Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum

Abstract

Phaeodactylum tricornutum is a pennate model diatom whose entire genome has been sequenced and an expressed sequence tag database established. This study aimed at developing a genetic tool kit to modify the genetic traits of P. tricornutum. A new expression system containing the elongation factor 2 (EF2) promoter fused to the luciferase gene was constructed, resulting in the pPhaT-EF2-Luc vector. This new vector was compared to the pPhaT-BP-Luc which harbors the fcpB promoter of P. tricornutum. After P. tricornutum transformation with these vectors, the presence of the exogenous luciferase gene inserted into the genomic DNA of the selected transformants was confirmed by Southern blot analysis. In the transformants, the fcpB promoter was activated dramatically upon 5h of light exposure and its activity was reduced in the dark. However, the expression level of the Luc gene regulated by the EF2 promoter was not significantly affected by light and was higher than that of the Luc gene regulated by the fcpB promoter. Transformants harboring the pEF2::Luc cassette had a high level of luciferase protein expression and constant luciferase activity throughout the light/dark cycle. In addition, the EF2 promoter of P. tricornutum was able to drive the expression of a heterologous gene in the green alga Chlamydomonas reinhardtii. These results indicate that the P. tricornutum EF2 promoter can be used for the expression of target genes in genetically transformed P. tricornutum and C. reinhardtii.