蜻蜓凤梨AfACO1基因的克隆及乙烯响应特性分析
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摘要
观赏凤梨是一种重要的热带花卉。生产上常人工施加外源乙烯或乙烯衍生物促进凤梨科植物开花,但作用的分子机制不清楚。以热带观赏花卉蜻蜓凤梨(Aechmea fasciata)为材料,利用转录组数据结合cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆了乙烯生物合成过程中限速酶1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1carboxylate,ACC)氧化酶(ACC oxidase,ACO)的一个编码基因,将其命名为AfACO1。AfACO1cDNA全长732bp,编码156个氨基酸。理化性质分析表明,该蛋白分子质量为17.47ku,等电点为8.46。AfACO1蛋白有保守的抗坏血酸和辅因子亚铁离子结合位点。进化树分析结果表明,AfACO1与水稻的2个OsACO1-likes蛋白亲缘关系最近,且进一步的结构域分析表明,它们拥有相似的保守基序。实时荧光定量PCR(quantitative real time PCR,qPCR)结果表明,AfACO1在抽薹39d后的成株心叶中表达量最高,且施加外源乙烯利后,AfACO1的表达量逐渐上调。结果表明AfACO1可能是蜻蜓凤梨成熟期乙烯生物合成的关键酶之一。研究结果为解析外源乙烯调控凤梨科植物开花的机制奠定了基础。
Ornamental bromeliads are important tropical flowering plant.Ethylene or substances that could release ethylene(i.e.ethephon,acetylene,etc)are widely used to promote flowering of bromeliads,however,the molecular mechanism is still unclear.With the ornamental bromeliads Aechmea fasciata as material,based on its transcriptome data and rapid amplification of cDNA ends(RACE)technology,a 1-aminocyclopropane-1carboxylate(ACC)oxidase(ACO)gene named as AfACO1 which might encode one of the rate-limiting enzyme was identified during ethylene biosynthesis.The length of AfACO1 cDNA was 732 bp,which could encode a 156-amino acids deduced protein.The molecular weight of the protein was 17.47 ku,with a theoretical pI of 8.46.AfACO1 contained both conserved ascorbic acid and cofactor Fe2+binding sites.Phylogenetic analysis indicated that AfACO1 was close to two OsACO1-likes.Furthermore,MEME motif analysis showed that it had similar conserved motifs.Quantitative real time PCR(qPCR)results showed that AfACO1 had highest expression level in central leaves of 39-day-after-flowering(DAF)adult plants.In addition,the expression of AfACO1 transcripts in central leaves of adult plants increased gradually before flowering after it was treated with exogenous ethephon.All the results above indicated that AfACO1 might be at least one of the ratelimiting enzyme during ethylene biosynthesis in Aechmea fasciata,which could provide a theoretical basis for flowering mechnism of bromeliads in the near future.
Ornamental bromeliads are important tropical flowering plant.Ethylene or substances that could release ethylene(i.e.ethephon,acetylene,etc)are widely used to promote flowering of bromeliads,however,the molecular mechanism is still unclear.With the ornamental bromeliads Aechmea fasciata as material,based on its transcriptome data and rapid amplification of cDNA ends(RACE)technology,a 1-aminocyclopropane-1carboxylate(ACC)oxidase(ACO)gene named as AfACO1 which might encode one of the rate-limiting enzyme was identified during ethylene biosynthesis.The length of AfACO1 cDNA was 732 bp,which could encode a 156-amino acids deduced protein.The molecular weight of the protein was 17.47 ku,with a theoretical pI of 8.46.AfACO1 contained both conserved ascorbic acid and cofactor Fe2+binding sites.Phylogenetic analysis indicated that AfACO1 was close to two OsACO1-likes.Furthermore,MEME motif analysis showed that it had similar conserved motifs.Quantitative real time PCR(qPCR)results showed that AfACO1 had highest expression level in central leaves of 39-day-after-flowering(DAF)adult plants.In addition,the expression of AfACO1 transcripts in central leaves of adult plants increased gradually before flowering after it was treated with exogenous ethephon.All the results above indicated that AfACO1 might be at least one of the ratelimiting enzyme during ethylene biosynthesis in Aechmea fasciata,which could provide a theoretical basis for flowering mechnism of bromeliads in the near future.
引文
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[18]ACHARD P,BAGHOUR M,CHAPPLE A,et al.The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristem-identity genes[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(15):6484-6489.
