玉米弯孢病病原菌ISSR-PCR反应体系的建立及优化

摘要

为了对玉米弯孢病菌的鉴定、区域发生关系以及遗传多样性等进行研究,采用单因素试验优化玉米弯孢叶斑病菌ISSR-PCR反应体系中模板DNA、引物、dNTPs、Taq酶、Mg2+的用量,并确定每条引物的最佳退火温度.结果表明,最佳反应体系为:模板DNA 1.6 μL(25 ng/ μL)、引物1.4 μL(5 μmol/L)、dNTPs 0.25 μL(2.5 mmol/L)、Taq酶0.4 μL(2.5 U/μL)、Mg2+0.8 μL(25 mmol/L)、10×Taq Buffer 2 μL、ddH2O 13.55 μL.在该体系下选用3个玉米弯孢病菌的全基因组DNA,分别对52条ISSR引物进行筛选,筛选出16条多态性高、扩增稳定的ISSR引物.玉米弯孢病菌ISSR-PCR反应体系的建立为利用ISSR分子标记技术对该病原菌进行遗传分析奠定了基础.%To study the identification,regional occurrence correlation and genetic diversity of Maize curvularia pathogens,single factor test was employed to optimize the quantities of the template DNA,primer,dNTPs,Taq poly-merase and Mg2+used in ISSR-PCR,as well as the determination of the optimum annealing temperature of each prim-er.The optimal reaction system was established in which template DNA,primer,dNTPs,Taq polymerase,Mg2+×10 Taq Buffer and ddH2O were 1. 6 μL(25 ng/μL),1. 4 μL (5 μmol/L),0. 25 μL (2. 5 mmol/L),0.4 μL (2.5 U/μL),0.8 μL (25 mmol/L),2 μL,13.55 μL respectively.52 primers for ISSR were screened with optimized system using genomic DNA of 3 Curvularia strains,and 16 primers with stable amplification and rich polymorphism were obtained.The study would provide the basis for the genetic analysis of maize Curvularia pathogens.