Original Paper

Molecular and General Genetics MGG

, Volume 252, Issue 5, pp 572-579

Stable nuclear transformation of the diatomPhaeodactylum tricornutum

  • K. E. AptAffiliated withDepartment of Plant Biology, Carnegie Institution of Washington
  • , A. R. GrossmanAffiliated withDepartment of Plant Biology, Carnegie Institution of Washington
  • , P. G. Kroth-PancicAffiliated withInstitut für Biochemie der Pflanzen, Heinrich-Heine Universität Düsseldorf

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Abstract

A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh 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 µg/mg total protein). Thecat reporter gene fused to afcp 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 inP. 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.

Key words

Bacillariophyta Biolistic transformation Phleomycin Reporter gene Selectable marker