免培养法对大鲵肠道微生物多样性的研究

Culture-independent analysis of intestinal microbial diversity from Chinese giant salamander

【目的】了解大鲵肠道内生细菌的组成及多样性。【方法】采用美国Mo Bio公司试剂盒提取大鲵肠道内容物总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,构建大鲵肠道内容物内生细菌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HaeⅢ酶切带谱不同的菌液进行测序,构建系统发育树。【结果】根据酶切带谱分析和测序结果的不同,将随机挑取的101个阳性克隆归为28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分别属于变形菌门(Proteobacteria)、梭菌门(Clostridia)、芽孢杆菌门(Bacilli)和衣原体门(Chlamydiae)4个门。其中,变形菌门(Proteobacteria,占克隆总数的92.08%)为最优势类群。序列比对结果表明这些克隆序列分别与已报道的20个属具有较高的相似性。此外,还有一个OTU在系统发育树上形成独立分支且未能确定其分类。【结论】大鲵肠道内生细菌多样性丰富,并且可能存在新的分类单元。

[Objective] The composition and diversity of intestinal microbial diversity of Chinese giant salamander （Andrias davidianus） were investigated. [Methods] The composition and diversity of intestinal microbial diversity of Chinese giant salamander were analyzed by culture-independent method. The total DNA were extracted by Fecal DNA Isolation Kit of Mo Bio company. The 16S rRNA gene of intestinal microbial diversity of Chinese giant salamander were amplified by universal PCR primers. Positive clones were analyzed by PCR restriction fragment length polymorphism （PCR-RFLP） and the clones with unique Hae Ⅲ patterns were selected for sequencing and constructing 16S rRNA gene phylogenetic tree. [Results] A total of 101 positive clones that randomly screened from the library were fall into 28 operational taxonomic units （OTUs） based on the result of Hae Ⅲ digestion and sequencing. These clone squences were divided into 4 phyla with phylogenetic analysis, which consisted of the Proteobacteria, Clostridia, Bacilli and Chlamydiae, of which Proteobacteria accounting for 92.08% of the total number of clones was the absolutely dominant group. Sequence alignment showed these clone sequences have high similarities with 20 genus that had been reported. In addition, one OTU on phylogenetic tree is independent and failed to determine its classification. [Conclusion] The intestinal microbial diversity of Chinese giant salamander harbors a diversity endophytic bacterial and maybe existence new OTUs.