论文部分内容阅读
目的分析我国云南中缅边境等地5种疟原虫18S rDNA的基因差异和种系发育。方法 2000-2015年收集来自我国云南中缅边境等地的疟疾患者血样(或疟原虫DNA),提取血样中的疟原虫DNA,对它们的18S rDNA基因进行扩增、测序分析,并与Gen Bank中疟原虫相应序列进行比较分析,利用邻接法(neighbor joining,NJ)、最大似然法(maximum likelihood,ML)和最大简约法(maximum parsimony,MP)构建系统进化树。结果共收集了94例疟疾患者血样或DNA,所有样品均扩增出18S rDNA基因序列。序列比对分析显示,恶性疟原虫(Plasmodium falciparum)、间日疟原虫(P.vivax)、三日疟原虫(P.malariae)、卵形疟原虫(P.ovale)和诺氏疟原虫(P.knowlesi)的种内差异分别为0~0.2%、0~0.1%、0~0.1%、0~0.1%和0。种系发育关系分析显示,NJ、MP和ML法构建的系统进化树的拓扑结构基本一致。本研究的疟原虫形成5个大的分支:恶性疟原虫形成一个分支,与Gen Bank中来自喀麦隆(KC428741、KC428742)、巴西(KC906718)和马来西亚(HQ283221)的分离株聚集在一起;间日疟原虫形成一个分支,其中大部分样品与来自喀麦隆(HF945443)、印度(HM014361、JQ627158)和哥伦比亚(U83877)的分离株形成一较小分支,小部分样本(Pv11、Pv18和Pv21)形成一个更小的分支后与食蟹猴疟原虫(P.cynomolgi)(L07559)相聚形成一较小分支;三日疟原虫形成一个分支,来自云南的三日疟原虫Pm1、Pm3和Pm4聚在一个小分支内,并与来自日本的分离株(AB250682)相聚,再与来自海南的分离株Pm5相聚,显示出地区差异性;卵形疟原虫形成一个分支,与来自越南的分离株(EU935736、AF387038)聚在一起;诺氏疟原虫形成一个分支,与来自缅甸的分离株(GU816250、GU816246)聚在一起。结论我国云南中缅边境等地5种疟原虫18S rDNA基因均无明显种内差异;种系发育关系分析显示,NJ、MP和ML法构建的系统进化树的拓扑结构基本一致。
Objective To analyze the genetic differences and phylogenetic relationships of 18S rDNA of five species of malaria parasites in China-Myanmar border in Yunnan Province, China. Methods Blood samples (or DNA of malaria) from malaria patients in Yunnan, China and Myanmar border areas were collected during 2000-2015. Plasmodium DNA in blood samples were extracted and their 18S rDNA genes were amplified, sequenced and compared with Gen Bank The corresponding sequences of Plasmodium were compared and analyzed. The phylogenetic tree was constructed by neighbor joining (NJ), maximum likelihood (ML) and maximum parsimony (MP). Results A total of 94 malaria patients were collected blood samples or DNA, all samples were amplified 18S rDNA gene sequence. Sequence alignment analysis showed that Plasmodium falciparum, P. vivax, P. malariae, P.ovale and P. nidulans (P .knowlesi) were 0 ~ 0.2%, 0 ~ 0.1%, 0 ~ 0.1%, 0 ~ 0.1% and 0 respectively. The analysis of phylogenetic relationships showed that the topological structures of phylogenetic trees constructed by NJ, MP and ML methods were basically the same. The Plasmodium of this study formed five major branches: P. falciparum formed a branch that aggregated with isolates from GenBank from Cameroon (KC428741, KC428742), Brazil (KC906718) and Malaysia (HQ283221); P. vivax Protozoa formed a branch where most of the samples formed a smaller branch with the isolates from Cameroon (HF945443), India (HM014361, JQ627158) and Colombia (U83877), and a small sample (Pv11, Pv18 and Pv21) formed a smaller And formed a smaller branch with P.cynomolgi (L07559); a branch of Plasmodium malaria formed a branch from which Plasmodium malariae Pm1, Pm3 and Pm4 from Yunnan gathered in a small branch , And clustered with an isolate from Japan (AB250682) and then with the isolate Pm5 from Hainan, showing regional variability; P. ovale formed a branch that converged with isolates from Vietnam (EU935736, AF387038) Together; Plasmodium knowlesi forms a branch that comes together with the isolate from Myanmar (GU816250, GU816246). Conclusion There were no significant intraspecific differences in the 18S rDNA genes of five species of malaria parasites between China and Myanmar border areas in Yunnan. Phylogenetic analysis showed that the topological structures of phylogenetic trees constructed by NJ, MP and ML methods were basically the same.