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Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial trans-acting small interfering RNA

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Abstract

Key message

TAS atasiRNA-producing region swapping used one-step, high efficiency, and high fidelity directional TC-cloning. Uniform silencing was achieved without lethality using miRNA trigger- TAS overexpression fusion cassettes to generate 21-nt atasiRNA.

Abstract

Plant transgenic technologies are very important for basic plant research and biotechnology. Artificial trans-acting small interfering RNA (atasiRNA) represents an attractive platform with certain advantages over other silencing approaches, such as hairpin RNA, artificial microRNA (amiRNA), and virus-induced gene silencing (VIGS). In this study, we developed two types of constructs for atasiRNA-mediated gene silencing in plants. To functionally validate our constructs, we chose TAS1a as a test model. Type 1 constructs had miR173-precursor sequence fused with TAS1a locus driven by single promoter–terminator cassette, which simplified the expression cassette and resulted in uniform gene silencing. Type 2 constructs contained two separate cassettes for miR173 and TAS1a co-expression. The constructs in each type were further improved by deploying the XcmI-based TC-cloning system for highly efficient directional cloning of short DNA fragments encoding atasiRNAs into TAS1a locus. The effectiveness of the constructs was demonstrated by cloning an atasiRNA DNA into the TC site of engineered TAS1a and silencing of CHLORINA 42 (CH42) gene in Arabidopsis. Our results show that the directional TC-cloning of the atasiRNA DNA into the engineered TAS1a is highly efficient and the miR173–TAS1a fusion system provides an attractive alternative to achieve moderate but more uniform gene silencing without lethality, as compared to conventional two separate cassettes for miR173 and TAS locus co-expression system. The design principles described here should be applicable to other TAS loci such as TAS1b, TAS1c, TAS2, or TAS3, and cloning of amiRNA into amiRNA stem-loop.

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Abbreviations

amiRNA:

Artificial microRNA

atasiRNA:

Artificial trans-acting small interfering RNA

DCL:

Dicer-like protein

dsRNA:

Double-stranded RNA

sRNA:

Small RNA

siRNA:

Small interfering RNA

TAS :

Trans-acting siRNA locus

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Acknowledgments

We thank Dr. David Setzer for allowing us to use his laboratory and the equipment for primer extension analysis. Thanks are also expended to: Dr. Joann R. De Tar for her technical assistance in Glycine max FAD2-1B atasiRNA design, and Mr. Neng Wang for his Arabidopsis planting and plant care as well as Dr. William Folk and Mrs. Theresa Musket for proof-reading. This work was supported by the Mid-America Research and Development Foundation and the Missouri Soybean Merchandising Council Grant (Project No. 11-338).

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Author notes

  1. Xinlu Chen

    Present address: Department of Plant Sciences, University of Tennessee, 347/359 Plant Biotech, Knoxville, TX, 37996, USA

Authors and Affiliations

  1. Plant Transformation Core Facility, Division of Plant Sciences, University of Missouri, 1-33 Agriculture Building, Columbia, MO, 65211, USA

    Ulku Baykal, Hua Liu, Xinlu Chen, Henry T. Nguyen & Zhanyuan J. Zhang

Authors
  1. Ulku Baykal
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  2. Hua Liu
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  3. Xinlu Chen
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  4. Henry T. Nguyen
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  5. Zhanyuan J. Zhang
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Corresponding author

Correspondence to Zhanyuan J. Zhang.

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The authors declare that they have no conflict of interest.

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Communicated by Z.-Y. Wang.

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Baykal, U., Liu, H., Chen, X. et al. Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial trans-acting small interfering RNA. Plant Cell Rep 35, 2137–2150 (2016). https://doi.org/10.1007/s00299-016-2024-9

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  • DOI: https://doi.org/10.1007/s00299-016-2024-9

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