卷丹百合RAPD-PCR反应体系的单因素逐项优化研究

Study on Gradual Optimization of RAPD-PCR Reaction System with Each Factor Design in Lilium lancifolium Thunb

[目的]探索建立卷丹百合RAPD-PCR反应的最适体系。[方法]用CTAB法提取卷丹百合的基因组DNA,通过采用单因素逐项优化的方法,对影响卷丹百合RAPD-PCR扩增的反应组分浓度进行优化。[结果]反应结果显示RAPD对模板的适应性很大;体系中Mg2＋在1.5～3.0μl都能产生较清晰的RAPD带;dNTP较适合的浓度范围为0.4～1.6μl;引物较适合的浓度范围为1.5～3.0μl;体系中TaqDNA聚合酶在0.1～0.8μl的浓度范围内均有良好的扩增效果。根据上述结果,卷丹百合RAPD-PCR反应最适的反应体系为：20μl的反应体系中含基因组DNA 2.0μl,Mg2＋2.0μl,dNTP 1.2μl,引物2.0μl,TaqDNA聚合酶0.2μl。[结论]该研究为在分子水平研究百合种质资源的分类及亲缘关系奠定了基础。

[Objective]The aim was to explore the establishment on the optimum system for the RAPD-PCR reaction of Lilium lancifolium.[Method] The genomic DNA of L.lancifolium was extracted with the method of CTAB and the concn.of components effecting the RAPD-PCR amplification reaction of L.lancifolium was optimized by the method of the gradual optimization of single factor.[Result]The reaction result showed that RAPD had large adaptability on template;the system with the Mg2＋ concn.between 1.5 μl and 3.0 μl could produce clearer RAPD bands;the suitable concn.range of dNTP was 0.4～1.6 μl;the suitable concn.range of primer was 1.5～3.0 μl;the system with the Taq DNA polymerase concn.between 0.1 μl and 0.8 μl had better amplification effect.According to the above result,the suitable reaction system of the RAPD-PCR reaction of L.lancifolium was that 2.0 μl genomic DNA,2.0 μl Mg2＋,1.2 μl dNTP,2.0 μl primer and 0.2 μl Taq DNA polymerase in the 20 μl reaction system.[Conclusion]The research laid the foundation for the study of the classification and the genetic relationship of the Lily germplasm conservation at the molecular level.