花生DNA的五种改良CTAB提取方法的比较分析及其应用
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摘要
花生是世界上重要的油料作物和经济作物之一,也是最重要的植物食用油来源之一,但花生分子标记和功能基因组学等分子生物学研究较落后。因此,本研究旨在建立适合自身的花生基因组DNA的提取方法,为开展花生分子标记研究和分子生物学研究提供帮助。本研究使用了5种改良CTAB法提取花生基因组DNA,所得DNA的纯度和浓度分别通过琼脂糖凝胶电泳和紫外分光光度计检测,再利用8种分子标记技术的48条单引物和4对转座子保守区扩增引物对提取获得的花生基因组DNA的质量进行扩增验证和应用。结果表明:(1)综合花生基因组DNA的质量检测数据以及花生分子标记技术和转座子保守区的扩增验证结果来看,五种改良CTAB法的DNA提取效果表现依次为:方法一>方法五>方法四>方法三>方法二,其中方法一为最佳首选,方法五和方法四的提取效果也好,但是由于其有利用到强腐蚀性的平衡酚,不安全且不环保,所以不推荐;(2)在花生上建立了8种分子标记技术和4类转座子保守区的扩增体系;(3)克隆获得了花生4类转座子保守区序列。本研究为今后开展花生分子标记研究及转座子的克隆鉴定利用提供了帮助。
Peanut is one of t he most important oil and economic crops in the world. It is also one of the most important sources of plant edible oil. However, researches such as peanut molecular markers and functional genomics are relatively lagging behind. Therefore, the purpose of this study was to establish suitable methods for peanut genomic DNA extraction, and offer help for the molecular marker studies and molecular biology studies in peanut. In this study, five improved CTAB methods were used to extract peanut genomic DNA. The purity and concentration of the obtained peanut DNA were detected by agarose gel electrophoresis and ultraviolet spectrophotometer, respectively. Then, the quality of the obtained peanut genomic DNA was verified through PCR with forty-eight single primers of eight kinds of single primer amplification reaction based molecular marker techniques and four pairs of primers designed according to the conserved region of transposons. The results showed that according to the comprehensive quality test data of peanut genomic DNA, the amplification results of molecular marker techniques and transposon homology-based cloning, the extraction effects of the five improved methods were as follows: method one>method five>method four>method three>method two, among which method one was the best one. Also, method five and method four were effective, but the highly corrosive balanced phenol which was not safe and environmentally friendly was used in these two methods, so they were not recommended.The amplification system of eight kinds of single primer amplification reaction based molecular marker techniques and four types of transposon homology-based cloning in peanut was established. The sequences of four types of transposon conserved region of peanut were obtained by cloning. In conclusion, this study would provide help for peanut molecular marker research and transposon cloning, identification and utilization in the future.
Peanut is one of t he most important oil and economic crops in the world. It is also one of the most important sources of plant edible oil. However, researches such as peanut molecular markers and functional genomics are relatively lagging behind. Therefore, the purpose of this study was to establish suitable methods for peanut genomic DNA extraction, and offer help for the molecular marker studies and molecular biology studies in peanut. In this study, five improved CTAB methods were used to extract peanut genomic DNA. The purity and concentration of the obtained peanut DNA were detected by agarose gel electrophoresis and ultraviolet spectrophotometer, respectively. Then, the quality of the obtained peanut genomic DNA was verified through PCR with forty-eight single primers of eight kinds of single primer amplification reaction based molecular marker techniques and four pairs of primers designed according to the conserved region of transposons. The results showed that according to the comprehensive quality test data of peanut genomic DNA, the amplification results of molecular marker techniques and transposon homology-based cloning, the extraction effects of the five improved methods were as follows: method one>method five>method four>method three>method two, among which method one was the best one. Also, method five and method four were effective, but the highly corrosive balanced phenol which was not safe and environmentally friendly was used in these two methods, so they were not recommended.The amplification system of eight kinds of single primer amplification reaction based molecular marker techniques and four types of transposon homology-based cloning in peanut was established. The sequences of four types of transposon conserved region of peanut were obtained by cloning. In conclusion, this study would provide help for peanut molecular marker research and transposon cloning, identification and utilization in the future.
