新疆断裂带含硫冷泉泉水细菌群落结构多样性

Bacterial community structure and diversity in a cold sulfur spring in Xinjiang faulting zone

【目的】为了解新疆断裂带含硫冷泉泉水中细菌群落结构的组成和物种多样性。【方法】采用免培养法直接从冷泉水中提取环境总DNA,采用细菌通用引物对泉水中细菌的16S rRNA基因进行PCR扩增,构建16S rRNA基因克隆文库。使用限制性内切酶HaeⅢ对随机挑选的阳性克隆子进行限制性片段长度多态性分析(Restriction Fragment Length Polymorphism,RFLP),选出具有不同酶切图谱的序列进行测序、BLAST比对和构建16S rRNA基因系统发育树。【结果】共从细菌16S rRNA基因文库中筛选了228个阳性克隆,RFLP分型得到33个不同的操作分类单元(Operational Taxonomic Unites,OTUs),覆盖度(Coverage C)为92%。BLAST比对、RDP归类及系统发育分析将这33个OTUs归为:变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)。变形菌门为绝对优势类群,占整个细菌克隆文库的98%,其中20%左右的类群与硫化物代谢相关的光合自养和化能自养类群纯培养菌具有高的相似性(>97%)。此外,还发现大量类群(总文库的64%,其中57%为军团菌属Legionella spp.,类群)与GenBank中已存细菌16SrRNA基因相似性小于96%。【结论】新疆断裂带含硫冷泉泉水中细菌类群的多样性较低,但可能存在大量潜在细菌新种和新分类。另外,该泉水可能是潜在的新军团菌病传播源,因而可能对下游人畜健康存在潜在威胁。

[Objective]In order to investigated composition and diversity of bacterial in a cold sulfur spring in Xinjiang faulting zone.[Methods]Environmental total DNA was directly extracted from the water of the No.10 cold sulfur spring.The 16S rRNA genes were amplified from the total DNA by PCR with bacteria-specific primers and construction a clone library.Positive clones were randomly selected from the library and identified by restriction fragment length polymorphism（RFLP）.The unique RFLP pattern corresponded sequences were sequenced,BLAST and then constructed phylogenetic tree.[Result]In total,228 positive clones were screened and grouped into 33 Operational Taxonomic Units（OTUs）.The clone coverage C value was 92%.33 Operational Taxonomic Units were divided into 3 phyla with Blast analysis and RDP classifer： Proteobacteria,Bacteroidetes,and Firmicutes.Proteobacteria（98%） was the absolutely dominant group,of which 20% of the clones were highly related to the known photoautotrophic and chemoautotrophic bacteria（ 97% sequence similarity）.Besides,64% of the clones showed less than 96% of sequence similarity with sequence deposited in GenBank database,of them 54% sequences were affiliated to genus Legionella spp.[Conclusion]Bacterial diversity in No.10 cold sulfur spring was low,but maybe have a diversity of novel species and lineages.In addition,large number of novel species of Legionella were detected in the spring water may suggest the water potentially a source of Legionnaires disease and may constitute a menace to the health of human and livestock that lived down the spring.