Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics.

基因功能的剖析需要复杂的工具来监测基因表达。使用表位肽和荧光蛋白标签进行基因标记是使用标签特异性抗体和蛋白质印迹法研究蛋白质表达的常规方法，其中包括繁琐的印迹和免疫检测步骤。纳米荧光素酶 (NanoLuc) 表现出极其明亮的生物发光，被设计为敏感的基因报告基因。由于其尺寸小和生物发光活性高，NanoLuc 可被设计为新型蛋白质标签，允许直接检测凝胶基质中的标记蛋白质（凝胶内检测）。在这项研究中，我们开发了 Gateway 兼容载体，用 NanoLuc 标记植物中的蛋白质。我们还定制了凝胶内检测条件，可以从低至 200 pg 的总蛋白提取物中检测 NanoLuc 标记的 MPK3。与 FLAG 标签和基于蛋白质印迹的检测相比，NanoLuc 标签和优化的凝胶内检测表现出更高的检测灵敏度，但省略了印迹和免疫检测步骤。我们还展示了基于 NanoLuc 的凝胶内检测方法在蛋白质表达分析、通过免疫共沉淀探测蛋白质-蛋白质相互作用以及通过 Phos-tag 凝胶电泳进行体内蛋白质磷酸化检测的多种应用。最后，NanoLuc 使用小麦矮化病毒复制子和 CRISPR/Cas9 介导的基因靶向来标记该基因的内源基因座。我们的数据表明 NanoLuc 标签和凝胶内检测可以以高灵敏度快速检测标记的蛋白质。多功能的 NanoLuc 工具包和方便的凝胶内检测方法有望促进植物功能基因组学的体外和体内蛋白质分析。