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

doi:10.13560/j.cnki.biotech.bull.1985.2017.05.012

摘要/Abstract

摘要： 研究蛋白质亚细胞定位的常规方法是构建由35S启动子驱动目的基因与绿色荧光蛋白基因（GFP）融合的表达载体,在细胞中瞬时或稳定表达来确定该蛋白质在细胞中的定位。35S启动子的优势是能够获得较强的GFP信号,但同时也可能因为蛋白质合成量过大,导致部分蛋白滞留在运输途径中或出现在其真实定位以外的区域。为了解决这一问题,以模式植物拟南芥中黄素单氧化酶FMO_GS-OX1为例,利用蛋白质抑制剂放线菌酮处理过量表达FMO_GS-OX1-GFP融合蛋白的烟草叶片。结果表明：未经放线菌酮处理的烟草叶片表皮细胞,细胞质和内质网中均呈现了较强的荧光信号,放线菌酮处理后,内质网上的信号消失,而细胞质则呈现出稳定的信号,因此判断FMO_GS-OX1合成后可能是经过内质网运输到细胞质中的。上述结果证明适当的放线菌酮处理,能够避免强启动子驱动报告基因造成的蛋白质合成过量的问题,可有效地提高蛋白质亚细胞定位的准确性。

关键词: GFP, FMOGS-OX1, 亚细胞定位, 放线菌酮, 内质网

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

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FMO_GS-OX1亚细胞定位的准确性[J]. 生物技术通报, 2017, 33(5): 83-88.

XIA Hong-yu KONG Wen-wen XU Rui CANG Wei LI Jing. Improving the Accuracy of FMO_GS-OX1Subcellular Localization Using Cycloheximide[J]. Biotechnology Bulletin, 2017, 33(5): 83-88.

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