昆明盐矿古老岩盐沉积中的原核生物多样性

Prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China

摘要:

应用PCR-DGGE和rRNA分析法研究了昆明盐矿古老岩盐沉积中的原核生物多样性。样品的细菌DGGE分析得到27条带,古菌得到18条带。样品与纯培养得到的19个属菌株的DGGE图谱对比分析发现,细菌18个属菌株,只有1个属菌株与样品中的1条带迁移位置都不一致;古菌1个属的菌株不与样品中任何条带迁移位置一致。表明纯培养所得菌株并非该环境中的优势类群。同时,建立了样品细菌和古菌的16S rDNA克隆文库,从中分别挑取36个细菌克隆和20个古菌克隆进行ARDRA分析。细菌可分为10个OTUs,其中3个OTUs是优势类群,分别占38.9%,25.0%,16.7%,其余7个OTUs各含有1个克隆。古菌分为8个OTUs,没有明显的优势类群。每个OTU的代表克隆16S rDNA序列分析表明,细菌分属3大类群:α-Proteobacteria,γ-Proteobacteria和Actinobacteria,以Pseudomonas属菌为优势,含有其它岩盐沉积中没有发现的Actinobacteria。古菌主要是Halorubrum属、Haloterrigena属菌和未培养古菌。本研究表明,昆明盐矿古老岩盐沉积具有较丰富的原核生物，但在原核生物物种组成和丰度上，免培养与此前的纯培养研究结果存在一定差异。因此，结合使用两类方法才能较全面地认识高盐极端环境微生物的多样性。

Abstract:

The prokaryotic microbial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China was investigated using PCR-DGGE and rRNA approaches. Total community DNA was extracted and purified by a direct method, which yielded amplified DNA of high molecular weight for samples. A variable region of 16S rRNA gene was then amplified by PCR with bacterial and archaeal primers and analyzed by denaturing gradient gel electrophoresis (DGGE). Twenty-seven major bands were detected in the bacterial DGGE profile of the sample, but only one band of pure culture strains of bacteria isolated from the Kunming Salt Mine matched with one band of sample. No band of pure culture strains of archaea isolated from the Kunming Salt Mine matched with 18 major bands of sample. The results indicated that most of microbes in this environment are likely uncultivable. Clones on the plate were not the predominant species in the community. Two 16S rRNA gene clone libraries (bacteria and archaea) were also constructed, and 36 and 20 clones were selected for amplified ribosomal DNA restriction analysis (ARDRA). ARDRA with enzymes AfaⅠ、HhaⅠ、HaeⅢ revealed 10 bacterial operational taxonomic units (OTUs), with three most abundant OTUs accounting for 38.9%, 25.0%, 16.7% of all the bacterial 16S rDNA clones, respectively. The remaining 7 OTUs presented at low levels, were represented by a single clone. Eight archaeal OTUs were obtained but no predominant OTUs. Some clones were sequenced and each sequence was compared with all nucleotide sequences in GenBank database. Examination of 16S rDNA clones showed that the ancient salt deposits in the Kunming Salt Mine contained a phylogenetically diverse population of organisms from the Bacteria domain with members of three major lineages represented: α-proteobacteria, γ-Proteobacteria and Actinobacteria, especially Pseudomonas. Surprisingly, we recovered a variety of sequence closely related to Actinobacteria which was not found in other salt deposits. All of archaeal clones are from Halorubrum, Haloterrigena and uncultured archaea. The results of DGGE and clone library profiling analysis both indicated that microbial community of the Kunming Salt Mine had higher diversity. In this initial survey, our polyphasic approaches demonstrated that novel and uncultured microbes thrive in the ancient salt deposits of the Kunming Salt Mine. Molecular analysis of the microbial diversity in salt deposits provides foundation for better application of microbial resources.