Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants.
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.
von Arnim
A G
AG
Department of Botany, University of Tennessee, Knoxville 37996-1100, USA. vonarnim@utk.edu
Deng
X W
XW
Stacey
M G
MG
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins; D002352:Carrier Proteins / Q000235:genetics; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D044767:Ubiquitin-Protein Ligases
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C471983:AT2G32950 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases