从苹果属腊叶标本中提取DNA的改良CTAB法研究

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

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。

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 freed buffer and improved 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.