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为满足高通量二代测序要求,本研究采用大豆黄花苗为试材,结合差速离心、蔗糖密度梯度离心及超速离心方法提取高纯度大豆线粒体基因组DNA(mt DNA)。结果表明,差速离心能够有效去除核基因组掺杂;超速离心与蔗糖密度梯度离心结合能够有效去除叶绿体污染。提取的mt DNA经琼脂糖凝胶电泳、紫外光度计检测及叶绿体和细胞核特异性引物检测表明,该方法提取的大豆mt DNA无叶绿体DNA及核DNA污染,且纯度高,可满足测序等对线粒体高纯度的要求,为研究大豆线粒体相关性状的机理奠定了坚实基础。
In order to meet the requirement of high-throughput second-generation sequencing, we used soybean yellow flower seedlings as experimental materials, and combined with differential centrifugation, sucrose density gradient centrifugation and ultracentrifugation to extract high-purity soybean mitochondrial DNA (mt DNA). The results showed that differential centrifugation can effectively remove nuclear genome doping. Ultracentrifugation and sucrose density gradient centrifugation can effectively remove chloroplast contamination. The extracted mt DNA was detected by agarose gel electrophoresis, ultraviolet photometer and chloroplast and nucleocapsid-specific primers. The mt DNA extracted from this method has no chloroplast DNA and nuclear DNA contamination and high purity, which can meet the requirements of mitochondria High purity requirements for the study of soybean mitochondrial traits related to the mechanism laid a solid foundation.