Dual-Luciferase Assay for Promoter Analysis

For the dual-luciferase (Dual-LUC) assay, the pGreenII 0800 LUC plasmid (Hellens et al., 2005 (link)) was modified as the effector vector. A Gateway R1R2 cassette and a 66 bp mini 35S promoter were PCR amplified and sequentially inserted into the multicloning site (MCS) upstream of the firefly luciferase gene in the effector vector, to make it compatible for the Gateway cloning technique and capable to test promoter sequences lacking a TATA-box or other essential elements of transcription. The Probe-1^MS1 sequence was first TA TOPO (Thermo Fisher Scientific) cloned into pCR8 and LR cloned into the effector vector. The MS188 coding sequence was Gateway cloned into the pUB-DEST vector (Grefen et al., 2010 (link)). All vectors were co-infiltrated into tobacco leaves with p19 of Tomato bushy stunt virus to enhance the transient expression. The infiltrated leaf tissues were collected 3 d after transfection and assayed for the LUC and REN levels using the Promega Dual-Glo® Luciferase Assay Kit (E2920) and BioTek Synergy LX Microplate Reader in accordance with the manufacturer’s instructions.