珍稀树种庙台槭不同组织DNA高效提取方法分析
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摘要
为探讨庙台槭不同组织DNA的高效提取方法,分别以叶片和果实为材料,以完全随机区组试验设计,设置不同浓度的β-巯基乙醇和不同浓度的PVP作为预处理,采用试剂盒法提取基因组DNA,提取后对基因组DNA进行浓度、分光光度值的测定以及PCR扩增,综合各项指标选择出效果最好的预处理方法,并进一步以CTAB法进行验证,之后应用于不同个体庙台槭的DNA提取中。结果发现:以庙台槭果实为材料,不需要预处理,提取的DNA即可满足下游实验需要;以叶片为材料,用0.5%β-巯基乙醇和1%PVP预处理能有效提高提取的DNA浓度和质量,经验证发现该预处理方法适用于CTAB法,且能够极显著地提高DNA提取的浓度和质量;进一步应用于所有132个庙台槭个体的总DNA提取,获得的DNA均能满足后续实验要求。本研究为进一步研究庙台槭遗传多样性等工作提供帮助,同时也能够为槭属其他植物的相关研究提供参考。
In order to study the effecient extraction methods of DNA from different tissues of Acer miaotaiense,DNA was extracted from leaves and fruits using kits, and completed randomized block design was used to set different concentrations of β-mercaptoethanol and PVP as pretreatments. Determination of concentrations,spectrophotometric values, and PCR amplification were performed on extracted genomic DNA and then these indicators were used to select the best pretreatment method. This pretreatment method was verified by the CTAB method, and applied in the DNA extraction of different individuals. The results showed that DNA extracted from fruits did not require pretreatment, and the extracted DNA could meet the needs of downstream experiments.Using leaves as material, 0.5% β-mercaptoethanol and 1% PVP were considered as effective to improve the concentration and quality of extracted DNA. It was found that the pretreatment method was suitable for CTAB and could significantly improve the concentration and quality of DNA extraction. It was further applied to the extraction of total DNA from all 132 individuals, and the obtained DNA all could meet the requirements of follow-up experiments. This study could lay a foundation for further research on the genetic diversity of Acer miaotaiense, and provide a reference for the related study on other Acer plants.
In order to study the effecient extraction methods of DNA from different tissues of Acer miaotaiense,DNA was extracted from leaves and fruits using kits, and completed randomized block design was used to set different concentrations of β-mercaptoethanol and PVP as pretreatments. Determination of concentrations,spectrophotometric values, and PCR amplification were performed on extracted genomic DNA and then these indicators were used to select the best pretreatment method. This pretreatment method was verified by the CTAB method, and applied in the DNA extraction of different individuals. The results showed that DNA extracted from fruits did not require pretreatment, and the extracted DNA could meet the needs of downstream experiments.Using leaves as material, 0.5% β-mercaptoethanol and 1% PVP were considered as effective to improve the concentration and quality of extracted DNA. It was found that the pretreatment method was suitable for CTAB and could significantly improve the concentration and quality of DNA extraction. It was further applied to the extraction of total DNA from all 132 individuals, and the obtained DNA all could meet the requirements of follow-up experiments. This study could lay a foundation for further research on the genetic diversity of Acer miaotaiense, and provide a reference for the related study on other Acer plants.
