Construction Of A System For The Transformation Of Foreign Genes In Dunaliella Salina

The research is on the basis of Dunaliella salina. The foreign gene transformation system is built for this alga. The result establishes the base to transform phytese gene to the alga. The main contents and results are as follows:Five bacteria are separated from the culture liquid of Dunaliella salina. Kanamycin, ampicillin, gentamicin, cefalothin, chloramphenicol, streptomycin and neomycin are used to test the sensitivities of bacteria for the seven antibiotics. Streptomycin, ampicillin, gentamicin, cefalothin and chloramphenicol are chose for they are sensitive to the bacteria. The function of two antibiotics are studied, the result proves that the function of antibiotics is added.The effect of streptomycin, ampicillin, gentamicin, cefalothin and chloramphenicol for the growth of Dunaliella salina is studied. The result is that the most effective antibiotic is chloramphenicol. While the concentration of other four antibiotics are 1600μg/mL, and cultured 10 days later, Dunaliella salina grows very well.Streptomycin, ampicillin, gentamicin and cefalothin are chose to combine to restrain the bacteria incidentally. The result is that when the concentration of each antibiotic is 800μg/mL and adds 3 times later the bacteria are restrained well, and then the axenic alga can be got from the solid medium, but the growth of Dunaliella salina is not affected.The effects of nutrient compositions such as nitrogen, phosphorus, carbon, vitamins B₁ and B₁₂ of axenic Dunaliella salina are optimized. The concentrations of nutrient compositions are optimized by orthogonal experiment as follows: NaNO₃ and NaH₂PO₄ are added as ten times as that of f/2 medium and [N]/[P] is the same as f/2 medium, NaHCO₃ 0.4g/L, V_B1100μg/L, V_B121.0μg/L. The others are added as the same as f/2 medium.Chloramphenicol, G418 and hygromycin are chose to do the experiment of selective marker. The result is that G418 and hygromycin restrain the culture of Dunaliella salina too little to use as the selective marker. But chloramphenicol is the most suitable regent if using CAT gene as selective marker for Dunaliella salina genetic engineering. Moreover, the selective concentration in solid is 80μg/mL.Transform GFP gene to Dunaliella salina by using electroporation. The effects of different growing stages and transfer conditions are studied. The results show that higher GFP expression can be gained when cells growing 7 days after inoculation, electroporated under 3 KV pulse voltage and 3ms pulse duration.