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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. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels ofmore » highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. Donald Danforth Plant Science Center, St. Louis MO 63132 USA
Publication Date:
Research Org.:
Donald Danforth Plant Science Center, St. Louis, MO (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1295954
Alternate Identifier(s):
OSTI ID: 1295955; OSTI ID: 1457329
Grant/Contract Number:  
SC0006627; MCB-1231726; MCB-1330562; AI043288
Resource Type:
Published Article
Journal Name:
The Plant Journal
Additional Journal Information:
Journal Name: The Plant Journal Journal Volume: 82 Journal Issue: 6; Journal ID: ISSN 0960-7412
Publisher:
Society for Experimental Biology
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; RNA silencing; artificial microRNA; MIRNA precursor; Brachypodium distachyon; monocot; Arabidopsis thaliana; technical advance

Citation Formats

Carbonell, Alberto, Fahlgren, Noah, Mitchell, Skyler, Cox, Jr, Kevin L., Reilly, Kevin C., Mockler, Todd C., and Carrington, James C. Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. United Kingdom: N. p., 2015. Web. doi:10.1111/tpj.12835.
Carbonell, Alberto, Fahlgren, Noah, Mitchell, Skyler, Cox, Jr, Kevin L., Reilly, Kevin C., Mockler, Todd C., & Carrington, James C. Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. United Kingdom. https://doi.org/10.1111/tpj.12835
Carbonell, Alberto, Fahlgren, Noah, Mitchell, Skyler, Cox, Jr, Kevin L., Reilly, Kevin C., Mockler, Todd C., and Carrington, James C. Wed . "Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors". United Kingdom. https://doi.org/10.1111/tpj.12835.
@article{osti_1295954,
title = {Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors},
author = {Carbonell, Alberto and Fahlgren, Noah and Mitchell, Skyler and Cox, Jr, Kevin L. and Reilly, Kevin C. and Mockler, Todd C. and Carrington, James C.},
abstractNote = {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. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.},
doi = {10.1111/tpj.12835},
journal = {The Plant Journal},
number = 6,
volume = 82,
place = {United Kingdom},
year = {Wed May 20 00:00:00 EDT 2015},
month = {Wed May 20 00:00:00 EDT 2015}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1111/tpj.12835

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Cited by: 32 works
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