花色素苷合成关键调节基因的研究进展
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摘要
花色素苷是一类广泛存在于植物体内的类黄酮化合物,在胞质内合成,积累于液泡中,是植物花色、果实、部分叶片呈色的主要原因,具有强抗氧化能力以及一定的消炎抗菌和抗病毒活性。目前对花色素苷合成途径的研究已较为成熟,一些重要的结构基因及调节基因在不同的物种中均被克隆,且对相关关键基因的研究也较为深入,但对某些调节基因在代谢通路的调节机制及其功能尚不清晰。现将近年来研究并不全面的某些调节基因在各种生命活动中的作用作总结概述,以便为后续相关研究提供参考。
Anthocyanin is a kind of flavonoids widely existing in plants. It is synthesized in cytoplasm and accumulated in vacuoles. The production of anthocyanin is the main reason for the colors of flowers, fruits and some leaves. At the same time, anthocyanin has strong antioxidant capacity, and a certain degree of anti-inflammatory,antibacterial and anti-virus activities. At present, the research on anthocyanin biosynthesis pathway has been mature. Some important structural genes and regulatory genes have been cloned in different species, and the related key genes have been studied deeply. However, the regulatory mechanism and functions of some regulatory genes in metabolic pathway are still unclear. This review summarized the roles of some regulatory genes in various life activities which have not been studied comprehensively in recent years, so as to provide reference for the follow-up related research.
Anthocyanin is a kind of flavonoids widely existing in plants. It is synthesized in cytoplasm and accumulated in vacuoles. The production of anthocyanin is the main reason for the colors of flowers, fruits and some leaves. At the same time, anthocyanin has strong antioxidant capacity, and a certain degree of anti-inflammatory,antibacterial and anti-virus activities. At present, the research on anthocyanin biosynthesis pathway has been mature. Some important structural genes and regulatory genes have been cloned in different species, and the related key genes have been studied deeply. However, the regulatory mechanism and functions of some regulatory genes in metabolic pathway are still unclear. This review summarized the roles of some regulatory genes in various life activities which have not been studied comprehensively in recent years, so as to provide reference for the follow-up related research.
引文
Bailey P.C.,Martin C.,Toledo-Ortiz G.,Quail P.H.,Huq E.,Heim M.A.,Jakoby M.,Werber M.,and Weisshaar B.,2003,Update on the Basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana,Plant Cell,15(11):2497-2502
Ben-Simhon Z.,Judeinstein S.,Nadler-Hassar T.,Trainin T.,Bar-Ya'akov I.,Borochov-Neori H.,and Holland D.,2011,A pomegranate(Punica granatum L.)WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development,Planta,234(5):865-881
Bernhardt C.,Zhao M.,Gonzalez A.,Lloyd A.,and Schiefelbein J.,2005,The b HLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis,Development,132(2):291-298
Chu H.,Jeong J.C.,Kim W.J.,Chung D.M.,Jeon H.K.,Ahn Y.O.,Kim S.H.,Lee H.S.,Kwak S.S.,and Kim C.Y.,2013,Expression of the sweetpotato R2R3-type Ib MYB1a gene induces anthocyanin accumulation in Arabidopsis,Physiol.Plant.,148(2):189
D'Amelia V.,Aversano R.,Batelli G.,Caruso I.,Castellano Moreno M.,Castro-Sanz A.B.,Chiaiese P.,Fasano C.,Palomba F.,and Carputo D.,2014,High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves,Plant J.,80(3):527-540
De Vetten N.,Quattrocchio F.,Mol J.,and Koes R.,1997,The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast,plants,and animals,Genes Dev.,11(11):1422-1434
Gao C.,Sun J.L.,Wang C.Q.,Dong Y.M.,Xiao S.,Xiao S.H.,Wang X.J.,and Jiao Z.G.,2017,Genome-wide analysis of basic/helix-loop-helix gene family in peanut and assessment of its roles in pod development,PLo S One,12(7):e0181843
