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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (1): 294-297.doi: 10.11924/j.issn.1000-6850.2011-2235

• 林学 园艺 园林 • 上一篇    下一篇

杨梅SRAP-PCR反应体系的建立与优化

林旗华 卢新坤 张泽煌   

  • 收稿日期:2011-08-01 修回日期:2011-10-08 出版日期:2012-01-05 发布日期:2012-01-05

Optimization of Sequence-Related Amplified Polymorphism Amplification System for Chinese Bayberry

  • Received:2011-08-01 Revised:2011-10-08 Online:2012-01-05 Published:2012-01-05

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摘要：

为了建立适宜杨梅基因组DNA的SRAP-PCR扩增体系。以杨梅基因组DNA为模板，通过正交试验设计，从Mg2+、模板DNA、dNTPs、Tap DNA聚合酶和引物5种因素4个水平对杨梅SRAP-PCR反应体系进行优化。各因素对杨梅SRAP-PCR反应的影响程度从大到小依次为：Mg2+，模板DNA，dNTP，引物和Taq DNA聚合酶；建立的杨梅SRAP-PCR最佳反应体系为25μL反应体系中含2.5 mmol/L Mg2+、50 ng DNA模板、0.25 mmol/L dNTPs、0.15 μmol/L引物和1.5 U Taq DNA聚合酶。这一体系的建立为今后利用SRAP-PCR技术开展杨梅分子遗传学研究打下了基础。

关键词: 烟草, 烟草, 花粉囊, 介质花粉, 种子

Abstract:

The aim was to establish SRAP-PCR amplification system which was suitable for Chinese Bayberry genome DNA. The optimized SRAP-PCR amplification system for Chinese bayberry was established by the orthogonal design. The results suggested that the order of factors which affect on the result of SRAP-PCR were Mg2+, template DNA, dNTPs, primers and Taq DNA polymerase. A suitable SRAP-PCR system for Chinese bayberry was that total 25 μL reaction system containing 2.5 mmol/L Mg2+, 50 ng template DNA, 0.25 mmol/L dNTPs, 0.15 μmol/L primers and 1.5 U Taq DNA polymerase could be able to amplified the most rich polymorphism and clear bands. The optimized SRAP-PCR system was tested on twelve Chinese bayberry germplasms and shown to be steady and reliable for molecular genetics research.