High-throughput Binary Vectors For Plant Gene Function Analysis

Gateway^TM cloning technology facilitates high-throughput cloning of target sequences by making use of the lambda bacteriophage site-specific recombination system. Target sequences are first shuttled into a commercially available 'entry vector' and are then recombined into various 'destination vectors' for expression in different experimental organisms. Gateway^TM 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. In this study, a series of high-throughput cloning binary vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing, and GFP fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for Gateway^TM recombinase, and in addition, unique restriction sites incorporated in the multiple cloning sites enable promoter replacement. In addition, various types of other Gateway? destination vectors that are currently available to the plant research community are reviewed and their uses and problems are discussed.