大白菜线粒体DNA有效、快速提取方法

目的:建立一种有效、快捷的大白菜线粒体DNA(mtDNA)提取方法。方法:采用差速离心和蔗糖衬垫相结合的方法先从大白菜胞质雄性不育系及保持系叶片中分离出线粒体,然后用SDS—蛋白酶K裂解法提取mtDNA。结果:琼脂糖凝胶电泳表明,采用该方法提取的mtDNA片段大小在30~45kb之间,电泳条带清晰,无明显降解发生;以提取的mtDNA为模板进行RAPD反应,不育系和保持系均扩增出较多的条带,而且呈现出丰富的多态性,大小分布在500bp-3000bp之间,且条带清晰,没有脱尾现象。结论:该方法提取mtDNA,省时、省钱,对实验设备要求较低,而且分离出的mtDNA质量较高,能满足后续分子生物学研究工作需要。

An Effective and Quick Protocol for Mitochondrial DNA Extraction from Chinese Cabbage

Objectives:To study and establish an effective and quick method for mitochondrial DNA(mtDNA)extraction from Chinese cabbage.Methods: Based on differential centrifugation and sucrose shelf method,the mitochondria was extracted from cytoplasmic male sterile lines and their maintainer lines of Chinese cabbage and the mtDNA was extracted successfully by SDS-Proteinase K method.Results:Agrose gel electrophoresis showed the extracted mtDNA fragments ranged between 30 and 45kb in size and was not destroyed by nucleases.Clear polymorphic fragments were obtained by RAPD-PCR with the templates of the extracted mtDNA and the polymorphic fragments ranged in size from 500 to 3000bp.Conclusion:Compared with other methods,this protocol costed less time and money and had higher efficiency and the extracted mtDNA with good quality can meet the need of the following research work.