|Other Abstract||Haematococcus pluvialis (Chlorohyceae) is generally regarded as one of the best biological source for astaxanthin which is widely used for health product, feed and medicine. However, the Haematococcus pluvialis through autotrophy is poor in biomass producty and costive when inudstrial production. Genetic engineering modification such as the construction of heterophic strains with glucose may solve the problems. This thesis had made an attempt to establish stable genetic transformation of Haematococcus pluvialis. Research work includes the analysis of the differentiation between motile cell and non-motile cell, the difference in cultivation and regeneration on solid plate for electrotransformation, biolistic transformation and PEG-mediated protoplast transformation.
Firstly, based on the morphology of two main cell type - motile cell and non-motile cell in its complicated life history, the cell differentiation of all Haematococcus pluvialis cells to be single type or the majority of cells to be single type during the cultivation were observed. Results showed that motile cell occupied more than 80% in the first 4 days and non-motile cell occupied more than 80% when cultivated for 12 days in BYA medium which contains 2 g/L sodium acetate, 2 g/L yeast extration in BG11. Meanwhile the maximm cells density was 8×105 cells/mL. Under the centrifugation condition of 500 g × 5 min , all types of cells will be collected. In Tris-HCl buffer with 0.2 mmol/L CaCl2, motile cells were more stable while non-motile cells did without CaCl2. In addition, compared with direct coating for regeneration, double-layer plate was proved to be significantly more effective while starch embedding showed no advantage. Futher experiments indicates that the regeneration rate of motile cell was higher than non-motile cell, and colonies in TAP and BYA in which sodium acetate was added formated more quickly than 8P-BG11 and 3N-BBM. BYA was the best medium for cell regeneration and the regeneration rate was 52.8% and 31.5% for motile cell and non-motile cell respectively.
Secondly, the sensibility to spectinomycin and zeomycin of Haematococcus pluvialis in solid plates and liuqid medium was analyzed, and electrotransformation and biolistic transformation were conducted. The results suggested that appropriate screening concentration of spectinomycin and zeomycin to Haematococcus pluvialis was 200 µg/mL, 8 µg/mL in solid palte respectively while 20 µg/mL, 1 µg/mL respectively in liquid medimu. Motile cells were more sensitive than non-motile cell to electricity, and the survival rate was 40% when the voltage was about 1000 v/cm for motile cell, but for non-motile cell, the voltage was 2000 v/cm to reach the same survial rate. Unofortunately, the experimental repeatability was poor and recombinant transformant was not observed via Ble gene to zeomycin, however, transformant was found via addA gene to spectinomycin. PCR analysis had proved that the transgene was integrated into the chloroplast genome and chromosome repestively by pHpluS1 plasmid and 18s-pHpluS1-28s plasmid.
Finally, based on the optimized protocol for protoplast preparation and regeneration, protoplast transformatioin of Haematococcus pluvialis was constructed for the first time. Protoplast viability was significantly increased when 0.5 mmol/L CaCl2 was added into the buffer solution. Protease-k was proved to be the most effective enzyme for protoplast preparation, and when the cell density was 5×106 cells/mL, preparation rate and viability was 78.5% and 78.4% respectively at 35C for 120 min. Transformant was found by electrotransformation based on protoplast, but the colonies also could’t grow in liquid medium. With PEG (polyethylene glycol) -mediated protoplast transformation, fluorescence was observed. Besides, transformant were found via addA gene to spectinomycin, and it is confirmed that the transgene was integrated into the chloroplast genome and chromosome repestively by pHpluS1 plasmid and 18s-pHpluS1-28s plasmid which were linearized by PCR analysis.|