四川白三叶根瘤菌遗传多样性及系统发育研究

Genetic diversity and phylogeny of rhizobia isolated from white clover in Sichuan Province

为阐明四川部分地区野生白三叶根瘤菌的遗传多样性及系统发育地位,对分离自四川雅安、康定、泸定、西昌、成都和乐山6个地区白三叶根瘤的69株菌进行系统研究。采用16SrDNA限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP)和16SrDNA基因、持家基因(recA、atpD、glnII)、结瘤基因(nodC)、固氮基因(nifH)系统发育分析的方法进行了研究。结果表明,16SrDNA PCR-RFLP中所有供试菌株产生了4种酶切图谱类型,表现出较为丰富的遗传多样性。持家基因与16SrDNA基因系统发育分析结果基本一致,9株代表菌株主要分布在α-变形菌纲(Alpha-Proteobacteria)的根瘤菌属(Rhizobium),并与豌豆根瘤菌三叶草生物型(R.leguminosarumbv.trifolii)ATCC 14480T的亲缘关系较近。PCR可扩增出nodC和nifH基因片段,但从属于土壤杆菌属(Agrobacterium)的菌株LS1105中则扩增不出这两个基因。所有供试菌株被鉴定到了种的水平,证实了68株为白三叶根瘤菌,并通过不同采样地点菌株之间的比较,发现白三叶与根瘤菌的共生关系因地理分布不同而具有多样性,对于丰富白三叶根瘤菌资源及其开发利用具有重要意义。

The obj ective of this study was to determine the genetic diversity and phylogeny of 6 9 isolates of sym-biotic bacteria associated with wild white clover obtained from six sites in Sichuan Province,China：Ya’an, Kangding,Luding,Xichang,Chengdu and Leshan.Polymerase chain reaction restriction fragment length poly-morphism （PCR-RFLP）and phylogenetic analyses of the 16S rDNA gene,housekeeping genes （recA,glnII andatpD）,nodulation gene （nodC）and nitrogen-fixing gene （nifH）were conducted in the research.Four PCR-RFLP fingerprint patterns were identified among the 69 isolates from the 16S rDNA analysis,indicating relatively high genetic diversity.The results of phylogenetic analysis of 16S rDNA and housekeeping genes were similar;nine representative strains mainly belonged to the genusRhizobiuminAlpha-Proteobacteriawere identified.In addition,nodCandnifH genes from strains from the Rhizobium genus were successfully ampli-fied by PCR,but strains belonging to the Agrobacterium genus were not able to be amplified.All of the iso-lates could be identified by species with sixty-eight strains confirmed as white clover rhizobia.The diversity of white clover rhizobia differed at different sites,indicating the potential to increase the availability of beneficial rhizobia strains .