响应面法优化胆木基因组DNA的提取方法
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摘要
为了得到较高质量的胆木基因组DNA,并将该方法应用于其他茜草科植物。本研究采用DX8Trial软件分别对PVP、β-巯基乙醇、Na Cl、CTAB、EDTA等胆木DNA提取因素进行设计,同时加入醋酸钠进行单因素试验,确定胆木基因组DNA的最佳提取方案,并将该方案应用到6种茜草科植物进行基因组DNA提取。结果得出最优提取方案为5%PVP、3.25%β-巯基乙醇、4.24%NaCl、2%CTAB、3%EDTA,提取过程中加入75%乙醇720μL以及醋酸钠90μL,该方案同时也适用于6种茜草科植物基因组DNA提取。响应面法的应用为胆木植物DNA提取提供了依据,为其分子生药学研究提供依据。
To obtain high-quality genome DNA from N. officinalis and apply this method to other Rubiaceae plants. The extraction factors of N. officinalis DNA, such as PVP, β-mercaptoethanol, Nacl, CTAB, EDTA, etc.,were designed by DX8 Trial software, respectively, and sodium acetate was added as the single factor test to determine the optimal extraction scheme for genome DNA from N. officinalis and apply the scheme to the extraction of genome DNA from 6 Rubiaceae plants. The results showed that the optimal extraction plan was 5%PVP, 3.25% beta-mercaptoethanol, 4.24% Nacl, 2% CTAB, and 3% EDTA. 720 μL 75% ethanol and 90 μL sodium acetate were added in the extraction process. The scheme was also suitable for the genome DNA extraction of other 6 kinds of Rubiaceae plants. The application of response surface methodology could provide a basis for DNA extraction of N. officinalis plants and for its molecular pharmacognosy research.
To obtain high-quality genome DNA from N. officinalis and apply this method to other Rubiaceae plants. The extraction factors of N. officinalis DNA, such as PVP, β-mercaptoethanol, Nacl, CTAB, EDTA, etc.,were designed by DX8 Trial software, respectively, and sodium acetate was added as the single factor test to determine the optimal extraction scheme for genome DNA from N. officinalis and apply the scheme to the extraction of genome DNA from 6 Rubiaceae plants. The results showed that the optimal extraction plan was 5%PVP, 3.25% beta-mercaptoethanol, 4.24% Nacl, 2% CTAB, and 3% EDTA. 720 μL 75% ethanol and 90 μL sodium acetate were added in the extraction process. The scheme was also suitable for the genome DNA extraction of other 6 kinds of Rubiaceae plants. The application of response surface methodology could provide a basis for DNA extraction of N. officinalis plants and for its molecular pharmacognosy research.
引文
Box G.E.P.,and Wilson K.B.,1951,On the experimental attainment of optimum conditions,Journal of the Royal Statistical Society Series B,(1):1-45
Dong Y.L.,Jia Q.C.,Ji Q.Y.,Xiao T.Z.,Jie S.,Cui H.J.,Jing W.,Hao Z.,Xiao B.J.,and Fen X.Z.,2017,Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard,and molecular mechanism of strictosamide via suppressing the NF-κB and MAPK signaling pathway in LPS-induced RAW264.7 macrophages,J.Ethnopharmacol.,196:66-74
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li N.,Cheng L.,Cheng Y.,Meng Z.Q.,Tang Z.H.,Liu W.J.,Wang Z.Z.,Ding G.,and Xiao W.,2014,In vivo anti-inflammatory and analgesic activities of strictosamide from Nauclea officinalis,Pharmaceutical Biology,52(11):1445-1450
Liew S.Y.,Khaw K.Y.,Murugaiyah V.,Looi C.Y.,Wong Y.L.,Mustafa M.R.,Litaudon M.,and Awang K.,2015,Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis,Phytomedicine,22(1):45-48
Xing S.S.,2015,Inter-specific association and vertical distribution among key medicinal plants of three countries in Southwest of Hainan Island,Thesis for M.S.,Hainan University,Supervisor:Yang X.B.,pp.91(邢莎莎,2015,海南省西南部三市县重点药用植物的垂直分布规律及其种间关联性研究,硕士学位论文,海南大学,导师:杨小波,pp.91)
Yan G.Z.,Zheng G.C.,Wang W.,and Liu P.,2009,Comparative study ofβ-mercaptoethanol and PVP on DNA extraction from red pepper,Neimenggu Minzu Daxue Xuebao(Ziran Kexue Ban)(Journal of Inner Mongolia University for Nationalities(Natural Sciences)),24(1):51-54,64(闫光照,郑根昌,王伟,刘鹏,2009,β-巯基乙醇与PVP在红干椒DNA提取中的比较研究,内蒙古民族大学学报(自然科学版),24(1):51-54,64)
Yan J.,Luo J.,and Wang X.G.,2011,A modified CTAB method for extraction of genome DNA from Glechoma longituba,Wuhan Daxue Xuebao(Medical Journal of Wuhan University),32(4):432-435(鄢嘉,罗娟,王小刚,2011,改良CTAB法提取连钱草基因组DNA,武汉大学学报,32(4):432-435)
