茶树CCoAOMT基因克隆及启动子的结构分析
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摘要
茶树咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)是甲基化EGCG生物合成的一种重要酶,为探明CCoAOMT基因的表达调控规律,进一步解析甲基化EGCG生物合成的调控机制。本研究采用同源克隆法获得了茶树CCoAOMT的cDNA全长序列,采用染色体步移技术(Genome walking)获得了该基因的启动子序列,并对其序列进行了生物信息学分析。结果表明,CCo AOMT全长cDNA为1 000 bp,其中开放阅读框长735 bp,编码245个氨基酸,含有caffeoyl-CoA O-methyltransferase和SAM功能结构域;进一步分离得到CCoAOMT基因上游调控序列1 624 bp,发现其含启动子核心元件TATA-box、CAAT-box及5'UTR Py-rich stretch (高水平转录顺式作用元件)、MYB (干旱诱导时的MYB结合位点)、G-box、GAG-motif、GATA-motif、GT1-motif、Sp1 (光响应元件)、CGTCA-motif、TGACG-motif (茉莉酸甲酯响应元件)等重要顺式作用元件。由结果推测,CCoAOMT基因在转录水平受各类转录因子的调控,该结论为进一步研究CCoAOMT基因的转录调控机制提供了理论指导。
Caffeoyl-CoA 3-O-methyltransferase(CCoAOMT) is an important enzyme in biosynthesis of methylated EGCG. In order to explore the regulation of CCoAOMT gene expression and the regulation of methylated EGCG biosynthesis, the full length c DNA of CCoAOMT sequence in Camellia sinensis was obtained by homology cloning. The promoter sequence of CCoAOMT was obtained by genome walking technology and its sequence was analyzed by bioinformatics method. The results showed that the full length of CCoAOMT c DNA was 1 000 bp,containing a 735 bp open reading frame(ORF), encoding 245 amino acids, including two functional domains,caffeoyl-CoA O-methyltransferase and SAM. The upstream regulatory sequence(1 624 bp) of CCoAOMT was obtained by further isolation. It was found that the sequence contained many cis-acting elements, such as the core promoter element TATA-box, CAAT-box and 5'UTR Py-rich stretch(cis-acting element conferring high transcription levels), MYB(MYB binding site involved in drought induction), G-box, GAG-motif, GATA-motif, GT1-motif and Sp1(light responsive element), CGTCA-motif, TGACG-motif(MeJA responsive element). The results suggested that CCoAOMT genes was regulated by various transcription factors at the transcriptional level, which would provide theoretical guidance for further study on the transcriptional regulation mechanism of CCoAOMT gene.
Caffeoyl-CoA 3-O-methyltransferase(CCoAOMT) is an important enzyme in biosynthesis of methylated EGCG. In order to explore the regulation of CCoAOMT gene expression and the regulation of methylated EGCG biosynthesis, the full length c DNA of CCoAOMT sequence in Camellia sinensis was obtained by homology cloning. The promoter sequence of CCoAOMT was obtained by genome walking technology and its sequence was analyzed by bioinformatics method. The results showed that the full length of CCoAOMT c DNA was 1 000 bp,containing a 735 bp open reading frame(ORF), encoding 245 amino acids, including two functional domains,caffeoyl-CoA O-methyltransferase and SAM. The upstream regulatory sequence(1 624 bp) of CCoAOMT was obtained by further isolation. It was found that the sequence contained many cis-acting elements, such as the core promoter element TATA-box, CAAT-box and 5'UTR Py-rich stretch(cis-acting element conferring high transcription levels), MYB(MYB binding site involved in drought induction), G-box, GAG-motif, GATA-motif, GT1-motif and Sp1(light responsive element), CGTCA-motif, TGACG-motif(MeJA responsive element). The results suggested that CCoAOMT genes was regulated by various transcription factors at the transcriptional level, which would provide theoretical guidance for further study on the transcriptional regulation mechanism of CCoAOMT gene.
