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
3Page: All 1 2 3 4 Related biology technology :1
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