摘要
用差速离心法自继代培养5个月的愈伤组织中分离叶绿体和线粒体.用饱和酚、氯仿和氯化铯梯度离心的方法纯化二种细胞器的DNA.同时,用同样的方法自供体植物叶中分离和纯化叶绿体和线粒体DNA.采用了6种限制性内切酶、凝胶电泳和Southern印迹杂交的方法.比较了供体植物和愈伤组织中叶绿体DNA和线粒体DNA酶切谱带的变异情况.在所用的EcoRⅠ、BamHⅠ、HindⅢ、BglⅠ、BglⅡ和DraⅠ等6种限制酶中,只有BamHⅠ、DraⅠ和BglⅡ可以获得愈伤组织叶绿体DNA和线粒体DNA的酶切谱带.可能是由于在长期的继代培养中,DNA甲基化限制了EcoRⅠ、HindⅢ和BglⅠ等酶的效能.从凝胶电泳的结果看,BamHⅠ切割的在7.4Kbp;DraⅠ切割的在5.9Kbp位置上,愈伤组织的叶绿体DNA比供体植物多出一条酶切谱带.在Southern印迹杂交中,用高梁线粒体DNA的片段为探针,DraⅠ酶切的愈伤组织线粒体DNA与供体植物在11.5Kbp、13Kbp和10Kbp的位置上出现明显差异;BamHⅠ酶切的,在9.5Kbp、6.3Kbp、5.2Kbp和4.6Kbp位置上呈现明显的差异.
Chloroplast DNA and mitochondrial DNA were isolated from leaves and callus of alfalfa. Those DNAs were digested with six restriction endonucleases(BamH Ⅰ,Bgl.Ⅰ ,Bgl Ⅱ, Dra Ⅰ, EcoR Ⅰ , Hind Ⅲ), electrophoresed and transfered to nylon membianes. Southern blots were hybridized with sorghum probes containing mitochondrial genes. Among the six restriction endonu-cleases,only BamH Ⅰ ,Dra Ⅰ and Bgl Ⅱ can cut the ctDNA and mtDNA isolated from callus.Electrophores and Southern blot hybridization analyses indicated different patterns between the ctDNA, mtDNA isolated from leaves and from callus. These results also demonstrated that the variations of organelle genome can be produced by tissue culture.
出处
《作物学报》
CAS
CSCD
北大核心
1994年第1期33-38,共6页
Acta Agronomica Sinica
关键词
苜蓿
组织培养
叶绿体
线粒体
DNA
Alfalfa, Tissue, culture, Chloroplast DNA, Mitochondrial DNA
