日本血吸虫线粒体基因序列的研究

发布时间：2018-06-12 11:00

本文选题：日本血吸虫 + 线粒体　；参考：《昆明医学院》2008年硕士论文

【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NC_002544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。
[Abstract]:[objective] to study the difference of mitochondrial CO1Cyt bND1 (ND1) ND6Nd4L gene in three natural isolated populations of Schistosoma japonicum in Yunnan Province, and to explore the genetic characteristics of Schistosoma japonicum. In order to clarify the genetic structure of Schistosoma japonicum population and further study the biological characteristics of Schistosoma japonicum in China. [methods] the ancient city of Dali, Yunnan Province, Dali Xizhou, was collected. Oncomelania hupensis was positive in Yongsheng three places and purchased from Jiangsu laboratory, and cercariae escaped. The rabbits were infected with 250 cercariae by abdominal attack (male, 200 cercariae per rabbit). The rabbits were killed 42 days after attack, and the adult worms were harvested by perfusion method. Genomic DNA was extracted by the kit and primers were designed using NC002544 as reference sequence. The whole gene of Schistosoma japonicum was amplified by PCR and sequenced. DNA Star software and Mega3.1 software were used to compare the similarity and analyze the phylogenetic tree. [results] the full-length gene fragment of Schistosoma japonicum was subcultured in three places of Yunnan Province and Jiangsu Province, respectively. The total length of the gene was 1527bp1 1116bpn891bp459bp264bp. In similarity analysis, there were slight differences in each sequence. There were two different base loci. The homology was 10. 99. 5 and 10. 0. 0. 10. 0. 0. 0. 99. 6 and 100. 99. 5 and 99. 0. 0. 99. 2. With the exception of ND4L gene, there was one missense mutation in all of the CO1C Cytb and two missense mutations in Nd1 and ND6. The phylogenetic tree shows that Schistosoma japonicum from three places in Yunnan and Jiangsu Province obviously gathered together, and had obvious branches with Schistosoma mansoni, Schistosoma japonicum, Schistosoma Mekong, Schistosoma Malay. But compared with the previously reported mitochondrial gene sequence of Schistosoma japonicum, it does not constitute an independent branch. [conclusion] there are slight differences among the sequences. Two missense mutations were found in each of the two missense mutants (Nd4L), and the samples collected from three places of Yunnan and one Jiangsu Province were all Schistosoma japonicum, and were closely related to each other and had no obvious branches. It does not constitute an intraspecific classification.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383

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