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Transient expression for functional gene analysis using Populus protoplasts

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Abstract

Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31100484, 31200507), the High-level Talents Program of Nanjing Forestry University (GXL201202), and the National High Technology Research and Development Program of China (2013AA102703).

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Authors and Affiliations

  1. Jiangsu Key Laboratory for Poplar Germplasm Enhancement and Variety Improvement, Nanjing Forestry University, Nanjing, 210037, China

    Biyue Tan, Meng Xu, Ying Chen & Minren Huang

  2. Key Laboratory of Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, 201137, China

    Biyue Tan, Meng Xu, Ying Chen & Minren Huang

Authors
  1. Biyue Tan
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  2. Meng Xu
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  3. Ying Chen
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  4. Minren Huang
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Corresponding authors

Correspondence to Meng Xu or Minren Huang.

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Biyue Tan and Meng Xu contributed equally to this work.

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Tan, B., Xu, M., Chen, Y. et al. Transient expression for functional gene analysis using Populus protoplasts. Plant Cell Tiss Organ Cult 114, 11–18 (2013). https://doi.org/10.1007/s11240-013-0299-x

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  • DOI: https://doi.org/10.1007/s11240-013-0299-x

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