Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: In-Vivo Detection of Transient Luciferase Activity in Tobacco Protoplasts

Authors
item Morey, Kevin
item Velten, Jeffrey

Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: July 16, 2000
Publication Date: N/A

Technical Abstract: We are interested in testing potential enhancer sequences using tobacco protoplasts and transient expression assays. We have constructed an expression vector containing a minimal (-90) CaMV 35S promoter upstream of a luciferase gene (obtained from clontech). The minimal 35S promoter has been engineered to accept candidate DNA sequence elements which would then enhance Luciferase activity in protoplasts. The expression vector also contains the GUS gene downstream of the Peanut Chlorotic Streak Virus promoter which is used to normalize Luciferase expression. In order to quantify enhancer activity we have characterized and fine tuned an in-vivo Luciferase assay that makes use of a scintillation counter. Transformation of Tobacco protoplasts with the full length CaMV 35S promoter in front of the Luciferase gene show that intact protoplasts will take up the luciferase substrate D-Luciferin (obtained from molecular probes) resulting gin the generation of photons which can be detected in a scintillation counter. We are able to normalize Luciferase expression using a MUG assay of the GUS gene contained within the expression vector. We have currently identified two potential transcriptional enhancers derived from the 5* intergenic regions of different geminiviruses; one restoring the minimal CaMV 35S promoter to 25% of full activity and the second restoring 70% activity. Our future goals are to scale up this technique in order to examine over 100 potential enhancer elements. The enhancers identified will be used to construct synthetic promoters that will provide alternative high level expression systems for plant transgenes.

Last Modified: 8/21/2014
Footer Content Back to Top of Page