DEVELOPMENT OF A CONSTITUTIVE PROMOTER FOR TRANSFORMATION OF THE DIATOM PHAEODACTYLUM TRICORNUTUM

Abstract

A marine algae Phaeodactylum tricornutum is a model diatom, whose whole genome was completely sequenced, and expressed sequence tag database is available. Its stable transformation method was established, so it is easy to transform it genetically. In order to develop a new and effective promoter for the constitutive expression of foreign genes, we have constructed a new expression system containing the EF2 promoter fused to a reporter luciferase gene, pPhaT-EF2-Luc, and this new vector system was compared to the existing vector which harbors the fcp promoter, pPhaT-BP-Luc, in P. tricornutum. After P. tricornutum was transformed with these vectors, it was confirmed by southern blot analysis that the exogenous luciferase gene was inserted into the genomic DNA of the selected transformants. In the transformants, the fcpB promoter was activated dramatically upon 5 h of light exposure and its activity was reduced in dark period. However, the expression level of Luc gene regulated by the EF2 promoter was steady regardless of light, and was higher than that of the Luc gene regulated by the fcpB promoter. Transformants harboring the pEF2::Luc cassette showed a high level of protein expression of luciferase and no significant change in the activity of luciferase throughout the light/dark cycle. In addition, the EF2 promoter in P. tricornutum was also shown to be able to express heterogeneous gene in green algae Chlamydomonas reinhardtii. These results indicate that the EF2 promoter is effective in heterogeneous overexpression in P. tricornutum transformants, and it is able to be used for expression of target gene in P. tricornutum and C. reinhardtii.