细菌聚类新方法:ddT聚类技术分析香根草联合固氮菌多样性

A novel grouping method:ddT clustering assessment of the diversity of associated nitrogen-fixing bacteria isolated from Vetiveria zizanioides

利用高分辨率的聚丙烯酰胺凝胶电泳技术,采用新型遗传分析仪Li-COR 4300L,结合测序的双脱氧链终止法原理,建立了基于16S rDNA序列T碱基分布特征的一种新的细菌多样性聚类技术——ddT聚类技术.应用该新的ddT聚类技术对分离自香根草的47株联合固氮菌多样性进行了聚类分析,获得了6个不同的主要类群,表明香根草联合固氮菌具有较大的遗传资源多样性.应用SDS-PAGE全细胞蛋白质电泳和DNA指纹图谱方法对该聚类结果进行验证,结果表明,该新聚类技术可以应用于细菌的多样性聚类分析.16SrDNA序列测定和系统发育分析表明,供试的联合固氮菌分别与γ-变形杆菌纲(γ-proteobacteria)的阴沟肠杆菌(Enterobacter cloacae),Enterobacter ludwigii,Pantoeaan anatis,恶臭假单胞菌(Pseudomonas putida),荧光假单胞菌(Pseudomonas fluorescens)和β-变形杆菌(β-proteobacteria)的中型假食酸菌(Pseudaci-dovorax intermedius),Mitsuaria chitosanitabida,越南伯克氏菌(Burkholderia vietnamiensis),佛莱辛草螺菌(Herbaspirillum frisingense)亲缘关系相近,其中肠杆菌属、伯克氏菌属和泛菌属是香根草联合固氮菌的优势类群,分别占分离菌株总数的45%,19%和13%,它们一起构成了分离菌株总数的77%.

A novel grouping method （ddT clustering） based on the T base diversity of 16S rDNA sequence of bacteria is presented by application of the high resolution of polyacrylamide gel electrophoresis and the genetic analysis system Li-COR 4300L.The novel ddT clustering method was used to group the 48 associated nitrogen-fixing bacteria isolated from Vetiveria zizanioides.Six different groups （group I to VI）,which showed the diversity of associated nitrogen-fixing bacteria isolated from the Vetiveria zizanioides,were obtained.The clustering methods of SDS-PAGE whole-cell protein patterns and DNA finger-printing patterns were also applied to verify ddT clustering results.16S rDNA sequences analysis indicated that the isolates mainly distributed in γ-proteobacteria （including Enterobacter cloacae,Enterobacter ludwigii,Pantoea ananatis,Pseudomonas putida,Pseudomonas fluorescens）,and β-proteobacteria members （Pseudacidovorax intermedius,Mitsuaria chitosanitabida,Burkholderia vietnamiensis,Herbaspirillum frisingense）.The predominant bacterial community in Vetiveria zizanioides are Enterobacter sp.,Burkholderia sp.and Pantoea sp.,which occupied 45%,19% and 13% of the total isolates respectively.