2.3.4 Enzyme activity measurement in Exp. 3
The methods for enzyme activity of starch synthesis were slightly modified from those described by Nakamura,Yuki, Park, & Ohya, 1989 and Zinselmeier et al. 1995. Four grains from each treatment were sampled at DAP 10, 25 d after vitro culture in Exp. 3, then frozen in liquid nitrogen for 2 min and stored at -80℃ for enzyme activity measurements. The extract contained the following: 50 mM Hepes; 2 mM MgCl2·6H2O; 3% (V/V) (CH2OH)2; 1 mM DTT; 1 mM EDTA-2 Na; 1 N NaCl.
ADP Glc pyrophosphorylase (EC 2.7.7.27) reaction system: 50 mM Hepes-NaOH (pH 7.2) 100 μL; 10 mM MgCl2·6H2O 50 μL; 15 mM DTT 100 μL; 1.5 mM ADPG 100 μL; 20 mM PPi 100 μL; Extract 200 μL.
Starch synthase (EC 2.4.1.21) reaction system: 50 mM Hepes-NaOH (pH7.2) 50 μL; 15 mM DTT 50 μL; 1.5 mM ADPG 50 μL; 1 mg Amylopectin; Extract 200 μL.
Sucrose invertase (EC 3.2.1.26) reaction system: CH3COOH-CH3COONa buffer (pH 4.8) 200 μL; 100 mM Sucrose 200 μL; Extract 100 μL.
Sucrose synthase (EC 2.4.1.13) reaction system: 50 mM Hepes-NaOH (pH7.2) 100 μL; 10 mM UDP 100 μL; 10 mM MgCl2·6H2O 50 μL; 100 mM Sucrose 150 μL; Extract 100 μL.