The Replication And Expression Of Rice Ragged Stunt Virus (RRSV) In Rice Protoplast

Rice ragged stunt virus (RRSV) is the member of Oryzavirus belonging to Reoviridae. The genome of RRSV is composed of 10 double-stranded RNAs, encoding 11 kinds of proteins containing 8 kinds of structural proteins and 3 k inds of non-structural proteins. Rice ragged stunt disease aroused by RRSV pos ed a serious threat to rice production in south china and became an important rice disease.Rice protoplast culture system that can be infected by RRSV has been est ablished. Making the rabbit antiserum directed towards the structural protein P8 and non-structural proteins of RRSV, the replication and expression of RRSV in rice protoplasts were detected employing immunofluorescence, fluorescent q uantitative PCR and Western blot. The results showed that:(1) The rabbit antis erums made through Prokaryotic expression were proved to be useful for virus detection of RRSV by the detection techniques of ELISA and Western blot; (2) RRSV Pns10 marked by immunofluorescence could form viroplasm-like incl usions in the rice protoplast and it gradually gathered as inclusion bodies after RRSV infection which also suggested that RRSV successfully infected rice pr otoplasts; (3) Taking rice protoplasts after inoculation in different period of tim e as experimental samples, the replication cycles of RRSV within rice protopla sts were detected employing Real-time PCR, we found that RRSV began to re plicate at 8 h after inoculation, S6、S7、S10 that express non-structural protein s of RRSV reached their climax at 24 h, S8 that express structural protein P 8 reached its climax at 32 h, and then gradually decreased but they still had a higher level. It indicated that 24-32 h after inoculation is the replication and transcription peak of RRSV; (4) Using the rabbit antiserum directed towards t he non-structural proteins and CP of RRSV as probes, the expression cycles af ter inoculation were detected applying Western blot, the results showed that RR SV in rice protoplasts non-structural proteins P7 and P10 began to express at 12 h, then reached their climax at 32 h, P8 as CP of RRSV began to expre ss at 16 h then reached its climax at 48 h; (5) RRSV genes were cloned emp loying transient expression system of tobacco to express the fusion YFP-RRSV proteions, localization of fluorescent protein in tobacco leaves was observed at 488 nm using confocal laser-scanning microscope, it was found that RRSV P ns6 targeted in cell membrane, P8 was observed into nucleus, Pns10 accumulat ed in large amounts of inclusion bodies in cytoplasm and formed viroplasm-lik e inclusions.