荸荠苯丙氨酸解氨酶基因的克隆和表达
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摘要
【目的】克隆荸荠Eleocharis tuberosa苯丙氨酸解氨酶基因(PAL),分析其序列特征及其在荸荠不同组织中和鲜切荸荠贮藏过程中的表达情况,为揭示鲜切荸荠黄化机理提供理论依据。【方法】通过RT-PCR和RACE技术从荸荠中克隆PAL基因的cDNA全长,采用生物信息学方法对其序列和所编码的蛋白进行预测分析,利用荧光定量PCR技术分析PAL基因在荸荠不同组织和鲜切荸荠贮藏过程中的表达情况。【结果】克隆得到荸荠PAL基因全长cDNA,将其命名为CwPAL,该序列长度为2 485 bp,含有1个2 142 bp的完整开放阅读框,共编码713个氨基酸。CwPAL蛋白分子式为C3437H5514N944O1058S34,相对分子质量为78 079,等电点为5.97,原子总数为10 987个。CwPAL包含PAL-HAL和PLN02457结构域及典型的PAL酶活性中心序列(GTITASGDLVPLSYIAG)。CwPAL的二级结构以α–螺旋为主,其三维结构模型呈典型的"海马状"结构。系统进化分析表明,CwPAL与菠萝Ananas comosus和海枣Phoenix dactylifera的PAL蛋白亲缘关系较近。荧光定量PCR分析表明,CwPAL基因在荸荠皮中的表达量最高,鲜切荸荠贮藏过程中CwPAL基因表达量快速上升,水杨酸处理显著抑制了CwPAL基因的表达。【结论】Cw PAL属于典型的苯丙氨酸解氨酶家族,该基因可能通过调控苯丙烷代谢从而影响鲜切荸荠的黄化。
【Objective】To clone full-length cDNA of phenylalanin ammonia-lyase(PAL) in Chinese water chestnut(Eleocharis tuberosa), investigate its sequence characteristics, analyze its expressions in different organs and during storage of fresh-cut E. tuberosa, and provide theoretical references for revealing the mechanism of E. tuberosa etiolation.【Method】The full sequence of PAL gene was cloned from E. tuberosa by RT-PCR and RACE techniques. Bioinformatics method was used to analyze the cDNA sequence and the encoded amino acid sequence. Real-time PCR was used to analyze the expression profile of PAL gene in different organs and in fresh-cut E. tuberosa during storage.【Result】The full-length cDNA of PAL gene in E.tuberosa was cloned and named CwPAL. The sequence consists of 2 485 bp with an intact open reading frame of 2 142 bp, encoding a polypeptide of 713 amino acids. The formula of CwPAL protein isC3437H5514N944O1058S34, the relative molecular weight is 78 079, pI is 5.97, and the total number of atoms is10 987. CwPAL protein contains two functional domains including PAL-HAL and PLN02457, and the typical PAL active site sequence(GTITASGDLVPLSYIAG). The main structural element in CwPAL secondary structure is α-Helix, the three-dimension structure of CwPAL protein shows a typical "sea horse" shape. The phylogenetic analysis showed that CwPAL was very closely related to PAL proteins of Ananas comosus and Phoenix dactylifera. The Q-PCR analysis showed that the expression level of CwPAL gene was the highest in peel,CwPAL gene expression quickly increased during storage of fresh-cut E. tuberosa, and salicylic acid treatment significantly inhibited CwPAL gene expression.【Conclusion】The cloned CwPAL gene is a typical member of PAL family, and CwPAL gene may affect E. tuberosa etiolation by regulating the phenylpropanoid pathway.
【Objective】To clone full-length cDNA of phenylalanin ammonia-lyase(PAL) in Chinese water chestnut(Eleocharis tuberosa), investigate its sequence characteristics, analyze its expressions in different organs and during storage of fresh-cut E. tuberosa, and provide theoretical references for revealing the mechanism of E. tuberosa etiolation.【Method】The full sequence of PAL gene was cloned from E. tuberosa by RT-PCR and RACE techniques. Bioinformatics method was used to analyze the cDNA sequence and the encoded amino acid sequence. Real-time PCR was used to analyze the expression profile of PAL gene in different organs and in fresh-cut E. tuberosa during storage.【Result】The full-length cDNA of PAL gene in E.tuberosa was cloned and named CwPAL. The sequence consists of 2 485 bp with an intact open reading frame of 2 142 bp, encoding a polypeptide of 713 amino acids. The formula of CwPAL protein isC3437H5514N944O1058S34, the relative molecular weight is 78 079, pI is 5.97, and the total number of atoms is10 987. CwPAL protein contains two functional domains including PAL-HAL and PLN02457, and the typical PAL active site sequence(GTITASGDLVPLSYIAG). The main structural element in CwPAL secondary structure is α-Helix, the three-dimension structure of CwPAL protein shows a typical "sea horse" shape. The phylogenetic analysis showed that CwPAL was very closely related to PAL proteins of Ananas comosus and Phoenix dactylifera. The Q-PCR analysis showed that the expression level of CwPAL gene was the highest in peel,CwPAL gene expression quickly increased during storage of fresh-cut E. tuberosa, and salicylic acid treatment significantly inhibited CwPAL gene expression.【Conclusion】The cloned CwPAL gene is a typical member of PAL family, and CwPAL gene may affect E. tuberosa etiolation by regulating the phenylpropanoid pathway.
