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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (16): 53-59.doi: 10.11924/j.issn.1000-6850.2011-3531

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

壳菜果ISSR-PCR反应体系的建立与优化

彭继庆 曹福祥 许若娴   

  • 收稿日期:2011-11-28 修回日期:2012-02-15 出版日期:2012-06-05 发布日期:2012-06-05
  • 基金资助:

    国家林业公益行业科研专项子项目“中国森林对气候变化的影响与林业适应对策研究”

Establishment and Optimization of ISSR-PCR Reaction System of Mytilaria laosensis

  • Received:2011-11-28 Revised:2012-02-15 Online:2012-06-05 Published:2012-06-05

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摘要：

为建立一个稳定、可重复的壳菜果ISSR-PCR反应体系，以壳菜果的总DNA为实验材料，通过单因素实验、正交试验和方差分析对模板DNA浓度、dNTPs浓度、镁离子浓度、引物浓度、Taq DNA聚合酶浓度5因素进行研究，确定了壳菜果ISSR-PCR反应的最优体系为：在20 μL的反应体系中模板DNA为30 ng，dNTPs浓度为0.20 mmol/L，Mg2+浓度为2.75 mmol/L，引物浓度为0.6 μmol/L，TaqDNA聚合酶为2.2 U。方差分析表明：5个因素中只有Mg2+浓度对反应体系影响最大，达到了显著性水平。

关键词: 阈强度, 阈强度

Abstract:

To establish a stable, repeatable ISSR-PCR reaction system of Mytilaria laosensis, those five factors, the concentration of DNA template, dNTPs, Mg2+, and primers and Taq DNA polymerase, were studied through the single factor tests, orthogonal tests and variance analysis with the total DNA of Mytilaria laosensis as experimental material. The optimal system of ISSR-PCR reaction was established as follows: 30 ng template DNA, 0.20 mmol/L dNTPs, 2.75 mmol/L Mg2+, 0.6 μmol/L primers, 2.20 U Taq DNA polymerase were contained in 20μL reaction system. Variance analysis showed that: only the Mg2+ concentration of five factors has the greatest impact on the reaction system and reached a significant level.

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