含有融合标记基因和LoxP/FRT位点的植物表达载体构建及应用

Construction and Application of the Vector Containing the Bar::gus Fusion Gene and LoxP/FRT Recognition Site

本研究利用化学合成法合成了包含pCAMBIA0390左右边界T-DNA和LoxP/FRT(LF)位点的DNA片段Ⅰ,利用SacⅡ和SphⅠ酶切位点,去除了pCAMBIA0390左右边界T-DNA和多克隆位点之间的序列,然后连接DNA片段Ⅰ和载体片段,构建了植物表达载体pGM323-LF-enTP。随后,再合成含有适合在单子叶植物中表达的由玉米Ubi-1启动子驱动的融合标记基因(Bar::gus)和水稻actin-1启动子驱动的Bt抗虫蛋白基因(Cry1Ab)表达元件的DNA片段Ⅳ,在pGM323-LF-enTP的基础上,利用SalⅠ和PstⅠ位点构建了同时含有LF位点、Bar::gus以及Cry1Ab基因表达元件的表达载体pGM626-LF-ABt。利用含有pGM626-LF-ABt的农杆菌遗传转化烟草和玉米,以草丁膦作为抗性筛选剂,非转化细胞得到了有效抑制,快速获得了转基因植株,利用GUS组织化学检测和RT-PCR分析了转基因植株中标记基因的表达,结果表明pGM626-LF-ABt可以用于农杆菌介导的单、双子叶植物遗传转化。本研究为培育安全抗虫转基因植物奠定了基础。

DNA fragment Ⅰ containing T-DNA ofpCAMBIA0390 vector and LoxP/FRT （LF） sites were synthesized with additional Sac＇ Ⅱ and Sph Ⅰ recognition sites. Then the vector was released from pCAMBIA0390, and the sequences between T-DNA and multiple cloning sites of T-DNA of pCAMBIA0390 vector were deleted with restriction enzyme Sac Ⅱ and Sph Ⅰ . Digested sequences were ligated to generate pGM323-LF-enTP plasmid. DNA fragment Ⅳ was synthesized with the fused Bar：：gus gene under maize ubiquitin promoter （Ubi- 1） and CrylA b under the rice actin-1 gene （Act-1） promoter. The vector containing LF and T-DNA sequences was released from pGM323-LF-enTP plasmid with Sa/I and Pst I sites. Then digested sequences were ligated to generate pGM626- LF-ABt plasmid. Transformed explants were cultured in selection medium containing bialaphos. Putatively transformed events were covered after selection cultivation. The expression of fused selectable marker gene was analyzed in different tissues of transgenic tobacco and transgenic maize. The results confirmed that the plasmid pGM626-LF-ABt could be used for plant transformation. These results would be a base for breeding transgenic plants with bio-safety.