一种快速高效提取花生叶片DNA的简化方法
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摘要
为获得适用于高通量测序的高质量花生叶片DNA,本方法取第三对真叶为实验材料,设计烧制圆球状枪头和离心管装置代替研钵研磨,并在CTAB法的基础上在杂质沉淀前快速分离DNA等简化方法提取DNA。琼脂糖凝胶电泳检测结果表明DNA条带清晰、完整性高。经超微量分光光度计检测,DNA浓度范围在104～131ng/μL,OD_(260)/OD_(280)为1.7～2.0,OD_(260)/OD_(230)大于2.0,说明杂质少,可获得高纯度,高浓度的DNA。此方法与常用的植物基因组DNA提取方法相比,具有成本低、时间短、质量高的优点,可用于基因组重测序、分子生物学技术以及分子标记筛选等后续研究,可提高花生分子育种与筛选工作效率。
In order to obtain the high-quality peanut DNA for high-throughput sequencing,the third pair of true leaves were used as experimental material,and the mortar was replaced with ballshaped pipet and a centrifuge tube.In addition,DNA was isolated from impurities.The concentration of DNA that detected by Nanodrop was 104～131 ng/μl,OD_(260)/OD_(280) was 1.7～2.0,and OD_(260)/OD_(230) was more than 2.0,which indicated that there were few impurities contaminated.Compared with the generally accepted plant genomic DNA extraction method,this method had the advantages of low cost,short time and high quality.It can be applied to the subsequent research such as genome re-sequencing,molecular marker screening,and enhance the efficiency of peanut molecular breeding and screening.
In order to obtain the high-quality peanut DNA for high-throughput sequencing,the third pair of true leaves were used as experimental material,and the mortar was replaced with ballshaped pipet and a centrifuge tube.In addition,DNA was isolated from impurities.The concentration of DNA that detected by Nanodrop was 104～131 ng/μl,OD_(260)/OD_(280) was 1.7～2.0,and OD_(260)/OD_(230) was more than 2.0,which indicated that there were few impurities contaminated.Compared with the generally accepted plant genomic DNA extraction method,this method had the advantages of low cost,short time and high quality.It can be applied to the subsequent research such as genome re-sequencing,molecular marker screening,and enhance the efficiency of peanut molecular breeding and screening.
引文
[1]FáVERO A P,PáDUA J G,COSTA T S,et al.New hybrids from peanut(Arachis hypogaea L.)and synthetic amphidiploid crosses show promise in increasing pest and disease tolerance[J].Genetics and Molecular Research,2015,14:16694-16703.
[2]MENG S,YANG X L,DANG P M,et al.Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut(Arachis hypogaea L.)[J].Genetics and Molecular Research,2016,15(3).DOI:10.4238/gmr.15038207.
[3]陈明娜,迟晓元,潘丽娟,等.中国花生育种的发展历程与展望[J].中国农学通报,2014,30(9):1-6.
[4]CLARKE J D.Cetyltrimethyl ammonium bromide(CTAB)DNA miniprep for plant DNA isolation[J].Cold Spring Harbor Protocols,2009(3).DOI:10.1101/pdb.prot5177.
[5]SCHNEIDERBAUER A,SANDERMANN J H,ERNST D.Isolation of functional RNA from plant tissues rich in phenolic compounds[J].Analytical Biochemistry,1991,197(1):91-95.
[6]徐宝梁,苏宁,陈颖,等.转基因棉籽检测中DNA模板提取方法研究[J].生物技术,2004,14(4):25-27.
[7]刘伟红,许文涛,商颖,等.果汁DNA提取方法比较及柑橘属植物分子生物学检测技术的研究[J].中国食品学报,2012,12(4):195-201.
[8]金晓玲,乌云塔娜,张智俊,等.榉属植物总DNA提取法研究[J].植物研究,2004,24(1):107-110.
[9]周贤达,孙辉,丁康芬,等.一种西瓜叶片DNA快速提取方法[J].中国瓜菜,2018,31(09):29-31,42.
[10]丁浩,郑唐春,梁德洋,等.一种简易高效提取多种植物纯净DNA的方法[J].植物研究,2015,35(3):457-461.
[11]陈林杨,宋敏舒,查红光,等.一种改良的植物基因组DNA通用提取方法[J].植物分类与资源学报,2014,36(3):375-380.
[12]FANG G,HAMMAR S,GRUMET R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,13(1):52-54,56.
[13]程运江,伊华林,庞晓明,等.几种木本果树DNA的有效提取[J].华中农业大学学报,2001(5):481-483.
[14]陈大明,张上隆,金勇丰.一种木本果树基因组DNA提取方法研究[J].浙江大学学报(农业与生命科学版),1997(6):621-624.
[2]MENG S,YANG X L,DANG P M,et al.Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut(Arachis hypogaea L.)[J].Genetics and Molecular Research,2016,15(3).DOI:10.4238/gmr.15038207.
[3]陈明娜,迟晓元,潘丽娟,等.中国花生育种的发展历程与展望[J].中国农学通报,2014,30(9):1-6.
[4]CLARKE J D.Cetyltrimethyl ammonium bromide(CTAB)DNA miniprep for plant DNA isolation[J].Cold Spring Harbor Protocols,2009(3).DOI:10.1101/pdb.prot5177.
[5]SCHNEIDERBAUER A,SANDERMANN J H,ERNST D.Isolation of functional RNA from plant tissues rich in phenolic compounds[J].Analytical Biochemistry,1991,197(1):91-95.
[6]徐宝梁,苏宁,陈颖,等.转基因棉籽检测中DNA模板提取方法研究[J].生物技术,2004,14(4):25-27.
[7]刘伟红,许文涛,商颖,等.果汁DNA提取方法比较及柑橘属植物分子生物学检测技术的研究[J].中国食品学报,2012,12(4):195-201.
[8]金晓玲,乌云塔娜,张智俊,等.榉属植物总DNA提取法研究[J].植物研究,2004,24(1):107-110.
[9]周贤达,孙辉,丁康芬,等.一种西瓜叶片DNA快速提取方法[J].中国瓜菜,2018,31(09):29-31,42.
[10]丁浩,郑唐春,梁德洋,等.一种简易高效提取多种植物纯净DNA的方法[J].植物研究,2015,35(3):457-461.
[11]陈林杨,宋敏舒,查红光,等.一种改良的植物基因组DNA通用提取方法[J].植物分类与资源学报,2014,36(3):375-380.
[12]FANG G,HAMMAR S,GRUMET R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,13(1):52-54,56.
[13]程运江,伊华林,庞晓明,等.几种木本果树DNA的有效提取[J].华中农业大学学报,2001(5):481-483.
[14]陈大明,张上隆,金勇丰.一种木本果树基因组DNA提取方法研究[J].浙江大学学报(农业与生命科学版),1997(6):621-624.