湖南长沙及湘西泡状带绦虫分离株线粒体cox1基因的克隆及序列分析

Cloning and Sequence Analysis of Mitochondrial Cytochrome c Oxidase Subunit 1 from Taenia hydatigena Isolates from Changsha and Xiangxi in Hunan Province

目的研究湖南省长沙及湘西泡状带绦虫分离株线粒体细胞色素c氧化酶第I亚基(cox1)基因部分序列(pcox1)的遗传变异情况,并用pcox1序列重构泡状带绦虫与其他带科绦虫的种群遗传关系。方法应用聚合酶链反应(PCR)扩增泡状带绦虫虫株的pcox1,应用ClustalX1.81程序对序列进行比对,然后用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树;同时利用DNAstar5.0中的Megalign程序进行同源性分析,并与GenBankTM中已知泡状带绦虫相应基因序列进行比较分析。结果所获得的pcox1序列长度一致,均为343bp,与GenBankTM公布的带科绦虫序列进行比较分析表明,湖南长沙分离株1和湘西分离株5与已知泡状带绦虫相应基因的相似性分别为99.4%和99.7%,与其它带科绦虫的相似性均小于90.0%;湖南长沙1和湘西分离株5与已知泡状带绦虫相应基因遗传距离分别为0.006和0.003,与其他带科绦虫的遗传距离均大于0.100。种系发育树表明,2个分离株与已知泡状带绦虫位于同一分枝,Bootstrap值在97%以上。结论由于泡状带绦虫pcox1序列种内相对保守,种间差异较大,故可作为种间遗传变异研究的标记,研究结果为进一步研究泡状带绦虫的群体遗传结构奠定了基础。

Objective To analyze whether there are any differences in the cytochrome c oxidase subunit 1 （cox1） gene of Taenia hydatigena strains from Changsha and Xiangxi in Hunan province, and to study the phylogenetic relationship among cestodes of the Taeniidae using cox 1 sequences. Method The partial cox1 （pcox1） was amplified from each T.hydatigena sample, and pcoxl sequences were aligned using the ClustalX 1.81. MP and NJ trees were constructed using the software Phylip 3.67 version 4.0 and Mage version 4.0, and ML tree was also constructed using Puzzle version 5.2. Sequence homology analysis was performed using the Megalign program of the software DNAstar version 5.0 by comparing with the corresponding sequence of T. hydatigena available in GenBank^TM. Result The lengths of all the pcoxl sequences was 343 bp. Sequence comparison revealed that the similarity in pcoxl sequences among Changsha, Xiangxi isolates with that of the T.hydatigena available in GenBank^TM were 99.4% and 99.7%, respectively. The two T. hydatigena strains grouped together with the reported T.hydatigena, with a bootstrap support of more than 97%. Conclusion There is no significant variation in pcox1 sequences within T.hydatigena, while inter-species difference is obvious. It is concluded that pcoxl sequences can be used as genetic marker for population genetic studies of cestodes. The Results of the present study provided foundation for further studies of population genetics of T. hydatigena.