A Protocol for VIGS in Arabidopsis Thaliana Using a One-Step TYMV-Derived Vector
互联网
830
Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants, which can be adapted for high-throughput functional genomics in model plant species such as Arabidopsis thaliana .
Here we describe the use of the Turnip yellow mosaic virus (TYMV)-derived vector pTY-S that has the ability to induce VIGS in Arabidopsis thaliana . This vector harbors a cDNA copy of the viral genome, in which a unique Sna BI restriction site has been engineered. This site allows the cloning of 80 bp synthetic oligonucleotides corresponding to inverted-repeat fragments of the target gene while retaining the ability of the virus to move systemically. Silencing requires plants to be simply inoculated by abrasion with a few micrograms of intact plasmid DNA, thus precluding the need for in vitro transcription, biolistic, or agroinoculation procedures. This one-step TYMV-based VIGS system is therefore simple to use, cost-effective, and highly consistent, which are important parameters to consider towards the development of high-throughput infection procedures. Another important characteristic of this viral vector is its capacity to infect and induce silencing in meristem tissues.
