Study Of The Function Of Peroxiredoxin Genes PrxⅡ In Rhizobium Leguminosarum

PeroxiredoxinⅡ(prxⅡ)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 prxⅡ genes recorded in NCBI database,named as RLPrxⅡA(RL1259),RLPrxⅡB(RL2440),and RLPrxⅡC(pRL120247).In this study,the roles of R.leguminosarum PrxⅡ in free-living bacteria and during N-fixing symbiosis on P.sativum by analyzing the phenotypes of three single PrxⅡ mutant strains,each lacking one to four of the putative sulfate.The PrxⅡA,PrxⅡB and PrxⅡC gene were cloned into the suicide vectors pJQ200 SK or pK19 mob to make the recombinant plasmids pJQPrxⅡA?Spec、pKPrxⅡB and pJQPrxⅡC?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 PrxⅡs to growth of R.leguminosarum,the growth of the PrxⅡ mutants were detected in the medium with or without oxides.All the PrxⅡ mutants have no effect on free-living growth.Howerver,compared to that of strain 3841,the inhibition zone of RLPrxⅡA was significantly larger in 20 mmol/L H2O2,and the inhibition zone of RLPrxⅡC 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 PrxⅡ 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 PrxⅡs in symbiotic process,the symbiotic capacity of the PrxⅡ 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 PrxⅡ mutants than on those inoculated with wt 3841.The effect of PrxⅡ 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 PrxⅡ mutants.By real-time PCR the expression of PrxⅡ genes under differen conditions was measured.The results showed that PrxⅡA gene was exclusively increased in 25-day-old nodules.PrxⅡB gene was significantly increased in the anaerobic condition.In conclusion,these results give a strong proof that PrxⅡs perform a undoubted role in antioxidation and symbiotic process.