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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (34): 53-57.

• 林学 园艺 园林 • 上一篇    下一篇

红叶石楠RAPD反应体系的优化

李妮 燕丽萍 刘翠兰 屈星 梁慧敏 夏阳   

  • 收稿日期:2012-07-26 修回日期:2012-09-11 出版日期:2012-12-05 发布日期:2012-12-05

Optimization of RAPD System for Photinia faseri

  • Received:2012-07-26 Revised:2012-09-11 Online:2012-12-05 Published:2012-12-05

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摘要：

建立并研究适合红叶石楠的RAPD的最佳反应体系，为以后开展红叶石楠的遗传多样性研究、物种资源研究和亲缘关系鉴定等提供重要的参考。采用改进后的CTAB法，成功地提取了红叶石楠基因组DNA，通过单因素试验，研究了Mg2+、dNTP、引物的浓度和模板DNA，Taq酶的用量对RAPD反应的影响，建立适合于红叶石楠的RAPD反应体系。结果表明，在25 μL的RAPD反应体系中最佳反应条件分别是10×PCR Buffer 2.5 μL、Mg2+ (25 mmol/L) 2.0 μL、引物(20 μmol/L) 2.0 μL、dNTPs (2.5 mmol/L) 1.8 μL、DNA模板(49.5 μg/mL) 2.0 μL、Taq聚合酶0.3 μL (5 U/μL)。适宜的PCR循环程序为94℃预变性4 min，然后44个循环（94℃变性30 s，36℃退火30 s，72℃延伸2 min）后，72℃延伸10 min，最后16℃保存。改进的CTAB法能成功地提取红叶石楠基因组DNA，利用单因素试验设计所建立的RAPD反应体系，通过重复试验证明了该体系稳定可靠，可应用于红叶石楠遗传多样性、亲缘关系等方面的研究中，为红叶石楠在分子生物学研究奠定了基础。

关键词: 相关性, 相关性

Abstract:

The reference for the research on the diversity and resource of Photinia faseri and the identification of the relation of its varieties were provided through establish and study the stable system of RAPD reaction. The genomic DNA of Photinia faseri was successfully extracted with the improved CTAB method and studied the concentrations of Mg2+, dNTPs and primer, and the dosages of template DNA and TaqDNA polymerase which could influence the results of RAPD by the single factor design, established the suitable system of RAPD reaction for Photinia faseri. The results showed that the optimized RAPD reaction conditions were 10×PCR Buffer 2.5 μL, Mg2+ (25mmol/L) 2.0 μL, primer (20 μmol/L) 2.0 μL, dNTPs(2.5 mmol/L) 1.8 μL, DNA (49.5 μg/mL) 2.0 μL, Taq polymerase 0.3 μL (5 U/μL) in a total of 25 μL reaction system. The appropriate PCR procedure was pre-denaturing at 94℃ for 4 min, followed 44 cycles (denaturing at 94℃ for 30 s，annealing at 36℃ for 30 s, extending at 72℃ for 2 min), extending at 72℃ for 10 min and preserving at 16℃. The genomic DNA of Photinia faseri was successfully extracted with the improved CTAB method and the reaction system was established for the procedure of RAPD by the single factor design, through the repeat tests showed that this system was stable and reliable, and could be used in the genetic diversity and genetic relationship of Photinia faseri, it laid the foundation to research Photinia faseri in molecular biology.