适用于SSR分析的广西莪术DNA提取方法考察  

Investigation of Extracton Methods of DNA from Curcuma kwangsiensis for Simple Sequence Repeat Analysis

作　　者：杨妮[1] 苏伟敏[1] 靳雅惠 方彬华 王建[1] 

机构地区：[1]广西中医药大学药学院,南宁530001

出　　处：《中国实验方剂学杂志》2015年第4期80-83,共4页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81160500)

摘　　要：目的：获取适片j于广两莪术简单序列重复（SSR）分析的高质量DNA，为陔品种的遗传多样性研究提供参考。方法：选择广西莪术嫩叶为材料，采用经典十六烷基三甲基溴化铵（CTAB）法和改良CTAB法提取广西莪术总DNA，考察手工和机器研磨的差异性，采用UV检测DNA浓度和纯度，DNA作为模板进行聚合酶链反应（PCR）扩增，引物选择clest SSR-01，clest SSR-03，clest SSR-06，clest SSR-08，clest SSR-14，clest SSR-15，利用聚丙烯酰胺凝胶电泳检测DNA扩增效果。结果：改良CTAB法所提DNA的A260nm／A280nm 1.8-2.0，蛋白质、多糖、RNA等物质去除较彻底，DNA符合PCR扩增结果要求，使用姜黄属通用引物能扩增出清晰条带。经典CTAB法所提DNA的A260nm／A280nm 1.50-1.60，含较多杂质，无法用于PCR扩增等分子生物学研究。结论：改良CTAB法提取所得DNA质量较好，可有效上除次生代谢产物对DNA的干扰，适用于广西莪术SSR分子标记和遗传多样性分析。Objective： To obtain high quality DNA from Curcuma kwangsiensis for simple sequence repeat （SSR） analysis, and provide a reference for research of genetic diversity of this variety. Method： Taking C. kwangsiensis leaves as experimental material, total DNA was extracted by the classic cetyl trimethyl ammonium bromide （CTAB） method and the modified CTAB method, difference between machine grinding and hand grinding was investigated, concentration and purity were determined by UV. DNA as a template tor polymerase chain reaction （PCR） amplification, taking clcst SSR-01, clest SSR-03, clest SSR-06, clest SSR-08, clest SSR-14 amt clest SSR-15 as primers, polyacrylamide gel electrophoresis was adopted to detect DNA amplification effect. Result： When DNA extracted by the modified CTAB method, A260nm/A280nm of them was 1.8-2.0, proteins, polysaccharides, RNA and other substances were removed completely, DNA confbrmed to requirements of PCR amplification result, it could amplify out clear bands by C. universal primers and was suitable for SSR analysis. A260nm/A280nm of DNA extracted by the classic CTAB method was 1.5-1.6, it contained some impurities, which cloud not be used for PCR amplification and other molecular biology researches. Conclusion： DNA quality is good which was extracted by the modified CTAB method, this method can effectively remove interferenceof the secondary metabolites for DNA, it is suitable for SSR molecular marker and genetic diversity analysis analysis of C.kwangsiensis.

关 键 词：广西莪术 基因组DNA 简单序列重复 引物 研磨方式 

分 类 号：R283.6[医药卫生—中药学] R282.5[医药卫生—中医学]