作者：唐祥凯1,2, 谭和平1,2, 沈兴中2, 李怀平1,2, 许洋1,2, 管驰1,2
作者单位：1. 茶叶标准与检测技术四川省重点实验室, 四川 成都 610021;
2. 中国测试技术研究院, 四川 成都 610021
关键词：DNA提取;茶树;方法论;稳定性分析
摘要：
建立一种从富含次生代谢产物的茶树嫩叶中高效提取基因组DNA的方法。提出使用冷冻研磨机来破碎细胞,在确保低温环境保护下,快速、均匀地破碎茶树细胞,解决细胞破碎后易氧化、褐变的关键难点;同时在提取液中加入-巯基乙醇等抗氧化剂进一步防止多酚类化合物的氧化;经过多次纯化有效去除多酚、多糖、蛋白质等杂质;所提取的基因组DNA经验证完整性好、纯度高,完全能进行酶切和聚合酶链式反应(PCR)。此外,进行系统性的方法学考察,结果表明该提取方法稳定、可靠、重现性好。该研究为茶树基因组DNA提取标准方法的建立奠定基础。
Efficient method for isolation of genomic DNA from tender tealeaves rich in secondary metabolites
TANG Xiangkai1,2, TAN Heping1,2, SHEN Xingzhong2, LI Huaiping1,2, XU Yang1,2, GUAN Chi1,2
1. Standard and Testing Technology of Tea Key Laboratory of Sichuan Province, Chengdu 610021, China;
2. National Institute of Measurement and Testing Technology, Chengdu 610021, China
Abstract: An efficient method for isolation of genomic DNA from tender tea leaves rich in secondary metabolites is established. First, the cells of tea leaves were grinded quickly and uniformly with a 6770 Freezer/Mill at low temperature. The difficulty of how to avoid the oxidation and browning of polyphenols has hence solved. Additionally, β-mercaptoethanol and other antioxidants were added into an extraction buffer to prevent the polyphenols from oxidizing. After several purification steps, impurities, such as polyphenols, polysaccharides, and proteins, were removed. The genomic DNA extracted from tea leaves is intact, pure and suitable for restriction digestion and polymerase chain reaction. Moreover, the systematic methodological study has indicated that the method is stable, reliable, and reproducible. The study has laid a foundation for the establishment of a standard method to extract genomic DNA from young leaves of tea.
Keywords: DNA extraction;tea;methodology;stability analysis
2015, 41(9): 47-50 收稿日期: 2015-3-9;收到修改稿日期: 2015-4-17
基金项目: 国家科技支撑计划项目(2008BAK41B01-7);四川省科技支撑计划项目(2009NZ0015)
作者简介: 唐祥凯(1986-),男,四川成都市人,硕士,主要从事茶树生物技术研究。
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