HPLC-MS/MS法测定烟叶中7种植物内源激素
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摘要
为获得烟草激素的质量分数数据,并分析其在抗性、生长发育等过程中的作用,采用高效液相色谱-三重四极杆质谱(HPLC-MS/MS)仪建立了同时分析烟叶中吲哚乙酸(IAA)、吲哚丁酸(IBA)、水杨酸(SA)、赤霉素(GA)、茉莉酸(JA)、脱落酸(ABA)和油菜素内酯(BR)等7种植物激素质量分数的方法。采用多离子反应检测模式,获得7种植物激素的特征碎片离子峰,并测定了烟草花叶病毒(TMV)感染的烟叶样品。结果表明:(1)7种植物激素在线性范围内的相关系数(r)为0.998 6～0.999 5,方法的检出限为0.07～3.00μg/L,日内日间相对标准偏差(RSD)为0.44%～9.76%,回收率为77.82%～117.63%;(2)烟草花叶病毒感染极显著(p<0.01)地增加了烟叶中SA、IBA、JA、ABA的质量分数,显著(p<0.05)增加了烟叶中IAA的质量分数,同时显著(p<0.05)降低了烟叶中GA的质量分数;TMV病毒感染对BR的影响不大。该方法具有良好的灵敏度、精密度和回收率,可用于植物相关激素的同时分析。
In order to determine the contents of endogenous phytohormones in tobacco and analyze the roles of phytohormones in the resistance,growth and development of tobacco,a method for simultaneously determining the contents of 7 endogenous phytohormones,including 3-indoleacetic acid(IAA),3-indolebutytic acid(IBA),salicylic acid(SA),gibberellin A3(GA),jasmonic acid(JA),abscisic acid(ABA) and brassinosteroids(BR) in tobacco was developed by high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The TMV(tobacco mosaic virus)-infected tobacco samples were determined by obtaining the characteristic fragment ion peaks of the 7 endogenous phytohormones under multiple reaction monitoring mode.The results showed that:1) The correlation coefficients(r) of the 7 phytohormones were from 0.998 6 to 0.999 5 in the linear ranges.The limits of quantification(LOQ) for all analytes were between0.07 and 3.00 μg/L,and the intra-and inter-day relative standard deviations(RSDs) were 0.44%-9.76% for all analytes.The recoveries were in the range of 77.82%-117.63%.2) The contents of SA,IBA,JA,ABA in TMV-infected tobacco were significantly higher(p<0.01) than the control sample,while which was significantly lower(p<0.05) for GA.However the content of BR was basically unchanged.With good sensitivity,precision and recovery,this method is suitable for determining various endogenous phytohormones.
In order to determine the contents of endogenous phytohormones in tobacco and analyze the roles of phytohormones in the resistance,growth and development of tobacco,a method for simultaneously determining the contents of 7 endogenous phytohormones,including 3-indoleacetic acid(IAA),3-indolebutytic acid(IBA),salicylic acid(SA),gibberellin A3(GA),jasmonic acid(JA),abscisic acid(ABA) and brassinosteroids(BR) in tobacco was developed by high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The TMV(tobacco mosaic virus)-infected tobacco samples were determined by obtaining the characteristic fragment ion peaks of the 7 endogenous phytohormones under multiple reaction monitoring mode.The results showed that:1) The correlation coefficients(r) of the 7 phytohormones were from 0.998 6 to 0.999 5 in the linear ranges.The limits of quantification(LOQ) for all analytes were between0.07 and 3.00 μg/L,and the intra-and inter-day relative standard deviations(RSDs) were 0.44%-9.76% for all analytes.The recoveries were in the range of 77.82%-117.63%.2) The contents of SA,IBA,JA,ABA in TMV-infected tobacco were significantly higher(p<0.01) than the control sample,while which was significantly lower(p<0.05) for GA.However the content of BR was basically unchanged.With good sensitivity,precision and recovery,this method is suitable for determining various endogenous phytohormones.
