Preliminary Functional Analysis And Characterization Of Heterotrophic Transformation Algal Strains Of Dunaliella Salina

Dunaliella salina (D. salina) is a kind of photoautotrophic unicellular green algae lacking rigid cell wall. D.salina itself has many advantages as following: growing in extreme environment with salinities ranging from 0.05 to 5 M sodium chloride, and thus strong anti-pollution ability; easy genetic manipulation; post-translational processing; simple culture conditions, inexpensive cultivation cost and relative short growth cycle; high nutritional value; non-toxic harmless, et al. Therefore, it is very important prospect to develop the bioreactor of D.salina to product many biological active compounds, such as pharmaceutical protein, angiostatin, antibodies and vaccine, and so on.At present, there are two patterns in D.salina cultivation, one is in open pond culture systems which often overrun by other algae or bacteria, in which the growth of D.salina are often limited by light, climate, nutrient and temperature; the other is in closed photobioreactor systems which was restrained by large starter inoculums grown and light limitation. So it is difficult to increase the biomass of D.salina in large-scale, which also limited the utilization of D.salina. Trophic conversion of D.salina from an obligate photoautotrophic to heterotrophic promote the biomass of D.salina to some extent through genetic engineering, which will provide the theoretical basis for solving the problem above.The growth rate and cell density of algae were markedly enhanced after single gene (including HUP1 from Chlorella, Glutl from human erythrocyte and Hxtl, Hxt2 and Hxt4 from Saccharomyces cerevisiae, respectively) was introduced into algae including Chlamydomonas, Chlorella, Diatoms and other algae Glutl, a member of glucose transporter protein family, located in cell membrane surface, is the main carrier of glucose transporter. Thus, in this study, we attempted to change the trophic pattern by introducing Glutl into D.salina, which will provide will provide the theoretical basis for improving the growth rate of D.salina, and the feasibility ideas for further development of D. Salina bioreactor. Our research group has cloned Glutl gene from human placenta tissue, and has confirmed its correction by sequencing, and then constructed inducible heterotrophic expression vector pMDDGN-Bar and constitutive heterotrophic expression vector G5Glutl-Bar of D.salina through gene recombination technology. Based on the study above, in this study, the two heterotrophic vectors were transformed into D.salina through optimized electroporation methods, heterotrophic algal strains of D.salina were selected used the PPT by liquid and solid screening procedures. We found that the wild algae all died on the concentration of 6μg/mL of PPT, while transformed algal strains grew well. Finally,3 constitutive heterotrophic algal strains and 2 inducible heterotrophic algal strains was screened out, and named as Cl, C2, C3, I1 and I2, respectively. Further, the results of RT-PCR demonstrated that 5 strains of transformants all appeared about 250 bp of specific DNA bands, suggesting that foreign Glutl gene had been successfully integrated into the genome of heterotrophic strains of D. salina.In transgenic organisms, the transgene copy number can greatly influence the expression level and genetic stability of the target gene, and thus the estimation of transgene copy number is one of the important contents in the study of genetically modified organisms(GMOs). Currently, Real-time PCR and Southern blotting techniques are ideal methods for estimating transgene copy number. Thus, we analyzed the transgene copy number of the 5 strain transformants using the two technologies, respectively. The results revealed that the copy numbers of C1, C2, C3 I1 and I2 were 2,1,3,4 and 4, respectively. Finally, the glucose transport of transformants was investigated. The results demonstrated that different concentrations of glucose have significant impact on photoautotrophic strains, constitutive heterotrophic strain (C2) and inducible heterotrophic strain (I2) in light conditions, and growth of the three different strains of D.salina was stronger on the concentration of 5 mm glucose than other glucose concentrations(P<0.05). The results also showed that combined light and glucose (5 mm) obviously increased growth rates of different algal strains; while under the condition of dark combined with glucose (5 mm), the growth ratios of constitutive heterotrophic strain (C2) were significantly higher than that of inducible heterotrophic strain (I2) and photoautotrophic strain (P<0.05), whereas, the growth ratios of inducible heterotrophic strain and photoautotrophic strain remained almost stagnant, which may be evoked by many copy number of Glutl gene randomly inserted into the genome DNA of D. salina, further interferes with D.salina genome stability systems and results in transgene silencing effect. In addition, growth ratios of constitutive heterotrophic strain under the condition of light combined with glucose were significantly higher than that under the condition of dark combined with glucose, suggesting that constitutive heterotrophic strain may be in the facultative heterophic, however, how to make it completely heterophy remains further studied, which will lay a foundation for large-scale fermentation culture of D.salina. In conclusion, D.salina heterotrophic strain was preliminarily established, which will provide theoretical basis for construction of D.salina bioreactor.