可可西里土壤样品中细菌多样性的分析

Diversity of culturable and unculturable bacteria in soil samples from Hoh Xil, China

摘要:

本实验采用纯培养和免培养相结合的方法对来源于可可西里的一份土壤样品中的细菌多样性进行了初步研究。纯培养实验使用了6种分离培养基, 共得到细菌19株, 其中放线菌7株, 非放线菌细菌12株。这些菌株分别属于叶杆菌属(Phyllobacterium)、贪食菌属(Variovorax)、假单胞菌属(Pseudomonas)、链霉菌属(Streptomyces)、小月菌属(Microlunatus)、原小单胞菌属(Promicromonospora)、韩国生工菌属(Kribbella)和芽孢杆菌属(Bacillus) 8个属。免培养分析采用基于通用引物PCR 扩增的细菌16S rRNA 基因文库的方法以及变性梯度凝胶电泳(DGGE)。16S rRNA基因文库分析结果表明, 该土壤样品细菌群落可划分为19个OTUs, 分属于5个不同纲, 优势顺序为β-Proteobacteria (75%), α-Proteobacteria (9%), γ-Proteobacteria (7%), Actinobacteria (7%), Firmicutes (2%)。变性梯度凝胶电泳分析表明, 该样品细菌多样性指数Shannon-wiener index为2.68, 表明其中微生物多样性较低, 这可能和其所处的极端环境有一定关系。比较纯培养和免培养的实验结果发现, 土壤中的一些优势细菌并没有被有效地分离, 需要在针对特定微生物设计特定培养基及培养条件进行选择性分离上做更多的探索研究。

Abstract:

Hoh Xil that is situated at the Yushu Tibetan Autonomous Region in southwest Qinghai, is one of the most primitive and well-preserved natural environment in the world. The microbial diversity in this area is largely unknown till date. In the present study an attempt has been made to explore the microbial diversity using both of cultivation-dependent and independent approaches. In the culture-dependent experiment, 19 isolates were obtained from the soil sample IMB08-049. 16S rRNA gene sequence analysis showed that the 19 strains belonged to 8 different genera, including Phyllobacterium, Variovorax, Pseudomonas, Streptomyces, Microlunatus, Kribbella, Promicromonospora and Bacillus. Denaturing gradient gel electrophoresis (DGGE) analysis revealed 17 V3 fragments of 16S rRNA gene from sample IMB08-049. Biodiversity was also assessed by amplified ribosomal DNA restriction analysis (ARDRA), DNA sequencing and phylogenetic analysis, respectively. 19 operational taxonomic units (OTUs) were clustered in the following phyla including Proteobacteria (91%), Actinobacteria (7%) and Firmicutes (2%). Comparison of the results from culture-dependent and culture-independent suggests that some dominant species were not isolated using the above culture-dependent method, and that new isolation method should be developed to discover more bacteria exciting.