甜菜TRAP-PCR反应体系的建立及引物开发策略

Establishment of TRAP-PCR Reaction System and Development Strategy of Primers for Beet

以糖用甜菜为材料,利用单因素实验探讨甜菜TRAP-PCR反应体系中d NTPs、引物、模板以及DNA聚合酶等4个因素的浓度变化对扩增结果的影响,最终建立糖用甜菜的最佳TRAP-PCR反应体系。结果表明,在20μL反应体系中,甜菜TRAP-PCR最优反应体系包含2.0μL的10×PCR buffer(含Mg2+)、10 ng的模板DNA、0.75 U的Taqase、10μmol/L固定引物及随机引物各0.8μL以及0.5μL的d NTPs(2.5 mmol/L each)。并建立了一种快速开发甜菜TRAP-PCR引物的方法,利用优化后的程序对15份糖甜菜品种进行扩增。结果表明,该体系扩增结果稳定、条带清晰,部分引物多态性丰富,可用于糖用甜菜品种指纹图谱的构建以及其他分子生物学领域的研究。

With sugar beet as the material, the influence of concentration changes of the four factors of d NTPs,primer, template and DNA polymerase in the beet TRAP-PCR reaction system on the amplification results wasdiscussed with single-factor experiment, and finally the optimal TRAP-PCR reaction system of sugar beet wasestablished. The results showed that in the 20 μL reaction system, the beet TRAP- PCR optimal reactionsystem contained 2.0 μL of 10 × PCR buffer（including Mg2 ＋）, 10 ng of template DNA, 0.75 U of Taqase,respectively 0.8 μL of 10 μmol/L fixed primer and random primer, and 0.5 μL of d NTPs（2.5 mmol/L each）. Inaddition, a method for rapid development of beet TRAP-PCR primer was proposed, 15 varieties of sugar beerwere amplified with the optimized program. The results show that the system has stable amplification results,clear bans and abundant polymorphism of partial primer, and can be used for the construction of sugar beetvariety fingerprint and research in other fields of molecular biology.