Hirai Sayaka, Oka Shin-ichiro, Adachi Eri, Kodama Hiroaki
Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, 263-8522 Japan.
Plant Cell Rep. 2007 May;26(5):651-9. doi: 10.1007/s00299-006-0277-4. Epub 2007 Jan 5.
RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the alpha-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum omega-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of alpha-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in alpha-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.
RNA干扰（RNAi）已被用于抑制各种真核生物中的基因表达。在植物中，通过导入转录自互补发夹RNA的RNAi载体可诱导RNAi。大多数基本的RNAi构建体都有一个被间隔序列打断的反向重复序列。为了测试RNAi构建体的沉默能力，我们开发了一种基于RNAi介导的毛状根中α-亚麻酸含量变化的体内检测方法。烟草内质网ω-3脂肪酸去饱和酶（NtFAD3）是根膜脂中α-亚麻酸产生的主要酶。用针对NtFAD3基因的RNAi载体转化的烟草毛状根显示α-亚麻酸含量降低。RNA沉默的频率受间隔序列的影响比间隔长度更大，至少在100至1800 bp之间是这样。由于在表现出弱沉默表型的转基因植物中仍存在大量针对NtFAD3基因的发夹RNA，低程度的沉默归因于类Dicer蛋白介导的发夹RNA加工效率低下。我们的结果表明，通过替换RNAi构建体的间隔序列，有可能产生广泛的RNAi诱导的沉默表型。
