正交设计优化紫萼藓科植物的ISSR-PCR反应体系

目的:建立紫萼藓科(Grimmiaceae)植物ISSR-PCR反应的最佳体系。方法:通过L16(45)正交试验,研究了dNTP浓度、镁离子浓度、模板DNA浓度、引物浓度、Taq DNA聚合酶浓度这5个因素在4个水平上对ISSR-PCR的影响。结果:建立了紫萼藓科ISSR-PCR反应的最佳反应体系,其中dNTPs浓度为0.8mmol/L、Mg2+浓度为3.0mmol/L、模板为15ng、引物浓度为1.4μmol/L、Taq酶量2U,总体积为25μl。反应程序为:扩增程序为:94℃预变性5min;94℃变性30s,48～52℃退火1min,72℃延伸2min,共40个循环;72℃延伸10min;4℃保存。采用引物UBC812等均能够得到有效扩增。结论:该优化体系的建立为下一步对紫萼藓进行ISSR分子标记奠定了基础。

Study on Optimization for ISSR- PCR Reaction System of Grimmiaceae using Orthogonal Design

Objective:Develop the most suitable ISSR-PCR system for Grimmiaceae.Method:The effects of various factors on ISSR-PCR reaction,such as concentration of dNTPs,Mg2+ and DNA template,primers and Taq DNA polymerase,were investigated to optimize the ISSR-PCR reaction system.Result:The most suitable ISSR-PCR system for Grimmiaceae was established,namely 25μl reaction system containing 3.0mmo1/ L Mg2+,0.8 mmo1/L dNTPs,10ng DNA template,1.4μmo1/L primer,and 2 U Taq DNA polymerase.Amplification procedure by PCR was pre-denaturation at 94℃ for 5 min,40cycles of denaturation at 94℃ for 30s,annealing at 48-52℃ for 1 min and extension at 72℃ for2min,and a anal extension at 72℃for 10min.Using the optimized reaction,Grimmiaceae materials were easy amplified with primer UBC812.Conclusion:The optimized system laid the groundwork for ISSR molecular analysis of Grimmiaceae.