【作者】 赵勇；

【导师】 潘迎捷； 赵立平；

【作者基本信息】 南京农业大学 ， 微生物学， 2005， 博士

【摘要】 土壤是微生物的大本营，蕴藏着丰富的微生物资源。土壤微生物在生物地球化学循环(biogeochemical cycles，BGC)过程中扮演着非常重要的角色，对地表生态系统乃至全球都存在重要的影响。土壤微生物除了自身存在着演替与变化之外，人类的活动，如土壤改良，也直接或间接地影响着土壤微生物的多样性。但由于理论与方法的限制，土壤微生物一直被认为是研究中的黑匣子(black box)。如何打开这个黑匣子，充分开发利用这丰富的土壤微生物资源，为人类、为社会造福，是摆在广大微生物学家面前的一个重要课题。传统的，针对土壤微生物的研究，大都是基于培养分离的方法。然而，近年来的许多研究表明，土壤中只有不到1％的微生物是可以在实验室条件下培养的，绝大部分的微生物在目前条件下还未能培养(uncultured)。突破传统的培养方法，发展不依赖于培养的微生物分子生态学方法，从根本上克服了传统培养方法的局限性，把人们的研究视野拓展到极其丰富的未培养微生物的世界。这一研究方法和理念在二十世纪八十年代中期被Pace及其同事提出来后，经过一段时期的发展，到二十世纪九十年代，即被广泛地应用到土壤微生物学的研究领域，为土壤微生物学这门古老而传统的学科注入了新的活力，并因此而逐渐形成了一个新的研究方向——土壤微生物分子生态，目前它的发展正方兴未艾。其基本理论基础就是，直接从土壤样品中提取微生物DNA，以基因(组)的多样性反映物种多样性，从而可以比较全面客观地展现土壤中微生物组成和多样性。 一、土壤微生物分子生态学研究中总DNA的提取 作为土壤微生物分子生态学方法的基础，最重要的一步就是从土壤样品中尽量毫无偏差地提取出高质量的、具有代表性的微生物总基因组DNA。本研究发展了一种土壤DNA提取方法，能从土壤中同时有效提取细菌和真菌总基因组DNA。根据DNA产量和纯度两个评价指标，从3种手提土壤DNA方法中优选出方法B为手提DNA方法(Lab method)，它包括样品预处理，细胞裂解，粗DNA纯化，其中细胞裂解组合了玻璃珠击打，SDS裂解，溶菌酶裂解。进一步应用PCR-RFLP及PCR-TGGE技更多还原

【Abstract】 Soil seems to be a microbial rain forest, and there stores plentiful resources of microorganisms. Soil microorganisms play pivotal roles in various biogeochemical cycles (BGC), and also influence aboveground ecosystems or even the planet. There are self-successions in soil microbial communities. While many anthropogenic activities, such as soil amendment practice can potentially affect soil microbial diversity. Our knowledge of soil microbial diversity is limited in part by our inability to study soil microorganisms. So soil microbial diversity is believed to be a black box. How to open the black box and sufficiently explore and make use of the plentiful resources of soil microorganisms for blessing the whole human being and society, is one of the important tasks for microbiologists.In general, soil microbial communities have often been studied by conventional methods based on cultivation and isolation techniques. Recently, it is well known that no more than 1% of the microorganisms existing in soil can be cultured under laboratory conditions, and the most remains are uncultured. Broken through the traditional cultivation techniques, microbial molecular ecology techniques based on cultivation-independent methods are developed, and they can overcome the drawbacks of the conventional techniques. Those techniques enlarged the research area to the uncultured microbial world. By the mid-1980s, Pace and his colleagues highlighted the idea of the need for nontraditional techniques to understand the microbial world. In 1990s, those techniques were used rapidly to study soil microbial diversity, and this activated soil microbiology. As a result, a new area, soil microbial molecular ecology, came into being. All in all, the idea is based on the fact that the diversity of microbial genomic DNA extracted directly from soil can reflect the diversity of microorganisms, and this can give a more real and更多还原