Zhou Tao, Liu Xuedong, Fan Zaifeng
State Key Laboratory for Agro-Biotechnology and Department of Plant Pathology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
Methods Mol Biol. 2018;1676:141-150. doi: 10.1007/978-1-4939-7315-6_8.
Virus-induced gene silencing (VIGS) is a genetic technology that exploits the RNA-mediated defense against virus. The method has great potential for plant reverse genetics as it could knock down gene expression in a rapid way, which is triggered by a replicating viral genome engineered to carry a fragment of host gene to be silenced. A number of efficient VIGS vectors are available for dicots, such as for model plant Nicotiana benthamiana; however, only a few of VIGS vectors for monocotyledonous cereal crops. Here, we describe the method for the use of a newly developed VIGS vector based on a maize-infecting Cucumber mosaic virus (CMV) strain ZMBJ-CMV for maize. The RNA2 of ZMBJ-CMV was modified as a vector pCMV201-2b having multiple cloning sites for the insert of 100-300 bp fragment of target gene. Using a method of vascular puncture inoculation of maize seeds with crude sap prepared from Agrobacterium-infiltrated N. benthamiana leaves, silencing of target genes could be obtained in 4 weeks.
病毒诱导的基因沉默（VIGS）是一种利用RNA介导的抗病毒防御机制的遗传技术。该方法在植物反向遗传学中具有巨大潜力，因为它能够快速敲低基因表达，这是由经过工程改造携带待沉默宿主基因片段的复制病毒基因组触发的。有许多有效的VIGS载体可用于双子叶植物，例如模式植物本氏烟草；然而，用于单子叶谷类作物的VIGS载体却很少。在此，我们描述了一种基于感染玉米的黄瓜花叶病毒（CMV）ZMBJ-CMV株系开发的新型VIGS载体用于玉米的方法。ZMBJ-CMV的RNA2被修饰为载体pCMV201-2b，其具有多个克隆位点，可插入100-300bp的目标基因片段。使用从农杆菌浸润的本氏烟草叶片制备的粗汁液通过血管穿刺接种玉米种子的方法，4周内即可实现目标基因的沉默。
