The Mechanism And Impact Factors Of Competitive Nodulation Of Soybean Rhizobia

In this study, 190 rhizobial strains, isolated from soybean in huanghuaihai area were characterized by IGS-RFLP. Strains matched well with soybean cultivar zhonghuang 13 were screened from representative isolates. Their competitive nodulation abilities were evaluated by the method of gfp and rfp marking. Two strains have significant differences in competitiveness were selected, and they were primarily analyzed by proteomic technique to research for the reasons of diversity in competitive nodulation abilities. This work will provide some useful information about the mechanism and impact factors of competitive nodulation of soybean rhizobial strains, and it will provide evidence for screening high effective rhizobial strains to make symbiotic nitrogen fixation more efficient.A total of 190 rhizobial strains were isolated from soybean nodules, which were collected from Henan, Shanxi and Anhui provinces in China. All of the strains were characterized by IGS-RFLP and they were divided into 28 types. 28 strains from each type were inoculated to soybean cultivar zhonghuang 13 for nodulation test, 5 strains, iucluded B. japonicum 4222, B. japonicum 4230, B. japonicum 4534, S. fredii USDA205 and S. fredii R25a, matched well with soybean were screened.The green-fluorescent protein gene (gfp) or red-fluorescent protein gene (rfp) was transferred into those 5 strains by double-parent hybridizing method respectively to obtain marked strains. The genetic stability of the foreign genes were detected and their effects on the strains'nodulation ability were evaluated. The foreign genes could auto-replicate and stably transfer to next generations, and it has no effect on the symbiotic nitrogen fixation activity of soybean. The results showed that this method could be used to evaluate the competitiveness of strains. Nine pairs of strains from the 5 strains with different fluorescent protein genes were inoculated on the soybean cultivar of Zhonghuang 13 for competitive nodulation test. The competitve nodulation ability of B. japonicum 4534 was highest, meanwhile, B. japonicum 4222 was lowest.The protein expression profiles of B. japonicum 4534 and B. japonicum 4222 were analyzed after they were treated with daidzein of zhonghuang 13. The results showed that: B. japonicum 4534 had 14 up-regulated proteins and 10 down-regulated proteins in expression with significant difference, while B. japonicum 4222 had 6 up-regulated proteins and 4 down-regulated proteins. Differentially displayed proteins from B. japonicum 4534 and B. japonicum 4222 were analyzed by MALDI-TOF-MS, and 10 of them were identified. The Differentially expressed proteins of B. japonicum 4534 were relevant to metabolism and transport, and which of B. japonicum 4222 were relevant to metabolism, transcription and translation.The protein expression profiles of B. japonicum 4534 was analyzed after it was treated with daidzein of zhonghuang 13 and the extracellular materials of B. japonicum 4222, it had 43 up-regulated proteins and 35 down-regulated proteins in expression with significant difference, which were relevant to flagellum, metabolism, transcription, translation, molecular chaperone, transport and defence. Meanwhile, B. japonicum 4222 was treated with daidzein of zhonghuang 13 and the extracellular materials of B. japonicum 4534, it had 25 up-regulated proteins and 22 down-regulated proteins in expression with significant difference, which were relevant to metabolism, translation and molecular chaperone.In both cases, the differential expression proteins of B. japonicum 4534 were more than B. japonicum 4222. Metabolism related proteins could provide energy and carbon skeleton for nodulation; transport related protein could enhance the secretion of the nodule factors from inner of the rhizobium cells, which heightened the appetency of rhizobium with their host plants; Flagellin could help improve the movement ability, than improve the competitive nodulation ability; Molecular chaperone and translation related proteins could enhance the expression amount of some proteins. The difference of rhizobia in responses to daizein and extracellular materials was one of the most important reasons that caused varieties in competitive nodulation ability.