杜氏盐藻2种碳酸酐酶基因5'上游的克隆与序列分析

目的：克隆盐藻2种碳酸酐酶基因(DCA1、CA1)5’上游区序列，并对其进行测序和序列分析。方法：利用Dra Ⅰ、EcoR Ⅴ、PvuⅡ和StuⅠ4种平端限制内切酶分别酶切盐藻基因组DNA，并与衔接头连接，构建成盐藻步行基因组文库GWL1、GWL2、GWL3和GWL4；巢式PCR方法从盐藻步行基因组文库中扩增DCA1和CA1基因5’上游区序列，双脱氧末端终止法测序。结果：DCA1基因，在GWL1、GWL3中分别扩增出约1．3kb和4、5kb的特异带，而CA1基因，则在GW12、GWL4中分别扩增出1．7kb和2．5kb的特异带；序列分析结果表明，所得序列的3’端与已知DCA1、CA1基因cDNA5’端序列完全一致。该2序列均有多个与转录调控有关的保守序列(如TATA—box、CAAT—box)和富含GT的重复序列等许多相似之处。结论：采用基因组步行方法从已知cDNA周围未知启动子或其他调控区域中克隆得到的DCA1、CA1基因的5’上游区序列，可能是2种新的杜氏盐藻碳酸酐酶基因的启动子区序列。

Cloning and sequencing of the 5''''upstream of the two carbonic anhydrase genes from Dunaliella salina

Aim: To clone and analyze sequences of the 5' upstream regions of the duplicated carbonic anhydrase (DCA1) and the carbonic anhydrase (CA1) from Dunaliella salina.Methods: The genomic DNA from Dunaliella salina was digested with Dra I,EcoR V,Pvu II and Stu I,respectively.A GenomeWalker Adaptor was ligated to the ends of the digested DNA fragments.Construction of GenomeWalker Libraries (GWL1,GWL2,GWL3 and GWL4) was finished.The 5' upstream regions of DCA1 and CA1 genes from Dunaliella salina GenomeWalker Libraries were amplified by nested PCR method.The DNA sequence was determined by dideoxy-mediated chain termination. Results: Single major PCR products of 1.3 kb and 4.5 kb fragments from the GWL1 and GWL3 in the DCA1 gene, and 1.7kb and 2.5kb fragments from the GWL2 and GWL4 in the CA1 gene were generated,respectively.The two specific fregments,1.3 kb and 1.7 kb,were cloned and sequenced.The partial sequences of 3' end in the 1.3 kb and 1.7 kb fragments were completely consistent with the partial sequences of 5' end of the DCA1 and CA1 gene cDNA, respectively.There were several conserved promoter motifs such as TATA-box,CAAT-box, and the tandem GT sequence in the sequences. Conclusion: The 5' upstream sequences obtained from the DCA1 and CA1 genes might be two novel promoters of carbonic anhydrase from Dunaliella salina,which were found in unknown genomic DNA sequences adjacent to a known sequence such as cDNA or other domain of regulation,by use of the GenomeWalker DNA walking.