黄灯笼辣椒肉桂酰辅酶A还原酶(CCR1)基因的克隆及序列分析
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摘要
CCR是调控苯丙烷代谢木质素合成支路的关键酶,其活性对辣椒辣味具有重要影响,本研究通过RT-PCR结合RACE法成功从海南黄灯笼椒茎尖克隆到一个全长CCR1,并通过DNAMAN、DNAStar等软件与网站对该序列进行生物信息学分析,结果表明:黄灯笼辣椒CCR1 cDNA序列全长1 362 bp,含有一个840 bp的完整的ORF,编码279个氨基酸;该基因编码蛋白无跨膜结构域,无信号肽,不是分泌蛋白,亚细胞定位于细胞质,为亲水蛋白;其氨基酸序列与拟南芥、巴西橡胶、番茄、烟草相似度达79.94%,存在一个CCR基因的典型保守序列—KNWYCYGK;CCR1基因与番茄、马铃薯、烟草等茄科植物亲缘关系较近。这为后续进一步分析CCR1功能,通过调控辣椒木质素路径来提高辣椒素含量提供依据。
C CR is a key enzyme regulating lignin biosynthesis in the branch of the biosynthesis pathway of phenylpropanoid metabolism, and its activity has an important effect on the pungency of pepper. The full length of CCR1 was obtained from the stem tip of Capsicum chinense Jacquin by combining the technology of RACE and RT-PCR. Then the characteristic of sequence was analyzed by online websites and software such as DNAMAN,DNAStar. The results revealed that the full-length CCR1 cDNA sequence was 1 362 bp, and contained a length of840 bp ORF encoding 279 amino acids. The encoding protein was not a secreted protein without transmembrane area or signal peptides and lied in cytoplasm mainly. The sequence alignment showed that the amino acid sequence of CCR1 has 79.94% homology with the sequences of CCRs in Arabidopsis thaliana, Hevea brasiliensis,Solanum lycopersicum and Nicotiana attenuate, and contained a typical conserved amino acid residues KNWYCYGK. It was found that the CCR1 protein had closely relationship with Solanum lycopersicum, Solanum tuberosum, Nicotiana attenuate and other Solanaceae plants. These results will be helpful to identify the function of CCR1 gene, and furthermore improve the content of capsaicin throuth regulating the pathway of lignin biosynthesis.
C CR is a key enzyme regulating lignin biosynthesis in the branch of the biosynthesis pathway of phenylpropanoid metabolism, and its activity has an important effect on the pungency of pepper. The full length of CCR1 was obtained from the stem tip of Capsicum chinense Jacquin by combining the technology of RACE and RT-PCR. Then the characteristic of sequence was analyzed by online websites and software such as DNAMAN,DNAStar. The results revealed that the full-length CCR1 cDNA sequence was 1 362 bp, and contained a length of840 bp ORF encoding 279 amino acids. The encoding protein was not a secreted protein without transmembrane area or signal peptides and lied in cytoplasm mainly. The sequence alignment showed that the amino acid sequence of CCR1 has 79.94% homology with the sequences of CCRs in Arabidopsis thaliana, Hevea brasiliensis,Solanum lycopersicum and Nicotiana attenuate, and contained a typical conserved amino acid residues KNWYCYGK. It was found that the CCR1 protein had closely relationship with Solanum lycopersicum, Solanum tuberosum, Nicotiana attenuate and other Solanaceae plants. These results will be helpful to identify the function of CCR1 gene, and furthermore improve the content of capsaicin throuth regulating the pathway of lignin biosynthesis.
