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芝麻高纯度线粒体DNA提取技术研究

DOI: 10.7668/hbnxb.2011.03.019, PP. 90-94

Keywords: 芝麻,mtDNA,提取,Dnase处理

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Abstract:

以2个芝麻品种黄化苗为试验材料,通过比较植物线粒体DNA(mtDNA)提取过程中的关键影响因素DNase处理时间,建立了一种经济、快速、高效的芝麻mtDNA提取方法.该方法分别通过匀浆化处理、2400g离心1min结合12000g离心12min的差速离心方法分离获得粗制线粒体,再经过DNase酶切处理1.h去除基因组DNA的污染,然后经0.6m0l/L蔗糖衬垫离心获得纯的线粒体,通过SDS-蛋白酶K方法裂解线粒体获得芝麻mtDNA.经过核酸蛋白分析仪分析表明:本技术每1g芝麻黄化苗能获得1P,g以上mtDNA;通过1.0%琼脂糖凝胶电泳检测表明获得mtDNA完整,无明显降解;并利用三类特异引物(核基因特异引物beta-actin、线粒体基因特异引物rrn26S以及叶绿体基因特异引物ARCP)进行PCR检测表明提取的mtDNA为高纯度.芝麻高纯度mtDNA提取技术的建立,为研究与芝麻线粒体基因有关的性状(如细胞质雄性不育、抗逆性等)奠定了技术基础.

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