Niu Yajie, Sheen Jen
Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.
Methods Mol Biol. 2012;876:195-206. doi: 10.1007/978-1-61779-809-2_16.
The protoplast transient expression system has become a powerful and popular tool for studying molecular mechanisms underlying various plant signal transduction pathways. Arabidopsis mesophyll protoplasts display intact and active physiological responses and are easy to isolate and transfect, which facilitate high-throughput screening and systematic and genome-wide characterization of gene functions. The system is suitable for most Arabidopsis accessions and mutant plants. Genetic complementation of mutant defective in sensor functions, gene expression, enzymatic activities, protein interactions, and protein trafficking can be easily designed and explored in cell-based assays. Here, we describe the detailed protocols for protoplast isolation, polyethylene glycol-calcium transfection, and different assays for quantifying the output of various signaling pathways.
原生质体瞬时表达系统已成为研究各种植物信号转导途径潜在分子机制的强大且常用的工具。拟南芥叶肉原生质体表现出完整且活跃的生理反应，易于分离和转染，这有利于高通量筛选以及基因功能的系统和全基因组表征。该系统适用于大多数拟南芥种质和突变体植物。在基于细胞的分析中，可以轻松设计和探索在传感器功能、基因表达、酶活性、蛋白质相互作用和蛋白质运输方面存在缺陷的突变体的遗传互补。在此，我们描述了原生质体分离、聚乙二醇-钙转染以及用于量化各种信号通路输出的不同分析方法的详细方案。
