Collings David A
Biomolecular Interaction Centre, School of Biological Sciences, The University of Canterbury, Christchurch, New Zealand.
Methods Mol Biol. 2013;1069:227-58. doi: 10.1007/978-1-62703-613-9_16.
The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.
植物基因组学数据近期的大规模扩展产生了大量的基因序列，对于这些序列有两个看似简单的问题需要回答：这些基因编码的蛋白质在细胞中的定位在哪里，以及它们发挥什么作用？一种广泛用于回答定位问题的方法是使用粒子轰击来瞬时表达用绿色荧光蛋白（GFP）或其众多衍生物标记的未知蛋白质。然后使用共聚焦荧光显微镜来监测荧光蛋白在细胞内的定位情况。如果融合蛋白的亚细胞定位不明显，那么可以通过与荧光蛋白与已知信号序列和蛋白质的融合所产生的定位进行比较，或者通过与荧光染料直接比较来确定。这篇综述旨在为想要踏上这条搭便车之路的研究人员以及希望理解他们旅行报告的审稿人和读者充当导游。它将描述一些可用于可视化蛋白质定位的技术，以及一些用于优化和确认在洋葱表皮细胞（最常用的实验系统）中通过粒子轰击产生的定位的实验方法。由于在诸如洋葱等异源表达系统中信号序列的不保守性以及随之而来的融合蛋白错误靶向始终是一个潜在问题，因此豌豆的银叶突变体的表皮细胞被提议作为一个模型系统。
