一平浪盐矿古老岩盐沉积中可培养细菌的系统发育多样性研究

Phylogenetic diversity of culturable bacteria in the ancient salt depositsof the Yipinglang Salt Mine,P.R.China

摘要:

运用纯培养法和基于16S rRNA基因序列的系统发育分析对云南省一平浪盐矿古老岩盐沉积中可培养细菌的多样性进行了研究。用补充0.5～3.5mol/L NaCl的MBA和ISP 2琼脂培养基从卤水、岩盐和盐土样品中分离到38株细菌，用细菌通用引物进行16S rRNA基因扩增和序列测定，用相关软件进行序列相似性搜索、比对和系统发育分析。结果表明，38个分离菌株可分为31个物种，属于4个大的系统发育类群(Proteobacteria，Bacteroidetes，Firmicutes，Actinobacteria)、17个科、24个属。多数菌株属于Proteobacteria门（18株，47.3%；Gamma-Proteobacteria，31.5%；Alpha-Proteobacteria，15.8%）和Firmicutes门（13株，34.2%）。这些分离菌株中，至少有3个菌株可能代表3个不同属的3个新物种：Y3、Y15和Y25分别代表Idiomarina属、Salinicoccus属和Saccharospirillum属的新物种；而菌株Y21有可能代表Staphylococcaceae科的一个新属。从以上结果可以看出，一平浪盐矿古老岩盐沉积中存在较为丰富的微生物物种多样性和系统发育多样性，并且潜藏着新的微生物资源。

Abstract:

The microbial diversity of cultivable bacteria，isolated from the ancient salt deposits from the Yipinglang Salt Mine（YPL）in the Yunnan Province，P. R. China，was investigated by using conventional culture-dependent method and phylogenetic analyses based on 16S rRNA gene sequence comparisons. 38 bacteria strains were isolated from the brine，halite and saline soil samples on MBA (marine broth agar 2216，Difco) and ISP 2 (International Streotomyces Project medium 2) media supplemented with 0.5～3.5mol/L NaCl. The genomic DNAs of the isolates were extracted and their 16S rRNA genes were amplified by PCR using bacterial universal primers. The resulting 16S rRNA gene sequences were compared with sequences obtained from public databases to find the most closely related species. Phylogenetic analyses were performed using the software packages MEGA after multiple alignment of sequence data by CLUSTAL X. The evolutional instances (corrected by Kimura's 2-parameter model) were calculated and clustering was performed with the neighbor-joining method. The results showed that the isolates are members of twenty-four genera (Acinetobacter，Agromyces，Arthrobacter，Bacillus，Brevundimonas，Chromohalobacter，Dietzia，Erythrobacter，Exiguobacterium，Halomonas，Idiomarina，Kocuria，Marinobacter，Micrococcus，Paracoccus，Planomicrbium，Porphyrobacter，Pseudomonas，Psychrobacter，Roseivivax，Saccharospirillum，Salegentibactor，Salinicoccus，Streptomyces) of seventeen families (Alteromonadaceae，Bacillaceae，Caulobacteraceae，Flavobacteriaceae，Halomonadaceae，Idiomarinaceae，Microbacteriaceae，Micrococcaceae，Moraxellaceae，Planococcaceae，Pseudomonadaceae，Rhodobacteraceae，Dietziaceae，Saccharospirillaceae，Sphingomonadaceae，Staphylococcaceae，Streptomycetaceae) in four major phylogenetic groups (Actinobacteria，Bacteroidetes，Firmicutes，Proteobacteria). The most abundant and diverse isolates were within the phyla of Proteobacteria（47.3%；Gamma-Proteobacteria，31.5%；Alpha-Proteobacteria，15.8%）and Firmicutes（34.2%）. The phylogenetic distance matrix results suggested that out of 38 isolates 32 are different strains of 27 known species，and that at least 3 stains represent new species within 3 characterized genera. Y3 (Accession No. EF177665) and Y25 (EF177670) represent new species of the genera Idiomarina and Saccharospirillum，respectively. Y15 (DQ837380)，Y16 (EF177680) and Y22 (EF177689) represent a new species of the genus Salinicoccus. And strain Y21 (EF177692) may represent a novel species of a possible new genus of the family Staphylococcaceae. The results presented above shown that there are abundant bacterial species diversity and phylogenetic diversity in the ancient salt deposits from the Yipinglang Salt Mine.