滇牡丹类黄酮3-O-葡萄糖基转移酶基因的鉴定及表达分析
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摘要
在植物中,类黄酮3-O-葡萄糖基转移酶(F3GT)将不稳定的花色素转变为稳定的花色素苷,是花色素苷形成的关键酶。本研究采样RT-PCR技术从滇牡丹中成功克隆获得一个完整的F3GT基因(Pd F3GT1),其含有1 371 bp的开放阅读框(ORF),编码456个氨基酸。该基因推断的蛋白与葡萄(Vitis vinifera)3GT蛋白的相似性为64%;与已知功能的3GT基因进行系统进化分析,结果显示Pd3GT1与葡萄3GT聚类到同一个分支。氨基酸比对显示Pd3GT1蛋白序列还有糖基转移酶家族特有的PSPG-box结构域。半定量PCR结果显示:Pd3GT1在花蕾,花开时期的花瓣中大量表达,在叶和花芽中有微弱表达;在根和茎中没有表达。说明Pd3GT基因表达具有组织表达特异性。该研究将为研究滇牡丹花色素形成机理,以及对滇牡丹类黄酮糖基转移酶的功能鉴定和利用基因工程手段改良花卉品质提供理论依据。
In plants,flavonoid 3-O-glucosyltransferas(F3GT),which transforms unstable flower pigment into stable flower pigment,is the key enzyme for forming anthocyanin in flowers.In this study,we used RT-PCR technology to clone a complete F3 GT gene from Paeonia delavayi.The gene sequence of Pd3GT1 consisted of 1371 bp open reading frames(ORF) and encoded 456 amino acids.The deduced Pd3 GT protein shared 64%similarity with that of Vitis vinifera 3GT.The system evolution analysis of 3GT gene with known function showed that Pd3GT1 and grape 3GT had the same branch.Amino acid alignment showed that Pd3GT1 protein sequence had PSPG-box structural domain which was peculiar to the family of glycosyl transferase.Semi-quantitative PCR results suggested that Pd3 GT was strongly expressed in petal during pre-blossom and bloom stage,slightly expressed in leaf and bud,but wasn't expressed in root and stem,which meant that the expression of Pd3GT1 gene was of tissue-specificity.This study would provide theoretical basis for the study of mechanism of flower pigment biosynthesis in P.delavayi,for functional identification of flavonoid 3-O-glucosyltransferase gene,and for improving flower quality by genetic engineering.
In plants,flavonoid 3-O-glucosyltransferas(F3GT),which transforms unstable flower pigment into stable flower pigment,is the key enzyme for forming anthocyanin in flowers.In this study,we used RT-PCR technology to clone a complete F3 GT gene from Paeonia delavayi.The gene sequence of Pd3GT1 consisted of 1371 bp open reading frames(ORF) and encoded 456 amino acids.The deduced Pd3 GT protein shared 64%similarity with that of Vitis vinifera 3GT.The system evolution analysis of 3GT gene with known function showed that Pd3GT1 and grape 3GT had the same branch.Amino acid alignment showed that Pd3GT1 protein sequence had PSPG-box structural domain which was peculiar to the family of glycosyl transferase.Semi-quantitative PCR results suggested that Pd3 GT was strongly expressed in petal during pre-blossom and bloom stage,slightly expressed in leaf and bud,but wasn't expressed in root and stem,which meant that the expression of Pd3GT1 gene was of tissue-specificity.This study would provide theoretical basis for the study of mechanism of flower pigment biosynthesis in P.delavayi,for functional identification of flavonoid 3-O-glucosyltransferase gene,and for improving flower quality by genetic engineering.
