Transient expression of proteins based on agro-infiltration techniques has proven very efficient and straightforward to study the intrinsic properties of proteins. The level of protein expression has been enhanced by the use of vector plasmids containing virus-derived sequences and the cloning step has been facilitated by recombination technologies. The pEAQ-HT-DEST series of vectors fulfilling these improvements are vectors of choice. However, they lack the possibility to directly and easily fuse the protein of interest to a fluorescent tag or to address it to the secretion pathway. In the present work we describe the production of 15 pEAQ-HT-DEST1-based plasmids designed to use the Gateway® cloning technology and to generate high levels of fluorescent fusion protein by agro-infiltration, in planta. This collection of plasmids includes binary vectors allowing N-terminal or C-terminal fusion to the bright tags EGFP or TagRFP for cytoplasmic accumulation or secretion and represents therefore a valuable tool for subcellular localization or biochemical studies. A viral protein, the blue fluorescent protein TagBFP, the green fluorescent protein variant T-Sapphire and an Arabidopsis protein were transiently expressed in N. benthamiana to demonstrate the potential of these vectors.

中文翻译：

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内部还是外部？Gateway载体的新集合，允许植物蛋白亚细胞定位或过度表达。

已经证明基于农业渗透技术的蛋白质瞬时表达非常有效且直接地研究了蛋白质的内在特性。通过使用包含病毒衍生序列的载体质粒，可以提高蛋白质表达水平，并且通过重组技术可以促进克隆步骤。实现这些改进的pEAQ-HT-DEST系列载体是选择的载体。但是，它们缺乏将目标蛋白直接轻松融合到荧光标签或将其定位到分泌途径的可能性。在本工作中，我们描述了15种基于pEAQ-HT-DEST1的质粒的生产，这些质粒设计用于在植物中使用Gateway®克隆技术并通过农业浸润产生高水平的荧光融合蛋白。质粒的收集包括允许N端或C端融合到明亮的标签EGFP或TagRFP的胞质积累或分泌的二元载体，因此代表了用于亚细胞定位或生化研究的有价值的工具。在本氏烟草中瞬时表达了一种病毒蛋白，蓝色荧光蛋白TagBFP，绿色荧光蛋白变体T-蓝宝石和拟南芥蛋白，以证明这些载体的潜力。