摘要
高效快速地分离提取高质量的线粒体DNA(mtDNA)是研究植物线粒体基因及其起源进化的重要前提。为获得高质量的mtDNA进行割手密资源的线粒体基因序列扩增及测序分析,本研究以割手密黄化苗为材料,通过简单差速离心分离得到线粒体,经DNaseⅠ消化处理,去除核DNA杂质得到较纯的线粒体,然后经过5%SDS和蛋白酶K充分裂解线粒体,利用饱和的苯酚/氯仿(1:1)和氯仿两次抽提除去蛋白质,并经过RNase A的消化处理除去RNA,无水乙醇沉淀等一系列操作得到mtDNA。所提取的mtDNA样品经紫外吸收光度测定,琼脂糖凝胶电泳和PCR扩增分析其浓度和纯度,结果表明利用该方法提取的mtDNA纯度高,完全能满足后续PCR分析及分子克隆测序的要求。该方法不仅DNA提取纯度高,操作简单、快速经济,可为今后开展甘蔗及其各野生种的研究提供技术支持。
Abs tract Origin and evolution analysis of plant based on mitochondrial genes is depends on the high quality mitochondrial DNA extracting rapidly and simply. To obtain the high purity mtDNA without nuclear DNA of Saccharum spontaneum to further study the mitochondrial gene amplification and sequence, the etiolated seedlings of S. spontaneum were used as experimental materials and mtDNA was extracted following a series procedures by differential centrifugation, DNaseⅠ treatment, lysis with 5% SDS and proteinase K treatment, removing protein by TE-saturated phenol/chloroform, chloroform extraction, RNase A treatment, and a final ethanol precipitation. The mtDNA samples were tested using spectrophotometry, agarose gel electrophoresis and PCR amplification,respectively. The result showed that the mtDNA isolated by this method had high purity and this high quality mtDNA could be used for PCR analysis and other molecular cloning or sequencing. This method is easy to operate to extract high purity mtDNA rapidly with low cost, it also provided the technical support for the study of sugarcane and its wild species in the future.
出处
《分子植物育种》
CAS
CSCD
北大核心
2015年第5期1141-1145,共5页
Molecular Plant Breeding
基金
云南省应用基础研究面上项目(2012FB150
2013FB037)
云南农业大学博士科研基金项目(A2002181)
云南省现代农业甘蔗产业技术体系建设项目(云财教[2014]105号)
云南省重点新产品开发计划项目(农业部分
2012BB014)
云南农业大学高原山地作物可持续生产系统研究省创新团队(云科人发[2012]18号)共同资助
关键词
甘蔗
割手密
线粒体DNA
提取方法
Sugarcane
Saccharum spontaneum
Mitochondrial DNA
Extraction method
作者简介
通讯作者，x．h_wang@163．com
通讯作者，lfs810@sina．com
