China Biotechnology. 2006, 26(11): 1-7.
Aim: Clone and characterize of the 5′- flanking region of the nitrate reductase (NR) gene derived from Dunaliella salina(D. salina ). Methods : The genomic DNA from D. salina was respectively digested with BamH I, EcoR I, Hind III, Pst I, Sal I and Xba I. A genomic walking cassette was ligated to the ends of the digested DNA fragments, and then genomic walking libraries comprising BL, EL, HL, PL, SL and XL were constructed. The 5′- flanking region of the NR gene from genomic walking libraries of D. salina was amplified by LA-PCR. The DNA sequences were analyzed with the software - Promoter Predictions. Isolated 5'-flanking regions fused to the GUS gene were tested for transient expression in the alga. Results: A single specific PCR product of about 1200bp in length from the HL library was generated. Also, we found several conserved motifs, such as CAAT-box, GAGA-box, which are related to regulation of transcription, and the putative binding sites of transcriptional factors such as EBP, EFII, NF-E1 and LV. BLAST showed that the DNA sequences shared high homology with 5′-upstream region of the NR gene from Dunaliella viridis. The isolated 5'-flanking regions were able to strongly drive GUS reporter gene expression , suggesting that it contains the promoter elements necessary for the transcription of the NR gene. The expression pattern of the GUS gene and the NR gene were similar, both ware induced by nitrate and repressed by ammonium. Conclusion: The cloned 5′- flanking sequences of NR gene derived from D. salina might be a specific promoter with the ability to"switch on or off " an expression of the heterologous gene in transgenic D. salina.
