Establishment Of The Quantative Systems Of Translational Frameshift Responsible For The Expression Of Helicases And Genome Replication Of Tobacco Bushy Top Virus

Tobacco bushy top virus (TBTV), a member of Umbravirus, and Tobacco vein distorting virus (TVDV), a member of Luteoviridae co-infected tobacco and caused tobacco bushy top disease.TBTV genome is a (+) ssRNAwith4152nucleotides, predictively encoding4ORFs. ORF1and ORF2, respectively encoding35kD and63kD proteins, have24nts overlapped region at the C-terminal region of ORF1and N-terminal region of ORF2. Based on the previous reports about other members of Umbravirus, it is speculated that ORF2may be expressed through post-translatinal frameshft mechanism and exist as ORF1-extended protein or ORF1/ORF2fused protein, which function as RNA dependent RNA polymerase (RdRp).In this thesis, the post-translational frameshift mechanism responsibe for the expression of ORF2was firstly determined as following. TBTV ORF1-encoded sequence were amplified by PCR and cloned into pEHISTEV to create the prokaryotic expression plasmid pEH-TB-ORF1. After transformed into E.coli Rosetta, the pEH-TB-ORFl expressed the ORF1protein with fused His-tag under the induction of IPTG. The purified His-ORF1proteins from PAGE-gel were used to immunize the rabbit and trigger the formation of antiserum against the TBTV ORF1protein with high titer at1:243,000. Through the antigen affinity purification, anti-ORF1polyclonal antibodys were purified from the antiserum against ORF1. This polyclonal antibody could detect the expression of TBTV ORF1from different sources, such as sample of tobacco bushy top disease in the field, in vivo translation product of TBTV-RNA in Arabidopsis protoplasts and in viro translation samples of TBTV-RNA in wheat germ extract (WGE). In WGE, ORF1extended protein was also detected at1:100ratio of ORF1and the former has the approximately equal molecular weight of the sum of ORF1and ORF2, which suggested that the ORF2is expressed as ORF1-extended protein through the translational frameshift mechanism.In order to quantitively investigate the cis-elements invlolved in the frameshift mechanism, Rluc was replaced the c-terminal region of ORF2with same reading frame to create TBTV-Rluc and its high sensitivity was used to reflect the expression of ORF2associated protein due to the low frameshift ratio of ORF1-extended protein/ORF1. Based on the TBTV-Rluc, a series of mutants (point mutations on candidate heptanucleotide slippery sequences) have been constructed to assess the useness. Meanwhile anti-Rluc polyclonal antibody was prepared to detect the Rluc-associated protein. Primary data showed that this Rluc-fused TBTV expressed multi-Rluc associated proteins including the ORF1-extended protein pattern. The applications of Rluc-fused TBTV vector on the research of frameshift mechanism relys the further optimization on this system.In order to construct the in vitro system of replication directed by TBTV RdRp, the soluble TBTV RdRp was purified as following. The sequence of ORF1/ORF2fused protein was amplified by over-lapped PCR and cloned into two types of prokaryotic expression vector pEHISTEV and pMAL-C2X respectively to construct plasmid pEH-TB-ORF1/ORF2and pMAL-TB-ORF1/ORF2. After transformed into E.coli Rosetta, pMAL-TB-ORFl/ORF2expressed ORF1/ORF2-associated at high effenciency under the induction of IPTG and the soluble MBP-TBTV ORF1/ORF2fused proteins were purified through affinity chromatography. The basic conditions of in vitro system of replication are in test.In addition, TBTV ORF3and ORF4proteins were expressed and purified to perform the project on protein-protein interaction or RNA-protein interaction about TBTV in the future.