利用酵母双杂系统筛选与春兰CgSEP1相互作用的蛋白
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摘要
兰花形态优雅,花香馥郁,花形奇特,具有高度特化的花器官,观赏价值较高。有关兰花花器官形成分子机理以及基因组测序正在不断研究中。为了探讨兰花基因间相互作用情况,本研究以春兰为材料进行基因克隆,获得了18个花发育相关基因的编码序列,经测序鉴定分别属于ABCDE类MADS-box家族基因。再以其中春兰AP/AGL9组的CgSEP1为诱饵蛋白,亚克隆至pGBKT7质粒,构建诱饵载体,将18个蛋白编码亚克隆至pGADT7质粒。通过酵母双杂系统筛选到了3个与其互作的蛋白:pGADT7-AG1、p GADT7-SEP2、pGADT7-AGL6-3。结果表明,C类AG-like和E类SEP-like、AGL-like家族基因参与SEP1基因春兰花发育过程,并形成多种蛋白混合物,共同调控花瓣和萼片的形成,进一步揭示了春兰的成花机理。
Orchids have elegant morphology, fragrant flowers, peculiar patterns, highly specialized flower organs,and high ornamental value. Molecular mechanism of organogenesis and genome sequencing of orchids' flower organ are being studied continuously. In order to investigate the interaction between genes in orchids, in this study,the coding sequence of 18 genes related to flower development was obtained by cloning from Cymbidium goeringii.The genes belonging to ABCDE class MADS-box family were identified by sequencing. Then CgSEP1 of AP/AGL9 group of Cymbidium goeringii was used as bait protein and subcloned into pGBKT7 plasmid to construct bait vector. Eighteen protein-coding subclones were cloned into pGADT7 plasmid. There were three proteins by yeast two-hybrid system: pGADT7-AG1, pGADT7-SEP2, p GADT7-AGL6-3, which interacted with it. This suggestd that Class C AG-like and Class E SEP-like and AGL-like family genes participate in the development of SEP1 gene Cymbidium goeringii and form a mixture of proteins, which jointly might regulate the formation of petals and sepals, further revealing the flowering mechanism of Cymbidium goeringii as well.
Orchids have elegant morphology, fragrant flowers, peculiar patterns, highly specialized flower organs,and high ornamental value. Molecular mechanism of organogenesis and genome sequencing of orchids' flower organ are being studied continuously. In order to investigate the interaction between genes in orchids, in this study,the coding sequence of 18 genes related to flower development was obtained by cloning from Cymbidium goeringii.The genes belonging to ABCDE class MADS-box family were identified by sequencing. Then CgSEP1 of AP/AGL9 group of Cymbidium goeringii was used as bait protein and subcloned into pGBKT7 plasmid to construct bait vector. Eighteen protein-coding subclones were cloned into pGADT7 plasmid. There were three proteins by yeast two-hybrid system: pGADT7-AG1, pGADT7-SEP2, p GADT7-AGL6-3, which interacted with it. This suggestd that Class C AG-like and Class E SEP-like and AGL-like family genes participate in the development of SEP1 gene Cymbidium goeringii and form a mixture of proteins, which jointly might regulate the formation of petals and sepals, further revealing the flowering mechanism of Cymbidium goeringii as well.
