Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): Expression of GUS in microalgae using heterologous promoter constructs

Abstract

Genetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptll) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1'2' promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5-24 transformants per 107 cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β-glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1'2' promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation.