London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON, N5V4T3, Canada.
London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON, N5V4T3, Canada; Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Plant Sci. 2018 Jun;271:117-126. doi: 10.1016/j.plantsci.2018.03.023. Epub 2018 Mar 23.
A major research topic nowadays is to study and understand the functions of the increasing number of predicted genes that have been discovered through the complete genome sequencing of many plant species. With the aim of developing tools for rapid and convenient gene function analysis, we have developed a set of "pGate" vectors based on the principle of Golden gate and Gateway cloning approaches. These vectors combine the positive aspects of both Golden gate and Gateway cloning strategies. pGate vectors can not only be used as Golden gate recipient vectors to assemble multiple DNA fragments in a pre-defined order, but they can also work as an entry vector to transfer the assembled DNA fragment(s) to a large number of already-existing, functionally diverse, Gateway compatible destination vectors without adding additional nucleotides during cloning. We show the pGate vectors are effective and convenient in several major aspects of gene function analyses, including BiFC (Bimolecular fluorescence complementation) to analyze protein-protein interaction, amiRNA (artificial microRNA) candidate screening and as assembly of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats, CRISPR-associated protein-9 nuclease) system elements together for genome editing. The pGate system is a practical and flexible tool which can facilitate plant gene function research.
目前的一个主要研究课题是研究和了解通过对许多植物物种的全基因组测序发现的越来越多的预测基因的功能。为了开发用于快速便捷地进行基因功能分析的工具，我们基于 Golden gate 和 Gateway 克隆方法的原理，开发了一套“pGate”载体。这些载体结合了 Golden gate 和 Gateway 克隆策略的优点。pGate 载体不仅可以用作 Golden gate 受体载体，按照预定的顺序组装多个 DNA 片段，还可以作为入口载体，在克隆过程中不添加额外核苷酸的情况下，将组装的 DNA 片段转移到大量已经存在的、功能多样的、兼容 Gateway 的目的载体上。我们展示了 pGate 载体在基因功能分析的几个主要方面的有效性和便利性，包括用于分析蛋白-蛋白相互作用的 BiFC（双分子荧光互补）、amiRNA（人工 microRNA）候选物筛选以及用于基因组编辑的 CRISPR/Cas9（成簇规律间隔短回文重复，CRISPR 相关蛋白 9 核酸酶）系统元件的组装。pGate 系统是一种实用且灵活的工具，可以促进植物基因功能研究。