引文
Chen G.H.,Ge J.T.,Yuan L.Y.,Zhu S.D.,Su Y.,Liu S.,and Wang C.G.,2017,Extraction of genomic DNA and construction o f RAPD molecular marker reaction system in Cucumis melo var.conomon group,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1575-1580(陈国户,葛继涛,袁凌云,朱世东,苏亚,刘姗,汪承刚,2017,酥瓜(Cucumis melo var.conomon Group)基因组DNA提取及RAPD分子标记反应体系的建立,基因组学与应用生物学,36(4):1575-1580)
Chen J.,Hu X.H.,Miao H.R.,Cui F.G.,and Yu S.L.,2008,Genome DNA extracted with CTAB method and its use for SSR and SRAP,Huasheng Xuebao(Journal of Peanut Science),37(1):29-31(陈静,胡晓辉,苗华荣,崔凤高,禹山林,2008,CTAB法提取花生总DNA在SSR和SRAP中的扩增效果,花生学报,37(1):29-31)
Collard B.C.Y.,and Mackill D.J.,2009,Start codon targeted(SCoT)polymorphism:a simple,novel DNA marker technique for generating gene-targeted markers in plants,Plant Mol.Biol.Rep.,27(1):86-93
Cui J.,Hu F.C.,Chen Z.,Guo L.J.,Duan L.,Fan H.Y.,and He F.,2017,Comparison of genomic DNA extraction methods and optimization of SCoT-PCR reaction systems for Artocarpus integer(Thunb.)Merr,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(4):1338-1346(崔健,胡福初,陈哲,郭利军,段恋,范鸿雁,何凡,2017,榴莲蜜基因组DNA提取方法比较及SCoT反应体系的优化,分子植物育种,15(4):1338-1346)
De Keukeleire P.,De Schepper S.,Gielis J.,and Gerats T.,2004,A PCR-based assay to detect hAT-like transposon sequences in plant,Chromosome Res.,12(2):117-123
Guillemaut P.,and Drouard L.M.,1992,Isolation of plant DNA:A fast,inexpensive and reliable method,Plant Mol.Biol.Rep.,10(1):60-65
Kalendar R.,Antonius K.,Sm伥kal P.,and Schulman A.H.,2010,i PBS:a universal method for DNA fingerprinting and retro transposon isolation,Theor.Appl.Genet.,121(8):1419-1430
Kang H.W.,Park D.S.,Go S.J.,and Eun M.Y.,2002,Fingerprint ing of diverse genomes using PCR with universal rice primers generated from repetitive sequence of Korean weedy rice,Mol.Cells,13(2):281-287
Kumar A.,Pearce S.R.,Mclean K.,Harrison G.,Heslop-Harrison J.S.,Waugh R.,and Flavell A.J.,1997,The Ty1-copia group of retrotransposons in plants:genomic organisation,evolution,and use as molecular markers,Genetica,100(1-3):205-217
Kumekawa N.,Ohtsubo E.,and Ohtsubo H.,1999,Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom,Genes Genet.Syst.,74(6):299-307
Lu Z.Z.,and Li F.,2018,Comparison of the DNA extraction effects of tree kits on the tobacco aft er baking,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural U-niversity(Natural Science)),33(1):172-175(陆铮铮,李芳,2018,3种试剂盒提取烤后烟叶DNA的效果比较,云南农业大学学报(自然科学),33(1):172-175)
Marmur J.,1961,A procedure for the isolation of deoxyribonucleic acid from microorganisms,J.Mol.Biol.,3(2):208-218
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nucleic Acids Res.,8(19):4321-4325
Singh A.K.,Rana M.K.,Singh S.,Kumar S.,Kumar R.,and Singh R.,2014,CAAT box-derived polymorphism(CBDP):a novel promoter-targeted molecular marker for plants,J.Plant Biochem.Biot.,23(2):175-183
Staginnus C.,Huettel B.,Desel C.,Schmidt T.,and Kahl G.,2001,A PCR-based assay to detect En/Spm-like transposon sequences in plants,Chromosome Res.,9(7):591-605
Williams J.G.K.,Kubelik A.R.,Livak K.J.,Rafalski J.A.,and Tingey S.V.,1990,DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,Nucleic Acids Res.,18(22):6531-6535
Xiong F.,Jiang J.,Han Z.,Zhong R.,He L.,Zhuang W.,and Tang R.,2011a,Molecular characterization of high plant species using PCR with primers designed from consensus branch point signal sequences,Biochem.Genet.,49(5-6):352-363