引文
Aceto S.,and Gaudio L.,2011,The MADS and the beauty:genes involved in the development of orchid flowers,Current Genomics,12(5):342-356
Acri-Nunes-Miranda R.,and Mondragon-Palomino M.,2014,Expression of paralogous SEP-,FUL-,AG-and STK-like MADS-box genes in wild-type and peloric Phalaenopsis flowers,Frontiers in Plant Science,5(5):76
Bemer M.,and Angenent G.C.,2010,Floral organ initiation and development,Springer Berlin Heidelberg,pp.173-194
Chang Y.Y.,Chiu Y.F.,Wu J.W.,and Yang C.H.,2009,Four orchid(Oncidium Gower Ramsey)AP1/AGL9-like%MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana,Plant and Cell Physiology,50(8):1425-1438
Chen J.Y.,and Cheng X.K.,eds.,2003,Chinese flower,Shanghai Culture Publishing House,Shanghai,China,pp.127-129(陈俊愉,程绪珂,主编,2003,中国花经,上海文化出版社,中国,上海,pp.127-129)
Coen E.S.,and Meyerowitz E.M.,1991,The war of whorls:genetic interactions controlling flower development,Nature,353(6339):31-37
De Bodt S.,Raes J.,Van de Peer Y.,and Theissen G.魬.,2003,And then there were many:MADS goes genomic,Trends in Plant Science,8(10):475-483
Ditta G.,Pinyopich A.,Robles P.,Pelas S.,and Yanofsky M.F.,2004,The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity,Current Biology,14(21):1935-1940
Fields S.,and Song O.,1989,A novel genetic system to detect protein-protein interactions,Nature,340(6230):245-246
Goto K.,Kyozuka J.,and Bowman J.L.,2001,Turning floral organs into leaves,leaves into floral organs,Current Opinion in Genetics and Development,11(4):449-456
Honma T.,and Goto K.,2001,Complexes of MADS-box proteins are sufficient to convert leaves into floral organs,Nature,409(6819):525-529
Hsiao Y.Y.,Pan Z.J.,Hsu C.C.,Yang Y.P.,Hsu Y.C.,Chuang Y.C.,Shih H.H.,Chen W.H.,Tsai W.C.,and Chen H.H.,2011,Research on orchid biology and biotechnology,Plant and Cell Physiology,52(9):1467-1486
Hsu H.F.,Hsu W.H.,Lee Y.I.,Mao W.T.,Yang J.Y.,Li J.Y.,and Yang C.H.,2015,Model for peranth formation in orchids,Nature Plants,46:1-8
Hsu H.F,Huang C.H.,Yang C.T.,and Chang H.Y.,2003,Ectopic expression of an orchid(Oncidium Gower Ramsey)AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana,Plant and Cell Physiology,44(8):783-794
Immink R.G.H.,Tonaco I.A.N.,de Folter S.,Shchennikova A.,van Dijk A.D.J.,Busscher-Lange J.,Borst J.W.,and Angenent G.C.,2009,SEPALLATA3:the'glue'for MADS box transcription factor complex formation,Genome Biology,10(2):R24
Jia C.L.,Xiang L.,Qin D.H.,Li X.B.,Wu C.,and Sun C.B.,2014,Cloning and expression analysis of CgSEP3 gene from Cymbidium goeringii,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),34(7):1305-1310(贾春蕾,向林,秦徳辉,李小白,吴超,孙崇波,2014,春兰CgSEP3基因的克隆和表达,西北植物学报,34(7):1305-1310)
Kaufmann K.,Melzer R.,and Theiβen G.,2005,MIKC-type MADS-domain proteins:structural modularity,protein interactions and network evolution in land plants,Gene,347(2):183-198
Lin C.S.,Hsu C.T.,Liao D.C.,Chang W.J.,Chou M.L.,Huang Y.T.,Chen J.W.,Ko S.S.,Chan M.T.,and Shih M.C.,2016,Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla,Plant Biotechnology Journal,14(1):284-298
