奶牛附红细胞体16SrRNA基因的克隆测序及系统发育进化树的建立

Cloning、sequencing and establishing the phylogenetic tree of E.wenyoni 16SrRNA gene

为了从分子水平上证实奶牛附红细胞体的存在及研究其分类学地位,利用原核生物16S rRNA基因通用引物对分离纯化的疑似奶牛附红细胞体进行16S rRNA基因的PCR扩增及克隆测序。结果扩增出长约1.5 kb的目的片段,测序结果表明:目的片段长度为1 439 bp,其核苷酸序列与国外已发表的牛温氏附红细胞体(E.wenyoni)的16SrRNA基因片段同源性高达97.1%,暂称为中国广西株(E.wenyoniCGX)。系统发育进化树显示:E.wenyoni和其他血营养菌在系统进化关系上组成了一个大的进化分支,与支原体科、支原体属的病原最接近(75%),而与立克次体科的病原较远(55%)。分析结果支持了Nei mark等和Messick等提出的将这类血营养菌划归支原体科、支原体属的建议。

In order to confirm that our region exist E. wenyoni and research it taxology, universal primer（27F and 1 492 R）was used to amplified 16SrRNA gene of separate pathogeny that suspiciosed E. wenyoni by polymerase chain reaction（PCR）, it was cloned and sequenced. A 1 500 bp segment was amplied, the result of sequence showed that the segment comprises 1 439 bp, share 97. 1% homology in bases with 16SrRNA gene of E. wenyoni that published abroad. However, there aiso exist Some difference, so we interimly call it E. wenyoni -CGX strain. By seting up the phylogenetic tree, E. wenyoni and other Haemotrophic baeterias have formed a large phylogenenetic branch, which close to the Mycoplasma genus of Mycoplasmatales （75%）, while far from the pathogeny of Rickettsiales（ 55%）. The result of analysis sustain the advice that the Eperythrozoom and haemobartonella genera should be reclassified in Mycoplasma genus of Mycoplasmatales.