Abstract: The conventional method of protein subcellular localization is to build the vector with the expression of fused target gene and green fluorescent protein gene（GFP）driven by 35S promoter. The subcellular localization of the target protein is then determined in the cells transiently expressing the fusion gene. Utilization of 35S promoter will lead to overexpression of the fusion gene and obtaining strong GFP signal. But sometimes the excessively synthesized protein will possibly remain in the transportation organelle or the areas exceeding the native protein location. The aim of this study is to solve this problem in the study of protein subcellular localization. To accurately determine the subcellular localization of Flavin-Containing Monooxygenase 1（FMO_GS-OX1）in model plant Arabidopsis,a protein inhibitor,cycloheximide was applied to repress the over-expression of FMO_GS-OX1-GFP fusion protein in tobacco epidermal cell. The results showed that before the treatment of cycloheximide,strong GFP signal was presented in both ER and cytosol. While after treated with cycloheximide,GFP signal disappeared in ER but remained in cytosol. This study demonstrated that proper treatment of cycloheximide may effectively avoid the excessive over-expression of the gene driven by 35S and thus is conducive to precisely determine the intercellular position where the protein facilitates its function.

Key words: GFP, FMOGS-OX1, subcellular localization, cycloheximide, endoplasmic reticulum