芜菁花青素合成关键酶DFR基因启动子的克隆及功能分析

Cloning and Functional Analysis of the Key Enzyme DFR Promoters in Turnip Anthocyanin Biosynthesis

在已经克隆的‘津田’芜菁和‘赤丸’芜菁二氢黄酮醇4–还原酶(DFR)基因的基础上,通过染色体步移法从两种芜菁基因组中克隆了BrDFR1和BrDFR2基因上游1 296和1 297 bp的启动子序列。生物信息学分析表明,启动子片段中包含TATA box、CAAT box、光调控元件、ABA应答元件、伤害应答元件、低温应答元件、防御与胁迫应答元件、MeJA应答元件等多个顺式作用元件。启动子BrDFR1P和BrDFR2P仅在15个核苷酸位点存在差异。将BrDFR1P和BrDFR2P分别连接到pCAMBIA1301植物表达载体上,构建Promoter::GUS载体,通过农杆菌介导遗传转化烟草。GUS组织化学染色检测表明,BrDFR1P和BrDFR2P均能驱动GUS基因表达。构建BrDFR1P和BrDFR2P的一系列缺失体,融合GUS基因后遗传转化烟草。染色结果表明,BrDFR1P和BrDFR2P缺失片段均具有相应的起始下游基因转录的活性特征。

Based on the gene sequences of dihydroflavonol 4-reductase(DFR)which were cloned from‘Tsuda'and‘Yurugi Akamaru'turnip,the 1 296 and 1 297 bp promoter fragments of BrDFR1 and BrDFR2 genes were obtained from these two turnips' genome by genome walking method. More cis-acting elements of BrDFR1 and BrDFR2 promoters,such as TATA box,CAAT box,light responsive elements,ABRE,WUN-motif,LTR,TC-rich repeats and MeJA responsive motif,were identified using bioinformatics method. The nucleotide sequences of BrDFR1 P and BrDFR2 P had 15 differences. The pCAMBIA1301 plant expression vectors containing these two promoter sequence respectively were constructed with the GUS reporter gene,which were called Promoter::GUS vectors,and then transformed into tobacco through the mediation of Agrobacterium tumefaciens. Histochemical staining analysis showed that the expression of GUS reporter gene could be driven by BrDFR1 P and BrDFR2 P. A series of 5′-end deleted fragments of these two promoters were constructed,which were fused with GUS gene and then transformed into tobacco. The stain result indicated that the deleted fragments of BrDFR1 P and BrDFR2 P had relatively active characteristic that could start the expression of GUS gene.