Analyzing GmPAP4 Expression in Soybean Nodules

To elucidate the expression pattern of GmPAP4 in soybean nodules, we cloned 2000 bp promoter sequences of GmPAP4 upstream of the translation start codon ATG, fused with β-glucuronidase (GUS) reporter gene (pGmPAP4:GUS). The resulting construct was introduced into the soybean hairy roots by Agrobacterium rhizogenes K599 and the transgenic hairy roots were then inoculated with rhizobia Bradyrhizobium diazoefficiens USDA110 for nodule development. After 4 weeks of rhizobia inoculation, transgenic hairy roots and nodules were stained as described previously and captured with a light microscope (Olympus U-TV0.5XC-3, Tokyo, Japan) [14 (link)]. For GUS activity, total nodule proteins were extracted and incubated in a mixture containing 10 mM 4-methylumbelliferyl β-D-glucuronide (MUG; Sigma Chemical Co., St.Louis, MO, USA) for 1 h at 37 °C. The fluorescence product of 4-methylumbelliferone (4-MU) was monitored using a VersaFluor™fluorometer (Bio-Rad, Hercules, CA, USA) with excitation at 365 nm and emission at 455 nm.