基于蛋白质组学分析跳枝梅花色差异
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摘要
本研究采用TCA/丙酮法提取‘复瓣跳枝’梅白色和红色花蕾的总蛋白进行2-DE和MALDI-TOF/TOF MS分析,初步研究其跳色的分子机理。结果显示:红花和白花相比有21个蛋白质点的相对丰度变化大于2倍,7个上调,14个下调。通过数据库鉴定出其中20个蛋白质,根据其功能分为:能量与代谢、转录、细胞结构、信号途径、次生代谢、蛋白合成、细胞生长以及防御。其中,核酮糖-1,5-二磷酸羧化酶、无机焦磷酸酶等在红花中为上调蛋白,可能为植物色素的合成运输提供能量。我们进一步采用qRT-PCR方法对编码12个差异蛋白的基因在转录水平进行了分析;同时对过氧化物酶蛋白的酶活性检测,结果显示在红花中该酶活性显著高于白花,这与蛋白趋势一致。基于蛋白质组分析,我们推测与信号转导相关的生长素结合蛋白和茉莉酸甲酯合成的关键酶丙二烯氧化物环化酶可能是造成跳枝梅花色变化的关键环节。
We used a combination of 2-DE and MALDI-TOF/TOF MS to identify the differential proteins between white flower(WF) and red flower(RF) of Japanese apricot Prunus mume ‘Fuban Tiaozhi'.The protein extraction followed the method of TCA/acetone precipitation. Twenty-one proteins showed a difference greater than 2 fold. Among them,seven proteins are up-regulated while fourteen downregulated. Using mass spectrometry and database searching, twenty proteins were demonstratively identified. Proteins identified were classified into eight functional types involved in energy and metabolism,transcription,cell structure,signaling pathway,secondary metabolism,protein synthesis,cell growth,and defence. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and inorganic pyrophosphatase are up-regulated in RF and provide material and energy for pigment biosynthesis and transport. The mRNA levels of twelve corresponding genes were detected using quantitative RT-PCR. The activity of peroxidase(POD) enzyme associated with defence was detected significantly higher in RF samples than in WF ones. The proteomic analysis suggests that auxin-binding protein involved in signaling pathway and allene oxide cyclase(AOC) which is an essential enzyme in methyl jasmonate(Me JA) biosynthesis are presumably key factors causing floral color variegation in Japanese apricot.
We used a combination of 2-DE and MALDI-TOF/TOF MS to identify the differential proteins between white flower(WF) and red flower(RF) of Japanese apricot Prunus mume ‘Fuban Tiaozhi'.The protein extraction followed the method of TCA/acetone precipitation. Twenty-one proteins showed a difference greater than 2 fold. Among them,seven proteins are up-regulated while fourteen downregulated. Using mass spectrometry and database searching, twenty proteins were demonstratively identified. Proteins identified were classified into eight functional types involved in energy and metabolism,transcription,cell structure,signaling pathway,secondary metabolism,protein synthesis,cell growth,and defence. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and inorganic pyrophosphatase are up-regulated in RF and provide material and energy for pigment biosynthesis and transport. The mRNA levels of twelve corresponding genes were detected using quantitative RT-PCR. The activity of peroxidase(POD) enzyme associated with defence was detected significantly higher in RF samples than in WF ones. The proteomic analysis suggests that auxin-binding protein involved in signaling pathway and allene oxide cyclase(AOC) which is an essential enzyme in methyl jasmonate(Me JA) biosynthesis are presumably key factors causing floral color variegation in Japanese apricot.
引文
[1]LI Y M,LIU Q L.Prunus mume:History and culture in China[J].Chronica Horticulturae,2011,51(3):28-35.
[2]TANAKA Y,SASAKI N,OHMIYA A.Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids[J].The Plant Journal,2008,54(4):733-749.
[3]李晓颖,余金保,王保根,等.南京梅花品种资源若干花性状的数量分布及评价[J].北京林业大学学报,2012,34(增刊1):45-49.LI X Y,YU J B,WANG B G,et al.Quantitative distribution and evaluation of some flower characteristics of Prunus mume cultivars[J].Journal of Beijing Forestry University,2012,34(Suppl.1):45--49.
[4]赵昶灵,郭维明,陈俊愉.梅花花色色素种类和含量的初步研究[J].北京林业大学学报,2004,26(2):68--73.ZHAO C L,GUO W M,CHEN J Y.Preliminary study on the categories and contents of the flower color pigments of Prunus mume Sieb.et Zucc[J].Journal of Beijing Forestry University,2004,26(2):68--73.
