中国生物工程杂志

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.