Liu C.,Xi W.Y.,Shen L.S.,Tan C.P.,and Yu H.,2009,Regulation of floral patterning by flowering time genes,Developmental Cell,16(5):711-722
Melzer S.,Lens F.,Gennen J.,Vanneste S.,Rohde A.,and Beeckman T.,2008,Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana,Nature Genetics,40(12):1489-1492
Münster T.,Wingen L.U.,Faigl W.,Wreth S.,Saedler H.,and Theiβen G.,2001,Charaterization of three GLOBOSA-like MADS-box genes from maize evidence for ancient paralogy in one class of floral homeotic B-function genes of grasses,Gene,262:1-13
Pan Z.J.,Chen Y.Y.,Du J.S.,Chen Y.Y.,Chung M.C.,Tsai W.C.,Wang C.N.,and Chen H.H.,2014,Flower development of Phalaenopsis orchid involves functionally divergent SEPAL-LATA-like genes,New Phytologist,202(3):1024-1042
Pelaz S.,Tapia-López R.,Alvarez-Buylla E.R.,and Yanofsky M.F.,2001,Conversion of leaves into petals in Arabidopsis,Current Biology,11(3):182-184
Shi J.,2014,Study on interaction between ankyrin ANK6 and SIG5 in Arabidopsis thaliana,Thesis for M.S.,Hunan Normal University,Supervisors:Li D.P.,and Luan S.,pp.6-7(史佳,2014,拟南芥锚蛋白ANK6和SIG5的相互作用研究,硕士学位论文,湖南师范大学,导师:李东屏,栾升,pp.6-7)
Sun C.B.,Xiang L.,Shi J.S.,Hu F.R.,Guo F.Q.,and Qin D.H.,2010,Cloning and real-time expression analysis of AGL6gene from Cymbidium goeringii,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(5):939-944(孙崇波,向林,施季森,胡凤荣,郭方其,秦徳辉,2010,春兰AGL6基因的克隆及实时定量表达分析,分子植物育种,8(5):939-944)
Theissen G.,and Saedler H.,2001,Plant biology:floral quartets,Nature,409(6819):469-471
Theiβen G.,2001,Development of floral organ identity:stories from the MADS house,Current Opinion in Plant Biology,4(1):75-85
Vandenbussche M.,Theissen G.E.,Van de Peer Y.,and Gerats T.,2003,Structural diversification and neo-functionalization during floral MADS-box gene evolution by C-terminal frameshift mutations,Nucleic Acids Research,31(15):4401-4409
Xiang L.,Sun C.B.,Guo F.Q.,Li B.J.,and Qin D.H.,2010,Cloning and sequence analysis of AGL6 gene from Cymbidium faberi,China Forestry Publishing,Beijing,China,pp.156-161(向林,孙崇波,郭方其,李伯钧,秦德辉,2010,蕙兰AGL6基因的克隆和序列分析,中国林业出版社,中国,北京,pp.156-161)
Xu Y.,Lai T.L.,Zhou J.,Kumar P.P.,and Yu H.,2006,Floral organ identity genes in the orchid Dendrobium crumenatum,Plant Journal for Cell and Molecular Biology,46(1):54-68
Yin X.,2013,Genetic structure of rice black-streaked dwarf virus(RBSDV)populations and identification of maize protein interacting with RBSDV P8,Dissertation Ph.D.,Shangdong Agricultural University,Supervisor:Li X.D.,pp.16-17(阴筱,2013,水稻黑条矮缩病毒(RBSDV)群体遗传结构分析及与RBSDV P8互作玉米蛋白的鉴定,博士学位论文,山东农业大学,导师:李向东,pp.16-17)
Yockteng R.,Almeida A.M.R.,Morioka K.,Alvarez-Buylla E.R.,and Specht C.D.,2013,Molecular evolution and patterns of duplication in the SEP/AGL6-like lineage of the Zingiberales:a proposed mechanism for floral diversification,Molecular Biology and Evolution,30(11):2401-2422
Yuan Y.X.,Wu J.,Sun R.F.,Zhang X.W.,Xu D.H.,Bonnema G.,and Wang X.W.,2009,A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time,Journal of Experimental Botany,60(4):1299-1308
Yu H.,and Goh C.J.,2000,Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition,Plant Physiology,123(4):1325-1336
