An optimized method and a dominant selectable marker for genetic engineering of an industrially promising microalga—Pavlova lutheri

Abstract

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.