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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (16): 176-179.doi: 10.11924/j.issn.1000-6850.2012-0279

所属专题： 生物技术； 园艺

• 林学 园艺 园林 • 上一篇    下一篇

从苹果属腊叶标本中提取DNA的改良CTAB法研究

丁芳兵 刘连芬 汤庚国 吴瑞姣 钱关泽   

  • 收稿日期:2012-02-06 修回日期:2012-04-08 出版日期:2012-06-05 发布日期:2012-06-05
  • 基金资助:

    苹果属（蔷薇科）植物分类修订;苹果属（蔷薇科）植物杂交起源研究;中国野生苹果砧木资源遗传多样性及嫁接亲和性差异的分子机理研究

DNA Extraction from Malus Leaves of Dried Specimens Based on the Modified Methods of CTAB

  • Received:2012-02-06 Revised:2012-04-08 Online:2012-06-05 Published:2012-06-05

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摘要：

为了探求从保存年代较长并富含多糖多酚的苹果属植物腊叶标本中提取基因组DNA的方法，在传统CTAB法的基础上加以改良，无液氮研磨材料后加入预冷CTAB free缓冲液和提高沉淀时盐浓度，所得模板直接用于扩增nrITS区和cpDNA matK基因。经紫外分光光度和琼脂糖凝胶电泳检测，结果表明用改良方法提取的DNA在质量和产量上优于常规方法并能满足后续扩增反应的要求。该方法不需要液氮研磨，节省了人力和成本，提前加入除杂缓冲液对去除多酚的效果良好，增加沉淀时盐的浓度能有效去除多糖，表明改良CTAB法适合苹果属腊叶标本叶片总DNA的提取。

关键词: 宁夏回族自治区, 宁夏回族自治区

Abstract:

In order to find methods for total DNA extraction from Malus herbarium specimens stored for many years and enriching polysaccharides and polyphenolics, modified CTAB methods were studied based on the conventional methods. The DNA templates were used directly in amplifying the whole nrITS region and cpDNA matK gene by adding precooling CTAB free buffer after treated without liquid nitrogen and improving the concentration of NaCl when precipitating. Utraviolet spectrophotometer and agarose gel electrophoresis methods were used to test the DNA quality. The results showed that modified CTAB methods were optimal to produce high quality and yields DNA and amplifiable in the polymerase chain reaction (PCR). This methods don’t need to grind with liquid nitrogen,saving manpower and reducing cost.In addition,adding CTAB free buffer and improving NaCl when precipitating were effective for the removement of polyphenolics and polysaccharides. The researches indicated that the modified CTAB was suitable for extracting genomic DNA from Malus herbarium leaves.