马蓝基因组DNA提取及RAPD反应体系的优化  

Extraction of Genomic DNA and Optimization of RAPD Reaction of Baphicacanthus cusia(Nees) Bremek

作　　者：黄以钟[1,2] 潘大仁[1,2] 王占成[2] 陈隽[1] 周以飞[1,2] 

机构地区：[1]福建农林大学作物学院,福建省植物分子与细胞生物学重点实验室,福州350002 [2]福建农林大学作物学院,教育部作物遗传育种与综合利用重点实验室,福州350002

出　　处：《中国农学通报》2009年第8期53-57,共5页Chinese Agricultural Science Bulletin

基　　金：福建省重大科技专项"福建省中药材GAP关键技术研究"(2004YZ02)

摘　　要：以马蓝为研究材料,对马蓝基因组DNA的提取以及RAPD-PCR反应体系进行了优化。结果表明,在提取液中加入PVP和β-巯基乙醇去除酚类物质,采用高盐沉淀DNA和多次洗涤去除糖类,可以得到高质量的马蓝基因组DNA。电泳检测结果表明,所获得的DNA完整,无降解,完全可以满足RAPD等分子标记分析的需要。同时对马蓝RAPD-PCR反应体系中各个影响因素进行了优化,建立了适合马蓝RAPD分子标记的最佳反应体系,即20μl反应体系中含有1.25U TaqDNA聚合酶,0.25mmol/Ld NTP,2.5mmol/L Mg2+,50ng模板DNA,1.0μmol/L引物;反应程序中最佳退火温度为38℃。The method for extraction genomic DNA from essential factors affecting the result of RAPD-PCR Baphicacanthus cusia （Nees） were investigated in the Bremek and some paper. PVP and β-mercaptoethanol were added to lyses buffer in order to inhibit oxidation of polyphenol, high concentration of salt solution and elution for many times were used to remove polysaccharide, using above methods ,high quality DNA of Baphicacanthus cusia （Nees） Bremek could be .obtained. Based on the high quality genomic DNA, some reaction conditions of PCR for RAPD was optimized. The results showed that the RAPD-PCR system with Taq DNA polymerase 1.25 U, dNTP 0.25 mmol/L, Mg2＋ 2.5 mmol/L, genomic DNA 50 ng and random primer 1.0 μmol/L in 20 μl solution was suitable for Baphicacanthus amplification reaction.

关 键 词：马蓝 RAPD DNA 反应体系 

分 类 号：S567.23[农业科学—中草药栽培]