Plant Biotechnology Research Group, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa.
Biotechniques. 2012 Apr;52(4):254-62. doi: 10.2144/0000113839.
RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.
RNA 沉默已被用于通过转录后基因沉默 (PTGS) 来生产对病毒病原体具有抗性的转基因植物。在某些情况下，由于反向重复 (IR) 构建体在克隆和植物转化过程中的不稳定性，该技术难以应用。尽管已经证明这种构建体可以通过内含子稳定化并有效地诱导 RNA 沉默，但我们发现 Pdk 内含子并没有稳定南非木薯花叶病毒 (SACMV) 沉默构建体。因此，我们开发了一种在组装 IR 之前，通过亚硫酸氢盐诱导 sense 臂上的碱基对错配来产生长 SACMV IR 构建体的方法。在模式测试植物烟草原生质体中表达 SACMV BC1 错配 IR 构建体，导致在 SACMV 感染后，病毒 BC1 转录本水平降低，从而降低病毒复制。在烟草原生质体愈伤组织系统中，错配的 SACMV AC1 IR 构建体比非错配 IR 构建体更有效地诱导 PTGS。我们生成 IR 构建体的新方法应该适用于许多过去证明难以生成这些构建体的序列。
