利用基于 CRISPR/Cas9 的基因编辑技术对水稻活性氧（ROS）稳态的遗传操作。

Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.

机构信息

Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu, China.

School of Food and Biological Engineering, Hefei University of Technology, Hefei, Anhui, China.

出版信息

Methods Mol Biol. 2022;2526:25-41. doi: 10.1007/978-1-0716-2469-2_3.

DOI:10.1007/978-1-0716-2469-2_3

PMID:35657510

Abstract

Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.

摘要

活性氧（ROS）现在被认为是植物应激反应中的关键信号。不利的环境条件要么促进 ROS 的产生，要么下调抗氧化酶，导致氧化还原平衡的改变和 ROS 相关应激信号的激活。为了揭示它们的信号机制并描述相关成分，ROS 动态平衡的遗传修饰是一种核心方法。基于 CRISPR/Cas9 的基因组编辑系统已成为包括植物在内的多种生物体中基因突变的有力工具。在本章中，我们描述了一种利用 CRISPR/Cas9 技术在水稻（Oryza sativa L.）中操纵 ROS 动态平衡的方法。描述了包括 Cas9/sgRNA 的设计和构建、农杆菌介导的转化以及突变特征的分步方案。还介绍了该系统在编辑水稻过氧化氢酶基因 CatC 中的应用，CatC 是控制 ROS 动态平衡的关键抗氧化酶。