Genome Structure Analysis On Segment 6 To Segment 10 Of Rice Ragged Stunt Virus Chinese Isolates

Rice ragged stunt virus(RRSV)is a typical member of the genus oryzavirus within the family Reoviridae,causing significant rice disease in South Asia and Southeast Asia.The dsRNA was extracted from RRSV infected rice tissue using CF-11 cellulose column chromatography and further purified through agarose gel electrophoresis.The PCR primers were designed according to coresponding sequence of the Thailand isolate(TH).The complete sequences of segment 6 to 10 of RRSV CT,SX and GD isolates were successfully amplified by RT-PCR and cloned into the pMD18-T vector. The cloned vectors were transformed into E.coli DH5α.The coloned genes were sequenced after verification using plasmid double degestion,plasmid PCR and single enzyme digestion of PCR products.Homology analysises of S₆-S₁₀segments of the CT,SX and GD isolates were performed.The variable sites were analysed.Priliminary analysis on the nucleotide sequences showed that S₁₀and S₇ of the three isolates were 99%homolgous to S₁₀ and S₇ of the TH isolate,respectively;SX-S₉ was 98%homolgous to the TH-S₉,CT-S₈ and GD-S₈ were 99%homolgous to the TH-S₈;SX-S₈ was 93%homolgous to the TH-S₈.The ORFs of the segments obtained in this study were the same as those of the coresponding TH isolate expect those of the SX-S₆,SX-S₇,SX-S₈ and GD-S₇.The difference was because of nucleotide deletions in these segments which resulted in framshifts.Bioinformatical analysises using the predicted amino acid sequences were also performed in virtue of biological software and correlative analysis tools online.The analysises included physicochemical properties,functional domains and secondary structure of the proteins.The results indicated:P6 possessed nuclear localization signal which were mapped to the 377 th(PNSPKKR)and the 380th(PKKREKA) animo acids;each of the NS10,P9and P8 has one R-2 motif respectively;P9 had a coiled-coil region;all five proteins have no signal peptides.