桦褐孔菌ISSR-PCR反应体系优化与建立  

Establishment and Optimization of ISSR-PCR Reaction System of Inonotus obliquus

作　　者：闫可[1] 尹勇刚[1] 傅常娥[1] 陈艳秋[1] 冉丽萍[1] 

机构地区：[1]延边大学农学院,吉林延吉133002

出　　处：《中国食用菌》2012年第2期37-40,共4页Edible Fungi of China

基　　金：国家自然基金项目部分内容;编号:31160408

摘　　要：采用改良CTAB法提取了桦褐孔菌总DNA，确定ISSR最适25μL反应体系为模板DNA浓度15ngμmol·L-1，dNTPs浓度150μmol·L-1，引物浓度25μmol·L-1，TaqDNA聚合酶浓度2．0U，Mg2＋浓度1．4mmol·L-1，10xbuffer2．5μL，其余用ddH20补足；确定ISSR扩增程序为：94℃预变性5min，35个循环：94℃变性1min、45℃～51℃退火1min（退火温度因不同引物而定）、72℃延伸1min，最后72℃延伸5min，4℃保存。筛选出16条ISSR引物，并成功应用引物UBC842完成了21株桦褐孔菌的ISSR—PCR反应。The high quality genome total DNA of Inonotus obliquus extracted fast through the CTAB law, was used in ISSR amplification. And the optimal 25μL ISSR reaction system of lnonotus obliquus was determined. The template DNA density was 15 ng.μL-1, dNTPs was 150 μmol.L-1, the primer density was 25μmol-L-1, the Taq DNA polymerase was 2.0 U, the Mg2＋ concentration was 1.4 mmol .L-1, the 10×buffer density is 2.5 μL, and the rest are supplemented with the double-distilled water. And the procedures of ISSR amplification are 5 rain at 94℃, followed by 35 cycles of 1 min at 94℃ min at 45℃-51℃ （depends on the primers） , 1 min at 72℃, and last extension at 72℃ for 5 rain. The amplified products were reserved at 4℃. Based on these reaction conditions, 16 ISSR primers were screened out and used in the further research. ISSR-PCR reactions of 21 Inonotus obliquus strains were conducted successfully with the using of primer UBC842.

关 键 词：桦褐孔菌 ISSR 反应体系 优化 

分 类 号：S567.39[农业科学—中草药栽培]