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目的:获取适用于广西莪术简单序列重复(SSR)分析的高质量DNA,为该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A260 nm/A280 nm1.8~2.0,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A260 nm/A280 nm1.50~1.60,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,可有效去除次生代谢产物对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 suitable for simple sequence repeat (SSR) analysis of Curcuma kwangsiensis and to provide reference for the genetic diversity of this variety. Methods: The young leaves of Curcuma zedoae from Guangxi were used as materials. The total DNA of Curcuma kwangtungensis was extracted by classic cetyltrimethylammonium bromide (CTAB) method and modified CTAB method. The difference of manual and machine grinding was investigated. The DNA concentration And purity, DNA was used as a template for polymerase chain reaction (PCR) amplification. Primer selection clest SSR-01, clest SSR-03, clest SSR-06, clest SSR-08, clest SSR-14, clest SSR- Polyacrylamide gel electrophoresis detection of DNA amplification. Results: The A260 nm / A280 nm1.8 ~ 2.0 DNA extracted by modified CTAB method could remove more proteins, polysaccharides and RNA, and the DNA conformed to the requirement of PCR amplification. A clear band . The DNA of A260 nm / A280 nm1.50 ~ 1.60 mentioned by the classic CTAB method contains more impurities and can not be used in molecular biology research such as PCR amplification. Conclusion: The DNA extracted by modified CTAB method is of good quality, which can effectively remove the interference of secondary metabolites to DNA and is suitable for the analysis of SSR molecular marker and genetic diversity of Curcuma.