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

Abstract

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.

Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems

“…After incubation for ~10 days, the mutant cells grew vigorously and had a greenish color in nitrate-containing liquid media with different concentrations of NaClO 3 compared to wild type D. salina cells, which became yellowish and eventually died. Furthermore, the mutant cells grew in medium containing up to 1600 mM NaClO 3, in agreement with our previous results (Lu et al, 2011). In the present study, growth experiments of wild type and mutant D. salina cells were performed on solid media with various nitrogen sources.…”

“…pMDDCA-NR, a recombinant plasmid containing the wild-type NR gene driven by the duplicated carbonic anhydrase (DCA) promoter from D. salina (Lu et al, 2011) was transfected into the mutant cells using electroporation. Specifically, the mutant D. salina cells were collected at the mid-exponential phase by centrifugation and thoroughly washed twice with the fresh nitrogen-free medium.…”

“…Because NR mutants with defects in the nitrogen assimilation pathway display chlorate resistance, screening for chlorate sensitivity has been successfully used for isolating mutants from several kinds of algae, such as Chlorella ellipsoidea (Wang et al, 2005) and Dunaliella viridis (Sun et al, 2006). We have previously demonstrated that wild-type D. salina can not grow in liquid media containing 200 mM chlorate or on culture plates containing 100 mM chlorate (Lu et al, 2011). Therefore, it may be possible to isolate D. salina mutants with defects in nitrate assimilation, which may further allow for the use of nitrate as a desirable selective agent.…”

A Review on Genetic Engineering of Microalgae with Respect to Genomes, Selectable Marker Genes, Promoters and Reporter Genes

“…Reports demonstrate the use of promoters of duplicated carbonicanhydrase1 (DCA1) in Dunaliella sp. (Li et al, 2010;Lu et al, 2011), fucoxanthin-chlorophyll a/c binding protein (fcp) gene in marine diatom (Apt et al, 1996;Miyagawa-Yamaguchi et al, 2011), actin1 (PyAct1) gene in Porphyra yezoensis (Takahashi et al, 2010), and violaxanthin/ chlorophyll a-binding protein (VCP) genes in Nannochloropsis sp. (Kilian et al, 2011 et al, 1992;Hallmann and Sumper, 1994).…”

Advances in genetic engineering of marine algae

“…The diatom fucoxanthin-chlorophyll a/c binding protein gene (fcp) promoter is effective in marine diatoms and other marine algae (Apt et al, 1996, MiyagawaYamaguchi et al, 2011, Qin et al, 2004, Zaslavskaia et al, 2000. The duplicated carbonic anhydrase 1 (DCY1) promoter was identified and used for stable nuclear transformation in D. salina (Li et al, 2010, Lu et al, 2011. The endogenesis PyAct1 (5′ upstream region of the actin1 gene from P. yezoensis) promoter was also confirmed to be effective in the transient gene expression of 12 red seaweed species (Hirata et al, 2011, Takahashi et al, 2010.…”