琯溪蜜柚高分量核dna提取方法的建立-园艺学报.pdfVIP

园 艺 学 报 2010 ，37（5）：835–840 Acta Horticulturae Sinica 琯溪蜜柚高分量核DNA提取方法的建立 潘腾飞，陈桂信，潘东明* （福建农林大学园艺产品贮运保鲜研究所，园艺学院，福州 350002 ） 摘 要：以琯溪蜜柚[Citrus grandis (L.) Osbeck ‘Guanximiyou ’]叶片为材料，比较了两种提取方法 对高分子量核 DNA （HMW-DNA ）制备效果的影响。结果表明，改良Zhang 法与 Peterson 法相比，前者 更适合于琯溪蜜柚 HMW-DNA 的提取。改良Zhang 法用温和的物理方法破碎细胞壁，在一定的渗透压下 保证细胞核的完整性，运用 miracloth 和低速离心去除大部分的多糖和细胞壁碎片，经显微观察，所提取 的细胞核完整、纯净、质量高；将这些细胞核用低熔点琼脂糖包埋，以琼脂糖小块的方式保存细胞核， 以保持核的稳定；琼脂糖小块的最适消化时间为48 h ，脉冲场凝胶电泳分析结果表明所获得的 HMW-DNA 大于 1 Mb，这些核 DNA 能够被限制性内切酶消化，适合于琯溪蜜柚 BAC 基因组文库的构建。 关键词：柚；高分子量核 DNA ；BAC 文库；脉冲场电泳 中图分类号：S 666.3 文献标识码：A 文章编号：0513-353X （2010 ）05-0835-06 Development of Method for Preparation of High Molecular Weight DNA from Citrus grandis (L.) Osbeck ‘Guanximiyou ’ * PAN Teng-fei ，CHEN Gui-xin，and PAN Dong-ming （Institute of Postharvest Science and Technology of Horticultural Products ，College of Horticulture，Fujian Agricultural and Forestry University ，Fuzhou 350002，China ） Abstract: High molecular weight nuclear DNA （HMW-DNA ）from leaves of Guanximiyou [Citrus grandis (L.) Osbeck ‘Guanximiyou’] was prepared by two methods （Zhang’s and Peterson’s method ）and the extracting effects of the two preparing methods were compared. The results showed that Zhang’s method was more suitable for preparation of HMW-DNA in Guanximiyou. The cell walls were broken down by gentle physical homogenization to keep integrity of nuclei under suitable osmolarity. Debris of cell walls and most of polysaccharides were removed by filtering with miracloth and centrifugation under slow speed. The isolated nuclei were proved to be intact，pure ，and high-quality by observation under microscope and were embedded with low-melting-point agarose to preserve the isolated nuclei in plugs