南海深海沉积物放线菌多样性分析

Actinobacterial diversity in the deep-sea sediments of the South China Sea

摘要:

【目的】免培养和纯培养相结合分析南海深海沉积物放线菌多样性。【方法】免培养方法通过提取沉积物宏基因组DNA，利用放线菌门特异性引物扩增放线菌16S rRNA基因序列，构建放线菌16S rRNA基因克隆文库，文库经RFLP (restriction fragment length polymorphism)分析后挑选代表序列测序并进行多样性指数分析和系统发育分析。可培养方法利用8种培养基进行菌株分离，对排重后的菌株进行16S rRNA基因序列多样性分析。【结果】构建的两个深海位点的16S rRNA基因克隆文库在放线菌门的放线菌纲(Actinobacteria)、酸微菌纲(Acidimicrobiia)、腈基降解菌纲(Nitriliruptoria)和嗜热油菌纲(Thermoleophilia) 4个纲中均有分布；两个位点中的种群结构有差异，N40-4位点的优势种群是放线菌纲的链霉菌目(Streptomycetales)；N63-4位点的优势种群是腈基降解菌纲的腈基降解菌目(Nitriliruptorales)。8种培养基共分离出41株放线菌，根据形态特征排重后得到的19株菌分布于10个不同的属，12个不同的种，其中稀有放线菌属比例较高，菌株OAct400为潜在的微杆菌属(Microbacterium)新种。【结论】南海深海沉积物蕴含着丰富的放线菌物种资源及大量未知种群，具有进一步研究的价值。

Abstract:

[Objective] The purpose of the study was to analyze the actinobacterial diversity in the deep-sea sediments of the South China Sea by using culture-independent and culture-dependent approach. [Methods] Actinobacteria-specific 16S rRNA gene clone library was constructed. Representative clones selected based on the restriction fragment length polymorphism analysis were sequenced and placed into operational taxonomic unit (OTU) groups according to the 16S rRNA gene sequence similarity. Diversity statistics were analyzed using SPADE analysis software. Eight media were used to isolate actinobacteria strains from 11 sediment samples. 16S rRNA gene sequences of the representatives were sequenced to analyze phylogenetic diversity of the actinobacteria. [Results] Forty and ninety-nine positive clones were obtained from the two libraries and placed into 16 and 34 OTUs respectively. Both two libraries contained class Actinobacteria, Acidimicrobiia, Nitriliruptoria and Thermoleophilia. The dominant population of site N40-4 and N63-4 were order Streptomycetales and Nitriliruptorales respectively. Forty-one strains were obtained by the culture-dependent approach. Analysis of the 16S rRNA gene sequences of 19 representative strains showed that these strains belong to 10 different genera and 12 different species. [Conclusion] The deep-sea sediments of the South China Sea harbor abundant and diverse actinobacteria. The result also implied that there may be large numbers of unknown actinobacterial groups.