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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (16): 227-232.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

无核葡萄SRAP-PCR反应体系的建立和优化

张旭彤 张朝红 肖 宇 王跃进   

  • 收稿日期:2010-11-01 修回日期:2010-12-11 出版日期:2011-07-05 发布日期:2011-07-05

Optimization of SRAP-PCR System in Seedless Grape

  • Received:2010-11-01 Revised:2010-12-11 Online:2011-07-05 Published:2011-07-05

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摘要：

以大粒无核葡萄‘皇家秋天’的基因组DNA为模板，对无核葡萄SRAP-PCR反应体系的主要成分及反应退火温度进行优化。获得的最优SRAP-PCR反应程序为94℃预变性5 min，94℃变性1 min，38℃复性1 min，72℃延伸1 min，5个循环；94℃变性1 min，56℃复性1 min，72℃延伸1 min，35个循环；72℃终延伸10 min。25 μL体系中，模板DNA 40 ng，Mg2+ 1.5 mmol/L，dNTPs 0.2 mmol/L，引物0.2 μmol/L，Taq DNA聚合酶0.6 U。实验结果表明，优化后的SRAP-PCR反应体系扩增多态性高，带型清晰，稳定性好。

关键词: 菌根侵染率, 菌根侵染率

Abstract:

In this research, annealing temperatures and several key parameters of the seedless grape SRAP-PCR reaction system were optimized. The result showed that for seedless grape, the most suitable protocol of SRAP-PCR was initially denaturing at 94℃ for 5 min, then 94℃, 1 min, 36℃ 1 min, and 72℃, 1 min for the first five cycles, and then the annealing temperature was raised to 53℃ for another 35 cycles, at last extending at 72℃ for 10 min. In this SRAP-PCR system, the optimum concentrations of Mg2+, dNTPs, Taq DNA polymerase, primers and DNA template were 1.5 mmol/L, 0.2 mmol/L, 1.0 U, 0.2 μmol/L, 40 ng/25 μL, respectively.