桔梗SRAP反应体系的优化

Optimization of SRAP Reaction System for Platycodon grandiflorus

目的:建立适合桔梗的比较稳定的SRAP反应体系,用于桔梗遗传多样性分析。方法:用改进的CTAB法提取桔梗叶片的总DNA,通过对不同镁离子浓度、dNTP浓度、模板DNA含量、引物浓度、DNA聚合酶量条件下的SRAP扩增反应的效果。结果:桔梗SRAP扩增反应的最佳体系:模板DNA 20ng,引物0.8μmol/L,dNTP150μmol/L,MgCl22.0mmol/L,TaqDNA聚合酶1unit,10×Buffer2.0μL;反应程序为94℃预变性5min;94℃变性1min,35℃退火1min,72℃延伸1min,5个循环;94℃变性1min,50℃退火1min,72℃延伸1min,35个循环;最后72℃延伸5min,4℃保存。结论:按此优化的SRAP条件进行实验,重现性良好,可用于桔梗遗传多样性分析。

Objective：The SRAP reaction systerm has been established for the analysis of genetic diversity Platycodon grandiflorus.Method：The total DNA in young leaves of Platycodon grandiflorus was successfully extracted by using the developed CTAB method.The optimum reaction system of SRAP for the P.grandiflorus was established by SRAP analysis of the different Mg2 ＋,DNA templates,primers and DNA polymerase.Result：The most optimal reaction system of SRAP is：20 ng template DNA,0.8 μmol/L primer,dNTPconcentration 150μL,MgCl2 2.0 mmol,1 U Taq polymerase,10 × Taq polymerase buffer at 2.0μL in the 20μL reaction volume.The optimum SRAP reaction procedure was：predenaturation at 94℃ for 5 min followed by 5 cycles of denaturation at 94℃ for 1 min,anneal at 35℃ for 1 min and extension at 72℃ for 1min;then 35cycles of 94℃ for 1 min,50℃ for 1 min and 72℃ for 1min;and a final extension at 72℃ for 5min;kept at 4℃.Conclusion：A high reproducibility was obtained with the optimized experiment condition.And it can be used to analysis of genetic diversit for Platycodon grandiflorus.