Total nuclease activity

Total nuclease activity was determined as described by Young and Gallie (2000), which was modified as follows. Thirty axes from seeds subjected to accelerated ageing for different periods of time were homogenized to a fine powder under liquid nitrogen. The soluble proteins were then extracted by grinding the powder in an extraction buffer composed of 150 mM tris-HCl at pH 6.8, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethanesulfonyl fluoride and 20 ||M leucin. The homogenate was centrifuged at 15,000g for 10 min, after which 50 |ll of the supernatant was added to 1.5 ml reaction medium including 50 mM tris-HCl at pH 6.8, 1 mM CaCl2, 1 mM MgCl2 and 1 mM ZnCl2. Then 50 |ll denatured (100 |g/ml) salmon sperm DNA was added to the reaction mixture, incubated at 37°C for 15 min and the absorbance of the mixture at 260 nm was determined. In this assay one unit of nuclease is defined as the amount required to decrease the absorbance of DNA at 260 nm by 0.01 AU. The specific activity of the nuclease was expressed as unit/mg protein/min.

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