von Arnim A G, Deng X W, Stacey M G
Department of Botany, University of Tennessee, Knoxville 37996-1100, USA.
Gene. 1998 Oct 9;221(1):35-43. doi: 10.1016/s0378-1119(98)00433-8.
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.
我们构建了一系列通用的克隆载体，这些载体有助于在植物细胞中表达与维多利亚水母绿色荧光蛋白（GFP）融合的蛋白质。通过落射荧光显微镜观察，在转基因拟南芥以及洋葱表皮细胞瞬时表达后，均已创建并可视化了氨基末端和羧基末端的蛋白质融合体。利用串联二聚体和其他与GFP的蛋白质融合体，我们发现先前描述的野生型GFP定位于细胞核，最有可能是由于GFP穿过核膜扩散所致，而非存在隐秘的核定位信号。本文描述了一种基于荧光的核定位信号定量检测方法。此外，我们还利用了先前已鉴定的突变体GFP-S65T和GFP-Y66H，以便分别表达红移和蓝色荧光蛋白，适用于双标记研究。GFP融合蛋白的表达由花椰菜花叶病毒35S启动子控制。以拟南芥COP1蛋白为模型，我们证实了天然COP1和GFP标记的COP1蛋白在亚细胞定位上非常相似。我们证明COP1定位于离散的亚核颗粒，并进一步证实与GFP融合不会损害野生型COP1蛋白的活性。
