瞬时表达介导的植物基因沉默及病毒抑制剂对基因沉默的抑制。
Transient Expression-Mediated Gene Silencing in Plants and Suppression of Gene Silencing with Viral Suppressors.
机构信息
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON, Canada.
出版信息
Methods Mol Biol. 2022;2400:33-41. doi: 10.1007/978-1-0716-1835-6_4.
Posttranscriptional gene silencing (PTGS) also known as RNA silencing or RNA interference is an evolutionarily conserved innate immunity in eukaryotes that targets the complementary RNA sequences to slice/degrade the target RNA or repress the translation of mRNA. In the past two decades, RNA silencing as an important antiviral mechanism has been studied extensively in plants. Intriguingly, almost every virus encodes at least a viral suppressor of RNA silencing (VSR) to counterattack RNA silencing with many strategies to interfere with different steps of RNA silencing. Therefore, the molecular identification of VSRs and elucidation of their functional mechanisms contribute to a better understanding of host resistance and viral pathogenicity. Here, we describe a protocol for the transient expression-induced gene silencing in 16c GFP transgenic and wild type Nicotiana benthamiana plants, and the suppression of single-stranded GFP and double-stranded GFP induced RNA silencing with a VSR in N. benthamiana plants. This protocol is simple and can serve as a standard for the identification and functional analysis of a VSR.
转录后基因沉默（PTGS），又称 RNA 沉默或 RNA 干扰，是真核生物中一种保守的先天免疫反应，其靶向互补的 RNA 序列，以切割/降解靶 RNA 或抑制 mRNA 的翻译。在过去的二十年中，RNA 沉默作为一种重要的抗病毒机制，在植物中得到了广泛的研究。有趣的是，几乎每种病毒都至少编码一种 RNA 沉默抑制子（VSR），通过多种策略来干扰 RNA 沉默的不同步骤，以对抗 RNA 沉默。因此，VSR 的分子鉴定及其功能机制的阐明有助于更好地理解宿主抗性和病毒致病性。在这里，我们描述了在 16c GFP 转基因和野生型 Nicotiana benthamiana 植物中瞬时表达诱导基因沉默的方法，以及在 N. benthamiana 植物中用 VSR 抑制单链 GFP 和双链 GFP 诱导的 RNA 沉默的方法。该方案简单易行，可作为鉴定和分析 VSR 的标准方法。
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