Assessment of some gene transformation methods in microalgae Dunaliella salina

Microalgae Dunaliella salina is a single-cell eukaryotic organism which is able to grow in high salt concentration. It has several advantages as a green bioreactor for producing of several pharmaceutical drugs including vaccines, hormones, growth factors, monoclonal antibodies and other recombinant proteins. In present study some gene transformation methods were applied for insertion of foreign genes into the microalgae cells. In the first step and to determine suitable selectable marker, some growth inhibitory compounds, such as kanamycin, hygromaicin, chloramphenicol and phosphinotricine were used. The results indicate the sensitivity of microalgae D. salina to phosphinotricine in concentration of 7 µg/ml. Then, the gene bar, encoding phosphinotricine transferase, was introduced as selectable marker in transformation process. The methods applied in gene transformation include agitation with glass beads, electroporation and gene gun bombardment. The constructs containing the GUS gene applied for optimizing of the transformation process. To evaluate the effect of different promoters, the cauliflower mosaic virus 35S and maize ubiquitine promoters were used for expression of the GUS reporter gen. The expression of GUS reporter gene was evaluated by histochemical staining method. The results obtained here proved efficiency of the glass beads method for successful expression of foreign gene. In stable nuclear transformation methods, the transformed D. salina cells were cultured on media containing phosphinotricine selectable marker. These results proved capability of 35S promoter in transformation using glass beads method. The construct harbouring HBsAg gene was applied for gene transformation into microalga by using biolistic, glass beads agitation and agrobacterium co-cultivation. The transformed cells were moved into selective media. The presence of foreign gene in the transformed cells was analyzed by molecular approaches including PCR and RT-PCR. These results prove capability of biolistic and glass bead approaches for successful gene transformation into D. salina.