引文
Chang A.,Singh S.,Phillips G.N.,and Throson J.S.,2011,Glycsyltransferase structural biology and its role in the design of catalysts for glycosylation,Current Opinion in Biotechnology,22:800-808
Feng H.,Yi S.L.,Zuo J.Q.,Xie J.H.,and Huang X.,2014,Progress of flower colour modification by genetic engineering and anthocyanin biosynthesis pathway,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):445-451(冯欢,易姝利,左佳琦,谢佳恒,黄萱,2014,基因工程及花色素合成途径在花色改良中的研究进展,基因组学与应用生物学,33(2):445-451)
Fu H.H.,Xin P.Y.,Xu Y.L.,Liu Y.,Wei Y.J.,Dong J.,Cao Y.L.,and Zhou J.,2010,Bioinformatics analysis of UFGTgene from several economics plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):92-102(付海辉,辛培尧,许玉兰,刘岩,韦援教,董娇,曹有龙,周军,2010,几种经济植物UFGT基因的生物信息学分析,基因组学与应用生物学,30(1):92-102)
Gao Y.,Wang Y.X.,Zhang L.,Dou T.H.,and Zhou Y.,2014,Impact of sequence depth to detectegene and expression value in RNA seq,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural University:Natural Edition),29(5):642-647(高原,王翌霞,张亮,窦同海,周雁,2014,转录组测序深度对表达基因检出及表达量估计的影响,云南农业大学学报:自然科学版,29(5):642-647)
Gong X.,Pan Y.Z.,and Yang Z.Y.,2003,The dibersities and value of present situation of Paeonia delavayi,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),23(2):218-223(龚洵,潘跃芝,杨志云,2003,滇牡丹的多样性和现状评估,西北植物学报,23(2):218-223)
Guo S.Q.,Yang Y.W.,and Ni W.C.,2009,Cloning and bioinformatics analysis of glucosyltransferase gene UGT76G2 from Stevia rebaudian,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(3):422-428(郭书巧,杨郁文,倪万潮,2009,甜叶菊葡糖基转移酶基因UGT76G2的克隆及生物信息学分析,基因组学与应用生物学,28(3):422-428)
Hong D.Y.,Pan K.Y.,and Hong Y.,1998,Taxonomy of the Paeonia delavayi complex(Paeoniaceae),Annals of the Missouri Botanical Garden,85(4):554-564
Huang J.X.,Wang L.S.,Li X.M.,and Lu Y.Q.,2006,Advances in molecular basis and evolution of floral color variation,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(4):321-333(黄金霞,王亮生,李晓梅,鲁迎青,2006,花色变异的分子基础与进化模式研究进展,植物学通报,23(4):321-333)
Lao J.,Oikawa A.,Bromley J.R,Mclnerney P.,Suttangkakul A.,Smith-Moritz A.M.,Plahar H.,Chiu T.Y.,González Fernández-Nino S.M.,Ebert B.,Yang F.,Christiansen K.M.,Hansen S.F.,Stonebloom S.,Adams P.D.,Ronald P.C.,Hillson N.J.,Hadi M.Z.,Vega-Sánchez M.E.,LoquéD.,Scheller H.V.,and Heazlewood J.L.,2014,The plant glycosyltransferase clone collection for functional genomics,The Plant Journal,79:517-529
Li C.H.,Du H.,Wang L.S.,Shu Q.Y.,Zheng Y.R.,Xu Y.J.,Zhang J.J.,Zhang J.,Yang R.Z.,and Ge Y.X.,2009,Flavonoid composition and antioxidant activity of tree peony(Paeonia section Moutan)yellow flowers,Journal of agricultural and food chemistry,57(18):8496-8503
Martin L.B.B.,Fei Z.G.,Giovannoni J.J.,and Rose J.K.C.,2013,Catalyzing plant science research with RNA-seq,Frontiers in Plant Science,4(6):1-10
Mol J.,Cornish E.,Mason J.,and Koes R.,1999,Novel coloured flowers,Current Opinion in Biotechnology,10(2):198-201
Offen W.,Martinez-Fleites C.,Yang M.,Kiat-Lim E.,Davis B.G.,Tarling C.A.,Ford C.M.,Bowles D.J.,and Davies G.J.,2006,Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification,The EMBO Journal,25(6):1396-1405
Ozsolak F.,and Milos P.M.,2011,RNA sequencing:advances,challenges and opportunities,Nature Reviews Genetics,12(2):87-98
