摘要： 以牡丹基因组DNA为模板,采用L16(45)正交试验设计对影响目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)的五因素(Taq酶用量、Mg2+浓度、模板DNA用量、dNTPs浓度和引物浓度)进行优化试验,建立了优化的牡丹SCoT-PCR反应体系:Mg2+2.50 mmol/L、dNTPs 0.25 mmol/L、引物0.60μmol/L、Taq DNA聚合酶0.50 U、模板DNA 1.00 ng/μL,1×PCR-Buffer,总体积20.00μL。比较各因素对扩增反应的结果,其中以Mg2+浓度的影响最大,DNA模板用量的影响最小。运用牡丹17个品种验证了该体系稳定可靠,并从36个SCoT引物中筛选出扩增条带清晰、多态性丰富的24个引物。这一体系的建立及多态性引物的筛选为今后利用SCoT标记技术对牡丹进行相关研究提供了科学依据。

关键词: 牡丹, SCoT-PCR, 反应体系, 正交设计, 引物筛选

Abstract: The orthogonal design table of L16(45)was used to optimize the five factors(Taq DNA polymerase,Mg2+,DNA template,dNTPs and primer concentrations)of SCoT-PCR(start codon targeted polymorphism)system in tree peony.The results showed that the optimized system was as follows:a total volume of 20 μL including 1.0 ng/μL DNA template,0.50 U Taq polymerase,0.60 μmol/L primer,2.50 mmol/L Mg2+,0.25 mmol/L dNTPs and 1 PCR Buffer.Each factor had different effect on the results.The concentration of Mg2+ was the key factor affecting the SCoT-PCR system.The optimized SCoT-PCR system was tested on seventeen peony genus,and the result was stable and reliable.From the 36 primer combinations tested,Twenty-four were selected with clear band patterns and abundant polymorphism.The optimized SCoT-PCR system and polymorphism primmer combinations provided the basis for further research on Paeonia suffruticosa.

Key words: SCoT-PCR, Reaction system, Orthognal design, Primers screening

中图分类号: 

• S685.11