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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (18): 143-147.

• 农学 农业基础科学 • 上一篇    下一篇

正交设计优化狭叶坡垒ISSR-PCR反应体系

代文娟 唐文秀 邓涛 丁莉 骆文华 黄仕训   

  • 收稿日期:2011-05-04 修回日期:2011-06-10 出版日期:2011-07-25 发布日期:2011-07-25
  • 基金资助:

    植物园迁地保护植物编目及信息标准化

Optimization for ISSR Reaction System of Hopea chinensis by Orthogonal Design

  • Received:2011-05-04 Revised:2011-06-10 Online:2011-07-25 Published:2011-07-25

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摘要：

以狭叶坡垒DNA为模板，利用正交试验分别对ISSR-PCR反应的MgCl2浓度、dNTPs浓度、Taq聚合酶浓度、引物浓度、模板DNA浓度进行了优化，并通过梯度PCR确定最佳退火温度和循环次数，最终确定狭叶坡垒最佳反应体系及扩增条件为：25 μL体系中1×PCR buffer，2 mmol/L MgCl2，0.25 mmol/L dNTPs，0.04 U/μL Taq聚合酶，0.2 μmol/L引物，4 ng/μL DNA模板；最佳扩增程序为：94℃预变性5 min；94℃变性45 s，53℃退火45 s，72℃延伸1.5 min，共35个循环；72℃最后延伸7 min。

关键词: 柑橘绿霉病菌, 柑橘绿霉病菌, 筛选, 拮抗作用, 放线菌, 鉴定

Abstract:

To optimize the inter simple sequence repeat(ISSR) reaction condition for Hopea chinensis genomic DNA, the concentrations of MgCl2, dNTPs, Taqpolymerase, primers and template DNA were studied with an orthogonal experimental design, and the optimal anneal temperature of primer and cycles were determined through gradient PCR. The optimal PCR system for ISSR analysis was 1×PCR buffer, 2 mmol/L MgCl2, 0.25 mmol/L dNTPs, 0.04 U/μL Taq polymerase, 0.2 μmol/L primer, 4 ng/μL template DNA in 25 μL reaction solution. And the augmentation procedure was pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 45 s, annealing at 53℃ for 45 s, extension at 72℃ for 1.5 min, reaction with 35 cycles, and extension at 72℃ for 7 min.

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