Submitted to: American Society of Plant Biologists
Publication Type: Abstract Only
Publication Acceptance Date: 3/20/2004
Publication Date: 7/24/2004
Citation: Tavva, V.S., Dinkins, R.D., Palli, S.R., Collins, G.B. 2004. Development of a two-hybrid ecdysone receptor-based gene switch for plants. American Society of Plant Biologists. Page 168, Abstract 750 7/24/04 Interpretive Summary: Expression of transgenes is becoming a powerful tool in many biotechnology applications including food product improvement. Constitutive promoters, those that are expressed all the time in all tissues, are presently the primary means used to express transgenes in plants. Metabolic energy waste, negative pleiotropic effects and potential gene escape are some of the disadvantages associated with the use of constitutive promoters. To counter these problems, gene switches that can regulate the expression of transgenes through hormone analogs and antibiotics have been developed. However, most of the compounds that are used to induce gene expression are impractical or inappropriate for regulation of transgenes for large-scale use in green houses and in the field. We have developed a gene regulation system to control expression of multiple genes simultaneously and independently in the same plant by using chemicals for regulation of transgenes for large-scale production and that are presently commercially available and registered for field use.
Technical Abstract: Expression of transgenes is becoming a powerful tool in many biotechnology applications. Constitutive promoters are the primary means used to express transgenes in plants. A number of chemical inducible gene switches have been developed to overcome certain disadvantages associated with the use of constitutive promoters. An ecdysone receptor-based gene switch is one of the best gene regulation systems available, because the chemical (methoxyfenozide) required for its regulation is registered for field use. The major limitation of this gene switch is that it requires M concentration of the ligand for activation. The main goal of this work is to develop an ecdysone receptor-based gene switch that works at nM concentrations of methoxyfenozide. Constructs were made by fusing the D, E and F domains of the ecdysone receptor to the GAL4 DNA-binding domain and the E and F domains of the retinoid x receptor (RxR) to the VP16 activation domain. These two constructs along with a reporter construct (luciferase gene placed under the control of 5x GAL4 response elements and a minimal promoter) were electroporated into protoplasts isolated from cell suspension cultures of tobacco. The electroporated protoplasts were exposed to varying concentrations of methoxyfenozide and luciferase activity was measured after 24h. Based on the transient expression studies, the two-hybrid gene switch is found to be more sensitive with low background and high induction levels in the presence of 16 to 400 nM of ligand, depending on the RxRs, compared to regular gene switch where it required 2 to 10 M of ligand for similar magnitude of induction. The efficiency of this two-hybrid gene switch is also tested in