山茶属三个F3H基因的分子特性、系统进化及蛋白结构差异分析
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摘要
黄烷酮3-羟化酶基因(flavanone 3-hydroxylase,F3H)在植物花青素合成途径中发挥重要作用,可作为山茶花色遗传育种的候选基因。本研究在前期从Gen Bank数据库中筛选得到白花茶树CsF3H、黄花金花茶CnF3H和红花浙江红山茶CcF3H三个基因。然而,它们的分子遗传与变异信息仍然缺乏,不利于最大正向效应基因的选择利用。本研究系统探讨了CsF3H、CnF3H和CcF3H基因的分子特征、系统进化和蛋白三维结构。结果发现CsF3H、CnF3H和CcF3H三者之间存在高度序列多样性,共含37个核苷酸和9个氨基酸差异,且与CsF3H和CnF3H相比,CcF3H序列发生了更多的变异。系统进化结果表明CsF3H、CnF3H和CcF3H蛋白与猕猴桃AcF3H具有共同祖先,但在后期进化过程中,CcF3H率先与CsF3H和CnF3H二者发生分化。序列比对和保守结构域分析发现CsF3H、CnF3H和CcF3H蛋白均含有一个保守的依赖2-酮戊二酸和二价铁离子的双加氧酶超家族特征区域,该区域内的组氨酸H~(218)和H~(276)以及天冬氨酸D220是Fe~(2+)结合位点,精氨酸R~(286)和丝氨酸S~(288)是2-酮戊二酸的重要结合位点。本研究还发现了3个茶属F3H蛋白的保守结构域中存在一个差异位点,与CsF3H和CnF3H第200位异亮氨酸I200相比,CcF3H对应氨基酸变异为缬氨酸V~(200),Web logo3.4分析揭示V200在植物F3H蛋白中更为保守。空间结构分析显示CcF3H中氨基酸V~(200的替换导致它和赖氨酸K197的分子作用力增强。本研究结果表明CcF3H是更为保守的植物F3H蛋白,缬氨酸V200可能是一个重要的功能位点。上述研究也为山茶属F3H基因提供了新的信息,为今后选择CcF3H基因作为山茶花色育种最适候选基因提供了理论支撑。
F3H, an ideal candidate gene for flow er color breeding of Camellia by genetic engineering, plays important roles in regulating plant anthocyanin biosynthesis. Three F3H genes including CsF3H from Camellia sinensis(white flower), CnF3H from Camellia nitidissima(yellow flower) and CcF3H from Camellia chekiangoleosa(red flower) have been obtained from Gen Bank database in our previous study. However, the information about their molecular properties and genetic divergence are still very limited, which is unfavorable to the utility of F3H gene with the largest positive effect. In this study, molecular characteristics, phylogeny and structure of CsF3H,CnF3H and CcF3H were analyzed. High levels of sequence polymorphisms containing 37 different nucleotides and9 amino acids were found among the CsF3H, CnF3H and CcF3H, and CcF3H harbors more sequence variation in comparison with CnF3H and CsF3H. Phylogeny results showed that CsF3H, CnF3H and CcF3H proteins have the same ancestor with Ac F3H from Actinidia chinensis, and CcF3H was firstly separated from CsF3H and CnF3Hduring the evolution process. Results of sequence alignments and structure analysis showed all the three F3H contain a conserved 2-oxoglutarate and Fe(Ⅱ) dependent oxygenase superfamily domain, which contains 5 functional sites including three ferrous iron(Ⅱ) binding residues(H~(218), D~(220), H~(276)) and two 2-oxoglutarate binding residues(R~(286), S~(288)). An Isoleucine(I~(200)) to Valine(V200) substitution was also observed at position 200 of CcF3H protein when compared with CsF3H and CnF3H, Web logo3.4 analysis showed V~(200) is more conserved than I~(200) in plant F3H proteins. Protein tertiary structure analysis showed the V~(200) substitution strengthened inter-residue interaction with Lycine(K~(197)) in CcF3H. All above results suggest CcF3H is more conservative as a plant F3H protein, the V200 is an important potentially functional site. This study also provides new insights into Camellia F3H genes, it also indicates that CcF3H gene can serve as a suitable candidate gene for Camellia flower genetic breeding in the future.
