Abstract：The low efficiency for recombined RNA viruses is the limiting factor in the study of plant RNA virus-based expression vectors. To enhance the accumulation levels of recombinant proteins in plants, we explored the application potential and effect of addition of multiple introns into the viral vector in plant virus RNA-based expression system. According to the characteristics of introns in higher plant, 11 introns were selected from the Arabidopsis thaliana genome. The intron sequences were synthesized in vitro and the intron-containing viral vectors were constructed based pTBSV-G, a Tomato bushy stunt virus (TBSV)-based vector. The NetPlantGene server was employed to predict coding and non-coding sequence features in synthetic viral vectors in vitro. And then reverse transcription PCR (RT-PCR) and Western blots were used to verify whether the introns in the viral vector could be correctly identified and processed by the nuclear pre-mRNA processing machinery in vivo. The expression levels of green fluorescent protein gene (gfp) in inoculated Nicotiana excelsiana leaves were examined by Western blots and enzyme linked immunosorbent assay (ELISA) respectively. Those data were gathered to evaluate the validity and regularity of introducing introns in the coding region of the viral genome. The NetPlantGene software could accurately identify all introns inserted into the viral coding region. The result indicated that it is a useful tool for prediction of coding and non-coding sequence features in designed viral vector before experimentally inoculated to plant. The results of RT-PCR showed that the sizes of amplification products in control group were larger than those in the experimental group and it was consistent with the theoretical prediction. Western blots analysis showed that the tagged proteins (P19, P33 and P92) could be detected by using the respective antibody. The results of RT-PCR and Western blots indicated the artificial introns in the viral vector can be correctly processed in the nucleus of the host cell. Compared with pTBSV-G, the GFP accumulation levels increase of about 2 fold when the viral vectors with 2 introns or 11 introns were inoculated. However, the viral vector with 9 introns does not significantly enhance the accumulation of GFP. In conclusion, the additions of introns in viral vector were able to increase the accumulation of recombinant proteins in plants. Nevertheless, the enhancement was not associated with the numbers of intron, whereas the position of introns might play a more important role on the accumulation of recombinant proteins in plants. The use of introns in plant virus RNA-based vectors will provide the basis for applications in molecular virology studies in the near future.
