红壤荒草地氨氧化细菌富集液16S rDNA文库的RFLP分析  

RFLP ANALYSIS OF AOB-SPECIFIC 16S rDNA LIBRARY FROM RED SOIL ENRICHMENT CULTURE

作　　者：张丽娜[1] 曹慧[1] 崔中利[1] 孙波[2] 李大力[3] 李顺鹏[1] 

机构地区：[1]南京农业大学农业部农业环境微生物工程重点开放实验室,南京210095 [2]中国科学院南京土壤研究所,南京210008 [3]南京理工大学化工学院,南京210094

出　　处：《土壤学报》2006年第4期635-641,共7页Acta Pedologica Sinica

基　　金：国家自然科学基金项目(40371069);中国科学院知识创新工程项目(KZCX3-SW-427);中国博士后科学基金项目(2003033495)资助

摘　　要：分析红壤荒草地富集液中氨氧化细菌的种群组成,选取氨氧化细菌16S rDNA特异性引物序列,利用PCR技术对从富集液中抽提的细菌总DNA进行扩增,并建立了氨氧化细菌特异性的16S rDNA文库。用酶HhaⅠ和RsaⅠ对该文库特异性片段进行了限制性酶切片断长度多态性分析(Restriction fragment lengthpolymorphism,RFLP),随机挑选的35个特异性克隆片段被分成3个不同的RFLP类型,其中优势型占了所有分析克隆子的94%,另两个型各占3%。从每个RFLP类型中挑取一定的转化子进行测序,测序结果经GenBank检索,发现在该富集液体系文库中存在大量亚硝化单胞菌属(Nitrosomonas)细菌序列,由此推测红壤荒草地中存在氨氧化细菌,Nitrosomonas属细菌能在富集条件下成为优势菌。Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. In order to analyze the ammonia-oxidizing bacteria population structure in red soil enrichment culture, a pair of AOB-specific primers was designed, and total microbial genomic DNA of enrichment culture was extracted by the direct method of PCR-amplification template. An AOB-specific 16S rDNA clone library was subsequently constructed. RFLP analysis of 16S rDNA was conducted using enzymes （ Hha Ⅰ and Rsa Ⅰ ） to digest 16S rDNA from the library. The randomly selected 35 cloned fragments were finally clustered into 3 different phylotypes, with the most abundant phylotype accounting for 94% of the selected 16S rDNA clones and the other two phylotypes for 3 %, respectively. Seven clones from 3 phenotypes were sequenced and the 16S rDNA sequences were compared with those from Gen- Bank. Based on the Blast analysis, a lot of Nitrosornonas-like sequences were found in this library. The result shows that there are ammonia-oxidizing bacteria in the red soil and Nitrosomortas sp. is a dominant bacterium in enrichment condition.

关 键 词：氨氧化细菌 16S rDNA基因文库 富集液 RFLP 红壤 

分 类 号：S154.34[农业科学—土壤学] Q938.1[农业科学—农业基础科学]