引文
Blum E.,Mazourek M.,O'Connell M.,Curry J.,Thorup T.,Liu K.,Jahn M.,and Paran I.,2003,Molecular mapping of capsaicinoid biosynthesis genes and quantitative trait loci analysis for capsaicinoid content in Capsicum,Theor.Appl.Genet.,108(1):79-86
Chen C.,Wang Z.J.,Cao X.L.,and Wang J.Z.,2011,Bioinformatics and expression analysis of cinnamoyl-CoA reductase gene from Salvia miltiorrhiza Bunge,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(10):1963-1968(陈尘,王政军,曹鑫林,王喆之,2011,丹参肉桂酰辅酶A还原酶基因克隆与生物信息学分析,西北植物学报,31(10):1963-1968)
Cheng S.H.,He S.K.,Chen W.B.,and Wu Y.G.,2009,Detection of capsaicin and dihydrocapsaicin content and analysis of pungency degree in different pepper genotypes,Hainan Daxue Xuebao(Natural Science Journal of Hainan University),27(1):38-42(成善汉,贺申魁,陈文斌,吴艳阁,2009,不同基因型辣椒的辣椒素含量测定和辣度级别分析,海南大学学报(自然科学版),27(1):38-42)
Estrada B.,Pomar F.,D觔Az J.,Merino F.,and Bernal M.A.,1998,Effects of m ineral fertilizer supplementation on fruit development and pungency in padron peppers,Journal of Pomology&Horticultural Science,73(4):493-497
Gao F.H.,Wu Z.S.,and Deng C.Z.,2009,High-yield cultural techniques for hotpepper with yellow lantern-shaped fruit in Hainan,Lajiao Zazhi(Journal of China Capsicum),7(2):18-20(高芳华,伍壮生,邓长智,2009,海南黄灯笼辣椒高产栽培技术,辣椒杂志,7(2):18-20)
Giordano A.,Liu Z.,Panter S.N.,Dimech A.M.,Shang Y.,Wijesinghe H.,Fulgu eras K.,Ran Y.,Mouradov A.,Rochfort S.,Patron J.N.,and Spangenberg G.C.,2014,Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum,by down-regulation of a cinnamoyl CoA reductase gene,Transgenic Research,23(3):503
Hoffmann L.,Besseau S.,Geoffroy P.,Ritzenthaler C.,Meyer D.,and Lapierre C.,2004,Silencing of hydroxycinnamoyl-coenzyme a shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis,Plant Cell,16(6):1446-1465
Kim S.,Park M.,Yeom S.I.,Kim Y.M.,Lee J.M.,Lee H.A.,Seo E.,Choi J.,Cheong K.,Kim K.,Jung K.,Lee G.,Oh S.,Bae C.,Kim S.,Lee H.,Kim S.,Kim M.,Kang B.,Jo Y.,Yang H.,Jeong H.,Kang W.,Kwon J.,Shin C.,Lim J.,Park J.H.,Huh J.H.,Kim J.,Kim B.,Cohen O.,Paran I.,Suh M.C.,Lee S.B.,Kim Y.,Shin Y.,Noh S.,Park J.,Seo Y.S.,Kwon S.,Kim H.A.,Park J.M.,Kim H.,Choi S.,Bosland P.W.,Reeves G.,Jo S.,Lee B.,Cho H.,Choi H.,Lee M.,Yu Y.,Choi Y.,Park B.,Deynze A.,Ashrafi H.,Hill T.,Kim W.T.,Pai H.,Ahn H.K.,Yeam I.,Giovannoni J.J.,Rose J.K.C.,S覬rensen I.,Lee S.,Kim R.W.,Choi I.,Choi B.,Lim J.,Lee Y.,and Choi D.,2014,Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species,Nat.Genet.,46(3):270-288
Lacombe E.,Hawkins S.,Van D.J.,Piquemal J.,Goffner D.O.P.,Boudet A.,and Grima J.,1997,Cinnamoyl Co A reductase,the first committed enzyme of the lignin branch biosynthetic pathway:cloning,expression and phylogenetic relationships,Plant J.,11(3):429-441
Lau J.K.,Brown K.C.,Dom A.M.,Witte T.R.,Thornhill B.A.,Crabtree C.M.,Perry H.E.J.,Brown M.,Ball J.G.,Creel R.G.,Damron C.L.,Rollyson W.D.,Stevenson C.D.,Hardman W.E.,Valentovic M.A.,Carpenter A.B.,and Dasgupta P.,2014,Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway,Apoptosis,19(8):1190-1201
Lauvergeat V.,Lacomme C.,Lacombe E.,Lasserreb E.,Robyb D.,and Grima-Pettenatia J.,2001,Two cinnamoyl-CoA reductase(CCR)genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria,Phytochemistry,57(7):1187-1195
