| Haematococcus pluvialis is an unicellular green algae, which could produce and accumulate plenty of valuable secondary metabolite—astaxanthin in stress culture situations. Elicitors could promote the synthesis of secondary metabolite in various plant cells, but the effects of elicitors on the algae, especially on the astaxanthin content of Haematococcus pluvialis had no further relevant reports. Therefore, the effects of different concentrations of methyl jasmonate (MeJA), arachidonic acid (AA) and cerium ammonium sulfate (CAS) on the cell growth and astaxanthin content of Haematococcus pluvialis were studied in this paper, in order to choose the best elicitor suited for the astaxanthin accumulation of Haematococcus pluvialis. The core fragment of dxs which was the first key enzyme gene of the astaxanthin biosynthetic pathway from Haematococcus pluvialis was cloned by using RT-PCR technology, and the bioinformation analysis was studied. The correlation between the astaxanthin content and the amount of dxs gene expression under the inducement of MeJA, AA and CAS were studied in this paper, in order to primarily elucidate the reason and mechanism of astaxanthin accumulation at the molecular level, which could provide a theoretical foundation for cosmically manufacturing astaxanthin.The effects of different concentrations of elicitors on the cell growth of Haematococcus pluvialis were studied. The result showed that the addition of different concentrations of MeJA and CAS both restrain the cell growth of Haematococcus pluvialis, and the effects on restraining during the high concentration elicitors are obvious, while appropriate concentration of AA can promote the cell growth of Haematococcus pluvialis. There was 23.04% higher in cell density at 62.5 mg/L AA treatment (12.661×105 cells/ml) compared with the controls (10.290×105 cells/ml) on 18th day.The effects of different concentrations of elicitors on the astaxanthin content of Haematococcus pluvialis were studied. The result showed that these three kinds of elicitors all promote the astaxanthin synthesis in the unit cell of Haematococcus pluvialis remarkably. At the concentration of 800μmol/L MeJA, the unit cell astaxanthin content in Haematococcus pluvialis could reach 1.75×10-4 mg/105cells, which is 23.24% higher than the control group (1.42×10-4 mg/105cells); at the concentration of 312.5 mg/L AA, the unit cell astaxanthin content in Haematococcus pluvialis could reach 2.68×10-4 mg/105cells, which is 88.73% higher than the control group; at the concentration of 2 mg/L CAS, the unit cell astaxanthin content in Haematococcus pluvialis could reach 1.46×10-4 mg/105cells, which is 1.41% higher than the control group.The effects of different concentrations of elicitors on the total astaxanthin production of Haematococcus pluvialis were studied. The result showed that the total astaxanthin production of treatment groups with the addition of different concentrations of MeJA and CAS were lower than that of the controls, while the addition of 0.1312.5 mg/L AA could increase the total astaxanthin production at different levels. There was 20 d earlier and 103.75% higher in total astaxanthin production at 312.5 mg/L AA treatment (2.987 mg/L) compared with the controls (1.466 mg/L) on 18th day, with obvious effect on production-increasing.The core fragment of dxs which was the first key enzyme gene of the astaxanthin synthesis from Haematococcus pluvialis was cloned by using the reverse transcription-polymerase chain reaction (RT-PCR), the core fragment is 827 bp in size including an incomplete open reading frame (ORF), which encodes 275 amino acids. Alignment of the DXS protein revealed that the deduced HpDXS protein had very high identity with other species of DXS protein (70%90%), the highest DXS protein identity (90%) is with Chlamydomonas reinhardtii DXS. Phylogenetic tree analysis showed that, according to the proximity relationships among species, it can be divided into three main branchs—plants, algae and bacteria. Haematococcus pluvialis clusted with Chlamydomonas reinhardtii from the Volvocales, there was very close genetic relationship between the two.The effects of different concentrations of elicitors on the dxs gene expression level of Haematococcus pluvialis were studied. The result showed that appropriate concentrations of MeJA, AA and CAS could enhance the dxs gene expression level. With the treatment of 800μmol/L MeJA, 312.5 mg/L AA and 2 mg/L CAS, dxs gene expression levels were the strongest.The effects of different concentrations of elicitors on the correlation between the astaxanthin content and the amount of dxs gene expression of Haematococcus pluvialis were studied. The result showed that, under the inducement of three kinds of elicitors, the astaxanthin content of Haematococcus pluvialis and the amount of dxs gene expression was linearly related, and the correlation coefficients were 0.8124, 0.9680, 0.9666, thus it can be seen that the dxs gene expression level can reflect the astaxanthin content of Haematococcus pluvialis, dxs was a key enzyme gene of astaxanthin synthesis of Haematococcus pluvialis.
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