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作 者 ：张玉宝;郭志鸿;李同祥;王亚军;谢忠奎

期 刊 ：武汉植物学研究 2009年 27卷 2期 页码：188-192

关键词：DRE顺式作用元件；DREB转录因子；双链DNA芯片；

Keywords:DRE cis-acting element, DREB transcription factors, Double-stranded DNA microarrys,

摘 要 ：DRE顺式作用元件能与DREB转录因子特异结合,在诱导逆境(干旱、高盐、低温)基因表达过程中起重要作用。dsDNA(double strand DNA)微阵列芯片技术能够有效地检测序列特异性DNA结合蛋白质(转录因子)与大量DNA靶点(顺式作用元件)的特异性结合,可有效分析生物分子结合作用。根据DRE顺式作用元件核心序列设计并化学合成含发夹结构的单链DNA探针,采用TaqDNA聚合酶在片延伸,并对其在片延伸体系的反应温度、Mg²⁺浓度以及单链探针是否变性等条件进行了优化。结果表明,50℃的反应温度,2.5mmol/L的Mg²⁺浓度和单链不变性是TaqDNA聚合酶在片延伸的最佳条件。优化方法制备的dsDNA芯片将更有利于DRE顺式作用元件与DREB抗逆转录因子相互作用的研究。

Abstract:DRE cis-acting element which could specifically bind DREB transcription factors plays important roles in inducing gene expression involved in dehydration,low-temperature,and high-salinity stresses.Double-stranded DNA microarrys could very effectively detect the interaction between DNA-cis-acting elements and protein-transcription factors in regulation of gene expression,and also could availably analyze the binding interaction of bio-molecular.In this study,we designed and chemically synthesized a hairpin structure single-stranded DNA probe according to the nuclear sequence of DRE cis-acting element.Furthermore,we adopted Taq DNA polymerase in the elongation system and optimized the new on-chip reaction conditions.Our experiments showed that the elongation temperature of 50℃,the Mg²⁺ concentration of 2.5 mmol/L and single-stranded,non-denatured probe are the optimal conditions in elongation with Taq DNA polymerase.These results provide further steps toward a better understanding of interaction mechanisms between DRE cis-acting element and DREB transcription factors.

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