Rodman Michele K, Yadav Narendra S, Artus Nancy N
BCS & E, Central R & D, DuPont Experimental Station, Wilmington, DE 19880, USA.
Biology Department, West Chester University, West Chester, PA 19383, USA.
New Phytol. 2002 Sep;155(3):461-468. doi: 10.1046/j.1469-8137.2002.00467.x.
• The association of viral-induced gene silencing (VIGS) elicited by a DNA virus with DNA methylation of the silenced transgene was studied. • 35S-Green fluorescent protein (GFP) transgenic Nicotiana benthamiana were treated with an inhibitor of DNA methylation, 5-azacytidine (5-Aza-C), and VIGS of the transgene was observed upon inoculation with tomato golden mosaic virus carrying the GFP coding sequence. • The onset of VIGS of the 35S-GFP transgene occurred 14-16 d after inoculation in both control and 5-Aza-C-treated plants. At this stage, the silencing was observed in localized regions. Silencing was uniform by 30 d after inoculation in plants that had methylated GFP-DNA, whereas plants that continued to display the same phenotype as seen at 14-15 d after inoculation had hypomethylated GFP-DNA. Viral expression of GFP persisted in pockets throughout the life of infected plants. • This is the first demonstration of a correlation between post transcriptional gene silencing induced by a DNA virus, and transgene methylation. The results suggest that, while DNA methylation is not necessary for the initiation of silencing, the progression of silencing is affected by inhibition of DNA methylation.
• 研究了由DNA病毒引发的病毒诱导基因沉默（VIGS）与沉默转基因的DNA甲基化之间的关联。
• 用DNA甲基化抑制剂5-氮杂胞苷（5-Aza-C）处理35S-绿色荧光蛋白（GFP）转基因本氏烟草，接种携带GFP编码序列的番茄金色花叶病毒后观察转基因的VIGS。
• 在对照植株和经5-Aza-C处理的植株中，35S-GFP转基因的VIGS在接种后14-16天开始出现。在此阶段，在局部区域观察到沉默现象。在接种后30天，GFP-DNA发生甲基化的植株中的沉默现象变得均匀，而在接种后14-15天继续表现出相同表型的植株中，GFP-DNA甲基化程度较低。在受感染植株的整个生命周期中，GFP的病毒表达在局部区域持续存在。
• 这是首次证明DNA病毒诱导的转录后基因沉默与转基因甲基化之间存在相关性。结果表明，虽然DNA甲基化对于沉默的起始不是必需的，但沉默的进展受到DNA甲基化抑制的影响。
