An optimized method and a dominant selectable marker for genetic engineering of an industrially promising microalga—Pavlova lutheri
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The marine microalga Pavlova lutheri has great nutritional value as it synthesizes and accumulates higher amounts of polyunsaturated fatty acids (PUFAs). It is commonly used in aquaculture. However, no transformation procedure has been realized for this industrially important microalga so far. Here, we report an efficient protocol for the nuclear transformation of P. lutheri. Agrobacterium-mediated transformation (AMT) of P. lutheri with a mutated genomic clone of phytoene desaturase (pds) gene, pds-L504R, from Haematococcus pluvialis yielded norflurazon-resistant P. lutheri cells. Ideal co-cultivation conditions for achieving higher numbers of transformants was found to be artificial seawater (ASW) medium, 100 μM acetosyringone, and a 24-h co-cultivation at 25 ± 1 °C. The integration of the introduced gene into the nuclear genome of P. lutheri was shown by PCR amplification of the T-DNA sequences from the genomic DNA of transformants and Southern blot analysis using T-DNA sequences as probes. The transgene expressed efficiently as evidenced by the results of stability and tolerance study, and the qRT-PCR analysis. Results clearly demonstrate the application of AMT approach and pds gene as a dominant selectable marker for the genetic engineering of P. lutheri for fundamental studies and biotechnological applications.
KeywordsPavlova lutheri Agrobacterium tumefaciens Transformation Norflurazon Phytoene desaturase
The authors are thankful to Dr. J. Steinbrenner (Universität Konstanz, Germany) for kindly providing the plasmid pPLAT-pds-L504R. The authors appreciate the Korea Institute of Advanced Technology, Korea and Federal Ministry of Education and Research, Germany for supporting the work. BP also acknowledges Lehrstuhl für Bioverfahrenstechnik, Friedrich-Alexander-University of Erlangen Nuremberg, Germany for the research support.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Allnutt FCT, Kyle DJ, Grossman AR, Apt KE (2000) Methods and tools for transformation of eukaryotic algae. USA Patent Number: 6027900Google Scholar
- Andersen RA, Berges JA, Harrison PJ, Watanabe MM (2005) Recipes for freshwater and seawater media. In: Andersen RA (ed) Algal culturing techniques. Elsevier, Amsterdam, pp 429–538Google Scholar
- Chomczynski P, Mackey K (1995) Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide and proteoglycan rich sources. Biotechniques 19:942–945Google Scholar
- Den Dulk-Ras A, Hooykaas PJ (1995) Electroporation of Agrobacterium tumefaciens. Methods Mol Biol 55:63–72Google Scholar
- Enzing C, Ploeg M, Barbosa M, Sijtsma L (2014) JRC Scientific and Policy Reports: Microalgae-based products for the food and feed sector: an outlook for Europe. Institute for Prospective Technological Studies. (eds Vigani M, Parisi C, Cerezo ER). Joint Research Centre. European CommissionGoogle Scholar
- Karami O (2008) Factors affecting Agrobacterium-mediated transformation of plants. Transgenic Plant J 2:127–137Google Scholar
- Ng I-S, Tan S-I, Kao P-H, Chang Y-K, Chang J-S (2017) Recent developments on genetic engineering of microalgae for biofuels and bio-based chemicals. Biotechnol J 12:10. https://doi.org/10.1002/biot.201600644
- Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091Google Scholar