Marine Biotechnology

, Volume 17, Issue 1, pp 1–7 | Cite as

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

Short Communication
First Online:
Received:
Accepted:

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.

Keywords

Artificial miRNA Carotenoids Phaeodactylum tricornutum Phytoene synthase Synthetic biology 
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Notes

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.

References

  1. Allen AE, Dupont CL, Obornik M, Horak A, Nunes-Nesi A, McCrow JP, Zheng H, Johnson DA, Hu H, Fernie AR, Bowler C (2011) Evolution and metabolic significance of the urea cycle in photosynthetic diatoms. Nature 473:203–207PubMedCrossRefGoogle Scholar
  2. Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A, Maheswari U, Martens C, Maumus F, Otillar RP, Rayko E, Salamov A, Vandepoele K, Beszteri B, Gruber A, Heijde M, Katinka M, Mock T, Valentin K, Verret F, Berges JA, Brownlee C, Cadoret J-P, Chiovitti A, Choi CJ, Coesel S, De Martino A, Detter JC, Durkin C, Falciatore A, Fournet J, Haruta M, Huysman MJJ, Jenkins BD, Jiroutova K, Jorgensen RE, Joubert Y, Kaplan A, Kroger N, Kroth PG, La Roche J, Lindquist E, Lommer M, Martin-Jezequel V, Lopez PJ, Lucas S, Mangogna M, McGinnis K, Medlin LK, Montsant A, Secq M-PO-L, Napoli C, Obornik M, Parker MS, Petit J-L, Porcel BM, Poulsen N, Robison M, Rychlewski L, Rynearson TA, Schmutz J, Shapiro H, Siaut M, Stanley M, Sussman MR, Taylor AR, Vardi A, von Dassow P, Vyverman W, Willis A, Wyrwicz LS, Rokhsar DS, Weissenbach J, Armbrust EV, Green BR, Van de Peer Y, Grigoriev IV (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456:239–244PubMedCrossRefGoogle Scholar
  3. Chen GaL J (2010) Use of quantitative polymerase chain reaction for determining copy numbers of transgenes in Lesquerella fendleri. Am J Agri Biol Sci 5:415–421CrossRefGoogle Scholar
  4. Darty K, Denise A, Ponty Y (2009) VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics 25:1974–1975PubMedCentralPubMedCrossRefGoogle Scholar
  5. De Riso V, Raniello R, Maumus F, Rogato A, Bowler C, Falciatore A (2009) Gene silencing in the marine diatom Phaeodactylum tricornutum. Nucleic Acids Res 37:e96PubMedCentralPubMedCrossRefGoogle Scholar
  6. Dunoyer P, Brosnan CA, Schott G, Wang Y, Jay F, Alioua A, Himber C, Voinnet O (2010) An endogenous, systemic RNAi pathway in plants. EMBO J 29:1699–1712PubMedCentralPubMedCrossRefGoogle Scholar
  7. Huang A, He L, Wang G (2011) Identification and characterization of microRNAs from Phaeodactylum tricornutum by high-throughput sequencing and bioinformatics analysis. BMC Genomics 12:1471–2164Google Scholar
  8. Khraiwesh B, Ossowski S, Weigel D, Reski R, Frank W (2008) Specific gene silencing by artificial microRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148:684–693PubMedCentralPubMedCrossRefGoogle Scholar
  9. Kroth PG (2007) Genetic transformation: a tool to study protein targeting in diatoms. Methods Mol Biol 390:257–267PubMedCrossRefGoogle Scholar
  10. Mathelier A, Carbone A (2010) MIReNA: finding microRNAs with high accuracy and no learning at genome scale and from deep sequencing data. Bioinformatics 26:2226–2234PubMedCrossRefGoogle Scholar
  11. McCarthy SS, Kobayashi MC, Niyogi KK (2004) White mutants of Chlamydomonas reinhardtii are defective in phytoene synthase. Genetics 168:1249–1257PubMedCentralPubMedCrossRefGoogle Scholar
  12. Molnar A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007) miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature 447:1126–1129PubMedCrossRefGoogle Scholar
  13. Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58:165–174PubMedCrossRefGoogle Scholar
  14. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690PubMedCrossRefGoogle Scholar
  15. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626PubMedCentralPubMedCrossRefGoogle Scholar
  16. Rohr J, Sarkar N, Balenger S, Jeong BR, Cerutti H (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J 40:611–621PubMedCrossRefGoogle Scholar
  17. Ryckebosch E, Muylaert K, Eeckhout M, Ruyssen T, Foubert I (2011) Influence of drying and storage on lipid and carotenoid stability of the microalga Phaeodactylum tricornutum. J Agric Food Chem 59:11063–11069PubMedCrossRefGoogle Scholar
  18. Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell Online 18:1121–1133CrossRefGoogle Scholar
  19. Xu P, Zhang Y, Kang L, Roossinck MJ, Mysore KS (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142:429–440PubMedCentralPubMedCrossRefGoogle Scholar
  20. Yamasaki T, Miyasaka H, Ohama T (2008) Unstable RNAi effects through epigenetic silencing of an inverted repeat transgene in Chlamydomonas reinhardtii. Genetics 180:1927–1944PubMedCentralPubMedCrossRefGoogle Scholar
  21. Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Genetics and Biotechnology Laboratory, Plant and AgriBiosciences Research Centre (PABC), School of Natural SciencesNational University of Ireland GalwayGalwayIreland

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