花生DNA的五种改良CTAB提取方法的比较分析及其应用

Comparative Analysis and Application of Five Improved CTAB Extraction Methods for Peanut DNA

花生是世界上重要的油料作物和经济作物之一,也是最重要的植物食用油来源之一,但花生分子标记和功能基因组学等分子生物学研究较落后。因此,本研究旨在建立适合自身的花生基因组DNA的提取方法,为开展花生分子标记研究和分子生物学研究提供帮助。本研究使用了5种改良CTAB法提取花生基因组DNA,所得DNA的纯度和浓度分别通过琼脂糖凝胶电泳和紫外分光光度计检测,再利用8种分子标记技术的48条单引物和4对转座子保守区扩增引物对提取获得的花生基因组DNA的质量进行扩增验证和应用。结果表明:(1)综合花生基因组DNA的质量检测数据以及花生分子标记技术和转座子保守区的扩增验证结果来看,五种改良CTAB法的DNA提取效果表现依次为:方法一>方法五>方法四>方法三>方法二,其中方法一为最佳首选,方法五和方法四的提取效果也好,但是由于其有利用到强腐蚀性的平衡酚,不安全且不环保,所以不推荐;(2)在花生上建立了8种分子标记技术和4类转座子保守区的扩增体系;(3)克隆获得了花生4类转座子保守区序列。本研究为今后开展花生分子标记研究及转座子的克隆鉴定利用提供了帮助。

Peanut is one of the most important oil and economic crops in the world. It is also one of the most important sources of plant edible oil. However, researches such as peanut molecular markers and functional genomics are relatively lagging behind. Therefore, the purpose of this study was to establish suitable methods for peanut genomic DNA extraction, and offer help for the molecular marker studies and molecular biology studies in peanut. In this study, five improved CTAB methods were used to extract peanut genomic DNA. The purity and concentration of the obtained peanut DNA were detected by agarose gel electrophoresis and ultraviolet spectrophotometer, respectively. Then, the quality of the obtained peanut genomic DNA was verified through PCR with forty-eight single primers of eight kinds of single primer amplification reaction based molecular marker techniques and four pairs of primers designed according to the conserved region of transposons. The results showed that according to the comprehensive quality test data of peanut genomic DNA, the amplification results of molecular marker techniques and transposon homology-based cloning, the extraction effects of the five improved methods were as follows: method one>method five>method four>method three>method two, among which method one was the best one. Also, method five and method four were effective, but the highly corrosive balanced phenol which was not safe and environmentally friendly was used in these two methods, so they were not recommended. The amplification system of eight kinds of single primer amplification reaction based molecular marker techniques and four types of transposon homology-based cloning in peanut was established. The sequences of four types of transposon conserved region of peanut were obtained by cloning. In conclusion, this study would provide help for peanut molecular marker research and transposon cloning, identification and utilization in the future.