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Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation

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Abstract

Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

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Availability of Data and Materials

The 18S rDNA sequence of strain NIES-4635 is available under DDBJ accession number LC776760. The sequences of N. palea fcp promoter and terminator are available under DDBJ accession numbers LC776935 and LC776936.

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Acknowledgements

We thank Dr. Wataru Sakamoto at the Institute of Plant Science and Resources, Okayama University, Japan, for his assistance with the microparticle bombardment. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics.

Funding

This study was supported by the Program to Disseminate Tenure Tracking System from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan; Grants-in-Aid for Scientific Research (C) (No. 18K05818); and the Asahi Glass Foundation to MN.

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Authors and Affiliations

  1. Graduate School of Environmental and Life Science, Okayama University, Okayama, 700-8530, Japan

    Koki Okada, Yu Morimoto & Yukine Shiraishi

  2. Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan

    Takashi Tamura & Michiko Nemoto

  3. The Advanced Support Center for Science Teachers, Tokyo Gakugei University, Tokyo, 184-8511, Japan

    Shigeki Mayama

  4. Tokyo Diatomology Lab, 2-3-2 Nukuikitamachi, Koganei, Tokyo, 184-0015, Japan

    Shigeki Mayama

  5. Faculty of Agriculture and Marine Science, Kochi University, Otsu-200, Monobe, Nankoku, Kochi, 783-8502, Japan

    Takashi Kadono & Masao Adachi

  6. Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake, Sakyo, Kyoto, 606-8502, Japan

    Kentaro Ifuku

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  1. Koki Okada
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  2. Yu Morimoto
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  3. Yukine Shiraishi
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  7. Masao Adachi
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  9. Michiko Nemoto
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Contributions

Michiko Nemoto designed the research; Koki Okada, Yu Morimoto, and Yukine Shiraishi performed the experiments; Koki Okada, Yu Morimoto, Yukine Shiraishi, Takashi Tamura, Shigeki Mayama, Takashi Kadono, Masao Adachi, Kentaro Ifuku and Michiko Nemoto analyzed the data; Koki Okada, Takashi Kadono, Masao Adachi, Kentaro Ifuku, and Michiko Nemoto wrote and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Michiko Nemoto.

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Okada, K., Morimoto, Y., Shiraishi, Y. et al. Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation. Mar Biotechnol 25, 1208–1219 (2023). https://doi.org/10.1007/s10126-023-10273-w

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