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. 2017 Mar 7;8:299. doi: 10.3389/fpls.2017.00299

FIGURE 4.

FIGURE 4

Sequence-specific binding of GmERF113 to the GCC-box. (A) Nucleotide sequences of the GCC-box and mGCC-box probes. The core GCC sequences are underlined and the mutated nucleotides in the mGCC probe are in italics. (B) SDS-PAGE analysis of the purified recombinant GmERF113 protein using His-Bind Kits. (C) EMSA showing sequence-specific binding to the GCC-box of the recombinant GmERF113 protein. Lane 1, titration with cold GCC sequence as a competitor; lane 2, labeled mGCC probe and GmERF113 protein; lane 3, labeled GCC probe and GmERF113 protein; lane 4, free GCC probe only. (D) The binding activity of GmERF113 to the GCC-box sequence motif in a yeast one-hybrid assay. Yeast cells were selected on SD (-Trp, -Leu) and SD (-Trp, -Leu, -His) media plates supplemented with 100 mM 3-amino-1,2,4-triazole (3-AT). (E) Schematic diagram of the reporter and effector plasmids. Reporter plasmids included four tandem copies of the GCC-box and 35Smini, and effector plasmids encoded GmERF113 under the control of the CaMV 35S promoter. (F) Relative GUS activities in transactivation assays. The numbers represent the fold increase in GUS activity compared with the control vector GCC-box/35Smini promoter (GCC 35SMini) alone. Results are presented as averages of three replicates ± standard deviation.