滇牡丹类黄酮7-O-葡萄糖基转移酶基因的鉴定与表达分析
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摘要
以野生滇牡丹为试验材料,以其转录组数据为基础,采用反转录PCR技术克隆得到一个具完整开放阅读框的类黄酮7-O-葡萄糖基转移酶基因(Pd7GT),并通过生物信息学分析手段、基因表达对该基因进行分析。结果显示:该基因全长1 446bp (Gen Bank登录号为KX394687),可编码481个蛋白氨基酸;生物信息学分析发现Pd7GT蛋白C末端含有典型糖基转移酶识别区(WAPQV)和UDP-葡萄糖基配体绑定位点(HCGWNS)的PSPG盒子,其蛋白序列GWAPQVMILEHEAVGGFVTHCGWNSTLEGISAGLPLVTWPIFAEQFYNEK,与可可(XP_007042481)、Herrannia umbratica (XP_021298085)、毛果杨(XP_006379195)、巨桉(XP_010066837)等以葡萄糖为糖基配体的类黄酮7-O-糖基转移酶聚为一类; Pd7GT基因在组织茎中表达量最高、不同花发育时期的花谢期表达量最高、不同颜色花瓣中黄花花瓣表达量最高。本研究为滇牡丹糖基转移酶异源表达、分子育种等研究提供一定的基础,为未来通过基因工程培育具新颖花色和抗性的滇牡丹新品种提供必要材料。
Taken wild Paeonia delavayi as experimental materials,we isolated and cloned a full length c DNA of flavonoid 7-O-glucosyltransferase gene through analyzing the transcriptome of P. delavayi by the methods of RT-PCR,which has a whole opening reading frame,and carried out analysis to Pd7 GT with bioinformatics and gene expression. The results showed that the full length of Pd7 GT gene 1 446 bp( Gen Bank ID: KX394687),could encode 481 amino acids; the results of bioinformatics analysis revealed there a PSPG box in the C-terminal of Pd7 GT,which includes typical identical region of glucosyltransferase( WAPQV) and UDP-glucose binding sites( HCGWNS),the proteinic sequence of Pd7 GT GWAPQVMILEHEAVGGFVTHCGWNSTLEGISAGLPLVTWPIFAEQFYNEK; the dendrogram of Pd7 GT clarified that Pd7 GT and Theobroma cacao( XP_007042481),Herrannia umbratica( XP_021298085),Populus trichocarpa( XP_006379195),Eucalyptus grandis( XP _010066837) clustered one clade,which clade takes UDP-glucose as sugar moiety and be flavonoid 7-O-glucosyltransferase. The present study some basis to heterologous expression of the glucosyltransferase and molecular breeding of P. delavayi,and culturing the new cultivar that has new color and resistance through genetic engineering.
Taken wild Paeonia delavayi as experimental materials,we isolated and cloned a full length c DNA of flavonoid 7-O-glucosyltransferase gene through analyzing the transcriptome of P. delavayi by the methods of RT-PCR,which has a whole opening reading frame,and carried out analysis to Pd7 GT with bioinformatics and gene expression. The results showed that the full length of Pd7 GT gene 1 446 bp( Gen Bank ID: KX394687),could encode 481 amino acids; the results of bioinformatics analysis revealed there a PSPG box in the C-terminal of Pd7 GT,which includes typical identical region of glucosyltransferase( WAPQV) and UDP-glucose binding sites( HCGWNS),the proteinic sequence of Pd7 GT GWAPQVMILEHEAVGGFVTHCGWNSTLEGISAGLPLVTWPIFAEQFYNEK; the dendrogram of Pd7 GT clarified that Pd7 GT and Theobroma cacao( XP_007042481),Herrannia umbratica( XP_021298085),Populus trichocarpa( XP_006379195),Eucalyptus grandis( XP _010066837) clustered one clade,which clade takes UDP-glucose as sugar moiety and be flavonoid 7-O-glucosyltransferase. The present study some basis to heterologous expression of the glucosyltransferase and molecular breeding of P. delavayi,and culturing the new cultivar that has new color and resistance through genetic engineering.
引文
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[2]龚洵,潘跃芝,杨志云.滇牡丹的多样性和现状评估[J].西北植物学报,2003,23(2):218-223.
[3]任秀霞,张盈,薛璟祺,等.滇牡丹天然居群的遗传多样性分析[J].植物遗传资源学报,2015,16(4):772-780.
[4]张艳丽,李文娟,王雁,等.滇牡丹花色类群性状变异分析[J].植物分类与资源学报,2011,33(2):183-190.
[5]周琳,王雁,律春燕,等.云南野生黄牡丹花色素成分的鉴定[J].东北林业大学学报,2011,39(8):52-54.
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[8]Keiko Y S. Functional genomics of family 1 glycosyltransferases in Arabidopsis[J]. Plant Biotechnology,2009,26(3):267-274.
[9]Gachon C M,Langlois-Meurinne M,Saindrenan P.Plant secondary metabolism glycosyltransferases:the emerging functional analysis[J]. Trends in Plant Science,2005,10(11):542-549.
