Screening And Identification Of OsRBR1 Interacting Proteins

Floral organ development is a complex biological process involving multiple genes in rice.In previous study,we isolated the OsRBR1 gene,a key gene for controlling flower organ formation in rice,by map-based cloning.However,the mechanism on how the gene specifically regulation floral organ formation remains unknow.On this basis,this study focused on screening and identification of the interaction proteins of OsRBR1,by using bioinformatics,yeast two hybrid,bimolecular fluorescence complementation analysis,and identification the function of OsMSI1 and OsYchF1,two interaction protein gene of OsRBR1,by gene knockout method.The main results were as follows:(1)Prediction the interaction proteins of OsRBR1.The interaction proteins of OsRBR1 were predicted through protein interaction analysis database,and nine proteins were predicted to interact with OsRBR1,including three OsE2F proteins,three OsDP proteins and three OsMSI proteins.The homology and phylogenetic analyses of the predicted proteins revealed that there is no direct interaction domain between OsE2F3 and RBR.It was speculated that the protein might be the same as the three OsDP proteins,and had an indirect interaction with OsRBR1.(2)Protein interaction identification between MSI1 and OsRBR1,and functional analysis of MSI1.It is now clear that,AtMSI1 and AtRBR1 are all involved in the regulation of fertility in Arabidopsis,and there is an interaction between the two proteins.The results of BIFC analysis showed that OsMSI1 interacts with OsRBR1.In order to further study the function of OsMSI1 gene,we constructed a double target knockout vector of OsMSI1 and transformed it into the rice variety Minghui 86.So far,some transgenic plants were obtained.(3)A CDNA Library of the young panicle of Minghui 86 was screened with full length OsRBR1-FL and truncated OsRBR1-B fusion protein which did not show self activation and toxicity,by yeast two hybrid methods.Eight protein genes showing interactions with OsRBR1 were screened out.Among them,the GTP binding protein OsYchF1,which encoded by LOC_Os08g09940,showed the homologous to OLA1 of mouse and human.It is now clear that,due to the attenuation of Rb phosphorylation,the mice of loss OLA1 function affects the process of cell cycle leading to its developmental delay and organ hypoplasia.It is hypothesized that OsYchF1 may participate in the regulation of cell cycle progression by affecting the phosphorylation of OsRBR1 protein,thus to affection spikelet development in rice.For further to study the function of OsYchF1,a single target knock-out vector of OsYchF1 was constructed and then transformed into rice cultivar Minghui 86,which is still in transformation.