GREEN-FLUORESCENT PROTEIN AS A NEW VITAL MARKER IN PLANT-CELLS
GREEN-FLUORESCENT PROTEIN AS A NEW VITAL MARKER IN PLANT-CELLS
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DOI：
10.1046/j.1365-313x.1995.08050777.x
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发表时间：
1995-11-01
期刊：
影响因子：
7.2
通讯作者：
GALBRAITH, DW
中科院分区：
文献类型：
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作者：
SHEEN, J;HWANG, SB;GALBRAITH, DW
水母的绿色荧光蛋白(GFP)在各种异源系统中已被用作一种方便的新的生命标志物。然而，GFP在高等真核生物中的表达一直存在问题，尤其是在高等植物中。本文报道了在玉米叶肉原生质体中很容易检测到的GFP基因的表达，无论是强的组成型启动子还是热激启动子。在这个单细胞系统中，GFP发出的亮绿色荧光在紫外光或蓝光激发下都是可见的，即使在液泡的蓝色荧光或叶绿体的红色自体荧光存在的情况下也是如此。不需要外源底物、辅因子或其他基因产物。GFP在植物细胞中非常稳定，几乎没有光漂白现象。基于绿色荧光蛋白的荧光激活细胞分选后可获得活细胞。本论文进一步报道了用基因枪法将GFP基因导入拟南芥的叶和根后，在完整的组织中检测到GFP。GFP在植物细胞中的成功检测依赖于利用来自玉米的通用转录增强子或来自烟草花叶病毒(TMV)的翻译增强子来促进其表达。这一新的报告可用于监测高等植物的基因表达、信号转导、共转染、转化、蛋白质运输和定位、蛋白质相互作用、细胞分离和纯化以及细胞谱系。
The green-fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat-shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single-cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co-factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence-activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co-transfection, transformation, protein trafficking and localization, protein-protein interaction, cell separation and purification, and cell lineage in higher plants.