黄鳝胃瘤线虫ITS及5.8S rDNA的克隆及序列分析

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

为了解渝西地区黄鳝胃瘤线虫的rDNA内转录间隔区（ITS）及5.8SrDNA 序列的遗传变异情况,本研究利用PCR扩增胃瘤线虫rDNA的片段,将目的片段克隆至pMDTM19-T Vector载体,对阳性克隆进行测序,序列用 BLAST和MEGA4.0进行相似性和种系发育分析.结果表明6株胃瘤线虫分离株扩增的序列总长存在差异（890-892bp）,其中ITS-1序列长度为350-351bp、5.8S序列长度为102-103bp及ITS-2序列长度为343bp.渝西地区胃瘤线虫ITS序列与不同宿主胃瘤线虫的相似性最高为98.3%,表明ITS可作为分子标记用于胃瘤线虫与其他线虫的种间鉴定.

In order to understand the genetic variation in the internal transcribed spacer （ITS） and 5.8S rDNA of Eustrongylides spp. from Monopterus albus in Western Chongqing, the ITS sequences were amplified by nested PCR from each Eustrongylides sample and the amplicons were cloned into pMDTM19-T vector. The in-serts were successfully sequenced. Homology and phylogeny analyses of these sequences were conducted with BLAST and MEGA 4.0 softwares. The results revealed that the total length of ITS and 5.8S in 6 Eu-strongylides isolates were different （890-892 bp）, and the ITS-1, 5.8S, ITS-2 were 350-351 bp, 102- 103 bp and 343 bp in length, respectively. The identity of ITS of Eustrongylides isolates from Western Chongqing was up to 98.3% compared with Eustrongylides from various hosts, suggesting that ITS can serve as a useful molecular marker for interspecific identification among Eustrongylides and other nematodes. The results of this study may lay down the foundation for further study on molecular epidemiology, species i-dentification and diagnostics of Eustrongylidesp.