玉米逆境诱导型启动子克隆及其植物表达载体构建

Isolation of Stress-inducible Promoter in Maize and Construction of Plant Expression Vector

设计特异引物,利用PCR方法从玉米(Zea mays)基因组DNA中克隆低温和盐相应蛋白(low temperature andsalt responsive protein,LS)基因上游1 735 bp,命名为Lsp。利用在线启动子预测工具PlantCARE分析表明,序列中含有TATA-box和CAAT-box等核心元件,还包含各种胁迫响应元件。以植物表达载体pCAMBIA1301为基础,将克隆得到的启动子片段与GUS报告基因融合构建了重组表达载体pCAM-Lsp,并用反复冻融法将其导入农杆菌EHA105,通过农杆菌介导法转化烟草,GUS组织化学染色显示出Lsp驱动GUS基因表达。结果表明,该Lsp启动子片段具备一定的启动活性,为探明玉米逆境胁迫启动子表达调控序列及其调控机制的研究奠定基础。

The upstream nucleotide sequence of maize low temperature and salt responsive,named Lsp,was isolated from the genomic DNA of maize by PCR.Promoter sequence analysis by PlantCARE showed that it had some typical cis-elements,including TATA box and CAAT box.In addition,there were light responsive elements,MYB binding sites,various phytohormone responsive elements and stress responsive elements in the promoter sequence.The fusion gene of Lsp promoter and GUS were constructed and named pCAM-Lsp.The vector was transformed into Agrobacterium tumefaciens.The vector containing Lsp promoter was transferred into tobacco by Agrobacterium tumefaciens mediated method,function of Lsp promoter was analyzed by identifying activation of GUS using histochemistry staining.The results indicated that the promoter activated expression of GUS report gene,which provided valuable information for the future research of regulatory sequence and regulatory mechanism of stress-inducible promoter.