不同16S rDNA靶序列PCR-DGGE分析油页岩细菌多样性

Analysis of Bacterial Diversity in Oil Shale by PCR-DGGE with Different 16S rDNA Target Sequences

为全面了解油页岩细菌群落组成结构,同时筛选出适于油页岩PCR-DGGE的最佳引物,采用改进SDS高盐提取法,提取抚顺西露天矿油页岩微生物总DNA,并用968F/1401R,338F/518R,341F/907R和1055F/1406R,对16S rDNA V6-V8,V3,V8和V9区进行PCR-DGGE分析.结果表明,4组引物均能较好扩增出目的基因,但不同靶序列对多样性检出有显著影响(P<0.001);与其他引物相比,968F/1401R和338F/518R获得的指纹图谱物种丰富度更高,检出细菌类群更丰富,较适合油页岩样品.油页岩细菌群落组成较简单,优势细菌包括不动杆菌属、假单胞菌属和埃希氏菌属,同时还有假单胞菌目和肠杆菌目尚未被鉴定的一些种类.

For a comprehensive understanding of bacterial community structure of the oil shale from Fushun west open-pit mine in China and screening out best prime for PCR-DGGE analysis of oil shale,an improved SDS-high-salt extraction method was used to extract microbial total DNA from the oil shale.Four-set primers（968 F /1401R,338 F /518R,341 F /907R and 1055 F /1406R）of 16 S rDNA high variable target regions,V6-V8,V3,V8,V9,were compared to obtain the optimal target sequences suitable for PCR-DGGE.The results from PCR-DGGE patterns showed that four-set primers can amplify the target sequences,but different target sequences of primers have a significant effect on the detection of the bacterial diversity（P〈0.001）.Compared with the other primers,968 F /1401R and 338 F /518R are more suitable for the bacterial diversity analysis of oil shale samples due to higher finger-print species richness and abundant bacteria group obtained from their PCR-DGGE.The community structure of bacteria in oil shale is not much rich.Acinetobacter,Pseudomonas and Escherichia are the dominant bacterial communities,as well as some unidentified bacterium which belong to Pseudomonadales and Enterobacteriales.