F3H, an ideal candidate gene for flow er color breeding of Camellia by genetic engineering, plays important roles in regulating plant anthocyanin biosynthesis. Three F3H genes including CsF3H from Camellia sinensis(white flower), CnF3H from Camellia nitidissima(yellow flower) and CcF3H from Camellia chekiangoleosa(red flower) have been obtained from Gen Bank database in our previous study. However, the information about their molecular properties and genetic divergence are still very limited, which is unfavorable to the utility of F3H gene with the largest positive effect. In this study, molecular characteristics, phylogeny and structure of CsF3H,CnF3H and CcF3H were analyzed. High levels of sequence polymorphisms containing 37 different nucleotides and9 amino acids were found among the CsF3H, CnF3H and CcF3H, and CcF3H harbors more sequence variation in comparison with CnF3H and CsF3H. Phylogeny results showed that CsF3H, CnF3H and CcF3H proteins have the same ancestor with Ac F3H from Actinidia chinensis, and CcF3H was firstly separated from CsF3H and CnF3Hduring the evolution process. Results of sequence alignments and structure analysis showed all the three F3H contain a conserved 2-oxoglutarate and Fe(Ⅱ) dependent oxygenase superfamily domain, which contains 5 functional sites including three ferrous iron(Ⅱ) binding residues(H~(218), D~(220), H~(276)) and two 2-oxoglutarate binding residues(R~(286), S~(288)). An Isoleucine(I~(200)) to Valine(V200) substitution was also observed at position 200 of CcF3H protein when compared with CsF3H and CnF3H, Web logo3.4 analysis showed V~(200) is more conserved than I~(200) in plant F3H proteins. Protein tertiary structure analysis showed the V~(200) substitution strengthened inter-residue interaction with Lycine(K~(197)) in CcF3H. All above results suggest CcF3H is more conservative as a plant F3H protein, the V200 is an important potentially functional site. This study also provides new insights into Camellia F3H genes, it also indicates that CcF3H gene can serve as a suitable candidate gene for Camellia flower genetic breeding in the future.
引文
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Freyre R.,Uzdevenes C.,Gu L.,and Quesenberry K.H.,2015,Genetics and anthocyanin analysis of flower color in Mexican petunia,Journal of the American Society for Horticultural Science,140(1):45-49
Ge C.L.,Huang C.H.,and Xu X.B.,2012,Research on anthocyanins biosynthesis in fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),39(9):1655-1664(葛翠莲,黄春辉,徐小彪,2012,果实花青素生物合成研究进展,园艺学报,39(9):1655-1664)
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Hu X.J.,Xu Y.J.,Gao L.P.,Xia T.,and Wang Y.S.,2014,Cloning and functional analysis of flavanone 3-hydroxylase gene(F3H)in tea(Camellia sinensis),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),22(3):309-316(胡晓婧,许玉娇,高丽萍,夏涛,王云生,2014,茶树黄烷酮3-羟化酶基因(F3H)的克隆及功能分析,农业生物技术学报,22(3):309-316)
Huang C.H.,Gao Y.H.,Zhu Y.Q.,Tong Z.K.,and Jiang X.F.,2013 Cloning and expression analysis of flavanone 3-hydroxylase gene Lr F3H from Lycoris radiata,Yuanyi Xuebao(Acta Horticulturae Sinica),40(5):960-970(黄春红,高燕会,朱玉球,童再康,姜小凤,2013,石蒜黄烷酮3-羟化酶基因Lr F3H的克隆及表达分析,园艺学报,40(5):960-970)
