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作 者:张玉宝[1] 郭志鸿[1] 李同祥[1,2] 王亚军[1] 谢忠奎[1]
机构地区:[1]中国科学院寒区旱区环境与工程研究所皋兰生态与农业综合试验站,兰州730000 [2]兰州大学生命科学学院,兰州730000
出 处:《武汉植物学研究》2009年第2期188-192,共5页Journal of Wuhan Botanical Research
基 金:中科院农办项目(CASN-115-06-08)资助
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。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 ele- ments 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 Mg^2+ 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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