In Vitro Replication System Mediated By RNA-dependent RNA Polymerase(RdRp) In Tobacco Bushy Top Virus

Tobacco bushy top virus(TBTV),a member of the genus of Umbravirus in the family of Tombusviridae,and Tobacco vein distorting virus(TVDV),a member in the family of Luteoviridae,co-infected tobacco causing tobacco bushy top disease.TBTV genome contains a positive single strand RNA with the length of 4 152 nts,which encodes four open reading frames(ORFs).ORF1 encodes 35 kDa protein,whose C-terminus has overlapped 8 amino acids with the N-terminus of ORF2.TBTV RdRp was expressed from ORF1 through-1 type translational frameshift mechanism with the moelcular weight equalling to the sum of ORF1 and ORF2.Based on the data of molecular variation assay,RdRp-coding region is the most variable within TBTV genome,which implied that the frequent mutation of the RdRp may be associated with the differential virulence of different TBTV isolates.The purpose of this study was focused on the construction of in vitro replication system mediated by TBTV RdRp and primary analysis of the core elements responsible for the regulation of in vitro replication.RdRp coding sequences of Tobacco bushy top virus(TBTV)China isolate were amplified through overlap PCR and inserted into the basic vector pMAL-C2 X to construct the prokaryotic expression plasmid pMAL-TB-RdRp.RdRp fused with MBP-tag with the molecular weight of 120 kDa was expressed through the induction of 0.5 mM IPTG.Based on data of the temperature courses assay,the soluble ratio of MBP-RdRp at 18 ? is about 17 %,which is enough for the following affinity chromatography analysis against MBP-tag.The purified MBP-RdRp can specifically recognize the 3'terminal region of TBTV plus or minus strand and perform in vitro replication.In addition,in vitro replication efficiency for the minus 3' region is remarkably higher than that for the plus 3' region.This in vitro replication system will facilitate the study of mechanism on TBTV replication.Based on the structure probing on the 3'UTR,12 mutants was constructed and performed by in vitro replication,which identified the core elements of responsible for the regulation of in vitro replication.Meanwhile,three prokaryotic expression vectors respectively containing RdRp coding sequences of TB-JC,TB-MD-I and TB-MD-II were constructed followed by prokaryotic expression and affinity chromatography.The three purified RdRp of different TBTV isolates also have the activity catalyzing in vitro replication of TBTV 3'UTR.This study facilitated the future work on the comparation among different TBTV RdRp and identification of key mutation amino acids in RdRp.