一种新的快速鉴定蛋白与靶DNA结合位点的方法

摘要

DREB转录因子在植物逆境胁迫响应中起非常重要的作用,对植物的生长发育起重要的调控作用.传统的DNA酶I足迹法应用同位素标记DNA,采用序列胶分离DNaseI酶切片段,操作较复杂,分辨率较低,不适用于高通量的样品检测.为阐明植物体内DREB转录因子的调控机制,本研究利用改进的DNA酶I足迹法(DNaseIfoot-printing)结合凝胶阻滞法(electrophoreticmobilityshiftassay,EMSA),选用荧光色素代替同位素标记,毛细管电泳技术代替序列胶检测DNaseI酶切片段,判定GmMYB1蛋白与GmDREB3启动子DNA结合的区域.采用限制性内切酶酶切GmDREB3启动子DNA验证以上结果.同时,在判定的GmMYB1结合区域的基础上,选择可能的DNA结合元件与GmMYB1进行EMSA试验,证明GmMYB1可以与相应元件结合.该方法比传统的DNA酶I足迹法快速、简便、准确并可靠,作为一种高通量的鉴定方法可用于大规模鉴定蛋白质的DNA结合位点.%DREB (dehydration-responsive element-binding protein) transcription factors play important roles in the stress response and regulation of plants growth and development. In traditional Dnase I Foot printing, DNA probes is labeled with isotope and then performed polyacrylamide gel electrophoresis to separate digested labeled-DNA fragments, which takes various steps, with low differentiation rate and to detect fewer samples. To explore the mechanism of transcriptional regulation of DREB3 in soybean, we used an improved Dnase I Foot-printing method combining with EMSA (electiophoretic mobility shift assay) to identify binding region of proteins and to find out the core element in binding site. In this research, DNA was labeled using fluorescence instead of isotope and automated capillary electrophoresis polyacrylamide gel electrophoresis was replaced to detect digested DNA fragments. Finally, DNA binding site of GmMYB 1 with GmDREB3 promoter was identified rapidly via the modified Dnase I Foot printing. On the other hand, restriction enzyme was used to validate this result. To further confirm binding element in GmDREB3 promoter, we used a putative DNA binding element of GmMYB 1 to complete EMSA, indicating that GmMYB 1 can bind target DNA element in vitro. In short, compared with classic Dnase I Foot printing, the modified method is more rapid, accurate and reliable, which will be advantageous as a high throughout method to largely identification of interaction between protein and target DNA sites in the future.