葡萄风信子MaGT1基因的克隆及其表达分析
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摘要
该研究以葡萄风信子(Muscari ameniacum)‘亚美尼亚’为试材,克隆了花青素合成途径过程中的类黄酮糖基转移酶基因MaGT1(GenBank登录号MK652470)。该基因开放阅读框全长1 338 bp,编码445个氨基酸,预测蛋白分子量为49.301 kD,理论等电点为5.40。结构分析显示,MaGT1蛋白具有PSPG保守结构域、UDP-糖基转移酶家族结构域和UDP-葡萄糖醛酸基/葡萄糖基转移酶保守域(UDPGT)。进化分析表明,MaGT1蛋白与油棕、海枣、葡萄亲缘关系相近,聚类于类黄酮糖苷糖基转移酶类分支,以UDP-葡萄糖/鼠李糖为主要糖供体。花青苷含量测定显示,花青苷仅在葡萄风信子着色的花中积累,在根、鳞茎和叶以及未着色的花蕾(S1)中几乎检测不到花青苷,且随着花的发育,花青苷含量不断增加,并在衰败期(S5)达到最高。荧光定量PCR分析表明,MaGT1基因的表达具有显著的时空特异性,并在花中优势表达,而在根、鳞茎和叶片中微量表达;在花发育不同阶段,MaGT1基因的表达量随着花发育不断增加,并在盛花期达到峰值。研究表明,MaGT1蛋白催化反应是花青素合成途径中的重要修饰步骤。该研究结果为进一步分析MaGT1基因在葡萄风信子花青素合成和调控中的功能提供了依据。
In this study, a flavonoid glycosyltransferase gene in the anthocyanin synthesis pathway was isolated from petals of Muscari ameniacum and designated as MaGT1(GenBank accession was MK652470). The full-length of ORF was 1 338 bp,encoding 445 amino acids. The molecular weight of the predicted enzyme was 49.301 kD and the pI value was 5.40. The results of structural analysis revealed that the deduced MaGT1 protein contains a typical conserved PSPG motif, an UDP-glycosyltransferase family domain and an UDP-glucuronosyltransferase/glucosyltransferase domain(UDPGT). The results of evolutionary analysis showed that MaGT1 protein is closely related to Elaeis guineensis, Phoenix dactylifera and Vitis vinifera and belongs to the flavonoid glycoside glycosyltransferase branch, with UDP-glucose/rhamnose as the main sugar donor. Anthocyanin content assay showed that anthocyanins only accumulate in the flowers of M. ameniacum, but almost no anthocyanins were accumulated in roots, bulbs and leaves, and uncolored flower buds(S1), and with the flower development process, the content of anthocyanins increased continuously and reached the highest in the decay period(S5). The results of real-time PCR showed that the expression of MaGT1 gene has significant space-time specificity. It is predominantly expressed in flower tissues and is rarely expressed in roots, bulbs and leaves. At different flower development stages, the expression level of MaGT1 gene increased with flower development and peaked at the fully opened petal stage. The results showed that the catalytic reaction of MaGT1 protein is an important modification step in the anthocyanin synthesis pathway. This study provides a basis for further study of the function of MaGT1 gene in the synthesis and regulation of anthocyanins in M. ameniacum.
In this study, a flavonoid glycosyltransferase gene in the anthocyanin synthesis pathway was isolated from petals of Muscari ameniacum and designated as MaGT1(GenBank accession was MK652470). The full-length of ORF was 1 338 bp,encoding 445 amino acids. The molecular weight of the predicted enzyme was 49.301 kD and the pI value was 5.40. The results of structural analysis revealed that the deduced MaGT1 protein contains a typical conserved PSPG motif, an UDP-glycosyltransferase family domain and an UDP-glucuronosyltransferase/glucosyltransferase domain(UDPGT). The results of evolutionary analysis showed that MaGT1 protein is closely related to Elaeis guineensis, Phoenix dactylifera and Vitis vinifera and belongs to the flavonoid glycoside glycosyltransferase branch, with UDP-glucose/rhamnose as the main sugar donor. Anthocyanin content assay showed that anthocyanins only accumulate in the flowers of M. ameniacum, but almost no anthocyanins were accumulated in roots, bulbs and leaves, and uncolored flower buds(S1), and with the flower development process, the content of anthocyanins increased continuously and reached the highest in the decay period(S5). The results of real-time PCR showed that the expression of MaGT1 gene has significant space-time specificity. It is predominantly expressed in flower tissues and is rarely expressed in roots, bulbs and leaves. At different flower development stages, the expression level of MaGT1 gene increased with flower development and peaked at the fully opened petal stage. The results showed that the catalytic reaction of MaGT1 protein is an important modification step in the anthocyanin synthesis pathway. This study provides a basis for further study of the function of MaGT1 gene in the synthesis and regulation of anthocyanins in M. ameniacum.