[5]TANAKA Y,BRUGLIERA F,CHANDLER S.Recent progress of flower colour modification by biotechnology[J].International Journal of Molecular Sciences,2009,10(12):5350--5369.
[6]JELLOULI N,SALEM A B,GHORBEL A,et al.Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis[J].Journal of Integrative Plant Biology,2010,52(10):933-940.
[7]胡东燕,张佐双.观赏桃[M].北京:中国林业出版社,2010.HU D Y,ZHANG Z S.Ornamental peach[M].Beijing:China Forestry Publishing House,2010.
[8]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[9]KATAYAMA H,NAGASU T,ODA Y.Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J].Rapid Communications in Mass Spectrometry,2001,15(16):1416--1421.
[10]张志良.植物生理学实验指导[M].北京:高等教育出版社,2001.ZHANG Z L.Experimental guide of plant physiology[M].Beijing:Higher Education Press,2001.
[11]NANNI V,ZANETTI M,BELLUCCI M,et al.The peach(Prunus persica)defensin Pp DFN1 displays antifungal activity through specific interactions with the membrane lipids[J].Plant Pathology,2013,62(2):393-403.
[12]熊晓然,陈蔚梅,冯胜彦,等.植物Rubisco活性中心的模拟分析[J].中国生物化学与分子生物学报,2003,19(4):493-498.XIONG X R,CHEN W M,FENG S Y,et al.Simulation on botanic Rubisco active center[J].Chinese Journal of Biochemistry and Molecular Biology,2003,19(4):493--498.
[13]JENSEN R G,BAHR J T.Ribulose 1,5-bisphosphate carboxylase-oxygenase[J].Annual Review of Plant Physiology,1977,28(1):379-400.
[14]GHANNOUM O,EVANS J R,CHOW W S,et al.Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4grasses[J].Plant Physiology,2005,137(2):638-650.
[15]许培磊,白吉刚,王秀娟,等.低温对不同基因型黄瓜叶片蛋白质组的影响[J].中国农业科学,2009,42(2):588-596.XU P L,BAI J G,WANG X J,et al.Effects of low temperature on leaf proteome from Cucumis sativus of different genotypes[J].Scientia Agricultura Sinica,2009,42(2):588-596.
[16]李小园,曾日中,校现周.植物焦磷酸酶(PPase)的研究进展[J].生命科学研究,2004,8(4):83-87.LI X Y,ZENG R Z,XIAO X Z.Advances on the research of pyrophosphatase in plants[J].Life Science Research,2004,8(4):83-87.
[17]WALKER R R,LEIGH R A.Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet(Beta vulgaris L.)[J].Planta,1981,153(2):140-149.
[18]MAESHIMA M.Tonoplast transporters:organization and function[J].Annual Review of Plant Biology,2001,52(1):469-497.
[19]ABDRAKHAMANOVA A,WANG Q Y,KHOKHLOVA L,et al.Is microtubule disassembly a trigger for cold acclimation[J]?Plant and Cell Physiology,2003,44(7):676-686.
[20]YOSHIKAWA M,YANG G,KAWAGUCHI K,et al.Expression analyses ofβ-tubulin isotype genes in rice[J].Plant and Cell Physiology,2003,44(11):1202-1207.
[21]饶国栋,张建国.植物微管蛋白基因研究进展[J].世界林业研究,2013,26(003):17--20.RAO G D,ZHANG J G.Advances of studies on plant tubulin gene[J].World Forestry Research,2013,26(003):17--20.
[22]皮妍.喜树中植物防御相关基因的克隆分析及对喜树碱含量的影响[D].上海:复旦大学,2007.PI Y.Molecular cloning and analysis of plant defence related genes from Camptothecaacuminata and the effect on camptothecin content[D].Shanghai:Fudan University,2007.
[23]徐伟,严善春.茉莉酸在植物诱导防御中的作用[J].生态学报,2005,25(8):2074-2082.XU W,YAN S C.The function of Jasmonic acid in induced plant defence[J].Acta Ecologica Sinica,2005,25(8):2074-2082.
[24]LIECHTI R,FARMER E E.The jasmonate pathway[J].Science,2002,296(5573):1649--1650.