Acri-Nunes-Miranda R.,and Mondragon-Palomino M.,2014,Expression of paralogous SEP-,FUL-,AG-and STK-like MADS-box genes in wild-type and peloric Phalaenopsis flowers,Frontiers in Plant Science,5(5):76
Bemer M.,and Angenent G.C.,2010,Floral organ initiation and development,Springer Berlin Heidelberg,pp.173-194
Chang Y.Y.,Chiu Y.F.,Wu J.W.,and Yang C.H.,2009,Four orchid(Oncidium Gower Ramsey)AP1/AGL9-like%MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana,Plant and Cell Physiology,50(8):1425-1438
Chen J.Y.,and Cheng X.K.,eds.,2003,Chinese flower,Shanghai Culture Publishing House,Shanghai,China,pp.127-129(陈俊愉,程绪珂,主编,2003,中国花经,上海文化出版社,中国,上海,pp.127-129)
Coen E.S.,and Meyerowitz E.M.,1991,The war of whorls:genetic interactions controlling flower development,Nature,353(6339):31-37
De Bodt S.,Raes J.,Van de Peer Y.,and Theissen G.魬.,2003,And then there were many:MADS goes genomic,Trends in Plant Science,8(10):475-483
Ditta G.,Pinyopich A.,Robles P.,Pelas S.,and Yanofsky M.F.,2004,The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity,Current Biology,14(21):1935-1940
Fields S.,and Song O.,1989,A novel genetic system to detect protein-protein interactions,Nature,340(6230):245-246
Goto K.,Kyozuka J.,and Bowman J.L.,2001,Turning floral organs into leaves,leaves into floral organs,Current Opinion in Genetics and Development,11(4):449-456
Honma T.,and Goto K.,2001,Complexes of MADS-box proteins are sufficient to convert leaves into floral organs,Nature,409(6819):525-529
Hsiao Y.Y.,Pan Z.J.,Hsu C.C.,Yang Y.P.,Hsu Y.C.,Chuang Y.C.,Shih H.H.,Chen W.H.,Tsai W.C.,and Chen H.H.,2011,Research on orchid biology and biotechnology,Plant and Cell Physiology,52(9):1467-1486
Hsu H.F.,Hsu W.H.,Lee Y.I.,Mao W.T.,Yang J.Y.,Li J.Y.,and Yang C.H.,2015,Model for peranth formation in orchids,Nature Plants,46:1-8
Hsu H.F,Huang C.H.,Yang C.T.,and Chang H.Y.,2003,Ectopic expression of an orchid(Oncidium Gower Ramsey)AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana,Plant and Cell Physiology,44(8):783-794
Immink R.G.H.,Tonaco I.A.N.,de Folter S.,Shchennikova A.,van Dijk A.D.J.,Busscher-Lange J.,Borst J.W.,and Angenent G.C.,2009,SEPALLATA3:the'glue'for MADS box transcription factor complex formation,Genome Biology,10(2):R24
Jia C.L.,Xiang L.,Qin D.H.,Li X.B.,Wu C.,and Sun C.B.,2014,Cloning and expression analysis of CgSEP3 gene from Cymbidium goeringii,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),34(7):1305-1310(贾春蕾,向林,秦徳辉,李小白,吴超,孙崇波,2014,春兰CgSEP3基因的克隆和表达,西北植物学报,34(7):1305-1310)
Kaufmann K.,Melzer R.,and Theiβen G.,2005,MIKC-type MADS-domain proteins:structural modularity,protein interactions and network evolution in land plants,Gene,347(2):183-198
Lin C.S.,Hsu C.T.,Liao D.C.,Chang W.J.,Chou M.L.,Huang Y.T.,Chen J.W.,Ko S.S.,Chan M.T.,and Shih M.C.,2016,Transcriptome-wide analysis of the MADS-box gene family in the orchid Erycina pusilla,Plant Biotechnology Journal,14(1):284-298
Liu C.,Xi W.Y.,Shen L.S.,Tan C.P.,and Yu H.,2009,Regulation of floral patterning by flowering time genes,Developmental Cell,16(5):711-722