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.,and Yao Y.N.,2018,Tomato Sl AN11 regulates flavonoid biosynthesis and seed dormancy by interaction with b HLH proteins but not with MYB proteins,Hortic.Res.,5:27
Heim M.A.,Jakoby M.,Werber M.,Martin C.,Weisshaar B.,and Bailey P.C.,2003,The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity,Mol.Biol.Evol.,20(5):735-747
Kavas M.,Balo lu M.C.,Atabay E.S.,Ziplar U.T.,Da gan H.Y.,and譈nver T.,2016,Genome-wide characterization and expression analysis of common bean b HLH transcription factors in response to excess salt concentration,Mol.Genet.Genomics,291(1):129-143
Lai B.,Li X.J.,Hu B.,Qin Y.H.,Huang X.M.,Wang H.C.,and Hu G.B.,2014,Lc MYB1 is a key determinant of differential anthocyanin accumulation among genotypes,tissues,developmental phases and ABA and light stimuli in Litchi chinensis,PLo S One,9(1):e86293
Li X.X.,Duan X.P.,Jiang H.X.,Sun Y.J.,Tang Y.P.,Yuan Z.,Guo J.K.,Liang W.Q.,Chen L.,Yin J.Y.,Ma H.,Wang J.,and Zhang D.B.,2006,Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis,Plant Physiol.,141(4):1167-1184
Lotkowska M.E.,Tohge T.,Fernie A.R.,Xue G.P.,Balazadeh S.,and Mueller-Roeber B.,2015,The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress,Plant Physiol.,169(3):1862-1880
Mao K.,Dong Q.L.,Li C.,Liu C.H.,and Ma F.W.,2017,Genome wide identification and characterization of apple b HLH transcription factors and expression analysis in response to drought and salt stress,Front.Plant Sci.,8:480
Massari M.E.,and Murre C.,2000,Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms,Mol.Cell.Biol.,20(2):429-440
Nakatsuka T.,Haruta K.S.,Pitaksutheepong C.,Abe Y.,Kakizaki Y.,Yamamoto K.,Shimada N.,Yamamura S.,and Nishihara M.,2008,Identification and characterization of R2R3-MYB and b HLH transcription factors regulating anthocyanin biosynthesis in gentian flowers,Plant Cell Physiol.,49(12):1818-1829
Ogo Y.,Itai R.N.,Nakanishi H.,Kobayashi T.,Takahashi M.,Mori S.,and Nishizawa N.K.,2007,The rice b HLH protein Os IRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions,Plant J.,51(3):366-377
Paz-Ares J.,Ghosal D.,Wienand U.,Peterson P.A.,and Saedler H.,1987,The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators,EM-BO J.,6(12):3553-3558
Qi T.C.,Huang H.,Song S.S.,and Xie D.X.,2015,Regulation of jasmonate-mediated stamen development and seed production by a b HLH-MYB complex in Arabidopsis,Plant Cell,27(6):1620-1633
Samira R.,Li B.H.,Kliebenstein D.,Li C.Y.,Davis E.,Gillikin J.W.,and Long T.A.,2018,The b HLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis,Plant Mol.Biol.,97(4-5):297-309
Schwinn K.E.,Ngo H.,Kenel F.,Brummell D.A.,Albert N.W.,Mc Callum J.A.,Pither-Joyce M.,Crowhurst R.N.,Eady C.,and Davies K.M.,2016,The onion(Allium cepa L.)R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis,Front.Plant Sci.,7:1865
Sun H.,Fan H.J.,and Ling H.Q.,2015,Genome-wide identification and characterization of the b HLH gene family in tomato,BMC Genomics,16(1):9
Szécsi J.,Joly C.,Bordji K.,Varaud E.,Cock J.M.,Dumas C.,and Bendahmane M.,2006,BIGPETALp,a b HLH transcription factor is involved in the control of Arabidopsis petal size,EMBO J.,25(16):3912-3920
Toledo-Ortiz G.,Huq E.,and Quail P.H.,2003,The Arabidopsis basic/helix-loop-helix transcription factor family,Plant Cell,15(8):1749-1770
Totsuka A.,Okamoto E.,Miyahara T.,Kouno T.,Cano E.A.,Sasaki N.,Watanabe A.,Tasaki K.,Nishihara M.,and Ozeki Y.,2018,Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation,Breeding Science,68(1):139-143
