title: Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors.
abstract: Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Alberto
authors: A
authors: Carbonell
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Noah
authors: N
authors: Fahlgren
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Skyler
authors: S
authors: Mitchell
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Kevin L
authors: KL
authors: Cox
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Kevin C
authors: KC
authors: Reilly
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: Todd C
authors: TC
authors: Mockler
authors: Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.
authors: James C
authors: JC
authors: Carrington
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D058431:Brachypodium / Q000235:genetics*; D003001:Cloning, Molecular; D020868:Gene Silencing; D005822:Genetic Vectors; D055029:Inverted Repeat Sequences; D035683:MicroRNAs / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D012322:RNA Precursors
chemical_list: C584086:MIRN390 microRNA, Arabidopsis; D035683:MicroRNAs; D012322:RNA Precursors
keywords: Arabidopsis thaliana; Brachypodium distachyon; MIRNA precursor; RNA silencing; artificial microRNA; monocot; technical advance