Establishment Of An Electroporation-mediated Transform System And Genetic Transformation Of A Foreign VgDGAT1a Gene In Dunaliella Salina

Dunaliella salina is a unicellular eukaryotic algae with higher carotenoids accumulation. It is the key to breed the oleaginous strain of D. salina for expanding its utilization to produce biodiesel by large-scale cultivation in saline land and the related water resources. Therefore, in this study, VgDGAT1a gene encoding high DGAT enzyme activity from high-oil plant Vernonia galamensis was genetically transformed into D. salina so as to breed excellent oleaginous strains of D. salina.The main results obtained in this study were described as the followings:(1) Sterile system of D. salina was established. Cefotaxime, chloramphenicol, streptomycin and ampicillin were investigated for their effects of bacteria elimination from D. salina cultures, indicating that their effects could be added. Combination treatment of two antibiotics showed much better in bacteria removal.(2) The test on susceptibility of D. salina to antibiotics showed that D. salina was more sensitive to chloramphenicol. After 5 days of cultivation with chloramphenicol, D. salina algae liquid color become yellow. Cells were breadown, and the cellular contents flowed out. Compared with the control groups, high dosage (1000?g/mL) of cefotaxime, streptomycin and ampicillin showed no inhibitory effects on D. salina growth.(3) The optimized electroporation system of D. salina was developed. This includes the cells grown for 7 days as the receptor,6?g/mL of plasmid for transfering, electroporation condition setting as 0.4 KV pulse voltage and 4 ms pulse duration. The transformation efficiency was increased up to 2.1‰, significantly higher than other conventional transformation methods used in D. salina.(4) Compared to the wild-type D. salina, Nile red staining showed that oil body in the transgenic cells significantly increased in size and amount.(5) Gravimetric method was used to determine the content of total oil in D. salina cells. the total oil content in the transgenic cell lines increased to 37% of cell dry weight compared with 21.7% in wild-type D. salina.(6) Kjeldahl apparatus and acetone methods were used to measure protein and carotenoids contents in transgenic D. salina and the wild-type, showing the transgene have no significant impact on these two traits.