玉米中与DRE元件结合的转录因子的克隆和结构分析

摘要

DREB类的转录因子特异性地与DRE 元件（脱水应答元件）结合，在植物感受干旱、高盐及低温等逆境条件时，激活一系列下游逆境应答基因的表达。进一步的研究发现，拟南芥DREB蛋白的DNA结合域（AP2区）中14位的缬氨酸和19位的谷氨酸对该类转录因子与DNA结合起着关键性的作用。利用酵母单杂交的方法，我们从玉米 (Zea mays L.) 的cDNA文库中分离到一个编码与DRE元件结合的蛋白的基因，命名为maDREB1。酵母体内的反式激活实验表明，该基因编码的蛋白能特异地与DRE元件结合并能激活下游报告基因的表达。对maDREB1蛋白14位和19位的氨基酸进行单点突变和双点突变实验，发现14位的缬氨酸突变为丙氨酸后maDREB1几乎丧失了其转录激活能力，而19位的谷氨酸突变为天门冬氨酸后maDREB1的转录激活能力也受到较大影响.%Dehydration-responsive element-binding (DREB) proteins specifically binding with dehydration-responsive element (DRE) have been identified as a kind of important transcription activator of plants under drought, high salt and cold stress. The conserved amino acid residues of Val (14th residue) and Glu (19th residue) in AP2/EREBP domain of DREB1A have been identified to be two key points in determining the binding ability of DREB gene with DRE element. Using the yeast one-hybrid system, we isolated one maize DREB gene named maDREB1 by screening cDNA library. Trans-activation experiment in yeast reporter strain demonstrated that maDREB1 protein could function as a DREB transcription factor activating target gene expression by specifically binding to the DRE cis-element. To assess the functional significance of these two residues in maDREB1, the V14 and E19 were substituted individually or doubly by Ala and Asp. Point mutation analysis showed that V14 substitution made significant loss of binding ability with DRE element, while point mutation of E19 had less effect. If the substitution happened simultaneously to these two residues, it would lead to great loss of the ability of binding with DRE element. It suggested that V14 and E19 were both important in protein-DNA interacting in maDREB1, though 14V was more essential. The copy number and expression pattern of maDREB1 was discussed.