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

Abstract

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.

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

“…According to this property of the NR gene switch, the NR gene promoter has been developed to drive inducible expression of foreign genes (Li et al 2008;Koblenz and Lechtreck 2005;Loppes et al 1999). In this study, an NR-deficient-rescue system was developed by complementing the NR-deficient mutants with the WT NR gene controlled by the DCA1 promoter.…”

Manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina using AccD and ME genes to enhance lipid content and to improve produced biodiesel quality

“…In other studies, transformation of some microalgae strains such as Chlamydomonas, Chlorella, Dunaliella with the Nit1 promoter is accomplished by electroporation (Champagne et al, 2009). Nit promoter previously was introduced into the D. salina genome as a inducible promoter to control expression of the bar and EGFP genes using the inexpensive inducer nitrate and repressor ammonium (Li et al, 2007;Li et al, 2008). On the other hand, the designed construct in the present study harbors 16S rRNA gene promoter as a constitutive promoter.…”

An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter

“…Hence, expression of the nitrate reductase gene is induced in the presence of nitrate ions and repressed in the presence of ammonium ions in a range of algae, including Cylindrotheca fusiformis, Chlamydomonas reinhardtii, Chlorella ellipsoidea, Dunaliella salina, Volvox carteri and Phaeodactylum triornutum (Wang et al 2004;Poulsen and Kröger 2005;Li et al 2007;Schmollinger et al 2010;Niu et al 2012;von der Heyde et al 2015). In addition, the promoter of the nitrate reductase gene has been used to regulate inducible expression of a reporter gene in several species of algae including C. reinhardtii (Schmollinger et al 2010), C. ellipsoidea (Wang et al 2004), D. salina (Li et al 2008;Li et al 2007) and P. triornutum (Niu et al 2012).…”