豆状带绦虫线粒体rrnL和nad5基因的克隆及种系发育研究

Cloning and Phylogenetic Analysis of Mitochondrial rrnL and nad5 Genes of Taenia pisiformis Isolates

本研究旨在阐明中国豆状带绦虫囊尾蚴河南分离株线粒体核糖体大亚基基因(rrnL)部分序列(prrnL)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位5基因(nad5)部分序列(pnad5)的遗传变异情况,并用prrnL和pnad5序列重构豆状带绦虫与其他带科绦虫的种系发育关系。采用聚合酶链式反应(PCR),扩增豆状带绦虫囊尾蚴分离株的线粒体prrnL和pnad5,将所测的序列用PAUP 4.0Beta 10程序MP法绘制种系发育树,利用DNAStar 5.0中的MegAlign程序进行同源性分析。所获得的豆状带绦虫线粒体prrnL长度约为847bp,pnad5为602bp。所获rrnL序列与GenBank中豆状带绦虫相应序列的相似性为99.1%～100%,nad5为99.2%～100%;河南分离株之间rrnL序列的相似性为99.65%,nad5为99.5%;与GenBank中其他带科绦虫序列的相似度rrnL低于83.4%,nad5低于85.7%。种系发育分析结果表明,所有豆状带绦虫分离株和已知豆状带绦虫位于同一分支。由于豆状带绦虫分离株的线粒体rrnL和nad5基因序列种内很保守,种间差异较大,故可作为带科绦虫种间遗传变异研究的标记。

The objectives of the present study were to examine sequence variation in the mitochondrial large subunit riboso- mal rRNA （rrnL） and NADH dehydrogenase subunit 5 （nadS） genes among Taenia pisiformis isolates from Henan province and study its phylogenetic relationships with other cestodes using rrnL and nad5 sequences. The partial rrnL （prrnL） and had5 （pnad5） genes sequences were amplified from each T. pisiformis sample by polymerase chain reaction （PCR）, and prrnL and pnad5 sequences were aligned using the ClustalX 1.81. Maximum parsimony （MP） tree was constructed using the software PAUP 4.0 Beta 10. The lengths of prrnL and pnad5 sequences were 847 and 602 bp, respectively. Sequence similarity in rrnL among the examined T. pisiforrnis samples were 99.65 %, and 99.5 % for nad5, whereas inter-specific similarity among taeniid cestodes were significantly lower than 83.4a/~ for rrnL, and lower than 85.7~ for nadS. Phylogenetic analysis showed that all the T. pisiforrnis isolates clustered in the same clade. It was concluded that prrnL and pnad5 sequences could be used as a genetic marker for the identification and differentiation of taeniid cestodes.