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Konishi S.,Izawa T.,Lin S.,Ebana K.,Fukuta Y.,Sasaki T.,and Yano M.,2006,An SNP caused loss of seed shattering during rice domestication,Science,312(5778):1392-1396
Liu F.P.,2008,Advance in research of camellia breeding,Guangxi Nongye Kexue(Guangxi Agricultural Sciences),39(6):815-819(刘福平,2008,茶花育种研究现状与趋势,广西农业科学,39(6):815-819)
Lu Z.H.,Duan X.M.,and Fan G.S.,2008,A review of the research on cultivar resources of Camellia reticulata,Shangdong Linye Keji(Shandong Forestry Science and Technology),1:88-90(卢珍红,段晓梅,樊国盛,2008,云南山茶品种资源研究进展,山东林业科技,1:88-90)
Mahajan M.,and Yadav S.,2014,Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco,Plant Mol.Biol.,85(6):551-573
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Nishihara M.,Yamada E.,Saito M.,Fujita K.,Takahashi H.,and Nakatsuka T.,2014,Molecular characterization of mutations in white-flowered Torenia plants,BMC Plant Biology,14(1):1-13
Qiao T.T.,Yao M.Z.,Zhou Y.H.,Ma C.L.,Wang X.C.,Chen L.,and Yang Y.J.,2009,Research progress of association analysis in plants and the application prospects in breeding of tea plant(Camellia sinensis(L.)O.Kuntze),Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),25(6):165-170(乔婷婷,姚明哲,周炎花,马春雷,王新超,陈亮,杨亚军,2009,植物关联分析的研究进展及其在茶树分子标记辅助育种上的应用前景,中国农学通报,25(6):165-170)
Singh K.,Rani A.,Kumar S.,Sood P.,Mahajan M.,Yadav S.,Singh B.,and Ahuja P.,2008,An early gene of the flavonoid pathway,flavanone 3-hydroxylase,exhibits a positive relationship with the concentration of catechins in tea(Camellia sinensis),Tree Physiol.,28(9):1349-1356
Tan J.,Tu L.,Deng F.,Hu H.,Nie Y.,and Zhang X.,2013,A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin,Plant Physiol.,162(1):86-95
Tanaka Y.,Tsuda S.,and Kusumi T.,1998,Metabolic engineering to modify flower color,Plant Cell Physiol.,39(11):1119-1126
Tsuda S.,Fukui Y.,Nakamura N.,Katsumoto Y.,YonekuraSakakibara K.,Fukuchi-Mizutani M.,Ohira K.,Ueyama Y.,Ohkawa H.,Holton TA.,Kusumi T.,and Tanaka Y.,2004,Flower color modification of Petunia hybrida commercial varieties by metabolic engineering,Plant Biotechnology,21(5):377-386
Wachira F.N.,Waugh R.,Powell W.,and Hackett C.A.,1995,Detection of genetic diversity in tea(Camellia sinensis)using RAPD Markers,Genome,38(2):201-210
Wang X.Q.,Ding Y.R.,Mou Z.L.,Cai Y.J.,2014,Research on the relationship between iron superoxide dismutase amino acid networks and thermostability,Shengwuwuli Xuebao(Acta Biophysica Sinica),30(2):146-156(王雪芹,丁彦蕊,牟兆琳,蔡宇杰,2014,超氧化物歧化酶氨基酸网络与耐热性的关系研究,生物物理学报,30(2):146-156)
Xiu M.L.,2014,New direction of Camellia breeding,Yuanlin(Garden),6:62-63(修美玲,2014,山茶育种新方向,园林,6:62-63)
Yang H.L.,Wang Y.C.,Jiang Z.W.,and Huang H.W.,2009,Construction of c DNA library of'Hongyang'kiwifruit and analysis of F3H expression,Yichuan(Hereditas),31(12):1265-1272(杨红丽,王彦昌,姜正旺,黄宏文,2009,红阳猕猴桃c DNA文库构建及F3H基因的表达初探,遗传,31(12):1265-1272)
