蒲公英ISSR-PCR反应体系及扩增程序的建立与优化

目的:优化蒲公英ISSR-PCR反应体系及扩增程序；筛选适合的引物,并确定最佳退火温度.方法:采用CTAB法提取蒲公英叶片的DNA,利用正交试验设计,从Taq酶MIX、模板DNA、引物3种因素2个水平,对蒲公英ISSR-PCR反应体系进行考察；采用单因素实验对其扩增程序进行优化.结果:确立了适合于蒲公英的最佳ISSR-PCR反应方法,即在25 μL反应体系中,内含Taq酶MIX 15 μL(1.25 UTaq DNA聚合酶,1.8 mmol·L-1 Mg2+,dNTPs各0.24 mmol·L-1),0.3 μmol·L-1引物,5 ng模板.在此基础上,从50条引物中筛选出16条扩增稳定、多态性丰富的ISSR引物,并通过梯度PCR试验,确定引物最佳退火温度.结论:采用单因子试验和正交设计方法可以快速建立ISSR-PCR反应体系,经过验证,证明该体系稳定可靠,可用于蒲公英遗传分析.

Optimization for Reaction System and PCR program of Taraxacum mongolicum ISSR-PCR

Objective: To establish and optimize ISSR-PCR reaction system for Taraxacum mongolicum.Method: Based on the analysis of orthogonal design test,an orthogonal design was used to optimize the ISSR-PCR amplification system on T.mongolicum by three factors(Taq polymerase Mix,DNA template and primer) at two concentration levels,respectively.PCR program was optimized by single factor experiments.Result: The suitable PCR reaction system contained 15 μL Taq polymerase MIX(1.25 U Taq DNA polymerase,1.8 mmol·L-1 Mg2+,dNTPs 0.24 mmol·L-1),0.3 μmol·L-1 primer and 5 ng template DNA in total 25 μL reaction solution.On this basis,16 primers were screened with stable amplification and rich polymorphism from 50 ISSR primers.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.Conclusion: It is a way to establisd the ISSR-PCR system for orthogonal design combining with single factor test.This optimized ISSR reaction system would provide the basis for the genetic analysis of T.mongolicum.