FIGURE 4.

The binding and regulatory activity of ZmDof30 on the p184 promoter. Analysis of ZmDof30 DNA-binding specificity with AAAG motifs located in p184 by EMSA (A) and a yeast one-hybrid system (B). E (p184) sequence containing two tandem AAAG core motifs; E (p184)-M sequence containing mutations from AAAG to AATC. The oligonucleotide probes with three tandem repeat E (p184) sequence and E (p184)-M sequence were synthesized and labeled with biotin for EMSA. Twin tandem E (p184) and E (p184)-M sequences were cloned into pAbAi vectors as bait. (C) Analysis of ZmDof30 regulatory activity on p184 in transient expression assays in tobacco leaves. The p184::GUS reporter plasmid and the pSuper::ZmDof30 effector plasmid were co-transformed into Nicotiana benthamiana leaves. The empty vector (pSuper1300) served as a control. GUS activity was normalized to LUC activity derived from an internal control plasmid. The average GUS/LUC value of the control was normalized as 1. The data are represented as the mean ± SD of the results from three independent experiments. Significant differences were assessed by the Student’s t-test using a P-value < 0.05.