Replication And Expression Of SRBSDV In Rice Protoplasts

Southern rice black-streaked dwarf virus(SRBSDV), transmitted by white-backed planthopper (WBPH) in a proliferative manner, has become one of significant rice viral diseases in our country. Impressively high efficiencies are achieved in this study by enzymatic digestion, and a rice protoplast system for studying virus was established and tested, using the theory of specific binding of antibody to antigen, we crosslink antibody and fluorescein, and observed the replication and expression of virus in protoplasts by immunofluorescence microscopy. Meanwhile, we detected virus related gene transcription and protein expression by real-time PCR and western blot.Firstly, to produce antiserum of structural protein P10 and nonstructural protein P9-1 of SRBSDV, prokaryotic expression was performed by a Gateway system. By an indirect ELISA method, the antiserum titer of P10 and P9-1 were determined to be 6400 times and 3200 times respectively. Western blot analysis showed that both the antibodies could detect the infected rice plants specifically. To test sensitivity of these two antibodies, IC-RT-PCR and Dot-blot ELISA were used, and a consistent result was obtained. Our results revealed that both P9-1 protein antibody and P10 protein antibody of SRBSDV have a high sensitivity.Protoplasts were infected by SRBSDV, using PEG-mediated inoculation. Detecting protoplasts with fluorescein crosslinking P9-1,P10 antibody incubation at 48h post-inoculation by immunofluorescence microscopy, we found that the protein of P10 expression quantity was less than the protein of P9-1. The result of real-time PCR indicated that SRBSDV genes of S5-1, S5-2,S6,S7-2,S9-1,S9-2 expression quantity reached peak at around 36h post-inoculation, and that SRBSDV genes of S7-1,S10 expression quantity reached peak at around 48h post-inoculation. The result of western blot detection indicated that SRBSDV proteins of P5-1,P6,P9-1 expression quantity reached peak at around 48h post-inoculation, and that SRBSDV proteins of P7-1.P10 expression quantity reached peak at around 60h post-inoculation.