【摘要】 对可能代表活性污泥中两个时期的主要苯酚降解菌群的 ERIC- PCR指纹图谱中的两条 2 .1 kb的条带 ( P1和 P8)中的 DNA片段进行克隆、转化 ,得到 1 93个转化子。 H inf I物理图谱分析得到 35种酶切类型 ,将两端各进行 1次测序后 ,同源性大于 90 %的克隆归为一类 ,共得到 1 0种序列类型。对各类型的代表克隆进行了全长测序 ,5种类型为 P1条带所有 ,3种类型为 P8所有 ,二者共享的类型为 2种 ,丰度最高的片段为 S3,P1条带中 76.3%的转化子和 P8条带中 66.7%转化子属该类型。对所得序列进行检索分析 ,它们与 Gen Bank中已知基因序列均无显著同源性。用 S3特异性引物对活性污泥样品及其它在 LB、d CGY、MP和 FWM培养基上的回收菌进行扩增 ,除 LB上的回收菌没有显示目的条带外 ,活性污泥和其回收菌中均检测到带有该基因组片段的菌种的存在。研究为专一性分离降酚活性污泥中的优势菌提供了分子探针更多还原

【Abstract】 One 2.1 kb signature DNA band whose intensity was correlated to phenol degrading efficiency of activated sludge was identified from ERIC PCR DNA fingerprints after one month monitoring of structural dynamics of microbial communities in a phenol degrading activated sludge system. The DNA fragments from the signature bands (P1and P8) of ERIC PCR profiles at first and eighth monitoring stage (three weeks apart) were eluted from the agarose gel and cloned into pGEM T Easy vector. A total of 193 transformants were obtained and physically mapped with Hinf I into 35 groups. One sequence reaction from each end of 35 representative clones was undertaken. The 35 physical mapping groups were grouped into 10 types after clones sharing 90% sequence homology were considered as the same type. Each representative clone from the ten groups was fully sequenced. It was found that the 10 representative DNA fragments showed no significant homology with all known sequences in the database after BLAST analysis against GenBank. Two types of fragments were shared between the P1 and P8 bands, while 5 types were only found in P1, other 3 only in P8. The change of DNA fragment composition in the two bands was a reflection of the dynamics of population composition in the phenol degrading consortium. Phenol removal efficiency remained similar between the two stages but population structures changed as a response to changes of pollutant concentrations in the feeding water. S3 was the most abundant fragment in all groups, accounting for 76.3% of all clones from P1 and 66.7% of that from P8 respectively. The specific amplification of DNA bands with S3 specific primers showed that the corresponding functional bacteria were detected in the activated sludge from M1 stage and its recovered colony mixtures from dCGY、MP and FWM media of the same sludge sample, but not from LB medium. Probes and primers designed to specifically detect the presence of the S3 containing bacteria can thus be used to isolate the potentially functional bacteria to realize the so called “sequence guided isolation” strategy.更多还原