【作者】 陈娟；

【导师】 杨红；

【作者基本信息】 华中师范大学 ， 微生物学， 2011， 硕士

【摘要】 木食性白蚁作为自然界木质纤维素的高效降解者,在能源日益缺乏的当今世界受到了科学界的广泛关注。木食性白蚁肠道中栖息着众多的共生微生物,这些微生物与白蚁的共生关系与共生机制长期以来一直是人们非常感兴趣的课题,对其共生关系的研究和探讨对深入了解木食性白蚁降解纤维素的机制,开发与利用有益的微生物资源于新型的生物质能源产业具有积极的意义。本文分别以黑胸散白蚁(Reticulitermes chinensis Snyder)和象白蚁Nasutitermessp.为对象研究了一种低等木食性白蚁和一种高等木食性白蚁肠道内共生细菌的多样性,并对结果进行了比较与分析,为进一步探讨共生细菌在不同类群木食性白蚁肠道中的作用与功能,阐明白蚁降解纤维素的机制奠定了基础。主要研究结果如下：1.以象白蚁Nasutitermes sp全肠DNA为模板,细菌16S rRNA基因的通用引物为引物扩增并构建了象白蚁肠道内共生细菌的16S rRNA基因文库。从随机挑取的419个克隆中得到73个细菌16S rRNA基因。对这些共生细菌16S rDNA的系统发育分析表明,象白蚁肠道内共生细菌主要有螺旋体(65.8%)、嗜纤维杆菌(16.5%)、拟杆菌(4.5%)这几大类,另外还包括一些浮霉菌,厚壁菌以及一些不能确认门类的非培养细菌。2.以黑胸散白蚁全肠DNA为模板,细菌16S rRNA基因的通用引物为引物扩增并构建了黑胸散白蚁肠道内共生细菌的16S rRNA基因文库。从随机挑取的602个克隆中得到150个细菌16S rRNA基因。对这些共生细菌16S rDNA的系统发育分析表明,黑胸散白蚁肠道内共生细菌主要类群有螺旋体(42.3%)、白蚁菌群1(19.3%)、变形菌(10.6%)、厚壁菌(7.1%)和拟杆菌(6.8%),此外,还包括一些浮霉菌和放线细菌。3.对黑胸散白蚁肠道内的好氧及兼性厌氧菌进行了MPN的测定,结果表明该白蚁肠道内好氧与兼性厌氧细菌的数量是9.25×105每条。4.对两个细菌16S rRNA基因文库克隆的Rarefaction分析表明,从两个文库所选的克隆数都能够较好地覆盖细菌的多样性。本研究的结果表明,象白蚁与黑胸散白蚁肠道内共生细菌的多样性与种群结构有明显的差异,推测这种差异与白蚁肠道内鞭毛虫的有无、消化道生理条件以及白蚁的代谢需要等生物学特性相关。更多还原

【Abstract】 Wood-feeding termites are one of the decomposers that can degrade cellulose effectively. As a typical symbiotic system in nature the symbiotic relationship between termites and their intestinal microorganisms are fasinating topics for researchers in the world all the time. The studies on the symbiotic microorganisms are not only useful for the declaration of the symbiotic mechanisms between termites and their gut microorganisms but also the declaration of mechamisms of cellulose degradation.In this paper, we studied the diversity of symbiotic bacteria in the gut of two kinds of wood-feeding termites:Reticulitermes chinensis Snyder that represents wood-feeding lower termites and Nasutitermes sp. that represents wood-feeding higher termites. The results form a basis for further studies on the clarification of the mechanisms of cellulose degradation in the future. The main results are as following:1. A bacterial 16S rRNA gene library was established from the PCR product amplified from the DNA extracted from the whole gut of Nasutitermes sp.with the universal bacterial 16S rRNA gene primers. In total 73 bacterial 16S rRNA genes were obtained from 419 random selected clones. Phylogenetic analysis of these 16S rRNA genes showed that the most dominant symbiotic bacterial group in the gut of Nasutitermes sp was Spirochaetes, which occupied 65.8% of the total bacterial clones. Fibrobacteres and Bacteroidales were also major bacterial groups that occupied 16.5% and 4.5%, respectively in total bacterial clones. In addition to these, there are some clones affiliated with Planctomycetes and Firmicutes.2. A bacterial 16S rRNA gene library was established from the PCR product amplified from the DNA extracted from the whole gut of Reticulitermes chinensis Snyder.with the universal bacterial 16S rRNA gene primers. In total 150 bacterial 16S rRNA genes were obtained from 602 random selected clones. Phylogenetic analysis of these 16S rRNA genes showed that the major symbiotic bacterial clones in the gut of Reticulitermes chinensis were affiliated with Spirochaetes (42.3%), Termite group 1 (19.3%), Proteobacteria (10.3%), Firmicutes (7.1%) and Bacteroidales (6.8%). In addition to those, there were still bacteria affiliated with Planctomycets and Actinobacteria, indicating a high baceterial diversity in the gut of Reticulitermes chinensis.3. MPN counting of the intestinal aerobic and facultive anaerobic bacteria in the gut of Reticulitermes chinensis showed that the number of aerobic and facultive anaerobic bacteria in every termite was 9.25 X 105 cells.4. Rarefaction analysis of the bacterial clones obtained from the two bacterial 16S rRNA genes showed that the number of selected clones were enough to cover the diversity of bacteria in the two termite guts, indicating the reliability of the diversity analysis.The results of this study showed that there were significant differences between the bacterial diversity and community structure in the gut of Nasutitermes sp.and Reticulitermes chinensis. In comparison with the bacterial diversity reported from the other termites, we speculated that the diversity and community structure of symbiotic bacteria in the intestinal tract of termites may be associated with the physiological conditions of digestive tract and the metabolic requirements of the termites, as well as whether or not exist flagellates in the gut of host termites.更多还原