Khosla Aashima, Nelson David C
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA.
Bio Protoc. 2020 Sep 5;10(17):e3747. doi: 10.21769/BioProtoc.3747.
Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are separated during translation by a 2A "self-cleaving" peptide. This approach enables changes in the relative abundance of a target protein to be detected sensitively, reducing variability in expression of the ratiometric reporter transgene that may occur across different tissues or transformation events. We recently developed a set of Gateway-compatible plant transformation vectors termed pRATIO that combine a variety of promoters, fluorescent and bioluminescent reporters, and 2A peptides derived from foot-and-mouth disease virus. Here, we describe in detail how to use the dual-fluorescent ratiometric reporter pRATIO3212 to examine the relative abundance of a target protein after transient expression in leaves. For this example, we analyze degradation of the SUPPRESSOR OF MAX2 1 (SMAX1) protein from in response to treatments with karrikins and -GR24. This protocol provides a simple, rapid, and readily scalable method for analysis of relative protein abundance in -infiltrated leaf tissues.
比率型报告基因是动态测量目标蛋白相对丰度的工具。在这些系统中，与荧光或生物发光报告基因融合的目标蛋白以固定的化学计量与与第二个报告基因融合的参考蛋白一起表达。两种融合蛋白都编码在单个转录本上，但在翻译过程中被一个2A“自切割”肽隔开。这种方法能够灵敏地检测目标蛋白相对丰度的变化，减少了比率型报告基因转基因在不同组织或转化事件中可能出现的表达变异性。我们最近开发了一组称为pRATIO的与Gateway兼容的植物转化载体，它们结合了多种启动子、荧光和生物发光报告基因以及源自口蹄疫病毒的2A肽。在这里，我们详细描述如何使用双荧光比率型报告基因pRATIO3212来检测叶片瞬时表达后目标蛋白的相对丰度。在这个例子中，我们分析了MAX2抑制因子1（SMAX1）蛋白在受到卡里金和-GR24处理后的降解情况。本方案提供了一种简单、快速且易于扩展的方法，用于分析农杆菌浸润的叶片组织中的相对蛋白丰度。