引文
[1]潘永贵,陈维信.鲜切荸荠中黄化物质研究[J].食品研究与开发,2008,29(2):33-36.
[2]PENG L T,JIANG Y M.Effects of chitosan coating onshelf life and quality of fresh-cut Chinese water chestnut[J].LWT-Food Sci Technol,2003,36(3):359-364.
[3]PAN Y G,Li Y X,YUAN M Q.Isolation,purification and identification of etiolation substrate from fresh-cut Chinese water-chestnut(Eleocharis tuberosa)[J].Food Chem,2015,186:119-122.
[4]HUANG J,GU M,LAI Z,et al.Functional analysis of the Arabidopsis PAL gene family in plant growth,development,and response to environmental stress[J].Plant Physiol,2010,153(4):1526-1538.
[5]KOUKOL J,CONNE E.The metabolism of aromatic compounds in higher plants[J].J Bio Chem,1961,236(10):2692-2698.
[6]ZHANG X,LIU C J.Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids[J].Mol Plant,2015,8(1):17-27.
[7]DIXONR A,PAIVAN L.Stress-induced phenylpropanoid metabolism[J].Plant Cell,1995,7(7):1085-1097.
[8]闫洪波,程玉豆,何近刚,等.鸭梨PAL克隆及其在果实发育和机械伤害过程中的表达[J].中国农业科学,2014,47(21):4341-4348.
[9]SALTVEITM E.Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock[J].Postharvest Biol Tec,2000,21(1):61-69.
[10]LOPEZ G,SALTVEITM E,CANTWELL M.Wound-induced phenylalanine ammonia lyase activity:Factors affecting it induction and correlation with the quality of minimally processed lettuce[J].Postharvest Biol Tec,1996,9(2):223-233.
[11]CAMPOS-VARGAS R,NONOGAKI H,SUSLOW T,et al.Heat shock treatments delay the increase in wound-induced phenylalanine ammonia-lyase activity by altering its expression,not its induction in Romaine lettuce(Lactuca sativa)tissue[J].Physiol Plantarum,2005,123(1):82-91.
[12]RU Z W,LAI Y Y,XU C J,et al.Polyphenol oxidase(PPO)in early stage of browning of phalaenopsis leaf explants[J].J Agr Sci,2013,5(9):57-64.
[13]许传俊,李红,李玲.蝴蝶兰叶片外植体褐变过程中PAL基因的表达变化[J].热带亚热带植物学报,2007,15(1):50-54.
[14]郑洁红,邱栋梁.龙眼叶片PAL基因的克隆与表达研究[J].热带作物学报,2012,33(1):79-83.
[15]GIBERTI S,BERTEA C M,NARAYANA R,et al.Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae[J].J Plant Physiol,2012,169(3):249-254.
[16]宋修鹏,黄杏,莫凤连,等.甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析[J].中国农业科学,2013,46(14):2856-2868.
[17]SHANG Q M,LI L,DONG C J.Multiple tandem duplic-ation of the phenylalanine ammonia-lyase genes in Cucumis sativus,L[J].Planta,2012,236(4):1093-1105.
[18]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析[J].中国农业科学,2014,47(6):1216-1226.
[19]YIN R,MESSNER B,FAUSKESSLER T,et al.Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation[J].J Exp Bot,2012,63(7):2465-2478.
[20]潘文,张卫华,杨会肖,等.尾叶桉苯丙氨酸解氨酶基因的克隆、表达与单核苷酸多态性分析[J].华南农业大学学报,2018,39(2):89-94.
[21]陈卫卫,唐然,吴昱煜,等.象草PAL基因克隆及其蛋白质结构与功能预测[J].草地学报,2016,24(1):137-145.
