Study Of The Function Of Thioredoxin Genes Trxs In Rhizobium Leguminosarum

Thioredoxin?Trx?is a kind of small thermostabilizing acidic protein,which changes the oxidation state of target protein with the help of the reversible oxidation of the dithiol of its active site.Trxs are the crucial redox protein in cells,and play an important role in regulating the oxidation resistance,stress resistance of cells and apoptosis,and also the DNA-binding DNA binding transcription factor activity.Rhizobia are aerobic,Gram-negative bacteria that interact symbiotically with Legumes to form nitrogen-fixing root nodules.In this symbiotic system,the nitrogenase enzyme in nodules complex is extremely sensitive to oxygen,rhizobia must therefore have means to prevent oxygen access to nitrogenase.In Rhizobium leguminosarum bv.Viciae3841,three genes,trxA?RL0025?,trxB?RL4571?and trxC?RL4663?were found to display significant sequence similarity to thioredoxins by the bioinformatics analysis.In this study,mutations in one,two or all three thioredoxin genes were constructed by homologous recombination and transduction.The role of R.leguminosarum trxs in free-living or aerobic-stress-living bacteria and during nitrogen-fixing symbiosis was further investigated by analyzing the phenotypes of mutant stains.The genes trxA,trxB and trxC were cloned into the suicide vectors pK19mob and pJQ200SK to make the recombinant plasmids pKtrxA,pJQtrxB?Tc and pJQtrxC?Spe.The recombinant plasmids were further transfered to strain R.leguminosarum 3841 by triparental mating.The single gene mutants RLtrxA,RLtrxB,RLtrxC and double gene mutant RLtrxAtrxB were constructed by screening test of antibiotics or using the sac mutagenesis strategy.The double mutants RLtrxAtrxC and RLtrxBtrxC,and triple gene mutant RLtrxAtrxBtrxC were generated by transduction.To achieve the expression of trx genes in Escherichia coli,the structural gene fragments of the three trx genes obtained by PCR were cloned into the prokaryotic expression vector pET-28a,and then the ligation products were transformed into E.coli BL21?DE3?strain.As demonstrated by SDS-PAGE,the relative molecular mass of the expressed proteins TrxA,TrxB,TrxC were approximately 12 kDa,35.1 kDa,25.7 kDa,respectively.Using 2-nitrobenzoic acid?DTNB?as substrate to measure enzyme kinetics parameters,the Km values calculated by the Michaelis-Menten equation and Lineweaver-Burk curve were 1.86mmol·L^-11 for TrxA,2.0 mmol·L^-1 for TrxB,and 1.79 mmol·L^-1 for TrxC,respectively,In AMS minimal media with glucose as a sole carbon source,the trx single and double mutants exhibited the same growth rate with the wild type strain RL3841.And the inhibition zone of all the trx single and double mutants were not increased or decreased significantly under various concentrations of inorganic H₂O₂ and organic hydroperoxide cumene hydroperoxide?CUOOH?compared to the control.However,the the triple gene mutant RLtrxAtrxBtrxC grew poorly compared to the wild-type RL3841,its inhibition zones were significantly larger under high concentrations of H₂O₂ and CuOOH.The nitrogen fixing capacity of the trx mutants was assessed after inoculation with Pisum sativum.Although all the trx mutants form pea nodules,they were severely impaired in abnormal nodules,and showed significantly lower nitrogen fixation capacity than plants inoculated with the wild type strain RL3841.The structure of 4-week-old mutant nodules was further observed by paraffin sections and transmission electron microscope,significantly fewer bacteroids and little senescences were observed on plants inoculated with all the trx mutants compared to those inoculated with wild type.These results showed that trxs play a critical role in bacteroid differentiation and preventing nodule premature decay.The expression of Trx relative genes under different conditions was measured by real-time fluorescence quantitative RT-PCR.In the presence of 1 mM H2O2,the expression of stress-responsive genes orhB,katE,katG were upregulated in the trx single mutants and triple mutant.The results suggest that the trx mutants might significantly modify cellular redox state and increase catalase gene expression.In contrast,the transcriptional activity of symbiotic genes nifD,hmuS and rirA was decreased in 4-week-old nodule inoculated by mutants RLtrxA,RLtrxB,RLtrxC,and RLtrxAtrxBtrxC than that in control nodules,which is the reason for the impairment of N₂-fxiation functioning observed in the trx mutants.