采用ARDRA研究双孢蘑菇培养料后发酵过程中的细菌群落结构(Ⅰ)——细菌16S rDNA全长文库的建立  

Analysis of Bacterial Communities Present in Agaricus bisporus Phase Ⅱ Compost Using Amplified Ribosomal DNA Restriction Analysis （ARDRA） （Ⅰ）——Construction of a Bacterial 16S rDNA Library

作　　者：何丽鸿[1,2] 于荣利[1] 陈明杰[1] 潘迎捷[3] 

机构地区：[1]中华人民共和国农业部应用真菌资源与利用重点开放实验室,上海市食用菌工程技术研究中心,上海市农业遗传育种重点实验室,上海市农业科学院食用菌研究所,上海201106 [2]南京农业大学生命科学院微生物系,南京210095 [3]上海海洋大学,上海200090

出　　处：《食用菌学报》2008年第4期11-19,共9页Acta Edulis Fungi

基　　金：农业部公益性行业科研专项项目“食用菌菌种质量评价与菌种信息系统研究与建立”(编号：nyhyzx07-008)的部分研究内容

摘　　要：采用化学裂解和酶解相结合的方法,以加入PVPP的高盐缓冲液作为细胞裂解反应体系,用PEG- 8000进行DNA沉淀.从双孢蘑菇堆肥后发酵的4个代表时期培养料样品中提取微生物总DNA,再以总DNA为模板,以专用引物F27和R1498进行PCR扩增,获得16S rDNA片段,经纯化后构建4个时期样品的细菌16S rDNA文库。试验结果表明,本试验获得的总DNA质量较好。采用PCR扩增可获得多个细菌、放线菌和真菌特异片段;细菌16S rDNA文库的目的片段插入效率在90%以上。An efficient and cost effective method for obtaining good quality DNA from composts containing high levels of organic matter has been developed. The protocol consisted of sample washing with sodium phosphate and EDTA, cell wall disruption using combined chemical and enzyme treatment prior to extraction, removal of humic acid and other inhibitors in high salt buffer containing polyvinyl polypyrrolidone （PVPP）, and precipitation of DNA with polyethylene glycol （PEG-8000）. The method was used to extract 16S ribosomal DNA from four samples taken at different stages in the preparation of Phase Ⅱ compost used for Agaricus bisporus cultivation. Extracted DNA was sufficiently free of contaminants to allow PCR amplification of products derived from bacteria, actinomycetes and fungi. Purified products derived from PCR amplification of the extracted 16S rDNA using primers F27 and R1498 were used to construct 16S rDNA libraries. PCR amplification of ten randomly selected positive clones using primer M13 revealed that the 16S rDNA insertion rate was higher than 90%.

关 键 词：双孢蘑菇 培养料后发酵 细菌16S rDNA文库 

分 类 号：S646.1[农业科学—蔬菜学]