将合成双链RNA直接导入拟南芥原生质体。

Direct transfer of synthetic double-stranded RNA into protoplasts of Arabidopsis thaliana.

作者信息

Jung Ha-Il, Zhai Zhiyang, Vatamaniuk Olena K

机构信息

Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853, USA.

出版信息

Methods Mol Biol. 2011;744:109-27. doi: 10.1007/978-1-61779-123-9_8.

DOI:10.1007/978-1-61779-123-9_8

PMID:21533689

Abstract

Double-stranded (ds) RNA interference (RNAi) is widely used as a reverse genetic approach for functional analysis of plant genes. Constitutive or transient RNAi effects in plants have been achieved via generating stable transformants expressing dsRNAs or artificial microRNAs (amiRNAs) in planta or by viral-induced gene silencing (VIGS). Although these tools provide outstanding resources for functional genomics, they require generation of vectors expressing dsRNAs or amiRNAs against targeted genes, transformation and propagation of transformed plants, or maintenance of multiple VIGS lines and thus impose time, labor, and space requirements. As we showed recently, these limitations can be circumvented by inducing RNAi effects in protoplasts via transfecting them with in vitro-synthesized dsRNAs. In this chapter we detail the procedure for transient gene silencing in protoplasts using synthetic dsRNAs and provide examples of approaches for subsequent functional analyses.

摘要

双链（ds）RNA干扰（RNAi）作为一种反向遗传学方法被广泛用于植物基因的功能分析。通过在植物中产生表达dsRNA或人工微小RNA（amiRNA）的稳定转化体，或通过病毒诱导的基因沉默（VIGS），已在植物中实现了组成型或瞬时RNAi效应。尽管这些工具为功能基因组学提供了出色的资源，但它们需要生成针对靶向基因表达dsRNA或amiRNA的载体、转化植物的转化和繁殖，或维持多个VIGS株系，因此对时间、劳动力和空间都有要求。正如我们最近所展示的，通过用体外合成的dsRNA转染原生质体来诱导RNAi效应，可以规避这些限制。在本章中，我们详细介绍了使用合成dsRNA在原生质体中进行瞬时基因沉默的程序，并提供了后续功能分析方法的示例。