枳抗寒性相关转录因子CBF启动子缺失表达载体的构建

Construction of Expression Vector of Trifoliate Orange Driven by CBF Promoter with Partial Deletion

试验针对枳[Poncirus trifoliata(L.)Raf.]CBF启动子序列顺式作用元件预测结果,设计特异引物并PCR扩增分离了元件顺序缺失片段,与基本表达载体pCAMBIA1391Z连接后转化大肠杆菌DH5α,提取质粒DNA后,通过双酶切、PCR及测序筛选出R0-7、R3-10、R4-8、R5-10、R6-10、R7-10和R8-10阳性转化子,成功构建了枳CBF启动子缺失表达载体,为枳CBF启动子顺式作用元件功能验证奠定了基础,为启动子核心元件筛选和转基因高效抗寒柑橘育种提供理论依据。

The transcription factor CBF known as master switch plays an important role during cold acclimation and responses to low temperature. In this study, the genomic DNA fragments with partial deletion in CBF promoter of trifoliate orange[Pon- cirus trifoliata（L.） Raf.] were isolated by PCR amplification, and ligated with expression vector pCAMBIA1391Z after column purification followed by double digestion, then transformed into E. coli DH5α. Finally, R0-7、R3-10、R4-8、R5-10、R6-10、 R7-10 and R8-10 were selected as positive transformants by double digestion and gene-specific PCR amplification with the plasmid DNAs extracted by alkaline lysis, then verified by nucleotide sequencing. The results are helpful for the further func- tional characterization of cis-action elements in trifoliate orange CBF promoter and high-efficiency transgenic citrus breeding for cold-resistant varieties.