TBSV Viral Vector Exploiting RNA Polymerase ⅠMediated Transcription

RNA plant virus-based expression system is a novel expression system developed in recent years. RNA polymerase II promoter (II promoter) are used in most of the existing RNA plant virus-based expression vector. Comparing with other plant RNA viruses, tomato bushy stunt virus (TBSV) has the potential advantages of bio-security, foreign gene carrying capacity and expression efficiency. TBSV has no cap structure, poly (A) tail and introns. If TBSV viral vectors are placed downstream of the RNA Pol I promoter, the transcripts is more like the wild-type virus more, then the virus infectivity could be effectively improved. In addition, plant RNA polymerase I-dependent transcription is species-specific; it is also helpful to improve the biological safety of virus-based expression system.we have had a preliminary study of the TBSV Viral Vector Exploiting RNA Polymerase I Mediated Transcription:514bp of the N. benthamiana type I promoter DNA sequences were cloned, which contains the plant RNA polymerase I promoter core sequence. The results of multiple sequence alignment showed its transcription initiation site located in the third adenine (A) of core sequence. The TBSV viral vectors QW which transcript by the N. benthamiana I promoter was built. Through Agrobacterium infiltration technology, it was inoculated in host plant N. benthamiana.5’-RACE technology which were measured N. benthamiana type I promoter and the CaMV35S promoter (type II promoter)-mediated transcription of TBSV virus5’end of the first base. The results indicated that we can get the full5’end of the viral genome RNA by two types of promoter transcription, and this results further validated the bioinformatic prediction. By comparing with the whole plant, cellular and molecular levels of comparative studies show that N. benthamiana I promoter mediated of TBSV viral vector’s Expression efficiency is higher than the type Ⅱ promoter (CaMV35S promoter) mediated of TBSV viral vectors. The results of virus vector mixed with the RNA Silencing Supressors (RSSs)2b inoculated showed that the strategy of inoculated RSSs that could improve plant RNA virus vector expression efficiency is not only for the type II promoter-mediated viral vector expression system, but also applies to the type I start viral vector-mediated expression system. We speculated that the improvement of efficiency for the type I promoter-mediated transcriptional expression vector is not due to avoid the virus-induced gene silencing (VIGS). Type I promoter for maintaining the integrity of the virus vector and the efficiency of transcription may be the key to the improvement of the efficiency for vector expression. We studied the TBSV virus type I promoter-mediated transcriptional expression vector host range, and the results indicated that the type I promoter could narrow TBSV virus expression vector’s host range. At a certain degree, the bio-safety of TBSV virus expression vector has been improved.Those results will help TBSV virus expression vectors to be a new expression vectors which are more suitable for Drug research. Meanwhile, the industrialization of production prospects exogenous gene expression will lay a solid theoretical foundation for the future guidance.