【作者】 任伟；

【导师】 牛安欧；

【作者基本信息】 华中科技大学 ， 病原生物学， 2006， 硕士

【摘要】 1.目的自从证实拟钉螺可作为湄公血吸虫病、中华血吸虫病和狸殖吸虫病的传播媒介以来,拟钉螺在医学上的重要性被人们所重视,且近年来我国东南沿海及内地的部分省市并殖吸虫病有此起彼伏﹑散在发生的趋势,作为狸殖吸虫的第一中间宿主,拟钉螺的分类研究既有生物学上的理论价值,又有医学上的实用价值。至今,有报道在亚洲发现属于拟钉螺亚科的种类已逾100种,以我国为最多﹑分布最广的国家,我国以往对拟钉螺的研究多限于传统方法,在拟钉螺的分类研究中存在如种名错定、同种异名等诸多问题。近年来分子生物学的发展为物种鉴定开辟了一条新路,本研究选用了RAPD技术对我国湖北、福建和广西3省5地的6种拟钉螺进行基因水平的研究,以了解其基因组DNA的多态性差异,并构建系统进化树。此外,形态学及地理生态环境比较一直是对螺种进行鉴定的基本方法,也是进行其它研究的基础,所以本研究还将RAPD分类结果与6种拟钉螺的外部形态和生态环境进行了综合分析和初步探讨。2.方法收集我国广西省武鸣县、福建省福州市新店以及湖北省五峰县沈家堡、十堰市许家沟、钟祥市客店3省5地的6种拟钉螺。其中福建新店,五峰沈家堡,钟祥客店的拟钉螺已有文献记载,分别命名为新店拟钉螺(T. xindiangensis),秉氏拟钉螺(T. pingi),向氏拟钉螺(T. hsiangi),洪山拟钉螺(T. hongshanensis)。广西武鸣,湖北十堰的拟钉螺暂以地名命名为武鸣拟钉螺和十堰拟钉螺。1.肉眼和体视显微镜系统观察拟钉螺的外部形态并记录,了解其生态环境;2.常规高盐法抽提上述拟钉螺的基因组DNA,采用RAPD技术对其进行PCR扩增,以方格短沟蜷作为种外对照。根据RAPD扩增的结果,计算各拟钉螺种间的遗传距离和相似系数,根据各分子标记的迁移率及其有或无,统计得所有位点的二元数据,采用SPSS11.0软件的Between-groups linkage法对所得二元数据进行聚类分析,绘制系统进化树。3.结果①6种拟钉螺形态特征观察及栖息环境的比较结果:秉氏拟钉螺和向氏拟钉螺均采自湖北五峰县沈家堡,两者的生态环境完全相同,外部形态上相似处颇多。洪山拟钉螺与新店拟钉螺在外部形态和生态环境方面较为相似。而其它螺种间则相对差异较大。②DNA提取: DNA溶液A260/A280值经测定在1.60～2.00之间,纯度符合要求。③RAPD:1)种外差异6条引物对拟钉螺与方格短沟蜷的DNA扩增的条带共计87条,对应基因组中的87个位点,其中拟钉螺的条带71条,方格短沟蜷14条;两者间相同条带为5条,相似系数为0.339,遗传距离大于0.66。2)种间差异本实验拟钉螺种间遗传距离在0.1301～0.3151间,平均值为0.2248,其中秉氏拟钉螺和向氏拟钉螺的扩增带型具有较大相似性,相似系数为0.8699,遗传距离仅为0.1301;新店拟钉螺与洪山拟钉螺的扩增带型具有较大相似性,相似系数为0.8558,遗传距离仅为0.1442,此两种与十堰拟钉螺扩增条带又具有一定相似性;而武鸣拟钉螺与其它螺种间则差异较显著,遗传距离为0.2234～0.3151。3)系统进化树在SPSS构建的系统进化树中6种拟钉螺被分为4类:①秉氏拟钉螺和向氏拟钉螺;②洪山拟钉螺和新店拟钉螺;③十堰拟钉螺;④武鸣拟钉螺。4.结论根据RAPD的实验结果,3省5地的6种拟钉螺在基因水平分为4类:①秉氏拟钉螺和向氏拟钉螺;②洪山拟钉螺和新店拟钉螺;③十堰拟钉螺;④武鸣拟钉螺。6种拟钉螺在基因水平上存在差异。结合外部形态和生态环境的观察比较结果发现,此DNA水平的差异与拟钉螺外部形态、生态因素上的差异存在一定关联。本实验首次将分子生物学水平的RAPD技术应用于对拟钉螺的遗传变异的研究,认为RAPD技术能有效的区分拟钉螺种间的差异,用于拟钉螺研究是行之有效的一种方法。更多还原

