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

作　　者：胡中会[1] 赵立华[1] 佟爱仔[1] 王连春[1] 孔宝华[1] 蔡红[1] 范静华[1] 陈海如[1] 秦西云[2] 

机构地区：[1]云南农业大学植物保护学院农业生物多样性与病虫害控制教育部重点实验室,云南昆明650201 [2]云南省烟草农业科学研究院,云南玉溪653100

出　　处：《云南农业大学学报》2011年第5期602-606,共5页Journal of Yunnan Agricultural University

基　　金：中烟公司科技计划项目(09YN005);中烟公司科技计划项目(2010YN18);中烟公司科技计划项目(2010YN19)

摘　　要：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.

关 键 词：烟草赤星病 ISSR-PCR 正交体系优化 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]