[10]Tanaka Y,Brugliera F,Chandler S. Recent progress of flower colour modification by biotechnology[J]. International Journal of Molecular Sciences,2009,10(12):5350-5369.
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[12]Hirotani M,Kuroda R,Suzuki H,et al. Cloning and expression of UDP-glucose:flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis[J]. Planta,2000,210(6):1006-1013.
[13]Nagashima S,Inagaki R,Kubo A,et al. c DNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures[J]. Planta,2004,218(3):456-459.
[14]Kramer C M,Prata R T N,Willits M G,et al. Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa[J]. Phytochemistry,2003,64(6):1069-1076.
[15]Kim J H,Kim B G,Park Y,et al. Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana[J]. Bioscience Biotechnology&Biochemistry,2006,70(6):1471-1477.
[16]Shi Q Q,Lin Z,Yan W,et al. Transcriptomic analysis of Paeonia delavayi wild population flowers to identify differentially expressed genes involved in purple-red and yellow petal pigmentation[J]. Plos One,2015,10(8):e0135038. doi:10.1371/journal. pone. 0135038
[17]王毅,赵能,原晓龙,等.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[J].基因组学与应用生物学,2017,36(4):1556-1562.
[18]Campbell J A,Davies G J,Bulone V,et al. A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities[J]. Biochemical Journal,1997,326(Pt3):929-939.
[19]Jones P,Vogt T. Glycosyltransferases in secondary plant metabolism:tranquilizers and stimulant controllers[J].Planta,2001,213(2):164-174.
[20]Mackenzie P I,Owens I S,Burchell B,et al. The UDP glycosyltransferase gene superfamily:recommended nomenclature update based on evolutionary divergence[J]. Pharmacogenetics,1997,7(4):255-269.
[21]Ishikura N,Mato M. Partial purification and some properties of flavonol 3-O-glycosyltransferases from seedlings of Vigna mungo,with special reference to the formation of Kaempferol 3-O-galactoside and 3-O-glucoside[J]. Plant&Cell Physiology,1993,34(2):329-335.
[22]Barpeled M,Lewinsohn E,Fluhr R,et al. UDP-rhamnose:flavanone-7-O-glucoside-2''-O-rhamnosyltransferase.Purification and characterization of an enzyme catalyzing the production of bitter compounds in citrus[J]. Journal of Biological Chemistry,1991,266(31):20953-20959.
[23]Lim E K,Bowles D J. A class of plant glycosyltransferases involved in cellular homeostasis[J]. The EMBO Journal,2014,23(15):2915-2922.
[24]Paquette S,Mller B L,Bak S. On the origin of family1 plant glycosyltransferases[J]. Phytochemistry,2003,62(3):399-413.
[25]姬向楠,何非,段长青,等.植物UDP-糖基转移酶生化特性和功能研究进展[J].食品科学,2013,34(9):316-323.
[26]Chang A,Singh S,Phillips G N,et al. Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation[J]. Current Opinion in Biotechnology,2011,22(6):800-808.
[27]Kapitonov D,Yu R K. Conserved domains of glycosyltransferases[J]. Glycobiology,1999,9(10):961-978.
[28]Coutinho P M,Deleury E,Davies G J,et al. An evolving hierarchical family classification for glycosyltransferases[J]. Journal of Molecular Biology,2003,328(2):307-317.
[29]Nakatsuka T,Sato K,Takahashi H,et al. Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian[J]. Journal of Experimental Botany,2008,59(6):1241-1252.
[30]Yoshihara N,Imayama T,Fukuchi-Mizutani M,et al.c DNA cloning and characterization of UDP-glucose:Anthocyanidin 3-O-glucosyltransferase in Iris hollandica[J]. Plant Science,2005,169(3):496-501.
[31]代畅,彭正松,杨在君,等.小麦糖基转移酶基因Ta UGT73D1的克隆及表达分析[J].分子植物育种,2017,15(6):2048-2057.
[32]陈娜,胡东青,潘丽娟,等.花生中UDP-葡萄糖基转移酶基因的克隆及在非生物胁迫下的表达研究[J].中国油料作物学报,2014,36(3):308-315.
[2]龚洵,潘跃芝,杨志云.滇牡丹的多样性和现状评估[J].西北植物学报,2003,23(2):218-223.
[3]任秀霞,张盈,薛璟祺,等.滇牡丹天然居群的遗传多样性分析[J].植物遗传资源学报,2015,16(4):772-780.
[4]张艳丽,李文娟,王雁,等.滇牡丹花色类群性状变异分析[J].植物分类与资源学报,2011,33(2):183-190.
[5]周琳,王雁,律春燕,等.云南野生黄牡丹花色素成分的鉴定[J].东北林业大学学报,2011,39(8):52-54.
[6]Togami J,Okuhara H,Nakamura N,et al. Isolation of c DNAs encoding tetrahydroxychalcone 2'-glucosyltransferase activity from carnation,cyclamen,and catharanthus[J]. Plant Biotechnology,2011,28(2):231-238.
