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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (28): 220-226.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

茶树TRAP反应体系的建立及正交设计优化

张丽群 成浩 王丽鸳 韦康   

  • 收稿日期:2012-05-02 修回日期:2012-06-05 出版日期:2012-10-05 发布日期:2012-10-05

Establishment of TRAP Reaction System for Camellia sinensis and Optimization Using Orthogonal Design

  • Received:2012-05-02 Revised:2012-06-05 Online:2012-10-05 Published:2012-10-05

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摘要：

TRAP标记技术因操作简单、重复性好、效率高等特点得到广泛应用，但该技术在茶树中的研究报道较少，且茶树TRAP-PCR体系优化尚未见报道。笔者采用正交设计的方法，对茶树TRAP-PCR反应中Mg2+、Taq酶、dNTP、固定引物、随机引物5个因素进行了优化研究，建立了茶树TRAP-PCR最佳反应体系。该20 μL反应体系含有2 μL 10×PCR Buffer (Mg2+ free)，50 ng模板DNA，1.75 mmo/L Mg2+，0.50 U Taq酶，0.20 mmol/L dNTP，0.20 mmol/L随机引物和0.20 mmol/L固定引物。根据优化结果，利用最佳反应体系，选用16个茶树品种对TRAP标记多态性进行了初步研究。

关键词: 药效试验, 药效试验

Abstract:

TRAP markers is widely used for its simplicity, repeatability and high efficiency, however, the technology is scarcely used in the study of tea plant, and tea tree TRAP-PCR system optimization has not been reported. Orthogonal design was applied for optimizing five factors (Mg2+, Taq DNA polymerase, dNTP, fixed primer, arbitrary primer) in the TRAP-PCR amplification system at four levels respectively. The best level of each factor were selected, a suitable TRAP-PCR reaction system was established, namely 20 μL reaction system containing 2μL 10×PCR Buffer(Mg2+ free)，50 ng DNA，1.75 mmo/L Mg2+，0.50 U Taq DNA polymerase, 0.20 mmol/L dNTP, 0.20 mmol/L arbitrary primer and 0.2 mmol/L fixed primer. TRAP polymorphism was studied using sixteen tea germplasm.