Ritsumeikan-Global Innovation Research Organization, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Plant Mol Biol. 2024 May 19;114(3):61. doi: 10.1007/s11103-024-01463-8.
Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.
农杆菌介导的瞬时表达和 RNA 沉默诱导是植物 RNA 生物学中的一种基本方法。在这里，我们介绍了一种新的报告基因检测方法，该方法使用 RUBY 作为多顺反子 mRNA，其编码甜菜色素生物合成途径的三个关键酶。甜菜色素赋予的红色着色使得无需组织破坏即可直观确认基因表达或沉默水平，并且沉默水平可以在短短几分钟内通过吸光度定量测量。与单独使用 RUBY 相比，在 p19（一种众所周知的 RNA 沉默抑制剂）存在的情况下共浸润 RUBY 可使 7 天后甜菜素的积累增加五倍。我们证明，与针对 RUBY 构建体内的 CYP76AD1 或糖基转移酶的两种 RNA 沉默诱导剂共浸润可有效降低 RUBY mRNA 和甜菜素水平，表明 RNA 沉默成功。因此，与传统的 RNA 沉默报告基因检测相比，基于 RUBY 的检测方法提供了一种简单快速的定量分析方法，而无需专门设备，使其成为广泛的 RNA 沉默研究的有用工具。
