摘要： 通过正交试验设计,对油菜黑胫病菌ISSR-PCR反应体系的各影响因素进行研究,即从Taq酶、Mg²⁺、dNTPs、引物以及模板浓度5因素4水平进行优化,筛选并建立了适合油菜黑胫病菌ISSR分子标记的最佳反应体系,即在25μL反应体系中包含Taq DNA酶1 U,Mg²⁺2.0 mmol/L,dNTPs 0.2 mmol/L,引物0.6μmol/L,模板40 ng以及2.5μL 10×Buffer。并以扩增条带丰富、清晰、稳定性强为原则,从所查找的110条引物中筛选出24条适于油菜黑胫病菌ISSR-PCR扩增的引物,同时对各引物的最佳退火温度进行了筛选确定,经对20株病原菌的稳定性检测,证实该优化体系可用于研究该病原菌的遗传多样性。

关键词: 油菜黑胫病原菌, ISSR-PCR, 体系优化

Abstract: An orthogonal design was used to optimize ISSR reaction system for Leptosphaeria biglobosa on oil- seed rape in five factors(Taq polymerase，Mg^{2 +}，dNTPs and the concentration of primer or template) at four levels． A suitable ISSR- PCR reaction system for Leptosphaeria biglobosa was established．The optimized reaction system consists of 1 U Taq DNA polymerase，2． 0 mmol /L Mg^{2 +}，0． 2 mmol /L dNTPs，0． 6 mol /L primer，40 ng template DNA，and 2． 5 L 10 × Buffer．24 primers were selected for the abundant and clear band from 110 random primers and their optimal annealing temperature was decided．This optimization of ISSR- PCR reaction system can be used to study the genetic diversity of the pathogen which was confirmed by testing on the stability of 20 strains of Leptospha- eria biglobosa．

Key words: Leptosphaeria biglobosa, ISSR- PCR, Optimization reaction system

中图分类号: 

• S435.654