2-Mercaptoethanol → 4-Methylumbelliferone

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To evaluate the activity of various promoters, GUS fluorescence was assayed by the method of Jefferson (1987). Maturing seeds at 17 DAF were homogenized in GUS extraction buffer (50 mM NaPO4 [pH 7.0], 10 mM 2-mercaptoethanol, 10 mM Na2-EDTA, 0.1% SDS, 0.1% Triton X-100). After centrifugation, 10 μl of the supernatant was mixed with 90 μl of assay buffer containing 1 mM 4-methylumbelliferyl-β-D-glucuronide (MUG). After incubating for one hour at 37° C., 900 μl of 0.2 M Na2CO3 was added to the mixture to terminate the reaction. Values obtained using a fluorometer were compared with those obtained from serial dilutions of 4-methylumbelliferone (4MU). The protein amount was determined using a BIG-RAD Protein Assay kit, with serum albumin as the standard. Three seeds were assayed for each transgenic plant.

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