| The molecular mechanisms that the extremely halo-tolerant unicellular green algae of the genus Dunaliella salina are capable of growing in salinities ranging from 0.05M to 5.0M of sodium chloride are intriguing. It has demonstrated that the levels of two plasma membrane proteins, internally duplicated carbonic anhydrase (DCA) and transferring-like protein (Ttf), were greatly elevated with increase of salt concentration. Furthermore, increasing salinities induced parallel increases in the levels of DCA, and its mRNA, and external carbonic anhydrase activity of Dunaliella salina. These results indicated that the DCA promoter might be an inducible promoter following hyperosmotic shock. However, no research on cloning and functional analysis of the hyperosmotically inducible promoter has been reported so far.Here, we report the cloning and functional analyses of the promoters of duplicated carbonic anhydrase (DCA1) and carbonic anhydrase (CA1) genes from Dunaliella salina. We also show that the promoters of DCA1 and CA1 genes can drive the expression of bar reporter gene, and that the promoter of DCA1 gene is able to confer sodium chloride concentration gradient regulation to the bar reporter gene in Dunaliella salina.PART ONE: Cloning and Sequencing of the Two Carbonic AnhydraseGenes from Dunaliella salina1. Methods1.1 Construction of Genome Walker libraries.The genomic DNA from Dunaliella salina was digested with Dra I, EcoR V, Pvu II and Stu I, respectively. A Genome Walker Adaptor was ligated to the ends of the digested DNA fragments. GenomeWalker Libraries, including GWL1,GWL2,GWL3 and GWL4, were constructed, respectively.1.2 Cloning of the 5'-flanking and 3'-flanking regions of the two carbonic Anhydrase genes from Dunaliella salinaGene-specific primers were made according to the 5'-UTR and 3'-UTR sequences of DCA1 and CA1 cDNA (The GenBank accession number is AF183939 for the DCA1 gene, and AF190735 for the CA1 gene) .The nested PCR amplification was performed with GenomeWalker Libraries DNA of Dunaliella salina as a template by using gene-specific primers and adapter primers. The specific PCR products obtained by using the nested PCR amplification were cloned into T-vector after recovering of PCR products was made on the gel.1.3 Cloning of the cross-intron genomic DNA of the two carbonic anhydrase genes from Dunaliella salinaThe sense and antisense primers of cross-intron PCR were made according to the 5'-UTR and 3'-UTR sequences of DCA1 and CA1 cDNA. The PCR amplification was performed with Dunaliella salina genome DNA as template. The specific PCR products were cloned into T-vector after PCR products were recovered on the gel.1.4 Analysis of sequenceThe DNA sequence was determined by dideoxy-mediated chain termination. Analyses of the DNA sequences obtained were processed by using Biosoft. 2. Results2.1 Cloning and Sequencing of the 5' Flanking of the Two Carbonic Anhydrase Genes from Dunaliella salinaSingle major PCR products of 1.3kb fragment from the GWL1 in theDCA1 gene, and 1.7kb fragment from the GWL2 in the CA1 gene were generated,respectively.The two specific fregments, 1.3kb and 1.7kb ,were cloned and sequenced. The partial sequences of 3'-end in the 1.3kb and 1.7kb fragment were completely consistent with the known sequences of 5'-end of the DCA1 and CA1 gene cDNAs, respectively. There were several conserved cis-acting elements ,such as TATA-box,CAAT-box,etc, and the tandem GT clusters in the sequences.2.2 Cloning and Sequencing of the 3' flanking of the two carbonic anhydrase genes from Dunaliella salinaSingle major PCR products of 1.7kb fragment from the GWL3 in the DCA1 gene, and 0.75kb from the GWL1 in the CA1 gene were generated,respectively. The two specific fregments, 1.7kb and 0.75kb ,were cloned and sequenced. Except for the very few bases, the partial sequences of 5'-end in the 1.7kb and 0.75kb fragments were completely consistent with the sequences of 3'-UTR of the DCA1 and CA1 gene cDNAs, respectively.2.3 Cloning...
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