Institute of Biosciences and Bioresources (IBBR), National Research Council of Italy (CNR), Naples, Italy.
Methods Mol Biol. 2021;2264:163-175. doi: 10.1007/978-1-0716-1201-9_11.
RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in many organisms. In tomato, RNAi technology has widely been used as a reverse genetic tool for functional genomics study. Generally, RNAi is often achieved through transgenes producing hairpin RNA molecules. RNAi lines have the advantage with respect to more modern CRISPR/Cas9 mutants of different levels of downregulation of target gene, and allow the characterization of life-essential genes that cannot be knocked out without killing the organism. Also, RNAi allows to suppress gene expression in multigene families in a regulated manner. In this chapter, an efficient approach to create RNAi stable knockdown-transformed tomato lines is reported. In order, it describes the choice of the target silencing fragment, a highly efficient cloning strategy for the hairpin RNA construct production, a relatively easy procedure to transform and regenerate tomato plants using Agrobacterium tumefaciens and a methodology to test the goodness of the transformation procedure.
RNA 干扰 (RNAi) 是一种由双链 RNA 诱导的转录后基因沉默现象。它已被广泛用作 knockdown 技术，用于分析许多生物体中的基因功能。在番茄中，RNAi 技术已广泛用作功能基因组学研究的反向遗传学工具。通常，RNAi 是通过产生发夹 RNA 分子的转基因来实现的。与不同程度下调靶基因的现代 CRISPR/Cas9 突变体相比，RNAi 线具有优势，并且允许对不能在不杀死生物体的情况下敲除的必需基因进行特征分析。此外，RNAi 允许以受调控的方式抑制多基因家族中的基因表达。在本章中，报告了一种创建 RNAi 稳定敲低转化番茄系的有效方法。它首先描述了选择靶沉默片段，用于发夹 RNA 构建体生产的高效克隆策略，使用根癌农杆菌转化和再生番茄植物的相对简单的程序，以及测试转化程序质量的方法。
