裸仁美洲南瓜ISSR-PCR反应体系的正交优化

Optimization for ISSR-PCR Reaction System in Hull-less Cucurbrita pepo Using Orthogonal Design

利用正交试验设计L16(44)对美洲南瓜ISSR-PCR反应体系的4因素(Mg2+,dNTPs,引物,Taq酶)在4个水平上进行优化试验,PCR结果用SPSS16.0统计软件分析,结果表明,各因素的不同水平对PCR反应结果都有显著影响,其中引物浓度的影响最大;本研究还筛选出各反应因素的最佳水平,建立美洲南瓜IS-SR-PCR反应的最佳体系(25μL)为:0.75 U Taq酶,2.0 mmol.L-1Mg2+,300μmol.L-1dNTPs,10μmol.L-1引物和DNA模板30 ng,2.5μL 10×buffer。

An orthogonal design combining with single factor experiment was applied to optimize ISSR（inter simple sequence repeat）-PCR（polymerase chain reaction） amplification system in Cucurbrita pepo.Four main reaction system elements（Mg2＋,Taq DNA polymerase,dNTPs,and primer） in ISSR-PCR were tested.The result was analyzed by software SPSS16.0,and showed that different levels of Mg2＋,Taq DNA polymerase,dNTPs,and primer had an significant effect on the PCR reaction result and the concentration of primer was the most effective factor;the modified ISSR-PCR system for Cucurbrita pepo was established,i.e.2.0 mmol·L-1 Mg2＋,300 μmol·L-1 dNTPs,10 μmol·L-1 primer,0.75 U Taq DNA polymerase,30 ng DNA template and 2.5 μL 10 ×buffer in the total volume of 25 μL.