Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin.

Promoter sequences of the cytomegalovirus (PCMV), the rous sarcoma virus long terminal repeat (PRSV-LTR) and the cauliflower mosaic virus 35s (PCaMV35s) were ligated with the beta-glucuronidase (GUS) gene, uidA, and were introduced into cells of the marine diatom, Phaeodactylum tricornutum. Transformants were selected on a 100 mg l(-1) Zeocin plate, and Zeocin-resistant clones were further selected by the occurrence of GUS activity. Two to 10 GUS-positive clones were obtained, and GUS activities in these transformants did not change in response to changes in ambient CO(2) concentration except that the PRSV-LTR was weakly activated in air. These results indicate that a wide spectrum of viral promoters originating from mammalian, avian and plant hosts can operate as constitutive promoters in a marine diatom. The CO(2) responsive promoter sequence of the chloroplastic carbonic anhydrase gene in P. tricornutum (Pptca1) with a deleted initiator region was ligated with the minimal region of the PCMV followed by uidA and was introduced into P. tricornutum. GUS expression in the resulting transformants was clearly regulated by CO(2), that is, GUS expression was stimulated in air to about 10-fold than that in cells grown in 5% CO(2). However, the CO(2) response disappeared when the core regulatory region of Pptca1 (-76 to -11 bp) was removed. The regulative function of the endogenous diatom promoter was thus maintained after fusion with an extrinsic viral promoter. These results indicate that diatom cells accommodate a wide range of transcriptional system from beyond the plant kingdom and that an efficient transcriptional system could potentially be constructed in marine diatoms by selecting an appropriate set of viral promoter and functional cis elements.