生物技术通报2017，Vol.33Issue(5)：83-88,6.DOI:10.13560/j.cnki.biotech.bull.1985.2017.05.012
利用放线菌酮提高黄素单氧化酶FMOGS-OX1亚细胞定位的准确性
Improving the Accuracy of FMOGS-OX1 Subcellular Localization Using Cycloheximide
摘要
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(FMOGS-OX1)in model plant Arabidopsis,a protein inhibitor,cycloheximide was applied to repress the over-expression of FMOGS-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.关键词
GFP/FMOGS-OX1/亚细胞定位/放线菌酮/内质网Key words
GFP/FMOGS-OX1/subcellular localization/cycloheximide/endoplasmic reticulum引用本文复制引用
夏洪宇,孔稳稳,徐蕊,苍炜,李晶..利用放线菌酮提高黄素单氧化酶FMOGS-OX1亚细胞定位的准确性[J].生物技术通报,2017,33(5):83-88,6.基金项目
国家自然科学基金基础科学人才培养基金项目(J1210069),黑龙江省教育厅科学技术研究项 目(12531003) （J1210069）
