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A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

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Abstract

Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

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Acknowledgments

This work was supported by the National Science Foundation of China (Grant No. 31171615, No. 31171543), China Agriculture Research System (CARS-05) and National Special Foundation for Transgenic Species of China (2014ZX08009-003-001; 2013ZX08009-003-001).

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Authors and Affiliations

  1. Institute of Genetics, College of Life Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China

    Yu Bai, Ning Han, Jinxia Wu, Yinong Yang, Junhui Wang, Muyuan Zhu & Hongwu Bian

  2. Department of Plant Pathology and Environmental Microbiology, and Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA

    Yinong Yang

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  1. Yu Bai
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  2. Ning Han
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  3. Jinxia Wu
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  4. Yinong Yang
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  5. Junhui Wang
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  6. Muyuan Zhu
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  7. Hongwu Bian
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Correspondence to Hongwu Bian.

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Yu Bai and Ning Han have contributed equally to this work.

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Bai, Y., Han, N., Wu, J. et al. A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes. Plant Cell Tiss Organ Cult 119, 211–219 (2014). https://doi.org/10.1007/s11240-014-0527-z

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