Xiong F.,Zhong R.,Han Z.,Jiang J.,He L.,Zhuang W.,and Tang R.,2011b,Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut(Arachis hypogaea L.)genotypes,Mol.Biol.Rep.,38(5):3487-3494
Xiong F.,Liu J.,Jiang J.,Zhong R.,He L.,Han Z.,Li Z.,Tang X.,and Tang R.,2013,Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAMD markers,Biochem.Genet.,51(11-12):889-900
Xiong F.Q.,Jiang J.,Zhong R.C.,Han Z.Q.,He L.Q.,Li Z.,Zhuang W.J.,and Tang R.H.,2010,Application of SCo Tmolecular markers in genus Arachis,Zuowu Xuebao(Acta Agronomica Sinica),36(12):2055-2061(熊发前,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华,2010,目标起始密码子多态性(SCoT)分子标记技术在花生属中的应用,作物学报,36(12):2055-2061)
Xiong F.Q.,Liu J.X.,He L.Q.,Han Z.Q.,Huang Z.P.,Tang X.M.,Jiang J.,Zhong R.C.,Wu H.N.,Li Z.,Luo S.Y.,Tang R.H.,and He X.H.,2017,Recent advances on the development and utilization of molecular markers based on LTR retrotransposons and MITE transposons from peanut(Arachis hypogaea L.),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(2):640-647(熊发前,刘俊仙,贺梁琼,韩柱强,黄志鹏,唐秀梅,蒋菁,钟瑞春,吴海宁,李忠,罗赛云,唐荣华,何新华,2017,花生LTR和MITE转座子及其分子标记开发利用研究进展,分子植物育种,15(2):640-647)
Yan J.Y.,Ma C.Q.,Chang B.,Fan X.G.,Li Z.,Yang Y.Z.,and Zhao Z.Y.,2017,A modified CTAB method for genomic DNA extraction from apple fruit,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(9):3610-3615(闫玖英,马长青,常博,范献光,李征,杨亚州,赵政阳,2017,改良CTAB法用于苹果果实基因组DNA的提取,分子植物育种,15(9):3610-3615)
Zhou H.,Yan C.X.,Guo L.C.,Liu Y.,and Shan S.H.,2012,Rapid extraction of genomic DNA from peanut by CTABmethod,Shandong Nongye Kexue(Shandong Agricultural Sciences),44(7):8-9,15(周浩,闫彩霞,郭凌超,刘宇,单世华,2012,CTAB法少量快速提取花生基因组DNA,山东农业科学,44(7):8-9,15)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored poly merase chain reaction amplification,Genomics,20(2):176-183
Chen J.,Hu X.H.,Miao H.R.,Cui F.G.,and Yu S.L.,2008,Genome DNA extracted with CTAB method and its use for SSR and SRAP,Huasheng Xuebao(Journal of Peanut Science),37(1):29-31(陈静,胡晓辉,苗华荣,崔凤高,禹山林,2008,CTAB法提取花生总DNA在SSR和SRAP中的扩增效果,花生学报,37(1):29-31)
Collard B.C.Y.,and Mackill D.J.,2009,Start codon targeted(SCoT)polymorphism:a simple,novel DNA marker technique for generating gene-targeted markers in plants,Plant Mol.Biol.Rep.,27(1):86-93
Cui J.,Hu F.C.,Chen Z.,Guo L.J.,Duan L.,Fan H.Y.,and He F.,2017,Comparison of genomic DNA extraction methods and optimization of SCoT-PCR reaction systems for Artocarpus integer(Thunb.)Merr,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(4):1338-1346(崔健,胡福初,陈哲,郭利军,段恋,范鸿雁,何凡,2017,榴莲蜜基因组DNA提取方法比较及SCoT反应体系的优化,分子植物育种,15(4):1338-1346)
De Keukeleire P.,De Schepper S.,Gielis J.,and Gerats T.,2004,A PCR-based assay to detect hAT-like transposon sequences in plant,Chromosome Res.,12(2):117-123
Guillemaut P.,and Drouard L.M.,1992,Isolation of plant DNA:A fast,inexpensive and reliable method,Plant Mol.Biol.Rep.,10(1):60-65
Kalendar R.,Antonius K.,Sm伥kal P.,and Schulman A.H.,2010,i PBS:a universal method for DNA fingerprinting and retro transposon isolation,Theor.Appl.Genet.,121(8):1419-1430
Kang H.W.,Park D.S.,Go S.J.,and Eun M.Y.,2002,Fingerprint ing of diverse genomes using PCR with universal rice primers generated from repetitive sequence of Korean weedy rice,Mol.Cells,13(2):281-287
