木槿ISSR-PCR反应体系的建立及优化

Establishment and Optimization of Hibiscus syriacus ISSR-PCR Reaction System

建立木槿ISSR-PCR反应体系,为木槿种质资源的创新与鉴定提供理论基础。采用正交试验法,对影响PCR结果的Taq酶用量、Mg2+浓度、dNTPs浓度、模板DNA用量及引物浓度5个因素进行分析,采用单因素试验对退火温度进行筛选,并利用最佳体系对24个木槿品种进行扩增验证。结果表明,最优反应体系(25μL)中,含10×buffer(Mg^2+free)2.5μL、Taq酶0.75U、Mg^2+2.0mmol/L、dNTPs0.10mmol/L、模板DNA100ng、引物0.5μmol/L。PCR反应程序为94℃预变性2min;94℃变性30s,50℃退火30s,72℃延伸1min,34个循环;72℃延伸6min,4℃保温。建立的体系对24个木槿品种能够扩增出清晰稳定、无拖带、多态性高的条带。

The ISSR-PCR reaction system of Hibiscus syriacus was established to provide theoretical basis for the Hibiscus germplasm resources innovation and identification.Orthogonal test was used to analyze five factors affecting the results of PCR: Taq enzyme dosage,Mg 2+,dNTPs concentration,template DNA dosage and primer concentration.Single factor design was used to screen annealing temperature,and 24 Hibiscus species were amplified and validated by this system.The results showed that the optimal reaction system(25 μL) contained 10×buffer (Mg^2+ free) 2.5 μL, Taq enzyme 0.75 U,Mg^2+ 2.0 mmol/L,dNTPs 0.10 mmol/L ,template DNA 100 ng,and primers 0.5 μmol/L.The procedure of PCR was 94 ℃ 2 min;94 ℃ 30 s,50 ℃ 30 s,72 ℃ 1 min,34 cycles;72 ℃ 6 min,4 ℃ for holding.Using this system,24 Hibiscus species could amplify clear and stable bands with high polymorphism without towing.