引文
Bai S.R.L.,Pang L.,Shi S.D.,and Wei L.,2014,Genomic DNAisolation from polysaccharides-rich Helianthemum soongoricum,Tianjin Nongye Kexue(Tianjin Agricultural Sciences),20(3):1-4(白沙如拉,庞磊,史树德,魏磊,2014,富含多糖的强旱生植物半日花基因组DNA提取方法,天津农业科学,20(3):1-4)
Chen P.,Li P.P.,Mai L.S.,Liu J.J.,He C.X.,Zhao S.J.,Wu X.Y.,and Chen Y.,2017,DNA molecular marker and its application in identification of seed varieties of beer barely,Liangshi Yu Siliao Gongye(Cereal&Feed Industry),(8):63-66(陈萍,李盼畔,麦丽珊,刘姣姣,何翠霞,赵淑嘉,吴小瑶,陈颖,2017,DNA分子标记技术及其在啤酒大麦品种鉴定中的应用,粮食与饲料工业,(8):63-66)
Cui C.,and Lin Y.D.,2008,The optimized method of extracting genomic DNA from leaves of Acer truncatum Bunge,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):118-121(崔翠,蔺银鼎,2008,元宝枫叶片总DNA提取方法的优化,生物技术通报,(4):118-121)
Du J.,Liu H.Y.,and Zou T.C.,2011,Analysis on main chemical components of seed and leaves of Acer guizhouense Y.K.Li,Zhongzi(Seed),30(4):94-96(杜娟,刘海燕,邹天才,2011,贵州槭种子和叶片主要化学成分分析,种子,30(4):94-96)
Healey A.,Furtado A.,Cooper T.,and Henry R.J.,2014,Protocol:a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species,Plant Methods,(10):21
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)
Li Q.Z.,Liu X.H.,Su J.L.,and Cong Y.,2009,Extraction of genomic DNA from Acer yangjuechi and system optimization of SRAP-PCR,Jiangsu Nongye Xuebao(Jiangsu Agricultural Journal),25(3):538-541(李倩中,刘晓宏,苏家乐,丛郁,2009,羊角槭基因组DNA提取及SRAP-PCR体系优化,江苏农业学报,25(3):538-541)
Li S.,Wang M.L.,and Zhao G.F.,2001,DNA extraction and characterization of Acer miaotaiense,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),21(2):232-236(李珊,王玛丽,赵桂仿,2001,庙台槭总DNA提取及鉴定,西北植物学报,21(2):232-236)
Li X.,Li M.,Hou L.,Zhang Z.Y.,Pang X.M.,and Li Y.Y.,2018,De novo transcriptome assembly and population genetic analyses for an endangered Chinese endemic Acer miaotaiense(Aceraceae),Genes,9(8):378
Ma X.H.,Wang L.Z.,and Wang S.Q.,1998,Advances in the researches of the chemical constituents of Aceraceous plants,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry College),13(1):87-95(马希汉,王兰珍,王姝清,1998,槭属植物化学成分研究评述,西北林学院学报,13(1):87-95)
Meng L.,Zhou L.,Zhang M.S.,and Dai S.L.,2006,An efficient and economic method for preparation total RNA of petals,Shengwu Jishu(Biotechnology),1(16):38-40(孟丽,周琳,张明姝,戴思兰,2006,一种有效的花瓣总RNA的提取方法,生物技术,1(16):38-40)
Meng Q.F.,Gao H.L.,Li H.X.,and Xu H.J.,2016,Biological and ecological characteristics of rare and endangered species of Acer miaotaiense,Henan Kexue(Henan Science),34(11):1830-1834(孟庆法,高红莉,李红喜,徐焕军,2016,珍稀濒危树种庙台槭的生物学及生态学特性研究,河南科学,34(11):1830-1834)
Meng Q.F.,Gao H.L.,and Wang H.Y.,2017,Fruiting characteristics and sowing seedlings of rare and endangered species of Acer miaotaiense,Henan Kexue(Henan Science),35(11):1773-1777(孟庆法,高红莉,王洪友,2017,珍稀濒危树种庙台槭结实特性及播种育苗试验研究,河南科学,35(11):1773-1777)
Sun H.L.,Lu Z.M.,and Zhang L.,2010,Advances in extraction and purification of plant genome DNA,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),25(6):102-106(孙璐宏,鲁周民,张丽,2010,植物基因组DNA提取与纯化研究进展,西北林学院学报,25(6):102-106)
Sun L.,2014,The genetic diversity and phylogeography of Litsea auriculata,an endemic and rare plant in China,Thesis for M.S.,Nanjing University,Supervisor:An S.Q.,pp.1-4(孙林,2014,中国特有珍稀植物天目木姜子遗传多样性与亲缘地理学研究,硕士学位论文,南京大学,导师:安树青,pp.1-4)
