Gateway-compatible vectors for plant functional genomics and proteomics
Gateway-compatible vectors for plant functional genomics and proteomics
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DOI：
10.1111/j.1365-313x.2005.02617.x
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发表时间：
2006-02-01
期刊：
影响因子：
7.2
通讯作者：
Pikaard, CS
中科院分区：
文献类型：
--
作者：
Earley, KW;Haag, JR;Pikaard, CS
Gateway 克隆技术通过利用噬菌体 lambda 位点特异性重组系统促进靶序列的高通量克隆。目标序列首先被捕获在市售的“进入载体”中，然后重组为各种“目标载体”，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所采用，这些实验室设计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、RNA 干扰基因敲除或亲和纯化实验的目的载体。我们回顾了植物研究界当前可用的各种类型的网关目标载体，并提供链接和参考，以便获得有关这些载体的其他信息。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，其将 FLAG、HA、cMyc、AcV5 或串联亲和纯化表位标签翻译融合到目标蛋白上，有或没有相邻的荧光蛋白。寡肽表位标签允许对体内表达的重组蛋白进行亲和纯化、免疫定位或免疫沉淀。我们展示了 pEarleyGate 目的载体在表达表位标记蛋白方面的实用性，这些蛋白可以通过免疫荧光显微镜进行亲和捕获或定位。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，表明标签和载体具有广泛的用途。
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.