茶树叶绿体DNA提取方法研究_陈春梅.pdfVIP

分子植物育种，2014 年， 第12 卷，第3 期，第562-566页 Molecular Plant Breeding, 2014, Vol.12, No.3, 562-566 技术主题 Technology Feature 茶树叶绿体DNA 提取方法研究 陈春梅 陈亮* 中国农业科学院茶叶研究所, 国家茶树改良中心, 农业部茶树生物学与资源利用重点实验室, 杭州, 310008 * 通讯作者, liangchen@ 摘 要 为了探究茶树叶绿体DNA (chloroplast DNA, cpDNA) 的提取方法，本研究采用改进的高盐-低pH 法和Percoll 密度梯度法提取cpDNA。结果表明，两种方法提取的cpDNA 经分光光度计检测，A260/A280 值在 1.80~1.90，高盐-低pH 法提取的cpDNA 产率为0.65 μg/g，Percoll 密度梯度法提取的cpDNA 产率为0.07 μg/g。 提取的cpDNA 经PCR 检测，均能扩增出目标片段，但高盐-低pH 法效果更好。研究结果为进一步研究茶 树叶绿体基因组结构、功能等方面奠定基础。 关键词 茶树(Camellia sinensis), 叶绿体DNA, 高盐-低pH 法, 密度梯度法 Extraction Method of Chloroplast DNA of Tea Plant (Camellia sinensis) Chen Chunmei Chen Liang * Tea Research Institute of the Chinese Academy of Agricultural Sciences, National Center for Tea Improvement, Key Laboratory of Tea Biology and Re- sources Utilization, Ministry of Agriculture, Hangzhou, 310008 * Corresponding author, liangchen@ DOI: 10.13271/j.mpb.012.000562 Abstract To explore an efficient method of extracting chloroplast DNA (cpDNA) from tea plant, the modified High-salt Low-pH and Percoll Density Gradient Centrifugation methods were compared and evaluated. The results showed that both methods successfully isolating good-quality cpDNA with an A260/A280 ratio between 1.80~1.90, however, a higher yield was obtained by the modified High-salt Low-pH method (0.65 μg/g) as compared to the other method (0.07 μg/g). In addition, although cpDNA extracted using both methods could serve as a template for PCR, better results were obtain with the modified High-salt Low-pH method. This research lays the groundwork for sequencing and analyzing of the entire chloroplast genome of tea plant. Keywords Tea plant ( Camellia sinensis), Chloroplast DNA, High-salt Low-pH method, Density Gradient Centrifugation method 叶绿体最早发现于自养生物蓝藻中，经进化成 前，已有401 例来自不同植物的叶绿体基因组全序 为植物细胞的一部分，以质体的形式存在于陆地植 列被公布(/nuccore) 。 物、藻类和部分原生生物中，是生物体内执行光合作 叶绿体基因组可用于细胞质