Grantee Research Project Results
2006 Progress Report: Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
EPA Grant Number: R829479C029Subproject: this is subproject number 029 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
Investigators: Palli, S. R. , Collins, G B
Institution: University of Kentucky
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2004 through September 30, 2007 (Extended to December 31, 2007)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
The objectives are to:
- Develop ecdysone receptor (EcR) gene switches that can support tight regulation of transgene expression in tobacco plants.
- Test the utility of the EcR gene switch in functional genomics studies.
Progress Summary:
Optimization of Reporter Gene Expression Cassette
In order to develop a tight EcR gene switch, we have conducted experiments to optimize the reporter gene expression cassette by manipulating the minimal promoter sequence used to drive the expression of transgene. We cloned the luciferase reporter gene under the control of -39 35S (including TATAA box and 8 bp upstream) and -31 35S (includes only TATAA box) minimal promoter sequences and tested the expression levels of the luciferase by electroporating only the reporter gene constructs into tobacco protoplasts. The performance of these new constructs was compared with our regular -46 35S reporter construct. Compared to the -46 35S reporter construct, there was about 20 percent reduction in the luciferase activity when the luciferase gene was placed under the control of -39 35S minimal promoter and 65 to 70 percent reduction in the activity when the luciferase gene was placed under -31 35S minimal promoter. These truncations were then tested in a two-hybrid gene switch (CfEcR+LmRXR) format. These experiments showed a reduction in the background levels of luciferase by 25 percent when -31 35S reporter construct was used in place of -46 35S reporter construct.
Optimization of Receptor Gene Expression Cassette
We are also testing the possibility of reducing the background activity of transgene in the absence of ligand by optimizing the receptor gene expression cassette. For this purpose we adopted the autoregulation format, in which the CfEcR and LmRXR receptors are placed under the control of GAL4 response elements and a minimal 35S promoter. This is in contrast to the standard format switch where the receptor expression cassettes were under the control of constitutive promoters. We have cloned both CfEcR and LmRXR cassettes under minimal promoters and tested their efficiency in lowering the reporter gene expression in the absence of ligand. We have used -300, -90, and -46 truncations of 35S promoter to drive the expression of receptor genes in autoregulation format switch. The -300 and -90 truncations of 35S promoter did not show much difference compared to the standard two-hybrid switch. By placing the receptor genes under -46 35S minimal promoter, we could see a reduction in the background expression levels of luciferase reporter gene in the absence of ligand. Further screening of these autoregulation format switches is in progress.
RXR Mutagenesis
We performed site-directed mutagenesis on LmRXR to change the amino acid residues responsible for the heterodimerization with EcR in the absence of ligand. We have generated and screened 5 LmRXR mutants (Lm-1, Lm-2, Lm-3, Lm-4, and Lm-5) in transient expression studies and compared the results with the wild-type LmRXR construct. Of the five mutants screened, two of them (Lm-4 and Lm-5) appear to work better than the wild-type LmRXR. Lm-4 showed low background expression in the absence of ligand and Lm-5 showed high induction levels compared to the wild-type LmRXR. Now, we are planning to make a new LmRXR construct by incorporating these two mutations together and test the luciferase reporter gene expression in a two-hybrid switch.
Optimization of Translational Start Site
Optimization of the translation start site may result in higher reporter activity. We have cloned several KOZAK sequences that were optimized for better transgene expression in plants, upstream of the luciferase reporter gene translation start site. Testing of these reporter constructs will be done in transient expression studies using tobacco protoplasts.
Future Activities:
The preliminary data obtained from the optimization of reporter and receptor constructs indicate that there is an improvement in lowering the background expression levels of reporter gene in the absence of ligand. During the next few months, we will complete the screening of different minimal promoters, autoregulation format switches, LmRXR mutants, and optimization of translational start site in transient assay studies. The outcomes of these different transient experiments will be used to make binary vector(s) for plant transformation. These constructs will be used for testing the utility of EcR gene switch in functional genomics applications.
Supplemental Keywords:
RFA, Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Genetics, Sustainable Environment, Treatment Technologies, Technology, Technology for Sustainable Environment, New/Innovative technologies, Ecology and Ecosystems, Agricultural Engineering, bioengineering, transgenic plants, plant genes, biotechnology, plant biotechnology, cloning, bioenergyProgress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R829479 The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R829479C001 Plant Genes and Agrobacterium T-DNA Integration
R829479C002 Designing Promoters for Precision Targeting of Gene Expression
R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
R829479C005 Development of Novel Plastics From Agricultural Oils
R829479C006 Conversion of Paper Sludge to Ethanol
R829479C007 Enhanced Production of Biodegradable Plastics in Plants
R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
R829479C011 Biological Effects of Epoxy Fatty Acids
R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
R829479C025 Chemical Induction of Disease Resistance in Trees
R829479C026 Development of Herbicide-Tolerant Hardwoods
R829479C027 Environmentally Superior Soybean Genome Development
R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
R829479C030 Engineered Plant Virus Proteins for Biotechnology
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.