引文
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[9] 王伟英,李海明,戴艺民,等.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[J].福建农业学报,2015,30(6):577-581.WANG W Y,LI H M,DAI Y M,et al.Cloning and expression analysis of flavonoid 3-O-glucosyltransferase gene from Narcissus tazetta.var.chinensis[J].Fujian Journal of Agricultural Sciences,2015,30(6):577-581.
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[12] 焦淑珍,刘雅莉,娄倩,等.葡萄风信子二氢黄酮醇4-还原酶基因(DFR)的克隆与表达分析[J].农业生物技术学报,2014,22(5):529-540.JIAO S Z,LIU Y L,LOU Q,et al.Cloning and expression analysis of dihydroflavonol 4-reductase gene (DFR) from grape hyacinth (Muscari armeniacum)[J].Journal of Agricultural Biotechnology,2014,22(5):529-540.
[13] 杜灵娟,陈凯利,刘雅莉,等.葡萄风信子花青素3-O-葡糖基转移酶基因的克隆及其表达分析[J].草业科学,2017,34(11):2 235-2 244.DU L J,CHEN K L,LIU Y L,et al.Cloning and expression analysis of anthocyanidin 3-O -glucosyltransferase gene in grape hyacinth[J].Pratacultural Science,2017,34(11):2 235-2 244.
[14] QI Y Y,LOU Q,LI H B,et al.Anatomical and biochemical studies of bicolored flower development in Muscari latifolium[J].Protoplasma,2013,250(6):1 273-1 281.
[15] SAWADA S,SUZUKI H,ICHIMAIDA F,et al.UDP-glucuronic acid:Anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers[J].Journal of Biological Chemistry,2005,280(2):899-906.
[16] ROJAS RODAS F,RODRIGUEZ T O,MURAI Y,et al.Linkage mapping,molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase[J].Plant Molecular Biology,2014,84(3):287-300.
[17] SAYAMA T,ONO E,TAKAGI K,et al.The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean[J].The Plant Cell,2012,24(5):2 123-2 138.
[18] BOSS P K,DAVIES C,ROBINSON S P.Expression of anthocyanin biosynthesis pathway genes in red and white grapes[J].Plant Molecular Biology,1996,32(3):565-569.
[19] ZHAO Z C,HU G B,HU F C,et al.The UDP glucose:Flavonoid-3-O-glucosyltransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn.) during fruit coloration[J].Molecular Biology Reports,2012,39(6):6 409-6 415.
[20] 王应丽,黄文俊,王瑛,等.箭叶淫羊藿EsUF3GT基因的克隆及表达分析[J].植物科学学报,2014,32(6):602-611.WANG Y L,HUANG W J,WANG Y,et al.Cloning and expression analysis of the EsUF3GT gene in Epimedium sagittatum (Sieb.et Zucc.) Maxim[J].Plant Science Journal,2014,32(6):602-611.
[21] GRIESSER M,HOFFMANN T,BELLIDO M L,et al.Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit[J].Plant Physiology,2008,146(4):1 528-1 539.
[22] 隋昕.香雪兰UDP-葡萄糖:类黄酮3-O-葡萄糖基转移酶基因的克隆及其功能鉴定[D].长春:东北师范大学,2011.
[2] 招雪晴,李勃,苑兆和,等.石榴PgUFGT基因克隆及表达分析[J].西北植物学报,2017,37(4):646-653.ZHAO X Q,LI B,YUAN Z H,et al.Cloning and expression analysis of PgUFGT gene from Punica granaum L.[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(4):646-653.
[3] HOU B K,LIM E K,HIGGINS G S,et al.N-glucosylation of cytokinins by glycosyltransferases of Arabidopsis thaliana[J].The Journal of Biological Chemistry,2004,279(46):47 822-47 832.
[4] KOBAYASHI S,ISHIMARU M,DING C K,et al.Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin[J].Plant Science:an International Journal of Experimental Plant Biology,2001,160(3):543-550.
[5] ALMEIDA J R,D?AMICO E,PREUSS A,et al.Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria xananassa)[J].Archives of Biochemistry and Biophysics,2007,465(1):61-71.
