杜氏盐藻rbcS启动子的克隆和功能分析  

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

作　　者：柴玉荣[1] 田芳[1] 刘红涛[1] 李杰 薛乐勋[1] 

机构地区：[1]郑州大学细胞生物学研究室

出　　处：《中国生物工程杂志》2008年第4期47-52,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(30540067)

摘　　要：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DraI、EcoR V、PvuII和StuI4种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL1、GWL2、GWL3和GWL4;设计特异引物从这4种文库中扩增rbcS基因的5′上游调控序列。在GWL1、GWL4中分别扩增出约1.2kb的片段。对该序列的分析表明,它的3′端与已知盐藻rbcScDNA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。To clone and analyze the 5＇ upstream region of the small subunit （rbcS） of ribulose-1,5- bisphosphate earboxylase/oxygenase （Rubisco）, genomic DNAs from Dunaliella salina were digested with Dra I, EcoR V, Pvu II and Stu I, respectively. Genome Walker 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. The resulting PCR product of about 1.2 kb from the GWL 4 was generated. The partial sequences of 3＇-end of it 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 repeats were found in the fragment. The sequences of about 800 bp located downstream of the EcoR I site of the 1.2 kb fragment was fused with bar-nos polyA to generate the expression vector pSP-B, pSP-B was transformed into the cells of D. salina by electroporation. PPT-resistant phenotype transformants 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 possessing the activity of promoter was the 5＇ upstream regulation region of the rbcS gene and it can be used in the construction of the bioreactor of transgenic D. salina.

关 键 词：盐藻 I  5-二磷酸核酮糖羧化酶/加氧酶 RBCS 启动子 

分 类 号：Q786[生物学—分子生物学]