花椒ISSR-PCR反应体系的建立与优化

采用单因子试验和正交设计2种方法对影响花椒ISSR-PCR反应体系的4个因素（TaqDNA聚合酶、Mg2＋、dNTP、引物）在3个水平上进行优化试验。选用L9（34）正交设计方案,从电泳结果中直观获得影响因素的最佳组合。单因子试验分别研究各影响因素不同含量对ISSR-PCR反应的影响情况,找出最佳反应水平。结果表明,适宜花椒ISSR-PCR反应的最佳体系为：20μL的反应体系中,Taq酶1.0U,Mg2＋2.0 mmol/L,dNTP 0.2mmol/L,引物1.0μmol/L,10×PCR buffer2.5μL,50 ng模板DNA,53℃退火,35个循环。

Establishment and optimization of ISSR-PCR system in Zanthoxylum bungeanum

Single factor test and orthogonal design methods were used to optimize ISSR-PCR amplification system in four factors（TaqDNA polymerase,Mg2＋,dNTP and primer） with three treatment levels on Zanthoxylum bungeanum.The best level of influence factors was obtained.The results showed that a suitable ISSR-PCR reaction system for Zanthoxylum bungeanum established was 20μL reaction system containing 1.0U TaqDNA polymerase,2.0mmol/L Mg2＋,0.1mmol/L dNTP,1.0μmol/L primer,10×PCR buffer 2.5μL and 50 ng DNA template.The annealing temperature was 53℃,cycle number was 35.