四川白三叶根瘤菌遗传多样性及系统发育研究

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16S rDNA限制性片段长度多态性分析(restriction fragment length polymorphism, RFLP)和16S rDNA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,16S rDNA 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16S rDNA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. leguminosarum bv. trifolii) ATCC 14480^T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。

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

The objective of this study was to determine the genetic diversity and phylogeny of 69 isolates of symbiotic 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 polymorphism (PCR-RFLP) and phylogenetic analyses of the 16S rDNA gene, housekeeping genes (recA, glnII and atpD), 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 genus Rhizobium in Alpha-Proteobacteria were identified. In addition, nodC and nifH genes from strains from the Rhizobium genus were successfully amplified by PCR, but strains belonging to the Agrobacterium genus were not able to be amplified. All of the isolates 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.