建兰AGL6基因的表达及功能分析
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摘要
采用文库稀释池法从建兰花器官全长均一化cDNA文库中筛选出1个AGL6类MADS-box基因CeAGL6,对其进行了信息学分析,并通过表达模式、互作蛋白和烟草的遗传转化研究其功能。研究结果发现,CeAGL6含有MADS结构域和K结构域,并具有AGL6类蛋白的典型基序。CeAGL6在建兰萼片中高水平表达,其次是花梗,在花瓣、唇瓣和合蕊柱中表达水平较低,不在叶片和根中表达。转CeAGL6烟草花期略微提前,出现四瓣花、六瓣花、雄蕊瓣化和多种不规则的五瓣花等表型。CeAGL6与CeAP1、CePI1、CeAG1和CeSEP3能互作,但自身不能形成同源二聚体。试验结果表明Ce AGL6参与了建兰成花诱导和花器官发育。
CeAGL6,a gene belongs to AGL6 class of the MADS-box gene family,was isolated from a normalized full-length cDNA library of Cymbidium ensifolium based on pooling strategy combined with PCR-based screening method. After bioinformatics analysis of CeAGL6,expression patterns,interacting protein and tobacco genetic transformation were performed to identify its functions in the flower development of Cymbidium ensifolium. Sequence analysis revealed the highly conserved MADS domain,K domain, and a typical motif of AGL6 class of MADS-box gene family in CeAGL6. The highest expression level of CeAGL6 was found in sepal,followed by pedicels,low level in petal,lip,and column,and expression in leaf and root was not detected. Overexpression of CeAGL6 caused an advanced flowering and novel phenotypes of four-petal flower,six-petal flower,petaloid stamens and a variety of irregular five-petal flower in transgenic tobacco. Yeast two-hybrid analysis indicated there were interactions between CeAGL6 and CeAP1,CePI1,CeAG1 or CeSEP3,but the homodimers cannot form by itself. The results showed that CeAGL6 was involved in promoting flowering and flower organ development of Cymbidium ensifolium.
CeAGL6,a gene belongs to AGL6 class of the MADS-box gene family,was isolated from a normalized full-length cDNA library of Cymbidium ensifolium based on pooling strategy combined with PCR-based screening method. After bioinformatics analysis of CeAGL6,expression patterns,interacting protein and tobacco genetic transformation were performed to identify its functions in the flower development of Cymbidium ensifolium. Sequence analysis revealed the highly conserved MADS domain,K domain, and a typical motif of AGL6 class of MADS-box gene family in CeAGL6. The highest expression level of CeAGL6 was found in sepal,followed by pedicels,low level in petal,lip,and column,and expression in leaf and root was not detected. Overexpression of CeAGL6 caused an advanced flowering and novel phenotypes of four-petal flower,six-petal flower,petaloid stamens and a variety of irregular five-petal flower in transgenic tobacco. Yeast two-hybrid analysis indicated there were interactions between CeAGL6 and CeAP1,CePI1,CeAG1 or CeSEP3,but the homodimers cannot form by itself. The results showed that CeAGL6 was involved in promoting flowering and flower organ development of Cymbidium ensifolium.
引文
Becker A,Theissen G.2003.The major clades of MADS-box genes and their role in the development and evolution of flowering plants.Mol Phylogenet Evol,29(3):464-489.
Boss P K,Sensi E,Hua C,Davies C,Thomas M R.2002.Cloning and characterisation of grapevine(Vitis vinifera)MADS-box genes expressed during inflorescence and berry development.Plant Science,162(6):887-895.
Carlsbecker A,Tandre K,Johanson U,Englund M,Engstr?m P.2004.The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce(Picea abies).Plant J,40(4):546-557.
Chanderbali A S,Kim S,Buzgo M,Zheng Z,Oppenheimer D G,Soltis D E,Soltis P S.2006.Genetic footprints of stamen ancestors guide perianth evolution in Persea(Lauraceae).Int J Plant Sci,167(6):1075-1089.
Chang Y Y,Chiu Y F,Wu J W,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.
Dirks-Mulder A,But?t R,Schaik P van,Wijnands J W P M,van den Berg R,Krol L,Doebar S,van Kooperen K,de Boer H,Kramer E M,Smets E F,Vos R A,Vrijdaghs A,Gravendeel B.2017.Exploring the evolutionary origin of floral organs of Erycina pusilla,an emerging orchid model system.BMC Evolutionary Biology,17(1):89.
Honma T,Goto K.2001.Complexes of MADS-box proteins are sufficient to convert leaves into floral organs.Nature.409(6819):525-529.
