Journal Article

effects of spacer sequences on silencing efficiency of plant RNAi vectors

Hirai, Sayaka, Kodama, Hiroaki, Oka, Shin-ichiro, Adachi, Eri

2007

Overview

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 the NtFAD3 gene 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 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.

• Publisher: Berlin/Heidelberg : Springer-Verlag,Springer,Springer Nature B.V
• Subject: alpha-linolenic acid / Binary vector / Biological and medical sciences / Biotechnology / Desaturase / DNA, Intergenic / DNA, Intergenic - genetics / Endoplasmic reticulum / Expression vectors / Fatty acids / Fundamental and applied biological sciences. Psychology / Gene expression / genes / Genetic engineering / Genetic technics / Genetic Vectors / Genetic Vectors - genetics / genetics / Hairpin construct / Hairy root / In vivo methods and tests / Introns / Introns - genetics / Inverted repeat / Linolenic acid / Lipids / Methods. Procedures. Technologies / Nicotiana - genetics / Nucleotide sequence / omega-3 fatty acids / phenotype / Phenotypes / Plants, Genetically Modified / Promoter Regions, Genetic / Promoter Regions, Genetic - genetics / proteins / Ribonucleic acid / RNA / RNA Interference / RNA processing / RNA-mediated interference / Roots / Spacer / stearoyl-CoA desaturase / Tobacco / Transgenic animals and transgenic plants / Transgenic plants