杜鹃花SCoT-PCR反应体系的优化及引物筛选

Optimization of SCoT-PCR Reaction System and Primer Screening in Rhododendron

本试验以马银花(R.ovatum)DNA为模板,采用L16(45)正交试验对目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)体系中5因素(Taq聚合酶用量,Mg^(2+)浓度,模板DNA用量,dNTPs浓度,引物浓度)进行了优化,建立了杜鹃花植物SCoT-PCR反应体系。结果表明,在20μL的反应体系中,模板DNA 1.00 ng/μL,Mg^(2+)为3.00 mmol/L,dNTPs为0.25 mmol/L,引物为0.50μmol/L,Taq DNA聚合酶为2.00 U。比较了各因素对反应体系的影响,发现引物物浓度的差异对体系影响显著,各因素影响的先后顺序为引物>Taq DNA聚合酶>dNTPs>Mg^(2+)>模板DNA。并运用了杜鹃属的4个不同种对最优体系进行了稳定性验证,选择了10个通用性的引物进行筛选,发现10个引物均能扩增出条带,其中有6个引物能够得到清晰可辨、多态性丰富的条带。该体系的建立将为杜鹃花遗传多样性、差异基因的表达分析,分子辅助育种、遗传图谱构建等研究提供技术支持。

In this experiment, the R. ovatum DNA was used as template, and the orthogonal experiment of L16（45）was designed to optimize the five factors（Taq DNA polymerase, Mg^2＋, template DNA, dNTPs and primer concentrations） of SCoT-PCR（start codon targeted polymorphism） system in Rhododendron plants. The results showed that the total volume of 20 μL system included template DNA 1.00 ng/μL, Mg^2＋3.00 mmol/L, dNTPs 0.25 mmol/L,primer 0.50 μmol/L and Taq DNA polymerase 2.00 U. The comparison of the effects of various factors on the reaction system indicated that different concentrations of primers had significantly different effect on reaction system, and the order of the effective degree was primer Taq DNA polymerase dNTPsMg^2＋template DNA.4 different species of Rhododendron were used to verify the stability of the optimal system, and 10 general-purpose primers were selected to screen. It was found that 10 primers all could amplify bands, in which 6 primers could amplify bands with clear and polymorphic strips. The establishment of the system could provide technical support for the study of Rhododendron genetic diversity, gene expression profile change, molecular assisted breeding,genetic map construction and other aspects.