魔芋DNA快速微量提取及其ISSR-PCR扩增  

A Rapid DNA Minipreparation for Amorphorphallus Blume and Suitable for ISSR-PCR

作　　者：杨朝柱[1,2] 潘娅妮 谢玲玲 牟方贵 赵青华 马作江 冉瑞林 李明辉[4] 滕建勋 陈永波 郭光耀 胡中立[2] 

机构地区：[1]恩施自治州农业科学院,恩施445000 [2]武汉大学生命科学院,武汉430072 [3]恩施市崔坝农技服务中心,恩施445018 [4]湖北省农业厅油料作物中心,武汉430071

出　　处：《基因组学与应用生物学》2009年第3期529-534,共6页Genomics and Applied Biology

基　　金：国家科技支撑计划项目(2007BAD73B03);湖北省科技攻关项目(2007AA201C51)共同资助

摘　　要：魔芋属天南星科魔芋属草本植物,其组织富含多糖和多酚等次生物质,这类物质的存在不仅使DNA提取变得困难,还会影响下游的分子生物学操作。采用改进的提取方法,主要包括在2mLEppendorf离心管中液氮研磨,CTAB-SDS裂解细胞,β-巯基乙醇浓度从2%提高到5%和添加PVP抑制多酚氧化,缓冲液去多糖等步骤,成功的提取了魔芋鲜叶的高质量DNA,其A260/A280位于1.80～2.00之间,产率超过470μg/g,ISSR-PCR扩增效果好。该方法具有快速、低廉、微量、稳定等特点,为深入研究魔芋资源遗传多样性、标记辅助育种和种芋纯度的鉴定奠定了基础。A morphophallus blume, a herb plant belonging to the genus Amorphophallas in the family of araceae, contained plenty of polysaccharides, polyphenols and other undefined secondary metabolites, which resulted that DNA isolation became a challenge and the DNA polymers chains reaction （PCR） was failed, subsequently. A rapid, inexpensive and reliable DNA minipreparation method was developed and the DNA extracted from the fresh leaf ofAmorphophallus blume was high quality and yield, and suitable for ISSR-PCR. The ratio of A260/A280 was ranged from 1.80 to 2.00, and the yield from 460μg/g to 502 μg/g （fresh weight）. The key steps modified in the DNA extraction protocol involved that fresh leaves were coiled and grounded into fine powder in 2 mL Eppendorf tube with liquid N2, DNA was released in CTAB-SDS solution, the concentration of β-mercaptoethanol was 5% instead of 2% and polyvinyl pyrrolideone （PVP） was used to remove the polyphenols, and the polysaccharides were precipitated and discharged with high salt buffer. It was demonstrated the method was suitable to extract DNA of a large amount of individuals and was applied to the further study of genetic diversity; Molecular marker assisted selecting （MAS） and identification of the parent corm purity in A morphophallus blume.

关 键 词：魔芋 组培苗 DNA提取 ISSR—PCR 

分 类 号：S632.9[农业科学—蔬菜学]