战骨ISSR-PCR反应条件的筛选与优化

Establishment and Optimization of the ISSR Reaction System for Premna fulva

采用正交和单因素试验设计方法,研究Mg2+、dNTP、Taq DNA聚合酶、引物、模板DNA、退火温度及循环次数对IS-SR-PCR扩增效果的影响,建立并优化战骨ISSR-PCR反应体系和程序,即25μl的体系中含Mg2+2.0 mmol/L,dNTP 0.2mmol/L,Taq DNA聚合酶1.0 U,引物0.8μmol/L,模板DNA 50 ng,10×Buffer 2.5μl。退火温度、循环次数分别为52℃、45次。结论:结合正交和单因素试验可快速建立ISSR-PCR反应体系。该体系稳定、可靠,可用于战骨遗传多样性分析。

Objective：Through orthoronal design and single factor test to establish ISSR-PCR program for Premna fulva and lay foundation for its genetic diversity anlysis.Method：Seven factors were test by orthogonal design and single factor test,including Mg2＋,dNTP,Taq DNA polymerase,primer,template DNA,annealing temperature,cycles.Results：ISSR-PCR reaction system of Premna fulva contained 2.0 mmol/L Mg2＋,0.2 mmol/L dNTP,1.0 U Taq DNA polymerase,0.8 μmol/L primer,50 ng template DNA.Primer annealing at 52 ℃,cycles was 45.The genetic diversity of Premna Fulva Crab population was analyzed by ISSR-PCR.It was a way to establish the ISSR-PCR system by orthogonal design and single factor test.The optimized reaction system was suitable for the study of genetic diversity.