摘要
目的:建立杜仲简单重复序列-聚合酶链式反应(SSR-PCR)的优化反应体系,为杜仲种质间的遗传差异及亲缘关系分析提供参考。方法:选择Taq DNA聚合酶,模板DNA,Mg2+,引物及d NTP浓度为考察因素,利用单因素试验和L16(45)正交试验优选杜仲的SSR-PCR反应体系,运用SPSS软件对试验结果进行分析。结果:各因素水平变化对反应体系影响顺序依次为Mg2+>Taq DNA聚合酶>引物>d NTP>模板DNA。优化的PCR反应体系为10×PCR缓冲液2μL,Taq DNA聚合酶0.5U,Mg2+浓度1.25 mmol·L-1,d NTP浓度0.2 mmol·L-1,引物浓度0.3μmol·L-1,模板DNA用量60 ng,定容至20μL。结论:利用该体系进行扩增,所得谱带明亮清晰,稳定性和重复性良好,适用于杜仲不同居群间亲缘关系和遗传多样性分析。
Objective：To optimize simple sequence repeat-polymerase chain reaction （SSR-PCR） system for providing a reference to genetic diversity and phylogenetic analysis among germplasms of Eucommiae Cortex.Method：Orthogonal design and single factor tests were adopted to optimize SSR-PCR system of Eucommiae Cortex with Taq DNA polymerase,template DNA,Mg2＋,primers and dNTP concentration as factors,test data were analyzed by SPSS software.Result：Effects of each factors were in the order of Mg^2＋ 〉 Taq DNA polymerase 〉 primers 〉 dNTP 〉 DNA template.Optimal reaction system was as follows：10 × PCR buffer of 2 μL,Taq DNA polymerase of 0.5 U,Mg^2＋ of 1.25 mmol ·L^-1,dNTP concentration of 0.2 mmol ·L^-1,primers of 0.3 μmol·L^-1 and DNA template of 60 ng in 20 μL reaction solution.Conclusion：Bands are bright and clear with good stability and reproducibility by amplification of this optimized SSR-PCR system,it is suitable for genetic diversity and phylogenetic analysis among germplasms of Eucommiae Cortex.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2015年第2期1-6,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家科技支撑计划项目(2011BAI01B08)
关键词
杜仲
微卫星标记
聚合酶链式反应
反应体系
引物
Eucommiae Cortex
microsatellite markers
polymerase chain reaction
reaction system
primer
