Research And Identification Of Bradyrhizobium Sp.GZL13-3 From Glycine Soja

Study on identify of strain isolated and purified from root nodule of Glycine soja, we used some methods, including identification of phynotype, analysis of 16S rDNA whole sequence, analysis of housekeeping genes, analysis of symbiotic nitrogen fixation genes and testing of nodulation in potted plant.We got one rhizobia GZL13-3 from root nodule of Glycine soja, it had typical rhizobia character of bacterial colony and physiology and biochemistry. Growth generations was 18h, the result of testing of BTB produced alkali, it was a slow-growing rhizobia; it can grow on the condition of pH5.0-8.0 and tolerate of 0.50% NaCl; it can tolerate 0.1% congo red or bromophenol blue; it can't utilize lactose, citrate and malonate; the sensitivity has disparity on different antibiotic. Rhizobia GZL13-3 G+C mol% was 61.6% used the method of heat denaturation temperature.The result of analysis of 16S rDNA whole sequence showed GZL13-3 was classified in the genus Bradyrhizobium, but the 16S rDNA sequence was very different with the other known species in this genus, GZL13-3 sequence similaritied with the close species B.yuanmingens were 95.3%. The research of housekeeping gene (recA and atpD) proved the result of analysis of 16S rDNA sequence, in the phylogency trees on basis of the housekeeping gene (recA and atpD), the tested strain GZL13-3 was also classified in the genus Bradyrhizobium.The phylogency trees on basis of the symbiotic nitrogen fixation genes (nodC and nifH) was different with the phylogency trees on basis of selfish genes. In the phylogency trees on basis of nodC gene and its homologous sequences, we found that the sequences of GZL13-3 nodC gene was phylogenetically close to the strain M.loti, that was possible the nodC gene may get from genus Mesorhizobium through horizontal transfer. In the phylogency trees on basis of nifH gene, GZL13-3 and B.jicamae made up a phyletic evolution branch, but had a farther genetic relationship with other strains in the genus Bradyrhizobium, this gene is one specific nifH gene.The results of testing of nodulation showed that GZL13-3 can form nodulation with Glycine max and Glycine soja. The SDS-PAGE analysis of whole protein showed the new isolated strains was same as GZL13-3, proved the nodulation ability of GZL13-3.All of these could illustrate that rhizobia GZL13-3 was belong to Bradyrhizobium, because of the low homology of selfish genes with known species in this genus, GZL13-3 may be one potential new strain. The additional test is necessary to identify the well-defined taxonomic status.