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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (31): 141-145.

• 林学 园艺 园林 • 上一篇    下一篇

胡椒SRAP反应体系的建立和优化

姜艳 刘进平   

  • 收稿日期:2012-03-28 修回日期:2012-05-23 出版日期:2012-11-05 发布日期:2012-11-05
  • 基金资助:

    海南野生胡椒种质资源遗传多样性分析和保护利用

Establishment and Optimization of Black Pepper SRAP Reaction System

  • Received:2012-03-28 Revised:2012-05-23 Online:2012-11-05 Published:2012-11-05

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摘要：

建立并优化胡椒SRAP分子标记体系，为海南胡椒属植物亲缘关系和遗传多态性分析、物种和品种鉴定等打下技术基础。利用单因素随机试验对胡椒SRAP-PCR反应体系中各组分（Taq DNA聚合酶、dNTP、模板DNA、引物和Mg2+）的浓度进行优化，同时筛选SRAP-PCR反应的循环数和最适退火温度。通过实验确定了SRAP-PCR反应体系为：反应总体系为20 μL，其中引物0.35 μmol/L，Taq DNA聚合酶1.0 U，dNTP 0.6 mmol/L，Mg2 + 1.5 mmol/L，模板DNA 25~200 ng，同时通过梯度PCR试验,确定引物最佳退火温度；最佳SRAP-PCR反应程序为：94℃预变性5 min；94℃变性30 s，35℃退火30 s，72℃延伸45 s，5个循环；然后94℃变性30 s，48℃退火30 s，72℃延伸45 s，40个循环；最后72℃延伸7 min，4℃保存。SRAP-PCR体系适为胡椒属植物遗传多样性分析奠定了基础，并成功地应用于海南胡椒属植物亲缘关系和遗传多态性分析。

关键词: 非点源污染, 非点源污染

Abstract:

The conditions of SRAP-PCR reaction were optimized using the method of single-factor experiment respectively. The optimal ISSR-PCR amplification was established as follows: Each 20-μL PCR mixture consisted of 1.0 U Taq DNA polymerase (TaKaRa Biotechnology), 6X PCR buffer, 0.6 mM dNTP, 0.35 μM primer, 1.5 mM Mg2+, and 25-200 ng template DNA. Thermal cycling (Biometra T1 Thermocycle) started with 5 min at 94°C for initial denaturing, and 5 cycles of 30 s at 94°C, 30 s at 35°C, and 45 s at 72°C, followed by 40 cycles of 30 s at 94°C, 30 s at 4°C and 45 s at 72°C. The last cycle was followed by a 7-min extension at 72°C. Amplified products were analyzed on 2% (w/v) gels and visualized with the Vilber Gel Documentation System.