[25]许建锋,张玉星,张江红,等.茉莉酸甲酯对苹果果实着色的影响[J].中国农学通报,2011,27(10):271-274.XU J F,ZHANG Y X,ZHANG J H,et al.Effect of methyl jasmonate on coloring of apple[J].Chinese Agricultural Science Bulletin,2011,27(10):271--274.
[26]BELHADJ A,TELEF N,SAIGNE C,et al.Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins,stilbene and anthocyanin accumulation in grapevine cell cultures[J].Plant Physiology and Biochemistry,2008,46(4):493--499.
[27]SHIMIZU Y,MAEDA K,KATO M,et al.Co-expression of Gb MYB1 and Gb MYC1 induces anthocyanin accumulation in roots of cultured Gynura bicolor DC.plantlet on methyl jasmonate treatment[J].Plant Physiology and Biochemistry,2011,49(2):159--167.
[28]田国忠,李怀方,裘维蕃.植物过氧化物酶研究进展[J].武汉植物学研究,2001,19(4):332--344.TIAN G Z,LI H F,QIU W F.Advances on research of plant peroxidases[J].Journal of Wuhan Botanical Research,2001,19(4):332--344.
[29]AUSTIN M J,MUSKETT P,KAHN K,et al.Regulatory role of SGT1 in early R gene-mediated plant defenses[J].Science,2002,295(5562):2077--2080.
[30]吴顺,萧浪涛,鲁旭东.植物的生长素结合蛋白[J].植物生理学通讯,2004,40(2):241--245.Wu S,Xiao L T,Lu X D.Auxin-binding proteins in plants[J].Plant Physiology Communications,2004,40(2):241-245.
[31]THIEL G,BLATT M R,FRICKER M D,et al.Modulation of K+channels in Vicia stomatal guard cells by peptide homologs to the auxin-binding protein C terminus[J].Proceedings of the National Academy of Sciences,1993,90(24):11493-11497.
[2]TANAKA Y,SASAKI N,OHMIYA A.Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids[J].The Plant Journal,2008,54(4):733-749.
[3]李晓颖,余金保,王保根,等.南京梅花品种资源若干花性状的数量分布及评价[J].北京林业大学学报,2012,34(增刊1):45-49.LI X Y,YU J B,WANG B G,et al.Quantitative distribution and evaluation of some flower characteristics of Prunus mume cultivars[J].Journal of Beijing Forestry University,2012,34(Suppl.1):45--49.
[4]赵昶灵,郭维明,陈俊愉.梅花花色色素种类和含量的初步研究[J].北京林业大学学报,2004,26(2):68--73.ZHAO C L,GUO W M,CHEN J Y.Preliminary study on the categories and contents of the flower color pigments of Prunus mume Sieb.et Zucc[J].Journal of Beijing Forestry University,2004,26(2):68--73.
[5]TANAKA Y,BRUGLIERA F,CHANDLER S.Recent progress of flower colour modification by biotechnology[J].International Journal of Molecular Sciences,2009,10(12):5350--5369.
[6]JELLOULI N,SALEM A B,GHORBEL A,et al.Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis[J].Journal of Integrative Plant Biology,2010,52(10):933-940.
[7]胡东燕,张佐双.观赏桃[M].北京:中国林业出版社,2010.HU D Y,ZHANG Z S.Ornamental peach[M].Beijing:China Forestry Publishing House,2010.
[8]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[9]KATAYAMA H,NAGASU T,ODA Y.Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J].Rapid Communications in Mass Spectrometry,2001,15(16):1416--1421.
[10]张志良.植物生理学实验指导[M].北京:高等教育出版社,2001.ZHANG Z L.Experimental guide of plant physiology[M].Beijing:Higher Education Press,2001.
[11]NANNI V,ZANETTI M,BELLUCCI M,et al.The peach(Prunus persica)defensin Pp DFN1 displays antifungal activity through specific interactions with the membrane lipids[J].Plant Pathology,2013,62(2):393-403.
[12]熊晓然,陈蔚梅,冯胜彦,等.植物Rubisco活性中心的模拟分析[J].中国生物化学与分子生物学报,2003,19(4):493-498.XIONG X R,CHEN W M,FENG S Y,et al.Simulation on botanic Rubisco active center[J].Chinese Journal of Biochemistry and Molecular Biology,2003,19(4):493--498.
[13]JENSEN R G,BAHR J T.Ribulose 1,5-bisphosphate carboxylase-oxygenase[J].Annual Review of Plant Physiology,1977,28(1):379-400.
