PDF(Pubmed)
Abstract：
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.
摘要：
通过农业浸润瞬时表达和诱导RNA沉默是植物RNA生物学的基本方法。这里,我们介绍了一种新的使用RUBY的报告分析，编码betalain生物合成途径的三个关键酶，作为多顺反子mRNA。甜菜碱赋予的红色色素沉着允许视觉确认基因表达或沉默水平，而不会破坏组织。和沉默水平可以定量测量吸光度在短短几分钟。与众所周知的RNA沉默抑制剂p19结合的RUBY的浸润，与单独的RUBY渗透相比，在渗透后7天诱导的甜菜素积累高五倍。我们证明了RUBY与两种RNA沉默诱导剂的共浸润，靶向CYP76AD1或RUBY构建体内的糖基转移酶，有效降低RUBYmRNA和betalain水平，表明成功的RNA沉默。因此,与常规的RNA沉默报告分析相比，基于RUBY的测定法提供了一种简单快速的定量分析方法，而无需专用设备，使其可用于广泛的RNA沉默研究。
