摘要
介绍了一种快速、大量提取大白菜线粒体DNA的方法。以大白菜黄化苗为试验材料,通过加入一定量的DNaseⅠ和RNase A酶,充分消除核基因组DNA和RNA的污染。通过加入蛋白酶K和SDS,并经2步变温处理,即先在50℃条件下温育1 h,接着在37℃条件下温育1 h,有效地提高了mtDNA产出率。经凝胶电泳和PCR等检测,结果表明:mtDNA电泳条带清晰,OD260与OD280比值为1.8左右,纯度较高;利用细胞核看家基因Actin设计引物,扩增mtDNA,没有获得扩增产物,表明mtDNA中没有核基因组污染;利用大白菜CMS96胞质不育系基因orf222设计引物,扩增不育系和保持系mtDNA,仅在不育系中扩增出670 bp的片段;利用5个随机引物进行RAPD分析,在保持系和不育系中可扩增出许多产物,且呈现出丰富的多态性,充分表明提取的mtDNA可以满足后续的分子生物学试验。该方法的创新之处在于,通过2步变温处理的方法充分裂解线粒体膜,释放大量mtDNA,有效地提高了mtDNA的产量。试验中获得的最高产出率为每克大白菜黄化苗获得18.83μg mtDNA。
A fast and high productivity method for mitochondrial DNA（mtDNA）isolation in Chinese cabbage was introduced.Yellow seedlings were used in order to remove chloroplast DNA（cpDNA）.DNase Ⅰ and RNase A enzyme were added to digest nuclear DNA（nDNA）and RNA in order to purify mitochondrion.Protein K and SDS were added to disrupt mitochondrial membranes and 2 steps of poecilothermal treatments with 50℃ for 1 h and 37℃ for 1 h were applied to release more mtDNA.Results showed electrophoretic band of mtDNA was clear and the purity was high.The ratio of OD260/OD280 was about 1.8.No products were amplified for mtDNA template by using primers deigned by nuclear housekeeping Actin genes.It meant mtDNA was not contaminated by nDNA.670 bp fragment could only be amplified in CMS lines by using primers deigned by orf222 gene special for Chinese cabbage CMS96.RAPD analysis with 5 primers showed many polymorphic bands were founded between CMS96 and maintainer lines.Therefore mtDNA isolated in this method is suitable for molecular manipulation.The innovation in this method is that 2 steps of poecilothermal treatments with 50℃ for 1 h and 37℃ for 1 h are used to disrupt mitochondrial membranes and make higher productivity of mtDNA.The highest yield in our study is 18.83 μg mtDNA for 1 gram yellow seedlings.
出处
《华北农学报》
CSCD
北大核心
2011年第2期138-142,共5页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金(30871710)
北京市自然科学基金(6092009)
国家科技支撑项目(2009BADB8B03)
现代农业产业技术体系(CARS-25)
