越橘花青苷合成相关基因VcTTG1的克隆与功能鉴定
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摘要
以‘Duke’越橘(Vacciniumcory mbosum ‘Duke’)为试材,从转录组数据库中克隆编码WD40蛋白的基因VcTTG1,分析其表达模式并鉴定其在花青苷合成过程中的作用功能,为进一步探讨越橘花青苷合成调控机理奠定理论基础。结果表明,克隆获得越橘VcTTG1(GenBank登录号为MH717246),ORF为1044bp,推测其编码348个氨基酸,含有典型的WD40结构域。系统发生分析表明,Vc TTG1与葡萄VvWDR1的同源性最高。VcTTG1在越橘根、枝条、幼叶、花和果实中均有表达,但表达量差异显著,在果实中较高,枝条中较低。在果实中随着VcTTG1表达的升高,花青苷含量呈递增的趋势。在拟南芥中超表达VcTTG1,其花青苷积累在VcTTG1转基因植株中显著增加。酵母双杂交试验结果表明,VcTTG1可与拟南芥bHLH蛋白AtTT8相互作用。由此推测,VcTTG1在调控花青苷合成过程中发挥重要作用。
The objective of this study is to isolate a blueberry(Vaccinium corymbosum) cultivar ‘Duke'VcTTG1 gene encoding WD40 protein by PCR technology from transcriptome data and study its expression,to identify its function in anthocyanin biosynthesis. This study laid the foundation for further study of the molecular mechanism of VcTTG1 affecting anthocyanin biosynthesis in blueberry. Sequence analysis showed that the open reading frame(ORF)of VcTTG1(GenBank accession No. MH717246) is1 044 bp,putatively encoded 348 amino acids. Protein structure analysis showed that VcTTG1 contained conserved WD40 domain. A phylogenetic tree indicated that the blueberry VcTTG1 exhibited the highestsequence similarity to Vitis vinifera VvWDR1. Expression analysis showed that VcTTG1 was expressed in roots,stems,young leaves,flowers and fruits. However,the expression levels varied,with the highest expression level in fruits and the relatively low transcript levels in stems. The content of anthocyanin in fruits was increased with the up-regulation of relative expression of VcTTG1. The VcTTG1 protein could interact with Arabidopsis bHLH protein AtTT8 and favorably contributes to anthocyanin accumulation in transgenic Arabidopsis. It is speculated that VcTTG1 plays a regulatory role in anthocyanin biosynthesis.
The objective of this study is to isolate a blueberry(Vaccinium corymbosum) cultivar ‘Duke'VcTTG1 gene encoding WD40 protein by PCR technology from transcriptome data and study its expression,to identify its function in anthocyanin biosynthesis. This study laid the foundation for further study of the molecular mechanism of VcTTG1 affecting anthocyanin biosynthesis in blueberry. Sequence analysis showed that the open reading frame(ORF)of VcTTG1(GenBank accession No. MH717246) is1 044 bp,putatively encoded 348 amino acids. Protein structure analysis showed that VcTTG1 contained conserved WD40 domain. A phylogenetic tree indicated that the blueberry VcTTG1 exhibited the highestsequence similarity to Vitis vinifera VvWDR1. Expression analysis showed that VcTTG1 was expressed in roots,stems,young leaves,flowers and fruits. However,the expression levels varied,with the highest expression level in fruits and the relatively low transcript levels in stems. The content of anthocyanin in fruits was increased with the up-regulation of relative expression of VcTTG1. The VcTTG1 protein could interact with Arabidopsis bHLH protein AtTT8 and favorably contributes to anthocyanin accumulation in transgenic Arabidopsis. It is speculated that VcTTG1 plays a regulatory role in anthocyanin biosynthesis.
引文
An X H,Tian Y,Chen K X,Liu X J,Liu D D,Xie X B,Cheng C G,Cong P H,Hao Y J.2015.MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.Plant and Cell Physiology,56(4):650-662.
An X H,Tian Y,Chen K X,Wang X F,Hao Y J.2012.The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.Journal of Plant Physiology,169:710-717.
