Figure 1
Analyses of the upstream regulator of ThbZIP1. A: E-box and its mutated sequences. B: The bindings of ThMYC6 / ThMYC4 to E-box and the mutated E-box sequences. a: pHIS2-p53 + pGAD-Rec2-p53 (positive control); b: pHIS2-E-box + pGAD-Rec2-ThMYC6/ThMYC4; c: pHIS2-E-M1 + pGAD-Rec2-ThMYC6/ThMYC4; d: pHIS2-E-M2 + pGAD-Rec2-ThMYC6/ThMYC4; e: pHIS2-E-M3 + pGAD-Rec2-ThMYC6/ThMYC4; f: pHIS2-p53 + pGAD-Rec2-ThMYC6 /ThMYC4 (negative control). pGAD-Rec2-53: p53 as a fusion with the GAL4 AD in pGAD-Rec. pHIS2-E-box, -E-M1, -E-M2, -E-M3: three tandem copies of E-box, its mutants E-M1, E-M2 and E-M3 (shown in Figure 1A) respectively inserted into pHIS2. pGAD-Rec2-ThMYC4, -ThMYC6: ThMYC4 and ThMYC6 as a fusion with the GAL4 AD in pGADT7-Rec2. The triangle indicates serial dilutions (1/1, 1/10, 1/100). C: The promoter fragments of ThbZIP1 with or without the cis-elements used in analyses of the yeast one-hybrid and coexpression of reporter and effector vectors. The black lines indicate the E-box, and the grey lines show the promoter fragments. D: The bindings of TFs to promoter fragments with or without the E-box motifs using yeast one-hybrid analysis (shown in Figure 1C). a: pHIS2- p53 + pGADT7-Rec2-p53(positive control); b: pHIS2-ProE(+) + pGADT7-Rec2-ThMYC6/ThMYC4; c: pHIS2-ProE(-) + pGADT7-Rec2-ThMYC6/ThMYC4; d: pHIS2-p53 + pGADT7-Rec2-ThMYC6/ThMYC4 (negative control). pHIS2-ProE(+), -ProE(-): pHIS2 harboring the promoter fragments containing E-box, or without E-box motifs. E: Diagrams of the reporter and effector vectors. F: The coexpression of reporter and effector vectors in tobacco leaves. pCAM-E-box, -E-M3: three tandem copies of E-box, or its mutant E-M3 were respectively fused to a minimal promoter to drive GUS. -E-boxp+, -E-boxp: one copy of promoter fragment containing E-box, or without E-box was respectively fused to a minimal promoter for driving GUS. pROKII-ThMYC6: the ORF of ThMYC6 was under the control of CaMV 35S promoter. G: The GUS activity assay of the coexpression of reporter and effector plasmid. Error bars indicate SE.