摘要
以春兰为研究材料,对春兰基因组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,可以得到高质量春兰基因组DNA。电泳检测表明,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。同时对春兰RAPD-PCR反应体系中各个影响因素进行了优化,建立了适合春兰RAPD分子标记的最佳反应体系,即20μl反应体系中含1UTaqDNA聚合酶,60μmol/LdNTPs,2.25mmol/LMg2+,1.0μmol/L引物,1.5ng/μl模板DNA。
The method for extraction genomic DNA from Cymbidium goeringii （Orchidaceae） and some essential factors affecting the result of RAPD-PCR were investigated in the paper. PVP and 2- 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 Cymbidium goeringii （Orchidaceae） could be obtained. Based on the high quality genomic DNA, some reaction conditions of PCR （Polymerase Chain Reaction） for RAPD were optimized. The results showed that the RAPD-PCR system with Taq DNA polymerase 1U, dNTP 60μmol/L, Mg^2＋ 2.25 mmol/L, random primer 1.0μmol/L and genomic DNA 1.5 ng/μL in 20μL solution was suitable for Cymbidium amplification reaction.
出处
《中国农学通报》
CSCD
2008年第1期68-73,共6页
Chinese Agricultural Science Bulletin
基金
上海市科技兴农重点攻关项目"花卉种质创新和产业发展关键技术研究"(沪农科攻字(2005)第1-1-8号)
