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海洋科学  2011 

杜氏盐藻叶绿体ATP 合酶α亚单位基因启动子的克隆及初步功能分析

, PP. 18-23

Keywords: 杜氏盐藻(Dunaliella,salina),叶绿体转化,ATP,合酶α,亚单位基因启动子

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Abstract:

采用DraⅠ,EcoRⅤ,PvuⅡ,ScaⅠ,SmaI和StuⅠ六种内切酶消化杜氏盐藻(Dunaliellasalina)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GenomeWalkerDNA文库。利用长距离PCR(longdistance-polymerasechainreaction,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1347bp。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1000bp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。

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