西藏扎布耶盐湖细菌多样性的免培养技术分析

Bacterial Diversity in Zabuye Salt Lake Tibet by Culture-Independent Approaches

摘要:

摘要：【目的】利用免培养技术，获得有关西藏高原高盐度、高海拔盐湖的细菌多样性认识。【方法】从西藏扎布耶盐湖沉积样品中提取微生物总DNA，利用细菌引物f530/r1492扩增16S rRNA基因，然后构建16S rRNA基因质粒文库。采用Hae Ⅲ和HhaⅠ两种内切酶对阳性克隆质粒DNA进行ARDRA分型分析，根据分型结果挑选克隆进行测序。得到它们的16S r RNA 部分序列， 根据获得的序列构建构建系统发育树。【结果】在系统发育树上，部分克隆（占总克隆数的57.14%）与已知细菌属归于同一分支，主要分布在γ-变形菌纲、α-变形菌纲、δ-变形菌纲、拟杆菌门（Bacteroidetes）、厚壁菌门(Firmicutes)和疣微菌门(Verrucomicrobia)的23个嗜盐细菌属之中。其余的克隆为未培养序列，与前者差异很大，在进化树上形成了独立的分支。【结论】研究结果显示出扎布耶茶卡湖中的细菌组成具有极其丰富的多样性。

Abstract:

Abstract: [Objective] To get knowledge about bacterial diversity in Zabuye salt lake With the culture-independent approaches. [Methods] Total DNA was extracted from sediments sample of Zabuye salt lake, Tibet. The 16S rRNA gene was amplified by PCR with Bacterial primer f530/r1492.Then the plasmid library of 16S rRNA genes was constructed. The positive clones were screened on plates with IPTG/X-gal/Ap. Amplified ribosomal DNA restriction analysis ( ARDRA) was carried out with restriction enzymes Hae III, Hha I. With the help of culture-independent approaches, DNA was directly extracted from the sediment samples of Zabuye salt lake. After construction of 16S rRNA gene library, ARDRA (amplified rDNA restriction analysis) analysis was carried out. Some clones were selected to be sequenced according to the ARDRA patterns. These partial sequences of 16S rRNA gene were used for construction of the phylogenetic tree.[Results] The phylogenetic tree showed that some clones (57.14% of total clones) belonged to 23 genera in γ-proteobacteria, ?-proteobacteria, δ-proteobacteria, bacteroidetes, firmicutes, and verrucomicrobia. The rest clones were quite different and were most related with uncultured sequences, forming special division in the tree. [Conclusion] These finding showed much prolific bacteria diversity in Zabuye salt lake.