[14]GHANNOUM O,EVANS J R,CHOW W S,et al.Faster Rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4grasses[J].Plant Physiology,2005,137(2):638-650.
[15]许培磊,白吉刚,王秀娟,等.低温对不同基因型黄瓜叶片蛋白质组的影响[J].中国农业科学,2009,42(2):588-596.XU P L,BAI J G,WANG X J,et al.Effects of low temperature on leaf proteome from Cucumis sativus of different genotypes[J].Scientia Agricultura Sinica,2009,42(2):588-596.
[16]李小园,曾日中,校现周.植物焦磷酸酶(PPase)的研究进展[J].生命科学研究,2004,8(4):83-87.LI X Y,ZENG R Z,XIAO X Z.Advances on the research of pyrophosphatase in plants[J].Life Science Research,2004,8(4):83-87.
[17]WALKER R R,LEIGH R A.Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet(Beta vulgaris L.)[J].Planta,1981,153(2):140-149.
[18]MAESHIMA M.Tonoplast transporters:organization and function[J].Annual Review of Plant Biology,2001,52(1):469-497.
[19]ABDRAKHAMANOVA A,WANG Q Y,KHOKHLOVA L,et al.Is microtubule disassembly a trigger for cold acclimation[J]?Plant and Cell Physiology,2003,44(7):676-686.
[20]YOSHIKAWA M,YANG G,KAWAGUCHI K,et al.Expression analyses ofβ-tubulin isotype genes in rice[J].Plant and Cell Physiology,2003,44(11):1202-1207.
[21]饶国栋,张建国.植物微管蛋白基因研究进展[J].世界林业研究,2013,26(003):17--20.RAO G D,ZHANG J G.Advances of studies on plant tubulin gene[J].World Forestry Research,2013,26(003):17--20.
[22]皮妍.喜树中植物防御相关基因的克隆分析及对喜树碱含量的影响[D].上海:复旦大学,2007.PI Y.Molecular cloning and analysis of plant defence related genes from Camptothecaacuminata and the effect on camptothecin content[D].Shanghai:Fudan University,2007.
[23]徐伟,严善春.茉莉酸在植物诱导防御中的作用[J].生态学报,2005,25(8):2074-2082.XU W,YAN S C.The function of Jasmonic acid in induced plant defence[J].Acta Ecologica Sinica,2005,25(8):2074-2082.
[24]LIECHTI R,FARMER E E.The jasmonate pathway[J].Science,2002,296(5573):1649--1650.
[25]许建锋,张玉星,张江红,等.茉莉酸甲酯对苹果果实着色的影响[J].中国农学通报,2011,27(10):271-274.XU J F,ZHANG Y X,ZHANG J H,et al.Effect of methyl jasmonate on coloring of apple[J].Chinese Agricultural Science Bulletin,2011,27(10):271--274.
[26]BELHADJ A,TELEF N,SAIGNE C,et al.Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins,stilbene and anthocyanin accumulation in grapevine cell cultures[J].Plant Physiology and Biochemistry,2008,46(4):493--499.
[27]SHIMIZU Y,MAEDA K,KATO M,et al.Co-expression of Gb MYB1 and Gb MYC1 induces anthocyanin accumulation in roots of cultured Gynura bicolor DC.plantlet on methyl jasmonate treatment[J].Plant Physiology and Biochemistry,2011,49(2):159--167.
[28]田国忠,李怀方,裘维蕃.植物过氧化物酶研究进展[J].武汉植物学研究,2001,19(4):332--344.TIAN G Z,LI H F,QIU W F.Advances on research of plant peroxidases[J].Journal of Wuhan Botanical Research,2001,19(4):332--344.
[29]AUSTIN M J,MUSKETT P,KAHN K,et al.Regulatory role of SGT1 in early R gene-mediated plant defenses[J].Science,2002,295(5562):2077--2080.
[30]吴顺,萧浪涛,鲁旭东.植物的生长素结合蛋白[J].植物生理学通讯,2004,40(2):241--245.Wu S,Xiao L T,Lu X D.Auxin-binding proteins in plants[J].Plant Physiology Communications,2004,40(2):241-245.
[31]THIEL G,BLATT M R,FRICKER M D,et al.Modulation of K+channels in Vicia stomatal guard cells by peptide homologs to the auxin-binding protein C terminus[J].Proceedings of the National Academy of Sciences,1993,90(24):11493-11497.