Characterization Of Methionine Sulfoxide Reductase Gene Family In Brachypodium Distachyon And Functional Analysis Of MSRB1.1

Abiotic stressor such as salt or drought,induced cells to generate a large mount of reactive oxygen species(Reactive Oxygen Species,ROS),and,if allowed to accumulated excessively,often caused intracelluar macromolecules oxidative damage.Proteins,especially those with rich methionine(methionine,Met),most susceptible of this oxidative damage.The methionine sulfoxide reductases(MSRs)catalyze the conversion of methionine sulfoxide to methionine and protect organisms against various abiotic stresses.In Brachypodium distachyon,it has been unclear.Here,six B.distachyon MSR(BdMSR)genes have been isolated;their structural characteristics,tolerance to salt,drought and oxidative stress were studied.In addition,the function of BdMSRB1.1 was further researched to discover the functional mechanism.The main research contents and results in our study were as follows.1.Cloning and function analysis of BdMSR gene family in abiotic stressAccording to the conservatism of homologous genes,six MSR genes in B.distachyon were obtained,both three from BdMSRA(BdMSRA2,-A4,-A5)and BdMSRB(BdMSRB1,-B3,-B5).Eight distinct cDNAs were cloned,since two of them(BdMSRB1 and-B5)produce a pair of alternatively spliced messages(BdMSRB 1.1/-B 1.2 and BdMSRB5.1/-B5.2).The known conserved domains and catalatic cysteine were found in these amino acid sequenses,showed a traditional structural feature of MSR.The genes were transcribed in the root,culm,leaf and during various stages of caryopsis development.Those induced by abiotic stress(salinity,drought,low temperature,CdCl2,H2O2 and abscisic acid)harbored known stress-responsive cis elements in their promoter sequences.The heterologous expression of five of the BdMSRs(-A2,-A4,-B1.1-B3 and-B5.1)in yeast revealed that their products gave a measure of protection against salinity,mannitol and oxidative stress.Substrate specificity analysis revealed that BdMSRB1.1 could reduce free Met-R-SO to Met.The enzymatic activities of BdMSRA4,-B1.1 and-B5.1 in transformed yeast under salt treatment have checked and increased obviously resulting in reducing more Met-SO to Met including the peptide and the free types under salt stress than those in control.2.Function and mechnism research of BdMSRB1.1 in abiotic stressMethionine sulfoxide redunctase MSRB1.1 in B.distachyon(BdMSRB1.1)was responded to abiotic stress such as salt,drought,H2O2 and ABA,suggested that it plays an important role in the process of response to abiotic stress in plants.The protein encoded by this gene was predicted to contain a highly conserved N-terminal low complexity region and a C-terminal SelR catalytic domain.It is noted that the final selenium Cysteine in C-terminal,bindind with selenium and zinc,showed methionine-R-sulfoxide reductase activity.Subcellular localization results showed that BdMSRB1.1 located in the chloroplast.Overexpression lines of BdMSRB1.1 in Arabidopsis(AtOE)and T-DNA insert mutant were obtained.The results of enzymatic activities showed that the MSR activity of AtOE lines were higher than that of the wild type(WT),and the mutant line was lower.Under the treatments of NaCl,Mannitol,H2O2 and ABA,the tolerance of AtOE lines was better than that of WT,while it was opposite in the mutant lines.The physiological results showed that overexpression of BdMSR23B1.1 could decrease the content of malondialdehyde(MDA)in AtOE lines,which could reduce the membrane damage.Besides,overexprssion of this gene could increase the content of soluble sugar and enhance the ability of cell tolerance to osmotic stress.The expression of marker genes in physiological pathway showed that BdMSRB1.1 increased the trancription of several key enzymes in ROS scavenging system,enhanced the activities of superoxide dismutase(SOD),catalase(CAT)correspondingly.Similarly,BdMSRB1.1 also induced up-regulated expression of Sucrose Synthase(SUS)and Sucrose Phosphate Synthase(SPS),two key enzymes in the soluble sugar synthesis pathway,and promote the sucrose synthesis.The above results confirmed that the BdMSRB1.1 gene was involved in the process of abiotic stress and played an positive role in plant.