引文
Bajic V.B.,Tan S.L.,and Sutuld Y.T.,2004,Promoter prediction analysis on the whole human genome,Nat.Biotechnol.,22(11):1467-1473
Beckers G.J.M.,and Spoel S.H.,2006,Fine-tuning plant defence signalling:salicylate versus jasmonate,Plant Biol.,8(1):1-10
Chen H.,He X.H.,Luo C.,Yang L.T.,Zhang B.Q.,and Song X.P.,2012,Molecular cloning of longan caffeoy(I)-Co AO-methyltransferase(DLCCoAOMT)and its expression analysis,Zhonguo Nongye Kexue(Scientia Agricultura Sinica),45(1):118-126(陈虎,何新华,罗聪,杨丽涛,张保青,宋修鹏,2012,龙眼咖啡酰辅酶A-O-甲基转移酶(DLCCoAOMT)基因的克隆和表达分析,中国农业科学,45(1):118-126)
Do C.T.,Pollet B.,Thévenin J.,Sibout R.,Denoue D.,Barrière Y.,Lapierre C.,and Jouanin L.,2007,Both caffeoyl coenzyme a 3-O-methyltransferase 1 and caffeic acid o-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis,Planta,226(5):1117-1129
Dou L.L.,Zhang X.H.,Pang C.Y.,Song M.Z.,Wei H.L.,Fan S.L.,and Yu S.X.,2014,Genome-wide analysis of the WRKY gene family in cotton,Mol.Genet.Genomics,289(6):1103-1121
Grimmig B.,and Matern U.,1997,Structure of the parsley caffeoyl-CoA-O-methyltrans-ferase gene harbouring a novel elicitor responsive cis-acting element,Plant Mol.Biol.,33(2):323-341
Higo K.,Iwamoto M.T.,and Ugawa Y.,1999,Plant cis-acting regulatory DNA elements(PLACE)database:1999,Nucleic Acids Res.,27(1):297-300
Hobo T.,Asada M.,Kowyama Y.,and Hattori T.,2010,ACGT-containing abscisic acid response element(ABRE)and coupling element 3(CE3)are functionally equivalent,Plant J.,19(6):679-689
Joshi C.P.,and Chiang V.L.,1998,Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases,Plant Mol.Biol.,37(4):663-674
Kirita M.,Honma D.,Tanaka Y.,Usui S.,and Maeda-Yamamoto M.,2010,Cloning of a novel o-methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity,J.Agric.Food Chem.,58(12):7196-7201
Kühnl T.,Koch U.,Heller W.,and Wellmann E.,1989,Elicitor induced s-adenosyl-l-methionine:caffeoyl-CoA 3-o-methyltransferase from carrot cell suspension cultures,Plant Sci.,60(1):21-25
Labruna G.,Pasanisi F.,Fortunato G.,Nardelli C.,Finelli C.,Farinaro E.,Contaldo F.,and Sacchetti L.,2011,Sequence analysis of the UCP1 gene in a severe obese population from southern Italy,J.Obesity,(12):269043
Li H.L.,Guo D.,Yang Z.P.,Tang X.,and Peng S.Q.,2014,Genome-wide identification and characterization of WRKYgene family in Hevea brasiliensis,Genomics,104(1):14-23
Li Y.H.,2013,Separation&purification of methylated EGCGand cloning&expression of related genes in Camellia sinensis,Dissertation for Ph.D.,Hunan Agricultural University,Supervisors:Liu Z.H.,Huang J.A.,pp.23-24(李银花,2013,茶树中甲基化EGCG的分离纯化与相关基因的克隆及其表达研究,博士学位论文,湖南农业大学,导师:刘仲华,黄建安,pp.23-24)
Lu J.,Zhao R.Y.,He Y.K.,and Song Y.R.,2014,Advances of the studies on plant promoter and its application,Ziran Kexue Jinzhan(Progress in Natural Science),14(8):856-862(路静,赵华燕,何奕昆,宋艳茹,2004,高等植物启动子及其应用研究进展,自然科学进展,14(8):856-862)
