The green fluorescent protein as a marker to visualize plant mitochondria in vivo
The green fluorescent protein as a marker to visualize plant mitochondria in vivo
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DOI：
10.1046/j.1365-313x.1997.11030613.x
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发表时间：
1997-03-01
期刊：
影响因子：
7.2
通讯作者：
Hanson, MR
中科院分区：
文献类型：
--
作者：
Kohler, RH;Zipfel, WR;Hanson, MR
为了确定如何利用绿色荧光蛋白（GFP）作为体内亚细胞定位的标记物和作为植物线粒体的标记物，产生了表达GFP的转基因悬浮细胞和烟草植物，其具有和不具有线粒体定位信号。所用的第一种GFP形式GFP 1在具有低自发荧光的细胞中容易观察到，例如悬浮细胞或毛状体，但在绿色组织中被掩盖。为了在具有高自发荧光的细胞和组织（如叶）中可视化GFP，需要使用非常强的启动子（35S35SAMV）、高表达的修饰的mGFP 4编码区和更亮的GFP突变形式（S65T）。共聚焦或双光子激光扫描显微镜揭示了表达GFP或coxIVGFP的细胞中荧光的独特亚细胞定位。在表达非靶向GFP的细胞中，荧光在核质中积累，但也分布在整个细胞质中。它被排除在液泡、核仁和可能是白细胞体的圆形体之外。相反，荧光特异性地定位于表达coxIVGFP融合蛋白的细胞中的线粒体，如通过与线粒体特异性染料共定位所示。这允许在整个植物发育过程中直接观察活植物细胞和组织中的线粒体和线粒体运动。单个细胞的三维重建可以提供有关线粒体分布和数量的额外信息。
To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.