Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Abstract

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

A Novel Biocompatible Polymer Derived from D-Mannitol Used as a Vector in the Field of Genetic Engineering of Eukaryotic Cells

“…Genetic transformation of plant cells is an important process that allows the deletion, overexpression or modulation of certain genes to improve nutritional qualities, to increase the resistance to pathogens and to improve tolerance to pollutants [6][7][8]. Unicellular microalgae have been widely used as plant model organisms and have received increasing attention to yield high-added value products including pigments, vitamins, polyunsaturated fatty acids or biofuels [9][10][11][12]. Although genetic engineering is a promising strategy for the Abbreviations: pDNA, plasmid of DNA.…”