Production and Detection of AhGLK1 Protein

This experiment was conducted using previously described methods^{56 (link)}. The AhGLK1 coding sequence were cloned in the pGEX4T-1 vector to allow production of GST-AhGLK1 fusion proteins after induction by isopropyl β-D-1-thiogalactopyranoside (IPTG). Expression of GST-AhGLK1 was induced in Escherichia coli BL21-Codon Plus-RP (Agilent Technologies) by adding IPTG to a final concentration of 0.5 mM at 37 °C for 9 h, after which the bacteria were transferred to 16 °C overnight. GST-AhGLK1 protein was purified and used for antibody production (polyclonal, rabbit). The antibody of PORA was obtained from Agrisera (http://www.agrisera.com). Detection of AhGLK1 and AhPORA proteins by immunoblot analysis was carried out as previously described^{57 (link)}. Leaves (100 mg) of peanut or Arabidopsis were ground in liquid nitrogen and homogenized with 1 mL of sample buffer (50 mM Tris, pH 6.8, 2 mM EDTA, 10% w/v glycerol, 2% SDS and 6% 2-mercaptoethanol), then denatured at 100 °C for 5 min, and finally subjected to SDS-PAGE.