Characterization Of OsWHIRLY Family In Oryza Sativa And Their Functional Study During Development And Magnaporthe Oryzae Infection

Rice is one of the three most important food crops in the world.How to improve its yield and grain quality has always been a hot issue.Rice diseases caused by Some fungi,viruses and bacteria pathogens often result in dramatic crop yield losses worldwide,for example the Magnaporthe oryzae(M.oryzae)caused rice blast disease is the most destructive threat to rice.Although the pathogenesis of M.oryzae has been widely discussed,it is still far form clear how rice defense against their infection.WHIRLY protein is one member of the plant-specfic transcription factors,which can bind to the single-strained DNA(ssDNA).In recent years,it was found that WHIRLY proteins have important roles in pathogen response signaling pathway in plants such as Arabidopsis,potato ect.Therefore,this study tries to find out the possible function of the two WHIRLY proteins of rice(OsWHIRLYl and OsWHIRLY2)during development and M.oryzae infection.The results were displayed as follows:1.After the infection of M.oryzae on rice for 24～108 hours,the expression of the two Os WHIRLY genes detected by quantitative real-time PCR did not show significant alterations compared to other defense responsive genes.It suggested that the OsWHIRLY genes maybe not take part in the immune responses of rice to M.oryzae.2.The expression of OsWHIRLY genes changes during rice senescence.The expression pattern of OsWHIRLY1 and OsWHIRLY2 does not appear obvious change in the first complete primary leaf,root and shoot at the rice early development stage from 5 to 30DAG(day after germination).However,they display clear up-regulation similar to some senescence-associated genes during flag leaf development.Thus the two OsWHIRLY genes are possibly involved in the flag leaf aging.3.OsWHIRLY1 and OsWHIRLY2 give different response to distinct phytohormone treatments.After exposure to 100 μM salicylic acid(SA)、50 μM Abscisic acid(ABA)、50 μM Gibberellin(GA)or 50 μM Methyl jasmonic acid(MeJA)for 6 days,their expression levels in the roots of rice seedlings were significantly elevated only in the case of ABA treatment.Further analysis of their expression patterns in rice seedlings in response to different concentrations of ABA(25、50 and 75 μM)for 1、2 and 6 days,it revealed that the expression variation of OsWHIRLY1 and OsWHIRLY2 was positively correlated with the ABA concentrations.Furthermore,they were strongly induced in rice shoots during the early stage of ABA treatment,whereas their cDNA were not evidently accumulated in rice roots until the late stage of ABA treatment.These results implied that they might both fulfill functions in ABA-dependent signaling pathways.4.Yeast two hybrid assay showed that OsWHIRLY2 interacted with OsWHILRY1 only when OsWHILRY1 performed as the bait protein.5.Transgenic rice lines overexpressing OsWHIRLY1 or Os WHIRLY2 as well as their RNAi mutant lines were identified by the selection of hygromycin resistance and qRT-PCR.We totally selected 29 positive OsWHIRLY overexpression transgenic plants,5 positive RNAi-OsWHIRLY1 and 13 positive RNAi-OsWHIRLY2 lines.But overexpression OsWHIRLY2 transgenic plants didn’t selected.These plants provide the possibility to further investigate OsWHIRLY1 and OsWHIRLY2 biological functions in future.Taken together,OsWHIRLY1 and OsWHIRLY2 may be integral to ABA-mediated leaf senescence in rice.