Yang W.G.,Xie G.H.,Yan W.H.,Zhou X.Y.,and Lu J.F.,2010,Optimization of wet-salting technology for Muraenesox cinereus using response surface analysis,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),10(1):133-139(杨文鸽,谢果凰,颜伟华,周星宇,卢佳芳,2010,响应面分析法优化海鳗的湿腌工艺,中国食品学报,10(1):133-139)
Yang W.L.,Lai W.Y.,Gao J.L.,and Liu X.,2012,Study on the determination method of total alkaloids in Nauclea officinalis,Zhongguo Dangdai Yiyao(China Modern Medicine),19(18):67-68(杨卫丽,赖伟勇,高靖淋,刘侠,2012,胆木总生物碱的含量测定方法研究,中国当代医药,19(18):67-68)
Yang W.L.,Liu M.S.,Mao C.N.,Lai W.Y.,and Zhang J.Q.,2008,Pharmacognostic studies on Nauclea officinalis,Zhongyaocai(Journal of Chinese Medicinal Materials),31(9):1324-1325(杨卫丽,刘明生,毛彩霓,赖伟勇,张俊清,2008,胆木的生药学研究,中药材,31(9):1324-1325)
Zhu T.T.,Jin L.,Du T.,Chen H.G.,Zhang Y.H.,Wang H.Z.,and Wang Y.,2012,Comparison of different methods for extraction of genomic DNA from Ephedra intermedia,Zhongguo Shiyan Fangjixue Zazhi(Chinese Journal of Experimental Traditional Medical Formulae),18(11):125-128(朱田田,晋玲,杜弢,陈红刚,张延红,王惠珍,王艳,2012,中麻黄基因组DNA不同提取方法的比较,中国实验方剂学杂志,18(11):125-128)
Dong Y.L.,Jia Q.C.,Ji Q.Y.,Xiao T.Z.,Jie S.,Cui H.J.,Jing W.,Hao Z.,Xiao B.J.,and Fen X.Z.,2017,Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard,and molecular mechanism of strictosamide via suppressing the NF-κB and MAPK signaling pathway in LPS-induced RAW264.7 macrophages,J.Ethnopharmacol.,196:66-74
Lan B.X.,Wang L.,Wu Z.K.,and Jiang B.L.,2015,Rapid miniprep extraction of genomic DNA from micro-endosperm maize with modified CTAB method,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):190-194(蓝碧秀,王凛,吴子恺,姜伯乐,2015,利用改良CTAB法快速小量提取微胚乳玉米基因组DNA,基因组学与应用生物学,34(1):190-194)
Li N.,Cheng L.,Cheng Y.,Meng Z.Q.,Tang Z.H.,Liu W.J.,Wang Z.Z.,Ding G.,and Xiao W.,2014,In vivo anti-inflammatory and analgesic activities of strictosamide from Nauclea officinalis,Pharmaceutical Biology,52(11):1445-1450
Liew S.Y.,Khaw K.Y.,Murugaiyah V.,Looi C.Y.,Wong Y.L.,Mustafa M.R.,Litaudon M.,and Awang K.,2015,Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis,Phytomedicine,22(1):45-48
Xing S.S.,2015,Inter-specific association and vertical distribution among key medicinal plants of three countries in Southwest of Hainan Island,Thesis for M.S.,Hainan University,Supervisor:Yang X.B.,pp.91(邢莎莎,2015,海南省西南部三市县重点药用植物的垂直分布规律及其种间关联性研究,硕士学位论文,海南大学,导师:杨小波,pp.91)
Yan G.Z.,Zheng G.C.,Wang W.,and Liu P.,2009,Comparative study ofβ-mercaptoethanol and PVP on DNA extraction from red pepper,Neimenggu Minzu Daxue Xuebao(Ziran Kexue Ban)(Journal of Inner Mongolia University for Nationalities(Natural Sciences)),24(1):51-54,64(闫光照,郑根昌,王伟,刘鹏,2009,β-巯基乙醇与PVP在红干椒DNA提取中的比较研究,内蒙古民族大学学报(自然科学版),24(1):51-54,64)
Yan J.,Luo J.,and Wang X.G.,2011,A modified CTAB method for extraction of genome DNA from Glechoma longituba,Wuhan Daxue Xuebao(Medical Journal of Wuhan University),32(4):432-435(鄢嘉,罗娟,王小刚,2011,改良CTAB法提取连钱草基因组DNA,武汉大学学报,32(4):432-435)
Yang W.G.,Xie G.H.,Yan W.H.,Zhou X.Y.,and Lu J.F.,2010,Optimization of wet-salting technology for Muraenesox cinereus using response surface analysis,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),10(1):133-139(杨文鸽,谢果凰,颜伟华,周星宇,卢佳芳,2010,响应面分析法优化海鳗的湿腌工艺,中国食品学报,10(1):133-139)
Yang W.L.,Lai W.Y.,Gao J.L.,and Liu X.,2012,Study on the determination method of total alkaloids in Nauclea officinalis,Zhongguo Dangdai Yiyao(China Modern Medicine),19(18):67-68(杨卫丽,赖伟勇,高靖淋,刘侠,2012,胆木总生物碱的含量测定方法研究,中国当代医药,19(18):67-68)
Yang W.L.,Liu M.S.,Mao C.N.,Lai W.Y.,and Zhang J.Q.,2008,Pharmacognostic studies on Nauclea officinalis,Zhongyaocai(Journal of Chinese Medicinal Materials),31(9):1324-1325(杨卫丽,刘明生,毛彩霓,赖伟勇,张俊清,2008,胆木的生药学研究,中药材,31(9):1324-1325)
Zhu T.T.,Jin L.,Du T.,Chen H.G.,Zhang Y.H.,Wang H.Z.,and Wang Y.,2012,Comparison of different methods for extraction of genomic DNA from Ephedra intermedia,Zhongguo Shiyan Fangjixue Zazhi(Chinese Journal of Experimental Traditional Medical Formulae),18(11):125-128(朱田田,晋玲,杜弢,陈红刚,张延红,王惠珍,王艳,2012,中麻黄基因组DNA不同提取方法的比较,中国实验方剂学杂志,18(11):125-128)