Wendrich Jos R, Liao Che-Yang, van den Berg Willy A M, De Rybel Bert, Weijers Dolf
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703HA, Wageningen, The Netherlands.
Methods Mol Biol. 2015;1284:421-31. doi: 10.1007/978-1-4939-2444-8_21.
Molecular cloning is a vital step in much of today's plant biological research. Particularly, when a species is amenable to transgenic manipulation, cloning enables detailed study of gene and protein function in vivo. Therefore, accurate, consistent, and efficient cloning methods have the potential to accelerate biological research. Traditional restriction-enzyme/ligase-based strategies are often inefficient, while novel alternative methods can be less economical. We have recently optimized a method for Ligation-Independent Cloning (LIC) that is both efficient and economical. We have developed a large set of LIC-compatible plasmids for application in plant research. These include dedicated vectors for gene expression analysis, protein localization studies, and protein misexpression. We describe a detailed protocol that allows the reliable generation of plant transformation-ready constructs from PCR fragments in 2-3 days.
分子克隆是当今许多植物生物学研究中的关键步骤。特别是当一个物种适合进行转基因操作时，克隆能够在体内对基因和蛋白质功能进行详细研究。因此，准确、一致且高效的克隆方法有可能加速生物学研究。传统的基于限制性酶/连接酶的策略往往效率低下，而新的替代方法可能成本更高。我们最近优化了一种高效且经济的不依赖连接酶克隆（LIC）方法。我们开发了大量适用于植物研究的LIC兼容质粒。这些包括用于基因表达分析、蛋白质定位研究和蛋白质错误表达的专用载体。我们描述了一个详细的方案，该方案能够在2至3天内从PCR片段可靠地生成可用于植物转化的构建体。
