【摘要】 采用DraⅠ,EcoRⅤ,PvuⅡ,ScaⅠ,Sma I和StuⅠ六种内切酶消化杜氏盐藻(Dunaliella salina)叶绿体基因组DNA,与相应DNA接头连接,构建成无载体连接的盐藻叶绿体GenomeWalker DNA文库。利用长距离PCR(long distance-polymerase chain reaction,LD-PCR)技术从该文库中克隆得到ATP合酶α亚单位(atpA)基因上游序列1 347bp。启动子特征分析显示该序列具有一般启动子的保守序列特征,根据这些特征设计引物,扩增4个5′端系列缺失的启动子片段,并用绿色荧光蛋白基因作为报告基因,构建启动子5′端系列缺失重组质粒,以期鉴定不同启动子片段的驱动报告基因转录的活性。基因枪转化结果显示,启动子长度约1 000 bp的重组载体转化盐藻后,其转化株中有黄绿色的盐藻细胞出现。说明克隆的序列具有启动子活性,能驱动绿色荧光蛋白基因在杜氏盐藻中有效表达,为建立盐藻叶绿体转化系统奠定了工作基础。更多还原

【Abstract】 To clone and identify the function of the promoter of the ATPase alpha subunit(atpA) gene from Dunaliella salina,a chloroplast GenomeWalker DNA Library from D.salina was constructed,from which the 1 347 bp upstream fragment of atpA gene was cloned by long distance-polymerase chain reaction(LD-PCR).According to the positions of the different promoter elements predicted by bioinformatics tools,5’-deleted se-ries primers were designed to amplify different promoter fragments.Series 5’-deleted expression vector con-taining the GFP gene was constructed and then transformed to cells of D.salina by particle bombardment.The transformed cells with p1000 recombinant vector produced green fluorescence,but not in other transformation groups,suggesting that the cloned region had the activity of the promoter and may drive the effective expres-sion of the GFP gene in D.salina.Our results have layed a foundation for the chloroplast transformation sys-tem of D.salina.更多还原