Study Of The Function Of Peroxiredoxin Genes Prx? In Rhizobium Leguminosarum

Peroxiredoxin?(prx?)is typical 2-Cys antioxidative enzyme of thioredoxin family,which can detoxification of hydrogen peroxide,alkyl hydroperoxides and peroxynitrites to protect cells against oxidative stress.During the catalytic process,the peroxide cysteine is oxidized to disulfides which reduced by Grx,so catalysis can be recycled.Rhizobia are aerobic,Gram-negative,nitrogen-fixing bacteria that live in soil rhizosphere as free-living cell or symbiosis relationship with leguminous plants under conditions of nitrogen limitation.In this symbiotic system,plant supplies the rhizobia with energy while Rhizobia provide valuable organic nitrogen to the plant.Strictly anaerobic environment is needed for nitrogen fixation as the nitrogenase in nodules is very sensitive to oxygen.In R.leguminosarum 3841,there are three prx? genes recorded in NCBI database,named as RLPrx?A(RL1259),RLPrx?B(RL2440),and RLPrx?C(pRL120247).In this study,the roles of R.leguminosarum Prx? in free-living bacteria and during N-fixing symbiosis on P.sativum by analyzing the phenotypes of three single Prx? mutant strains,each lacking one to four of the putative sulfate.The Prx?A,Prx?B and Prx?C gene were cloned into the suicide vectors pJQ200 SK or pK19 mob to make the recombinant plasmids pJQPrx?A?Spec?pKPrx?B and pJQPrx?C?Tc.By triparental mating,these recombinant plasmids were conjugated into strain 3841.The mutants were isolated by selecting for recombination using the sac mutagenesis strategy,and mapped by PCR.To investigate the contribution of the Prx?s to growth of R.leguminosarum,the growth of the Prx? mutants were detected in the medium with or without oxides.All the Prx? mutants have no effect on free-living growth.Howerver,compared to that of strain 3841,the inhibition zone of RLPrx?A was significantly larger in 20 mmol/L H2O2,and the inhibition zone of RLPrx?C was increased significantly in all concentrations of H2O2.And the inhibition zone of all mutants were increased significantly in 20 mmol/L CuOOH.The results showed that All the Prx? genes have no function of free-living growth,while Prx II played a role in defense again oxidative stress.As investigating the role of three Prx?s in symbiotic process,the symbiotic capacity of the Prx? mutants were assessed after inoculation with Pisum sativum.Plants inoculated with either of the PrxII 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 PrxII mutants.The structure of 4-week-old nodules was further observed by paraffin sections and transmission electron microscope.Significantly fewer nodules were observed on plants inoculated with all the Prx? mutants than on those inoculated with wt 3841.The effect of Prx? mutants on nodule structure by microscopic analyses of 4-week-old nodules.The nodules induced by induced by the RLPrxIIC undergo early abortion.This result confirms the impairment of bacteroid differentiation and nodule functioning observed in the Prx? mutants.By real-time PCR the expression of Prx? genes under differen conditions was measured.The results showed that Prx?A gene was exclusively increased in 25-day-old nodules.Prx?B gene was significantly increased in the anaerobic condition.In conclusion,these results give a strong proof that Prx?s perform a undoubted role in antioxidation and symbiotic process.