番茄叶霉病菌ISSR-PCR体系的建立

Establishment of ISSR-PCR System for Passalora fulva

以番茄叶霉病菌(Passalora fulva)基因组DNA为模板,采用单因素试验和正交设计试验对该菌ISSR-PCR体系中的一些重要参数(Mg2+、dNTPs、引物、模板DNA、TaqDNA聚合酶、缓冲液、循环次数)和引物进行筛选和优化,并对退火温度进行了梯度优化,建立了番茄叶霉病菌ISSR-PCR的最佳反应体系(20μL):Mg2+1.5 mmol/L,dNTPs 0.4 mmol/L,引物1.5μmol/L,模板DNA45 ng,TaqDNA聚合酶1.0 U,1倍的PCR缓冲液,循环40次,退火温度50℃。

The single factor test and orthogonal design test were used to optimize some important parameters of ISSR-PCR system（Mg2＋,dNTPs,primer,DNA template,Taq DNA polymerase,Buffer and cycles） and to screen the primers based on genome DNA of Passalora fulva.The optimal annealing temperature of ISSRPCR was optimized by gradient PCR.A suitable ISSR-PCR system（20 μL） for P.fulva was established as follows： 1.5 mmol/L Mg^2＋,0.4 mmol/L dNTPs,1.5 μmol/L primer,45 ng DNA templates,1.5 U TaqDNA polymerase,1 times of PCR-Buffer,the appropriate cycles of ISSR-PCR are 40 and the annealing temperature is 50℃.