Stable nuclear transformation of the diatom Phaeodactylum tricornutum

Abstract

A nuclear transformation system has been developed for the diatom Phaeodactylum tricornutum using microparticle bombardment to introduce the sh ble gene from Streptoalloteichus hindustanus into cells. The sh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from the P. tricornutum fcp genes were used to drive expression of sh ble. Between 10-100 transformants were recovered/10(8) cells. Transformants were able to grow on at least 500 micrograms/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations the sh ble gene was present in 1-3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1-2.0 micrograms/mg total protein). The cat reporter gene fused to a fcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed in P. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.