[6] 韩振云,宋婷婷,田佶,等.苹果属观赏海棠McUFGT的克隆及其在不同叶色品种间的表达差异分析[J].园艺学报,2014,41(2):301-310.HAN Z Y,SONG T T,TIAN J,et al.Cloning and expression analysis of McUFGT in different cultivars of crabapple[J].Acta Horticulturae Sinica,2014,41(2):301-310.
[7] TANAKA Y,YONEKURA K,FUKUCHI-MIZUTANI M,et al.Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora[J].Plant & Cell Physiology,1996,37(5):711-716.
[8] SUI X,GAO X,AO M,et al.cDNA cloning and characterization of UDP-glucose:Anthocyanidin 3-O-glucosyltransferase in Freesia hybrida[J].Plant Cell Reports,2011,30(7):1 209-1 218.
[9] 王伟英,李海明,戴艺民,等.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[J].福建农业学报,2015,30(6):577-581.WANG W Y,LI H M,DAI Y M,et al.Cloning and expression analysis of flavonoid 3-O-glucosyltransferase gene from Narcissus tazetta.var.chinensis[J].Fujian Journal of Agricultural Sciences,2015,30(6):577-581.
[10] MULHOLLAND D A,SCHWIKKARD S L,CROUCH N R.The chemistry and biological activity of the Hyacinthaceae[J].Natural Product Reports,2013,30(9):1 165-1 210.
[11] 安维忠,刘雅莉,刘红利,等.葡萄风信子MaANS基因及启动子的克隆与分析[J].西北植物学报,2015,35(9):1 728-1 734.AN W Z,LIU Y L,LIU H L,et al.Isolation and analysis of Muscari armeniacum MaANS gene and its promoter[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(9):1 728-1 734.
[12] 焦淑珍,刘雅莉,娄倩,等.葡萄风信子二氢黄酮醇4-还原酶基因(DFR)的克隆与表达分析[J].农业生物技术学报,2014,22(5):529-540.JIAO S Z,LIU Y L,LOU Q,et al.Cloning and expression analysis of dihydroflavonol 4-reductase gene (DFR) from grape hyacinth (Muscari armeniacum)[J].Journal of Agricultural Biotechnology,2014,22(5):529-540.
[13] 杜灵娟,陈凯利,刘雅莉,等.葡萄风信子花青素3-O-葡糖基转移酶基因的克隆及其表达分析[J].草业科学,2017,34(11):2 235-2 244.DU L J,CHEN K L,LIU Y L,et al.Cloning and expression analysis of anthocyanidin 3-O -glucosyltransferase gene in grape hyacinth[J].Pratacultural Science,2017,34(11):2 235-2 244.
[14] QI Y Y,LOU Q,LI H B,et al.Anatomical and biochemical studies of bicolored flower development in Muscari latifolium[J].Protoplasma,2013,250(6):1 273-1 281.
[15] SAWADA S,SUZUKI H,ICHIMAIDA F,et al.UDP-glucuronic acid:Anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers[J].Journal of Biological Chemistry,2005,280(2):899-906.
[16] ROJAS RODAS F,RODRIGUEZ T O,MURAI Y,et al.Linkage mapping,molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase[J].Plant Molecular Biology,2014,84(3):287-300.
[17] SAYAMA T,ONO E,TAKAGI K,et al.The Sg-1 glycosyltransferase locus regulates structural diversity of triterpenoid saponins of soybean[J].The Plant Cell,2012,24(5):2 123-2 138.
[18] BOSS P K,DAVIES C,ROBINSON S P.Expression of anthocyanin biosynthesis pathway genes in red and white grapes[J].Plant Molecular Biology,1996,32(3):565-569.
[19] ZHAO Z C,HU G B,HU F C,et al.The UDP glucose:Flavonoid-3-O-glucosyltransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn.) during fruit coloration[J].Molecular Biology Reports,2012,39(6):6 409-6 415.
[20] 王应丽,黄文俊,王瑛,等.箭叶淫羊藿EsUF3GT基因的克隆及表达分析[J].植物科学学报,2014,32(6):602-611.WANG Y L,HUANG W J,WANG Y,et al.Cloning and expression analysis of the EsUF3GT gene in Epimedium sagittatum (Sieb.et Zucc.) Maxim[J].Plant Science Journal,2014,32(6):602-611.
[21] GRIESSER M,HOFFMANN T,BELLIDO M L,et al.Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit[J].Plant Physiology,2008,146(4):1 528-1 539.
[22] 隋昕.香雪兰UDP-葡萄糖:类黄酮3-O-葡萄糖基转移酶基因的克隆及其功能鉴定[D].长春:东北师范大学,2011.