Biochemistry and Physiology of Plants, Faculty of Biology, Bielefeld University, Bielefeld, Germany.
Methods Mol Biol. 2024;2798:235-263. doi: 10.1007/978-1-0716-3826-2_17.
A set of peroxidases detoxifies HO and mediates HO-dependent signal propagation. The peroxidases include peroxiredoxins, glutathione peroxidases, ascorbate peroxidases, and catalases. This at least partial redundancy impedes addressing individual proteins in living plant cells so that the protein functions are often studied by biochemical assays in vitro. In vivo analysis frequently relies on transgenic insertion lines resulting in the knockdown or knockout of the protein of interest. However, many proteins have multiple isoforms in close genomic arrangement so that even crossing of transgenic lines does not allow for a knockdown of all isoforms. The genes encoding for the three cytosolic peroxiredoxins PRXIIB, C, and D in Arabidopsis thaliana are located in close vicinity on chromosome 1 so that crossing over between the genes most rarely occurs and successful crossing of the plants appears impossible. Genome editing instead allows targeting of multiple isoforms and knocks out several genes at once. This chapter describes how to inactivate the three cytosolic peroxiredoxins by CRISPR/Cas9 in A. thaliana.
一组过氧化物酶可使 HO 解毒并介导 HO 依赖的信号转导。过氧化物酶包括过氧化物酶体、谷胱甘肽过氧化物酶、抗坏血酸过氧化物酶和过氧化氢酶。这种至少部分冗余性阻碍了在活植物细胞中针对单个蛋白质进行寻址，因此蛋白质功能通常通过体外生化测定来研究。体内分析通常依赖于转基因插入系，导致感兴趣的蛋白质的敲低或敲除。然而，许多蛋白质具有紧密基因组排列的多个同工型，即使转基因系的杂交也不能使所有同工型的表达量降低。拟南芥中三种细胞质过氧化物酶 PRXIIB、C 和 D 的编码基因位于染色体 1 上的紧密附近位置，因此基因之间的交叉很少发生，成功的植物杂交似乎是不可能的。基因组编辑可靶向多个同工型并同时敲除多个基因。本章描述了如何在拟南芥中通过 CRISPR/Cas9 使三种细胞质过氧化物酶失活。
