| Two fragments were amplified and cloned in plasmid vectors with the strong promoter of Dunaliella salina respectively. Therefore two plasmid vectors with gfp, named as p215t and p115t, were constructed in this experiment. The identification of recombinant plasmids by endonuclease digestion proved the successful construction in molecular biology. In order to explore the expression of gfp gene in prokaryotic host, plasmids were transformed into cells of Escherichia coli MC4100A. gfp gene expressed successfully in strain MC4100A, however, the products of gfp showed different distribution in host cells. The fluorescence appeared as a halo or a polar localization in cell with p215t. In p215t, there is a RR- signal peptide sequence in the upstream of gfp gene, which can help to transport the Gfp protein to the periplasmic space. With p115t, the fluorescence in transformed cell was uniform because of the lack of RR- signal peptide sequence.The method electroporation was adopted to transform the plasmids into D. salina cells. Three parameters of electroporation, that is cell age, buffer and electric-field intensity, were test in order to find the optimal condition of electroporation for D. salina. The results showed 7days'cell age, 1.0mmol·L-1 HEPES+0.5mol·L-1 glycerol and 6 kV·cm-1 was the optimal condition for higher expression. The highest rates of electroporation were 1.00‰and 1.02‰for p215t and p115t respectively.Because of the lack of wall, cells of D. salina are sensitive to osmotic pressure. Therefore maintaining the osmotic pressure may be an effective way to improve the rate of electroporation. As an osmotic stabilizer, glycerol was added to HEPES buffer. Two effects of glycerol were investigated to improve the survival rate of cells before electroporation and to promote the growth of the cells. Highist survival rate, about 86%, was abtained when HEPES buffer with 0.5mol·L-1 glycerol was used to treat cells before electroporation. When added to the algal medium, glycerol stimulated the growth of cells in lower concentration, and inhibited cells growths in higher concentration. 5 mmol·L-1 glycerol was advantage to holding the highest number of active cells and to promoting the accumulation of total biomass. Therefore 0.5 mol·L-1 glycerol might be a suitable osmotic stabilizer for electroporation of D. salina.Gene gfp expressed successfully in D. salina cells, and is expected to become a better selecting mark. The fluorescence from transformants was uniform and brighter with p115t, while the fluorescence was a little dim with p215t.Several unusual cells were observed in the transformed sample with p215t, and it was deduced preliminarily that RR- signal peptide might responsible for the phenomenon.This experiment may provide useful parameters for establishing a new bio-reactor of D. salina and developing the transgenetic system of D. salina further.
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