引文
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[9]肖颖.液相色谱-串联质谱联用技术在食品安全分析中的应用[D].重庆:西南大学,2013.XIAO Ying.Application of LC-MS/MS in food safety analysis[D].Chongqing:Southwest University,2013.
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[12]彭妙,朱列书,陈建国,等.烟草胞质雄性不育系与其保持系内源激素的平衡关系[J].烟草科技,2016,49(9):15-21.PENG Miao,ZHU Lieshu,CHEN Jianguo,et al.Equilibrium relationship of endogenous hormone between tobacco cytoplasmic male-sterile lines and maintainer lines[J].Tobacco Science&Technology,2016,49(9):15-21.
[13]姜骁,吴拥军,杨亚,等.烟草钾外流通道基因NTORK1的启动子克隆及激素诱导分析[J].江苏农[[[[[[[业学报,2016,32(2):274-277.JIANG Xiao,WU Yongjun,YANG Ya,et al.Cloning and phytohormone induction of the promotor of potassium outward channel gene NTORK1 from tobacco[J].Jiangsu Journal of Agricultural Sciences,2016,32(2):274-277.
[14]杨亚.调控烟草钾吸收的激素筛选及初步应用[D].贵阳:贵州大学,2016.YANG Ya.The screening and preliminary application of the hormone on regulation tobacco potassium uptake specialty[D].Guiyang:Guizhou University,2016.
[15]何俊龙,赵世钦,王彦楠,等.烟草内源激素对干旱胁迫响应的研究进展[J].中国农学通报,2015,31(30):205-209.HE Junlong,ZHAO Shiqin,WANG Yannan,et al.Response of endogenous hormones of tobacco to drought stress[J].Chinese Agricultural Science Bulletin,2015,31(30):205-209.
[16]袁祖丽,吴中红.镉胁迫对烟草根抗氧化能力和激素含量的影响[J].生态学报,2010,30(15):4109-4118.YUAN Zuli,WU Zhonghong.Effect of cadmium on antioxidative capability and phytohormone level in tobacco roots[J].Acta Ecologica Sinica,2010,30(15):4109-4118.
[17]杨洁,胡日生,童建华,等.打顶对烟草腋芽生长及植物激素含量的影响[J].烟草科技,2013(10):72-75.YANG Jie,HU Risheng,TONG Jianhua,et al.Effects of topping on axillary bud growth of and plant hormone contents in tobacco[J].Tobacco Science&Technology,2013(10):72-75.
[18]赫冬梅,胡国公,穆琳.烟草内源激素的酶联免疫吸附法(ELISA)测定[J].烟草科技,2000(5):41-42.HE Dongmei,HU Guogong,MU Lin.Determination of endogenous hormones in tobacco by enzyme-linked immunosorbent assay(ELISA)[J].Tobacco Science&Technology,2000(5):41-42.
[19]商耀,郭承芳,杜咏梅,等.烟草主要内源激素的分析方法研究[J].烟草科技,2000(4):41-43.SHANG Yao,GUO Chengfang,DU Yongmei,et al.Analysis of major endogenous hormones in tobacco[J].Tobacco Science&Technology,2000(4):41-43.
[20]邱雄雄,钟启升,詹松,等.超高效液相色谱-串联三重四极杆质谱测定烟草中的六种植物激素[C]//第二十届全国色谱学术报告会及仪器展览会会议论文集.西安:中国化学会、中国色谱学会,2015:90.QIU Xiongxiong,ZHONG Qisheng,ZHAN Song,et al.Ultra performance liquid chromatography determination of six kinds of plant hormones of tobacco in the three tandem triple quadrupole mass spectrometry[C]//Proceedings of the Twentieth National Symposium on Chromatography and Instrument Exhibition.Xi’an:China Chemical Society and China Chromatographic Society,2015:90.
[21]邓克勤.植物凝集素类受体蛋白激酶LecRK-b2的生物信息学分析及基因功能研究[D].长沙:湖南大学,2009.DENG Keqin.Bioinformatic analysis and function investigation of Arabidopsis lectin receptor like kinase LecRK-b2[D].Changsha:Hunan University,2009.
