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植物研究 ›› 2009, Vol. 29 ›› Issue (1): 80-85.doi: 10.7525/j.issn.1673-5102.2009.01.017

• 论文 • 上一篇    下一篇

利用正交设计优化兴安落叶松RAPD-PCR反应体系

李雪峰;张含国*;贯春雨;张瑶   

  1. 东北林业大学林木遗传育种与生物技术教育部重点实验室,哈尔滨 150040
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-01-20 发布日期:2009-01-20
  • 通讯作者: 张含国
  • 基金资助:
     

Establishment and Optimization of RAPD-PCR Reaction System for Larix gmelnii(Rupr.) Rupr Using Orthogonal Design

LI Xue-Feng;ZHANG Han-Guo*;GUAN Chun-Yu;ZHANG Yao   

  1. Northeast Forestry University,Key Laboratory of Forest Tree Genetic Improvement and Biotechnology,Ministry of Education,Harbin 150040
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-01-20 Published:2009-01-20
  • Contact: ZHANG Han-Guo
  • Supported by:
     

摘要: 以兴安落叶松针叶DNA为模板,对影响落叶松RAPD-PCR 扩增的重要参数进行了优化试验,以期建立兴安落叶松RAPDPCR反应的最佳体系。通过采用正交设计L16(45)对兴安落叶松RAPD-PCR反应的5因素(Taq酶、Mg2+、dNTP、模板DNA、引物)在4个水平上进行优化试验,结果表明兴安落叶松最佳的RAPD-PCR的反应体系(20 μL)中含有模板90 ng,0.5 μmol·L-1的引物,1×反应缓冲液,DNTP各为0.25 mmol·L-1,1 U的Taq DNA聚合酶,Mg2+ 2.5 mmol·L-1。在此基础上筛选出20个扩增稳定、多态性丰富的RAPD引物,并通过梯度 PCR试验,确定了引物最佳退火温度。

关键词: 兴安落叶松, RAPD-PCR, 正交设计, 反应体系

Abstract: The orthogonal design was used to optimize RAPD amplification system of Larix gmelnii(Rupr.) Rupr with five factors (Taq polymerase, Mg2+, dNTP, primer, DNA templet) at four levels, respectively. Through the deep analysis, a suitable RAPD-PCR reaction system was established, namely 20 μL reaction system containing 1.0 U Taq DNA polymerase, 2.5 mmol·L-1 Mg2+,0.25 mmol·L-1 dNTP, 0.5 μmol·L-1 primer, 1×PCR buffer, 90 ng DNA template. 20 primers with stable amplification and rich polymorphism for RAPD were screened. The optimal annealing temperature for RAPD-PCR reaction was proposed by gradient PCR. The result provided a standardizing RAPD-PCR program for the analysis of genetic diversity of L. gmelnii(Rupr.) Rupr.

Key words: Larix gmelnii(Rupr.)Rupr, RAPD-PCR, orthogonal design, reaction system

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