华南虎绦虫ITS及5.8S rDNA序列扩增与序列分析

Amplification and Sequence Analysis of the ITS and 5.8S rDNA of Cestodes From Panthera tigris amoyensis in China

以从广州动物园华南虎体内采集的2条绦虫作为研究对象,利用核糖体DNA(rDNA)内转录间隔区序列(ITS)的遗传标记特点,用保守引物BD1和BD2对提取的DNA进行PCR扩增,经胶回收纯化后,连接到pGEM-T Easy载体上,然后转化并鉴定出阳性菌落,对菌落进行PCR扩增并测序,将测序结果与GenBank公布的相关绦虫ITS序列进行对比分析,结果显示,采自广州动物园华南虎的2条绦虫ITS及5.8S rDNA序列总长均为1387bp,序列相似性为100%,与猬迭宫绦虫(Spirometra erinaceieuropaei)、舌形属绦虫(Diphyllobothrium ligula)、宽节双叶槽绦虫(Diphyllobothriumlatum)的ITS及5.8S rDNA序列相似性分别为96.7%、66.5%、71.5%,结果显示,此次分离的绦虫属于迭宫属的猬迭宫绦虫,所测得的华南虎猬迭宫绦虫的ITS序列为首次报道。

Utilizing the first and second internal transcribed spacers of ribosomal DNA（ITS rDNA） as the genetic marker,the specific identity of the cestodes from a South China tiger（Panthera tigris amoyensis） in Guangzhou Zoo was performed in the present study.The ITS rDNA was amplified by PCR using the conserved primer pair BD1 and BD2.The PCR products were purified,cloned into pGEM-T Easy vector,and then the positive clones were selected for sequencing.DNA sequences were compared with relevant sequences previously reported in GenBank.The results showed that the ITS and 5.8S rDNA of the cestodes from Panthera tigris amoyensis was 1387 bp in length and have no sequence variation existed between two samples.The sequence similarities were 96.7%,66.5%,and 71.5% respectively for ITS and 5.8S rDNA compared to those of Spirometra erinaceieuropaei,Diphyllobothrium ligula,and Diphyllobothrium latum.This indicates that the cestodes from Panthera tigris amoyensis in China represents the S.Erinaceieuropaei.