Preliminary Study On The Relationship Between OsRbohB,OsRbohE And OsDMI3 In ABA Signal Transduction

ABA is a major phytohormone that regulates a broad range of plant traits and is especially important for adaptation to environmental conditions.It accumulates in plant cells under water stress,promotes stomatal closure in guard cells and regulates the expression of many genes,the products of which may protect vegetative tissues from dehydration or high osmotic pressure.Plant NADPH oxidase is one of the most important component in ABA signaling network.OsDMI3 plays an significant role as well.It has been shown that ABA induce the expression of OsRbohB and OsRbohE in rice protoplasts.The results as follows:Coding sequence of OsRbohB were cloned and inserted into sites of pXZP008 vector for the study of subcellular location.The relative level of OsDMI3 gene expression was detected by RT-PCR.It showed that transient expression of OsRbohB increases the gene expression of OsDAMI3 and the expression of OsDMI3 would be more increasing after the treatment of ABA.While,RNA interference(RNAi)silencing of OsRbohB decreased the OsDMI3 gene expression.It indicated OsRbohB regulates the ABA-induced expression of OsDMI3 at the transcription level.For further study,we use the methods of BiFC and yeast two-hybrid system and GST-pull down to investigate the interaction between OsRbohs and OsDMI3.OsRbohE was cloned into the pGADT7 vector.The yeast cells transformated the OsRbohs-AD and OsDMI3-BD activate the downstream promoters could make colonies turn blue.Clone OsrbohB into YFPN and YFPC vector and transform constructs into epidermal cell of onion,then detect fluorescence by confocal.There was yellow fluorescence from cells that contain OsRbohB-YFPN and OsDMI3-YFPC constructs.The conclusions indicate it may have interaction between OsRbohs and OsDMI3.Transient expression or RNA interference(RNAi)silencing of OsRbohs in rice protoplasts to investigate the ABA-induced production of H2O2.Connect the CDS region of OsRbohs to pXZP008-mcherry vector.Transform OsRbohB and OsRbohE into rice protoplasts.With treatment of ABA,transient expression of OsRbohs increases ABA-induced production of H2O2-Both OsRbohB and OsRbohE were expressed make further increasing.While,gene silencing of single OsRbohs decreases ABA-induced production of H2O2-When both OsRbohB and OsRbohE were silent,production of H2O2 was significantly reduced,ABA treatment can not restore H2O2 production.