Hu Li-xia,Xu Jing,Pang Ji-liang,Wag Li-lin,Wang Hui-zhong,Yu Hong.2011.Cloning and functional research of an AGL6 homologous gene in hybird of Cymbidium goeringii and Cymbidium hybirdium.Acta Horticulture Sinica,38(2):317-326.(in Chinese)胡立霞,徐京,庞基良,王利琳,王慧中,余红.2011.春兰×大花蕙兰杂种AGL6同源基因的克隆及其功能研究.园艺学报,38(2):317-326.
Hu Yue-miao,Sun Chong-bo,Xiang Lin,Hu Feng-rong.2016.Cloning and real-time expression of AGL6-3 gene from Cymbidium goeringii.Acta Botanica Boreali-Occidentalia Sinica,36(2):225-230.(in Chinese)胡月苗,孙崇波,向林,胡凤荣.2016.春兰AGL6-3基因的克隆及实时定量表达分析.西北植物学报,36(2):225-230.
Hsu H F,Huang C H,Chou L T,Yang C H.2003.Ectopic expression of an orchid(Oncidium Gower Ramsey)AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana.Plant Cell Physiol,44(8):783-794.
Hsu H F,Hsu W H,Lee Y I,Mao W T,Yang J Y,Li J Y,Yang C H.2015.Model for perianth formation in orchids.Nat Plants,1(5):15046.
Kim S,Koh J,Yoo M,Kong H,Hu Y,Ma H,Soltis P S,Soltis D E.2005.Expression of floral MADS-box genes in basal angiosperms:implications for the evolution of floral regulators.The Plant Journal,43(5):724-744.
Li Lai-yun.2017.Molecular mechanism of AGL6 homologous genes involving in regulation of floral development in Manglietia patungensis Hu[M.D.Dissertation].Jingzhou:Yangtze University.(in Chinese)李来运.2017.巴东木莲AGL6同源基因参与调控花发育的分子机制[硕士论文].荆州:长江大学.
Losa A,Caporali E,Spada A,Martinelli S,Marziani G.2004.AOM3 and AOM4:two MADS box genes expressed in reproductive structures of Asparagus officinalis.Sex Plant Reprod,16(5):215-221.
Ma H,Yanofsky M F,Meyerowitz E M.1991.AGL1-AGL6,an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes.Genes Dev,5(3):484-95.
Moon Y H,Kang H G,Jung J Y,Jeon J S,Sung S K,An G.1999.Determination of the motif responsible for interaction between the rice APTELA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system.Plant Physiol,120(4):1193-1203
Mouradov A,Glassick TV,Hamdorf B A,Murphy L C,Marla S S,Yang Y,Teasdale R D.1998.Family of MADS-box genes expressed early in male and female reproductive structures of monterey pine.Plant Physiol,117(1):55-62.
Ohmori S,Kimizu M,Sugita M,Miyao A,Hirochika H,Uchida E,Nagato Y,Yoshida H.2009.MOSAIC FLORAL ORGANS1,an AGL6-Like MADS box gene,regulates floral organ identity and meristem fate in rice.The Plant Cell,21(10):3008-3025.
Petersen K,Didion T,Andersen C H,Nielsen K K.2004.MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth.J Plant Physiol,161(4):439-447.
Purugganan M D,Rounsley S D,Schmidt R J,Yanofsky M F.1995.Molecular evolution of flower development:diversification of the plant mads-box regulatory gene family.Genetics,140(1):345-356.
Qiu A,Liu Z,Li J,Guan D,He S.2016.The ectopic expression of carop1 modulates the response of tobacco plants to Ralstonia solanacearum and aphids.Frontiers in Plant Science,7(e1904).
Reinheimer R,Kellogg E A.2009.Evolution of AGL6-like MADS-box genes in grasses(Poaceae):ovule expression is ancient and palea expression is new.Plant Cell,21(9):2591-2605.
Rijpkema A S,Zethof J,Gerats T,Vandenbussche M.2010.The petunia AGL6 gene has a SEPALLATA-like function in floral patternin.Plant Journal,60(1):1-9.
Rounsley S D,Ditta G S,Yanofsky M F.1995.Diverse roles for MADS box genes in Arabidopsis development.Plant Cell,7(8):1259-1269.
Shindo S,Ito M,Ueda K,Kato M,Hasebe M.1999.Characterization of MADS genes in the gymnosperm Gnetum parvifolium and its implication on the evolution of reproductive organs in seed plants.Evolution&Development,1(3):180-190.
Sun Chong-bo,Xiang Lin,Shi Ji-sen,Hu Feng-rong,Guo Fang-qi,Qin De-hui.2010.Cloning and real-time expression analysis of AGL6 gene from Cymbidium goeringii.Molecular Plant Beeeding,8(5):939-944.(in Chinese)孙崇波,向林,施季森,胡凤荣,郭方其,秦德辉.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.