Vicient C.M.,and Delseny M.,1999,Isolation of total RNA from Arabidopsis thaliana seeds,Anal.Biochem.,268(2):412-413
Wang F.,2008,Analysis of community ecology and genetic diversity in the relic and rare plant Euptelea pleiospermum Hook.F.et Thoms.,Thesis for M.S.,Southwest University,Supervisor:He P.,pp.1-2(王芳,2008,孑遗珍稀植物领春木群落生态和遗传多样性研究,硕士学位论文,西南大学,导师:何平,pp.1-2)
Wang J.H.,Wang L.,and Wang J.S.,2015,Comparative on extraction method of total DNA from sun-dried fruits and dried leaves of Forsythia suspense,Shanxi Nongye Kexue(Journal of Shanxi Agricultural Sciences),43(12):1594-1597(王嘉荟,王琳,王金胜,2015,连翘生晒果实和干燥叶片总DNA提取方法比较,山西农业科学,43(12):1594-1597)
Wang Y.,2017,Wood identification for Lauraceae species by DNA technique,Thesis for M.S.,Nanjing Forestry University,Supervisor:Luo J.Y.,pp.36-37(王英,2017,基于DNA技术识别樟科木材,硕士学位论文,南京林业大学,导师:骆嘉言,pp.36-37)
Wang Z.W.,Yu M.D.,and Lu C.,2001,Application of PVP in total DNA extraction in mulberry,Xinan Nongye Daxue Xuebao(Journal of Southwest Agricultural University),23(1):61-65(王卓伟,余茂德,鲁成,2001,PVP在桑叶总DNA提取中的应用,西南农业大学学报,23(1):61-65)
Wei D.L.,2017,Study on sowing and seedling rising of rare species of Acer miaotaiense,Gansu Linye Keji(Journal of Gansu Forestry Science and Technology),42(2):37-39(魏等郎,2017,珍稀树种庙台槭播种育苗研究,甘肃林业科技,42(2):37-39)
Yan G.Q.,Ren Y.,Zhang B.H.,and Li Z.Q.,2004,The extraction and purity characterization of genome DNA from leaf-tissue of three ligneous plants,Shanxi Shifan Daxue Xuebao(Journal of Shanxi Normal University(Natural Science Edition)),18(1):71-77(闫桂琴,任鹰,张变红,李钻青,2004,三种木本植物基因组DNA的提取及纯度检测,山西师范大学学报(自然科学版),18(1):71-77)
Yang H.Y.,2017,Protocol for automated DNA extraction and detection of QTLs related with lignin in Eucalyptus,Thesis for M.S.,Chinese Academy of Forestry Sciences,Supervisor:Gan S.M.,pp.3(杨合宇,2017,桉树DNA自动提取及木素相关QTL定位,硕士学位论文,中国林业科学研究院,导师:甘四明,pp.3)
Ye Z.Y.,2017,Studies on genetic diversity and mating system of Elaeagusmollis diels,Thesis for M.S.,Chinese Academy of Forestry Sciences,Supervisor:Zhang J.G.,pp.1-4(叶占洋,2017,翅果油树遗传多样性和交配系统研究,硕士学位论文,中国林业科学研究院,导师:张建国,pp.1-4)
Yi Q.P.,2006,Establishment of DNA extraction,purification and quantification technique system in Diospyros spp.,Thesis for M.S.,Huazhong Agri-cultural University,Supervisor:Luo Z.R.,pp.29-34(易庆平,2006,柿属植物DNA提取纯化检测技术体系的建立,硕士学位论文,华中农业大学,导师:罗正荣,pp.29-34)
Zhang B.,Zhang L.,Zhu G.Q.,Wang M.X.,and Huang M.R.,2004,A rapid and simple method of total DNA extraction from tree,Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University(Natural Sciences Edition),28(1):13-16(张博,张露,诸葛强,王明庥,黄敏仁,2004,一种高效的树木总DNA提取方法,南京林业大学学报(自然科学版),28(1):13-16)
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Zhang J.H.,2006,Resources and protection of rare and endangered tree species in Xiaolongshan forest area in west section of Qinling district,Gansu Linye Keji(Journal of Gansu Forestry Science and Technology),31(2):12-15(张建华,2006,秦岭西段小陇山林区珍稀濒危树种资源及保护,甘肃林业科技,31(2):12-15)
Zheng Y.K.,Hu X.Y.,Song X.Q.,and Wang J.,2015,Optimized extraction method for genomic DNA from Dendrobium species(Orchidaceae),Redai Shengwu Xuebao(Journal of Tropical Organisms),6(2):168-172(郑云柯,胡翔宇,宋希强,王健,2015,石斛属植物基因组DNA提取方法的对比,热带生物学报,6(2):168-172)
