A duplicated carbonic anhydrase 1 (DCA1) promoter mediates the nitrate reductase gene switch of Dunaliella salina

The unicellular Dunaliella salina, the most extremely halotolerant eukaryote, is able to survive in salt concentrations from 0.05 to 5 M NaCl, whose halotolerant property encouraged us to develop salt-inducible promoters for research on transgenic bioreactors. A duplicated carbonic anhydrase 1 (DCA1) promoter from D. salina has been identified to drive the inducible expression of the bar gene under the induction of different NaCl concentrations. The purpose of this study was to identify whether the DCA1 promoter could drive and modulate the expression of the nitrate reductase (NR) gene in NR-deficient mutants of D. salina. Here, the wild-type NR gene under the control of the DCA1 promoter was transformed into the NR-deficient mutant (J1) of D. salina by electroporation, and the complemented transformant T1-J1 was screened by nitrate, in which NR activity was restored up to 48% of the wild-type (WT) cells. Different from the natural NR gene switch in WT cells where NR activity disappeared in ammonium medium, NR activity was detected in the T1-J1 cells despite changes of the nitrogen sources. Furthermore, both the NR transcripts and NR activity were increased with rising NaCl concentration from 0.5 to 2.0 M in the T1-J1 cells but not in the WT cells. These findings strongly support that the DCA1 promoter not only drives the inducible expression of the NR gene effectively, but also mediates lost of the natural NR gene switch in D. salina.