10株火鸡组织滴虫18S rRNA基因的克隆及进化分析

Cloning and Phylogenetic Analysis of the 18S rRNA of Histomonas meleagridis Isolates from Hunan Province

应用聚合酶链反应(PCR)鉴定湖南娄底(简称HMLD1-3)、湘潭(HMXT1-2)、永州(HMYZ1-3)和宁乡(HMNX1-2)共10株火鸡组织滴虫,并用18SrRNA部分序列重构火鸡组织滴虫与其它相近原虫的种群遗传关系。作者通过PCR扩增火鸡组织滴虫虫株的18S rRNA部分序列,将PCR扩增出的片段纯化后克隆至pGEM-T Easy载体,重组质粒通过菌落PCR鉴定后,对阳性菌落进行序列测定并进行序列分析。结果表明,来自湖南省的10株火鸡组织滴虫18SrRNA序列长度一致,均为532bp,与GenBankTM公布的火鸡组织滴虫序列相似性在97.5%以上,与其它原虫差异均大于20%。种系发育树分析表明,10个分离株与已知火鸡组织滴虫位于同一分枝。本研究证实该病原为火鸡组织滴虫,从分子生物学水平证明了湖南省存在火鸡组织滴虫。本研究为火鸡组织滴虫病诊断及火鸡组织滴虫种群体遗传学研究奠定了良好基础。

Histomonas meleagridis were identified by PCR form Hunan Province,and their phylogenetic relationship was analysed by using 18S rRNA sequences.The 18S rRNA sequences were amplified from each H.meleagridis sample and the amplicons were cloned into pGEM-T Easy vector,respectively.The inserts were successfully sequenced,and the length of 18S rRNA of the 10 H.meleagridis isolates were 532 bp.Sequence comparison revealed that the similarity in18S rRNA sequences between Hunan isolates and the H.meleagridis available in GenBankTM were more than 97.5%,respectively.The 10 isolated strains grouped together with the reported H.meleagridis.The existence of H.meleagridis in Hunan Province was confirmed at the level of molecular biology.The results indicated that PCR was suitable for the diagnosis and population genetics of H.meleagridis.