【作者】 李梅；

【导师】 李飞； 严新民； 王红； 贾雪梅；

【作者基本信息】 昆明医学院 ， 病原生物学， 2008， 硕士

【摘要】 [目的]研究云南省三个自然隔离群日本血吸虫线粒体CO1、cytb、ND1、ND6、ND4L基因差异,探讨日本血吸虫种内遗传特点,为阐明我国日本血吸虫的种群遗传结构及进一步研究日本血吸虫生物学特性奠定基础。[方法]采集云南省大理古城、大理喜洲、永胜三地阳性钉螺及购买江苏省实验室传代阳性钉螺,逸出尾蚴,经腹部攻击感染健康家兔（雄性）,每兔感染尾蚴200条～250条,攻击感染后42天剖杀家兔,利用灌注法取成虫。试剂盒抽提基因组DNA,以NC₀02544为参考序列设计引物,采用PCR技术对云南三地以及江苏实验室传代的日本血吸虫的CO1、Cytb、tb、ND1、ND6、ND4L全基因进行扩增、测序。用DNAStar软件及Mega3.1软件进行相似性比较和进化树分析。[结果]分别获得云南三地及江苏省实验室传代日本血吸虫全长CO1、Cytb,ND1、ND6、ND4L基因片段,全长分别为1527bp、1116bp、891bp、459bp、264bp。相似性分析,各序列有微小差异,分别有10、6、6、6、2个碱基位点不同,同源性分别为:99.5%～100.0%、99.6%～100%、99.5%～100.0%、99.1%～100%、99.2%～100%。除ND4L基因外其CO1、Cytb均有一个错义突变,ND1、ND6各有两个错义突变。进化树显示云南三地及江苏省实验室传代日本血吸虫明显聚集在一起,同曼氏血吸虫、埃及血吸虫、湄公血吸虫、马来血吸虫有明显的分支,但与所检获的以往发表的日本血吸虫线粒体相同基因序列比较不构成独立的分支。[结论]各序列有微小差异,同源性较高;CO1、cytb均有一个错义突变,ND1、ND6各有两个错义突变,ND4L无错义突变;本研究所选云南三地及一江苏省实验室传代的样本均属于日本血吸虫,且亲缘关系密切,无明显分支,不能构成种内分类。更多还原

【Abstract】 [Objective]To study genomic diversity among the three natural isolates of Schistosoma japonicum in Yunnan province.To investigate intraspecific genetic characteristic about Schistosoma japonicum.To establish foundation for elucidate the population hereditary constitution of Schistosoma japonicum in China and do further study about biologic characteristics of Schistosoma japonicum.[Methods]Cercariaeum of Schistosoma japonicum were obtained from masccline Oncomelania hupensis from Dali,Xizhou,and Yongsheng in Yunnan Province and one laboratory maintained isolates in Jiangsu Province.Then every male rabbit is infected with 200～250 cercariae of Schisosoma japonicum via abdominal skin. Rabbits were killed on the 42nd after infection.Schistosome japonicurn adults were collected from rabbits through perfusion method.Genomic DNA was isolated with kit, and the complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b(Cytb)and NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L (ND4L)were amplified by PCR using specific primers and sequenced.Program DNAStar and Mega3.1 were used for sequence analysis.[Results]The complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1),cytochrome b,NADH dehydrogenase subunit 1(ND1),NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L(ND4L)of Schistosoma japonicum amplified from different geographic strains were respectively obtained,the length of complete gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1),cytochrome b,NADH dehydrogenase subunit 1(ND1), NADH dehydrogenase subunit 6(ND6)and NADH dehydrogenase subunit 4L (ND4L)of Schistosoma japonicum amplified from different geographic strains were 1527bp,1116bp,891bp,459bp and 264bp,respectively.Comparison of sequences representing the isolates revealed small inter-isolate sequence variations of 10,6,6, 6 and 2 basic group sites,respectively.The identity were 99.5%～100.0%,99.6 %～100%,99.5%～100.0%,99.1%～100%and 99.2%～100%, respectively.There was one missense mutant in the gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b respectively, and two missense mutant in the gene fragments of the mitochondrial NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6) respectively,except NADH dehydrogenase subunit 4L(ND4L).The phylogenetic tree demonstrate:Schistosoma japonicum from three natural isolates in Yunnan Province and one laboratory maintained isolates in Jiangsu Province obviously assemble,have obvious branch with Schistosoma mansoni,Schistosoma haematobium,Schistosoma mekongi and Schistosoma malay,but can’t be constituted independent branch with the published sequences of the same mitochondrial gene were obtained from GeneBank.[Conclusion]The sequences have small difference and higher autoploidy;There was one missense mutant in the gene fragments of the mitochondrial cytochrome C oxidase subunit 1(CO1)and cytochrome b,and two missense mutant in the gene fragments of the mitochondrial NADH dehydrogenase subunit 1(ND1)and NADH dehydrogenase subunit 6(ND6),and no missense mutant in the gene fragment of the mitochondrial NADH dehydrogenase subunit 4L(ND4L);The samples still belong to Schistosoma japonicum in this study and have intimate genetic relationship,but no obvious branch,so can’t be constituted intraspecies categorization.更多还原