| Selenium is a trace element discovered by Swedish chemist Berzelius. It is crucial for of human and animal health, and mainly involved in anti-oxidation, anti-aging and physiological activities. Inorganic selenium has a strong toxicity to cell, and the body can not intake inorganic selenium directly. Only organic selenium can be utilized by human. Many plants and yeast can bioaccumulate selenium, so the organic selenium products focused on yeast, rice, wheat and other organisms. Haematococcus pluvialis is an autotrophic unicellular green alga, and under stress conditions it can synthesize astaxanthin, which is a natural health products and shows a strong oxidation resistance. Using H. pluvialis to enrich selenium has excellent market prospects. There are few studies about Se bioaccumulation mechanisms on H. pluvialis. Traditional researches were focused on physiology and biochemistry of plants under stress conditions, analyses and determination of expression levels of genes. But the mechanisms of the function variation under various stress conditions can not be well defined yet. Proteins as the direct executors of life, can reflect the physical activities under different conditions, and represent genes that control living organisms. The proteomics method was adopted in order to study the physiological and biochemical impact of sodium selenite in H. pluvialis, and the intracellular mechanisms of selenium enriched in H. pluvialis.In this study, H. pluvialis SAG 192.80 was selected as the experimental material, and cultured for 10 days under different concentrations of sodium selenite. The growth of algal cells was analyzed to determine the appropriate selenium concentration gradients. Then growth rat e under stress, physiological and biochemical characteristics of algae, Se contents and best conditions were analyzed. Further we set normal growth of algae as a control, and studied the differentiation of expression of proteins under selenium stress. The main results are:(1) In the semi-lethal experiments, the sodium selenite lethal concentration was 23.32 mg/L, with confidence interval for [19.27, 28.23] mg/L.(2) Different concentrations of sodium selenite were utilized in the culture medium, which is(0 mg/L 3 mg/L, 13 mg/L, 23 mg/L and 33 mg/L). Low concentration of 3 mg/L sodium selenite did not affect the growth rate. The more increase of concentrations, the more obviously growth rate inhibited. All algae in the first five days was not affected.(3) The photosynthetic efficiency and chlorophyll content of selenium treated algal cells were consistent with the control group in initial five days. Then the high Se concentration group had a significant decline, as the concentration increased. This suggests that selenium could affect growth of algal cells after it reach a certain density.(4) SOD and CAT activities in algae were remarkably increased after selenium treatment. This indicates that the strong oxidative substances were generated under selenium-stress conditions, and thereby normal physiological activities of algae were damaged.(5) The total selenium and organic selenium treated with different concentrations of selenium were measured. We found that total selenium and organic selenium within algae rea ched maximum after 13mg/L of selenite treatment, which are 514.76 ?g/g and 340.19 ?g/g respectively. Se treatment at the first day of culture is the best timespot.(6) TCA / acetone extraction of protein, and two-dimensional electrophoresis gels were analyzed using PDquest 8.0 software. A good repeatability of data were shown. Comparative analyses revealed 40 proteins with significant changes, including 25 up-regulated and 15 down-regulated proteins, and 31 proteins identified via MS.(7) After BLAST with protein database in the NCBI, 27 proteins were annotated to GO database. The main functions of identified proteins involved photosynthesis, antioxidant, regulation of a series of cellular and molecular level of amino acid metabolism, energy metabolism, ion transport and signal transduction, cell division and cell repair. |
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