Abstract:

A bead beating plus column(RBB+C) method has been performed to extract and purify metegenomic DNA from the organophosphorus (OP) polluted soil and activated sludge. According to the conservative domains of the organophosphorus hydrolase, the degenerate primers were designed to amplify the partial organophosphorus hydrolase genes. The DNA library of the partial organophosphorus hydrolase genes was constructed by pEASY-T3. The DNA sequences of the positive clones selected randomly from the library were analyzed to evaluate the diversity of organophosphorus hydrolase genes in polluted soil and activated sludge. The results showed that in DNA library of the partial organophosphorus hydrolase genes, 88% from the OP-polluted soil and 97% from the activated sludge shared 44%~65% and 23%~77% similarities respectively with genes reported in GenBank. It indicated that there were diverse and novel organophosphorus hydrolase genes in OP-polluted soil and activated sludge. The microorganism diversity was also studied by PCR-DGGE.

Key words: organophosphorus pesticide pollution, soil, activated sludge, DNA fragment library, PCR-DGGE

摘要：

采用珠磨法直接从受有机磷污染的土壤和活性泥样品中提取和纯化宏基因组DNA。根据有机磷降解酶基因保守区设计引物,从宏基因组中扩增有机磷降解酶基因片段。回收扩增产物后,构建了以pEASY-T3为载体的有机磷降解酶基因片段文库。从文库中随机挑选克隆进行DNA序列测定,并分析测序结果,评价有机磷污染土壤和活性泥中有机磷降解酶基因的多样性和新颖性。结果显示,88%土壤来源序列与GenBank中同类酶序列相似性在44%~65%之间,97%活性泥来源序列与GenBank中同类酶的相似性在23%~77%之间,且序列多样性极为丰富,显示在有机磷污染土壤和活性泥中含有丰富和新颖的有机磷降解酶基因资源。同时采用PCR-DGGE的方法对有机磷污染土壤和活性泥中微生物多样性进行了初步研究。

关键词: 有机磷污染；土壤；活性泥；基因片段文库；PCR-DGGE