RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants
RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants
复制标题
DOI：
10.1007/s10059-013-2203-2
复制
发表时间：
2013-03-01
影响因子：
3.8
通讯作者：
Lee, Kyun Oh
中科院分区：
文献类型：
--
作者：
Harmoko, Rikno;Fanata, Wahyu Indra Duwi;Lee, Kyun Oh
在植物中，具有反向重复序列的转基因用于诱导有效的RNA沉默，这也经常由高度转录的有义转基因诱导。由正义转基因诱导的RNA沉默依赖于RNA依赖性RNA聚合酶6（RDR 6），其将单链（ss）RNA转化为双链（ds）RNA。相比之下，已经提出由自身互补发夹RNA（hpRNA）诱导的RNA沉默不需要RDR 6，因为hpRNA可以直接折叠回自身以形成dsRNA。然而，目前尚不清楚RDR 6是否通过扩增dsRNA在植物内传播RNA沉默而在hpRNA诱导的RNA沉默中发挥作用。为了解决在存在或不存在RDR 6的情况下hpRNA诱导的RNA沉默的效率，产生了表达绿色荧光蛋白（GFP）的野生型（WT，Col-0）和rdr 6 -11拟南芥系，并用GFP-RNA干扰（RNAi）构建体转化。尽管大多数GFP-RNAi转化的WT系在T1代中表现出GFP表达的几乎完全沉默，但在GFP-RNAi转化的rdr 6 -11系中仍保持不同水平的GFP表达。T3代中GFP-RNAi的纯合表达不足以在几个rdr 6 -11系中诱导完全GFP沉默。我们的研究结果表明RDR 6是高效hpRNA诱导的RNA沉默所必需的。
In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.