Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency
Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency
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DOI：
10.1073/pnas.97.7.3718
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发表时间：
2000-03-28
影响因子：
11.1
通讯作者：
Somerville, CR
中科院分区：
文献类型：
--
作者：
Cutler, SR;Ehrhardt, DW;Somerville, CR
我们描述了一个通用的方法，用于确定组件的亚细胞结构在多细胞生物体中利用的能力，产生数千个独立的转化体在拟南芥。构建拟南芥cDNA文库，使cDNA插入绿色荧光蛋白（GFP）编码序列的3'末端。通过农杆菌介导的转化将该文库直接导入拟南芥中。5，700株转基因植物的荧光成像表明，约2%的株系表达具有与可溶性GFP不同的亚细胞分布的融合蛋白。约一半鉴定的标记物通过非天然编码序列靶向过氧化物酶体或其他亚细胞目的地（即，框外cDNA）。这一观察结果表明，一些靶向信号是足够的法律信息内容，他们可以经常偶然产生的，该方法的潜力，以独特的动态过程中识别标志物证明了通过鉴定的GFP融合蛋白，显示细胞周期调节的变化，在亚细胞分布。我们的研究结果表明，筛选绿色荧光蛋白融合蛋白库是一个有用的方法，用于识别和可视化组件的亚细胞结构及其相关的动力学在高等植物细胞。
We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that approximate to 2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP, About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently law information content that they can be generated frequently by chance, The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.