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Zuker A.,Tzfira T.,Ben-Meir H.,Ovadis M.,Shklarman E.,Itzhaki H.,Forkmann G.,Martens S.,Neta-Sharir I.,Weiss D.,and Vainstein A.,2002,Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene,Molecular Breeding,9(1):33-41
Bordoli L.,and Schwede T.,2012,Automated protein structure modeling with Swiss-Model workspace and the protein model portal,Methods Mol.Biol.,857:107-136
Britsch L.,Ruhnau-Brich B.,and Forkmann G.,1992,Molecular cloning,sequence analysis,and in vitro expression of flavanone 3 Beta-hydroxylase from Petunia hybrida,J.Biol.Chem.,267(8):5380-5387
Fan J.,Dai J.,Hu J.P.,Liang Z.,and Huang M.Y.,2009,Bioinformatics research on the tomato Le NHX1 gene,Shengwu Jishu Tongbao(Biotechnology Bulletin),12:87-91(范晶,代娟,胡建平,梁梓,黄明远,2009,生物信息学对番茄Le NHX1基因的研究,生物技术通报,12:87-91)
Fan J.,Hu J.P.,Gao L.Y.,Liao J.H.,and Huang M.Y.,2015,Molecular character,phylogeny and expression of tomato Le NHX3 gene involved in multiple adverse stress responses,The Open Biotechnology Journal,9:243-249
Faure G.,Bornot A.,and de Brevern A.,2008,Protein contacts,inter-residue interactions and side-chain modelling,Biochimie,90(4):626-639
Forkmann G.,Heller W.,and Grisebach H.,1980,Anthocyanin biosynthesis in flowers of matthiola incana flavanone 3-and flavonoid 3'-hydroxylases,Zeitschrift für Naturforschung C,35(9):691-695
Freyre R.,Uzdevenes C.,Gu L.,and Quesenberry K.H.,2015,Genetics and anthocyanin analysis of flower color in Mexican petunia,Journal of the American Society for Horticultural Science,140(1):45-49
Ge C.L.,Huang C.H.,and Xu X.B.,2012,Research on anthocyanins biosynthesis in fruit,Yuanyi Xuebao(Acta Horticulturae Sinica),39(9):1655-1664(葛翠莲,黄春辉,徐小彪,2012,果实花青素生物合成研究进展,园艺学报,39(9):1655-1664)
Hu X.C.,2015,Management and development of Ningbo Jin Zhong tea city,Zhongguo Chaye(China Tea),4:14-15(胡旭成,2015,宁波金钟茶城经营实践及发展愿景,中国茶叶,4:14-15)
Hu X.J.,Xu Y.J.,Gao L.P.,Xia T.,and Wang Y.S.,2014,Cloning and functional analysis of flavanone 3-hydroxylase gene(F3H)in tea(Camellia sinensis),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),22(3):309-316(胡晓婧,许玉娇,高丽萍,夏涛,王云生,2014,茶树黄烷酮3-羟化酶基因(F3H)的克隆及功能分析,农业生物技术学报,22(3):309-316)
Huang C.H.,Gao Y.H.,Zhu Y.Q.,Tong Z.K.,and Jiang X.F.,2013 Cloning and expression analysis of flavanone 3-hydroxylase gene Lr F3H from Lycoris radiata,Yuanyi Xuebao(Acta Horticulturae Sinica),40(5):960-970(黄春红,高燕会,朱玉球,童再康,姜小凤,2013,石蒜黄烷酮3-羟化酶基因Lr F3H的克隆及表达分析,园艺学报,40(5):960-970)
Jiang C.,Wen Q.,Chen Y.,Xu L.A.,and Huang M.R.,2011,Efficient extraction of RNA from various Camellia species rich in secondary metabolites for deep transcriptome sequencing and gene expression analysis,African Journal of Biotechnology,10(74):16769-16773
Kapil R.,and Sethi S.,1963,Development of male and female gametophytes in Camellia Sinensis(L.)O.Kuntze,Proc.Natl.Inst.Sci.India B,29:567-574
Kim J.,Lee Y.,Kim B.,Lim Y.,and Hoon Ahn J.,2008,Flavanone 3β-hydroxylases from rice:key enzymes for favonol and anthocyanin biosynthesis,Mol.Cells,25(2):312-316
Konishi S.,Izawa T.,Lin S.,Ebana K.,Fukuta Y.,Sasaki T.,and Yano M.,2006,An SNP caused loss of seed shattering during rice domestication,Science,312(5778):1392-1396