Li B.,Liang Y.,and Cai Y.R.,2006,Achievements in research on plant cinnamoyl-CoA reductase(CCR)genes,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),4(S):55-65(李波,梁颖,柴友荣,2006,植物肉桂酰辅酶A还原酶(CCR)基因的研究进展,分子植物育种,4(S):55-65)
Liu K.,Cui S.,Yang F.Y.,Han X.D.,Wang R.G.,and Zhang Z.Y.,2018,Clone and functional identification of cinnamoyl CoAreductase genes from Caragana intermedia,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),38(2):18-29(刘坤,崔爽,杨飞芸,韩晓东,王瑞刚,张子义,2018,中间锦鸡儿CCR2和CCR3基因的克隆和功能鉴定,中国生物工程杂志,38(2):18-29)
Liu S.,and Zhao D.G.,2015,Cloning and expression analysis of RksAGL gene in seedless chestnut rose(Rosa kweichonensis var.sterilis),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):579-586(刘松,赵德刚,2015,无籽刺梨(Rosa kweichonensis var.sterilis)RksAGL基因克隆及表达分析,基因组学与应用生物学,34(3):579-586)
Mazourek M.,Pujar A.,Borovsky Y.,Paran I.,Mueller L.,and Jahn M.M.,2009,A dynamic interface for capsaicinoid systems biology,Plant Physiol.,150(4):1806-1821
Pichon M.,Courbou I.,Beckert M.,Boudet A.,and Grima-Pettenati J.,1998,Cloning and characterization of two maize cD-NAs encoding cinnamoyl-CoA reductase(CCR)and differential expression of the corresponding genes,Plant Plant Mol.Biol.,38(4):671-676
Piquemal J.,Lapierre C.,Myton K.,O'connell A.,Schuch W.,Grima-pettenati J.,and Boudet A.,1998,Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants,Plant J.,13(1):71-83
Rest B.V.D.,and Rochange S.F.,2006,Down-regulation of cinnamoyl-CoA reductase in tomato(Solanum lycopersicum L.)induces dramatic changes in soluble phenolic pools,J.Exp.Bot.,57(6):1399-1411
Tamasloukht B.,and Pichon M.,2011,Characterization of a cinnamoyl-CoA reductase 1(CCR1)mutant in maize:effects on lignification,fiber development,and global gene expression,J.Exp.Bot.,62(11):3837
Tu Y.,Rochfort S.,Liu Z.,Ran Y.,Griffith M.,Badenhorst P.,Louie G.V.,Bowman M.E.,Smith K.F.,Noel J.P.,Mouradov A.,and Spangenberg G.,2010,Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genesfrom perennial ryegrass(Lolium perenne),Plant Cell,22(10):3357-3373
Whiting S.,Derbyshire E.J.,and Tiwari B.,2014,Could capsaicinoids help to support weight management?A systematic review and meta-analysis of energy intake data,Appetite,73(73):183-188
Zhang W.,Wei R.,Chen S.,Jiang J.,Li H.,Huang H.,Yang G.,Wang S.,Wei H.,and Liu G.,2015,Functional characterization of CCR in birch(Betula platyphylla×Betula pendula)through overexpression and suppression analysis,Physiol.Plant.,154(2):283
Zhang Y.,Zhang B.,Zhao Z.C.,Gao A.P.,Chen Y.Y.,Huang J.F.,Dang Z.G.,and Luo R.X.,2014,Cloning and sequence analysis of CCR gene from mango,Huabei Nongxuebao(Acta A-griculturae Boreali-Sinica),29(12):16-19(张宇,张波,赵志常,高爱平,陈业渊,黄建峰,党志国,罗睿雄,2014,芒果CCR基因的克隆及其序列分析,华北农学报,29(12):16-19)
Zhang Z.X.,2015,Resources identification of extremely hot pepper and analysis on expression profiling related to spicy genes,Thesis for M.S.,Hainan University,Supervisor:Cheng S.H.,pp.3-6(张子昕,2015,极辣辣椒资源鉴定及辣味基因的表达谱分析,硕士学位论文,海南大学,导师:成善汉,pp.3-6)
