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Dunaliella (Dunaliella salina, D. salina) is a single-celled eukaryotic algae, no cell wall, are photosynthetic autotrophs. Dunaliella itself has many advantages: in 0.05-5 M NaCl survive in extreme environments and thus giving it a strong anti-pollution; genetic easy to operate; has translational processing and modification; culture conditions of simple, low cost, short growth cycle ; their high nutritional value; nontoxic, etc., making salina become an expression of exogenous genes good host. Therefore, the development salina as a bioreactor to produce a variety of biologically active proteins such as medicinal exogenous protein, angiostatin, antibodies and vaccines has great application prospects. Salina culture in two ways: one is the open pool-type large-scale cultivation, but susceptible to microbial contamination, climate, light, nutrition and diurnal temperature and other factors; another is a closed bioreactor illumination unit training, but the use of light exist bioreactor inoculation volume and light suppression effect and other shortcomings. Thus making salt algae growth rate and biomass difficult to significantly improved, thereby salina use has been greatly limited. By means of genetic engineering to change the nutritional pattern salina (from photosynthetic autotrophic to heterotrophic transition) to some extent can maximize salt algae biomass, in order to fundamentally solve the above problems may provide a theoretical basis. Chlamydomonas reinhardtii, Chlorella, diatoms and other algae heterotrophic study found that a single foreign gene transfer, such as chlorella HUP1 gene (H / hexose cotransporter 1), human erythrocytes Glutl gene (Glucose transporterl) and wine yeast Hxtl, Hxt2 and Hxt4 genes algae nutrition transition mode, these algae can use an external source HETEROTROPHIC saccharides, the growth rate and cell density were significantly improved. Glucose transporter (Glut1) glucose transporter protein family in one channel transport protein in the cell membrane surface, is the main carrier of the glucose transporter. Therefore, in this study we try to Glut1 gene into the cells of Dunaliella salina to change the nutritional patterns, so as to raise the growth rate of Dunaliella to provide a theoretical basis for the further development of Dunaliella bioreactor to provide feasibility ideas. Our previous been cloned from human placenta of human Glut1 genes and confirmed by sequencing the cloned gene sequence Glut1 correctness, further use of recombinant DNA technology to build Dunaliella constitutive and inducible expression vector heterotrophic. This topic is on this basis, the electroporation method by optimizing the two heterotrophic expression vectors into Dunaliella cells, the use of glufosinate (Phosphinothricin, PPT) through the liquid and the solid filtered salina heterotrophic algae conversion strains. The results showed that the concentration of 6μg/mL PPT wild algae in control all died, while transforming algae strains grow well on this basis were screened out of the three constitutive heterotrophic algae strains and two inducible heterotrophic algae strains, named: C1, C2, C3, I1 and I2. Further screened five heterotrophic algae strains were transformed by RT-PCR, the results show that five transforming algae strains in the appropriate position size have emerged about 250bp of the more specific DNA bands, suggesting that exogenous gene Glutl has been successfully integrated into the Dunaliella algae strains of heterotrophic into the genome. Genetically modified organisms in transgenic copy number of the target gene expression level and have a great impact on genetic stability, which makes estimating transgene copy number in GMO research to become an important and one of the topics. Real-time quantitative PCR (Real-time Quantitative PCR) and Southern botting transgenic technology is ideal method copy number, so we use both technical analysis of five strains of heterotrophic algae transformed the number of copies, and found three salt constitutive heterotrophic algae algae strains transformed C1, C2 and C3 Glut1 gene copy number, respectively: 2,1 and 3; 2 Dunaliella inducible heterotrophic algae strains transformed I1 and I2 Glut1 gene copy number was: 4 and 4. Southern blotting detection C1, C2 and C3, copy number and Real-time PCR results are consistent, but in I1 and I2 whose copy number is 3 and 2, respectively, slightly lower than the Real-time PCR results. Finally heterotrophic algae strains transformed glucose transporter were analyzed, the results show that a variety of different lighting conditions, the glucose concentration on autotrophic strains constitutively heterotrophic strains (C2) and inducible heterotrophic strains (I2) growth have significant impact, and the different strains of D. salina in glucose concentration of 5 mm under the action of the growth was stronger than the other glucose concentration (P lt; 0.05); also found that in the light and glucose (5 mm) together can significantly enhance the different strains of D. salina algae strain growth rate (P lt; 0.05), and glucose in the dark together, constitutive strains of heterotrophic growth rate significantly higher than that induced autotrophic and heterotrophic strains strains (P lt; 0.05), autotrophic plants and heterotrophic strains induced growth is almost stagnant, the reason may be inducible heterotrophic strains Glutl gene copy number as many randomly inserted into the chromosomal DNA of Dunaliella loci not OK interfere Dunaliella cell genome homeostatic system which led to lower growth or stagnation, or appear transgene silencing effect. The constitutive heterotrophic strains in the light of the joint action of glucose and the growth rate is also significantly higher than the dark and under the action of glucose in the growth rate, suggesting that the heterotrophic algae strains may be in the facultative heterotrophic, how to make it in a completely heterotrophic state, to further lay the foundation for large-scale fermentation, still need further study. In conclusion, this study initially established heterotrophic algae strains transformed for salina bioreactor to provide a theoretical basis for the establishment.
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