A Protocol for the Fluorometric Quantification of mGFP5-ER and sGFP(S65T) in Transgenic Plants |
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Authors: | Remans Tony Schenk Peer M Manners John M Grof Christopher PL Elliott Adrian R |
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Institution: | (1) Department of Biochemistry, The University of Queensland, Brisbane, QLD, 4072, Australia;(2) Department of Biochemistry and Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Brisbane, QLD, 4072;(3) CSIRO Tropical Agriculture, 120 Meiers Road, Indooroopilly, QLD, 4068, Australia |
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Abstract: | The Green Fluorescent Protein (GFP) from Aequorea victoria has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluorTM Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 ± 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 g and 2.11 g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 g mGFP5-ER per mg extractable protein. |
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Keywords: | green fluorescent protein sugarcane tobacco transgene expression level |
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