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A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis

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Abstract

Chaetoceros gracilis belongs to the centric diatoms, and has recently been used in basic research on photosynthesis. In addition, it has been commercially used in fisheries and is also attracting interest as a feedstock for biofuels production and biorefinery. In this study, we developed an efficient genetic transformation system for C. gracilis. The diatom cells were transformed via multi-pulse electroporation using plasmids containing various promoters to drive expression of the nourseothricin acetyltransferase gene (nat) as a selectable marker. The transformation efficiency reached ~400 positive transgenic clones per 108 recipient cells, which is the first example of successful transformation with electroporation in a centric diatom species. We further produced two expression vectors: the vector pCgLhcr5p contains the light-dependent promoter of a fucoxanthin chlorophyll a/c binding protein gene and the vector pCgNRp contains the inducible promoter of a nitrate reductase gene to drive the expression of introduced genes. In both vectors, an acetyl-CoA acetyltransferase promoter drives nat gene expression for antibiotic selection. Stable integration and expression of reporter genes, such as the firefly luciferase and green fluorescent protein Azami–Green genes, were observed in transformed C. gracilis cells. This efficient and stable transformation system for C. gracilis will enable both functional analysis of diatom-specific genes and strain improvement for further biotechnological applications.

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Acknowledgments

We thank Dr. N. Poulsen and Dr. N. Kröger at the Technical University of Dresden, Germany, for the gift of the vectors pTpNR-GFP/fcpNat, and Dr. J. Hotta, Yamagata Univ. for his help in microscopic analysis of the diatoms. We also appreciate the excellent technical assistance of Ms. N. Inui. This work was supported by the Japan Science and Technology Agency, Advanced Low Carbon Technology Research and Development Program (to K. I., Y. Y., and Y. K.).

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Authors and Affiliations

  1. Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

    Kentaro Ifuku, Dongyi Yan & Mado Miyahara

  2. Graduate School of Life Science, University of Hyogo, 3-2-1 Kohto, Kamigohri, Ako-gun, Hyogo, 678-1297, Japan

    Natsuko Inoue-Kashino & Yasuhiro Kashino

  3. Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu City, 501-1193, Japan

    Yoshiharu Y. Yamamoto

Authors
  1. Kentaro Ifuku
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  2. Dongyi Yan
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  3. Mado Miyahara
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  4. Natsuko Inoue-Kashino
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  5. Yoshiharu Y. Yamamoto
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  6. Yasuhiro Kashino
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Correspondence to Kentaro Ifuku.

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Ifuku, K., Yan, D., Miyahara, M. et al. A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis . Photosynth Res 123, 203–211 (2015). https://doi.org/10.1007/s11120-014-0048-y

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  • DOI: https://doi.org/10.1007/s11120-014-0048-y

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