用于克隆及分子标记分析的甘蔗高质量基因组DNA提取方法
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摘要
甘蔗是世界上重要的糖料作物,其遗传背景复杂,基因组庞大复杂。但基因组测序尚未完成,严重制约了甘蔗分子生物学研究进展。为构建成熟完善的甘蔗高质量基因组DNA提取方法,本研究采用四种改良CTAB法提取甘蔗基因组DNA,先通过紫外分光光度计和琼脂糖凝胶电泳检测提取所得甘蔗基因组DNA的质量,再使用8对转座子保守区同源克隆扩增引物和8种基于单引物扩增反应的分子标记技术的78条单引物对提取的甘蔗基因组DNA的质量进行扩增验证。结果表明:(1)结合甘蔗基因组DNA的质量数据以及甘蔗转座子保守区同源克隆扩增和分子标记技术研究的验证结果来看,四种改良CTAB法的DNA提取效果表现依次为:方法三>方法四>方法二>方法一。但方法三和方法四均使用到强腐蚀性的平衡酚而不安全环保,而方法二和方法一则分别使用了二次和一次氯仿抽提,根据样品的实际状态,本研究认为后两者中的一种可作为甘蔗基因组DNA的最佳提取方法;(2)建立了甘蔗6类转座子保守区同源克隆和8种基于单引物扩增反应的分子标记技术的扩增体系。本研究为以后开展甘蔗转座子的克隆鉴定利用和分子标记研究提供了帮助。
Sugarcane is an important sugar crop in the world. Its genetic background is complex and its genome is huge and complex. However, due to incomplete genome sequencing, the progress of molecular biology in sugarcane has been restricted seriously. In order to build a mature and perfect method for extracting sugarcane genomic DNA with high quality, four improved CTAB methods were used to extract sugarcane genomic DNA. The quality of the extracted sugarcane genomic DNA was first detected by UV spectrophotometer and agarose gel electrophoresis,respectively. Then, we used 8 pairs of transposon conserved region homolog cloning and amplification primers and78 single primers of 8 kinds of molecule marker technologies based on single primer amplification reaction to amplify and verify the quality of the obtained sugarcane genomic DNA. The results showed as follows:(1) According to the quality data of sugarcane genomic DNA, the verification results of homolog cloning and amplification of sugarcane transposon conserved region and molecular marker techniques, the DNA extraction effects of the four improved CTAB methods were as follows: Method three>Method four>Method two>Method one, but the use of highly corrosive equilibrium phenol in the method three and four was not safe and environmental protection, while method two and one used chloroform extraction twice and once respectively. Finally, the author thought that we should select method two or one as the optimum extraction method according to the actual state of sugarcane sample;(2) The amplification system of six types of homolog cloning and amplification of transposon conserved region and eight kinds of molecular marker technologies based on single primer amplification reaction in sugarcane has been established. This study would be helpful for the cloning, identification and utilization of sugarcane transposon and molecular marker research.
Sugarcane is an important sugar crop in the world. Its genetic background is complex and its genome is huge and complex. However, due to incomplete genome sequencing, the progress of molecular biology in sugarcane has been restricted seriously. In order to build a mature and perfect method for extracting sugarcane genomic DNA with high quality, four improved CTAB methods were used to extract sugarcane genomic DNA. The quality of the extracted sugarcane genomic DNA was first detected by UV spectrophotometer and agarose gel electrophoresis,respectively. Then, we used 8 pairs of transposon conserved region homolog cloning and amplification primers and78 single primers of 8 kinds of molecule marker technologies based on single primer amplification reaction to amplify and verify the quality of the obtained sugarcane genomic DNA. The results showed as follows:(1) According to the quality data of sugarcane genomic DNA, the verification results of homolog cloning and amplification of sugarcane transposon conserved region and molecular marker techniques, the DNA extraction effects of the four improved CTAB methods were as follows: Method three>Method four>Method two>Method one, but the use of highly corrosive equilibrium phenol in the method three and four was not safe and environmental protection, while method two and one used chloroform extraction twice and once respectively. Finally, the author thought that we should select method two or one as the optimum extraction method according to the actual state of sugarcane sample;(2) The amplification system of six types of homolog cloning and amplification of transposon conserved region and eight kinds of molecular marker technologies based on single primer amplification reaction in sugarcane has been established. This study would be helpful for the cloning, identification and utilization of sugarcane transposon and molecular marker research.
引文
Chen Z.L.,Qin C.X.,Guo Y.Y.,Yang C.F.,Luo H.B.,and He H.B.,2010,A rapid DNA extraction method from sugarcane leaves for SSR-PCR,Guangxi Nongye Kexue(Guangxi A-gricultural Sciences),41(11):1155-1157(陈忠良,秦翠鲜,郭元元,杨翠芳,罗海斌,何海波,2010,一种适用于甘蔗SSR-PCR的DNA快速提取方法,广西农业科学,41(11):1155-1157)
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 Molecular Biology Reporter,27(1):86-93
Feschotte C.,and Wessler S.R.,2002,Mariner-like transposases are widespread and diverse in flowering plants,Proc.Natl.Acad.Sci.USA,99(1):280-285
Guillemaut P.,and Drouard L.M.,1992,Isolation of plant DNA:A fast,inexpensive and reliable method,Plant Molecular Biology Reporter,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 retrotransposon isolation,Theor.Appl.Genet.,121(8):1419-1430
Kang H.W.,Park D.S.,Go S.J.,and Eun M.Y.,2002,Fingerprinting 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
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,Journal of Plant Biochemistry and Biotechnology,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.,2011,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.,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
Zhang X.,Feschotte C.,Zhang Q.,Jiang N.,Eggleston W.B.,and Wessler S.R.,2001,P instability factor:an active maize transposon system associated with the amplification of tourist-like MITEs and a new superfamily of transposases,Proc.Natl.Acad.Sci.USA,98(22):12572-12577
Zhang X.,Jiang N.,Feschotte C.,and Wessler S.R.,2004,PIF-and Pong-Like transposable elements:distribution,evolution and relationship with tourist-like miniature inverted-repeat transposable elements,Genetics,166(2):971-986
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
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 Molecular Biology Reporter,27(1):86-93
Feschotte C.,and Wessler S.R.,2002,Mariner-like transposases are widespread and diverse in flowering plants,Proc.Natl.Acad.Sci.USA,99(1):280-285
Guillemaut P.,and Drouard L.M.,1992,Isolation of plant DNA:A fast,inexpensive and reliable method,Plant Molecular Biology Reporter,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 retrotransposon isolation,Theor.Appl.Genet.,121(8):1419-1430
Kang H.W.,Park D.S.,Go S.J.,and Eun M.Y.,2002,Fingerprinting 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
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,Journal of Plant Biochemistry and Biotechnology,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.,2011,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.,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
Zhang X.,Feschotte C.,Zhang Q.,Jiang N.,Eggleston W.B.,and Wessler S.R.,2001,P instability factor:an active maize transposon system associated with the amplification of tourist-like MITEs and a new superfamily of transposases,Proc.Natl.Acad.Sci.USA,98(22):12572-12577
Zhang X.,Jiang N.,Feschotte C.,and Wessler S.R.,2004,PIF-and Pong-Like transposable elements:distribution,evolution and relationship with tourist-like miniature inverted-repeat transposable elements,Genetics,166(2):971-986
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
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