[7]Vogt T,Jones P. Glycosyltransferases in plant natural product synthesis characterization of a supergene family[J].Trends in Plant Science,2000,5(9):380-386.
[8]Keiko Y S. Functional genomics of family 1 glycosyltransferases in Arabidopsis[J]. Plant Biotechnology,2009,26(3):267-274.
[9]Gachon C M,Langlois-Meurinne M,Saindrenan P.Plant secondary metabolism glycosyltransferases:the emerging functional analysis[J]. Trends in Plant Science,2005,10(11):542-549.
[10]Tanaka Y,Brugliera F,Chandler S. Recent progress of flower colour modification by biotechnology[J]. International Journal of Molecular Sciences,2009,10(12):5350-5369.
[11]Tanaka Y,Sasaki N,Ohmiya A. Biosynthesis of plant pigments:anthocyanins,betalains and carotenoids[J]. Plant Journal,2008,54(4):733-749.
[12]Hirotani M,Kuroda R,Suzuki H,et al. Cloning and expression of UDP-glucose:flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis[J]. Planta,2000,210(6):1006-1013.
[13]Nagashima S,Inagaki R,Kubo A,et al. c DNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures[J]. Planta,2004,218(3):456-459.
[14]Kramer C M,Prata R T N,Willits M G,et al. Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Allium cepa[J]. Phytochemistry,2003,64(6):1069-1076.
[15]Kim J H,Kim B G,Park Y,et al. Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana[J]. Bioscience Biotechnology&Biochemistry,2006,70(6):1471-1477.
[16]Shi Q Q,Lin Z,Yan W,et al. Transcriptomic analysis of Paeonia delavayi wild population flowers to identify differentially expressed genes involved in purple-red and yellow petal pigmentation[J]. Plos One,2015,10(8):e0135038. doi:10.1371/journal. pone. 0135038
[17]王毅,赵能,原晓龙,等.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[J].基因组学与应用生物学,2017,36(4):1556-1562.
[18]Campbell J A,Davies G J,Bulone V,et al. A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities[J]. Biochemical Journal,1997,326(Pt3):929-939.
[19]Jones P,Vogt T. Glycosyltransferases in secondary plant metabolism:tranquilizers and stimulant controllers[J].Planta,2001,213(2):164-174.
[20]Mackenzie P I,Owens I S,Burchell B,et al. The UDP glycosyltransferase gene superfamily:recommended nomenclature update based on evolutionary divergence[J]. Pharmacogenetics,1997,7(4):255-269.
[21]Ishikura N,Mato M. Partial purification and some properties of flavonol 3-O-glycosyltransferases from seedlings of Vigna mungo,with special reference to the formation of Kaempferol 3-O-galactoside and 3-O-glucoside[J]. Plant&Cell Physiology,1993,34(2):329-335.
[22]Barpeled M,Lewinsohn E,Fluhr R,et al. UDP-rhamnose:flavanone-7-O-glucoside-2''-O-rhamnosyltransferase.Purification and characterization of an enzyme catalyzing the production of bitter compounds in citrus[J]. Journal of Biological Chemistry,1991,266(31):20953-20959.
[23]Lim E K,Bowles D J. A class of plant glycosyltransferases involved in cellular homeostasis[J]. The EMBO Journal,2014,23(15):2915-2922.
[24]Paquette S,Mller B L,Bak S. On the origin of family1 plant glycosyltransferases[J]. Phytochemistry,2003,62(3):399-413.
[25]姬向楠,何非,段长青,等.植物UDP-糖基转移酶生化特性和功能研究进展[J].食品科学,2013,34(9):316-323.
[26]Chang A,Singh S,Phillips G N,et al. Glycosyltransferase structural biology and its role in the design of catalysts for glycosylation[J]. Current Opinion in Biotechnology,2011,22(6):800-808.
[27]Kapitonov D,Yu R K. Conserved domains of glycosyltransferases[J]. Glycobiology,1999,9(10):961-978.
[28]Coutinho P M,Deleury E,Davies G J,et al. An evolving hierarchical family classification for glycosyltransferases[J]. Journal of Molecular Biology,2003,328(2):307-317.
[29]Nakatsuka T,Sato K,Takahashi H,et al. Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian[J]. Journal of Experimental Botany,2008,59(6):1241-1252.
[30]Yoshihara N,Imayama T,Fukuchi-Mizutani M,et al.c DNA cloning and characterization of UDP-glucose:Anthocyanidin 3-O-glucosyltransferase in Iris hollandica[J]. Plant Science,2005,169(3):496-501.
[31]代畅,彭正松,杨在君,等.小麦糖基转移酶基因Ta UGT73D1的克隆及表达分析[J].分子植物育种,2017,15(6):2048-2057.
[32]陈娜,胡东青,潘丽娟,等.花生中UDP-葡萄糖基转移酶基因的克隆及在非生物胁迫下的表达研究[J].中国油料作物学报,2014,36(3):308-315.