LüM.,Ni Z.Y.,Wang J.,Li B.,Luo S.P.,and Fan L.,2010,Tissue specificity express analysis of methyltransferase,Henongxue Bao(Journal of Nuclear Agricultural Sciences),24(4):713-719(吕萌,倪志勇,王娟,李波,罗淑萍,范玲,2010,棉花甲基化酶基因CCOMT和CCoAOMT的组织特异性表达分析,核农学报,24(4):713-719)
Martz F.,Maury S.,Pincon G.,and Legrand M.,1998,cDNAcloning,substrate specificity and expression study of tobacco caffeoyl-coA 3-O-methyltransferase,a lignin biosynthetic enzyme,Plant Mol.Biol.,36(3):427-437
McConn M.,Creelman RA.,Bell E.,Mullet J.E.,and Browse J.,1997,Jasmonate is essential for insect defense Arabidopsis,Proc.Natl.Acad.Sci.USA,94(10):5473-5477
Michèle R.C.,Elisa C.,Thérèse T.L.,and Hirel B.,2002,A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene(GS15)promoter,Plant Cell Physiol.,43(9):1006-1016
Nejad E.S.,Askari H.,and Soltani S.,2012,Regulatory TGACG-motif may elicit the secondary metabolite production through inhibition of active cyclin-dependent kinase/cyclin complex,Plant Omics,5(6):553-558
Schmitt D.,Pakusch A.E.,and Matern U.,1991,Molecularcloning,induction,and taxonomic distribution of caffeoyl-coa 3-o-methyltransferase,an enzyme involved in disease resistance,J.Biol.Chem.,266(26):17416-17423
Shepard A.R.,Zhang W.,and Eberhardt N.L.,1994,Two CGT-CA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells,J.Biol.Chem.,269(3):1804-1814
Singh M.,Bag S.K.,Bhardwaj A.,Ranjan A.,Mantri S.,Nigam D.,Sharma Y.K.,and Sawant S.V.,2015,Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana,BMC Plant Biol.,15(1):1-21
Wang Y.N.,Zhu L.,Ye A.H.,Tao S.Q.,and Qin B.,2013,The gene cloning of ATP sulfurylase of tea piant,the key enzyme related to seleniam metabolism and the structure analysis of it's promoter,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),34(4):654-661(王雅楠,朱林,叶爱华,陶少强,秦冰,2013,茶树硒营养代谢关键酶ATP硫化酶基因克隆及启动子结构分析,热带作物学报,34(4):654-661)
Wei J.H.,and Song Y.R.,2001,Recent advances in study of lignin biosynthesis and manipulation,Zhiwu Xuebao(Acta Botanica Sinica),43(8):771-779(魏建华,宋艳茹,2001,木质素生物合成途径及调控的研究进展,植物学报,43(8):771-779)
Ye Z.H.,1997,Association of caffeoyl coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants,Plant Physiol.,115(4):1341-1350
Yi M.,Zhang S.G.,Xie Y.H.,and Sun X.M.,2013,Isolation and single nucleotide polymorphisms analysis of caffeic acid O-methyltransferase(LkCOMT)in Larix kaempferi,Linye Kexue Yanjiu(Forest Research),26(S):52-59(易敏,张守攻,谢允慧,孙晓梅,2013,日本落叶松咖啡酸-O-甲基转移酶基因LkCOMT的克隆及单核苷酸多态性分析,林业科学研究,26(S):52-59)
Zhang Y.,Lv H.P.,Ma C.Y.,Guo L.,Tan J.F.,Peng Q.H.,and Lin Z.,2015,Cloning of a caffeoyl-coenzyme a o-methyl transferase from Camellia sinensis and analysis of its catalytic activity,J.Zhejiang Univ.Sci.B,16(2):103-112
Zhao J.,Gao Y.,Zhang Z.,Chen T.,Guo W.,and Zhang T.,2013,A receptor-like kinase gene(GbRLK)from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis,BMC Plant Biol.,13(1):110
Zhao J.P.,Jiang X.L.,Zhang B.Y.,and Su X.H.,2012,Involvement of microRNA-mediated gene expression regulation in the pathological development of stem canker disease in Populus trichocarpa,PLoS One,7(9):44968
