烟草赤星病菌ISSR-PCR反应体系的优化

Optimization of ISSR-PCR Reaction System for Alternaria alternate in Tobacco

采用改良的CTAB法提取烟草赤星病菌丝基因组DNA,通过正交与单因素变量设计试验,对烟草赤星病菌ISSR-PCR反应体系的各个影响因素进行优化,建立了适合烟草赤星病菌ISSR分子标记的最佳反应体系,即在25μL反应体系中包含10×buffer 2.5μL,模板20 ng,Mg2+浓度2.0 mmol/L,引物浓度0.8μmol/L,dNTPs浓度0.2 mmol/L,Taq DNA酶0.75 U。利用所建立的体系对采自云南昆明、德宏、楚雄、曲靖、玉溪等地8份材料进行检验,其结果表明优化后的体系适合烟草赤星病菌的ISSR-PCR反应。

The genomic DNA was extracted from Alternaria alternata mycelium infecting Tobacco using modified CTAB method.Using the genomic DNA of A.alternate,the factors influencing ISSR-PCR system were optimized with the orthogonal and single factor variable design.The results show that the system,which contains 2.5 μL 10 × buffer,20ng template,2.0 mmol/L Mg2+,0.8 μmol/L primer,0.2mmol/L dNTPs,0.75 U Taq DNA polymerase in 25μL reaction is suitable for A.alternata ISSR molecular marker analysis,DNA of eight samples collected from Kunming,Dehong,Chuxiong,Qujing,Yuxi and other areas were used in ISSR-PCR application with this system and results indicate it is suitable for this reaction system.