Gull Iram, Jander Georg
Boyce Thompson Institute, Ithaca, NY 14853, USA.
Bio Protoc. 2023 Jul 20;13(14):e4760. doi: 10.21769/BioProtoc.4760.
Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm () gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview.
病毒介导的瞬时基因过表达和基因表达沉默可用于筛选植物中的基因功能。甘蔗花叶病毒（SCMV）是马铃薯Y病毒科的一种正链RNA病毒，已被改造用作载体，以感染包括玉米在内的单子叶植物，用于瞬时基因过表达和基因表达沉默。相对于稳定转化，SCMV介导的玉米瞬时表达具有速度更快、成本更低的优点。在这里，我们描述了一种将构建体克隆到质粒载体pSCMV-CS3中的方法。通过粒子轰击用pSCMV-CS3构建体转化玉米幼苗后，病毒在植物中复制并系统传播。随后可以通过用来自感染SCMV的植物的汁液摩擦接种来完成玉米幼苗的感染。作为该方法实际应用的一个例子，我们还描述了对草地贪夜蛾基因表达的病毒诱导基因沉默（VIGS）。在玉米转化之前，通过将草地贪夜蛾靶基因的一段克隆到pSCMV-CS3中来创建转基因病毒。让毛虫取食病毒感染的玉米植株，这些植株在毛虫摄入后产生dsRNA以沉默草地贪夜蛾靶基因的表达。当毛虫取食其寄主植物时，利用SCMV进行植物介导的昆虫VIGS能够快速筛选基因功能。图形概述。
