安徽省不同地区日本血吸虫群体线粒体基因遗传变异研究

Investigation on the genetic variation of mitochondrial genes in Schistosoma japonicum species in different areas of Anhui Province

目的探讨安徽省不同地区日本血吸虫种群的遗传进化关系。方法分别从安徽省山丘型流行区(池州市石台县)和湖沼型流行区(安庆市大观区、铜陵市枞阳县)采集钉螺,逸出尾蚴后感染小鼠,每只小鼠感染(50±3)条尾蚴。35 d后解剖小鼠,门静脉灌注法收集成虫,提取成虫DNA,PCR扩增、克隆和测序成虫的线粒体NADH(还原型烟酰胺腺嘌呤二核苷酸)脱氢酶1(ND1)和细胞色素C氧化酶亚基Ⅰ(COⅠ)基因,利用生物信息学软件Dnasp 6.0和Mega X分析所得序列基本特征并构建系统进化树作遗传进化分析。结果安徽省三个地区日本血吸虫样本的ND1基因序列存在15个变异位点,COⅠ序列存在74个变异位点;ND1基因和COⅠ基因的核苷酸多样性(Pi)分别为0.0047、0.0173,样本间COⅠ基因序列差异高于ND1基因;来自湖沼型流行区的样本ND1基因和COⅠ基因的变异位点数、核苷酸多样性(Pi)均高于来自山丘型流行区样本。来自山丘型流行区的样本在ND1系统进化树中与来自湖沼型流行区的样本聚集在一起,而在COⅠ基因系统进化树中单独成簇。结论来自湖沼型流行区的日本血吸虫样本群体的基因多样性高于来自山丘型流行区的样本。安徽省三个地区日本血吸虫群体的COⅠ基因存在遗传分化,而ND1基因尚未产生较明显的遗传分化。

Objective To investigate the genetic evolutionary relationship among Schistosoma japonicum populations in different areas within Anhui Province.Methods Naturally Oncomelania hupensis were collected form the hilly/mountainous regions(Shitai County in Chizhou City)and swamp/lake areas(Daguan district in Anqing City,Zongyang County in Tongling City).Cercariae escaped from the infected snails obtained in diverse locality were used to infect laboratory-raised female BALB/c mice by dose of(50±3)cercariae in individual mouse.Then the mice were dissected 35 days after infection to collect the adult worms via portal vein perfusion technique.DNA was extracted from the adults to undergo PCR amplification,cloning and sequencing of the mitochondrial dehydrogenase 1(ND1)and cytochrome C oxidase subunitⅠ(COⅠ)gene fragments.The sequences were analyzed by bioinformatics software Dnasp 6.0 and Mega X.Evolutionary trees were constructed using the neighbor-joining method with MEGA software(version 10.2.2)to observe the evolution clues.Results Fifteen nucleotide variation sites in ND1 gene and 74 in COⅠgene were identified in the samples obtained in three districts.The nucleotide diversity(Pi)of ND1 and COⅠgene was 0.0047 and 0.0173,respectively.The variation of COⅠbetween these samples was a little higher than that of ND1 gene.Variable sites of ND1 and COⅠgenes and Pi were higher in the samples from swamp/lake areas than in those from hilly/mountainous regions.Samples from Shitai County were independent in the tree based on COⅠgene,yet all of the samples were gathered together in the tree at the foundation of ND1 gene.Conclusion The genetic diversity is higher in S.japonicum from marshy regions than in that from hilly regions.The results indicated that there was some differences between the COⅠgene of S.japonicum from hilly region and marshy region in Anhui province,and meanwhile there was no significant differences between the ND1 gene of these S.japonicum worms by now.