Inducible EGFP expression under the control of the nitrate reductase gene promoter in transgenic Dunaliella salina

The upstream region of the nitrate reductase (NR) gene is able to provide an ideal promoter for inducible expression of the heterologous genes because NR transcripts are induced by nitrate and repressed by ammonium. To improve the expression of heterologous proteins in transgenic Dunaliella salina, we developed a novel vector with the ability to switch on or off the expression of the heterologous genes using the upstream region of the NR gene. In the present study, a 1.2-kb fragment containing the promoter (Pnr) and a 0.9-kb fragment containing the terminator (Tnr) of the NR gene were cloned from the genome of D. salina. The results of mobility shift assays indicated that a GATA element in the isolated Pnr was involved in the nitrogen-dependent regulation. Additionally, the transcription start site of the D. salina NR gene was located at 133 bp upstream of the start codon ATG. Gene splicing by overlap extension (SOEing) was employed to construct a recombinant vector, p7NET, containing a Pnr-EGFP-Tnr expression cassette. On day 2 after the p7NET was introduced into the D. salina cells, EGFP-expressing transformants of D. salina appeared, in which expression of EGFP was induced by nitrate but repressed by ammonium. RT-PCR and PCR-Southern blots showed that the EGFP gene was correctly transcribed in transgenic D. salina. The findings of the present study suggest that the promoter of the NR gene from D. salina may be used to drive the inducible expression of heterologous proteins in transgenic D. salina.