【Abstract】 1. ObjectiveSince it was proved that the Tricula.sp can be the snail hosts of Schistosomiasis mekongi, Schistosomiasis sinensium and Paragonimiasis skrjabini, the significance of Tricula has been recognized. Recently, some reports said the Paragonimiasis has a resurgent trend in southeast China and some inland cities. As the first intermediate hosts of Pagumogonimus skrjabini, the classification of Tricula not only has great significance in biology, but also beneficial to the medicine practice. Until now, more than 100 kinds of species belong to Triculinae were reported in Asia and half of them located in China. The Tricula species were identified by the traditional means. But there are many disputes existed in such a classification. With the development of molecule biological, some new techniques were provided to us. The RAPD technique was used in this study to analyze the genetic variation of 6 populations of Tricula collected from 5 areas of 3 provinces. The exterior morphological features and ecological environment are fundamental factors to identify a new species, so the exterior morphological features were observed and the ecological environment were analyzed in this research also.2. Materials and methods6 tricula populations were collected from 5 areas of 3 provinces in China, such as Guangxi-Wuming, Fujian-Fuzhou, Hubei-Wufeng, Hubei-Shiyan and Hubei-Zhongxiang. Tricula species collected from Fujian-Fuzhou, Hubei-Wufeng and Hubei-Zhongxiang were reported formally and named T.xindiangensis, T.pingi, T.hsiangi and T.hongshanensis respectively. The populations collected from Guangxi-Wuming and Hubei-Shiyan was called Guangxi-Wuming isolate and Hubei-Shiyan isolate in this research. 1. Make a systematic observation of the exterior morphological features by naked eyes and stereo microscope, comparing the ecological environment of the snails and record the results. 2. High-Content-NaCl-Solution method was used to extract the genomic DNA. RAPD technique was used to amplified the genomic DNA. Then, the genetic distance & genetic similarity of the tricula from various regions were calculated according to the types of bands of the amplified products on the agarose gel electrophoresis. The phylogenetic tree was thereby mapped, and the DNA polymorphic bands detected were analyzed by clustering analysis with spss11.0 software.3. Results①Observational and comparative of exterior morphological features and ecological environments of different population of Tricula:There are much similarity between T. pingi and T. hsiangi, T. hongshanensis and T. xindiangensis in the exterior morphological features and the habitat environments. While other species show much diversity.②DNA extraction:The A260/A280 value of the DNA solution ranged from 1.60～2.00, the purity is qualified.③RAPD:1) The difference out of species: The RAPD products of 6 Tricula populations and Semisulcospira cancellata showed obvious dissimilarity. 87 bands, which were homologous to 87 loci of the genome, were amplified with 6 random primers. There are 5 bands of the 87 bands were the same between the two species. The genetic distance between them was larger than 0.66, and the genetic similarity was only 0.339.2) The difference between species: Genetic distances among the 6 Tricula populations ranged from 0.1301～0.3151, and the average was 0.2248. The type of bands of amplified products between T.pingi and T.hsiangi were much similar. The genetic similarity was 0.8699 and the genetic distance was only 0.1301.The bands type of T. hongshanensis and T. xindiangensis were much similar. The genetic similarity was 0.8558 and the genetic distance was only 0.1442. The bands type of these two snails had some similarity with Hubei-Shiyan isolate in some degree. While Guangxi-Wuming isolate had much diversity to others. The genetic distance among them ranged from 0.2234～0.3151.3) The populations of Tricula involved in this article can be divided into four groups:①T. pingi and T. hsiangi;②T. hongshanensis and T. xindiangensis;③Hubei-Shiyan isolates;④G uangxi-Wuming isolates.4. ConclusionAccording to the results of RAPD-PCR, 6 populations of Tricula involved in this article can be divided into four groups in terms of genetic level:①T. pingi and T. hsiangi;②T. hongshanensis and T. xindiangensis;③Hubei-Shiyan isolates;④G uangxi-Wuming isolates. Differences at genetic level exist in Tricula from different areas in China, such genetic differences in accord with the exterior morphological features and the ecological environment of the Tricula to some degree.This research firstly used the RAPD technique to analyze the genetic variation of Tricula. In addition, exterior morphological features were observed and the ecological environments of different population of Tricula were analyzed. The classification by the RAPD in this research was in accord with the traditional classification in principle. The RAPD technique was an effective method to analyze the genetic variation of Tricula.更多还原