Silencing on the spot.: Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system
Silencing on the spot.: Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system
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DOI：
10.1104/pp.126.3.930
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发表时间：
2001-07-01
期刊：
影响因子：
7.4
通讯作者：
Carrington, JC
中科院分区：
文献类型：
--
作者：
Johansen, LK;Carrington, JC
农杆菌介导的植物组织瞬时表达技术是一种快速、有效的分析植物基因及其产物的方法。在许多情况下，不需要生产转基因植物就可以生产高水平的活性蛋白。在这项研究中，开发了一系列的工具，使强或弱的诱导RNA沉默和抑制RNA沉默的情况下，稳定的转基因。瞬时递送指导双链绿色荧光蛋白（GFP）转录物产生的基因快速诱导共同递送的GFP报告基因的RNA沉默，有效地防止GFP蛋白和mRNA的积累。烟草蚀纹病毒沉默抑制子P1/HC-Pro部分抑制了强dsGFP诱导剂引发的RNA沉默。在不存在强双链GFP诱导物的情况下，功能性GFP基因在瞬时测定中充当弱RNA沉默诱导物，严重限制GFP mRNA随时间的积累。P1/HC-Pro抑制了GFP基因诱导的弱沉默。这些结果表明，RNA沉默可以由多种诱导剂触发，并使用瞬时基因递送系统进行全面分析。它们还表明，RNA沉默可能是农杆菌介导的瞬时测定中基因表达的显著限制，但这种限制可以通过使用RNA沉默抑制剂来克服。
The Agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants. In many Cases, high levels of active protein can be produced without the need to produce transgenic plants. In this study, a series of tools were developed to enable strong or weak induction of RNA silencing and to suppress RNA silencing in the absence of stable transgenes. Transient delivery of a gene directing production of a double-stranded green fluorescent protein (GFP) transcript rapidly induced RNA silencing of a codelivered GFP reporter gene, effectively preventing accumulation of GFP protein and mRNA. RNA silencing triggered by the strong dsGFP inducer was partially inhibited by the tobacco etch virus silencing suppressor, P1/HC-Pro. In the absence of the strong- double-stranded GFP inducer, the functional GFP gene served as a weak RNA silencing inducer in the transient assay, severely Limiting accumulation of the GFP mRNA over time. The weak silencing induced by the GFP gene was suppressed by P1/HC-Pro. These results indicate RNA silencing can be triggered by a variety of inducers and analyzed entirely using transient gene delivery systems. They also indicate that RNA silencing may be a significant limitation to expression of genes in the Agrobacterium-mediated transient assay but that this limitation can be overcome by using RNA silencing suppressors.