Basu A,Rhone M,Lyons T J.2010.Berries:emerging impact on cardiovascular health.Nutrition Reviews,68:168-177.
Baudry A,Heim M A,Dubreucq B,Caboche M,Weisshaar B,Lepiniec L.2004.TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana.The Plant Journal,39(3):366-380.
Debeaujon I,Nesi N,Perez P,Devic M,Grandjean O,Caboche M,Lepiniec L.2003.Proanthocyanidin-accumulating cells in Arabidopsis testa:regulation of differentiation and role in seed development.The Plant Cell,11(15):2514-2531.
Gao Y F,Liu J K,Chen Y F,Tang H,Wang Y,He Y M,Ou Y B,Sun X C,Wang S H,Yao Y N.2018.Tomato SIAN11 regulates flavonoid biosynthesis and seed dormancy by interaction with bHLH proteins but not with MYB proteins.Horticulture Research,5:27.
Gordillo G,Fang H,Khanna S,Harper J,Philips G,Sen C K.2009.Oral administration of blueberry inhibits angiogenic tumor growth and enhances survival of mice with endothelial cell neoplasm.Antioxid Redox Signal,11:47-58.
Grotewold E,Sainz M B,Tagliani L,Hernandez J M,Bowen B,Chandler V L.2000.Identification of the residues in the Myb domain of maize C1that specify the interaction with the bHLH cofactor R.Proceedings of the National Academy of Sciences of the United States of America,97:13579-13584.
He S L,Ho S L.2018.Functional characterization of a WD-repeat protein gene(OsWD1)in rice.American Journal of Agriculture and Forestry,6(2):18-27.
Hernandez J M,Heine G F,Irani N G,Feller A,Kim M G,Matulnik T,Chandler V L,Grotewold E.2004.Different mechanisms participate in the R-dependent activity of the R2R3 MYB transcription factor C1.Journal of Biological Chemistry,279(46):48205-48213.
Hsiao Y C,Hsu Y F,Chen Y C,Chang Y L,Wang C S.2016.A WD40 protein,AtGHS40,negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions.Journal of Plant Research,129(6):1127-1140.
Koornneef M.1981.The complex syndrome of ttg mutants.Arabidopsis Inf Serv,18:45-51.
Li D,Roberts R.2001.WD-repeat protein:structure characteristics,biological function,and their involvement in human disease.Cellular and Molecular Life Sciences,58:2085-2097.
Li Mao-fu,Yang Yuan,Wang Hua,Liu Jia-shen,Jin Wan-mei.2017.Cloning and expression of bHLH gene from Rosa chinensis‘Slater’s Crimson China’and its interaction with transcription factors MYB and WD40.Acta Horticulturae Sinica,44(10):1949-1958.(in Chinese)李茂福，杨媛，王华，刘佳棽，金万梅.2017.月季bHLH基因的克隆、表达及其与MYB和WD40的互作分析.园艺学报，44(10):1949-1958.
Liu Xin-yu,Han Hong-qiang,Ge Hai-yan,Jiang Ming-min,Chen Huo-ying.2014.Cloning,expression and interaction of anthocyanin-related transcription factors SmTTG1,SmGL3 and SmTT8 in eggplant.Acta Horticulturae Sinica,41(11):2241-2249.(in Chinese)刘新宇，韩洪强，葛海燕，将明敏，陈火英.2014.茄子花青素合成中SmTTG1,SmGL3和SmTT8的表达及其蛋白质间的相互作用.园艺学报，41(11):2241-2249.
Liu Y J,Hou H,Jiang X L,Wang P Q,Dai X L,Chen W,Gao L P,Xia T.2018.A WD40 repeat protein from Camellia sinensis regulates anthocyanin and proanthocyanidin accumulation through the formation of MYB-bHLH-WD40 ternary complexes.International Journal of Molecular Sciences,19:1686.
Maes L,InzéD.2008.Functional specialization of the TRANSPARENT TESTA GLABRA 1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves.Plant Physiology,148:1453-1464.
