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中国农学通报

中国农学通报 ›› 2009, Vol. 25 ›› Issue (2): 250-254.

所属专题： 生物技术

• 农学 农业基础科学 • 上一篇    下一篇

白木香基因组DNA提取与ISSR反应体系的优化

邹枚伶,夏志强,王文泉   

  • 收稿日期:2008-10-13 修回日期:2008-11-20 出版日期:2009-01-20 发布日期:2009-01-20

Optimization for Extracting Total DNA and ISSR-PCR System in Aquilaria sinensis (Lour.) Spreng

Meiling Zou   

  • Received:2008-10-13 Revised:2008-11-20 Online:2009-01-20 Published:2009-01-20

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854

摘要： 白木香为瑞香科沉香属植物，是我国特有的珍贵药用植物，也是国产沉香药材唯一资源植物，现已濒临灭绝，被载入《中国植物红皮书》。采用改良CTAB法提取白木香DNA较传统方法简单高效。通过琼脂糖凝胶电泳和OD260/OD280比值测定，所得基因组DNA纯度高，可作为ISSR等以PCR为基础的DNA多态性标记。本实验还通过优化影响白木香ISSR-PCR的主要参数，确立了适用于白木香的ISSR反应体系和扩增条件。结果表明在20µL反应体中，模板DNA、引物、Mg2+、dNTP和Taq DNA聚合酶五种主要成分的最适浓度分别为25ng、0.8µmol/L、2.5mmol/L、0.2mmo1/L、1.0U，扩增出足量产物至少需要35个循环。

关键词: 参与式发展方法, 参与式发展方法, 两渔村, 发展规划

Abstract: Aquilaria sinensis(Lour.)Gilg classified as Thymelaeaceae, is one of the valuble medicinal plants in our country. But its wilding resource has been profoundly damaged, and has been recorded in《China Plants Red Book》. The improved CTAB method which can extracting high-quality genome DNA of Aquilaria sinensis(Lour.) Spreng was much more simple and efficient than the conventional methods. And the extracted DNA was suitable for the polymorphic analysis based on PCR, such as ISSR-PCR. The optimal ISSR-PCR reaction conditions in Aquilaria sinensis(Lour.) Spreng was established by studying the main parameters. Results showed that the optimum concentration of five important components i.e. template DNA, primer, Mg2+, dNTP, Taq DNA polymerase in 20 µl reaction mixture was 25ng、0.8µmol/L、2.5mmol/L、0.2mmo1/L、1.0U, respectively, and at least 35 PCR cycles should be carried out to ensure sufficient PCR products.