Ren X.X.,Zhang Y.,Xue J.Q.,Zhu F.Y.,Shi F.R.,Wang S.L.,and Zhang X.X.,2015,Genetic diversity analysis of natural populations in Paeonia delavayi,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(4):772-780(任秀霞,张盈,薛璟祺,朱富勇,石丰瑞,王顺利,张秀新,2015,滇牡丹天然居群的遗传多样性分析,植物遗传资源学报,16(4):772-780)
Shi Q.,Zhou L.,Wang Y.,Li K.,Zheng B.Q.,and Miao K.,2015,Transcriptomic analysis of Paeonia delavayi wild population flowers to identify differentially expressed genes involved in Purple-Red and yellow petal pigmentation,PLo S One,10(8):1-20
Vogt T.,and Jones P.,2000,Glycosyltransferases in plant natural product synthesis:characterization of a supergene family,Trends in Plant Science,5(9):380-386
Wang L.,Li Z.C.,and He C.Y.,2012,Transcriptome-wide mining of the differentially expressed transcripts for natural variation of floral organ size in Physalis philadelphica,Journal of experimental botany,63:6457-6465
Wang W.Y.,Li H.M.,Dai Y.M.,Zou H.,Wu S.J.,Li Y.S.,and Lin J.B.,2015,Cloning and expression analysis of flavonoid3-O-glucosyltrnasferase gene from Narcissus tazetta.var.Chinensis,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),30(6):577-581(王伟英,李海明,戴艺民,邹晖,吴水金,李跃森,林江波,2015,中国水仙类黄酮3-氧-葡糖基转移酶基因的克隆与序列分析,福建农业学报,30(6):577-581)
Wang Y.,Wang C.C.,Zhou X.,Bi W.,Yang Y.M.,and Wang J.,2015,Identification and characterization of a flavonoid3-O-glucosyltransferas gene from Indosasa hispida,Guangxi Zhiwu(Guihaia),35(2):244-249(王毅,王晨晨,周旭,毕玮,杨宇明,王娟,2015,七彩红竹类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析,广西植物,35(2):244-249)
Xiao J.P.,Li J.,and Guo H.C.,2015,Bioinformatical and expression analysis of UDP-glucose:flavonoid 3-3-O-glucosyltransferase gene from pigmented potato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(5):1017-1026(肖继坪,李俊,郭华春,彩色马铃薯类黄酮3-O-葡萄糖基转移酶基因(3GT)的生物信息学和表达分析,分子植物育种,13(5):1017-1026)
Yoshida K.,Toyama Y.,Kameda K.,and Kondo T.,2000,Contribution of each caffeoyl residue of the pigment molecule of gentiodelphin to blue color development,Phytochemistry,54(1):85-92
Zhang Y.L.,Li Z.H.,Ma H.,Wang Y.,Li W.J.,Liu X.X.,and Wan Y.M.,2011,Character variation of flower color groups in Paeonia delavayi(Paeoniaceae),Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),33(2):183-190(张艳丽,李正红,马宏,王雁,李文娟,刘秀贤,万友名,2011,滇牡丹花色类群性状变异分析,植物分类与资源学报,33(2):183-190)
Zhao D.,Hao Z.,and Tao J.,2012,Effects of shade on plant growth and flower quality in the herbaceous peony(Paeonia lactiflora Pall),Plant Physiology and Biochemistry,61:187-196
Zhao Y.P.,Chen F.D.,and Guo W.M.,2003,Advances in genetic engineering of flower color of ornamental plants,Zhiwuxue Tongbao(Chinese Bullrtin of Botany),20(1):51-58(赵云鹏,陈发棣,郭维明,2003,观赏植物花色基因工程研究进展,植物学通报,20(1):51-58)
Zhao Z.C.,Hu G.B.,Hu F.C.,Wang H.C.,Yang Z.Y.,and Lai B.,2012,The UDP glucose:flavonoid-3-O-glucosyltransferase(UFGT)gene regulates anthocyanin biosynthesis in litchi(Litchi chinesis Sonn.)during fruit coloration,Mol.Biol.Rep.,39(6):6409-6415
Zhou L.,Wang Y.,Lv C.Y.,and Peng Z.H.,2011,Identification of components of flower pigments in petals of Paeonia lutea wild population in Yunnan,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),39(8):52-54(周琳,王雁,律春燕,彭镇华,2011,云南野生黄牡丹花色素成分的鉴定,东北林业大学学报,39(8):52-54)