Kumar A.,Pearce S.R.,Mclean K.,Harrison G.,Heslop-Harrison J.S.,Waugh R.,and Flavell A.J.,1997,The Ty1-copia group of retrotransposons in plants:genomic organisation,evolution,and use as molecular markers,Genetica,100(1-3):205-217
Kumekawa N.,Ohtsubo E.,and Ohtsubo H.,1999,Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom,Genes Genet.Syst.,74(6):299-307
Lu Z.Z.,and Li F.,2018,Comparison of the DNA extraction effects of tree kits on the tobacco aft er baking,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural U-niversity(Natural Science)),33(1):172-175(陆铮铮,李芳,2018,3种试剂盒提取烤后烟叶DNA的效果比较,云南农业大学学报(自然科学),33(1):172-175)
Marmur J.,1961,A procedure for the isolation of deoxyribonucleic acid from microorganisms,J.Mol.Biol.,3(2):208-218
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nucleic Acids Res.,8(19):4321-4325
Singh A.K.,Rana M.K.,Singh S.,Kumar S.,Kumar R.,and Singh R.,2014,CAAT box-derived polymorphism(CBDP):a novel promoter-targeted molecular marker for plants,J.Plant Biochem.Biot.,23(2):175-183
Staginnus C.,Huettel B.,Desel C.,Schmidt T.,and Kahl G.,2001,A PCR-based assay to detect En/Spm-like transposon sequences in plants,Chromosome Res.,9(7):591-605
Williams J.G.K.,Kubelik A.R.,Livak K.J.,Rafalski J.A.,and Tingey S.V.,1990,DNA polymorphisms amplified by arbitrary primers are useful as genetic markers,Nucleic Acids Res.,18(22):6531-6535
Xiong F.,Jiang J.,Han Z.,Zhong R.,He L.,Zhuang W.,and Tang R.,2011a,Molecular characterization of high plant species using PCR with primers designed from consensus branch point signal sequences,Biochem.Genet.,49(5-6):352-363
Xiong F.,Zhong R.,Han Z.,Jiang J.,He L.,Zhuang W.,and Tang R.,2011b,Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut(Arachis hypogaea L.)genotypes,Mol.Biol.Rep.,38(5):3487-3494
Xiong F.,Liu J.,Jiang J.,Zhong R.,He L.,Han Z.,Li Z.,Tang X.,and Tang R.,2013,Molecular profiling of genetic variability in domesticated groundnut(Arachis hypogaea L.)based on ISJ,URP,and DAMD markers,Biochem.Genet.,51(11-12):889-900
Xiong F.Q.,Jiang J.,Zhong R.C.,Han Z.Q.,He L.Q.,Li Z.,Zhuang W.J.,and Tang R.H.,2010,Application of SCo Tmolecular markers in genus Arachis,Zuowu Xuebao(Acta Agronomica Sinica),36(12):2055-2061(熊发前,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华,2010,目标起始密码子多态性(SCoT)分子标记技术在花生属中的应用,作物学报,36(12):2055-2061)
Xiong F.Q.,Liu J.X.,He L.Q.,Han Z.Q.,Huang Z.P.,Tang X.M.,Jiang J.,Zhong R.C.,Wu H.N.,Li Z.,Luo S.Y.,Tang R.H.,and He X.H.,2017,Recent advances on the development and utilization of molecular markers based on LTR retrotransposons and MITE transposons from peanut(Arachis hypogaea L.),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(2):640-647(熊发前,刘俊仙,贺梁琼,韩柱强,黄志鹏,唐秀梅,蒋菁,钟瑞春,吴海宁,李忠,罗赛云,唐荣华,何新华,2017,花生LTR和MITE转座子及其分子标记开发利用研究进展,分子植物育种,15(2):640-647)
Yan J.Y.,Ma C.Q.,Chang B.,Fan X.G.,Li Z.,Yang Y.Z.,and Zhao Z.Y.,2017,A modified CTAB method for genomic DNA extraction from apple fruit,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(9):3610-3615(闫玖英,马长青,常博,范献光,李征,杨亚州,赵政阳,2017,改良CTAB法用于苹果果实基因组DNA的提取,分子植物育种,15(9):3610-3615)
Zhou H.,Yan C.X.,Guo L.C.,Liu Y.,and Shan S.H.,2012,Rapid extraction of genomic DNA from peanut by CTABmethod,Shandong Nongye Kexue(Shandong Agricultural Sciences),44(7):8-9,15(周浩,闫彩霞,郭凌超,刘宇,单世华,2012,CTAB法少量快速提取花生基因组DNA,山东农业科学,44(7):8-9,15)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored poly merase chain reaction amplification,Genomics,20(2):176-183