Efficient virus-induced gene silencing in Arabidopsis using a 'one-step' TYMV-derived vector
Efficient virus-induced gene silencing in Arabidopsis using a 'one-step' TYMV-derived vector
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DOI：
10.1111/j.1365-313x.2008.03620.x
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发表时间：
2008-11-01
期刊：
影响因子：
7.2
通讯作者：
Jupin, Isabelle
中科院分区：
文献类型：
--
作者：
Pflieger, Stephanie;Blanchet, Sandrine;Jupin, Isabelle
病毒诱导的基因沉默（VIGS）是分析植物基因功能的重要工具。该技术利用重组病毒载体，在其基因组中含有植物基因片段，产生双链rna，启动同源依赖的靶基因沉默。几种病毒VIGS载体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。在这里，我们证明了芜菁黄花叶病毒（TYMV）衍生的病毒载体具有在拟南芥中诱导VIGS的能力，加入col0，只要它携带目标基因的倒置重复片段。当用含有病毒基因组cDNA拷贝的完整质粒DNA进行简单的磨擦接种植物时，观察到强大而可靠的基因沉默，从而排除了体外转录、生物或农接种程序的需要。我们的研究结果表明，一个76 bp的片段足以导致叶、茎和花中的基因沉默，并且tymv衍生的载体也具有靶向分生组织中表达的基因的能力。因此，本文描述的VIGS载体可能是提高拟南芥高通量功能基因组学的理想工具。
Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. This technique exploits recombinant viral vectors harbouring fragments of plant genes in their genome to generate double-stranded RNAs that initiate homology-dependent silencing of the target gene. Several viral VIGS vectors have already been successfully used in reverse-genetics studies of a variety of processes occurring in plants. Here, we show that a viral vector derived from Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana, accession Col-0, provided that it carries an inverted-repeat fragment of the target gene. Robust and reliable gene silencing was observed when plants were inoculated simply by abrasion with intact plasmid DNA harbouring a cDNA copy of the viral genome, thus precluding the need for in vitro transcription, biolistic or agroinoculation procedures. Our results indicate that a 76 bp fragment is sufficient to cause gene silencing in leaves, stems and flowers, and that the TYMV-derived vector also has the ability to target genes expressed in meristematic tissues. The VIGS vector described here may thus represent an ideal tool for improving high-throughput functional genomics in Arabidopsis.