Bernacki Steven, Karimi Mansour, Hilson Pierre, Robertson Niki
Department of Plant Biology, North Carolina State University, Raleigh, NC, USA.
Methods Mol Biol. 2010;655:27-45. doi: 10.1007/978-1-60761-765-5_3.
Reverse genetics has proven to be a powerful approach to elucidating gene function in plants, particularly in Arabidopsis. Virus-induced gene silencing (VIGS) is one such method and achieves reductions in target gene expression as the vector moves into newly formed tissues of inoculated plants. VIGS is especially useful for plants that are recalcitrant for transformation and for genes that cause embryo lethality. VIGS provides rapid, transient knockdowns as a complement to other reverse genetics tools and can be used to screen sequences for RNAi prior to stable transformation. High-throughput, forward genetic screening is also possible by cloning libraries of short gene fragments directly into a VIGS plasmid DNA vector, inoculating, and then looking for a phenotype of interest. VIGS is especially useful for studying genes in crop species, which currently have few genetic resources. VIGS facilitates a rapid comparison of knockdown phenotypes of the same gene in different breeding lines or mutant backgrounds, as the same vector is easily inoculated into different plants. In this chapter, we briefly discuss how to choose or construct a VIGS vector and then how to design and carry out effective experiments using VIGS.
反向遗传学已被证明是阐明植物基因功能的一种强大方法，尤其是在拟南芥中。病毒诱导的基因沉默（VIGS）就是这样一种方法，随着载体进入接种植物的新形成组织，它能实现靶基因表达的降低。VIGS对于难以转化的植物以及导致胚胎致死的基因特别有用。VIGS可提供快速、瞬时的基因敲低，作为其他反向遗传学工具的补充，并且可用于在稳定转化之前筛选RNAi的序列。通过将短基因片段文库直接克隆到VIGS质粒DNA载体中、接种，然后寻找感兴趣的表型，高通量的正向遗传筛选也是可行的。VIGS对于研究目前遗传资源很少的作物物种中的基因特别有用。由于同一载体很容易接种到不同植物中，VIGS有助于快速比较同一基因在不同育种系或突变背景下的基因敲低表型。在本章中，我们简要讨论如何选择或构建VIGS载体，然后如何使用VIGS设计和开展有效的实验。
