一种快速高效的油茶叶片DNA提取方法
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摘要
油茶叶片含有较多的多糖和多酚等次生代谢产物是提取DNA的重要障碍。本研究以油茶叶片为材料研究高质量的DNA提取方法。建立了一种改良的CTAB法,运用2×CTAB缓冲液来裂解细胞,在裂解细胞后的溶液中加入终浓度为2 mol/L Li Cl,用氯仿异戊醇抽提溶液,用等体积的异丙醇沉淀DNA,用预热的70%乙醇洗涤DNA沉淀。运用紫外分析和琼脂糖凝胶电泳分析测定了DNA的产率和质量,并与经典SDS法、经典CTAB法、高盐低p H法进行了对比。结果表明,改良的CTAB法提取的DNA产率比经典SDS的少,与经典CTAB法和高盐低pH法的相似,达到39.0μg/g,且纯度高,无RNA的污染。改良的CTAB法提取的叶片DNA可用于油茶后续的PCR分析研究。
The secondary met abolites existed in leaves of Camellia oleifera, such as polysaccharides and polyphenols, are important obstacles in the extraction of DNA. Using Camellia oleifera leaves as materials, a method to extract high quality DNA was studied in this research. An improved CTAB method was provided to lyse cells in2 ×CTAB solution. The final concentration of 2 mol/L Li Cl was added into the lysate. The lysate solution was extracted with chloroform iso-amyl alcohol, and DNA was precipitated by isopropanol of equal volume. The DNA precipitation was washed by preheated 70% ethanol. The yield and quality of DNA were determined through ultraviolet analysis and agarose gel electrophoresis analysis, and were compared with the results obtained from the classic CTAB method, classic SDS method, classic CTAB method, high salt and low pH method. The results showed that the yield of DNA extracted by the improved CTAB method was less than that by classic SDS method,but was similar to that by classic CTAB method and high salt and low p H method, which was about 39.0 μg/g with high purity and no RNA pollution. The leaf DNA extracted by the improved CTAB method is suitable for the following PCR works for Camellia oleifera.
The secondary met abolites existed in leaves of Camellia oleifera, such as polysaccharides and polyphenols, are important obstacles in the extraction of DNA. Using Camellia oleifera leaves as materials, a method to extract high quality DNA was studied in this research. An improved CTAB method was provided to lyse cells in2 ×CTAB solution. The final concentration of 2 mol/L Li Cl was added into the lysate. The lysate solution was extracted with chloroform iso-amyl alcohol, and DNA was precipitated by isopropanol of equal volume. The DNA precipitation was washed by preheated 70% ethanol. The yield and quality of DNA were determined through ultraviolet analysis and agarose gel electrophoresis analysis, and were compared with the results obtained from the classic CTAB method, classic SDS method, classic CTAB method, high salt and low pH method. The results showed that the yield of DNA extracted by the improved CTAB method was less than that by classic SDS method,but was similar to that by classic CTAB method and high salt and low p H method, which was about 39.0 μg/g with high purity and no RNA pollution. The leaf DNA extracted by the improved CTAB method is suitable for the following PCR works for Camellia oleifera.
