MOLECULAR CHARACTERIZATION OF THE ASSIMILATORY NITRATE REDUCTASE GENE AND ITS EXPRESSION IN THE MARINE GREEN ALGA DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

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Abstract
Nitrate is used as a nutrient for growth by many organisms through the nitrate assimilation pathway. The key enzyme involved in nitrate assimilation is nitrate reductase (NR). Transcriptional regulation of NR genes has been studied in various phototrophic eukaryotes from freshwater or terrestrial habitats. Here, we describe the first NR gene from a marine phytoplankton, the green alga Dunaliella tertiolecta Butcher. Its sequence is very similar to that of the other green algae, but its intron structure and transcriptional regulation differ significantly. A partial genomic NR gene fragment was cloned using direct PCR amplification with degenerate primers, and rapid amplification of cDNA ends was performed to obtain a complete NR gene from a cDNA of D. tertiolecta. The complete NR gene of D. tertiolecta is 2.7 kb in size and is similar at the amino acid level to the NRs from freshwater green algae Chlorella vulgaris (50.6%), Chlamydomonas reinhardtii (58.4%), and Volvox carteri (59.4%). Transcriptional regulation of D. tertiolecta NR was examined in response to different nitrogen sources and during the diel cycle. Dunaliella tertiolecta NR transcript was induced by nitrate and repressed by ammonium. In addition, transcription was not induced under nitrogen depletion. Induction of NR transcript responded to the diel cycle, and transcript levels were maximized within an hour after the beginning of the light period. The induction and repression of NR transcript were much slower than reported in freshwater green algae, which may be related to the size of internal nitrogen pools.

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