Nicholas N. Lyssenko and Mark L. Tucker, Soybean and Alfalfa Research Lab, USDA, ARS, Bldg. 006, BARC-West, Beltsville, MD, USA 20705
In 1987 Jefferson et al. described the use of β-glucuronidase (GUS) as a marker to study gene expression in plants. Expression of GUS activity in plant tissue can be quantified by fluorometry or assayed qualitatively by histochemistry. The GUS enzyme cleaves the commercially available substrate 4-methylumbelliferyl-β-D-glucuronic acid (MUG) to produce fluorescent methylumbelliferone (MU). The VersaFluor fluorometer, when equipped with the simple-to-install 360 excitation and 460 emission filters, has the necessary sensitivity to accurately measure MU in plant tissues. Quantification of GUS activity using the VersaFluor fluorometer is relatively inexpensive and reliable. We have used the instrument to quantify transient expression of a CaMV 35S-GUS construct in bean abscission zones. Abscission is the process by which plants shed organs. The bean abscission cellulase (BAC) accumulates tissue specifically in induced abscission zones. GUS expression is used as an internal control in experiments designed to identify regulatory regions in the BAC gene promoter. An example of the approach used and results obtained is described below. In addition to quantification of GUS activity, we use the VersaFluor fluorometer equipped with the same set of filters to quantify the DNA preparations. The fluorescent dye for DNA quantification is Hoechst 33258 from Bio-Rad.
Materials and Methods
The promoter to be tested for hormonal or tissue-specific expression is fused to the luciferase gene taken from the pDO432