The Study Of The Function Of Glutaredoxin Genes Grxs In Rhizobium Leguminosarum

Glutaredoxins(Grxs) are small ubiquitous oxidoreductases of the thiorexoin superfamily that reduce protein disulfide bonds or glutathione-protein-mixed disulphides bonds. Grxs control the redox status of protein and play a crucial role in bacteria response to oxidative stress. Rhizobia are aerobic, Gram-negative nitrogen-fixing, bacteria that form nodules with leguminous plants under conditions of nitrogen limitation. The nitrogenase in the nodules is very sensitive to oxygen, so a non-aerobic environment is needed for nitrogen fixation. In R. leguminosarum 3841, there genes, RLgrx1(RL4289), RLgrx2(RL2615) and RLgrx3(RL4261), were found to display significant sequence similarity to glutaredoxins by bioinformatics analysis. This study investigated the role of R. leguminosarum grxs in free-living or aerobic-stress-living bacteria and during nitrogen-fixing symbiosis, by analyzing the phenotypes of mutant stains.This study cloned the grx1, grx2 and grx3 gene to the suicide vector pJQ200 SK or pK19 mob to make the recombinant plasmids(pJQgrx1?Spec, pKgrx2 and pJQgrx3?Tc). These recombinant plasmids were transfer to Rlv3841 by tri-parental mating, and the grx single, double mutants were constructed by screening test of antibiotics or the sucrose with high concentration. And then grx double mutants and tri-mutant were generated by transduction.In order to investigate the contribution of the various grxs to growth of R. leguminosarum, the free-living experiment was conducted. Under the free-living condition, all of the grx mutants exhibited no generation time extension. The zone of inhibition assays was carried out to investigated the oxidative stress funcation among the various grx genes, cells of all the grx mutant stains were deficient in consumption of high concentrations of H2O2, but not in that of CuOOH. The real-time qRT-PCR results showed that grx1 was expressed in dependently of exogenous H2O2.In order to investigated the role of these Grxs in symbiotic process, the nitrogen fixing capacity of the grx mutants were assessed after inoculation with Pisum sativum. No significant difference in the number of nodules was observed between plants inoculated with the RLgrx2 and plants inoculated with the wt RL3841 strain. On the contrast, significantly fewer nodules were observed on plants inoculated with RLgrx3, RLgrx1grx3 and RLgrx1grx2grx3 than those inoculated with RL3841. The nitrogen-fixing ability of the mutants was further measured by acetylene reduction. Plants inoculated with either of the grx mutants had a significantly lower nitrogen fixation capacity than plants inoculated with the wt RL3841 strain. However, there were no significant difference in nitrogen fixation capacity among plants inoculated with grx mutants. Morever, the effect of grx gene deletion on nodule structure were investigated. Nodules infected with the grx mutants were more senescent cells, and nodules infected with RLgrx1grx2grx3 had a significant change in the shape.In conclusion, this study had generated the grx single mutants, double mutants and tri-mutants. Although the deletion of grx in RL3841 had no signigicant different under the free-living condition and in consumption of CuOOH,but the grx strains were deficient in consumption of high concentrations of H2O2.Under symbiosis condition,grx mutants could accelerate the senescence of nodules,and lower the nitrogen-fixing ability.