[22]李敏.植物激素和过氧化氢在烟草响应TMV中的作用[D].南京:南京农业大学,2001.LI Min.The roles of plant hormones and H2O2in the response of tobacco to TMV infection[D].Nanjing:Nanjing Agricultural University,2001.
[23]梁俊峰,谢丙炎,张宝玺,等.β-氨基丁酸、茉莉酸及其甲酯诱导辣椒抗TMV作用的研究[J].中国蔬菜,2003(3):4-7.LIANG Junfeng,XIE Bingyan,ZHANG Baoxi,et al.Induction of resistance to TMV in pepper plants by DL-β-aminobutyric(BABA),jasmonic acid(JA)and methyl jasmonate(MeJA)[J].China Vegetables,2003(3):4-7.
[2]Santner A,Calderon-Villalobos L I,Estelle M.Plant hormones are versatile chemical regulators of plant growth[J].Nature Chemical Biology,2009,5(5):301-307.
[3]ZHANG Shanshan,CAI Zhenying,WANG Xuelu.The primary signaling outputs of brassinosteroids are regulated by abscisic acid signaling[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(11):4543-4548.
[4]吴倩,王璐,吴大朋,等.植物激素样品前处理方法的研究进展[J].色谱,2014,32(4):319-329.WU Qian,WANG Lu,WU Dapeng,et al.Recent advances in sample preparation methods of plant hormones[J].Chinese Journal of Chromatography,2014,32(4):319-329.
[5]夏群,辛培勇,褚金芳.全自动在线固相萃取-液相色谱-串联质谱法同时检测水稻中6种内源性植物激素[J].分析化学,2016,44(3):409-415.XIA Qun,XIN Peiyong,CHU Jinfang.An automated on-line solid phase extraction-liquid chromatographytandem mass spectrometric method for six endogenous phytohormones analysis in rice[J].Chinese Journal of Analytical Chemistry,2016,44(3):409-415.
[6]刘志勇,沈春章,董元彦.气相色谱法速测油菜中的乙烯释放量[J].化学与生物工程,2006,23(2):55-56.LIU Zhiyong,SHEN Chunzhang,DONG Yuanyan.Determination of ethylene release rate in rape by gas chromatography[J].Chemistry&Bioengineering,2006,23(2):55-56.
[7]曾少华,高洁莹.植物激素理化检测方法的研究进展[J].农产品加工(学刊),2013(5):62-66.ZENG Shaohua,GAO Jieying.Recent development in physical and chemical determination of phytohormones[J].Academic Periodical of Farm Products Processing,2013(5):62-66.
[8]Chiwocha S D S,Cutler A J,Abrams S R,et al.The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid,auxin,cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy,moist-chilling and germination[J].The Plant Journal,2005,42:35-48.
[9]肖颖.液相色谱-串联质谱联用技术在食品安全分析中的应用[D].重庆:西南大学,2013.XIAO Ying.Application of LC-MS/MS in food safety analysis[D].Chongqing:Southwest University,2013.
[10]张文君,李敏敏,张凤.液质联用技术及其应用[J].山东化工,2014,43(11):121-122.ZHANG Wenjun,LI Minmin,ZHANG Feng.Liquid mass coupling technique and its application[J].Shandong Chemical Industry,2014,43(11):121-122.
[11]李振华.烟草种子萌发的激素效应研究进展[J].云南农业大学学报,2013,28(3):416-423.LI Zhenhua.Advances in hormones during tobacco seed germination[J].Journal of Yunnan Agricultural University,2013,28(3):416-423.
[12]彭妙,朱列书,陈建国,等.烟草胞质雄性不育系与其保持系内源激素的平衡关系[J].烟草科技,2016,49(9):15-21.PENG Miao,ZHU Lieshu,CHEN Jianguo,et al.Equilibrium relationship of endogenous hormone between tobacco cytoplasmic male-sterile lines and maintainer lines[J].Tobacco Science&Technology,2016,49(9):15-21.