Upadhyay A.,Gaonkar T.,Upadhyay A.K.,Jogaiah S.,Shinde M.P.,Kadoo N.Y.,and Gupta V.S.,2018,Global transcriptome analysis of grapevine(Vitis vinifera L.)leaves under salt stress reveals differential response at early and late stages of stress in table grape cv.thompson seedless,Plant Physiol.Biochem.,129:168-179
Wada T.,Kunihiro A.,and Tominaga-Wada R.,2014,Arabidopsis CAPRICE(MYB)and GLABRA3(b HLH)control tomato(Solanum lycopersicum)anthocyanin biosynthesis,PLo SOne,9(9):e109093
Walker A.R.,Davison P.A.,Bolognesi-Winfield A.C.,James C.M.,Srinivasan N.,Blundell T.L.,Esch J.J.,Marks M.D.,and Gray J.C.,1999,The TRANSPARENT TESTA GLABRA1locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40 repeat protein,Plant Cell,11(7):1337-1350
Wang J.Y.,Hu Z.Z.,Zhao T.M.,Yang Y.W.,Chen T.Z.,Yang M.L.,Yu W.G.,and Zhang B.L.,2015,Genome-wide analysis of b HLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato(Solanum lycopersicum),BMC Genomics,16(1):39
Xu W.J.,Grain D.,Bobet S.,Le Gourrierec J.,Thévenin J.,Kelemen Z.,Lepiniec L.,and Dubos C.,2014,Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-b HLH-WDR complexes and their targets in Arabidopsis seed,New Phytol.,202(1):132-144
Zhang L.C.,2013,Functional study of wheat MYB transcription factors,Dissertation Ph.D.,Chinese Academy of Agricultural Science,Supervisors:Liu X.,and Kong X.Y.,pp.1-55(张立超,2013,小麦MYB转录因子的功能研究,博士学位论文,中国农业科学院,导师:刘旭,孔秀英,pp.1-55)
Zhang N.,Hu Z.L.,Chen X.Q.,Hou X.S.,Li Y.,and Chen G.P.,2008,Analysis of metabolic pathway and establishment of regulating model of anthocyanin synthesis,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),28(1):97-105(张宁,胡宗利,陈绪清,侯晓姝,李勇,陈国平,2008,植物花青素代谢途径分析及调控模型建立,中国生物工程杂志,28(1):97-105)
Zhang X.,Luo H.M.,Xu Z.C.,Zhu Y.J.,Ji A.J.,Song J.Y.,and Chen S.L.,2015,Genome-wide characterisation and analysis of b HLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza,Sci.Rep.,5(11244):11244
Zhang X.Y.,Zhang S.L.,Luo J.,Ye Z.W.,and Li S.C.,2004,Advances in research on fruit anthoyanin synthesis,Guoshu Xuebao(Journal of Fruit Science),21(5):456-460(张学英,张上隆,骆军,叶正文,李世诚,2004,果实花色素苷合成研究进展,果树学报,21(5):456-460)
Zhou L.L.,Shi M.Z.,and Xie D.Y.,2012,Regulation of anthocyanin biosynthesis by nitrogen in TTG1-GL3/TT8-PAP1-programmed red cells of Arabidopsis thaliana,Planta,236(3):825-837
Zhou Y.,Zhou H.,Lin-wang K.L.,Vimolmangkang S.,Espley R.V.,Wang L.,Allan A.C.,and Han Y.P.,2014,Transcriptome analysis and transient transformation suggest an ancient duplicated MYB transcription factor as a candidate gene for leaf red coloration in peach,BMC Plant Biol.,14(1):388
Zhu Q.L.,Sui S.Z.,Lei X.H.,Yang Z.F.,Lu K.,Liu G.D.,Liu Y.G.,and Li M.Y.,2015,Ectopic expression of the coleus R2R3 MYB-type proanthocyanidin regulator gene Ss MYB3alters the flower color in transgenic tobacco,PLo S One,10(10):e0139392
Ben-Simhon Z.,Judeinstein S.,Nadler-Hassar T.,Trainin T.,Bar-Ya'akov I.,Borochov-Neori H.,and Holland D.,2011,A pomegranate(Punica granatum L.)WD40-repeat gene is a functional homologue of Arabidopsis TTG1 and is involved in the regulation of anthocyanin biosynthesis during pomegranate fruit development,Planta,234(5):865-881
Bernhardt C.,Zhao M.,Gonzalez A.,Lloyd A.,and Schiefelbein J.,2005,The b HLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis,Development,132(2):291-298
Chu H.,Jeong J.C.,Kim W.J.,Chung D.M.,Jeon H.K.,Ahn Y.O.,Kim S.H.,Lee H.S.,Kwak S.S.,and Kim C.Y.,2013,Expression of the sweetpotato R2R3-type Ib MYB1a gene induces anthocyanin accumulation in Arabidopsis,Physiol.Plant.,148(2):189
