石斛属部分植物SRAP-PCR反应体系的建立与优化

Establishment and Optimization of SRAP-PCR Reaction System inSome Species of Dendrobium

建立石斛属(Dendrobium)植物SRAP-PCR反应体系,为石斛的品种鉴定与分类提供理论和技术基础。采用单因素及正交试验对反应体系中的各因子进行优化,确定最优退火温度,并筛选有效引物。结果表明:适用于石斛属植物SRAP最佳的反应体系为:Mg^2+2.5 mmol/L,dNTPs 0.25 mmol/L,Taq DNA聚合酶1.25 U,引物0.4μmol/L,模板DNA 30 ng,10×PCR buffer 2.5μL,其余用灭菌后的ddH2O补足至25μL。最优扩增程序为:94℃预变性5 min;94℃变性1 min,36℃复性1 min,72℃延伸1 min,5次循环;94℃变性1 min,根据不同引物的不同退火温度复性1 min,72℃延伸1 min,30次循环;最终72℃延伸7 min,保存于4℃恒温箱中。应用所建立的优化反应体系成功地筛选出15条多态性较高的引物。

The purpose of this study was to establish an optimized SRAP system for some species of Dendrobium.Single factor and orthogonal test designs were applied to optimize five factors,Mg^2+,dNTPs,Taq DNA polymerase,primer and template DNA concentration,in the SRAP amplification system.Due to different species,the concentrations of Mg^2+,dNTPs,Taq DNA polymerase,primers and template DNA in the SRAP reaction system were different,and the annealing temperature and the number of cycles in the amplification program also affected the experimental results,and finally the selection and establishment of Dendrobium SRAP optimal reaction system and amplification procedure was:Mg2+2.5 mmol/L,dNTPs 0.25 mmol/L,Taq DNA polymerase 1.25 U,primer 0.4μmol/L,template DNA 30 ng,and the rest were filled with ddH2O.A total of 25μL of amplification system was formed.The optimal amplification procedure was:pre-denaturation for 5 min at 94℃,denaturation for 1 min at 94℃,renaturation for 1 min at 36℃,extension for 1 min at 72℃,5 cycles,denaturation at 94℃for 1 min,depending on the annealing temperature of the different primers refolding for 1 min,extending at 72℃for 1 min,30 cycles,and finally extending at 72℃for 7 min,stored at 4℃constant temperature.A total of 15 primers with high polymorphism were successfully screened out by the optimized reaction system.