Cloning of nitrite reductase gene from Haematococcus pluvialis and transcription and enzymatic activity analysis at different nitrate and phosphorus concentration

Abstract

Haematococcus pluvialis is an economic microalga to produce astaxathin. To study the nitrogen metabolic process of H. pluvialis, the transcription level and enzyme content of nitrite reductase at different nitrate and phosphorus concentrations were studied. In this research, nitrite reductase gene (nir) was first cloned from H. pluvialis, which consists of 5592 nucleotides and includes 12 introns. The cDNA ORF is 1776 bp, encoding a 592 amino acid protein with two conserved domains. Phylogenetic analysis showed that the nir gene in H. pluvialis had the highest affinity with other freshwater green algae. Nitrogen and phosphorus play an important role in the growth of H. pluvialis. The single factor experiments of nitrogen on growth conditions showed that the group with 0.2 g/L NaNO₃ had a relative high biomass. The single factor experiments of phosphorus on growth conditions showed that the group with 0.06 g/L K₂HPO₄ had a relative high biomass. The transcription level and enzymatic activity of nitrite reductase were detected at different nitrate and phosphorus concentrations. In the absence of nitrogen and phosphorus in the medium, nitrite reductase activity is the highest. This research provides theoretical guidance for optimization of culture medium for H. pluvialis and also provides an experimental basis for understanding of nitrogen metabolism pathway in H. pluvialis.

Keywords: Enzymatic activity; Haematococcus pluvialis; Nitrate concentration; Phosphorus concentration; Transcriptional level; nir.