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【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。
【Objective】 To understand the composition and diversity of endophytic bacteria in Taxus chinensis. 【Method】 Total DNA was extracted from Taxus chinensis. 16S rDNA specific amplification of total DNA was performed using bacterial universal primers 799F and 1492R. The 16S rDNA clone library of endophytic bacterium Taxus was constructed and the positive clones were subjected to PCR-RFLP Enzyme fragment length polymorphism) analysis, and digested with different bands were sequenced to establish phylogenetic tree. 【Result】 Based on the results of restriction enzyme digestion and sequencing, 158 randomly selected positive clones were classified into 26 different operable taxonomic units (OUTs). Phylogenetic analysis showed that these cloned sequences belong to Proteobacteria Proteobacteria (Alpha, Beta, Gamma, Delta subgroups), Firmicutes, Actinobacteria and Bacteroidetes. Among them, Proteobacteria (58.86% of the total number of clones) was the most dominant group. Sequence alignment results showed that these cloned sequences have higher similarity with the reported 20 genera. In addition, there is also an OUTs that form independent branches in the phylogenetic tree and are unable to determine their classification. 【Conclusion】 Endophytic bacteria are abundant in Taxus chinensis and may have new taxonomic units.