中文摘要：

CBF（C-repeat binding factor）是调控植物冷驯化相关基因表达的调控转录激活因子,可与下游冷应答基因启动子的核心序列CBT/DRE元件（CCGAC）特异性地结合,启动耐寒基因的表达,提高植物的抗寒能力.为揭示播娘蒿（Descurainia sophia）的抗寒机制,研究播娘蒿转录因子Ds CBF1蛋白AP2区域编码氨基酸对启动下游抗寒基因表达的作用.首先分析Ds CBF1蛋白AP2区域编码氨基酸的疏水性,设计重叠延伸PCR引物,定点突变Ds CBF1基因;然后构建p ET32a-Ds CBF1突变体原核表达载体,通过冻融法转化入宿主大肠杆菌BL21（DE3）中,进行原核表达,并通过镍离子亲和层析纯化Ds CBF1突变体蛋白;最后利用化学发光法EMSA试验（Electrophoretic mobility shift assay）方法分析Ds CBF1蛋白与含有CRT/DRE元件的DNA探针之间的相互作用.EMSA试验分析显示,播娘蒿Ds CBF1的AP2区域的57位苯丙氨酸F（TTT）和68位缬氨酸V（GTT）分别突变为精氨酸R（CGT）和谷氨酸E（GAA）,导致播娘蒿顺式调控因子Ds CBF1蛋白与下游冷响应基因的CRT/DRE元件结合效率出现明显下降趋势,说明播娘蒿Ds CBF1蛋白AP2区域编码氨基酸位点的突变影响了播娘蒿的抗寒能力.本研究表明播娘蒿Ds CBF1的AP2区域的57位苯丙氨酸F（TTT）和68位缬氨酸V（GTT）位点对于播娘蒿的冷驯化具有十分重要的作用.

英文摘要：

CBF（C-repeat binding factor） is a transcriptional activator related to the regulation of plant cold acclimation gene expression, and AP2 domain of CBF protein can specifically bind with CBT/DRE element of cold-responsive gene promoter, which switches on the expression of anti-cold gene and improves plant cold hardiness. To reveal the anti-cold mechanism of Descurainia sophia and the mechanism of how AP2 domain of CBF protein activates the expression of its downstream genes, we analyzed the hydrophobic amino acids of AP2 domain of CBF protein, designed PCR primer to mutant Ds CBF1 gene, constructed mutant p ET32a-Ds CBF1 prokaryotic expression vector, transformed it into E. coli BL21（DE3） by freeze-thaw method, expressed and purified Ds CBF1 mutant proteins by nickel ion affinity chromatography. After that we analyzed using EMSA the interaction between Ds CBF1 mutant protein and DNA probe. The EMSA results showed that the mutation of the 57 th and the 68 th amino acid sites in the AP2 region of D. sophia from F（TTT） and V（GTT） to R（CGT） and E（GAA） respectively, leading to an obvious decline of binding Ds CBF1 with its downstream genes, thereby affecting the freeze resistance of D. sophia. The reults suggested that the 57 th amino acid site F and the 68 th amino acid site V play an important role in the cold acclimation of D. sophia.