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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (4): 146-150.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

甜瓜ISSR-PCR反应体系的正交优化研究

赵春梅 金荣荣 刘英 汪磊   

  • 收稿日期:2011-08-31 修回日期:2011-11-21 出版日期:2012-02-05 发布日期:2012-02-05

Optimization for ISSR-PCR System of Cucumis melo L. Using Orthogonal Design

  • Received:2011-08-31 Revised:2011-11-21 Online:2012-02-05 Published:2012-02-05

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摘要：

为在最短时间内找到甜瓜ISSR-PCR反应四个主要因素的最优水平，确定甜瓜ISSR-PCR最优反应体系进行本研究。采用CTAB法提取甜瓜基因组DNA，获得完整性好且纯度较高的DNA样品。利用统计软件MINTAB创建4因素3水平的正交试验设计，PCR结果用MINITAB进行方差分析。结果显示引物浓度对PCR反应结果影响最大，Taq DNA聚合酶对PCR结果影响最小，并对各因素水平间进行因素内多重比较分析，最终建立最优化的反应体系（20 μL）：1×buffer，100 ng DNA模板，Taq DNA聚合酶1 U，引物0.4 mmol/L，dNTP为0.1 mmol/L。

关键词: 土壤酶活性, 土壤酶活性

Abstract:

In order to find out the optimal level of four factors of melon ISSR-PCR reaction and to determine the optimum reaction system within the shortest time this study was done. The genomic DNA of melon was extracted by CTAB method and DNA sample with high purity and good integrity was obtained. In this study orthogonal design with three levels of four factors was created by software MINTAB and the analysis of variance of PCR result was carried out by MINITAB. The results showed that the primer concentration was the greatest impact on the result of the PCR reaction, Taq DNA polymerase was minimal impact on the PCR result. By the use of multiple comparison between all pairs of level a better amplification system of ISSR PCR reaction was established: 1×PCR buffer, 100 ng templates DNA, 1U Taq DNA polymerase, 0.4 μmol/L primers, 0.1mmol/L dNTP in the total volume of 20 μL.