Max-Planck Institute for Plant Breeding Research, Cologne, Germany.
Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet, Bangladesh.
Methods Mol Biol. 2022;2479:71-84. doi: 10.1007/978-1-0716-2233-9_6.
Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second "turbo"-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.
利用噬菌体 λ Red 重组功能的重组方法广泛用于在大肠杆菌中对携带细菌人工染色体 (BAC) 的真核基因进行多功能修饰。虽然 BAC 转化为将修饰的基因整合到动物细胞的基因组中以生成敲入和敲除系提供了一种简单的方法，但该策略的成功应用受到高等植物中同源重组频率低的限制。然而，使用农杆菌的转移 DNA (T-DNA) 可以以高频率转化植物细胞，T-DNA 稳定且随机地整合到植物基因组中。因此，通过 T-DNA 插入或基于 T-DNA 的 Crisp/Cas9 构建体的基因组编辑诱导的敲除突变的遗传互补，可以适当地研究通过重组方法修饰并通过农杆菌 T-DNA 载体转移到植物细胞中的植物基因的功能。在这里，我们描述了两种从 BAC 修饰和转移植物基因到农杆菌 T-DNA 植物转化载体的重组方法。第一个方案使用条件性自杀 ccdB 基因盒来辅助遗传互补测定，以在任何基因位置产生点突变、缺失和插入。第二个“涡轮增压”重组方案利用各种 I-SceI 插入盒将荧光蛋白标签融合到植物基因产物上，以通过亲和纯化、质谱分析和细胞定位研究促进其体内相互作用伙伴的表征。
