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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (27): 239-244.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

橡胶草基因组DNA提取及RAPD反应体系的优化

吴坤鑫 李若霖 崔百明 王颖 张秀春 陈雄庭   

  • 收稿日期:2011-08-08 修回日期:2011-08-27 出版日期:2011-10-25 发布日期:2011-10-25
  • 基金资助:

    国家自然科学基金“巴西橡胶树橡胶粒子膜蛋白复合物的研究”;中央级公益性科研院所基本科研业务费专项资金“橡胶草野生资源的调查和产胶性评价”

Extraction of Genomic DNA and Optimization of RAPD Reaction of Taraxacum kok-saghyz Rodin

  • Received:2011-08-08 Revised:2011-08-27 Online:2011-10-25 Published:2011-10-25

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摘要：

为探索适合橡胶草基因组DNA的提取方法和RAPD反应体系。采用改良CTAB法提取橡胶草叶片总DNA，得到的DNA能满足RAPD分析需要。通过单因子实验法分析DNA浓度、引物浓度、dNTPs浓度、Taq聚合酶以及Mg2+浓度对RAPD扩增结果的影响，建立了适合于橡胶草RAPD分子标记的最佳反应体系，即20 μL体系中包括：模板DNA30 ng、Taq DNA聚合酶1.75 U、dNTPs浓度0.25 mmol/L、Mg2+浓度2.0 mmol/L、引物浓度0.5 μmol/L、10×PCR Buffer 2.0 μL。RAPD扩增反应程序为：94℃预变性5 min，94℃ 30 s，37℃ 30 s，72℃ 70 s，45个循环，最后72℃延伸10 min。该反应体系具有较好的稳定性及可重复性。

关键词: 体细胞胚发生, 体细胞胚发生, 植物激素, 内因, 外因

Abstract:

The purpose of this study was to obtain the best method for extracting genomic DNA from Taraxacum kok-saghyz Rodin and to establish the best reaction condition of the RAPD amplification system. The high quality genomic DNA of Taraxacum kok-saghyz Rodin could be obtained from young leaves by the improved CTAB method. Some reaction conditions of PCR for RAPD were optimized by setting the gradient with template DNA concentration, primer concentration, dNTPs concentration, Taq DNA Polymerase concentration and Mg2+ concentration through a single factor experiment. And the optimized reaction system and program of RAPD were as follows. The reactions were performed in a volume of 20 μL, which containing 30 ng DNA template、1.75U Taq DNA polymerase、0.25 mmol/ L dNTPs、2.0 mmol/L Mg2+、0.5 μmoL/L random primer、2.0 μL 10×PCR Buffer. The RAPD reaction was programmed by 1 cycle for 5 min at 94℃,followed by 45cycles for 30 s at 94℃,30 s at 37℃, and 70 s at 72℃and finally, followed by 1 cycle for 10 min at 72℃. The reaction system was reproducible and highly reliable, and it could be effectively for RAPD analysis in Taraxacum kok-saghyz Rodin.