Zhou H.L.,Li S.P.,Li W.L.,He J.H.,Bao D.H.,and Li M.F.,2016,DNA extraction,optimization of SSR-PCR reaction system and primer screening of Persea americana,Shengwu Jishu Tongbao(Biotechnology Bulletin),32(4):143-150(周海兰,李绍鹏,李卫亮,贺军虎,包冬红,李茂富,2016,油梨基因组DNA提取、SSR-PCR反应体系优化及引物筛选,生物技术通报,32(4):143-150)
Zhou X.W.,Liu H.Y.,Wang N.,Nan H.,Zhao S.W.,Wei Z.P.,Yue W.Y.,Zhang Y.H.,Song J.R.,Cao J.R.,and Cao S.Q.,2017,Application review of DNA molecular markers technique in wheat genetic breeding,Gansu Nongye Keji(Gansu Agricultural Science and Technology),(5):64-68(周喜旺,刘鸿燕,王娜,南海,赵尚文,魏志平,岳维云,张耀辉,宋建荣,曹世勤,2017,DNA分子标记技术在小麦遗传育种中的应用综述,甘肃农业科技,(5):64-68)
Chen P.,Li P.P.,Mai L.S.,Liu J.J.,He C.X.,Zhao S.J.,Wu X.Y.,and Chen Y.,2017,DNA molecular marker and its application in identification of seed varieties of beer barely,Liangshi Yu Siliao Gongye(Cereal&Feed Industry),(8):63-66(陈萍,李盼畔,麦丽珊,刘姣姣,何翠霞,赵淑嘉,吴小瑶,陈颖,2017,DNA分子标记技术及其在啤酒大麦品种鉴定中的应用,粮食与饲料工业,(8):63-66)
Cui C.,and Lin Y.D.,2008,The optimized method of extracting genomic DNA from leaves of Acer truncatum Bunge,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):118-121(崔翠,蔺银鼎,2008,元宝枫叶片总DNA提取方法的优化,生物技术通报,(4):118-121)
Du J.,Liu H.Y.,and Zou T.C.,2011,Analysis on main chemical components of seed and leaves of Acer guizhouense Y.K.Li,Zhongzi(Seed),30(4):94-96(杜娟,刘海燕,邹天才,2011,贵州槭种子和叶片主要化学成分分析,种子,30(4):94-96)
Healey A.,Furtado A.,Cooper T.,and Henry R.J.,2014,Protocol:a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species,Plant Methods,(10):21
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)
Li Q.Z.,Liu X.H.,Su J.L.,and Cong Y.,2009,Extraction of genomic DNA from Acer yangjuechi and system optimization of SRAP-PCR,Jiangsu Nongye Xuebao(Jiangsu Agricultural Journal),25(3):538-541(李倩中,刘晓宏,苏家乐,丛郁,2009,羊角槭基因组DNA提取及SRAP-PCR体系优化,江苏农业学报,25(3):538-541)
Li S.,Wang M.L.,and Zhao G.F.,2001,DNA extraction and characterization of Acer miaotaiense,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),21(2):232-236(李珊,王玛丽,赵桂仿,2001,庙台槭总DNA提取及鉴定,西北植物学报,21(2):232-236)
Li X.,Li M.,Hou L.,Zhang Z.Y.,Pang X.M.,and Li Y.Y.,2018,De novo transcriptome assembly and population genetic analyses for an endangered Chinese endemic Acer miaotaiense(Aceraceae),Genes,9(8):378
Ma X.H.,Wang L.Z.,and Wang S.Q.,1998,Advances in the researches of the chemical constituents of Aceraceous plants,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry College),13(1):87-95(马希汉,王兰珍,王姝清,1998,槭属植物化学成分研究评述,西北林学院学报,13(1):87-95)
Meng L.,Zhou L.,Zhang M.S.,and Dai S.L.,2006,An efficient and economic method for preparation total RNA of petals,Shengwu Jishu(Biotechnology),1(16):38-40(孟丽,周琳,张明姝,戴思兰,2006,一种有效的花瓣总RNA的提取方法,生物技术,1(16):38-40)
Meng Q.F.,Gao H.L.,Li H.X.,and Xu H.J.,2016,Biological and ecological characteristics of rare and endangered species of Acer miaotaiense,Henan Kexue(Henan Science),34(11):1830-1834(孟庆法,高红莉,李红喜,徐焕军,2016,珍稀濒危树种庙台槭的生物学及生态学特性研究,河南科学,34(11):1830-1834)
Meng Q.F.,Gao H.L.,and Wang H.Y.,2017,Fruiting characteristics and sowing seedlings of rare and endangered species of Acer miaotaiense,Henan Kexue(Henan Science),35(11):1773-1777(孟庆法,高红莉,王洪友,2017,珍稀濒危树种庙台槭结实特性及播种育苗试验研究,河南科学,35(11):1773-1777)
