Effect of the hydrophobin HFBI-fusion tag on exogenous protein accumulation in tobacco plant / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in plants.</p><p><b>METHODS</b>The HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were examined by Western blotting, ELISA and RT-PCR.</p><p><b>RESULTS</b>The HFBI fusion tag significantly enhanced the accumulation of GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of spherical protein particles in the plant cells.</p><p><b>CONCLUSION</b>HFBI fusions can increase the accumulation of its fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in plants from agroinfection-compatible expression vectors.</p>

Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana / 南方医科大学学报

<p><b>OBJECTIVE</b>To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana.</p><p><b>METHODS</b>Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement.</p><p><b>RESULTS</b>The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the p19 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus P1 protein produced no obvious enhancement GFP expression.</p><p><b>CONCLUSION</b>Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.</p>