Assessment of the Effectiveness of a Nuclear-Launched TMV-Based Replicon as a Tool for Foreign Gene Expression in Plants in Comparison to Direct Gene Expression from a Nuclear Promoter |
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Authors: | Michal Man Bernard L Epel |
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Institution: | (1) Department of Plant Sciences, the George S. Wise Faculty of Life Sciences, Tel Aviv University, 69778 Tel Aviv, Israel |
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Abstract: | An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses
the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between
an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal
nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a
control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually
expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast,
GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold
less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the
TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based
replicon was ca. 66-fold lower than produced by the control. |
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