新疆沙湾冷泉沉积物中免培养古菌多样性初步研究

【目的】了解新疆沙湾冷泉沉积物的古菌组成及多样性。【方法】采用免培养法,液氮研磨提取冷泉沉积物总DNA,使用古菌通用引物进行16S rRNA基因扩增,构建16S rRNA基因文库。对阳性克隆进行HhaI限制性酶切分型,选出具有不同酶切图谱的序列进行测序,将所得序列与GenBank数据库中序列比对并构建16S rRNA基因系统发育树。【结果】从冷泉沉积物古菌16S rRNA基因文库中随机挑选了121个阳性克隆,共得到22个不同的可操作分类单元,BLAST结果表明全部克隆子归属于泉古菌门(Crenarchaeote)中免培养类群。系统发育分析归类为Soil-Freshwater-subsurface group和MarinegroupI,2个亚群并且各占整个文库的50%。其中40%左右的克隆子与具有无机碳和硝酸盐同化能力的泉古菌有高的相似性。此外还发现40%的克隆子与低温泉古菌类群具有很高的相似性。【结论】新疆沙湾冷泉沉积物中古菌类群多样性较低,但存有大量高度适应此低温、贫营养环境的泉古菌类群。

Uncultured archaeal diversity in cold spring sediment of Shawan, Xinjiang, China

Abstract: [Objective] We surveyed the composition and diversity of archaea in Xinjiang Shawan cold spring sediment. [Methods] We studied the archaeal diversity in the cold spring sediment by direct extracting environmental DNA with liquid nitrogen grinding method and constructing clone libraries of 16S rRNA gene amplified with archaeal-speci?c primers. Amplified 16S rRNA gene fragments were analyzed by Restriction Fragment Length Polymorphism (RFLP), and the unique RFLP patterns were selected for sequencing, alignment and constructing 16S rRNA gene phylogenetic tree. [Results] A total of 121 positive clones were randomly screened from the library and 22 Operational Taxonomic Units (OTUs) were determined. BLAST analysis indicated that all OTUs were affiliated with the phylum Crenarchaeota. Phylogenetic analysis classified them into two subgroups of Soil-Freshwater-subsurface group, and Marine group I and represented 50% of total clones, respectively. Of them, clones with the potential to assimilate nitrate accounted for 40% of the total archaeal clones. In addition, 40% of clones were related to the cold-loving Crenarchaeota. [Conclusion] These results suggested that the spring sediment harbors a low diversity of archaea, however, a large fraction of Crenarchaeota indigenous species might exist and well adapted to the cold and oligotraphic environment.