列总长存在一定差异(922～927 bp),其中包含部分的18S、28 S及全部的ITS-1 (350～351 bp)、5.8S(102 bp)及ITS-2 (340～344 bp)序列.本研究系国内首次报道胃瘤线虫的ITS序列,其结果为黄鳝胃瘤线虫的分类鉴定以及进一步的分子流行病学调查和群体遗传研究奠定了基础.

Cloning and Sequence Analysis of the ITS and 5.8 S rDNA of Larval Eustrongylides spp.Isolates from Monopterus albus (zuiew)

The objectives of the present study were to examine sequence variation in the internal transcribed spacer(ITS) and 5.8S rDNA of Eustrongylides spp.isolates from Monopterus albus,and to reconstruct their phylogenetic relationship using ITS sequences.The ITS sequences were amplified from each Eustrongylides spp.samples and the amplicons were cloned into pGEM-T Easy vector,respectively.The inserts were successfully sequenced,and the length of ITS in 32 Eustrongylides spp.isolates were different(922-927 bp).Sequence analysis revealed that the ITS-1,5.8S and ITS-2 rDNA of these samples were 350-351 bp,102 bp and 340-344 bp in length,respectively.The results indicate that the ITS sequences provide useful genetic marker for molecular identification of Eustrongylides spp.It was the first time that the complete sequence of ITS and 5.8 S rDNA of Eustrongylides spp.were reported.The results of this study lay down the foundation for further study on molecular epidemiology and diagnostics of Eustrongylides spp.