引文
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Allen G.C.,Flores-vergara M.A.,Krasynanski S.,Kumar S.,and Thomps W.F.,2006,A modified protocol for rapid DNAisolation from plant tissues using cetyltrimethylammonium bromide,Nat.Protoc.,1(5):2320-2325
Ding H.,Zheng T.C.,Lang D.Y.,and Qu G.Z.,2015,A Simple and efficient method of DNA extraction from different plant samples,Zhiwu Yanjiu(Bulletin of Botanical Research),35(3):457-461(丁浩,郑唐春,梁德洋,曲冠证,2015,一种简易高效提取多种植物纯净DNA的方法,植物研究,35(3):457-461)
Huang D.L.,Qin X.L.,Liao Q.,Gao Y.J.,and Fang F.X.,2010,Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):101-106(黄东亮,覃肖良,廖青,高轶静,方锋学,2010,高质量甘蔗基因组DNA的简便快速提取方法研究,生物技术通报,(5):101-106)
Huang Y.F.,Chen X.M.,Lei Z.G.,Chen Y.Z.,and Peng S.F.,2004,Analysis of Camellia oleifera germplasm resources by RAPDⅠ.DNA extracting and establishment of PCR amplified condition,Henan Nongye Kexue(Henan agricultural sciences),(12):22-25(黄永芳,陈锡沐,雷治国,陈永忠,彭邵峰,2004,油茶种质资源RAPD分析Ⅰ.DNA提取和PCR扩增条件建立,河南农业科学,(12):22-25)
Huang Y.F.,Liao G.C.,Wu X.H.,An X.,Ruan Y.Y.,and Tan Y.K.,2009,DNA extraction of AFLP reaction system for Camellia oleifera,Guangdong Linye Keji(Guangdong Forestry Science and Technology),25(1):23-26(黄永芳,廖国春,吴雪辉,安星,阮瑶瑶,谭裕开,2009,一种适合AFLP分析的油茶DNA提取方法,广东林业科技,25(1):23-26)
Kim C.S.,Lee C.H.,Shin J.S.,Chung Y.S.,and Hyung N.I.,1997,A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP,Nucleic Acids Res.,25(5):1085-1086
Li J.J.,Zhou G.L.,Peng J.C.,Chen X.F.,Wang Z.X.,Ma H.Y.,Zhang Q.H.,and Cheng G.W.,2015,A fast process for cot ton DNA extraction,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):670-674(李婧婧,周桂林,彭家成,陈先锋,王兆贤,马惠应,张庆虎,程国旺,2015,一种棉花DNA快速提取方法,分子植物育种,13(3):670-674)
Li J.L.,Wang S.,Yu J.,Wang L.,and Zhou S.L.,2013,A modified CTAB protocol for plant DNA extraction,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):72-78(李金璐,王硕,于婧,王玲,周世良,2013,一种改良的植物DNA提取方法,植物学报,48(1):72-78)
Liu S.H.,Liu W.,Liu M.F.,Yang F.T.,Liu Q.S.,and Liang H.,2011,An efficient method for extracting DNA and RNAfrom kiwifruit leaves and flower buds,Shengwu Jishu Tongbao(Biotechnology Bulletin),(9):171-175(刘胜洪,刘文,刘明峰,杨凤婷,刘庆生,梁红,2011,一种高效提取猕猴桃DNA和RNA的方法,生物技术通报,(9):171-175)
Ma L.,2007,Comparison of camellia oil and olive oil in nutri tional value,Liangshi Yu Shipin Gongye(Cereal and Food Industry),14(6):19-21(马力,2007,茶油与橄榄油营养价值的比较,粮食与食品工业,14(6):19-21)
Pang X.A.,Bai B.W.,Jiang X.,Chen J.L.,and Jiao P.P.,2015,Total DNA extraction and RAPD system optimization of tamarix plants in tarim basin desert,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):2015-2023(庞新安,白宝伟,姜喜,陈加利,焦培培,2015,塔里木盆地荒漠区柽柳属植物总DNA提取及RAPD体系优化,基因组学与应用生物学,34(9):2015-2023)
Pich U.,and Schubert I.,1993,Miniprep method for isolation ofDNA from pla nts with a high content of polphenolics,Nucleic Acids Res.,21(14):3328-3332
Sangannavar P.A.,Vanti G.L.,Anupama S.,Methre R.M.,Bhute N.B.,Savita S.G.,Vamadevaiah,H.M.,and Katageri I.S.,2013,Economic,easy and fast method for isolating high quantity and quality DNA from cotton(gossypium spp.)suitable for molecular studies,J.Cell Tiss.Res.,13(1):3503-3506
Shepherd L.D.,and Mc Lay T.G.B.,2011,Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue,J.Plant Res.,124(2):311-314
