【作者】 谭小艳； 马耀华； 明东风； 马丽； 侯乐峰； 牛善壮；

【Author】 TAN Xiao-yan;MA Yao-hua;MING Dong-feng;MA Li;HOU Lefeng;NIU Shan-zhuang;Department of Life Science,Zaozhuang College;Pomegranate Research Center;

【机构】 枣庄学院生命科学系； 枣庄市石榴研究中心；

【摘要】 [目的]研究从成熟石榴叶片中提取高质量基因组DNA的方法。[方法]根据成熟石榴叶片组织中富含多酚、多糖的特点,以峄城中国石榴种质资源圃20种石榴成熟叶片为材料,分别采用改良CTAB法（Ⅰ、Ⅱ、Ⅲ、Ⅳ）4种方法提取成熟石榴基因组DNA,通过琼脂糖凝胶、紫外分光光度计分析、ISSR扩增等方法检测所提取DNA的质量。[结果]（1）改良的CTABⅣ法能从各个石榴品种的成熟叶片中提取出基因组DNA,琼脂糖电泳胶孔干净,无拖带,且DNA的纯度和完整性都较好,OD_260nm/OD_280nm的比值均在1.8～2.0之间,无降解现象,其质量、产量都高于其他改良CTAB法。（2）经ISSR-PCR扩增结果表明,此方法提取的基因组总DNA比较适于ISSR分析。其主要步骤是:在石榴叶片的研磨中添加Vc、PVP等抗氧化物质,加入CTAB提取液后65℃水浴30min后冷却至室温,离心条件为15℃,10000r/min,1Omin,DNA粗提液用氯仿抽提3次,可获得高质量的DNA。[结论]改良的CTABⅣ法能有效地从石榴成熟叶片中提取到高质量的基因组DNA。更多还原

【Abstract】 [Objective] To develop an efficient and stable method for extracting high-quality genomic DNA from mature leaves of pomegranate.[Method] The mature leaves of pomegranate are rich in polyphenols,polysaccharides,20 pomegranate cultivars leaves from China germplasm nursery at Yicheng of Zaozhuang were used as studying materials.The genomic DNA was extracted using the improved CTAB method（Ⅰ,Ⅱ,Ⅲ,Ⅳ）,and the purity and quantity of each DNA sample obtained was evaluated by agarose gel electrophoresis,UV scanning and ISSR polymorphic analysis.[Results]（1） Each of the genomic DNA extracted from mature leaves of20 pomegranate cultivars by this method was pure,integral,and the sample hole was clean and no pulling,the value of OD_{260 nm}/OD_{280 nm} was 1.8 to 2.0,no degradation phenomena.And its quality and yield was high than that extracted by other improved CTAB methods.（2） The ISSR-PCR amplification results showed that this method of extracting total genomic DNA meet the analysis requirements.The main steps are:add antioxidants:Vc and PVP in the milling process of the leaves,65℃ water bath for 30 min after add the CTAB buffer,cooling to room temperature,centrifugation conditions are 15℃,10 000 r/min and10 min.The crude DNA was extracted 3 times with chloroform,can high-quality DNA is obtained.[Conclusion] The improved CTAB Ⅳ method could be used for extracting high-quality genomic DNA from matured pomegranate leaves effectively.更多还原