核盘菌分枝酸变位酶同源效应蛋白启动子的克隆及功能分析

Cloning and Analyzing the Function of the Promoter of an Effector Homologous of Chorismate Mutase in Sclerotinia sclerotiorum

为了进一步研究分枝酸变位酶同源效应蛋白在核盘菌与植物互作过程中的作用机制,对其启动子进行了克隆和功能分析。采用启动子在线软件Promoter 2. 0和Promoter scan分析分枝酸变位酶同源基因的上游序列,寻找其启动子位点和顺式作用元件,然后通过PCR技术克隆预测启动子的DNA序列,构建荧光蛋白GFP融合载体,转化核盘菌,最后通过检测GFP荧光信号来验证启动子功能。经生物信息学分析发现,该效应蛋白基因上游具有启动子序列特征,包含TATA盒和CAAT盒等顺式元件,采用引物XS1-1和XS1-2进行PCR扩增,得到了733 bp启动子序列,序列包含相关的启动元件,然后以质粒pBlunt NAT-GFP为基础,将经PCR克隆得到的启动子(N-Pro)片段连入载体中,构建得到启动子N-Pro+GFP融合载体,通过REMI技术转化核盘菌原生质体,潮霉素筛选得到了一些转化子,PCR验证融合载体整合到了核盘菌基因组DNA上,最后经荧光显微镜检测到了菌丝GFP荧光信号,结果表明,克隆的分枝酸变位酶同源基因ATG上游733 bp的DNA序列具有启动子功能。

In order to investigate the mechanism of the effector homologous of chorismate mutase in Sclerotinia sclerotiorum,the promoter was cloned and analyzed.The promoter element and the Cis element were analyzed through the online software,Promoter 2.0 and Promoter scan,then the DNA sequence of the promoter was cloned by the method of PCR and the GFP fusion vector were constructed,transformed in the protoplast of Sclerotinia sclerotiorum.In the end,the GFP fluorescence were detected through confocal microscope.As a result,TATA box and CAAT box were found in the upstream of the gene,and a 733 bp DNA sequence upstream of ATG was cloned by PCR using the primer XS1-1 and XS1-2,then the GFP fusion vector were constructed based on the plasmid of pBluntNAT-GFP,through transforming the protoplast of Sclerotinia sclerotiorum,some transformants were obtained in which the GFP fluorescence were detected through confocal microscope.All these indicated that the 733 bp DNA sequence upstream of ATG could act as the promoter of the gene.