非培养方法解析烟草根部内生细菌的群落结构

为了解烟草根部内生细菌多样性,采用改进的CTAB法提取烟草根部总DNA,利用细菌16 S rDNA基因通用引物对烟草根总DNA进行16 S rDNA扩增,构建烟草根部内生细菌16 S rDNA克隆文库;挑取具有不同酶切谱型的克隆进行测序、比对并构建16 S rDNA基因系统发育树。构建的烟草根部内生细菌16 S rDNA克隆文库中,155个克隆分属于36个不同的分类单元,Blast结果表明,大部分克隆与已知细菌的16 S rDNA序列相似性较高,分别属于变形菌门(Proteobacteria)的Alpha、Gamma、Beta亚群,放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Fir-micutes)中的肠杆菌属(Enterobacter)、不动杆菌属(Acinetobacter)、寡养食单胞菌属(Stenotrophomonas)、假单胞菌属(Pseudomonas)、嗜甲基菌属(Methylobacillus)、食酸菌属(Acidovorax)、芽孢杆菌属(Bacillus)等16个属,其中13.5%的克隆序列与非可培养细菌(Uncultured bacteria)的相似性在93%～99%之间,假单胞菌属细菌是烟草根部内生细菌的优势种群。这些研究结果说明烟草根部存在较为丰富的内生细菌系统发育多样性,并且潜藏着未知的内生细菌资源。

Investigation of Endophytic Bacterial Community Structure within the Tobacco Roots Using Culture-independent Techniques

To the aim of study was to investigate endophytic bacterial diversity in tobacco root,to lay a foundation for furthering to tap.We investigated endophytic bacterial diversity in tobacco root from Yunnan by culture-independent method.Total DNA genome of tobacco root sample was extracted using CTAB procedure with some modifications.A pair of bacterial PCR primers was used for endophytic bacterial 16 S rDNA amplification and a clone library was constructed for the tobacco root DNA samples.Clones screened from clone library on the basis of MspⅠdigestion patterns were sequenced and compared,flowed by constructing Neighbor-Joining tree.The results show that in total 155 clones were grouped to 36 operational taxonomic units,most of the clones showed high similarity to the known cultured bacteria.Sequence analysis revealed diverse phyla of bacteria in the 16 S rDNA library,which consisted of alpha,gamma,Beta subclasses of the Proteobacteria,Actinobacteria,Bacteroides,Firmicutes and Uncultured bacteria;86.2% of the clones were highly related to the known bacteria in the genus Enterobacter,Acinetobacter,Stenotrophomonas,Pseudomonas,Methylobacillus,Acidovorax,Bacillus etc;whereas 13.5% of the clones showed higher affiliation with uncultured bacteria(93%-99% sequence similarity) and may represent novel taxa.The dominant bacterial groups of tobacco endophytic bacteria belong to Pseudomonas genera.There was abundant endophytic bacterial diversity in tobacco root from Yunnan,and there were large number of unknown endophytic bacterial groups here.