Screening Protein Factors In Laodelphax Striatellus Which Interact With Rbsdv P10and Analysis Of RNA Silencing Suppressor Activity Of Rbsdv P9-1

Rice black-streaked dwarf virus (RBSDV), a member of group2of the genus Fijivirus within the family Reoviridac, is transmitted to cereal crops including rice, corn, wheat and barley via Laodelphax sreiatellus Fallen, and causing severe economic losses. RBSDV genome has10double-stranded RNA segments which contains13open reading frames (ORF) and encodes13proteins.In order to obtain insect factor required for RBSDV transmission in Laodelphax striatellus, the coat protein P10encoded by RBSDV S10was chosen as the bait using yeast two-hybrid system to screen Laodelphax striatellus cDNA library. First, RBSDV coat protein gene P10was amplified by RT-PCR and inserted into yeast two-hybrid system bait vector pGBKT7, then screened Laodelphax striatellus cDNA library by sequence transformation method. The cDNA insert fragments of positive clones were identified by PCR. The plasmids containing different cDNA inserts were transformed into E. coli DH5a and then sequenced. The sequencing results were blasted in the GenBank library.455positive clones were acquired by screening L. striatellus cDNA, and377clones were sequenced. By alignment analysis, these377clones encoded15different kinds of proteins, such as actin, serpin peptidase inhibitor, RACK and GAPDH3. The result promotes the study of obtaining the insect factors for RBSDV transmission.Now, there are nearly no rice varieties that have clear resistance to RBSDV and Southern rice black-streaked dwarf virus (SRBSDV). RBSDV S6gene segment (R6) and SRBSDV S6gene segment (SR6) were fused by recombinant PCR, and the fused fragment S6-SR6was obtained. RBSDV S10gene segment (R10) and SRBSDV S10gene segment (SR10) were also fused, and obtained the fusion gene fragment S10-SR10. Fusion gene R6-SR6and R10-SR10were ligated into pBS vector in a revert repeat manner, then inserted into binary expression vector pCAMBIA1301. The pCAMBIA1301-hp (R6-SR6) and pCAMBIA1301-hp (R10-SR10) containing hairpin structure were constructed. The contructed vectors proved to be consistent with the expected design by enzyme digestion and PCR. This study provides the foundation for production of transgenic plants that are resistant to RBSDV and SRBSDV.Our preliminary studies showed that RBSDV P9-1protein could inhibit local silencing triggered by sense GFP RNA. In this study, we further analyzed the suppressor activity of P9-1protein encoded by RBSDV S9-1By co-infiltration method, the results showed that P9-1protein could suppress local silencing mediated by sense strand RNA, but couldn’t suppress the silencing caused by dsGFP. P9-1couldn’t suppress systemic silencing mediated by GFP, but can stop the transduction of silencing signal. Previousely published studies indicated that the expression of virus gene which encoded virus suppressors was much higher than the other genes in genome. In order to determine the relative expression abundance of P9-1gene in the genome of RBSDV, the relative gene expression abundance of P9-1were analyzed by Real-Time PCR. The results showed that the relative gene expression of P9-1was not the highest when compared to other genome segments in rice or in maize.