Cloning and Functional Analyses of Promoters of Two Carbonic Anhydrase Genes from Dunaliella salina

The cloning vectors pMD-DCA1 and pMD-CA containing the promoters of duplicated carbonic anhydrase 1 (DCA1) and carbonic anhydrase (CA) genes,respectively,from Dunaliella salina,and expression vector pDM307 containing bar-NOS polyA fragment were digested with EcoRⅠ.The bar-NOS polyA fragment was fused,respectively,to the fragments of the vectors pMD-DCA1 and pMD-CA to form transgenic D.salina expression vectors pMDDC-B and pMDC-B.The micro-shots were prepared by coating two constructs (pMDDC-B and pMDC-B) with gold particle.Each sample was bombarded once,twice,and thrice,respectively,with micro-projectile gun at a rupture pressure of 690 kPa in helium gas.The screening culture of the bombarded alga cells was performed in PKS liquid and solid medium containing 3 mg/L phosphinothricin (PPT) to develop the transformed cells of D.salina.Analyses of the transformed cells were carried out through PCR,Southern blotting,and Northern blotting.The results of screening culture showed that the expression of the external bar gene of vectors pMDDC-B and pMDC-B was stable and transient,respectively,in the transformed D.salina cells.In the meantime,the transformed efficiency of particle bombardment twice was higher than that of once or thrice particle bombardment at a rupture pressure of 690 kPa in helium gas.PCR and Southern blotting analyses indicated that the external bar gene was integrated into the genome of the cells.Northern analysis indicated that expression efficiency of the bar gene driven by DCA1 promoter was regulated by the gradient concentration of sodium chloride,and the positive blotting signal intensity of the bar mRNA was highest in the medium containing 2 mol/L of sodium chloride.The findings of the present study suggest that promoter of the DCA1 gene may be an inducible promoter following a hyperosmotic shock with high activity and safety in the research of transgenic D.salina .The tandem GT sequences of the promoter region of DCA1 and CA genes may be related to the molecular mechanisms of the extreme halo-toleration of the unicellular green alga,D.salina.