适合于基因组测序的红麻高纯度线粒体DNA提取  

Extraction of high purity mitochondrial DNA from kenaf for genome sequencing

作　　者：赵杨[1] 陈鹏[1] 周瑞阳[1] 

机构地区：[1]广西大学农学院,南宁530004

出　　处：《东北农业大学学报》2011年第4期98-101,共4页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(30900912;31060199);教育部科学技术研究重点项目(209095)

摘　　要：采用红麻黄化苗为试材,结合密度梯度离心和差速离心的方法提取线粒体DNA(mtDNA)以满足测序要求。结果表明,蔗糖密度梯度离心法比Percoll密度梯度离心法更适合于红麻线粒体的分离。分离后的线粒体经DNaseI消化核DNA,并采用Jannus绿检测线粒体的完整性,SDS和蛋白酶K裂解线粒体,并用酚/氯仿抽提除去蛋白,乙醇沉淀得到线粒体DNA,再经RNaseA消化去除RNA。提取的mtDNA经1.0%琼脂糖凝胶电泳、紫外分光光度计检测,并设计叶绿体和核特异性基因引物检测是否有污染存在。结果表明,此方法提取的mtDNA纯度高,没有污染,满足基因组测序要求。An efficient method for extraction of mitochondrial DNA（mtDNA） from etiolated tissues of kenaf for sequencing was developed.The results showed that sucrose gradient centrifugation was better than Percoll＇ s as for mitochondria extraction of kenaf.The mitochondria was digested with DNase I to remove the nuclear DNA,then was extracted with phenol/chloroform,followed by ethanol deposition,and got rid of RNA contamination by treating RNase A.The mtDNA was detected by gelose electro-phoresis and ultraviolet spectrophotometer,furthermore,a chloroplast and nuclear-specific primers were designed to detect mtDNA purity.The results showed that the mtDNA extracted in this way had high purity and no contamination,then could be used as genome sequencing.

关 键 词：红麻 线粒体 MTDNA 测序 

分 类 号：S563.5[农业科学—作物学] Q751[生物学—分子生物学]