核糖体小亚基RNA和细胞色素氧化酶亚单位Ⅱ基因在中国利什曼原虫系统发育学分析中应用的比较  

Comparison on the difference between the analysis of phylogenetic relationships of Leishmania isolates from China with small subunit ribosomal RNA and cytochrome oxidase Ⅱ gene

作　　者：曹得萍[1] 廖琳[2] 陈达丽[2] 陈建平[2] 

机构地区：[1]青海大学医学院病原生物学教研室,西宁810001 [2]四川大学华西医学中心寄生虫学教研室,成都261004

出　　处：《国际医学寄生虫病杂志》2014年第1期20-24,共5页International JOurnal of Medical Parasitic Diseases

基　　金：基金项目：国家自然科学基金（81171607、30800094、30771883）;国家十一五重大科技专项（2008-ZX10004-011）

摘　　要：目的比较核糖体小亚基RNA（small subunit ribosomal RNA．SSUrRNA）和细胞色素氧化酶亚单位Ⅱ（cytochrome oxidaseⅡ，COXⅡ）这2种分子标志在中国利什曼原虫系统发育学分析中的不同。方法提取利什曼原虫各虫株核基因组和线粒体基因组．PCR方法扩增中国不同地区利什曼原虫SSUrRNA和COXⅡ基因．扩增产物送上海生工生物工程技术服务有限公司测序．序列碱基通过Clustal X软件比对，用DAMBE软件进行单倍型分析、Mega4软件计算遗传距离，Mrbayes3．1．2软件构建贝叶斯进化树。结果COXⅡ分析结果表明．流行于中国的利什曼原虫虫株未形成一个单系群：GS7和XJ771属于杜氏利什曼原虫复合体；10株利什曼原虫形成的6个单倍型（MHOM／CN/93/GS7,IPHL／CN／77／XJ771,MHOM／CN／84／JS1和MGER／CN／60／GS．GER20除外）形成1个单系群：而SSUrRNA分析结果表明：11株利什曼原虫（IPHL／CN／77／XJ771，MHOM/CN，93／GS7，MRH0／CN／88／KXG-2，MHOM／CN／84／JS1．MGER／CN／60／GS．GER20除外）形成一个独立枝；江苏株JS1和热带利什曼原虫聚在一起，不与杜氏利什曼原虫聚在一起。结论SSUrRNA和COXⅡ得出的结果较一致．但COXⅡ基因在分析利什曼原虫的系统发育关系时．可能是一个更可靠的遗传标志。Objective To compare the difference between the analysis on the phylogenetic relationships of Leishmania isolates from different foci in China with partial fragment of small subunit ribosomal RNA （SSUrRNA） gene sequences or kinetoplast eytochrome oxidase Ⅱ （COXⅡ ） gene sequences. Methods Ge- nomic DNA and kinetoplast DNA were extracted from the promastigotes. The COⅡ and SSUrRNA genes were amplified using respective primers. Products of PCR were sent to a commercial company for sequencing using the same PCR primer. Combined sequences matrix was aligned using Clustal X （1.8） with default settings and refined manually. Bayesian tree was constructed using Mrbayes-3.1.2. Results Leishmania isolates from different foci in China does not form a mono-phyletic group. The isolate MHOM/CN/93/GS7 and the isolate IPHL/CN/77/XJ771 belong to Leishmania donovani complex. Six haplotypes representing 10 Chinese Leishmania isolates（except isolates MHOM/CN/93/GS7, IPHL/CN/77/XJ771, MHOM/CN/84/JS1 and MGER/CN/60/GSGER20） form a monophyletic group （results from CO Ⅱ genes database）. Eleven Chinese Leishmania isolates （except IPHL/CN/77/XJ771,MHOM/CN/93/GS7 ,MRHO/CN/88/KXG-2,MHOM/CN/84/JS1,MGER/CN/60/GSGER20） form a subclade,near to Leishmania tarentolae （results from SSUrRNA genes database）; the isolateMHOM/CN/84/JS1 is sister of L. tropica representing L. tropica complex with high posterior probability （PP= 1.00）, instead of clustering with L. donovani complex or with other 10 Chinese isolates. Conclusions The analysis results with either SSUrRNA or COX Ⅱ seemed consistent. However kinetoplast cytochrome oxidase Ⅱ gene might be a more reliable genetic marker than SSUrRNA to analyze the phylogentic relationship of Chinese Leishmania isolates.

关 键 词：中国利什曼原虫 前鞭毛体 系统发育学 核糖体小亚基RNA 细胞色素氧化酶亚单位Ⅱ 

分 类 号：R382.22[医药卫生—医学寄生虫学]