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Nicholas N. Lyssenko and Mark L. Tucker, Soybean and Alfalfa Research Lab, USDA, ARS, Bldg. 006, BARC-West, Beltsville, MD, USA 20705
Introduction
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 plasmid (Ow et al.,
1986; see Koehler et al., 1996, for details of promoter construction).
The GUS gene is fused to an enhanced 35S promoter (Kay et al., 1987).
An accurate measure of the concentration of the gene constructs is determined
with the VersaFluor fluorometer using the Hoechst 33258 dye from Bio-Rad.
The two constructs are then co-precipitated at constant and equal-molar
concentrations onto gold particles (Figure 1A). Plant material is bombarded
with the gold particles using the Biolistic PDS-1000/He particle gun (Bio-Rad)
as depicted in Figure 1B.
Following particle bombardment and a 48-hour incubation period in ethylene (a plant hormone that induces abscission), approximately 1 mm of tissue is harvested from the upper surface of the bombarded explants (abscission zones, petioles and stems), and frozen separately in liquid nitrogen (Figure 1B). The protein extraction procedure is modified from the original protocol described by Jefferson et al. (1987) to allow the assay of cellulase, luciferase, and GUS activity from the same tissue extract. Approximately 150 mg of plant tissue is ground in liquid nitrogen, thawed in 450 l of extraction buffer (0.1 M sodium phosphate, pH 7.8, 1% Triton X-100, 2 mM EDTA, 1 mM DTT and 0.1% BSA), vortexed and then centrifuged at 10,000 rpm in a microcentrifuge for 4 minutes.
The supernatant is then transferred to new tubes in 75 l aliquots for measurement of cellulase and luciferase activity as described elsewhere (Koehler et al., 1996). For GUS assay, 75 l of the supernatant is combined with 25 l of a GUS adjustment buffer (0.4 M citrate buffer pH 5.5, 34 mM EDTA and 17 mM DTT) and the combined mixtures incubated at 55 C for 30 minutes. The GUS marker enzyme, originally from E. coli, is resistant to heat inactivation at 55 C; however, heating significantly reduces endogenous GUS-like activity in plant extracts. Following incubation, the mixture is combined with 20 l of methanol, which further reduces background GUS-like activity (Kosugi et al., 1990), and 12.5 l of 10 mM MUG. Immediately after addition of MUG, 10 l of the mixture is removed and combined with 2 ml of 0.2 M sodium carbonate to stop GUS conversion of MUG to MU. These tubes are labeled T0. The remaining mixture is incubated at 37 C overnight and the T0 tubes stored in the dark at room temperature. Following incubation at 37 C, 10 l of the mixture is combined with 2 ml of 0.2 M sodium carbonate and the tube labeled T12.
MU standards are prepared at 50, 100 and 500 nM in 0.2 M sodium carbonate. The VersaFluor fluorometer is warmed for 15 minutes before conducting the assay. The instrument has low, medium and high gains. The range of MU standards described above is best measured at a medium gain.
The instrument is zeroed with a blank consisting of 0.2 M sodium carbonate. First, the highest standard, 500 mM, is measured and set to equal 5,000 relative units with the range option of the fluorometer. Next, the two remaining standards are measured to ascertain a linear response. Finally, the fluorescence of extracts in the T0 and T12 tubes is measured and recorded.
Results and Discussion
Fusion of the enhanced CaMV 35S promoter (Kay et al., 1987) to the GUS
reporter gene creates a gene construct (D35S) that expresses GUS constitutively
in transformed plant tissues. Inclusion of this construct is used as an
internal control to reduce variability in all our particle bombardment
experiments (Figure 1A). The expression results (Figure 2B) are reported
as the ratio of luciferase to GUS activity normalized to 100% for BKm3
expression in abscission zones. Expression of luciferase from the test
promoter, the BKm3 construct (Figure 1A), relative to GUS expression from
D35S (i.e., luciferase to GUS ratio) in stems and petioles is approximately
10% of the expression in abscission zones (Figure 2B). This pattern of
expression is very similar to that for native cellulase activity measured
in the same extracts collected from particle bombarded stems, petioles
and leaf abscission zones (LAZ) (Figure 2A).
The VersaFluor fluorometer is inexpensive and easy to use. It has the necessary sensitivity to reproducibly measure transient GUS activity in particle gun bombarded plant tissues. In addition, using the Hoechst 33258 dye to quantitate DNA by the VersaFluor fluorometer is reliable, simple, and accurate.
References
1. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., EMBO 6, 3901-3907
(1987).
2. Kay, R., Chan, A., Daly, M. and McPherson, J., Science, 236, 1299-1302 (1987).
3. Koehler, S. M., Matters, G. L., Nath, P., Kemmerer, E. C. and Tucker, M. L., Plant Molec. Biol., 31, 595-606 (1996).
4. Ow, D. W., Wood, K. V., DeLuca, M., DeWet, J. R., Helinski, D. R., and Howell, S. H., Science, 234, 856-859 (1986).
Triton is a trademark of Rohm and Haas. Biolistic is a trademark of E.I.
Du Pont de Nemours and Co. Biolistic technology is exclusively licensed to
Bio-Rad Laboratories.
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