杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率，利用基因组步行方法和巢式PCR，从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列，并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA，并与接头连接，构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4；设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明，它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致，说明是该基因的5'端上游区，并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box），富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合，构建表达载体，电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测，表明该区域能驱动外源基因bar在转基因盐藻中的表达，推断是盐藻rbcS基因的启动子调控区。

Isolation and Functional Analyse of Promoter of the rbcS Gene from Dunaliella salina

To clone and analyze the 5’ upstream region of the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco), The genomic DNAs from Dunaliella salina were digested with Dra I, EcoR V, Pvu II and Stu I, respectively. GenomeWalker Adaptors were then ligated to the ends of the digested DNA fragments. Accordingly, GenomeWalker Libraries including GWL 1, GWL 2, GWL 3 and GWL 4 were constructed. The 5’ upstream regions of rbcS were amplified from the above 4 GenomeWalker Libraries by nested PCR. Single major PCR product of about 1.2 kb from the GWL 1 and GWL 4 was generated. The partial sequences of 3’-end in the 1.2 kb fragment from GWL 4 was completely consistent with the sequences of 5’-end of the rbcS cDNA, indicating that the fragment was located the upstream of the rbcS cDNA. Several conserved promoter motifs, such as TATA-like box, CAAT-like box, etc, and the tandem GT were found in the fragment. The sequences 0f about 800 bp located downstream of the EcoR I site in the 1.2 kb fragment was fused with bar-nos polyA to generate the expression vector pSP-B. pSP-B was trandformed into the cells of D. salina by electroporation. PPT-resistant phenotype tranaformants were isolated, The results of PCR and Southern bolts of the transformed D. salina showed bar gene had been integrated into the genome of D. salina. It is concluded that the sequence we isolated from D. salina was the 5’ upstream region of the rbcS gene and possessing activity of promoter, thus it can be used in the construction of the bio-reactor of transgenic D. salina.