Zhao W.C.,and Xue Y.C.,2009,The sequence analysis and protein structure prediction of lignin biosynthesis CCR gene in poplar,Shengwu Xinxixue(China Journal of Bioinformatics),7(2):81-84(赵文超,薛永常,2009,杨树木质素合成酶CCR基因的序列分析及蛋白结构预测,生物信息学,7(2):81-84)
Chen C.,Wang Z.J.,Cao X.L.,and Wang J.Z.,2011,Bioinformatics and expression analysis of cinnamoyl-CoA reductase gene from Salvia miltiorrhiza Bunge,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(10):1963-1968(陈尘,王政军,曹鑫林,王喆之,2011,丹参肉桂酰辅酶A还原酶基因克隆与生物信息学分析,西北植物学报,31(10):1963-1968)
Cheng S.H.,He S.K.,Chen W.B.,and Wu Y.G.,2009,Detection of capsaicin and dihydrocapsaicin content and analysis of pungency degree in different pepper genotypes,Hainan Daxue Xuebao(Natural Science Journal of Hainan University),27(1):38-42(成善汉,贺申魁,陈文斌,吴艳阁,2009,不同基因型辣椒的辣椒素含量测定和辣度级别分析,海南大学学报(自然科学版),27(1):38-42)
Estrada B.,Pomar F.,D觔Az J.,Merino F.,and Bernal M.A.,1998,Effects of m ineral fertilizer supplementation on fruit development and pungency in padron peppers,Journal of Pomology&Horticultural Science,73(4):493-497
Gao F.H.,Wu Z.S.,and Deng C.Z.,2009,High-yield cultural techniques for hotpepper with yellow lantern-shaped fruit in Hainan,Lajiao Zazhi(Journal of China Capsicum),7(2):18-20(高芳华,伍壮生,邓长智,2009,海南黄灯笼辣椒高产栽培技术,辣椒杂志,7(2):18-20)
Giordano A.,Liu Z.,Panter S.N.,Dimech A.M.,Shang Y.,Wijesinghe H.,Fulgu eras K.,Ran Y.,Mouradov A.,Rochfort S.,Patron J.N.,and Spangenberg G.C.,2014,Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum,by down-regulation of a cinnamoyl CoA reductase gene,Transgenic Research,23(3):503
Hoffmann L.,Besseau S.,Geoffroy P.,Ritzenthaler C.,Meyer D.,and Lapierre C.,2004,Silencing of hydroxycinnamoyl-coenzyme a shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis,Plant Cell,16(6):1446-1465
Kim S.,Park M.,Yeom S.I.,Kim Y.M.,Lee J.M.,Lee H.A.,Seo E.,Choi J.,Cheong K.,Kim K.,Jung K.,Lee G.,Oh S.,Bae C.,Kim S.,Lee H.,Kim S.,Kim M.,Kang B.,Jo Y.,Yang H.,Jeong H.,Kang W.,Kwon J.,Shin C.,Lim J.,Park J.H.,Huh J.H.,Kim J.,Kim B.,Cohen O.,Paran I.,Suh M.C.,Lee S.B.,Kim Y.,Shin Y.,Noh S.,Park J.,Seo Y.S.,Kwon S.,Kim H.A.,Park J.M.,Kim H.,Choi S.,Bosland P.W.,Reeves G.,Jo S.,Lee B.,Cho H.,Choi H.,Lee M.,Yu Y.,Choi Y.,Park B.,Deynze A.,Ashrafi H.,Hill T.,Kim W.T.,Pai H.,Ahn H.K.,Yeam I.,Giovannoni J.J.,Rose J.K.C.,S覬rensen I.,Lee S.,Kim R.W.,Choi I.,Choi B.,Lim J.,Lee Y.,and Choi D.,2014,Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species,Nat.Genet.,46(3):270-288
Lacombe E.,Hawkins S.,Van D.J.,Piquemal J.,Goffner D.O.P.,Boudet A.,and Grima J.,1997,Cinnamoyl Co A reductase,the first committed enzyme of the lignin branch biosynthetic pathway:cloning,expression and phylogenetic relationships,Plant J.,11(3):429-441
Lau J.K.,Brown K.C.,Dom A.M.,Witte T.R.,Thornhill B.A.,Crabtree C.M.,Perry H.E.J.,Brown M.,Ball J.G.,Creel R.G.,Damron C.L.,Rollyson W.D.,Stevenson C.D.,Hardman W.E.,Valentovic M.A.,Carpenter A.B.,and Dasgupta P.,2014,Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway,Apoptosis,19(8):1190-1201
Lauvergeat V.,Lacomme C.,Lacombe E.,Lasserreb E.,Robyb D.,and Grima-Pettenatia J.,2001,Two cinnamoyl-CoA reductase(CCR)genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria,Phytochemistry,57(7):1187-1195
