中国野生葡萄叶绿体分离及叶绿体DNA提取的研究  

An Optimized Chloroplast Isolation and Chloroplast DNA Extraction Protocol for Chinese Wild Grapes

作　　者：谢海坤 焦健[1] 樊秀彩[1] 张颖[1] 姜建福[1] 孙海生[1] 刘崇怀[1] XIE Haikun JIAO Jian FAN Xiucai ZHANG Ying JIANG Jianfu SUN Haisheng LIU Chonghuai(Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, Chin)

机构地区：[1]中国农业科学院郑州果树研究所,郑州450009

出　　处：《西北植物学报》2016年第7期1464-1469,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：现代农业产业技术体系建设专项资金(CARS-30-yz-1);中国农业科学院科技创新工程专项(CAAS-ASTIP-2015-ZFRI);农业部物种保护项目(2130135-34)

摘　　要：以中国野生刺葡萄、山葡萄、桑叶葡萄和东南葡萄的成熟叶片为材料,比较柱式植物叶绿体DNAout试剂盒和改良的高盐-低pH法分离叶绿体及提取cpDNA效果。结果显示:(1)2种方法均分离得到了中国野生葡萄的叶绿体,但与柱式植物叶绿体DNAout试剂盒相比,改良的高盐-低pH法得到的叶绿体浓度高、杂质少,更适合中国野生葡萄叶绿体分离。(2)柱式植物叶绿体DNAout试剂盒提取的cpDNA,其OD260/OD280在1.28~1.36之间,质量浓度为4.2~7.8ng·μL^(-1),质量低,不能满足后续测序要求;而改良的高盐-低pH法提取的cpDNA,其OD260/OD280值在1.84~1.90之间,质量浓度为2 514.4~4 133.7ng·μL^(-1),质量高,纯度好,能满足后续测序要求。研究表明,改良的高盐-低pH法能简便、快速获得中国野生葡萄完整叶绿体及高质量cpDNA,并且提取的cpDNA可满足后续测序要求,为葡萄属植物叶绿体基因组的深入研究奠定了基础。Mature leaves collected from Vitis davidii ,V .amurensis ,V .heyneana and V .chunganensis were used for chloroplast isolation and cpDNA extraction in this study .The two methods were the column plant chloroplast DNAout and modified high-salt low-pH method ,and the results were compared with each other .（1） Both methods had separated the chloroplast of Chinese wild grapes ,but the modified high-salt low-pH method obtained higher concentration and less impurity of chloroplast than that of column plant chloroplast DNAout .So the modified high-salt low-pH method was more suitable for chloroplast isolation . （2） The value of OD260/OD280 of cpDNA extracted by the column plant chloroplast DNAout was between 1 .28 and 1 .36 ,and the concentration was between 4 .2 ng？μL -1 and 7 .8 ng？μL -1 ,which did not meet the demand of subsequent chloroplast genome sequencing .In contrast ,the value of OD260/OD280 of cpDNA extracted by the modified high-salt low-pH method was between 1 .84 and 1 .90 and the concentration was＆amp;nbsp;between 2 514 .4 ng ？ μL -1 and 4 133 .7 ng？ μL -1 ,so the cpDNA extracted in this way was extremely high-quality and pure .As a result ,the cpDNA extracted by the modified high-salt low-pH method meet the demand of subsequent chloroplast genome sequencing .As a conclusion ,the modified high-salt low-pH method isolated intact chloroplast and extract high-quality cpDNA of Chinese wild grapes simply and quickly .And the cpDNA meet the demand of subsequent chloroplast genome sequencing .It was also a critical step to make further research of chloroplast genomes of V itis L .

关 键 词：中国野生葡萄 叶绿体分离 cpDNA提取 柱式植物叶绿体DNAout 改良的高盐-低pH法 

分 类 号：Q781[生物学—分子生物学] S663.1[农业科学—果树学]