胡萝卜叶片线粒体DNA的提取方法

The Study on the Method for Isolating Mitochondrial DNA from the Carrot Leaf

提取高质量的线粒体DNA,对植物细胞质雄性不育的研究起着关键性的作用。以胡萝卜叶片为材料,通过高盐差速离心和蔗糖沉淀差速离心2种方法提取线粒体DNA。通过改变分离和沉淀线粒体离心力大小、使用DNaseⅠ处理得到了无核DNA污染的线粒体。用SDS和蛋白酶K裂解线粒体经酚/氯仿/异戊醇抽提除去蛋白,并用RNaseA消化从而得到单纯线粒体DNA。还特别设计了叶绿体特,异性引物检测线粒体DNA纯度。结果表明:利用优化后的蔗糖沉淀差速离心法提取胡萝卜线粒体DNA,不仅操作简单,而且所得线粒体DNA纯度高、产量高,每克叶片能够得到34.46μg线粒体DNA。

In this experiment, high-salt differential centrifugation and differential centrifugation of sucrose sedimentating were used to extract mitochondrial DNA from carrot leaves. No nuclear DNA-contamined mitochondrial DNA was obtained by adding DNase I and changing the force of centrifuging and sedimentating of mitochondrial. To crack mitochondria, SDS and proteinase K were used. Pure mitochondrial DNA was successfully obtained by phenol/chloroform/isoamyl alcohol extraction to remove protein, and digestion with RNase A. Chloroplast-specific primers were designed to detect mitochondrial DNA purity. The results showed that using the optimized method of differential centrifugation of sucrose sedimentating extracting carrot mitochondrial DNA not only operate simpler but also obtain mitochondrial DNA with higher purity and higher yield（34.46 μg mitochondrial DNA per gram of leaf）.