Liu F.P.,2008,Advance in research of camellia breeding,Guangxi Nongye Kexue(Guangxi Agricultural Sciences),39(6):815-819(刘福平,2008,茶花育种研究现状与趋势,广西农业科学,39(6):815-819)
Lu Z.H.,Duan X.M.,and Fan G.S.,2008,A review of the research on cultivar resources of Camellia reticulata,Shangdong Linye Keji(Shandong Forestry Science and Technology),1:88-90(卢珍红,段晓梅,樊国盛,2008,云南山茶品种资源研究进展,山东林业科技,1:88-90)
Mahajan M.,and Yadav S.,2014,Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco,Plant Mol.Biol.,85(6):551-573
Mahmood T.,Akhtar N.,and Khan B.,2010,The morphology,characteristics,and medicinal properties of Camellia sinensis'tea,Journal of Medicinal Plants Research,4(19):2028-2033
Marchler-Bauer A.,Lu S.,Anderson J.B.,Chitsaz F.,Derbyshire M.K.,De Weese-Scott C.,Fong J.H.,Geer L.Y.,Geer R.C.,Gonzales N.R.,Gwadz M.,Hurwitz D.I.,Jackson J.D.,Ke Z.,Lanczycki C.J.,Lu F.,Marchler G.H.,Mullokandov M.,Omelchenko M.V.,Robertson C.L.,Song J.S.,Thanki N.,Yamashita R.A.,Zhang D.,Zhang N.,Zheng C.,and Bryan S.H.,2011,CDD:a conserved domain database for the functional annotation of proteins,Nucleic Acids Res.,39:225-229
Nishihara M.,Yamada E.,Saito M.,Fujita K.,Takahashi H.,and Nakatsuka T.,2014,Molecular characterization of mutations in white-flowered Torenia plants,BMC Plant Biology,14(1):1-13
Qiao T.T.,Yao M.Z.,Zhou Y.H.,Ma C.L.,Wang X.C.,Chen L.,and Yang Y.J.,2009,Research progress of association analysis in plants and the application prospects in breeding of tea plant(Camellia sinensis(L.)O.Kuntze),Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),25(6):165-170(乔婷婷,姚明哲,周炎花,马春雷,王新超,陈亮,杨亚军,2009,植物关联分析的研究进展及其在茶树分子标记辅助育种上的应用前景,中国农学通报,25(6):165-170)
Singh K.,Rani A.,Kumar S.,Sood P.,Mahajan M.,Yadav S.,Singh B.,and Ahuja P.,2008,An early gene of the flavonoid pathway,flavanone 3-hydroxylase,exhibits a positive relationship with the concentration of catechins in tea(Camellia sinensis),Tree Physiol.,28(9):1349-1356
Tan J.,Tu L.,Deng F.,Hu H.,Nie Y.,and Zhang X.,2013,A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin,Plant Physiol.,162(1):86-95
Tanaka Y.,Tsuda S.,and Kusumi T.,1998,Metabolic engineering to modify flower color,Plant Cell Physiol.,39(11):1119-1126
Tsuda S.,Fukui Y.,Nakamura N.,Katsumoto Y.,YonekuraSakakibara K.,Fukuchi-Mizutani M.,Ohira K.,Ueyama Y.,Ohkawa H.,Holton TA.,Kusumi T.,and Tanaka Y.,2004,Flower color modification of Petunia hybrida commercial varieties by metabolic engineering,Plant Biotechnology,21(5):377-386
Wachira F.N.,Waugh R.,Powell W.,and Hackett C.A.,1995,Detection of genetic diversity in tea(Camellia sinensis)using RAPD Markers,Genome,38(2):201-210
Wang X.Q.,Ding Y.R.,Mou Z.L.,Cai Y.J.,2014,Research on the relationship between iron superoxide dismutase amino acid networks and thermostability,Shengwuwuli Xuebao(Acta Biophysica Sinica),30(2):146-156(王雪芹,丁彦蕊,牟兆琳,蔡宇杰,2014,超氧化物歧化酶氨基酸网络与耐热性的关系研究,生物物理学报,30(2):146-156)
Xiu M.L.,2014,New direction of Camellia breeding,Yuanlin(Garden),6:62-63(修美玲,2014,山茶育种新方向,园林,6:62-63)
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