【作者】 王凤超； 于珊珊； 陈冠军； 刘巍峰；

【Author】 Wang Feng-Chao Yu Shan-Shan Liu Wei-Feng (Sate Key Laboratory of Microbial Technology, School of Life Science. Shandong University)

【机构】 山东大学生命科学学院微生物技术国家重点实验室；

【摘要】 微生物在生物圈中分布广泛,其种类和数量是别的生物无法比拟的.但是约99% 的微生物是不能通过传统的培养方法进行培养的.随着分子生物学在微生物研究中的运用,使我们可以初步的了解这些非可培养微生物,也促进了一门新兴学科-环境基因组学的兴起和发展.所谓的环境基因组学是指一定环境样品中所有微小生物遗传物质的总和. 人们通过构建环境基因组文库,可以从中筛选得到一些新的有生物活性的物质,及编码这种物质的基因,也可以通过克隆16SrRNA对非可培养微生物进行分类.而分子生物学在环境基因组学中的应用首先要求得到有广泛代表性的高质量的DNA样品.我们采取了各种提取和纯化的方法,得到了DNA样品,通过凝胶电泳,限制性内切酶酶切,及PCR扩增等实验,可以得出液氮研磨和CTAB抽提缓冲液相结合可以得到大量DNA样品,Sepharose 4B spin columns可以有效的去除腐殖酸等抑制限制性酶切及PCR反应的杂质.进一步利用纯化的环境样品DNA构建基因组文库筛选新功能基因及特定环境微生物菌系的分析工作正在进行。更多还原

【Abstract】 The biosphere is dominated by microorganisms, yet about 99 % of the microorganisms have not been or can not be cultured by traditional methods. With the development of molecular biology, and its application in microbiology, we have started to know some of those uncultured microorganisms, and accordingly a new subject-environmental genomics, which is the tall microbial genomics in a environmental sample, has since developed. By constructing the environmental genomics library, we can get new bioactive substance, and it’s encoding gene. We can also clone the 16SrRNA to analyze the phylogeny of related microorganisms. In this respect, high quality DNA sample need to be recovered from environmental samples without culturing microorganisms first. We compared several methods to extract and purify DNA sample from soil. By using agarose gel electrophoresis, restriction endonucleases, and PCR amplification, an extraction method based on grinding under liquid nitrogen and CTAB extraction buffer can obtain large amount of DNA. After purification with Sepharose 4B spin columns which was shown to effectively remove humic acids and other contaminants which inhibit the following restriction enzyme digestions and PCR from crude DNA samples. Further work to construct DNA libraries to screening functional genes or phylogenetically analyze microorganism constituents in specific environment is under way.更多还原