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Reduction in Carotenoid Levels in the Marine Diatom Phaeodactylum tricornutum by Artificial MicroRNAs Targeted Against the Endogenous Phytoene Synthase Gene

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Abstract

MicroRNAs (miRNAs) are key regulators of gene expression in eukaryotes where they can function to downregulate expression levels or functioning of messenger RNAs (mRNAs) that are targeted by mature miRNAs displaying sequence homology. The ‘active’ mature miRNA forms are short RNAs which are processed from longer precursor miRNA molecules that have a stem-loop structure. While artificial miRNAs have been developed for gene knockdown experiments in a range of eukaryotes, it is not known whether artificial or endogenous miRNAs can functionally knockdown mRNA levels in the model marine diatom Phaeodactylum tricornutum. Here, we investigate the potential use of artificial microRNAs (amiRNAs) for targeted gene knockdowns in P. tricornutum, by generation of transformants harbouring a transgene cassette for the generation of amiRNAs designed to target the endogenous phytoene synthase (PSY) gene. In P. tricornutum, the amiRNA stem-loop precursor was processed to produce a mature amiRNA that successfully targeted the PSY mRNA and reduced PSY mRNA levels. As the PSY gene is a key component of the carotenoid biosynthetic pathway, the levels of carotenoids in the P. tricornutum amiRNA knockdown lines were reduced relative to untransformed control lines. This study demonstrates that artificial miRNAs can be successfully deployed for gene knockdown experiments in the model diatom P. tricornutum, providing a powerful tool for future metabolic engineering and synthetic biology experimentation in this model marine diatom.

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Acknowledgments

We thank Phil Dix (National University of Ireland, Maynooth (NUIM), Ireland) for provision of PDS-1000/He particle delivery system for biolistic transformation. CS and SK are grateful to Ireland’s Environment Protection Agency (EPA) STRIVE Programme (2009-PhD-ET-8) for funding this research. We are thankful to Ronan Sulpice and Peter McKeown for their comments on earlier drafts of this manuscript.

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  1. Genetics and Biotechnology Laboratory, Plant and AgriBiosciences Research Centre (PABC), School of Natural Sciences, National University of Ireland Galway, University Road, Galway, Ireland

    Simrat Kaur & Charles Spillane

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  1. Simrat Kaur
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  2. Charles Spillane
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Correspondence to Charles Spillane.

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Kaur, S., Spillane, C. Reduction in Carotenoid Levels in the Marine Diatom Phaeodactylum tricornutum by Artificial MicroRNAs Targeted Against the Endogenous Phytoene Synthase Gene. Mar Biotechnol 17, 1–7 (2015). https://doi.org/10.1007/s10126-014-9593-9

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  • DOI: https://doi.org/10.1007/s10126-014-9593-9

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