[22]郭新红,喻达时,王婕,等.6种植物中木质纤维素含量的比较研究[J].湖南大学学报(自科版),2008,35(9):76-78.
[23]COCHRANEF C,DAVINL B,LEWISN G.The Arabidopsis phenylalanine ammonia lyase gene family:Kinetic characterization of the four PAL isoforms[J].Phytochemistry,2004,65(11):1557-1564.
[24]R?SLER J,SCHMID J.Maize phenylalanine ammonialyase has tyrosine ammonia-lyase activity[J].Plant Physiol,1997,113(1):175-179.
[25]CALABRESEJ C,JORDAN D B,BOODHOO A,et al.Crystal structure of phenylalanine ammonia lyase:Multiple helix dipoles implicated incatalysis[J].Biochemistry,2004,43(36):11403-11416.
[26]RITTER H,SCHULZG E.Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[J].Plant Cell,2004,16(12):3426-3436.
[27]LUO Y,LI X,HE J,et al.Isolation,characterisation,and antioxidant activities of flavonoids from chufa(Eleochar-is tuberosa)peels[J].Food Chem,2014,164(3):30-35.
[28]DEHGHAN S,SADEGHI M,P?PPEL A,et al.Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding,salicylic acid treatment,and salinity stress in safflower,Carthamus tinctorius[J].Bioscience Rep,2014,34(3):689-699.
[29]DIALLINAS G,KANELLIS A K.A phenylalanine ammonia-lyase gene from melon fruit:cDNA cloning,sequence and expression in response to development and wounding[J].Plant Mol Biol,1994,26(1):473-479.
[30]BECERRA-MORENO A,REDONDO-GIL M,BENAVIDES J,et al.Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot[J].Front Plant Sci,2015,6:836-837.
[31]CHEN J Y,WEN P F,KONG W F,et al.Effect of salicylic acid on phenylpropanoids and phenylalanine ammonia-lyase in harvested grape berries[J].Postharvest Biol Tech,2006,40(1):64-72.
[32]LUO Z,WU X,XIE Y,et al.Alleviation of chilling injury and browning of postharvest bamboo shoot by salicylic acid treatment[J].Food Chem,2012,131(2):456-461.
[33]LU X,SUN D,LI Y,et al.Pre-and post-harvest salicylic acid treatments alleviate internal browning and maintain quality of winter pineapple fruit[J].Sci Hortic-Amsterdam,2011,130(1):97-101.
[34]SAYYARI M,BABALAR M,KALANTARI S,et al.Effect of salicylic acid treatment on reducing chilling injury in stored pomegranates[J].Postharvest Biol Tec,2009,53(3):152-154.
[35]PENG L T,JIANG Y M.Exogenous salicylic acid inhibits browning of fresh-cut Chinese water chestnut[J].Food Chem,2006,94(4):535-540.
[2]PENG L T,JIANG Y M.Effects of chitosan coating onshelf life and quality of fresh-cut Chinese water chestnut[J].LWT-Food Sci Technol,2003,36(3):359-364.
[3]PAN Y G,Li Y X,YUAN M Q.Isolation,purification and identification of etiolation substrate from fresh-cut Chinese water-chestnut(Eleocharis tuberosa)[J].Food Chem,2015,186:119-122.
[4]HUANG J,GU M,LAI Z,et al.Functional analysis of the Arabidopsis PAL gene family in plant growth,development,and response to environmental stress[J].Plant Physiol,2010,153(4):1526-1538.
[5]KOUKOL J,CONNE E.The metabolism of aromatic compounds in higher plants[J].J Bio Chem,1961,236(10):2692-2698.
[6]ZHANG X,LIU C J.Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids[J].Mol Plant,2015,8(1):17-27.
[7]DIXONR A,PAIVAN L.Stress-induced phenylpropanoid metabolism[J].Plant Cell,1995,7(7):1085-1097.
[8]闫洪波,程玉豆,何近刚,等.鸭梨PAL克隆及其在果实发育和机械伤害过程中的表达[J].中国农业科学,2014,47(21):4341-4348.
[9]SALTVEITM E.Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock[J].Postharvest Biol Tec,2000,21(1):61-69.
[10]LOPEZ G,SALTVEITM E,CANTWELL M.Wound-induced phenylalanine ammonia lyase activity:Factors affecting it induction and correlation with the quality of minimally processed lettuce[J].Postharvest Biol Tec,1996,9(2):223-233.
[11]CAMPOS-VARGAS R,NONOGAKI H,SUSLOW T,et al.Heat shock treatments delay the increase in wound-induced phenylalanine ammonia-lyase activity by altering its expression,not its induction in Romaine lettuce(Lactuca sativa)tissue[J].Physiol Plantarum,2005,123(1):82-91.
