红豆杉免培养内生细菌多样性的初步研究

Culture-independent analysis of endophytic bacterial diversity in Taxus chinensis

摘要:

【目的】了解红豆杉(Taxus chinensis)内生细菌的组成及多样性。【方法】提取红豆杉组织总DNA, 选用细菌通用引物799F和1492R对总DNA进行16S rDNA特异性扩增, 构建红豆杉内生细菌16S rDNA克隆文库, 对阳性克隆进行PCR-RFLP (限制性内切酶片段长度多态性)分析, 并对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158个阳性克隆归为26个不同的可操作分类单元(OUTs), 系统发育分析表明这些克隆序列分别属于变形菌门(Proteobacteria, 包含Alpha、Beta、Gamma、Delta亚群)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes) 4个门。其中, 变形菌门(Proteobacteria, 占克隆总数的58.86%)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OUTs在系统发育树上形成独立分支且未能确定其分类。【结论】红豆杉内生细菌多样性丰富, 并且可能存在新的分类单元。

Abstract:

[Objective] We investigated the composition and diversity of endophytic bacterial in Taxus chinensis. [Methods] The composition and diversity of endophytic bacterial in Taxus chinensis were analyzed by culture-independent method. When total community DNA were extracted, a pair of bacterial PCR primers were used for endophytic bacterial 16S rDNA gene amplification and a clone library was constructed for the Taxus chinensis DNA samples. Positive clones were analyzed by PCR restriction fragment length polymorphism (PCR-RFLP) and the clones with unique patterns were selected for sequencing and constructing 16S rRNA gene phylogenetic tree. [Results] Based on Hae Ⅲ digestion and sequencing result, a total of 158 positive clones that randomly screened from the library were fall into 26 operational taxonomic units (OTUs). These clone squences were divided into 4 phyla with phylogenetic analysis, which consisted of Alpha, Beta, Gamma and delta subclasses of the Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, of which Proteobacteria accounting for 58.86% 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 OUTs on phylogenetic tree is independent and failed to determine its classification. [Conclusion] Taxus chinensis harbors a diversity endophytic bacterial and maybe existence new taxon.