Preliminary Study On The Roles Of Rice Receptor-like Protein Kinase FLR1 In Grain Storability

The quality of rice grain after storage is determined not only by the environment and technology of storage but also by genes that control the quality of mature seeds.Previously,our lab found out that rice FERONIA-like receptor(FLR)affected rice starch synthesis,seed size,and structure of starch.In this study,we further compared seed germination rate of wild type and FER mutant seeds after artificial aging.Preliminary data showed that Arabidopsis FER and rice FLR1 affected seed germination after artificial aging.Because FLR1 is a protein kinase,potential FLR1 interacting proteins were screened using yeast two-hybrid method and some interacting proteins were verified.To further investigate mechanisms of FLR1 in grain storage,FLR1 antibody was prepared by expression and purification of a recombinant FLR1 protein from bacteria and immunization of rats with the recombinant FLR1.The main findings are as follows:(1)After artificial aging,the germination rate of a mutant Arabidopsis fer4 seeds was lower than that of wild type seeds;the germination rate of rice seeds overexpressing FLR1 gene was higher than that of wild type seeds.These results collectively indicating that FER/FLR1 gene product positively regulate seed storability.(2)Through the yeast two-hybrid library screening,28 rice FLR1 binding proteins were identified and seven of the proteins were verified to interact with FLR1 in vitro using one-to-one method.(3)The N-terminal(FLR1-BW,1-450 aa)and C-terminal(FLR1-BN,474-892 aa)portions of FLR1 were cloned into protein expression vectors pET-32a,pGEX-4T-1,pET-22b,and pTriEx-4,respectively.One recombinant protein FLR1-BW was successfully expressed in E.coli,purified to homogeneity,and used for antibody production in rats.The anti-FLRl antibody specifically recognized the purified FLR1 protein and identified a protein of 130kD from total proteins of rice seeds,indicating the high quality of the antibody.In summary,these preliminary data suggest that rice FLR1 is positively linked to seed storage.The potential mechanisms of FLR1 in seed storage are through its interaction with other proteins as 28 proteins were identified as FLR1 binding proteins.The production of anti-FLRl antibody will surely enhance the further study on the roles of FLR1 regulating rice storage.