Submitted to: Society for In Vitro Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2007
Publication Date: 6/10/2007
Citation: Palli, S.R., Tavva, V.S., Singh, A.K., Dinkins, R.D., Collins, G.B. 2007. Ecdysone Receptor Gene Switch Technology for Inducible Gene Expression in Plants. Society for In Vitro Biology Proceedings.
Technical Abstract: Inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and in the basic understanding of gene function. As a result several gene switches have been developed. However, the properties of the chemicals used in most of these switches make their use limited to research purposes. An ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide required for its regualtion is registed for field use. An EcR gene switch with a potential for use in large-scale field applications and its applicability to a variety of plant species has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to LBD of Locust migratoria retinoid X receptor (LmRXR) or Homo sapiensretinoid X receptor (HsRXR). Upon application of methoxyfenozide, the heterodimer of these two fusion proteins transactivates the luciferase reporter gene placed under the control of multiple copies of cis acting elements and a minimal 35S promoter. The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid combination and a reduction in the background expression levels was achieved by using the CfEcR:HsRXR two-hybrid combination. The performance of EcR gene switch was improved further using Hs-LmRXR chimeras and/or CfEcR mutants. The efficiency of EcR gene switches in inducing the target gene expression was also tested in functional genomic studies by regurlating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco plants. We have also carried out microarray analysis of gene expression in Arabidopsis plants containing switch components and determines that neither gene switch components nor chemical ligands cause significant changes in gene expression.