【作者】 李利；

【导师】 王春仁；

【作者基本信息】 黑龙江八一农垦大学 ， 预防兽医学， 2008， 硕士

【摘要】 土耳其斯坦东毕吸虫(Orientobilharzia turkestanicum)是寄生于牛、羊等多种动物门静脉和肠系膜静脉的一种血吸虫。它与所在属内的其它血吸虫寄生于动物导致的疾病称为东毕吸虫病(orientobilharziasis),主要分布于亚洲和欧洲的一些国家和地区,常呈地方性流行,对畜牧业危害十分严重。近年来,土耳其斯坦东毕吸虫的研究主要集中在其形态特征描述、生活史研究及东毕吸虫病的流行病学调查、诊断、药物防治和预防措施等方面,关于其分子分类和种系发生的基础研究少见报道。核糖体DNA是细胞核内编码核糖体RNA的基因,为一类中度重复序列,以串联多拷贝的形式存在于细胞核内染色体DNA中,每个重复单位由非转录间隔区(non-transcribed spacer regions, NTS)、内部转录间隔区(internal transcribed spacer region, ITS)和3种核糖体基因编码区(18S、5.8S、28S)组成,不同区域其进化速率不同。线粒体为母系遗传,其基因间很少重组,所以可以反映出母系的进化历史,这样线粒体一个基因就可以代表整个线粒体基因组的变异情况。因此,我们以土耳其斯坦东毕吸虫核糖体内转录间隔区(ITS)序列、28S核糖体DNA大亚基序列(28S ribosomal DNA large-subunit sequences, 28S rDNA-LSU)、线粒体细胞色素c氧化酶亚基1(cytochrome c oxidase subunit 1 gene, cox1)基因和烟酰胺腺嘌呤二核苷酸脱氢酶亚基1(nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene, nad1)基因为研究对象,对目的片段进行克隆和序列测定,构建分子系统发生树,探讨土耳其斯坦东毕吸虫在裂体科中的系统发生位置。从自然感染黄牛、绵羊、绒山羊和山羊的肝门静脉及肠系膜静脉内采集虫体,经形态学鉴定为土耳其斯坦东毕吸虫。以SDS-蛋白酶K法抽提不同终末宿主的土耳其斯坦东毕吸虫基因组DNA,根据GenBank上发表的土耳其斯坦东毕吸虫及相关血吸虫序列,利用Oligo6.0软件设计特异引物,PCR扩增ITS区序列、28S rDNA-LSU序列、线粒体cox1基因和nad1基因,扩增产物经纯化后克隆于pMD18-T载体,转化到JM109感受态细胞,提取质粒DNA,经PCR和双酶切鉴定后,阳性质粒测序,应用DNAStar软件比较不同终末宿主土耳其斯坦东毕吸虫核苷酸序列的同源性,并应用DNAMAN软件分析28S rDNA-LSU序列RNA二级结构的改变情况。检索GenBank,查找血吸虫相关基因序列,应用Clustal X1.83软件对相关序列进行排序,提交引导树,使用MEGA软件采用邻接法(neighbor-joining method, NJ)和最大简约法(maximum parsimony method, MP),绘制系统发生树。序列分析结果表明,土耳其斯坦东毕吸虫核糖体ITS区序列、28S rDNA-LSU序列、线粒体cox1编码基因、nad1编码基因大小分别为874 bp,1 304 bp,1 125 bp和518 bp;不同终末宿主土耳其斯坦东毕吸虫ITS区序列、28S rDNA-LSU序列、cox1基因序列和nad1基因序列存不同程度的差异,绵羊、绒山羊和山羊源土耳其斯坦东毕吸虫28S rDNA-LSU序列RNA二级结构相同或相似,黄牛源与羊源土耳其斯坦东毕吸虫28S rDNA-LSU序列RNA二级结构存在较大差异。以肝片形吸虫作外群,基于核糖体ITS区序列、28S rDNA-LSU序列、线粒体cox1基因序列和nad1基因序列采用NJ法和MP法构建的系统发生树,其结果一致,均显示土耳其斯坦东毕吸虫的分类地位处于裂体属内,同时土耳其斯坦东毕吸虫归属非洲血吸虫种群。更多还原

【Abstract】 Adult worms of Orientobilharzia turkestanicum live in the portal veins or intestinal veins of cattle, sheep and other animals and cause orientobilharziasis, which have an important impact on livestock industry and have a large distribution in Asia and several areas of Europe. In recent years, researches are mainly focused on the morphology, life cycle of this pathogen, and epidemiology, diagnosis, treatment, prevention of orientobilharziasis. However, little has been done for the classification and molecular phylogeny of O. turkestanicum. Ribosomal DNA (rDNA) was generally existed in the living nature, it has numerous characters for using as the DNA molecular maker, and for example it has a lot of multicopy and different revolutionary rates in different regions; it is suitable for re-constructing the deep phylogenic trees due to its revolutionary rate is slow in the coding region of the rDNA; it is easy to contrive the universal primers, align and amplify, in hence, it is widely used as the molecular maker in the phylogenetic analysis. The ribosomal DNA contains non-transcribed spacer (NTS), internal transcribed spacer region (ITS), three ribosomal gene coding regions (18S, 5.8S and 28S), which be applied in molecular phylogeny. Since mitochondrial DNA (mtDNA) is maternal inheritance, seldom recombination between them, and one of the mitochondrial genomes may represent the whole variation, they can be used to determine the molecular phylogeny. The ITS, 28S rDNA-LSU(28S ribosomal DNA large-subunit sequences)in the ribosomal DNA and the cox1 (cytochrome c oxidase subunit 1 gene) and nad1 (nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene) genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and analyzed, and compared with other sequences of schistosome, counstucted phylogeny tree then determined the molecular phylogeny.O. turkestanicum was isolated from the portal veins or intestinal veins of cattle, sheep, cashmere goat and goat, and identified by morphology. Genomic DNA was extracted from parasites isolated from individual host by SDS-proteinase K method. The ITS, 28S rDNA-LSU, cox1 and nad1 genes were amplified by PCR and cloned into pMD18-T easy vector and then sequenced and compared with by Chromas and DNAStar software, and the RNA secondary structure of 28S-LSU were analyzed by DNAMAN software. The sequences were compared with other schistosomes published in GenBankTM using the Clustal X1.83 software and the phylogenic trees were generated using the MEGA software. Phylogenetic relationships between them were reconstructed using the neighbor-joining method (NJ) and the maximum parsimony method (MP). Sequencing results showed that ITS, 28S rDNA-LSU, cox1 and nad1 were 874 bp, 1 304 bp, 1 125 bp and 518 bp, respectively. The sequences from different definitive hosts exist nucleotide variations to some extent and the RNA secondary structure of 28S rDNA-LSU from caprine are identical or resemble, which have large variation compared with that of bovine. The phylogenetic tree revealed that O. turkestanicum is placed within the African schistosomes, and O. turkestanicum should be considered a sister species of Schistosoma spp.更多还原