D'Amelia V.,Aversano R.,Batelli G.,Caruso I.,Castellano Moreno M.,Castro-Sanz A.B.,Chiaiese P.,Fasano C.,Palomba F.,and Carputo D.,2014,High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves,Plant J.,80(3):527-540
De Vetten N.,Quattrocchio F.,Mol J.,and Koes R.,1997,The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast,plants,and animals,Genes Dev.,11(11):1422-1434
Gao C.,Sun J.L.,Wang C.Q.,Dong Y.M.,Xiao S.,Xiao S.H.,Wang X.J.,and Jiao Z.G.,2017,Genome-wide analysis of basic/helix-loop-helix gene family in peanut and assessment of its roles in pod development,PLo S One,12(7):e0181843
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.,and Yao Y.N.,2018,Tomato Sl AN11 regulates flavonoid biosynthesis and seed dormancy by interaction with b HLH proteins but not with MYB proteins,Hortic.Res.,5:27
Heim M.A.,Jakoby M.,Werber M.,Martin C.,Weisshaar B.,and Bailey P.C.,2003,The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity,Mol.Biol.Evol.,20(5):735-747
Kavas M.,Balo lu M.C.,Atabay E.S.,Ziplar U.T.,Da gan H.Y.,and譈nver T.,2016,Genome-wide characterization and expression analysis of common bean b HLH transcription factors in response to excess salt concentration,Mol.Genet.Genomics,291(1):129-143
Lai B.,Li X.J.,Hu B.,Qin Y.H.,Huang X.M.,Wang H.C.,and Hu G.B.,2014,Lc MYB1 is a key determinant of differential anthocyanin accumulation among genotypes,tissues,developmental phases and ABA and light stimuli in Litchi chinensis,PLo S One,9(1):e86293
Li X.X.,Duan X.P.,Jiang H.X.,Sun Y.J.,Tang Y.P.,Yuan Z.,Guo J.K.,Liang W.Q.,Chen L.,Yin J.Y.,Ma H.,Wang J.,and Zhang D.B.,2006,Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis,Plant Physiol.,141(4):1167-1184
Lotkowska M.E.,Tohge T.,Fernie A.R.,Xue G.P.,Balazadeh S.,and Mueller-Roeber B.,2015,The Arabidopsis transcription factor MYB112 promotes anthocyanin formation during salinity and under high light stress,Plant Physiol.,169(3):1862-1880
Mao K.,Dong Q.L.,Li C.,Liu C.H.,and Ma F.W.,2017,Genome wide identification and characterization of apple b HLH transcription factors and expression analysis in response to drought and salt stress,Front.Plant Sci.,8:480
Massari M.E.,and Murre C.,2000,Helix-loop-helix proteins:regulators of transcription in eucaryotic organisms,Mol.Cell.Biol.,20(2):429-440
Nakatsuka T.,Haruta K.S.,Pitaksutheepong C.,Abe Y.,Kakizaki Y.,Yamamoto K.,Shimada N.,Yamamura S.,and Nishihara M.,2008,Identification and characterization of R2R3-MYB and b HLH transcription factors regulating anthocyanin biosynthesis in gentian flowers,Plant Cell Physiol.,49(12):1818-1829
Ogo Y.,Itai R.N.,Nakanishi H.,Kobayashi T.,Takahashi M.,Mori S.,and Nishizawa N.K.,2007,The rice b HLH protein Os IRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions,Plant J.,51(3):366-377
Paz-Ares J.,Ghosal D.,Wienand U.,Peterson P.A.,and Saedler H.,1987,The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators,EM-BO J.,6(12):3553-3558
Qi T.C.,Huang H.,Song S.S.,and Xie D.X.,2015,Regulation of jasmonate-mediated stamen development and seed production by a b HLH-MYB complex in Arabidopsis,Plant Cell,27(6):1620-1633
Samira R.,Li B.H.,Kliebenstein D.,Li C.Y.,Davis E.,Gillikin J.W.,and Long T.A.,2018,The b HLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis,Plant Mol.Biol.,97(4-5):297-309
Schwinn K.E.,Ngo H.,Kenel F.,Brummell D.A.,Albert N.W.,Mc Callum J.A.,Pither-Joyce M.,Crowhurst R.N.,Eady C.,and Davies K.M.,2016,The onion(Allium cepa L.)R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis,Front.Plant Sci.,7:1865
