黑籽南瓜ISSR-PCR反应体系的正交设计优化

Optimization for ISSR-PCR Reaction System of Cucurbita ficifolia Bouche by Orthogonal Design

以云南个旧黑籽南瓜叶片基因组DNA为模板,固定反应程序,采用L_(16)(4~5)正交试验设计的方法,对影响ISSR-PCR反应的五因素(dNTPs,Mg^(2+),引物,Taq DNA聚合酶,模板DNA)在四个水平上进行优化试验。研究建立起了适于云南黑籽南瓜ISSR-PCR的最佳反应体系:25μl反应体系中含10×buffer 2.5μl、0.15mmol/L dNTPs、2.5mmol/L Mg2+、0.40μmol/L引物、1.5U Taq DNA聚合酶、DNA模板15ng。扩增程序为:95℃预变性5min,94℃变性45s,52℃退火45s,72℃延伸90s,进行35个循环,最后72℃延伸7min。该优化体系的建立为今后用ISSR标记技术进行黑籽南瓜种质资源的分类鉴定和遗传多样性研究奠定了基础。

In this study,the orthogonal design was applied to optimize ISSR-PCR amplification system on Cucurbita ficifolia in four levels of five factors(dNTPs,Mg^(2+),primers,Taq DNA polymerase and DNA template).The most suitable ISSR-PCR system for Cucurbita ficifolia was established that is total 25μL reaction system containing 1.5U Taq DNA polymerase,2.5mmol/L Mg^(2+),15ng DNA template,0.15 mmol/L dNTPs,and 0.40μmol/L primer.The PCR amplification procedure used in this study was as follow:predenature at 95℃for 5min,then 94℃for 45s,52℃for 45s,72℃for 90s,for 35 cycles,finally extended at 72℃for 7min.This optimized system would play an important role in further research of classification,identification and genetic diversity on Cucurbita ficifolia by ISSR molecular marker.