马褂木ISSR-PCR扩增体系的优化

摘要

[Objective]A stable ISSR reaction system suitable for Liriodendron chinese was established to provide technical support for ISSR diversity analysis of Liriodendron chinese population.[Method]In the single factor experiment,main factors of ISSR reaction system including MgCl2,dNTPs,primer concentration,Taq DNA polymerase,DNA template amount and annealing temperature were optimized and screened.[Result]Using kit to extract DNA in Liriodendron chinese leaves was better than using the improved CTAB method.The optimal PCR reaction system was:20 μL reaction volume containing 2.0 μL10× buffer,1.8 mmol/L MgC12,0.2 mmol/L dNTP,05 μmol/L primer,1U Taq polymerase,60 ng template DNA,and 59.1℃ annealing temperature.[Conclusion]Through the optimized ISSR-PCR reaction system,clear and stable stripes could be attained,thus it's proper for ISSR diversity analysis of Liriodendron chinese population.%[目的]建立稳定的马褂木ISSR-PCR扩增体系,为马褂木种群ISSR多样性分析提供技术支持.[方法]采用单因素试验对ISSR-PCR扩增体系中的MgC12、dNTPs、引物浓度、TaqDNA聚合酶、DNA模板用量和退火温度等主要因素进行筛选优化.[结果]采用试剂盒方法提取马褂木叶片DNA的效果优于改良CTAB法,最优PCR反应体系为:总体积20.0 μL中含有l0x Buffer 2.0 μL、MgC12 1.8 mmol/L、dNTPs 0.2 mmol/L、引物0.5 μmol/L、Taq DNA聚合酶1.00 U、DNA模板60 ng、退火温度59.1℃.[结论]优化后的马褂木ISSR-PCR扩增体系能够扩增获得清晰、稳定的条带,可满足马褂木种群ISSR多样性分析的要求.