[12]RU Z W,LAI Y Y,XU C J,et al.Polyphenol oxidase(PPO)in early stage of browning of phalaenopsis leaf explants[J].J Agr Sci,2013,5(9):57-64.
[13]许传俊,李红,李玲.蝴蝶兰叶片外植体褐变过程中PAL基因的表达变化[J].热带亚热带植物学报,2007,15(1):50-54.
[14]郑洁红,邱栋梁.龙眼叶片PAL基因的克隆与表达研究[J].热带作物学报,2012,33(1):79-83.
[15]GIBERTI S,BERTEA C M,NARAYANA R,et al.Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae[J].J Plant Physiol,2012,169(3):249-254.
[16]宋修鹏,黄杏,莫凤连,等.甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析[J].中国农业科学,2013,46(14):2856-2868.
[17]SHANG Q M,LI L,DONG C J.Multiple tandem duplic-ation of the phenylalanine ammonia-lyase genes in Cucumis sativus,L[J].Planta,2012,236(4):1093-1105.
[18]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析[J].中国农业科学,2014,47(6):1216-1226.
[19]YIN R,MESSNER B,FAUSKESSLER T,et al.Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation[J].J Exp Bot,2012,63(7):2465-2478.
[20]潘文,张卫华,杨会肖,等.尾叶桉苯丙氨酸解氨酶基因的克隆、表达与单核苷酸多态性分析[J].华南农业大学学报,2018,39(2):89-94.
[21]陈卫卫,唐然,吴昱煜,等.象草PAL基因克隆及其蛋白质结构与功能预测[J].草地学报,2016,24(1):137-145.
[22]郭新红,喻达时,王婕,等.6种植物中木质纤维素含量的比较研究[J].湖南大学学报(自科版),2008,35(9):76-78.
[23]COCHRANEF C,DAVINL B,LEWISN G.The Arabidopsis phenylalanine ammonia lyase gene family:Kinetic characterization of the four PAL isoforms[J].Phytochemistry,2004,65(11):1557-1564.
[24]R?SLER J,SCHMID J.Maize phenylalanine ammonialyase has tyrosine ammonia-lyase activity[J].Plant Physiol,1997,113(1):175-179.
[25]CALABRESEJ C,JORDAN D B,BOODHOO A,et al.Crystal structure of phenylalanine ammonia lyase:Multiple helix dipoles implicated incatalysis[J].Biochemistry,2004,43(36):11403-11416.
[26]RITTER H,SCHULZG E.Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[J].Plant Cell,2004,16(12):3426-3436.
[27]LUO Y,LI X,HE J,et al.Isolation,characterisation,and antioxidant activities of flavonoids from chufa(Eleochar-is tuberosa)peels[J].Food Chem,2014,164(3):30-35.
[28]DEHGHAN S,SADEGHI M,P?PPEL A,et al.Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding,salicylic acid treatment,and salinity stress in safflower,Carthamus tinctorius[J].Bioscience Rep,2014,34(3):689-699.
[29]DIALLINAS G,KANELLIS A K.A phenylalanine ammonia-lyase gene from melon fruit:cDNA cloning,sequence and expression in response to development and wounding[J].Plant Mol Biol,1994,26(1):473-479.
[30]BECERRA-MORENO A,REDONDO-GIL M,BENAVIDES J,et al.Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot[J].Front Plant Sci,2015,6:836-837.
[31]CHEN J Y,WEN P F,KONG W F,et al.Effect of salicylic acid on phenylpropanoids and phenylalanine ammonia-lyase in harvested grape berries[J].Postharvest Biol Tech,2006,40(1):64-72.
[32]LUO Z,WU X,XIE Y,et al.Alleviation of chilling injury and browning of postharvest bamboo shoot by salicylic acid treatment[J].Food Chem,2012,131(2):456-461.
[33]LU X,SUN D,LI Y,et al.Pre-and post-harvest salicylic acid treatments alleviate internal browning and maintain quality of winter pineapple fruit[J].Sci Hortic-Amsterdam,2011,130(1):97-101.
[34]SAYYARI M,BABALAR M,KALANTARI S,et al.Effect of salicylic acid treatment on reducing chilling injury in stored pomegranates[J].Postharvest Biol Tec,2009,53(3):152-154.
[35]PENG L T,JIANG Y M.Exogenous salicylic acid inhibits browning of fresh-cut Chinese water chestnut[J].Food Chem,2006,94(4):535-540.