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利用烟草脆裂病毒快速筛选桃蚜RNA干扰靶点

Rapid Screening of Myzus persicae (Green Peach Aphid) RNAi Targets Using Tobacco Rattle Virus.

作者信息

Feng Honglin, Jander Georg

机构信息

Boyce Thompson Institute, Ithaca, NY, USA.

出版信息

Methods Mol Biol. 2022;2360:105-117. doi: 10.1007/978-1-0716-1633-8_10.

Abstract

Plant-mediated RNA interference (RNAi) can be used to reduce the growth of insect pests, including Myzus persicae (green peach aphid), a prolific pest of numerous dicot crop species. In one approach, viruses that have been engineered to carry an aphid gene fragment are used to infect plants and thereby silence target gene expression in the aphids feeding on these plants, a process called virus-induced gene silencing, or VIGS. Tobacco Rattle Virus (TRV) in the model plant, Nicotiana benthamiana, was the first of many VIGS systems that have been developed for different plant species. In this chapter, we describe a method for silencing M. persicae gene expression using an established TRV-VIGS vector that infects and spreads in N. benthamiana. The two parts of the TRV genome, RNA1 and RNA2, have been cloned into Agrobacterium T-DNA vectors for initiation of plant infections. The RNA2 construct is modified with a Gateway-compatible cloning site to allow insertion of aphid genes. When feeding on TRV-infected N. benthamiana plants, aphids ingest dsRNAs that silence specific target genes. TRV-VIGS of aphid genes allows rapid identification of essential gene targets that can be used for the control of M. persicae by this and other RNAi methods.

摘要

植物介导的RNA干扰(RNAi)可用于抑制害虫生长,包括桃蚜(烟蚜),它是多种双子叶作物的常见害虫。一种方法是,利用经过基因工程改造携带蚜虫基因片段的病毒感染植物,从而使取食这些植物的蚜虫体内的靶基因表达沉默,这一过程称为病毒诱导的基因沉默(VIGS)。模式植物本氏烟草中的烟草脆裂病毒(TRV)是首个为不同植物物种开发的众多VIGS系统之一。在本章中,我们描述了一种使用已建立的TRV-VIGS载体沉默桃蚜基因表达的方法,该载体可在本氏烟草中感染并传播。TRV基因组的两个部分RNA1和RNA2已被克隆到农杆菌T-DNA载体中,用于引发植物感染。RNA2构建体用一个与Gateway兼容的克隆位点进行了修饰,以便插入蚜虫基因。当蚜虫取食感染了TRV的本氏烟草植株时,会摄取使特定靶基因沉默的双链RNA。对蚜虫基因进行TRV-VIGS可快速鉴定出重要的基因靶点,这些靶点可用于通过这种及其他RNAi方法来防治桃蚜。

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