Cloning and heterologous expression of nitrate reductase genes from Dunaliella salina

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5'/3')-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5'-untranslated region of 151 bp and a 3'-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.