柑橘冷诱导基因及其启动子表达载体构建与瞬时表达分析

Construction of plant vectors with promoter and cold-induced genes in citrus and transient expression verification

【目的】以YFP为报告基因,构建柑橘冷诱导基因及其启动子的植物表达载体。【方法】克隆获得枳冷诱导基因Ptcor8及其启动子p Ptcor8,柠檬中同源基因Clcor8及其启动子p Clcor8;双酶切含启动子的克隆载体和植物表达原始载体p1D4,连接获得含启动子的中间载体;再双酶切含冷诱导基因的克隆载体和中间载体,连接后获得重组质粒;通过冻融法将重组质粒导入农杆菌EHA105中。【结果】构建了p1D4/p Ptcor8-Ptcor8::YFP,p1D4/p Ptcor8-Clcor8::YFP和p1D4/p Clcor8-Ptcor8::YFP 3个融合表达载体,瞬时表达发现3个融合基因均可在冰糖橙叶片中表达。【结论】3个融合表达载体的成功构建为下一步转化柠檬,分析枳冷诱导基因Ptcor8及其启动子p Ptcor8的功能奠定了基础。

【Objective】The plant expression vectors of citrus cold-induced genes and promoters was con-structed fused with YFP reporter gene.【Methods】Ptcor8 gene and its promoter p Ptcor8 in Poncirus trifoliate,homologous gene Clcor8 and its promoter p Clcor8 in Citrus limon were cloned respectively. Then thecloning vectors containing promoters and the precursor plant expression vector p ID4 were digested withtwo kinds of restriction endonucleases,and the digested products was ligated to get intermediate vector.Next,the intermediate vectors and the cloning vectors containing the cold-induced genes were digestedwith two types of restriction endonucleases and were ligated consequently to contruct recombinant plas-mid. Finally,freeze-thawing was used to transform the vector plasmid into Agrobacterium EHA105.【Re-sults】p1D4/p Ptcor8-Ptcor8::YFP,p1D4/p Ptcor8-Clcor8::YFP and p1D4/p Clcor8-Ptcor8::YFP fusion ex-pression vectors were constructed,and further transient expression analysis showed that the fused genewas successfully expressed in C. sinensis leaves.【Conclusion】The successful construction of three fusionexpression vectors will provide a basis for transforming C. limon and analyzing the function of cold-in-duced gene Ptcor8 and its promoter p Ptcor8.