Mutation Breeding Of Haematococcus Pluvialis And Optimizing The Nitrogen And Phosphorus Of Its Medium

Haematococeus pluvialis is a single cell green alga and has the ability to accumulate astaxanthin under the environmental or nutritional stresses. Due to the richest content of natural astaxanthin, H. pluvialis is considered to have high economic value. Astaxanthin has the remarkable ability of coloring, anti-cancer, anti-oxidation, resistance to ultraviolet radiation and improvement of immunity, etc. so astaxanthin has wide development prospects in many areas such as health products cosmetics, medicine, and aquaculture. In the process of producing astaxanthin by the large-scale cultivation of H.pluvialis, breeding of good strains has always been the focus of the researches. Mutation breeding of algae strains, which can grow fast and have high content of astaxanthin, has practical significance for the industry.In order to improve the astaxanthin content of H. Pluvialis. this paper applied the method of ultraviolet radiation to the mutation breeding of H. Pluvialis. The wild type strain (HO) of H.Pluvialis was irradiated with ultraviolet and 8 min was determined to be the appropriate time of ultraviolet irradiation according to the fatality rate. After mutation, the algae were screened and tested the characteristics, and two strains (H7 and H11) which have high yield of astaxanthin were selected. Compared with the original strains, the growth rate of the two mutagenic strains has increased by 18.9% and 17.0%and the astaxanthin content has increased by 9.0% and 4.5%.On the basis of breeding the algae strains, the nitrogen source and the optimal concentration of the nitrogen and phosphorus of the medium were studied thoroughly. By comparing the influence of sodium nitrate, ammonium bicarbonate and ammonium chloride on the growth of H.Pluvialis, the suitable nitrogen source is sodium nitrate and the optimum concentration is 1.0 g/L. H.Pluvialis was cultured in the medium containing 1.0 g/L sodium nitrate for 12 days, and the biomass can reach 4.85×105 cell/ml. Then astaxanthin can accumulate effectively and the content is 3.49%. The optimal concentration of phosphorus was 0.04g/L. H.Pluvialis was cultured under the optimal concentration of phosphorus for 12 days, the biomass can reach 5.9×103 cell/ml, astaxanthin content can up to 3.82%. In the orthogonal experiment, the optimal concentration of NaN03, K2HPO4·3H2O and the pH were 0.8g/L,0.04g/L and 7, in the green growth stage. In astaxanthin accumulation stage, the optimal initial concentration of NaNO3, K2HPO4·3H2O and pH were 0.4 g/L,0.06g/L, and 6.Finally, the nitrate reductase is the key enzyme of nitrogen metabolism, which was studied. The DNA and cDNA sequences of nitrate reductase were cloned firstly. The DNA sequence has 5636bp. including 15 introns and 14 exons.Thc cDNA sequence has 2718bp, encoding 905 amino acids. The amino acid sequence of the nitrate reductase was compared using BlastP. three conserved domains, including Mo-Co superfamily. cyt-b5 super and FNR-like superfamily were found. Finally, the real-time fluorescent quantitative PCR was carried out to analyze the transcription of nitrate reductase gene. The results showed that when the concentration of sodium nitrate in the culture medium was between Og/L and 1.0g/L, the transcription of nitrate reductase increased with the increase of the nitrate concentration. The nitrate reductase is the key enzyme of nitrate assimilation, so the higher nitrate concentration in the culture medium will need more nitrate reductase and the transcription of nitrate reductase gene is increased. As a result, the growth of H. pluvialis is increased.To sum up, this study applied ultraviolet mutagenesis to H.phivialis and two strains with higher grow speed and higher content of astaxanthin were selected. Then the suitable nitrogen source and the concentration of nitrogen and phosphorus were determined preliminarily, which laid a foundation for industrial production. Cloning of nitrate reductase gene and analysis of its transcription under different nitrogen levels provide a molecular basis for the study of the process of nitrogen metabolism and optimization nitrogen source.