Grantee Research Project Results
2003 Progress Report: Designing Promoters for Precision Targeting of Gene Expression
EPA Grant Number: R829479C002Subproject: this is subproject number 002 , 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: Designing Promoters for Precision Targeting of Gene Expression
Investigators: Harding, Scott A. , Tsai, Chung-Jui
Institution: Michigan Technological University
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2003 through December 12, 2004
Project Period Covered by this Report: January 1, 2003 through December 12, 2004
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text | Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation
Objective:
The goals of this research project are to: (1) construct nested deletion series for Pt4CL1 and Pt4CL2 promoters, and prepare promoter deletion::glucuronidase (GUS) fusion gene constructs—generation of approximately 20 deletion constructs; (2) conduct aspen transformation with Pt4CL1 and Pt4CL2 promoter deletion::GUS constructs—generation of 5-10 independent transgenic lines per construct, a total of 100 to 200 transgenic aspen plants; and (3) characterize transgenic plants via molecular and histochemical analyses—greenhouse maintenance and characterization of all transgenic plants.
Progress Summary:
To date, 9 months after the project started in January 2003, most of the progress toward the listed objectives, specifically as stated, has been limited to preparation of the nested deletion series of the Pt4CL1 gene promoter sequence, and preparation of the vector into which each promoter deletion eventually will be ligated. Regarding construction of nested deletions of 4CLI promoter, the following applies:
(1) Twelve sense primers and one reverse primer were designed to PCR-amplify a nested series of 5' truncated sections of the 4CLI promoter sequence. With respect to the transcription start site (position 1), and with 5' to 3' numbering, these span the bases: -801/+77, -688/+77, -531/+77, -464/+77, -414/+77, -355/+77, -278/+77, -265/+77, -243/+77, -176/+77, -148/+77, and –52/+77.
(2) The resulting promoter fragments were sequenced for verification, gel purified, and stored at -20°C for ligation into the binary vector pBI121.
(3) Ligation of the 4CLI promoter series into the binary vector pBI121 is ongoing. A working quantity of pBI121 was obtained by transforming competent TOP10F1 Escherichia coli cells with limited existing quantities of the vector. This increased the vector yield from alkaline lysis plasmid preparations over that obtained from previously used in-house lines of E. coli. After vector purification, the restriction enzymes HindIII and XbaI were used to remove the 35S promoter from the vector backbone. The prepared vector was gel purified, and ligation with the 4CL1 promoter deletions is underway.
(4) Promoter deletion constructs -688/+77::GUS and -278/+77::GUS have been obtained successfully. Mobilization of the binary vector into Agrobacterium tumefaciens soon will be initiated for aspen transformation.
Aspen Transformation
Transformation of aspen with the full-length 4CL1 promoter::GUS and full-length 4CL2 promoter::GUS has been initiated. So far, more than 10 independent callus lines (putative transformants) have been obtained for each construct, and are maintained on TDZ containing media for shoot regeneration. Whole plant regeneration is anticipated within 2 months. In addition to the progress described above, one of the students working on this project has developed considerable skill at the histochemical detection of GUS reporter activity using previously generated GUS-transformed aspen plants. This work is particularly time consuming, but already has prepared the student for assaying GUS expression driven by the 4CL1 promoter fragments in Objective 3 of the project.
Future Activities:
Complete all stated objectives of the project.
Supplemental Keywords:
promoters, aspen, transgenic, gene expression, molecular analysis, histochemical analysis, sense primer, reverse primer, GUS., Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Geochemistry, Technology, New/Innovative technologies, Environmental Engineering, Agricultural Engineering, bioengineering, genetics, T-DNA, transgenic plants, plant genes, biotechnology, remediation, gene expression, promoters, histochemical analysisProgress 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.