Chakrabarty Romit, Banerjee Rituparna, Chung Sang-Min, Farman Mark, Citovsky Vitaly, Hogenhout Saskia A, Tzfira Tzvi, Goodin Michael
Department of Plant Pathology, University of Kentucky, Lexington 40546, USA.
Mol Plant Microbe Interact. 2007 Jul;20(7):740-50. doi: 10.1094/MPMI-20-7-0740.
Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.
植物功能蛋白质组学研究越来越依赖于能够促进高通量基因克隆以及与自发荧光蛋白融合表达的载体。在此，我们描述了pSITE质粒家族，这是一组新型的农杆菌双元载体，适用于多种自发荧光蛋白融合体在植物细胞中的稳定整合或瞬时表达。pSITE载体允许通过单步Gateway介导的重组克隆来构建双元载体，这些双元载体可直接用于瞬时表达研究，或用于在含有卡那霉素的培养基上筛选转基因植物。这些载体可用于表达天然蛋白或与单体红色荧光蛋白、增强型绿色荧光蛋白及其蓝移和黄移光谱变体的融合体。我们已经验证了这些载体可用于瞬时表达分析以及转基因植物的产生。此外，我们还生成了用于对肌动蛋白丝、染色质、内质网和核仁进行荧光标记的标记物。最后，我们表明pSITE载体可用于病毒感染细胞中的靶向基因表达这将有助于高通量表征宿主-病毒相互作用中的蛋白质动态。
