Elsevier

Gene

Volume 221, Issue 1, 9 October 1998, Pages 35-43
Gene

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

https://doi.org/10.1016/S0378-1119(98)00433-8Get rights and content
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Abstract

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.

Keywords

GFP
Nuclear targeting
Subnuclear architecture
COP1

Abbreviations

CaMV, cauliflower mosaic virus
CCD, charge coupled device
FRET, fluorescence resonance energy tranfer
GFP, green fluorescent protein
gfp, gene encoding GFP
GUS, β-glucuronidase
NLS, nuclear localization signal
TEV, tobacco etch virus
UV, ultraviolet light
wt, wild type

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