摘要
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、2 400 g离心15min结合12 000 g离心12 min的差速离心方法分离获得粗制线粒体,再经过DNase酶切处理1.5 h去除基因组DNA的污染,然后经0.6 mol/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1 g芝麻黄化苗能获得1μg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ARCP5)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。
Using etiolated seedlings of 2 sesame varieties,an economic,rapid,efficient sesame seed mitochondria DNA（mtDNA） extraction method was established through the key factor DNase digestion time,including homogenizing processing,differential centrifugation comprising 2 400 g centrifugation for 15 min and 12 000 g centrifugation for 12 min to acquire primary mitochondria,DNase digestion for 1.5 h to eliminate the pollution of genome DNA,centrifugation with 0.6 mol/L sucrose padding to get pure mitochondria,and SDS-proteinase K lysis to obtain pure mtDNA.The mtDNA samples were tested using DNA/Protein Analyzer,PCR with genome-special markers（nuclear gene-specific primer beta-actin,mitochondrial gene-specific primer rrn26S and chloroplast gene-specific primer ARCP5） and 1.0%agarose gel electrophoresis.It was proved that the mtDNA isolated by this method not only has the yield more than 1 μg per gram material,but also characterized of high purity.The results showed that this high quality mtDNA can be successfully used in genetic studies as sesame cytoplasmic male sterility and stress resistance.
出处
《华北农学报》
CSCD
北大核心
2011年第3期90-94,共5页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金项目(30871552)
芝麻现代农业产业技术体系建设专项(nycytx-20)
中国农业科学院油料作物研究所所长基金资助项目
