The effects of spacer sequences on silencing efficiency of plant RNAi vectors
The effects of spacer sequences on silencing efficiency of plant RNAi vectors
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DOI：
10.1007/s00299-006-0277-4
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发表时间：
2007-01
影响因子：
6.2
通讯作者：
Sayaka Hirai;S. Oka;E. Adachi;H. Kodama
中科院分区：
文献类型：
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作者：
Sayaka Hirai;S. Oka;E. Adachi;H. Kodama
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，可以通过引入转录自身互补发夹RNA的RNAi载体来诱导RNAi。大多数基本的RNAi构建体具有被间隔序列中断的反向重复序列。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测定。烟草内质网ω-3脂肪酸去饱和酶（NtFAD 3）是烟草根膜脂α-亚麻酸合成的主要酶。用抗NtFAD 3基因的RNAi载体转化烟草毛状根后，α-亚麻酸含量下降。RNA沉默的频率受间隔序列的影响大于间隔长度，至少在100和1800 bp之间。由于大量的针对NtFAD 3基因的发夹RNA仍然存在于表现出弱沉默表型的转基因植物中，因此低程度的沉默归因于Dicer样蛋白介导的发夹RNA加工效率低。我们的研究结果表明，通过替换RNAi构建体的间隔序列，可以产生广泛的RNAi诱导的沉默表型。
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 α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against theNtFAD3gene showed a decrease in α-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 theNtFAD3gene 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.