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为有效利用SSR-PCR技术对药用植物苦参进行遗传多样性分析,采用正交设计L16(45)对影响苦参SSRPCR体系的5个因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化,PCR结果用DPS数据处理软件分析,筛选出各因素的最佳水平,建立适宜苦参SSR-PCR的反应体系和扩增程序,并选用内蒙古苦参对该体系进行稳定性验证。结果表明:在10μL的苦参SSR-PCR体系中,模板DNA的用量为20.0ng,Taq DNA聚合酶的用量为0.2~0.6U,Mg2+的浓度为2.5mmol/L,dNTPs浓度为0.1mmol/L,引物的浓度为0.6μmol/L。扩增程序为:94℃预变性3min;94℃变性1min,56℃退火45s,72℃1min,35个循环;72℃延伸10min。4℃保存。选用12份苦参DNA对该体系进行稳定性验证,该体系具有较高的扩增稳定性,可用于药用植物苦参SSR标记的研究。
In order to effectively analyze the genetic diversity of Sophora flavescens by using SSR-PCR technique, five factors affecting the Sophora flavescens SSRPCR system (Taq enzyme, Mg2 +, template DNA, dNTP, primer) The PCR results were analyzed by DPS data processing software, the best level of each factor was screened out, and the reaction system and amplification program suitable for Sophora flavescens SSR-PCR were established. The system was selected by Inner Mongolia Sophora flavescens Stability verification. The results showed that the amount of template DNA was 20.0ng, the amount of Taq DNA polymerase was 0.2 ~ 0.6U, the concentration of Mg2 + was 2.5mmol / L, the concentration of dNTPs was 0.1mmol / L , Primer concentration of 0.6μmol / L. Amplification procedures: 94 ℃ denaturation 3min; 94 ℃ denaturation 1min, 56 ℃ annealing 45s, 72 ℃ 1min, 35 cycles; 72 ℃ extended 10min. 4 ℃ save. The stability of this system was verified by using 12 parts of Sophora flavescens DNA. The system has high stability and can be used for the study of SSR markers of Sophora flavescens in medicinal plants.