新疆野生胀果甘草内生细菌多样性的非培养初步分析

Endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang by culture-independent method

摘要:

摘要：【目的】了解新疆野生甘草内生细菌多样性，为开发新的微生物资源奠定基础。【方法】采用改进的CTAB (十六烷基三甲基溴化铵)法提取新疆野生胀果甘草根部总DNA，利用细菌16S rDNA 基因通用引物对甘草总DNA 进行16S rDNA 基因扩增，构建甘草内生细菌16S rDNA基因文库；挑选具有不同酶切图谱的克隆进行测序、比对并构建16S rDNA 基因系统发育树。【结果】构建的甘草内生细菌16S rDNA基因文库中, 150个克隆分属于32个不同的分类单元，Blast结果表明大部分克隆与已知细菌的16S rDNA基因序列相似性较高，分别归属于变形杆菌门(Proteobacteria)的alpha、gamma亚群，厚壁菌门(Firmicutes)，放线菌门(Actinobacteria)，拟杆菌门(Bacteroidetes)中的鞘脂菌属(Sphingobium)，叶杆菌属(Phyllobacterium)，生丝单胞菌属(Hyphomonas)，土壤杆菌属(Agrobacterium)等14个属, 其中26%的克隆与已知细菌16S rDNA 基因相似性小于96%，可能代表新的分类单元.【结论】甘草内生细菌多样性丰富且存在尚未被认识的新物种。

Abstract:

Abstract: [Objective] We investigated endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang. [Methods] We investigated endophytic bacterial diversity in root of Glycyrrhiza inflata Bat. from Xingjian by culture-independent method. Total DNA genome of Glycyrrhiza sample was extracted using CTAB (Hexadecyl trimethyl ammonium Bromide) procedure with some modifications. A pair of bacterial PCR primers were used for endophytic bacterial 16S rDNA gene amplification and a clone library was constructed for the Glycyrrhiza DNA samples. Clones screened from clone library on the basis of Hae III digestion patterns were sequenced and compared, flowed by constructing Neighbor-Joining tree. [Results] In total 150 clones were grouped to 32 operational taxonomic units, most of the clones showed high similarity to the known cultured bacteria. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, gamma subclasses of the Proteobacteria, Bacteroides, Firmicutes, Actinobacteria, and Uncultured bacteria; 74% of the clones were highly related to the known bacteria in the genus Sphingobium, Phyllobacterium, Hyphomonas, Agrobacterium etc (>96% sequence similarity); whereas 26% of the clones showed lower affiliation with known genus (<96% sequence similarity) and may represent novel taxa. [Conclusion] There was abundant endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang as well as many unknown organisms.