Sun H.L.,Lu Z.M.,and Zhang L.,2010,Advances in extraction and purification of plant genome DNA,Xibei Linxueyuan Xuebao(Journal of Northwest Forestry University),25(6):102-106(孙璐宏,鲁周民,张丽,2010,植物基因组DNA提取与纯化研究进展,西北林学院学报,25(6):102-106)
Sun L.,2014,The genetic diversity and phylogeography of Litsea auriculata,an endemic and rare plant in China,Thesis for M.S.,Nanjing University,Supervisor:An S.Q.,pp.1-4(孙林,2014,中国特有珍稀植物天目木姜子遗传多样性与亲缘地理学研究,硕士学位论文,南京大学,导师:安树青,pp.1-4)
Vicient C.M.,and Delseny M.,1999,Isolation of total RNA from Arabidopsis thaliana seeds,Anal.Biochem.,268(2):412-413
Wang F.,2008,Analysis of community ecology and genetic diversity in the relic and rare plant Euptelea pleiospermum Hook.F.et Thoms.,Thesis for M.S.,Southwest University,Supervisor:He P.,pp.1-2(王芳,2008,孑遗珍稀植物领春木群落生态和遗传多样性研究,硕士学位论文,西南大学,导师:何平,pp.1-2)
Wang J.H.,Wang L.,and Wang J.S.,2015,Comparative on extraction method of total DNA from sun-dried fruits and dried leaves of Forsythia suspense,Shanxi Nongye Kexue(Journal of Shanxi Agricultural Sciences),43(12):1594-1597(王嘉荟,王琳,王金胜,2015,连翘生晒果实和干燥叶片总DNA提取方法比较,山西农业科学,43(12):1594-1597)
Wang Y.,2017,Wood identification for Lauraceae species by DNA technique,Thesis for M.S.,Nanjing Forestry University,Supervisor:Luo J.Y.,pp.36-37(王英,2017,基于DNA技术识别樟科木材,硕士学位论文,南京林业大学,导师:骆嘉言,pp.36-37)
Wang Z.W.,Yu M.D.,and Lu C.,2001,Application of PVP in total DNA extraction in mulberry,Xinan Nongye Daxue Xuebao(Journal of Southwest Agricultural University),23(1):61-65(王卓伟,余茂德,鲁成,2001,PVP在桑叶总DNA提取中的应用,西南农业大学学报,23(1):61-65)
Wei D.L.,2017,Study on sowing and seedling rising of rare species of Acer miaotaiense,Gansu Linye Keji(Journal of Gansu Forestry Science and Technology),42(2):37-39(魏等郎,2017,珍稀树种庙台槭播种育苗研究,甘肃林业科技,42(2):37-39)
Yan G.Q.,Ren Y.,Zhang B.H.,and Li Z.Q.,2004,The extraction and purity characterization of genome DNA from leaf-tissue of three ligneous plants,Shanxi Shifan Daxue Xuebao(Journal of Shanxi Normal University(Natural Science Edition)),18(1):71-77(闫桂琴,任鹰,张变红,李钻青,2004,三种木本植物基因组DNA的提取及纯度检测,山西师范大学学报(自然科学版),18(1):71-77)
Yang H.Y.,2017,Protocol for automated DNA extraction and detection of QTLs related with lignin in Eucalyptus,Thesis for M.S.,Chinese Academy of Forestry Sciences,Supervisor:Gan S.M.,pp.3(杨合宇,2017,桉树DNA自动提取及木素相关QTL定位,硕士学位论文,中国林业科学研究院,导师:甘四明,pp.3)
Ye Z.Y.,2017,Studies on genetic diversity and mating system of Elaeagusmollis diels,Thesis for M.S.,Chinese Academy of Forestry Sciences,Supervisor:Zhang J.G.,pp.1-4(叶占洋,2017,翅果油树遗传多样性和交配系统研究,硕士学位论文,中国林业科学研究院,导师:张建国,pp.1-4)
Yi Q.P.,2006,Establishment of DNA extraction,purification and quantification technique system in Diospyros spp.,Thesis for M.S.,Huazhong Agri-cultural University,Supervisor:Luo Z.R.,pp.29-34(易庆平,2006,柿属植物DNA提取纯化检测技术体系的建立,硕士学位论文,华中农业大学,导师:罗正荣,pp.29-34)
Zhang B.,Zhang L.,Zhu G.Q.,Wang M.X.,and Huang M.R.,2004,A rapid and simple method of total DNA extraction from tree,Nanjing Linye Daxue Xuebao(Journal of Nanjing Forestry University(Natural Sciences Edition),28(1):13-16(张博,张露,诸葛强,王明庥,黄敏仁,2004,一种高效的树木总DNA提取方法,南京林业大学学报(自然科学版),28(1):13-16)
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