The Replication And Expression Of Rice Dwarf Virus In Rice Protoplast

Rice dwarf virus(RDV)is the member of Phytoreovirus belonging to Reoviridae.The genome of RDV is composed of 12 double-stranded RNAs,encoding two categories of proteins containg structural proteins and non-structual proteins.The disease generally occurs in the south of our country in Yunnan,Hainan and Fujian area etc.,which can cause serious reduction of output at the outbreak of year.Firstly,prokaryotic expression of non-structual protein Pns6 and structural protein P8 was performed by a Gateway system in this study,and expression vectors pDEST17-Pns6 and pDEST17-P8 were obtained.Then they were transformed into E.coli Rosetta strain respectively,and the HIS-tag fusion protein were obtained with induction of IPTG.Both fusion proteins were used to immunize New Zealand Rabbits respectively as an antigen for production of polyclonal antibodies.Western blot analysis showed that both the antibodies could detect infected rice plants specifically.By an indirect ELISA method,the antiserum titer of Pns6 and P8 were determined to be 6400 times.Dot-blot ELISA was established for reliable,sensitive and specific detection of RDV.These results indicated that both antibodies against non-structural protein and structural protein of RDV could be applied to large-scale investigation and detection of the virus in the fields.Secondly,a infection system of rice protoplast was established,and the replication and expression of virus were studied in rice protoplast by using fluorescence quantitative technique,immunofluorescence technique,western blot technique and electron microscope technique.(1)Fluorescence quantitative assay showed that virus reached the peak in 24-36 hours after infected rice protoplasts,while S6 and S12 gene reached the peak at 24 h,and S8,S10,S11 gene reached the peak at 36 h.(2)After 48 hours of virus infected protoplasts,immunofluorescence labeling found Pns6 can form punctate inclusions similar to viroplasms in the cytoplasm.Our results indicated that the Pns6 may be a component of the viroplasms matrix in the plant host.Meanwhile,the coat protein P8 carried out a large number of replication after 48 hours of virus infected,and there is a movement trend toward the outside of the cell.(3)Using the western blot analysis,expression quantity of the coat protein P8 in protoplast was lower than that of movement protein Pns6,and the maximum value appeared late.Our results indicated that,in the infected host cells,Pns6 first involved in the synthesis of viroplasm,then were the assembly and release of virus particles.(4)Electron microscopic section observed the structure of inclusion bodies were formed in rice protoplast,and a similar double capsid structure of virus particles can be seen in the inclusion bodies.