Green fluorescent protein quantification in whole plants.
As future biotechnology applications utilize recombinant proteins as commercial products, nondestructive assays will be necessary to determine protein concentrations accurately within plant tissues. Green fluorescent protein (GFP) has been proposed as a potential marker for the monitoring of transgenic plants and quantifying recombinant protein levels under field conditions. This chapter discusses the utility of using GFP fluorescence as an indicator of protein concentrations and the methods used to quantify GFP fluorescence in whole plant tissues. Furthermore, we discuss the accuracy and effectiveness of the portable General Fluorescence Plant Meter (GFP Meter, Opti-Sciences, Inc.) compared to a laboratory-based spectrofluorometer (Fluoro-Max2, Jobin Yvon & Glen Spectra). In whole plants, GFP fluorescence was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence, and subsequently, its fluorescence intensity decreased over time. Younger leaves were significantly more fluorescent than older leaves on the same plant. GFP fluorescence intensity was directly correlated with the concentration of soluble protein per unit wet mass and with another genetically linked recombinant protein (Bacillus thuringiensis [Bt] cry1Ac endotoxin protein).
Department of Plant Sciences, University of Tennessee, Knoxville, USA.
Matthew D
MD
Halfhill
Reginald J
RJ
Millwood
C Neal
CN
Stewart
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D017930:Genes, Reporter; D007202:Indicators and Reagents; D030821:Plants, Genetically Modified / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D007202:Indicators and Reagents; D011994:Recombinant Proteins