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[20]LEI M,LI Z Y,WANG J B,et al.AfAP2-1,an age-dependent gene of Ananas comosus,responds to exogenous ethylene treatment[J].International Journal of Molecular Sciences,2016,17(3):303.
[21]LI Y H,WU Q S,HUANG X,et al.Molecular cloning and characterization of four genes encoding ethylene receptors associated with pineapple(Ananas comosus L.)flowering[J].Frontiers in Plant Science,2016,7:710.
[22]TRUSOV Y,BOTELLA J R.Silencing of the ACC synthase gene ACACS2causes delayed flowering in pineapple[Ananas comosus(L.)Merr.][J].Journal of Experimental Botany,2006,57(14):3953-3960.
[23]HAMILTON A J,BOUZAYEN M,GRIERSON D.Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(16):7434-7437.
[24]HOFFMAN N E,KO K,MILKOWSKI D,et al.Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3[J].Plant Molecular Biology,1991,17(6):1189-1201.
[25]SPANU P,REINHARDT D,BOLLER T.Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes[J].The EMBO Journal,1991,10(8):2007-2013.
[26]HAMILTON A J,LYCETT G W,GRIERSON D.Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants[J].Nature,1990,342(19):.284-287.
[27]KAHANA A,SILBERSTEIN L,KESSLER N,et al.Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber[J].Plant Molecular Biology,1999,41(4):517-528.
[28]RUPERTI B,BONGHI C,RASORI A,et al.Characterization and expression of two members of the peach 1-aminocyclopropane-1-carboxylate oxidase gene family[J].Physiologia Plantarum,2001,111(3):336-344.
[29]BABULA D,MISZTAL L H,JAKUBOWICZ M,et al.Genes involved in biosynthesis and signalisation of ethylene in Brassica oleracea and Arabidopsis thaliana:identification and genome comparative mapping of specific gene homologues[J].Theoretical and Applied Genetics,2006,112(3):410-420.
[30]BINNIE J E,McMANUS M T.Characterization of the 1-aminocyclopropane-1-carboxylic acid(ACC)oxidase multigene family of Malus domestica Borkh[J].Phytochemistry,2009,70(3):348-360.
[31]CHEN H,SUN J,LI S,et al.An ACC oxidase gene essential for cucumber carpel development[J].Molecular Plant,2016,9(9):1315-1327.
[32]SOMYONG S,POOPEAR S,SUNNER S K,et al.ACCoxidase and miRNA 159a,and their involvement in fresh fruit bunch yield(FFB)via sex ratio determination in oil palm[J].Molecular Genetics&Genomics,2016,291(3):1243-1257.
[33]ROCKLIN A M,TIERNEY D L,KOFMAN V,et al.Role of the nonheme Fe(II)center in the biosynthesis of the plant hormone ethylene[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(14):7905-7909.
[34]THROWER J S,BLALOCK R,KLINMAN J P.Steadystate kinetics of substrate binding and iron release in tomato ACC oxidase[J].Biochemistry,2001,40(32):9717-9724.
[35]ROCKLIN A M,KATO K,LIU H W,et al.Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase:single turnover reaction[J].Journal of Biological Inorganic Chemistry,2004,9(2):171-182.
[36]ZHANG Z,REN J S,CLIFTON I J,et al.Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase--the ethylene-forming enzyme[J].Chemistry&Biology,2004,11(10):1383-1394.
[37]SUN X,LI Y,HE W,et al.Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase[J].Nature Communications,2017,8:15758.
[38]NIE X,SINGH R P,TAI G C.Molecular characterization and expression analysis of 1-aminocyclopropane-1-carboxylate oxidase homologs from potato under abiotic and biotic stresses[J].Genome,2002,45(5):905-913.
[39]LASSERRE E,GODARD F,BOUQUIN T,et al.Differential activation of two ACC oxidase gene promoters from melon during plant development and in response to pathogen attack[J].Molecular&General Genetics,1997,256(3):211-222.
[40]BARRY C S,BLUME B,BOUZAYEN M,et al.Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[J].The Plant Journal,1996,9(4):525-535.
[41]ZHANG M,YUAN B,LENG P.The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit[J].Journal of Experimental Botany,2009,60(6):1579-1588.
[42]CHAE H S,CHO Y G,PARK M Y,et al.Hormonal cross-talk between auxin and ethylene differentially regulates the expression of two members of the 1-aminocyclopropane-l-carboxylate oxidase gene family in rice(Oryza sativa L.)[J].Plant Cell Physiology,2000,41(3):354-362.
