Dual-luciferase assay
Dual-luciferase (Dua-Luc) assays were carried out as described by He et
al (2021). The promoter regions of CsJAZ6 and CsANS were
sub-cloned into the pGreenII 0800-LUC vector as reporters. The cDNA
sequences of CsHSFA1b , CsHSFA2 , CsJAZ6 ,CsAN1 , CsEGL3 , and CsTTG1 were inserted into the
pCambia2300 vector as effectors. The pCambia2300 empty vector was used
as a negative control. Tobacco leaves were sprayed with 100 mmol/L
fluorescein (Promega) and left in the dark for 5 min. Then, a low-light
cooled charge-coupled device imaging device (NightOWL II LB983) were
used to capture Luc images. LUC and REN activities of the CsJAZ6and CsANS promoters were measured with the Dual-Luciferase
Reporter Assay kit (Promega Corporation, Wisconsin, USA).