Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/3/2002
Publication Date: 8/7/2002
Citation: Cazzonelli, C.I., Velten, J.P. 2002. A transient in vivo luciferase-based promoter assay in tobacco leaves[abstract]. American Society of Plant Biologists, August 3-7, 2002, Denver, Colorado. CDROM.
Technical Abstract: An in vivo dual-luciferase reporter gene assay for analyzing transient promoter regulation in leaf tissue is described. The assay makes use of agrobacteria harboring two compatible binary plasmids containing either an intron-modified firefly (Photinus pyralis) luciferase gene regulated by a test promoter or an intron-modified sea pansy (Renilla reniformis) luciferase gene controlled by the constitutive plant SUPER promoter. Agrobacteria were infused into tobacco leaf tissues through stomatal openings and 24 hours after infection leaf discs were incubated in coelenterazine, followed by beetle luciferin, and relative light emissions continuously measured. Maximum levels of light emission from the firefly luciferase assay were normalized against those from the sea pansy luciferase assay, providing a ratio that allowed comparison of the strength of several test promoters. Applications of the described assay in screening for transcriptional and post-transcriptional enhancer elements and characterizing plant promoters are discussed.