Earley Keith W, Haag Jeremy R, Pontes Olga, Opper Kristen, Juehne Tom, Song Keming, Pikaard Craig S
Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA.
Plant J. 2006 Feb;45(4):616-29. doi: 10.1111/j.1365-313X.2005.02617.x.
Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.
Gateway克隆技术利用噬菌体λ位点特异性重组系统，促进了目标序列的高通量克隆。目标序列首先被捕获到市售的“入门载体”中，然后再重组到各种“目的载体”中，以便在不同的实验生物体中表达。Gateway技术已被许多植物实验室采用，这些实验室构建了用于启动子特异性分析、蛋白质定位研究、蛋白质/蛋白质相互作用研究、组成型或诱导型蛋白质表达研究、通过RNA干扰进行基因敲低或亲和纯化实验的目的载体。我们综述了植物研究界目前可用的各种类型的Gateway目的载体，并提供链接和参考文献，以便获取有关这些载体的更多信息。我们还描述了一组用于农杆菌介导的植物转化的“pEarleyGate”质粒载体，这些载体可将FLAG、HA、cMyc、AcV5或串联亲和纯化表位标签翻译融合到目标蛋白质上，有或没有相邻的荧光蛋白。寡肽表位标签可用于体内表达的重组蛋白的亲和纯化、免疫定位或免疫沉淀。我们展示了pEarleyGate目的载体在表达可通过亲和捕获或免疫荧光显微镜定位的表位标签蛋白方面的实用性。检测FLAG、HA、cMyc和AcV5标签的抗体与多种单子叶和双子叶植物中的内源蛋白的交叉反应相对较少，这表明这些标签和载体具有广泛的实用性。
