不同宿主来源猬迭宫绦虫线粒体pcox1、pcox3和prrn S基因的系统发育分析

Sequence and phylogenetic analysis of mitochondrial pcox1,pcox3 and prrn S genes for Spirometra erinaceieuropaei

为了分析不同宿主猬迭宫绦虫的遗传多样性和种系发育关系,本研究通过PCR方法扩增了湖南省7种不同宿主体内采集的7株猬迭宫绦虫(其中3株为裂头蚴)的线粒体pcox1、pcox3和prrn S基因并测序,采用DNAStar 5.0软件分析其与其他绦虫相应基因序列的同源性;利用DnaSP V6.12软件计算并分析各基因序列的多样性;利用邻接法绘制pcox1、pcox3和prrn S基因的遗传进化树。PCR扩增及测序结果显示,7株虫株均扩增出了分别为396 bp(pcox1)、362 bp(pcox3)和280 bp(prrn S)的目的片段,且3种基因碱基差异率分别为0~2.27%、0~1.10%和0~1.79%。同源性分析结果显示,本研究的7株猬迭宫绦虫与已报道的其他猬迭宫绦虫线粒体pcox1、pcox3和prrn S基因序列的同源性分别为97.20%~100.00%、98.90%~100.00%和97.90%~100.00%;与其他绦虫线粒体pcox1、pcox3和prrn S基因序列同源性分别为66.70%~84.10%、51.70%~72.10%和63.80%~87.10%。基于基因序列单倍型数(H)、单倍型多样性(Hd)、核苷酸多样性(Pi)、平均核苷酸差异(K)数据的基因序列多样性分析结果显示,3个基因变异率为pcox1>prrn S>pcox3。系统进化树结果显示,7株虫株的pcox1、pcox3和prrn S基因均与已知猬迭宫绦虫相应基因聚类在同一分支,与其他绦虫所属分支相距较远,且基于pcox1、pcox3和prrn S基因均可以将猬迭宫绦虫聚为两个分支。上述结果表明,上述3种基因尤其是pcox3基因均可以作为研究不同宿主来源猬迭宫绦虫遗传变异的分子标记及用于其虫种的鉴定,且猬迭宫绦虫的遗传变异与宿主无关。该研究是国内首次通过线粒体基因探究不同宿主猬迭宫绦虫的遗传进化关系,为猬迭宫绦虫的分子鉴定、分类和群体遗传研究奠定了基础。

To analyze the genetic diversity and phylogenetic relationship of 7 Spirometra erinaceieuropaei collected from 7differential hosts in Hunan province, the pcox1, pcox3 and prrn S genes of S. erinaceieuropaei were amplified by normal PCR and sequenced. The acquired sequences were subjected to the analysis of nucleic acid sequence homology, genetic diversity and phylogenetic relationship compared with reported S. erinaceieuropaei sequences. In this study, PCR products at the length of 396bp,362bp and 280bp were amplified based on pcox1, pcox3 and prrn S genes, respectively. Nucleotide difference within three genes were ranged 0-2.27%, 0-1.10% and 0-1.79%, respectively. Homology analysis showed that the pcox1, pcox3 and prrn S genes from 7 isolates in this study had 97.20%-100.00%, 98.90%-100.00% and 97.90%-100.00%, respectively, similarity with reported S. erinaceieuropaei, and 66.70%-84.10%, 51.70%-72.10% and 63.80%-87.10%, respectively, similarity compared with other tapeworms. The result of genetic diversity analysis, based on the number of haplotypes(H), haplotype diversity(Hd), nucleotide diversity(Pi) and average nucleotide differences(K) of gene sequences, showed that the mutation rate of each gene was pcox1>prrn S>pcox3. The phylogenetic tree based on pcox1, pcox3 and prrn S showed that all 7 isolates in this study were clustered into the same branch with reported S. erinaceieuropaei, respectively, and these 7 isolates were belonged to 2 branches. In conclusion, the results indicated that pcox1, pcox3 and prrn S genes, especially the pcox3 gene, could be used as molecular markers for studying of genetic variation in tapeworm. This study is the first report to investigate the genetic diversity by amplify three genes of mitochondrial, providing the fundamental data for identification, classification and population genetic structure of S. erinaceieuropaei.