斯氏并殖吸虫线粒体Cytb基因的扩增、测序以及遗传进化分析

Amplification,sequencing and phylogenetic analysis on mitochondrial Cytbgene of Pagumogonimus skrjabini

目的扩增出斯氏并殖吸虫线粒体Cytb(cytochrome B)基因片段并测序,分析该基因对吸虫遗传分类的意义。方法提取斯氏并殖吸虫成虫基因组DNA,PCR扩增线粒体Cytb基因,克隆质粒并测序。在NCBI的GeneBank中作相似性和同源性检索,用DNAsis等软件分析这些吸虫的线粒体Cytb基因,采用最大简约法(MP)和邻位相连法(NJ)构建系统进化树,用MEGA4软件对遗传距离进行计算分析。结果获得542bp的斯氏并殖吸虫线粒体Cytb基因片段,通过在GeneBank数据库中Blast发现,斯氏并殖吸虫的Cytb序列与日本血吸虫(中国流行区虫种)、异盘并殖吸虫、哈氏并殖吸虫以及巨睾吸虫等7种吸虫有相似性,斯氏并殖吸虫与日本血吸虫的遗传距离最近。结论线粒体Cytb基因可作为吸虫遗传进化分类的依据之一,也是斯氏并殖吸虫与血吸虫等易出现较高的交叉反应率的原因之一。

In order to analyze the evolutionary significance of the mitochondrial Cytb gene of Pagumogonimus skrjabini, genomic DNA of that was extracted and amplified with polymerase chain reaction （PCR） and subsequently sequenced. The mi- tochondrial Cytb gene sequences used for comparison with those obtained in this study were downloaded from the NCBI data- base. The sequence was compared to the Cytb gene sequence of Paragonimus siamensis, Paragonirnus hokuoensis, Paragoni- mus miyazakii , Paragonirnus vietnamensis , Paragonimus macrorchis , and Schistosoma japonicurn. Phylogenetie tree were constructed using the Maximum parsimony （MP）and the Neighbor-Joining （NJ） method. Sequence homology analysis was performed and the nucleotide composition were accounted using MEGA4 in the DNAStar software package. The results indica- ted that Cytb gene of Pagumogonimus skrjabini was 542 bp. The Cytb gene was similar with that of Pagumogonimus skrjabi- hi, Paragonimus siamensis , Paragonimus hokuoensis , Paragonirnus miyazakii , Paragonimus vietnarnensis , Paragonimus macrorchis, and Schistosoma japonicum. Phylogenetic analysis of Cytb gene revealed that Pagumogonimus skrjabini was the most closely related to Schistosoma japonicum. The mitochondrial Cytb gene can be one of the basis of the phylogenetic classifi- cation of trematoda. This is one of the reason of cross-reactivity between Pagumogonimus skrjabini and Schistosoma.