An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants.
Despite the success of the MUG fluorometric assay for quantitative analysis of beta-glucuronidase (GUS) activity within a vast array of transgenic plant species and tissues, attempts to apply this protocol for analysis of woody plants has been found to be problematic, primarily due to the interfering effects of phenolics and other secondary metabolites. Our analysis of transgenic spruce needles and poplar leaves illustrates that low tissue mass to extract volume, along with the inclusion of polyvinylpolypyrolidone and either beta-mercaptoethanol or metabisulphite, are essential for producing reliable results. The primary action of these additives was found to involve increased GUS extractability and the preservation of GUS activity during extract manipulation, but they were not completely effective in eliminating GUS enzymatic inhibitors. Normalization of GUS activity upon DNA concentration was also found to be an effective alternative to protein concentration, providing the ability to make cross-species and inter-tissue comparisons of gusA transgene activity.
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Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 3800, G1V 4C7 Sainte-Foy, Quebec, Canada.
Rutledge
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D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D028222:Picea / Q000235:genetics* / Q000378:metabolism; D010936:Plant Extracts / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D032107:Populus / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results
D010936:Plant Extracts; D005966:Glucuronidase