广西两地异盘并殖吸虫分子系统发生研究

发布时间：2018-11-06 10:32

【摘要】： 目的:通过研究广西两地(灵川、那坡)异盘并殖吸虫分离株的核糖体DNA第二内部转录间隔区基因(ITS2)和细胞色素c氧化酶亚单位Ⅰ基因(CO1)序列,比较分析两地虫体的基因差异,揭示它们之间的亲缘关系和分类学地位。方法:采集灵川和那坡两地的异盘并殖吸虫囊蚴接种适宜终宿主大鼠,获得成虫后提取基因组DNA,PCR扩增其ITS2和CO1基因序列并测序。从Genebank检索到与它们同源性相近的并殖吸虫基因序列,应用Megalign软件对基因序列进行比较和同源性分析,然后用Mega及Phylip软件以最小进化法(ME)、邻接法(N-J)、最大似然法(ML)、最大简约法(MP)构建系统发生树,其中ME和N-J法用Kimura双参数法计算遗传距离,自举检验副本数为1000,ML和MP法自举检验副本数为500。结果:那坡异盘并殖吸虫分离株与灵川异盘并殖吸虫分离株的ITS2基因序列相似度为100%,CO1基因序列相似度为97.5%,同源性较高;而那坡与泰国一个分离株的ITS2和CO1基因序列相似度高达100%,具有高度同源性。从ITS2序列系统发生树来看,来自那坡、灵川、越南、泰国和印度的分离株均处于异盘并殖吸虫的种群下,但印度分离株是个相对独立的分枝,亲缘关系较远。从CO1序列系统发生树来看,不同的发生树中那坡分离株均与泰国、越南分离株的亲缘关系较近,以较高的自举检验置信值形成单系群,而灵川分离株的分枝均处在这个群之外,印度分离株的亲缘关系则更远一些。结论:那坡分离株与灵川分离株的ITS2基因和CO1基因序列的同源性比较高,而那坡分离株与泰国分离株的2个基因序列的同源性更高。虽然那坡和灵川分离株在形态学上有一定差异,但在不同方法构建的系统发生树中都位于异盘并殖吸虫的种群分枝下。CO1系统发生树中那坡分离株均与泰国、越南分离株的亲缘关系较近,形成有共同起源的单系群,而灵川分离株的分枝均处在这个群之外。据此认为,灵川和那坡的分离株可能属于不同的地理株。
[Abstract]:Objective: to study the sequence of ribosomal DNA second internal transcriptional spacer (ITS2) and cytochrome c oxidase subunit I (CO1) gene of paragonimus xenostroma isolated from Lingchuan, Napo, Guangxi. The genetic differences between the two species were analyzed, and the phylogenetic relationship and taxonomic status between them were revealed. Methods: the metacercariae of Lingchuan and Napo were collected and inoculated into the rats suitable for the final host. The genomic DNA,PCR was extracted from adult worms to amplify the sequence of ITS2 and CO1 genes and sequenced. The homologous gene sequences of paragonimus were retrieved from Genebank. The sequence of paragonimus was compared and homologous analyzed by Megalign software. Then, the sequence of paragonimus gene was analyzed by Mega and Phylip using (ME), adjacency method of minimal evolution (N-J). The phylogenetic tree was constructed by maximum likelihood method (ML),) and maximum reduction method (MP), in which ME and N-J methods were used to calculate genetic distance by Kimura two-parameter method, and the number of bootstrap test replicas was 1000m L and 500 by MP method. Results: the similarity of ITS2 gene sequence between paragonimiasis natapo isolate and Lingchuan paragonimiasis isolate was 97.5, and the similarity of ITS2 gene sequence was 97.5. The sequence similarity of ITS2 and CO1 between Napo and a Thai isolate is as high as 100, with high homology. From the phylogenetic tree of ITS2 sequence, the isolates from Napo, Lingchuan, Vietnam, Thailand and India are all under the population of paragonimus heterodendens, but the Indian isolate is a relatively independent branch, and the genetic relationship is relatively distant. According to the phylogenetic tree of CO1 sequence, Napo isolates in different phylogenetic trees were closely related to Thai and Vietnamese isolates, and monophyletic groups were formed with higher confidence values of bootstrap test, but the branches of Lingchuan isolates were all outside this group. The Indian isolates were more closely related. Conclusion: the homology of ITS2 gene and CO1 gene sequence between Napo isolate and Lingchuan isolate is higher than that between Napo isolate and Thai isolate. Although there are some differences in morphology between Napo and Lingchuan isolates, the phylogenetic trees constructed by different methods are all located under the branches of the population of paragonimus heterophylla. The Napo isolates in the phylogenetic tree of CO1 are all different from those in Thailand. Vietnamese isolates were closely related to each other, forming a monophyletic group with a common origin, while the branches of Lingchuan isolates were all located outside this group. It is concluded that the isolates of Lingchuan and Napo may belong to different geographical strains.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R383.23

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