Li B.,Liang Y.,and Cai Y.R.,2006,Achievements in research on plant cinnamoyl-CoA reductase(CCR)genes,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),4(S):55-65(李波,梁颖,柴友荣,2006,植物肉桂酰辅酶A还原酶(CCR)基因的研究进展,分子植物育种,4(S):55-65)
Liu K.,Cui S.,Yang F.Y.,Han X.D.,Wang R.G.,and Zhang Z.Y.,2018,Clone and functional identification of cinnamoyl CoAreductase genes from Caragana intermedia,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),38(2):18-29(刘坤,崔爽,杨飞芸,韩晓东,王瑞刚,张子义,2018,中间锦鸡儿CCR2和CCR3基因的克隆和功能鉴定,中国生物工程杂志,38(2):18-29)
Liu S.,and Zhao D.G.,2015,Cloning and expression analysis of RksAGL gene in seedless chestnut rose(Rosa kweichonensis var.sterilis),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(3):579-586(刘松,赵德刚,2015,无籽刺梨(Rosa kweichonensis var.sterilis)RksAGL基因克隆及表达分析,基因组学与应用生物学,34(3):579-586)
Mazourek M.,Pujar A.,Borovsky Y.,Paran I.,Mueller L.,and Jahn M.M.,2009,A dynamic interface for capsaicinoid systems biology,Plant Physiol.,150(4):1806-1821
Pichon M.,Courbou I.,Beckert M.,Boudet A.,and Grima-Pettenati J.,1998,Cloning and characterization of two maize cD-NAs encoding cinnamoyl-CoA reductase(CCR)and differential expression of the corresponding genes,Plant Plant Mol.Biol.,38(4):671-676
Piquemal J.,Lapierre C.,Myton K.,O'connell A.,Schuch W.,Grima-pettenati J.,and Boudet A.,1998,Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants,Plant J.,13(1):71-83
Rest B.V.D.,and Rochange S.F.,2006,Down-regulation of cinnamoyl-CoA reductase in tomato(Solanum lycopersicum L.)induces dramatic changes in soluble phenolic pools,J.Exp.Bot.,57(6):1399-1411
Tamasloukht B.,and Pichon M.,2011,Characterization of a cinnamoyl-CoA reductase 1(CCR1)mutant in maize:effects on lignification,fiber development,and global gene expression,J.Exp.Bot.,62(11):3837
Tu Y.,Rochfort S.,Liu Z.,Ran Y.,Griffith M.,Badenhorst P.,Louie G.V.,Bowman M.E.,Smith K.F.,Noel J.P.,Mouradov A.,and Spangenberg G.,2010,Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genesfrom perennial ryegrass(Lolium perenne),Plant Cell,22(10):3357-3373
Whiting S.,Derbyshire E.J.,and Tiwari B.,2014,Could capsaicinoids help to support weight management?A systematic review and meta-analysis of energy intake data,Appetite,73(73):183-188
Zhang W.,Wei R.,Chen S.,Jiang J.,Li H.,Huang H.,Yang G.,Wang S.,Wei H.,and Liu G.,2015,Functional characterization of CCR in birch(Betula platyphylla×Betula pendula)through overexpression and suppression analysis,Physiol.Plant.,154(2):283
Zhang Y.,Zhang B.,Zhao Z.C.,Gao A.P.,Chen Y.Y.,Huang J.F.,Dang Z.G.,and Luo R.X.,2014,Cloning and sequence analysis of CCR gene from mango,Huabei Nongxuebao(Acta A-griculturae Boreali-Sinica),29(12):16-19(张宇,张波,赵志常,高爱平,陈业渊,黄建峰,党志国,罗睿雄,2014,芒果CCR基因的克隆及其序列分析,华北农学报,29(12):16-19)
Zhang Z.X.,2015,Resources identification of extremely hot pepper and analysis on expression profiling related to spicy genes,Thesis for M.S.,Hainan University,Supervisor:Cheng S.H.,pp.3-6(张子昕,2015,极辣辣椒资源鉴定及辣味基因的表达谱分析,硕士学位论文,海南大学,导师:成善汉,pp.3-6)
Zhao W.C.,and Xue Y.C.,2009,The sequence analysis and protein structure prediction of lignin biosynthesis CCR gene in poplar,Shengwu Xinxixue(China Journal of Bioinformatics),7(2):81-84(赵文超,薛永常,2009,杨树木质素合成酶CCR基因的序列分析及蛋白结构预测,生物信息学,7(2):81-84)