益智ISSR-PCR反应体系建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Alpinia oxyphylla Miq

目的:获得适合的益智ISSR-PCR扩增反应体系。方法:利用正交设计L16(45)和单因素试验对益智ISSR-PCR反应体系的5因素(Taq酶,Mg2+,模板DNA,dNTPs,引物)在4个水平上进行优化试验,将二者所得结果进行综合比较分析。结果:最终获得益智ISSR-PCR反应的最优体系(20μl)为:模板DNA100ng,Mg2+3.0mmol/L,引物0.8μmol/L,dNTPs0.25mmol/L,TaqDNA聚合酶1.0U。最后,对益智ISSR-PCR最佳反应体系进行梯度退火,得到最佳退火温度为50℃。结论:这一优化系统的建立为今后利用ISSR标记技术研究分析益智遗传多样性奠定基础。

Objective： To obtain a suitable ISSR-PCR reaction system of Alpinia axyphylla Miq.. Method： It was used to optimize ISSR-PCR amplification system on Alpinia oxyphylla Miq. in four levels of five factors （Taq DNA polymerase, Mg^2＋ , DNA template, dNTPs, primer） by single factor test and orthogonal design in this study, and then, the two methods gained different results. Result： Through the deep analyze and compare, a suitable ISSR-PCR reaction system was established, namely 20μl reaction system containing 100ng DNA template, 3.0 mmol/L Mg^2＋, 0.8μmol/L primer, 0.25mmol/L dNTPs, 1.0U Taq DNA polymerase. The optimal annealing temperature was 50℃ for ISSR-PCR reaction which was proposed by gradient PCR. Conclusion： The result result a foundation for the analysis of genetic diversity of Alpinia oxyphylla Miq..