Matus J T,Poupin M J,Ca?ón P,Bordeu E,Alcalde J A,Arce-Johnson P.2010.Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine(Vitis vinifera L.).Plant Molecular Biology,72(6):607-620.
Pang Y Z,Wenger J P,Saathoff K,Peel G J,Wen J Q,Huhman D,Allen S N,Tang Y H,Cheng X F,Tadege M,Ratet P,Mysore K S,Sumner L W,Marks M D,Dixon R A.2009.A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis bur not for trichome development.Plant Physiology,151:1114-1129.
Payne C T,Zhang F,Lloyd A M.2000.GL3 encodes bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1and TTG1.Genetics,156:1349-1362.
Pertuzatti P B,Barcia M T,Rebello L P G,Gómez-Alonso S,Duarte R M T,Duarte M C T,Godoy H T,Hermosín-Gutiérrez I.2016.Antimicrobial activity and differentiation of anthocyanin profiles of rabbiteye and highbush blueberry using HPLC-DAD-ESI-MS and multivariate analysis.Journal of Functional Foods,26:506-516.
Smith T F,Gaitatzes C,Saxena K,Neer E J.1999.The WD repeat:a common architecture for diverse functions.Trends in Biochemical Sciences,24:181-185.
Song Y,Liu H D,Zhou Q,Zhang H J,Zhang Z D,Li Y D,Wang H B,Liu F Z.2017.High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors.Journal of Integrative Agriculture,16(3):591-604.
Walker A R,Davison P A,Bolognesi-Winfield A C,James C M,Srinivasan N,Blundell T L,Esch J J,Marks M D,Gray J C.1999.The TRANSPARENT TESTA GLABRA1 Locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40 repeat protein.The Plant Cell,11:1337-1349.
Zhang F,Gonzalez A,Zhao M,Payne C T,Lloyd A.2003.A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis.Development,130:4859-4869.
An X H,Tian Y,Chen K X,Wang X F,Hao Y J.2012.The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation.Journal of Plant Physiology,169:710-717.
Basu A,Rhone M,Lyons T J.2010.Berries:emerging impact on cardiovascular health.Nutrition Reviews,68:168-177.
Baudry A,Heim M A,Dubreucq B,Caboche M,Weisshaar B,Lepiniec L.2004.TT2,TT8,and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana.The Plant Journal,39(3):366-380.
Debeaujon I,Nesi N,Perez P,Devic M,Grandjean O,Caboche M,Lepiniec L.2003.Proanthocyanidin-accumulating cells in Arabidopsis testa:regulation of differentiation and role in seed development.The Plant Cell,11(15):2514-2531.
Gao Y F,Liu J K,Chen Y F,Tang H,Wang Y,He Y M,Ou Y B,Sun X C,Wang S H,Yao Y N.2018.Tomato SIAN11 regulates flavonoid biosynthesis and seed dormancy by interaction with bHLH proteins but not with MYB proteins.Horticulture Research,5:27.
Gordillo G,Fang H,Khanna S,Harper J,Philips G,Sen C K.2009.Oral administration of blueberry inhibits angiogenic tumor growth and enhances survival of mice with endothelial cell neoplasm.Antioxid Redox Signal,11:47-58.
Grotewold E,Sainz M B,Tagliani L,Hernandez J M,Bowen B,Chandler V L.2000.Identification of the residues in the Myb domain of maize C1that specify the interaction with the bHLH cofactor R.Proceedings of the National Academy of Sciences of the United States of America,97:13579-13584.
He S L,Ho S L.2018.Functional characterization of a WD-repeat protein gene(OsWD1)in rice.American Journal of Agriculture and Forestry,6(2):18-27.
Hernandez J M,Heine G F,Irani N G,Feller A,Kim M G,Matulnik T,Chandler V L,Grotewold E.2004.Different mechanisms participate in the R-dependent activity of the R2R3 MYB transcription factor C1.Journal of Biological Chemistry,279(46):48205-48213.