Feng H.,Yi S.L.,Zuo J.Q.,Xie J.H.,and Huang X.,2014,Progress of flower colour modification by genetic engineering and anthocyanin biosynthesis pathway,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),33(2):445-451(冯欢,易姝利,左佳琦,谢佳恒,黄萱,2014,基因工程及花色素合成途径在花色改良中的研究进展,基因组学与应用生物学,33(2):445-451)
Fu H.H.,Xin P.Y.,Xu Y.L.,Liu Y.,Wei Y.J.,Dong J.,Cao Y.L.,and Zhou J.,2010,Bioinformatics analysis of UFGTgene from several economics plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),30(1):92-102(付海辉,辛培尧,许玉兰,刘岩,韦援教,董娇,曹有龙,周军,2010,几种经济植物UFGT基因的生物信息学分析,基因组学与应用生物学,30(1):92-102)
Gao Y.,Wang Y.X.,Zhang L.,Dou T.H.,and Zhou Y.,2014,Impact of sequence depth to detectegene and expression value in RNA seq,Yunnan Nongye Daxue Xuebao(Journal of Yunnan Agricultural University:Natural Edition),29(5):642-647(高原,王翌霞,张亮,窦同海,周雁,2014,转录组测序深度对表达基因检出及表达量估计的影响,云南农业大学学报:自然科学版,29(5):642-647)
Gong X.,Pan Y.Z.,and Yang Z.Y.,2003,The dibersities and value of present situation of Paeonia delavayi,Xibei Zhiwu Xuebao(Acta Botanica Boreali-occidentalia Sinica),23(2):218-223(龚洵,潘跃芝,杨志云,2003,滇牡丹的多样性和现状评估,西北植物学报,23(2):218-223)
Guo S.Q.,Yang Y.W.,and Ni W.C.,2009,Cloning and bioinformatics analysis of glucosyltransferase gene UGT76G2 from Stevia rebaudian,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),28(3):422-428(郭书巧,杨郁文,倪万潮,2009,甜叶菊葡糖基转移酶基因UGT76G2的克隆及生物信息学分析,基因组学与应用生物学,28(3):422-428)
Hong D.Y.,Pan K.Y.,and Hong Y.,1998,Taxonomy of the Paeonia delavayi complex(Paeoniaceae),Annals of the Missouri Botanical Garden,85(4):554-564
Huang J.X.,Wang L.S.,Li X.M.,and Lu Y.Q.,2006,Advances in molecular basis and evolution of floral color variation,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(4):321-333(黄金霞,王亮生,李晓梅,鲁迎青,2006,花色变异的分子基础与进化模式研究进展,植物学通报,23(4):321-333)
Lao J.,Oikawa A.,Bromley J.R,Mclnerney P.,Suttangkakul A.,Smith-Moritz A.M.,Plahar H.,Chiu T.Y.,González Fernández-Nino S.M.,Ebert B.,Yang F.,Christiansen K.M.,Hansen S.F.,Stonebloom S.,Adams P.D.,Ronald P.C.,Hillson N.J.,Hadi M.Z.,Vega-Sánchez M.E.,LoquéD.,Scheller H.V.,and Heazlewood J.L.,2014,The plant glycosyltransferase clone collection for functional genomics,The Plant Journal,79:517-529
Li C.H.,Du H.,Wang L.S.,Shu Q.Y.,Zheng Y.R.,Xu Y.J.,Zhang J.J.,Zhang J.,Yang R.Z.,and Ge Y.X.,2009,Flavonoid composition and antioxidant activity of tree peony(Paeonia section Moutan)yellow flowers,Journal of agricultural and food chemistry,57(18):8496-8503
Martin L.B.B.,Fei Z.G.,Giovannoni J.J.,and Rose J.K.C.,2013,Catalyzing plant science research with RNA-seq,Frontiers in Plant Science,4(6):1-10
Mol J.,Cornish E.,Mason J.,and Koes R.,1999,Novel coloured flowers,Current Opinion in Biotechnology,10(2):198-201
Offen W.,Martinez-Fleites C.,Yang M.,Kiat-Lim E.,Davis B.G.,Tarling C.A.,Ford C.M.,Bowles D.J.,and Davies G.J.,2006,Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification,The EMBO Journal,25(6):1396-1405
Ozsolak F.,and Milos P.M.,2011,RNA sequencing:advances,challenges and opportunities,Nature Reviews Genetics,12(2):87-98
