期刊
PLANT JOURNAL
卷 82, 期 6, 页码 1061-1075出版社
WILEY
DOI: 10.1111/tpj.12835
关键词
RNA silencing; artificial microRNA; MIRNA precursor; Brachypodium distachyon; monocot; Arabidopsis thaliana; technical advance
资金
- USDA AFRI NIFA Postdoctoral Fellowship [MOW-2012-01361]
- National Science Foundation [MCB-1231726, MCB-1330562]
- National Institutes of Health [AI043288]
- Department of Energy [DOE DE-SC0006627]
- U.S. Department of Energy (DOE) [DE-SC0006627] Funding Source: U.S. Department of Energy (DOE)
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1330562] Funding Source: National Science Foundation
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. 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.
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