垃圾填埋场渗滤液中古细菌群落16S rRNA基因的ARDRA分析

ARDRA Analysis of 16S rRNA Genes from the Archaeal Community in Landfill Leachate

利用特异性的引物对 ,选择性扩增垃圾填埋场渗滤液中古细菌群落的 1 6 S r RNA基因片断 ,在此基础上建立 1 6 Sr DNA克隆文库。经古细菌通用寡核苷酸探针的原位杂交筛选后 ,克隆文库内古细菌 1 6 S r DNA扩增片断的多样性通过ARDRA分析 ( amplified r DNA restriction analysis)而获得。利用 PCR将各重组克隆子内的 1 6 S r DNA外源片断再扩增出来后 ,两种限制性内切酶 -H ha 和 H ae -被分别用于 1 6 Sr DNA克隆片断的限制酶切分析。结果表明 ,随机选出的 70个古细菌 1 6 S r DNA克隆片断被分为 2 1个不同的 ARDRA型 (组 ) ,其中的两个优势型总共占了所有被分析克隆子的6 0 % ,而其余 1 9个型的相对丰度均处于较低的水平 ,当中的 1 4个型更仅含有 1个克隆子。通过对 1 6 Sr RNA基因的 PCR扩增、克隆及其 ARDRA分析 。

Using PCR and a pair of specific primers, archaeal small subunit 16S rRNA genes were selectively amplified from total genomic DNA extracted from the microbial community in the leachate of a full-scale sanitary landfill, and an archaeal 16S rDNA clone library was subsequently established. Clones were screened by colony hybridization with an archaeal oligonucleotide probe, and diversity of amplified 16S rDNA fragments in the library was gained by a restriction fragment length analysis (ARDRA). The cloned 16S rDNA fragments were reamplified, and restriction analysis was performed following separate digestion with enzymes Hha Ⅰ and Hae Ⅲ. A total of 70 cloned 16S rDNA fragments were analyzed, and they were finally clustered into 21 different groups (ARDRA patterns), with two most abundant groups accounting for 60% of all the 16S rDNA clones. The remaining 19 groups presented at low levels, of which a total of 14 groups were represented by a single clone. By the method of ARDRA, we could rapidly estimate the population structure of Archaea in the landfill leachate.