Preliminary Study On Replication And Translation Of Bean Common Mosaic Virus Peanut Stripe Strain

Bean common mosaic virus(BCMV)belongs to the genus Potyvirus in the family Potyviridae and has filamentous virion containing a positive single stranded RNA.BCMV genomic RNA lacks the 5' cap structure and contains a VPg at the 5' end and poly(A)at the3'end.BCMV genomic RNA is 10 kb in length and encodes a polyprotein,which be cleaved into 10 functional proteins including Nib protein.Nib protein has RNA-dependent RNA polymerase(RdRp)activity,which is responsible for the replication between genomic RNA and its complementary strand.5'UTR and 3'UTR of BCMV peanut stripe strains from different regions in shandong province were cloned using 5'RACE and 3'RACE and further analyzed using DNAMAN and MEGA software.5'UTR or 3'UTR of BCMV peanut stripe strains have high identities,however 3'UTR have different length of poly(A).The effect of 5'UTR and 3'UTR of BCMV on translation were evaluated through constructing the Firefly luciferase(Fluc)fused vectors and in vitro translation in wheat germ extract(WGE).5'UTR of BCMV showed remarkable positive effect on translation of reporter gene and it is suggested that BCMV 5'UTR has IRES activity.The core region of IRES activity is located at the 5' terminal big stem-loop structure within 5'UTR of BCMV.3'UTR inhibited the positive of BCMV 5'UTR on translation although sole 3'UTR failed to regulate the translation.It is suggested that 3'UTR and 5'UTR have long-distance RNA-RNA interaction.We also cloned Nib coding sequences of BCMV peanut stripe strains from different regions in shandong province,which have high identities.It is suggested that different BCMV peanut stripe strains may not have different RdRp activities.In this study,BCMV Nib coding sequences were inserted into pMAL-c2 x to construct prokaryotic expression vector pMALBC-Nib can express target protein(BCMV Nib fused with MBP tag)under different IPTG.In addition,soluable analysis showed that 60 % of BCMV Nib fused with MBP tag existed in supernatant,In vitro replication experiments showed that this pro-teincan specifically recognize the 3' end fragments of BCMV genomic and complementary strands.It was demonstrated thar BCMV NIv possesses RdRp activity and has specificity of region,and a preliminary establishment of BCMV Nib in vitro replication system.