十字花科蔬菜黑腐病菌(Xanthomonas campestris pv. campestris)AFLP分析体系的建立及优化

Construction and Optimization of an efficient System for Xanthomonas campestris pv. campestris AFLP Analysis

以英国华威大学收集的十字花科蔬菜黑腐病菌野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris)的6个生理小种菌株为材料,优化了该类菌种的AFLP分析体系包括DNA的提取、MseI/EcoRI酶切时间、扩增反应体系的组成及染色方法等步骤,建立了一套适于该菌种的AFLP分析体系。该体系中各最优因素为:酶切体系为50μL,模板DNA用量为600ng,酶切体系中MseI和EcoRI各加入5U,酶切温度为37℃,反应时间为4～6h;预扩增产物稀释10倍进行选择性扩增;检测方法为银染法,银染液中硝酸银含量为8g/L且染色时间是15min时效果最佳,该体系的建立为该类菌种的分子水平遗传多样性研究提供了技术支撑。

6 isolates of Xanthomonas campestris pv.campestris collected by University of Warwick was used as the materials.The affect factors of AFLP were optimized,which were DNA extraction method,digestion time of Mse I/EcoR I,compositions of PCR reaction system and staining method,and an efficient system for X.campestris pv.campestris AFLP analysis was established.The optimized factors were：the digestion system was 50 μL,the DNA template was 600 ng,the Mse I and EcoR I were both 5 U,the temperature was 37℃,reaction time was 4-6 h,pre-amplified products were diluted 10-fold for next selective amplification,the detection method was silver staining（AgNO 3 content was 8 g/L and staining time was 15 min or so）.The system was a powerful support for molecular level of genetic diversity in X.campestris pv.campestris.