Efficient virus-induced gene silencing in Arabidopsis
Efficient virus-induced gene silencing in Arabidopsis
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DOI：
10.1104/pp.106.084624
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发表时间：
2006-09-01
期刊：
影响因子：
7.4
通讯作者：
Dinesh-Kumar, S. P.
中科院分区：
文献类型：
--
作者：
Burch-Smith, Tessa M.;Schiff, Michael;Dinesh-Kumar, S. P.
病毒诱导的基因沉默(VIGS)是一种植物RNA沉默技术，它利用携带目的基因片段的病毒载体产生双链RNA，从而启动靶基因的沉默。已经开发了几种针对VIGS的病毒载体，它们已经成功地用于植物中发生的各种过程的反向遗传学研究。这一方法还没有被模式双子叶植物拟南芥(Arabitopsis Thaliana)广泛采用，可能是因为到目前为止，还没有在这个物种中有效的VIGS的简单协议。在这里，我们证明了一种广泛使用的基于烟草骚动病毒的VIGS载体可以用于沉默拟南芥生态型哥伦比亚-0的基因。该协议涉及将携带目的基因片段的VIGS载体农业渗透到2-3叶期的幼苗中，并且只需对现有的VIGS协议进行最小程度的修改，即可在其他物种中使用烟草摇铃病毒载体，如烟草和番茄(Lycopersicon Esculentum)。本文描述的方法在拟南芥生态型Columbia-0中提供了有效的沉默。我们发现VIGS可以用来沉默与一般代谢和防御有关的基因，也可以有效地抑制高表达的转基因的表达。还描述了一种监控VIGS进度和效率的标记系统。
Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.