Hsiao Y C,Hsu Y F,Chen Y C,Chang Y L,Wang C S.2016.A WD40 protein,AtGHS40,negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions.Journal of Plant Research,129(6):1127-1140.
Koornneef M.1981.The complex syndrome of ttg mutants.Arabidopsis Inf Serv,18:45-51.
Li D,Roberts R.2001.WD-repeat protein:structure characteristics,biological function,and their involvement in human disease.Cellular and Molecular Life Sciences,58:2085-2097.
Li Mao-fu,Yang Yuan,Wang Hua,Liu Jia-shen,Jin Wan-mei.2017.Cloning and expression of bHLH gene from Rosa chinensis‘Slater’s Crimson China’and its interaction with transcription factors MYB and WD40.Acta Horticulturae Sinica,44(10):1949-1958.(in Chinese)李茂福，杨媛，王华，刘佳棽，金万梅.2017.月季bHLH基因的克隆、表达及其与MYB和WD40的互作分析.园艺学报，44(10):1949-1958.
Liu Xin-yu,Han Hong-qiang,Ge Hai-yan,Jiang Ming-min,Chen Huo-ying.2014.Cloning,expression and interaction of anthocyanin-related transcription factors SmTTG1,SmGL3 and SmTT8 in eggplant.Acta Horticulturae Sinica,41(11):2241-2249.(in Chinese)刘新宇，韩洪强，葛海燕，将明敏，陈火英.2014.茄子花青素合成中SmTTG1,SmGL3和SmTT8的表达及其蛋白质间的相互作用.园艺学报，41(11):2241-2249.
Liu Y J,Hou H,Jiang X L,Wang P Q,Dai X L,Chen W,Gao L P,Xia T.2018.A WD40 repeat protein from Camellia sinensis regulates anthocyanin and proanthocyanidin accumulation through the formation of MYB-bHLH-WD40 ternary complexes.International Journal of Molecular Sciences,19:1686.
Maes L,InzéD.2008.Functional specialization of the TRANSPARENT TESTA GLABRA 1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves.Plant Physiology,148:1453-1464.
Matus J T,Poupin M J,Ca?ón P,Bordeu E,Alcalde J A,Arce-Johnson P.2010.Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine(Vitis vinifera L.).Plant Molecular Biology,72(6):607-620.
Pang Y Z,Wenger J P,Saathoff K,Peel G J,Wen J Q,Huhman D,Allen S N,Tang Y H,Cheng X F,Tadege M,Ratet P,Mysore K S,Sumner L W,Marks M D,Dixon R A.2009.A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis bur not for trichome development.Plant Physiology,151:1114-1129.
Payne C T,Zhang F,Lloyd A M.2000.GL3 encodes bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1and TTG1.Genetics,156:1349-1362.
Pertuzatti P B,Barcia M T,Rebello L P G,Gómez-Alonso S,Duarte R M T,Duarte M C T,Godoy H T,Hermosín-Gutiérrez I.2016.Antimicrobial activity and differentiation of anthocyanin profiles of rabbiteye and highbush blueberry using HPLC-DAD-ESI-MS and multivariate analysis.Journal of Functional Foods,26:506-516.
Smith T F,Gaitatzes C,Saxena K,Neer E J.1999.The WD repeat:a common architecture for diverse functions.Trends in Biochemical Sciences,24:181-185.
Song Y,Liu H D,Zhou Q,Zhang H J,Zhang Z D,Li Y D,Wang H B,Liu F Z.2017.High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors.Journal of Integrative Agriculture,16(3):591-604.
Walker A R,Davison P A,Bolognesi-Winfield A C,James C M,Srinivasan N,Blundell T L,Esch J J,Marks M D,Gray J C.1999.The TRANSPARENT TESTA GLABRA1 Locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40 repeat protein.The Plant Cell,11:1337-1349.
Zhang F,Gonzalez A,Zhao M,Payne C T,Lloyd A.2003.A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis.Development,130:4859-4869.