Ukoskit K.,Thompson P.G.,Watson C.E.,and Lawrence G.W.,1997,Identifying a randomly amplified polymorphic DNA(RAPD)markers linked to gene for root knot nematode resistance in sweetpotato,J.Am.Sco.Hortic.Sci.,122(6):818-821
Whitlock R.,Hipperson H.,Mannarelli M,and Burke T.,2008,Ahigh-throughput protocol for extracting high-purity genomic DNA from plants and animals:technical advances,Mol.Ecol.Resour.,8(4):736-741
Xu L.W.,Wang F.G.,Zhao J.R.,and Yi H.M.,2009,Studies on genomic DNA extraction of maize with high salt,low p Hmethod,Yumi Kexue(Journal of Maize Science),17(1):59-61,70(许理文,王凤格,赵久然,易红梅,2009,高盐低p H值法提取玉米基因组DNA的研究,玉米科学,17(1):59-61,70)
Yang C.F.,Chen B.L.,Huang C.M.,and LüW.L.,2011,Isolation of genomic DNA and establishhment of ISSR reaction system for Camellia crepnelliana Tutch,Journal of Southern Agriculture,42(3):233-235
Yao D.,Yan W.,Guan S.Y.,Chang W.Y.,and Wang P.W.,2009,Extraction effect of genomic DNA from different tissues of Soybean with high-salt low-p H methods,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),(12):50-53(姚丹,闫伟,关淑艳,常唯一,王丕武,2009,高盐低p H值法提取大豆不同组织DNA的效果,河南农业科学,(12):50-53)
Yun Z.T.,Liao X.Y.,and Weng X.C.,2011,Comparison of physicochemical properties and fatty acids composition of tea seed oil Camellia Oleifera seed oil,Shipin Gongye Keji(Science and Technology of Food Industry),32(6):136-138(恽卓婷,廖鲜艳,翁新楚,2011,茶叶籽油与油茶籽油理化性质及脂肪酸组成比较,食品工业科技,32(6):136-138)
Zhang Z.J.,Tan X.F.,and Chen Y.Z.,2003,A simple method of simultaneous extraction of DNA and RNA from Camellia oleifera,Shengwu Jishu(Biotechnology),13(3):23-24(张智俊,谭晓风,陈永忠,2003,同时提取油茶中DNA和RNA的简便方法,生物技术,13(3):23-24)
Allen G.C.,Flores-vergara M.A.,Krasynanski S.,Kumar S.,and Thomps W.F.,2006,A modified protocol for rapid DNAisolation from plant tissues using cetyltrimethylammonium bromide,Nat.Protoc.,1(5):2320-2325
Ding H.,Zheng T.C.,Lang D.Y.,and Qu G.Z.,2015,A Simple and efficient method of DNA extraction from different plant samples,Zhiwu Yanjiu(Bulletin of Botanical Research),35(3):457-461(丁浩,郑唐春,梁德洋,曲冠证,2015,一种简易高效提取多种植物纯净DNA的方法,植物研究,35(3):457-461)
Huang D.L.,Qin X.L.,Liao Q.,Gao Y.J.,and Fang F.X.,2010,Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane,Shengwu Jishu Tongbao(Biotechnology Bulletin),(5):101-106(黄东亮,覃肖良,廖青,高轶静,方锋学,2010,高质量甘蔗基因组DNA的简便快速提取方法研究,生物技术通报,(5):101-106)
Huang Y.F.,Chen X.M.,Lei Z.G.,Chen Y.Z.,and Peng S.F.,2004,Analysis of Camellia oleifera germplasm resources by RAPDⅠ.DNA extracting and establishment of PCR amplified condition,Henan Nongye Kexue(Henan agricultural sciences),(12):22-25(黄永芳,陈锡沐,雷治国,陈永忠,彭邵峰,2004,油茶种质资源RAPD分析Ⅰ.DNA提取和PCR扩增条件建立,河南农业科学,(12):22-25)
Huang Y.F.,Liao G.C.,Wu X.H.,An X.,Ruan Y.Y.,and Tan Y.K.,2009,DNA extraction of AFLP reaction system for Camellia oleifera,Guangdong Linye Keji(Guangdong Forestry Science and Technology),25(1):23-26(黄永芳,廖国春,吴雪辉,安星,阮瑶瑶,谭裕开,2009,一种适合AFLP分析的油茶DNA提取方法,广东林业科技,25(1):23-26)
Kim C.S.,Lee C.H.,Shin J.S.,Chung Y.S.,and Hyung N.I.,1997,A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP,Nucleic Acids Res.,25(5):1085-1086
Li J.J.,Zhou G.L.,Peng J.C.,Chen X.F.,Wang Z.X.,Ma H.Y.,Zhang Q.H.,and Cheng G.W.,2015,A fast process for cot ton DNA extraction,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):670-674(李婧婧,周桂林,彭家成,陈先锋,王兆贤,马惠应,张庆虎,程国旺,2015,一种棉花DNA快速提取方法,分子植物育种,13(3):670-674)
