1] Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA. [2] National Institute of Plant Genome Research, New Delhi, India.
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.
Nat Protoc. 2014 Jul;9(7):1549-62. doi: 10.1038/nprot.2014.092. Epub 2014 Jun 5.
Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2-3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.
基于烟草蚀纹病毒（TRV）的病毒诱导基因沉默（VIGS）广泛应用于各种植物物种中，以下调目标植物基因的表达。TRV 是一种具有 TRV1 和 TRV2 基因组的双分、正链 RNA 病毒。为了诱导转录后基因沉默（PTGS），TRV2 基因组经过遗传修饰，携带目标基因的一个片段，并通过农杆菌接种进入植物（同时携带 TRV1 基因组）。然后，携带 TRV1 和 TRV2 的农杆菌菌株通过三种方法之一共同接种到 3 周龄的植物叶片中：无针注射器、农杆菌浸蘸法或牙签刺法。在 TRV 接种后 2-3 周内，新发育的非接种叶片中会发生目标基因沉默。本文描述的 TRV-VIGS 方案仅需 4 周即可实施，并且比目前可用的其他基因沉默技术更快、更容易执行。虽然我们以 Nicotiana benthamiana 为例，但该方案可适用于其他植物物种。