[13]姜骁,吴拥军,杨亚,等.烟草钾外流通道基因NTORK1的启动子克隆及激素诱导分析[J].江苏农[[[[[[[业学报,2016,32(2):274-277.JIANG Xiao,WU Yongjun,YANG Ya,et al.Cloning and phytohormone induction of the promotor of potassium outward channel gene NTORK1 from tobacco[J].Jiangsu Journal of Agricultural Sciences,2016,32(2):274-277.
[14]杨亚.调控烟草钾吸收的激素筛选及初步应用[D].贵阳:贵州大学,2016.YANG Ya.The screening and preliminary application of the hormone on regulation tobacco potassium uptake specialty[D].Guiyang:Guizhou University,2016.
[15]何俊龙,赵世钦,王彦楠,等.烟草内源激素对干旱胁迫响应的研究进展[J].中国农学通报,2015,31(30):205-209.HE Junlong,ZHAO Shiqin,WANG Yannan,et al.Response of endogenous hormones of tobacco to drought stress[J].Chinese Agricultural Science Bulletin,2015,31(30):205-209.
[16]袁祖丽,吴中红.镉胁迫对烟草根抗氧化能力和激素含量的影响[J].生态学报,2010,30(15):4109-4118.YUAN Zuli,WU Zhonghong.Effect of cadmium on antioxidative capability and phytohormone level in tobacco roots[J].Acta Ecologica Sinica,2010,30(15):4109-4118.
[17]杨洁,胡日生,童建华,等.打顶对烟草腋芽生长及植物激素含量的影响[J].烟草科技,2013(10):72-75.YANG Jie,HU Risheng,TONG Jianhua,et al.Effects of topping on axillary bud growth of and plant hormone contents in tobacco[J].Tobacco Science&Technology,2013(10):72-75.
[18]赫冬梅,胡国公,穆琳.烟草内源激素的酶联免疫吸附法(ELISA)测定[J].烟草科技,2000(5):41-42.HE Dongmei,HU Guogong,MU Lin.Determination of endogenous hormones in tobacco by enzyme-linked immunosorbent assay(ELISA)[J].Tobacco Science&Technology,2000(5):41-42.
[19]商耀,郭承芳,杜咏梅,等.烟草主要内源激素的分析方法研究[J].烟草科技,2000(4):41-43.SHANG Yao,GUO Chengfang,DU Yongmei,et al.Analysis of major endogenous hormones in tobacco[J].Tobacco Science&Technology,2000(4):41-43.
[20]邱雄雄,钟启升,詹松,等.超高效液相色谱-串联三重四极杆质谱测定烟草中的六种植物激素[C]//第二十届全国色谱学术报告会及仪器展览会会议论文集.西安:中国化学会、中国色谱学会,2015:90.QIU Xiongxiong,ZHONG Qisheng,ZHAN Song,et al.Ultra performance liquid chromatography determination of six kinds of plant hormones of tobacco in the three tandem triple quadrupole mass spectrometry[C]//Proceedings of the Twentieth National Symposium on Chromatography and Instrument Exhibition.Xi’an:China Chemical Society and China Chromatographic Society,2015:90.
[21]邓克勤.植物凝集素类受体蛋白激酶LecRK-b2的生物信息学分析及基因功能研究[D].长沙:湖南大学,2009.DENG Keqin.Bioinformatic analysis and function investigation of Arabidopsis lectin receptor like kinase LecRK-b2[D].Changsha:Hunan University,2009.
[22]李敏.植物激素和过氧化氢在烟草响应TMV中的作用[D].南京:南京农业大学,2001.LI Min.The roles of plant hormones and H2O2in the response of tobacco to TMV infection[D].Nanjing:Nanjing Agricultural University,2001.
[23]梁俊峰,谢丙炎,张宝玺,等.β-氨基丁酸、茉莉酸及其甲酯诱导辣椒抗TMV作用的研究[J].中国蔬菜,2003(3):4-7.LIANG Junfeng,XIE Bingyan,ZHANG Baoxi,et al.Induction of resistance to TMV in pepper plants by DL-β-aminobutyric(BABA),jasmonic acid(JA)and methyl jasmonate(MeJA)[J].China Vegetables,2003(3):4-7.