The Key Laboratory of Marine Reservoir Evolution and Hydrocarbon Accumulation Mechanism, Ministry of Education, Beijing, China.
Appl Microbiol Biotechnol. 2010 Apr;86(4):1183-94. doi: 10.1007/s00253-009-2426-5. Epub 2010 Jan 28.
Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.
目前，分子生物学技术在土壤样品研究中取得了巨大的发展。本研究的目的是通过应用非培养技术从已知的油气田上方采集的土壤样本，改进油气微生物勘探方法。首先，基于 16S rRNA 基因克隆文库，分析了班 876 油气田上方土壤细菌的群落结构。土壤细菌群落沿深度始终存在差异；然而，DGS1 和 DGS2 两个细菌文库中均以绿弯菌门和芽单胞菌门为主，产甲烷菌较少。其次，使用基于培养的程序和基于培养的组特异性实时 PCR（RT-PCR）分别定量甲烷氧化菌的数量，其数量与一个或两个数量级的定量方差不一致。特别强调了基于甲烷氧化菌 pmoA 基因的 RT-PCR 计数优势。最后，通过构建功能基因克隆文库，分析了土壤样品中甲烷氧化菌群落的多样性和分布。克隆中 508-bp 的插入片段在系统发育上均属于甲烷氧化菌 pmoA 基因，相似度为 83%至 100%。然而，大多数相似度都低于 96%。五个甲烷氧化菌克隆文库清楚地表明，异常甲烷氧化菌（Methylosinus 和 Methylocystis）占据了研究区域。
