Li C, Zhang Z C, Ghebremariam K M, Wang L H, Wu L, Liang Y
State Key Laboratory of Crop Stress Biology in Arid Region, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China.
State Key Laboratory of Crop Stress Biology in Arid Region, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China
Genet Mol Res. 2014 Nov 11;13(4):9443-52. doi: 10.4238/2014.November.11.9.
Virus-induced gene silencing (VIGS) is an important tool for studying gene function. However, a number of factors highly restrict the application of VIGS, such as unstable efficiency and tissue-specific silencing. We developed a novel evaluation method for improving the applicability of VIGS vectors. In this method, 4 indexes were defined and utilized to evaluate VIGS efficiency by silencing the endogenous phytoene desaturase (PDS) gene with a tobacco rattle virus-based VIGS vector. To illustrate the reliability of this evaluation method, we assessed the silencing efficiency of SpPDS and SpMPK1 in Solanum pimpinellifolium. The silencing results of SpPDS showed that an optical density at 600 nm of 2.0 was more suitable than 1.0 for VIGS in S. pimpinellifolium. This suggests that the proposed evaluation method is a valid technique for optimizing the VIGS system of plants. Moreover, the SpMPK1 gene was highly silenced in the 4th-9th leaves with a 50-95% reduction in transcription levels, further demonstrating that this method can be used to select highly silenced candidates for further experiments, particularly when the target gene shows no phenotypic change after being silenced.
病毒诱导的基因沉默（VIGS）是研究基因功能的重要工具。然而，许多因素严重限制了VIGS的应用，如效率不稳定和组织特异性沉默。我们开发了一种新的评估方法来提高VIGS载体的适用性。在该方法中，定义并利用4个指标，通过基于烟草脆裂病毒的VIGS载体沉默内源性八氢番茄红素去饱和酶（PDS）基因来评估VIGS效率。为了说明该评估方法的可靠性，我们评估了野生型醋栗番茄中SpPDS和SpMPK1的沉默效率。SpPDS的沉默结果表明，在野生型醋栗番茄中，600nm处光密度为2.0比1.0更适合用于VIGS。这表明所提出的评估方法是优化植物VIGS系统的有效技术。此外，SpMPK1基因在第4至第9片叶子中高度沉默，转录水平降低了50-95%，进一步证明该方法可用于选择高度沉默的候选基因进行进一步实验，特别是当目标基因沉默后无表型变化时。