Sun H.,Fan H.J.,and Ling H.Q.,2015,Genome-wide identification and characterization of the b HLH gene family in tomato,BMC Genomics,16(1):9
Szécsi J.,Joly C.,Bordji K.,Varaud E.,Cock J.M.,Dumas C.,and Bendahmane M.,2006,BIGPETALp,a b HLH transcription factor is involved in the control of Arabidopsis petal size,EMBO J.,25(16):3912-3920
Toledo-Ortiz G.,Huq E.,and Quail P.H.,2003,The Arabidopsis basic/helix-loop-helix transcription factor family,Plant Cell,15(8):1749-1770
Totsuka A.,Okamoto E.,Miyahara T.,Kouno T.,Cano E.A.,Sasaki N.,Watanabe A.,Tasaki K.,Nishihara M.,and Ozeki Y.,2018,Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation,Breeding Science,68(1):139-143
Upadhyay A.,Gaonkar T.,Upadhyay A.K.,Jogaiah S.,Shinde M.P.,Kadoo N.Y.,and Gupta V.S.,2018,Global transcriptome analysis of grapevine(Vitis vinifera L.)leaves under salt stress reveals differential response at early and late stages of stress in table grape cv.thompson seedless,Plant Physiol.Biochem.,129:168-179
Wada T.,Kunihiro A.,and Tominaga-Wada R.,2014,Arabidopsis CAPRICE(MYB)and GLABRA3(b HLH)control tomato(Solanum lycopersicum)anthocyanin biosynthesis,PLo SOne,9(9):e109093
Walker A.R.,Davison P.A.,Bolognesi-Winfield A.C.,James C.M.,Srinivasan N.,Blundell T.L.,Esch J.J.,Marks M.D.,and Gray J.C.,1999,The TRANSPARENT TESTA GLABRA1locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40 repeat protein,Plant Cell,11(7):1337-1350
Wang J.Y.,Hu Z.Z.,Zhao T.M.,Yang Y.W.,Chen T.Z.,Yang M.L.,Yu W.G.,and Zhang B.L.,2015,Genome-wide analysis of b HLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato(Solanum lycopersicum),BMC Genomics,16(1):39
Xu W.J.,Grain D.,Bobet S.,Le Gourrierec J.,Thévenin J.,Kelemen Z.,Lepiniec L.,and Dubos C.,2014,Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-b HLH-WDR complexes and their targets in Arabidopsis seed,New Phytol.,202(1):132-144
Zhang L.C.,2013,Functional study of wheat MYB transcription factors,Dissertation Ph.D.,Chinese Academy of Agricultural Science,Supervisors:Liu X.,and Kong X.Y.,pp.1-55(张立超,2013,小麦MYB转录因子的功能研究,博士学位论文,中国农业科学院,导师:刘旭,孔秀英,pp.1-55)
Zhang N.,Hu Z.L.,Chen X.Q.,Hou X.S.,Li Y.,and Chen G.P.,2008,Analysis of metabolic pathway and establishment of regulating model of anthocyanin synthesis,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),28(1):97-105(张宁,胡宗利,陈绪清,侯晓姝,李勇,陈国平,2008,植物花青素代谢途径分析及调控模型建立,中国生物工程杂志,28(1):97-105)
Zhang X.,Luo H.M.,Xu Z.C.,Zhu Y.J.,Ji A.J.,Song J.Y.,and Chen S.L.,2015,Genome-wide characterisation and analysis of b HLH transcription factors related to tanshinone biosynthesis in Salvia miltiorrhiza,Sci.Rep.,5(11244):11244
Zhang X.Y.,Zhang S.L.,Luo J.,Ye Z.W.,and Li S.C.,2004,Advances in research on fruit anthoyanin synthesis,Guoshu Xuebao(Journal of Fruit Science),21(5):456-460(张学英,张上隆,骆军,叶正文,李世诚,2004,果实花色素苷合成研究进展,果树学报,21(5):456-460)
Zhou L.L.,Shi M.Z.,and Xie D.Y.,2012,Regulation of anthocyanin biosynthesis by nitrogen in TTG1-GL3/TT8-PAP1-programmed red cells of Arabidopsis thaliana,Planta,236(3):825-837
Zhou Y.,Zhou H.,Lin-wang K.L.,Vimolmangkang S.,Espley R.V.,Wang L.,Allan A.C.,and Han Y.P.,2014,Transcriptome analysis and transient transformation suggest an ancient duplicated MYB transcription factor as a candidate gene for leaf red coloration in peach,BMC Plant Biol.,14(1):388
Zhu Q.L.,Sui S.Z.,Lei X.H.,Yang Z.F.,Lu K.,Liu G.D.,Liu Y.G.,and Li M.Y.,2015,Ectopic expression of the coleus R2R3 MYB-type proanthocyanidin regulator gene Ss MYB3alters the flower color in transgenic tobacco,PLo S One,10(10):e0139392