[2]VERSIEUX L M,BARBARA T,WANDERLEY M,et al.Molecular phylogenetics of the Brazilian giant bromeliads(Alcantarea,Bromeliaceae):implications for morphological evolution and biogeography[J].Molecular Phylogenetic Evolution,2012,64(1):177-189.
[3]MULLER M,MUNNE-BOSCH S.Ethylene response factors:a key regulatory hub in hormone and stress signaling[J].Plant Physiology,2015,169(1):32-41.
[4]SHAKEEL S N,GAO Z,AMIR M,et al.Ethylene regulates levels of ethylene receptor/CTR1signaling complexes in Arabidopsis thaliana[J].The Journal of Biological Chemistry,2015,290(19):12415-12424.
[5]LIU X,SHIOMI S,NAKATSUKA A,et al.Characterization of ethylene biosynthesis associated with ripening in banana fruit[J].Plant Physiology,1999,121(4):1257-1266.
[6]CHOUDHURY S R,ROY S,SAHA P P,et al.Characterization of differential ripening pattern in association with ethylene biosynthesis in the fruits of five naturally occurring banana cultivars and detection of a GCC-box-specific DNA-binding protein[J].Plant Cell Reports,2008,27(7):1235-1249.
[7]QIN Y M,HU C Y,PANG Y,et al.Saturated very-longchain fatty acids promote cotton fiber and Arabidopsis cell elongation by activating ethylene biosynthesis[J].The Plant Cell,2007,19(11):3692-3704.
[8]LINKIES A,MULLER K,MORRIS K,et al.Ethylene interacts with abscisic acid to regulate endosperm rupture during germination:a comparative approach using Lepidium sativumand Arabidopsis thaliana[J].The Plant Cell,2009,21(12):3803-3822.
[9]RAUF M,ARIF M,FISAHN J,et al.NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis[J].The Plant Cell,2013,25(12):4941-4955.
[10]ROSE N R,McDONOUGH M A,KING O N,et al.Inhibition of 2-oxoglutarate dependent oxygenases[J].Chemical Society Reviews,2011,40(8):4364-4397.
[11]MARKOLOVIC S,WILKINS S E,SCHOFIELD C J.Protein hydroxylation catalyzed by 2-oxoglutarate-dependent oxygenases[J].The Journal of Biological Chemistry,2015,290(34):20712-20722.
[12]MARTINEZ S,HAUSINGER R P.Catalytic mechanisms of Fe(II)-and 2-oxoglutarate-dependent oxygenases[J].The Journal of Biological Chemistry,2015,290(34):20702-20711.
[13]DONG J G,FERNANDEZ-MACULET J C,YANG S F.Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(20):9789-9793.
[14]TURNBULL C G,SINCLAIR E R,ANDERSON K L,et al.Routes of ethephon uptake in pineapple(Ananas comosus)and reasons for failure of flower induction[J].Journal of Plant Growth Regulation,1999,18(4):145-152.
[15]DUKOVSKI D,BERNATZKY R,HAN S.Flowering induction of Guzmania by ethylene[J].Scientia Horticulturae,2006,110(1):104-108.
[16]KUAN C,YU C,LIN M,et al.Foliar application of aviglycine reduces natural flowering in pineapple[J].Horticulture Science,2005,40:123-126.
[17]WANG R,HSU Y,BARTHOLOMEW D.Delaying natural flowering in pineapple through foliar application of aviglycine,an inhibitor of ethylene biosynthesis[J].Horticulture Science,2007,42:1188-1191.
[18]ACHARD P,BAGHOUR M,CHAPPLE A,et al.The plant stress hormone ethylene controls floral transition via DELLA-dependent regulation of floral meristem-identity genes[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(15):6484-6489.
[19]SWITZENBERG J A,LITTLE H A,HAMMAR S A,et al.Floral primordia-targeted ACS(1-aminocyclopropane-1-carboxylate synthase)expression in transgenic Cucumis melo implicates fine tuning of ethylene production mediating unisexual flower development[J].Planta,2014,240(4):797-808.
[20]LEI M,LI Z Y,WANG J B,et al.AfAP2-1,an age-dependent gene of Ananas comosus,responds to exogenous ethylene treatment[J].International Journal of Molecular Sciences,2016,17(3):303.
