Protein Production in Tobacco from an Amplified Inducible System

This Phase I proposal will test a UV-B light inducible system in tobacco to produce exceptionally large amounts of protein. There are three principal components to the system: 1) a tightly regulated, UV-B inducible promoter fused to a geminivirus replication associated protein (Rep) coding region, 2) a geminivirus-derived vector that is rapidly amplified by the Rep protein to produce very high yields of the target protein, and 3) plant growth under light-emitting diode (LED) lighting for precise control of the expression system. After sufficient plant biomass accumulation, exposure to UV-B will trigger production of Rep. The Gemini vector DNA will then be precisely excised from the plant genome and replicated episomally, leading to very high intracellular concentrations of transgene mRNA and the encoded transgenic protein product. The specific aims of the Phase I proposal are to: 1 Assemble UV-B inducible genetic constructs to express Rep protein and introduce them into tobacco plants. 2 Test a prototype LED-based lighting system for plant biomass production. 3 Test background expression levels and upon UV-B activation, test the ability of the constructs to induce the geminivirus based expression system in tobacco. 4 Determine the total amount of protein produced per gram of plant biomass.