Cloning and characterization of the nitrate reductase gene from Dunaliella salina

摘要

A cDNA corresponding to the nitrate reductase gene was isolated from Dunaliella salina by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence (3694 bp) contained an open reading frame of 2700 bp encoding 900 amino acids, a 5′untranslated region of a 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 60%, 58% and 50% identity in amino acids sequence to the corresponding genes of Dunaliella tertiolector, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. 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 nitrate reductases from other higher plants. The full-length cDNA was overexpressed in E. coli as a fusion protein and the recombinant protein was visualized by SDS-PAGE. The recombinant protein, after induction with IPTG, accumulated to about 21% of total bacterial protein. Analysis of the enzymatic activity of the protein synthesized in E. coli showed that the nitrate reductase was functional even though it was expressed together with the maltose binding protein.