Beckers G.J.M.,and Spoel S.H.,2006,Fine-tuning plant defence signalling:salicylate versus jasmonate,Plant Biol.,8(1):1-10
Chen H.,He X.H.,Luo C.,Yang L.T.,Zhang B.Q.,and Song X.P.,2012,Molecular cloning of longan caffeoy(I)-Co AO-methyltransferase(DLCCoAOMT)and its expression analysis,Zhonguo Nongye Kexue(Scientia Agricultura Sinica),45(1):118-126(陈虎,何新华,罗聪,杨丽涛,张保青,宋修鹏,2012,龙眼咖啡酰辅酶A-O-甲基转移酶(DLCCoAOMT)基因的克隆和表达分析,中国农业科学,45(1):118-126)
Do C.T.,Pollet B.,Thévenin J.,Sibout R.,Denoue D.,Barrière Y.,Lapierre C.,and Jouanin L.,2007,Both caffeoyl coenzyme a 3-O-methyltransferase 1 and caffeic acid o-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis,Planta,226(5):1117-1129
Dou L.L.,Zhang X.H.,Pang C.Y.,Song M.Z.,Wei H.L.,Fan S.L.,and Yu S.X.,2014,Genome-wide analysis of the WRKY gene family in cotton,Mol.Genet.Genomics,289(6):1103-1121
Grimmig B.,and Matern U.,1997,Structure of the parsley caffeoyl-CoA-O-methyltrans-ferase gene harbouring a novel elicitor responsive cis-acting element,Plant Mol.Biol.,33(2):323-341
Higo K.,Iwamoto M.T.,and Ugawa Y.,1999,Plant cis-acting regulatory DNA elements(PLACE)database:1999,Nucleic Acids Res.,27(1):297-300
Hobo T.,Asada M.,Kowyama Y.,and Hattori T.,2010,ACGT-containing abscisic acid response element(ABRE)and coupling element 3(CE3)are functionally equivalent,Plant J.,19(6):679-689
Joshi C.P.,and Chiang V.L.,1998,Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases,Plant Mol.Biol.,37(4):663-674
Kirita M.,Honma D.,Tanaka Y.,Usui S.,and Maeda-Yamamoto M.,2010,Cloning of a novel o-methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity,J.Agric.Food Chem.,58(12):7196-7201
Kühnl T.,Koch U.,Heller W.,and Wellmann E.,1989,Elicitor induced s-adenosyl-l-methionine:caffeoyl-CoA 3-o-methyltransferase from carrot cell suspension cultures,Plant Sci.,60(1):21-25
Labruna G.,Pasanisi F.,Fortunato G.,Nardelli C.,Finelli C.,Farinaro E.,Contaldo F.,and Sacchetti L.,2011,Sequence analysis of the UCP1 gene in a severe obese population from southern Italy,J.Obesity,(12):269043
Li H.L.,Guo D.,Yang Z.P.,Tang X.,and Peng S.Q.,2014,Genome-wide identification and characterization of WRKYgene family in Hevea brasiliensis,Genomics,104(1):14-23
Li Y.H.,2013,Separation&purification of methylated EGCGand cloning&expression of related genes in Camellia sinensis,Dissertation for Ph.D.,Hunan Agricultural University,Supervisors:Liu Z.H.,Huang J.A.,pp.23-24(李银花,2013,茶树中甲基化EGCG的分离纯化与相关基因的克隆及其表达研究,博士学位论文,湖南农业大学,导师:刘仲华,黄建安,pp.23-24)
Lu J.,Zhao R.Y.,He Y.K.,and Song Y.R.,2014,Advances of the studies on plant promoter and its application,Ziran Kexue Jinzhan(Progress in Natural Science),14(8):856-862(路静,赵华燕,何奕昆,宋艳茹,2004,高等植物启动子及其应用研究进展,自然科学进展,14(8):856-862)
LüM.,Ni Z.Y.,Wang J.,Li B.,Luo S.P.,and Fan L.,2010,Tissue specificity express analysis of methyltransferase,Henongxue Bao(Journal of Nuclear Agricultural Sciences),24(4):713-719(吕萌,倪志勇,王娟,李波,罗淑萍,范玲,2010,棉花甲基化酶基因CCOMT和CCoAOMT的组织特异性表达分析,核农学报,24(4):713-719)
