盐藻启动子活性片段的克隆及序列分析

Cloning and Sequence Analysis of a Fragment with Promoter Activity in Dunaliella salina (Chlorophyceae)

报道了以启动子探测质粒 pECE7为载体克隆盐藻 (Dunaliellasalina)中具有启动子活性DNA片段的研究 .经限制性内切酶EcoRⅠ分别消化盐藻基因组DNA和质粒pECE7,并进行体外重组及转化筛选 ,得到一具有启动子活性的DNA片段Ped .经进一步氯霉素抗性筛选 ,证明此启动子具有较高的活性 .测序结果显示Ped长 2 4 3bp .对其序列分析表明它具有启动子初级结构的基本特征 .Southern杂交显示此启动子片段确实来自于盐藻基因组 ,且其广泛存在于整个基因组中启动盐藻基因的表达 .经推测 ,Ped中含有多种转录因子结合位点的同源序列 。

A DNA fragment with promoter activity was cloned from Dunaliella salina using promoter probing plasmid pECE7. After being digested by Eco R Ⅰ, the D. salina genomic DNA and pECE7 were recombined and then transferred E. coli DH5 α . After several rounds of selection, a fragment Ped with promoter activity was obtained. A high activity was detected in Ped according to the selection with different chloromycetin concentrations. The nucleotide sequence of Ped is composed of 243 bp. Sequence analysis showed that it contained the basic characteristic of primary structure of promoter. Southern blot analysis suggested that Ped was harbored in D. salina genome, and it was widely spread in the whole genome to promote the gene expression in alga. When searching for the binding sites of transcriptional factors, it was predicted that there were several homologues of varies binding sites of transcriptional factor and Ped might involve in the regulation of multi gene expression.