芥菜型油菜BjuA09DFR基因及其启动子的克隆与转化和表达

Expression of BjuA09DFR Gene in Brassica juncea andIts Promoter Cloning,Transformation and Analysis

该研究利用实时荧光定量(qRT-PCR)检测了BjuA09 DFR基因的时空表达特异性,并通过克隆BjuA09 DFR基因启动子片段,构建该基因的启动子GUS融合表达载体,利用农杆菌介导法将重组质粒转入野生型拟南芥,最后对拟南芥转基因材料不同发育时期的不同组织部位进行GUS组织化学染色,分析BjuA09 DFR基因启动子的表达模式,为BjuA09 DFR基因启动子功能的进一步研究提供理论依据。结果表明:(1)BjuA09 DFR基因在芥菜型油菜的多个组织部位都有表达,尤其是在叶、花、角果和授粉后15d种子中表达量较高。(2)成功构建了BjuA09 DFR基因启动子和GUS基因融合表达载体(pBjuA09 DFR∷GUS),采用农杆菌介导法将重组质粒转入野生型拟南芥,经卡那霉素筛选和PCR检测抗性苗,获得转基因拟南芥阳性苗。(3)GUS组织化学分析结果显示,转基因拟南芥材料的GUS活性具有明显的时空特异性,在叶、花、角果和种子中的染色较深,具有很强的GUS活性。

In this study,qRT-PCR was applied to detect the temporal and spatial expression specificity of BjuA 09 DFR gene.In addition,the BjuA 09 DFR gene promoter was cloned,and the promoter GUS fusion expression vector of BjuA 09 DFR gene was constructed.We transferred the fusion expression vector into Arabidopsis thaliana by Agrobacterium -mediated floral dip method.The expression pattern of BjuA 09 DFR gene promoter was analyzed by GUS histochemical staining in different tissues during the different development periods,which provided theoretical basis for further study of the function of BjuA 09 DFR gene promoter.The results showed that:(1) BjuA 09 DFR gene could express in multiple tissues of Brassica juncea ,especially highly expressed in leaves,flowers,silicles and seeds of 15 days after pollination.(2) The fusion expression vector of GUS gene drived by BjuA 09 DFR gene promoter (pBjuA 09 DFR ∷GUS) was successfully constructed,and transformed into wild-type A.thaliana by Agrobacterium -mediated method.We successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection.(3) Histochemical analysis of GUS showed that the GUS activity of transgenic A.thaliana displayed obviously temporal and spatial specificity,with the deeper staining in leaves,flowers,silicles and seeds of 15 days after pollination.