Chuah Jo-Ann, Horii Yoko, Numata Keiji
Enzyme Research Team, RIKEN Center for Sustainable Resource Science.
Enzyme Research Team, RIKEN Center for Sustainable Resource Science;
J Vis Exp. 2016 Dec 16(118):54972. doi: 10.3791/54972.
The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.
在植物中导入外源蛋白并表达（或下调）特定基因的能力，为基础研究以及植物生物技术领域的新应用提供了强大工具。目前存在的将蛋白质或基因导入植物细胞的可行方法，即农杆菌介导法和微粒轰击法，具有转化频率低、宿主范围有限或设备和微载体成本高的缺点。以下方案概述了一种简单且通用的方法，该方法采用合理设计的肽作为多种基于核酸和蛋白质的货物进入植物的递送剂。选择肽作为该系统开发的工具是因为它们具有生物可降解性、尺寸减小、性质多样且可调节以及能够进入细胞内/细胞器的能力。描述了针对广泛递送货物（质粒DNA、双链DNA或RNA以及蛋白质）中每种类型的优化制剂的制备、表征和应用。还讨论了该方案中的关键步骤、可能的修改以及该方法现有的局限性。