Martz F.,Maury S.,Pincon G.,and Legrand M.,1998,cDNAcloning,substrate specificity and expression study of tobacco caffeoyl-coA 3-O-methyltransferase,a lignin biosynthetic enzyme,Plant Mol.Biol.,36(3):427-437
McConn M.,Creelman RA.,Bell E.,Mullet J.E.,and Browse J.,1997,Jasmonate is essential for insect defense Arabidopsis,Proc.Natl.Acad.Sci.USA,94(10):5473-5477
Michèle R.C.,Elisa C.,Thérèse T.L.,and Hirel B.,2002,A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene(GS15)promoter,Plant Cell Physiol.,43(9):1006-1016
Nejad E.S.,Askari H.,and Soltani S.,2012,Regulatory TGACG-motif may elicit the secondary metabolite production through inhibition of active cyclin-dependent kinase/cyclin complex,Plant Omics,5(6):553-558
Schmitt D.,Pakusch A.E.,and Matern U.,1991,Molecularcloning,induction,and taxonomic distribution of caffeoyl-coa 3-o-methyltransferase,an enzyme involved in disease resistance,J.Biol.Chem.,266(26):17416-17423
Shepard A.R.,Zhang W.,and Eberhardt N.L.,1994,Two CGT-CA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells,J.Biol.Chem.,269(3):1804-1814
Singh M.,Bag S.K.,Bhardwaj A.,Ranjan A.,Mantri S.,Nigam D.,Sharma Y.K.,and Sawant S.V.,2015,Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana,BMC Plant Biol.,15(1):1-21
Wang Y.N.,Zhu L.,Ye A.H.,Tao S.Q.,and Qin B.,2013,The gene cloning of ATP sulfurylase of tea piant,the key enzyme related to seleniam metabolism and the structure analysis of it's promoter,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),34(4):654-661(王雅楠,朱林,叶爱华,陶少强,秦冰,2013,茶树硒营养代谢关键酶ATP硫化酶基因克隆及启动子结构分析,热带作物学报,34(4):654-661)
Wei J.H.,and Song Y.R.,2001,Recent advances in study of lignin biosynthesis and manipulation,Zhiwu Xuebao(Acta Botanica Sinica),43(8):771-779(魏建华,宋艳茹,2001,木质素生物合成途径及调控的研究进展,植物学报,43(8):771-779)
Ye Z.H.,1997,Association of caffeoyl coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants,Plant Physiol.,115(4):1341-1350
Yi M.,Zhang S.G.,Xie Y.H.,and Sun X.M.,2013,Isolation and single nucleotide polymorphisms analysis of caffeic acid O-methyltransferase(LkCOMT)in Larix kaempferi,Linye Kexue Yanjiu(Forest Research),26(S):52-59(易敏,张守攻,谢允慧,孙晓梅,2013,日本落叶松咖啡酸-O-甲基转移酶基因LkCOMT的克隆及单核苷酸多态性分析,林业科学研究,26(S):52-59)
Zhang Y.,Lv H.P.,Ma C.Y.,Guo L.,Tan J.F.,Peng Q.H.,and Lin Z.,2015,Cloning of a caffeoyl-coenzyme a o-methyl transferase from Camellia sinensis and analysis of its catalytic activity,J.Zhejiang Univ.Sci.B,16(2):103-112
Zhao J.,Gao Y.,Zhang Z.,Chen T.,Guo W.,and Zhang T.,2013,A receptor-like kinase gene(GbRLK)from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis,BMC Plant Biol.,13(1):110
Zhao J.P.,Jiang X.L.,Zhang B.Y.,and Su X.H.,2012,Involvement of microRNA-mediated gene expression regulation in the pathological development of stem canker disease in Populus trichocarpa,PLoS One,7(9):44968