细粒棘球蚴线粒体CO1基因的克隆与序列分析

Cloning and sequence analysis of the partial CO1 gene within mitochondrial DNA of Echinococcus granulosus

目的研究细粒棘球蚴(Echinococcus granulasus)种间遗传标记特点,为该虫种的分子分类学研究提供基础。方法在内蒙古地区从羊肝脏采集细粒棘球蚴,肝包虫病患者手术剥离囊包后,签署知情同意书,采集细粒棘球蚴。提取虫体DNA,扩增线粒体细胞色素氧化酶1(CO1)基因,将其克隆到PGM-T载体后,用PCR技术鉴定阳性菌落,并进行测序,运用DNAstar5.0软件计算序列间的相似性,计算遗传距离,同时,应用MEGA4.0软件的最大似然法(ML-Maximum Likelihood)和邻接法(NJ-Neighbor Joining)构建系统发育树,进行聚类分析。结果细粒棘球蚴DNA扩增出的羊株、人株CO1基因序列片段长度为936bp。同其他亲缘关系较近的属CO1基因序列比对:羊株、人株细粒棘球蚴CO1序列与黄花棘球绦虫的同源性最高,分别为91.9%、91.2%;羊株、人株细粒棘球蚴CO1序列与黄花棘球绦虫位于同一分支,自展值(Boostrap)最高,均为100%。羊株、人株细粒棘球蚴CO1序列所属分支与原头目的 Proteocephalus macrocephalus所属分支相隔较远。结论CO1基因序列稳定保守,无宿主特异性,可作为细粒棘球蚴理想的种间遗传标记。

We investigated the inter-specific genetic marker of Echinococcus granulosus,which was used for further study in molecular classification.The genomic DNA of Echinococcus granulosus collected from the infected sheep and people in Inner Mongolia were extracted.The mitochondrial cytochrome oxidase subunit 1（CO1）gene was amplified by PCR using universal primers,and then the PCR product was cloned into pGM-T vectors.The insert was sequenced successfully and compared with other cestodes sequences by DNAStar 5.0and MEGA 4.0.Results showed that the PCR product was 936 bp in length.Compared with other CO1 gene sequences of related cestodes,the homology among Echinococcus granulosus and Echinococcus canadensis were 91.9% and 91.2%.Echinococcus granulosus and Echinococcus canadensis were in the same branch,and bootstrap values（Boostrap）in the highest was 100%.Echinococcus granulosus affiliated branch of the original leaders of the branch belonged to Proteocephalus macrocephalus far apart.It suggested that the CO1 gene sequences were stably conservative and nonhost-specific,which can be used as an ideal species among Echinococcus granulosus genetic markers.