Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. (20th May 2015)

• Record Type: Journal Article
• Authors: Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; Cox, Kevin L.; Reilly, Kevin C.; Mockler, Todd C.; Carrington, James C.
• Is Part Of: Plant journal. Volume 82:Number 6(2015:Jun.)
• Page Start: 1061
• Page End: 1075
• Publication Date: 2015-05-20
• Subjects: Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.12835
• Languages: English
• ISSNs: 0960-7412

Abstract

<abstract abstract-type="main" id="tpj12835-abs-0001"> <title>Summary</title> <p>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 <italic>Oryza sativa MIR390</italic> (<italic>OsMIR390</italic>) 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 <italic>OsMIR390‐</italic>based vectors were validated in transgenic <italic>Brachypodium distachyon</italic> plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric <italic>OsMIR390</italic>‐based precursors that include distal stem–loop sequences from <italic>Arabidopsis thaliana MIR390a (AtMIR390a)</italic>. 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.</p> </abstract>