摘要
油茶叶片含有较多的多糖和多酚等次生代谢产物是提取DNA的重要障碍。本研究以油茶叶片为材料研究高质量的DNA提取方法。建立了一种改良的CTAB法,运用2×CTAB缓冲液来裂解细胞,在裂解细胞后的溶液中加入终浓度为2 mol/L Li Cl,用氯仿异戊醇抽提溶液,用等体积的异丙醇沉淀DNA,用预热的70%乙醇洗涤DNA沉淀。运用紫外分析和琼脂糖凝胶电泳分析测定了DNA的产率和质量,并与经典SDS法、经典CTAB法、高盐低p H法进行了对比。结果表明,改良的CTAB法提取的DNA产率比经典SDS的少,与经典CTAB法和高盐低pH法的相似,达到39.0μg/g,且纯度高,无RNA的污染。改良的CTAB法提取的叶片DNA可用于油茶后续的PCR分析研究。
The secondary met abolites existed in leaves of Camellia oleifera, such as polysaccharides and polyphenols, are important obstacles in the extraction of DNA. Using Camellia oleifera leaves as materials, a method to extract high quality DNA was studied in this research. An improved CTAB method was provided to lyse cells in2 ×CTAB solution. The final concentration of 2 mol/L Li Cl was added into the lysate. The lysate solution was extracted with chloroform iso-amyl alcohol, and DNA was precipitated by isopropanol of equal volume. The DNA precipitation was washed by preheated 70% ethanol. The yield and quality of DNA were determined through ultraviolet analysis and agarose gel electrophoresis analysis, and were compared with the results obtained from the classic CTAB method, classic SDS method, classic CTAB method, high salt and low pH method. The results showed that the yield of DNA extracted by the improved CTAB method was less than that by classic SDS method,but was similar to that by classic CTAB method and high salt and low p H method, which was about 39.0 μg/g with high purity and no RNA pollution. The leaf DNA extracted by the improved CTAB method is suitable for the following PCR works for Camellia oleifera.
作者
黄国文
管天球
赵雨云
陈莫林
刘宏辉
Huang Guowen;Guan Tianqiu;Zhao Yuyun;Chen Molin;Liu Honghui(College of Chemistry and Bioengineering, Hu＇nan University of Science and Technology, Yongzhou, 425199)
出处
《分子植物育种》
CAS
CSCD
北大核心
2018年第13期4350-4354,共5页
Molecular Plant Breeding
基金
湖南省科技计划项目(2014NK2021)
湘南优势植物资源综合利用湖南省重点实验室(XNZW15C19)共同资助
关键词
油茶
叶片
DNA提取
改良CTAB法
Camellia oleifera
Leaves
DNA extraction
Improved CTAB method
