免培养法研究野生川金丝猴肠道内生细菌多样性

Culture-independent analysis of intestinal microbial diversity from Rhinopithecus roxellana

摘要:

【目的】了解野生川金丝猴(Rhinopithecus roxellana)肠道内生细菌的组成及其多样性。【方法】提取川金丝猴肠道内生细菌总DNA，选用细菌通用引物799F和1492R对总DNA进行16S rRNA基因特异性扩增，构建川金丝猴肠道内生细菌16S rRNA基因克隆文库，对阳性克隆进行限制性内切酶片段长度多态性(PCR-RFLP)分析，并对Hae Ⅲ酶切带谱菌株进行测序，构建系统发育树。【结果】根据酶切带谱分析和测序结果，将随机挑取的157个阳性克隆归为27个不同的可操作分类单元(OTUs)。系统发育分析表明这些克隆序列有62.10%属于厚壁菌门(Firmicutes)，其中包括梭菌属(Clostridium)、Cellulosilyticum属、Robinsoniella属、Anaerofustis属、Blautia属和Anaerovorax属，有37.90%属于未培养细菌。【结论】川金丝猴肠道内生细菌多样性丰富，并且可能存在新的分类单元。

Abstract:

[Objective] The composition and diversity of intestinal microbial diversity of wild Sichuan snub-nosed monkey (Rhinopithecus roxellana) were investigated. [Methods] The total DNA extracted from endophytic bacterial was performed 16S rRNA gene specific amplification by used bacteria-specific primers 799F and 1492R and constructed a clone library. Then, 16S rRNA gene phylogenetic tree was established by analyzed positive clones used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by sequenced some of the clones. [Results] The 157 clones analyzed were grouped into 27 operational taxonomic units (OTUs), where the clone distribution was as follows: 62.10% of Firmicutes which contained Clostridium, Cellulosilyticum, Robinsoniella, Anaerofustis, Anaerovorax and Blautia, 37.90% of uncultured bacteria. [Conclusion] The intestine bacterial of wild Sichuan snub-nosed monkey has a rich diversity and maybe exist new taxon.