Li J.L.,Wang S.,Yu J.,Wang L.,and Zhou S.L.,2013,A modified CTAB protocol for plant DNA extraction,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):72-78(李金璐,王硕,于婧,王玲,周世良,2013,一种改良的植物DNA提取方法,植物学报,48(1):72-78)
Liu S.H.,Liu W.,Liu M.F.,Yang F.T.,Liu Q.S.,and Liang H.,2011,An efficient method for extracting DNA and RNAfrom kiwifruit leaves and flower buds,Shengwu Jishu Tongbao(Biotechnology Bulletin),(9):171-175(刘胜洪,刘文,刘明峰,杨凤婷,刘庆生,梁红,2011,一种高效提取猕猴桃DNA和RNA的方法,生物技术通报,(9):171-175)
Ma L.,2007,Comparison of camellia oil and olive oil in nutri tional value,Liangshi Yu Shipin Gongye(Cereal and Food Industry),14(6):19-21(马力,2007,茶油与橄榄油营养价值的比较,粮食与食品工业,14(6):19-21)
Pang X.A.,Bai B.W.,Jiang X.,Chen J.L.,and Jiao P.P.,2015,Total DNA extraction and RAPD system optimization of tamarix plants in tarim basin desert,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):2015-2023(庞新安,白宝伟,姜喜,陈加利,焦培培,2015,塔里木盆地荒漠区柽柳属植物总DNA提取及RAPD体系优化,基因组学与应用生物学,34(9):2015-2023)
Pich U.,and Schubert I.,1993,Miniprep method for isolation ofDNA from pla nts with a high content of polphenolics,Nucleic Acids Res.,21(14):3328-3332
Sangannavar P.A.,Vanti G.L.,Anupama S.,Methre R.M.,Bhute N.B.,Savita S.G.,Vamadevaiah,H.M.,and Katageri I.S.,2013,Economic,easy and fast method for isolating high quantity and quality DNA from cotton(gossypium spp.)suitable for molecular studies,J.Cell Tiss.Res.,13(1):3503-3506
Shepherd L.D.,and Mc Lay T.G.B.,2011,Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue,J.Plant Res.,124(2):311-314
Ukoskit K.,Thompson P.G.,Watson C.E.,and Lawrence G.W.,1997,Identifying a randomly amplified polymorphic DNA(RAPD)markers linked to gene for root knot nematode resistance in sweetpotato,J.Am.Sco.Hortic.Sci.,122(6):818-821
Whitlock R.,Hipperson H.,Mannarelli M,and Burke T.,2008,Ahigh-throughput protocol for extracting high-purity genomic DNA from plants and animals:technical advances,Mol.Ecol.Resour.,8(4):736-741
Xu L.W.,Wang F.G.,Zhao J.R.,and Yi H.M.,2009,Studies on genomic DNA extraction of maize with high salt,low p Hmethod,Yumi Kexue(Journal of Maize Science),17(1):59-61,70(许理文,王凤格,赵久然,易红梅,2009,高盐低p H值法提取玉米基因组DNA的研究,玉米科学,17(1):59-61,70)
Yang C.F.,Chen B.L.,Huang C.M.,and LüW.L.,2011,Isolation of genomic DNA and establishhment of ISSR reaction system for Camellia crepnelliana Tutch,Journal of Southern Agriculture,42(3):233-235
Yao D.,Yan W.,Guan S.Y.,Chang W.Y.,and Wang P.W.,2009,Extraction effect of genomic DNA from different tissues of Soybean with high-salt low-p H methods,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),(12):50-53(姚丹,闫伟,关淑艳,常唯一,王丕武,2009,高盐低p H值法提取大豆不同组织DNA的效果,河南农业科学,(12):50-53)
Yun Z.T.,Liao X.Y.,and Weng X.C.,2011,Comparison of physicochemical properties and fatty acids composition of tea seed oil Camellia Oleifera seed oil,Shipin Gongye Keji(Science and Technology of Food Industry),32(6):136-138(恽卓婷,廖鲜艳,翁新楚,2011,茶叶籽油与油茶籽油理化性质及脂肪酸组成比较,食品工业科技,32(6):136-138)
Zhang Z.J.,Tan X.F.,and Chen Y.Z.,2003,A simple method of simultaneous extraction of DNA and RNA from Camellia oleifera,Shengwu Jishu(Biotechnology),13(3):23-24(张智俊,谭晓风,陈永忠,2003,同时提取油茶中DNA和RNA的简便方法,生物技术,13(3):23-24)