菠菜叶片线粒体DNA的提取及RAPD分析

以菠菜叶片为实验材料,采用差速离心法、DNaseI处理得到无核污染的线粒体、以SDS法去除蛋白,消化RNA得到单纯的菠菜叶片mtDNA,通过对mtDNA纯度的测定,从而建立了菠菜叶mtDNA快速、高效的提取技术体系;同时选取30个引物对三种菠菜mtDNA进行RAPD扩增,对提取的mtDNA体系进行了RAPD分析,结果表明,由引物S7、S16、S21、S24扩增出的条带清晰、多态性较好,片段大小在200~2000 bp之间,表明菠菜各品种的mtDNA各碱基序列上的多态性,是各品种之间进化凸现的原因之一。本研究建立的试验方法耗时短、对离心机的要求不高,并且提取的mtDNA产率高、结构也比较完整。

Extraction and RAPD Analysis of Mitochondrial DNA in Spinach Leaves

Mitochondria in spinach leaves without nuclear pollution were obtained with differential centrifuga -tion and DNase I method .Furthermore , protein was removed with SDS so as to digest RNA , thus pure mtDNA of spinach leaves was gotten .Besides , the purity of mtDNA was tested .30 primers were selected for RAPD amplifica-tion of the mtDNA of three kinds of spinach .The results of RAPD analysis showed that the stripes amplified by primers S7 , S16 , S21 and S24 were clear and had good polymorphism with fragments between 200 and 2000 bp.This demonstrates that the polymorphism of mtDNA in base sequences is the reasons for evolution of every species of spinach .With efficiency and low demand for centrifugal machine , the method established in this study has mtDNA high yields and structural integrity .