Ren X.X.,Zhang Y.,Xue J.Q.,Zhu F.Y.,Shi F.R.,Wang S.L.,and Zhang X.X.,2015,Genetic diversity analysis of natural populations in Paeonia delavayi,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),16(4):772-780(任秀霞,张盈,薛璟祺,朱富勇,石丰瑞,王顺利,张秀新,2015,滇牡丹天然居群的遗传多样性分析,植物遗传资源学报,16(4):772-780)
Shi Q.,Zhou L.,Wang Y.,Li K.,Zheng B.Q.,and Miao K.,2015,Transcriptomic analysis of Paeonia delavayi wild population flowers to identify differentially expressed genes involved in Purple-Red and yellow petal pigmentation,PLo S One,10(8):1-20
Vogt T.,and Jones P.,2000,Glycosyltransferases in plant natural product synthesis:characterization of a supergene family,Trends in Plant Science,5(9):380-386
Wang L.,Li Z.C.,and He C.Y.,2012,Transcriptome-wide mining of the differentially expressed transcripts for natural variation of floral organ size in Physalis philadelphica,Journal of experimental botany,63:6457-6465
Wang W.Y.,Li H.M.,Dai Y.M.,Zou H.,Wu S.J.,Li Y.S.,and Lin J.B.,2015,Cloning and expression analysis of flavonoid3-O-glucosyltrnasferase gene from Narcissus tazetta.var.Chinensis,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),30(6):577-581(王伟英,李海明,戴艺民,邹晖,吴水金,李跃森,林江波,2015,中国水仙类黄酮3-氧-葡糖基转移酶基因的克隆与序列分析,福建农业学报,30(6):577-581)
Wang Y.,Wang C.C.,Zhou X.,Bi W.,Yang Y.M.,and Wang J.,2015,Identification and characterization of a flavonoid3-O-glucosyltransferas gene from Indosasa hispida,Guangxi Zhiwu(Guihaia),35(2):244-249(王毅,王晨晨,周旭,毕玮,杨宇明,王娟,2015,七彩红竹类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析,广西植物,35(2):244-249)
Xiao J.P.,Li J.,and Guo H.C.,2015,Bioinformatical and expression analysis of UDP-glucose:flavonoid 3-3-O-glucosyltransferase gene from pigmented potato,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(5):1017-1026(肖继坪,李俊,郭华春,彩色马铃薯类黄酮3-O-葡萄糖基转移酶基因(3GT)的生物信息学和表达分析,分子植物育种,13(5):1017-1026)
Yoshida K.,Toyama Y.,Kameda K.,and Kondo T.,2000,Contribution of each caffeoyl residue of the pigment molecule of gentiodelphin to blue color development,Phytochemistry,54(1):85-92
Zhang Y.L.,Li Z.H.,Ma H.,Wang Y.,Li W.J.,Liu X.X.,and Wan Y.M.,2011,Character variation of flower color groups in Paeonia delavayi(Paeoniaceae),Zhiwu Fenlei Yu Ziyuan Xuebao(Plant Diversity and Resources),33(2):183-190(张艳丽,李正红,马宏,王雁,李文娟,刘秀贤,万友名,2011,滇牡丹花色类群性状变异分析,植物分类与资源学报,33(2):183-190)
Zhao D.,Hao Z.,and Tao J.,2012,Effects of shade on plant growth and flower quality in the herbaceous peony(Paeonia lactiflora Pall),Plant Physiology and Biochemistry,61:187-196
Zhao Y.P.,Chen F.D.,and Guo W.M.,2003,Advances in genetic engineering of flower color of ornamental plants,Zhiwuxue Tongbao(Chinese Bullrtin of Botany),20(1):51-58(赵云鹏,陈发棣,郭维明,2003,观赏植物花色基因工程研究进展,植物学通报,20(1):51-58)
Zhao Z.C.,Hu G.B.,Hu F.C.,Wang H.C.,Yang Z.Y.,and Lai B.,2012,The UDP glucose:flavonoid-3-O-glucosyltransferase(UFGT)gene regulates anthocyanin biosynthesis in litchi(Litchi chinesis Sonn.)during fruit coloration,Mol.Biol.Rep.,39(6):6409-6415
Zhou L.,Wang Y.,Lv C.Y.,and Peng Z.H.,2011,Identification of components of flower pigments in petals of Paeonia lutea wild population in Yunnan,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),39(8):52-54(周琳,王雁,律春燕,彭镇华,2011,云南野生黄牡丹花色素成分的鉴定,东北林业大学学报,39(8):52-54)