适于基因组测序的高质量桃线粒体DNA的高效提取方法

Extraction of High-quality Mitochondrial DNA of Prunus persica for Genome Sequencing

高质量线粒体DNA(mtDNA)是线粒体基因组测序及序列分析的重要前提。为获得高质量桃mtDNA,以‘21世纪’桃叶片为外植体,24℃完全避光条件下进行愈伤组织诱导及增殖。对获得的愈伤组织进行匀浆化处理,结合差速离心、蔗糖衬垫和SDS裂解等方法提取并纯化线粒体。结果表明:MS培养基添加3.0 mg·L-1 6-BA和1.0 mg·L-1 2,4-D可高效诱导并增殖桃叶片愈伤组织,诱导率可达到100%。荧光显微镜检测显示纯化后的线粒体完整且具有活性。提取mtDNA的产率为13.10μg·g-1,无RNA和蛋白质等其它杂质污染。mtDNA电泳检测条带大小约为23 kb,无降解。特异性基因PCR检测表明获得的mtDNA不存在核DNA和叶绿体DNA污染,其纯度可满足后续分子生物学试验及高通量基因组测序的要求。该研究建立了一个适合桃mtDNA快速高效提取方法,为后续相关分子生物学研究提供了参考依据。

High quality of mitochondrial DNA(mtDNA)is a fundamental basis for mitochondrial genome sequencing and sequencing analysis.Callus from‘21st Century’peach leaves were induced at 24℃under completely dark conditions callus induction and proliferution.The obtained callus was homogenized and mtDNA was extracted and purified by differential centrifugation,sucrose liner and SDS pyrolysis.The results showed that,the callus,of which the induction rate could reach 100%,could be effectively induced and proliferated with 3.0 mg·L-1 6-BA and 1.0 mg·L-1 2,4-D.The purified mitochondria,detected by fluorescence microscopy,were complete and active.The yield of purified mtDNA extracted from callus was 13.10μg·g-1 without RNA and proteins.The band size of mtDNA electrophoresis was about 23 kb without degradation.The obtained mtDNA was free of nuclear DNA and chloroplast DNA contamination by specific gene PCR.In this study,a rapid and efficient extraction technology system suitable for high quality of mitochondrial DNA in Prunus persica was established.It laid a reference for the subsequent research on relevant molecular biology.