Promoter activity analysis using dual luciferase assay

A dual luciferase assay vector pGreenII0800-LUC was used to analyze the activity of the target promoter. This vector contains firefly luciferase (LUC) reporter gene that can be driven by the target promoter and Renilla luciferase (REN) reporter gene driven by 35S. The purified DNA fragment of the target promoter was fused with LUC reporter gene in the vector digested with HindIII and Sa1I enzymes to construct the recombinant vector. The vector pGreenII0800-LUC without promoter insertion before LUC reporter gene was used as negative control. The recombinant and negative control vectors were individually transformed into maize and rice protoplasts or tobacco (Nicotiana benthamiana) leaves. In the protoplast transient expression experiments, the isolation of protoplasts from maize or rice green leaves, PEG-calcium transfection of plasmid DNA, and protoplast culture were performed according to standard protocols [52]. In the leaf transient expression experiment, tobacco plants were grown until at least six leaves were available. The transfection of plasmid DNA into tobacco leaves by infiltration with Agrobacterium and transient expression assay followed standard protocols [53].

The ratio of LUC and REN activity (LUC/REN) was used to reflect the activity of the target promoter. The LUC/REN value was determined using the dual luciferase reporter assay system (Promega). Briefly, the transformed protoplasts or homogenate prepared from transformed tobacco leaves were centrifuged at 12000×g for 15 s at room temperature, and the supernatant was removed. Next, 100 μl of passive lysis buffer was added for further homogenization. Twenty microliters of lysate was mixed with 100 μl of LAR II, and then the LUC activity was measured using a GloMax 20/20 luminometer (Promega). Finally, 100 μl of Top & Glo reagent was added to the reaction, and the REN activity was measured.