[21]LI Y H,WU Q S,HUANG X,et al.Molecular cloning and characterization of four genes encoding ethylene receptors associated with pineapple(Ananas comosus L.)flowering[J].Frontiers in Plant Science,2016,7:710.
[22]TRUSOV Y,BOTELLA J R.Silencing of the ACC synthase gene ACACS2causes delayed flowering in pineapple[Ananas comosus(L.)Merr.][J].Journal of Experimental Botany,2006,57(14):3953-3960.
[23]HAMILTON A J,BOUZAYEN M,GRIERSON D.Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(16):7434-7437.
[24]HOFFMAN N E,KO K,MILKOWSKI D,et al.Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3[J].Plant Molecular Biology,1991,17(6):1189-1201.
[25]SPANU P,REINHARDT D,BOLLER T.Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes[J].The EMBO Journal,1991,10(8):2007-2013.
[26]HAMILTON A J,LYCETT G W,GRIERSON D.Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants[J].Nature,1990,342(19):.284-287.
[27]KAHANA A,SILBERSTEIN L,KESSLER N,et al.Expression of ACC oxidase genes differs among sex genotypes and sex phases in cucumber[J].Plant Molecular Biology,1999,41(4):517-528.
[28]RUPERTI B,BONGHI C,RASORI A,et al.Characterization and expression of two members of the peach 1-aminocyclopropane-1-carboxylate oxidase gene family[J].Physiologia Plantarum,2001,111(3):336-344.
[29]BABULA D,MISZTAL L H,JAKUBOWICZ M,et al.Genes involved in biosynthesis and signalisation of ethylene in Brassica oleracea and Arabidopsis thaliana:identification and genome comparative mapping of specific gene homologues[J].Theoretical and Applied Genetics,2006,112(3):410-420.
[30]BINNIE J E,McMANUS M T.Characterization of the 1-aminocyclopropane-1-carboxylic acid(ACC)oxidase multigene family of Malus domestica Borkh[J].Phytochemistry,2009,70(3):348-360.
[31]CHEN H,SUN J,LI S,et al.An ACC oxidase gene essential for cucumber carpel development[J].Molecular Plant,2016,9(9):1315-1327.
[32]SOMYONG S,POOPEAR S,SUNNER S K,et al.ACCoxidase and miRNA 159a,and their involvement in fresh fruit bunch yield(FFB)via sex ratio determination in oil palm[J].Molecular Genetics&Genomics,2016,291(3):1243-1257.
[33]ROCKLIN A M,TIERNEY D L,KOFMAN V,et al.Role of the nonheme Fe(II)center in the biosynthesis of the plant hormone ethylene[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(14):7905-7909.
[34]THROWER J S,BLALOCK R,KLINMAN J P.Steadystate kinetics of substrate binding and iron release in tomato ACC oxidase[J].Biochemistry,2001,40(32):9717-9724.
[35]ROCKLIN A M,KATO K,LIU H W,et al.Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase:single turnover reaction[J].Journal of Biological Inorganic Chemistry,2004,9(2):171-182.
[36]ZHANG Z,REN J S,CLIFTON I J,et al.Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase--the ethylene-forming enzyme[J].Chemistry&Biology,2004,11(10):1383-1394.
[37]SUN X,LI Y,HE W,et al.Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase[J].Nature Communications,2017,8:15758.
[38]NIE X,SINGH R P,TAI G C.Molecular characterization and expression analysis of 1-aminocyclopropane-1-carboxylate oxidase homologs from potato under abiotic and biotic stresses[J].Genome,2002,45(5):905-913.
[39]LASSERRE E,GODARD F,BOUQUIN T,et al.Differential activation of two ACC oxidase gene promoters from melon during plant development and in response to pathogen attack[J].Molecular&General Genetics,1997,256(3):211-222.
[40]BARRY C S,BLUME B,BOUZAYEN M,et al.Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato[J].The Plant Journal,1996,9(4):525-535.
[41]ZHANG M,YUAN B,LENG P.The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit[J].Journal of Experimental Botany,2009,60(6):1579-1588.
[42]CHAE H S,CHO Y G,PARK M Y,et al.Hormonal cross-talk between auxin and ethylene differentially regulates the expression of two members of the 1-aminocyclopropane-l-carboxylate oxidase gene family in rice(Oryza sativa L.)[J].Plant Cell Physiology,2000,41(3):354-362.