Melzer S.,Lens F.,Gennen J.,Vanneste S.,Rohde A.,and Beeckman T.,2008,Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana,Nature Genetics,40(12):1489-1492
Münster T.,Wingen L.U.,Faigl W.,Wreth S.,Saedler H.,and Theiβen G.,2001,Charaterization of three GLOBOSA-like MADS-box genes from maize evidence for ancient paralogy in one class of floral homeotic B-function genes of grasses,Gene,262:1-13
Pan Z.J.,Chen Y.Y.,Du J.S.,Chen Y.Y.,Chung M.C.,Tsai W.C.,Wang C.N.,and Chen H.H.,2014,Flower development of Phalaenopsis orchid involves functionally divergent SEPAL-LATA-like genes,New Phytologist,202(3):1024-1042
Pelaz S.,Tapia-López R.,Alvarez-Buylla E.R.,and Yanofsky M.F.,2001,Conversion of leaves into petals in Arabidopsis,Current Biology,11(3):182-184
Shi J.,2014,Study on interaction between ankyrin ANK6 and SIG5 in Arabidopsis thaliana,Thesis for M.S.,Hunan Normal University,Supervisors:Li D.P.,and Luan S.,pp.6-7(史佳,2014,拟南芥锚蛋白ANK6和SIG5的相互作用研究,硕士学位论文,湖南师范大学,导师:李东屏,栾升,pp.6-7)
Sun C.B.,Xiang L.,Shi J.S.,Hu F.R.,Guo F.Q.,and Qin D.H.,2010,Cloning and real-time expression analysis of AGL6gene from Cymbidium goeringii,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(5):939-944(孙崇波,向林,施季森,胡凤荣,郭方其,秦徳辉,2010,春兰AGL6基因的克隆及实时定量表达分析,分子植物育种,8(5):939-944)
Theissen G.,and Saedler H.,2001,Plant biology:floral quartets,Nature,409(6819):469-471
Theiβen G.,2001,Development of floral organ identity:stories from the MADS house,Current Opinion in Plant Biology,4(1):75-85
Vandenbussche M.,Theissen G.E.,Van de Peer Y.,and Gerats T.,2003,Structural diversification and neo-functionalization during floral MADS-box gene evolution by C-terminal frameshift mutations,Nucleic Acids Research,31(15):4401-4409
Xiang L.,Sun C.B.,Guo F.Q.,Li B.J.,and Qin D.H.,2010,Cloning and sequence analysis of AGL6 gene from Cymbidium faberi,China Forestry Publishing,Beijing,China,pp.156-161(向林,孙崇波,郭方其,李伯钧,秦德辉,2010,蕙兰AGL6基因的克隆和序列分析,中国林业出版社,中国,北京,pp.156-161)
Xu Y.,Lai T.L.,Zhou J.,Kumar P.P.,and Yu H.,2006,Floral organ identity genes in the orchid Dendrobium crumenatum,Plant Journal for Cell and Molecular Biology,46(1):54-68
Yin X.,2013,Genetic structure of rice black-streaked dwarf virus(RBSDV)populations and identification of maize protein interacting with RBSDV P8,Dissertation Ph.D.,Shangdong Agricultural University,Supervisor:Li X.D.,pp.16-17(阴筱,2013,水稻黑条矮缩病毒(RBSDV)群体遗传结构分析及与RBSDV P8互作玉米蛋白的鉴定,博士学位论文,山东农业大学,导师:李向东,pp.16-17)
Yockteng R.,Almeida A.M.R.,Morioka K.,Alvarez-Buylla E.R.,and Specht C.D.,2013,Molecular evolution and patterns of duplication in the SEP/AGL6-like lineage of the Zingiberales:a proposed mechanism for floral diversification,Molecular Biology and Evolution,30(11):2401-2422
Yuan Y.X.,Wu J.,Sun R.F.,Zhang X.W.,Xu D.H.,Bonnema G.,and Wang X.W.,2009,A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time,Journal of Experimental Botany,60(4):1299-1308
Yu H.,and Goh C.J.,2000,Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition,Plant Physiology,123(4):1325-1336