葡萄5BB品种SRAP-PCR反应体系影响因素

Influence Factors of the SRAP-PCR Reaction System on Grape 5BB

为建立适合葡萄5BB品种的SRAP-PCR反应体系,利用正交设计对葡萄SRAP-PCR反应体系5种因素(Taq DNA聚合酶,Mg2+,模板DNA,dNTP,引物)4个水平进行优化。结果表明,各因素水平变化对PCR反应的影响从大到小顺序为:Mg2+,引物,dNTP,Taq DNA聚合酶,模板DNA;筛选出各因素的最佳水平,建立了葡萄5BB品种SRAP-PCR反应的最佳体系(20μL)为:Taq DNA聚合酶2U,Mg2+2.0mmol/L,模板DNA60ng,dNTP0.25mmol/L,引物0.10μmol/L。这一优化系统的建立为今后利用SRAP标记技术对葡萄进行相关研究提供了帮助。

To established a SRAP-PCR reaction system for the grape 5BB. In this paper,the orthogonal design was used to optimize SRAP-PCR amplification system on grape on four levels of five factors （Taq DNA polymerase,Mg2＋,DNA template,dNTPs,and primer,respectively）. The results showed the order of each factor in different levels affected on the result of PCR was Mg2 ＋ ,primer,dNTPs,Taq DNA polymerase and DNA template. A most suitable SRAP-PCR system for grape 5BB was established that was total 20 μL reaction system containing 2 UTaq DNA polymerase,2.0 mmol/L Mg2＋,60 ng DNA template,0.25 mmol/L dNTPs,and 0.10 μmol/L primer. This optimized system for SRAP marker would become one of the protocols for further research on grape.