Sun W,Huang W,Li Z,Song C,Liu D,Liu Y,Hayward A,Liu Y,Huang H,Wang Y.2014.Functional and evolutionary analysis of the AP1/SEP/AGL6 superclade of MADS-box genes in the basal eudicot Epimedium sagittatum.Ann Bot,113(4):653-668.
Tang Wen-long,Zhao Peng-fei,Li Yong-hua,He Dan,Lu Jiu-xing.2018.Cloning and expression analysis of transcription factor gene PsAGL6 in tree peony(Paeonia suffruticosa L.).Acta Botanica Boreali Occidentalia Sinica,37(3):439-444.(in Chinese)唐文龙,赵鹏飞,李永华,贺丹,逯久幸.2018.牡丹PsAGL6基因克隆与表达分析.西北植物学报,37(3):439-444.
Theissen G,Becker A,Di Rosa A,Kanno A,Kim J T,Münster T,Winter K U,Saedler H.2000.A short history of MADS-box genes in plants.Plant Mol Biol,42(1):115-149.
Thompson B E,Bartling L,Whipple C,Hall D H,Sakai H,Schmidt R,Hake S.2009.Bearded-ear encodes a MADS-box transcription factor critical for maize floral development.Plant Cell,21(9):2578-2590.
Tsuchimoto S,Mayama T,Van Der Krol A,Ohtsubo E.2000.The whorl-specific action of a petunia class B floral homeotic gene.Genes to Cells,5(2):89-99.
Viaene T,Vekemans D,Becker A,Melzer S,Geuten K.2010.Exspression divergence of the AGL6 MADS domain transcription factor lineage after a core eudicot duplication suggests functional diversification.BMC Plant Biology,10:148.
Wang P P,Liao H,Kong H Z.2015.Flexibility in the structure of spiral flowers and its underlying mechanisms.Nature Plants,15188.
Wu Jing-hua,Wu Shao-hua,Yang Chao,Zhang Zhi-zhong.2013.Cloning and expression analysis on AP1 homologous gene from Cymbidium ensifolium and interaction analysis between AP1 and MADS-box transcription factors.Acta Horticulturae Sinica,40(10):1935-1942.(in Chinese)吴菁华,吴少华,杨超,张志忠.2013.建兰AP1基因的克隆、表达及其与MADS-box转录因子相互作用的分析.园艺学报,40(10):1935-1942.
Xiang Lin,ChenYue,Chen Li-ping,Sun Chong-bo.2018.Expression analysis of B,C and E class MADS-box genes in Cymbidium ensifolium.Acta Horticulturae Sinica,45(8):1595-1604.(in Chinese)向林,陈跃,陈丽萍,孙崇波.2018.建兰花发育相关B、C和E类MADS-box基因的表达分析.园艺学报,45(8):1595-1604.
Yim Y S,Moak P,Sanchez-Villeda H,Musket T A,Close P,Klein P E,Mullet J E,McMullen M D,Fang Z,Schaeffer M L,Gardiner J M,Coe E H Jr,Davis G L.2007.A BAC pooling strategy combined with PCR-based screenings in a large,highly repetitive genome enables integration of the maize genetic and physical maps.BMC Genomics,8(1):47.
Yockteng R,Almeida AM,Morioka K,Alvarez-Buylla E R,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.Mol Biol Evol,30(11):2401-2422.
Yu X,Chen G,Guo X,Lu Y,Zhang J,Hu J T,Tian S B,Hu Z L.2017.Silencing SlAGL6,a tomato AGAMOUS-LIKE 6 lineage gene,generates fused sepal and green petal.Plant Cell Reports,36(6):1-11.
Zahn L M,Kong H,Leebens-Mack J H,Kim S,Soltis P S,Landherr L L,Soltis D E,Depamphilis C W,Ma H.2005.The evolution of the SEPALLATA subfamily of MADS-Box genes:a preangiosperm origin with multiple duplications throughout angiosperm history.Genetics,169(4):2209-2223.
Boss P K,Sensi E,Hua C,Davies C,Thomas M R.2002.Cloning and characterisation of grapevine(Vitis vinifera)MADS-box genes expressed during inflorescence and berry development.Plant Science,162(6):887-895.
Carlsbecker A,Tandre K,Johanson U,Englund M,Engstr?m P.2004.The MADS-box gene DAL1 is a potential mediator of the juvenile-to-adult transition in Norway spruce(Picea abies).Plant J,40(4):546-557.
Chanderbali A S,Kim S,Buzgo M,Zheng Z,Oppenheimer D G,Soltis D E,Soltis P S.2006.Genetic footprints of stamen ancestors guide perianth evolution in Persea(Lauraceae).Int J Plant Sci,167(6):1075-1089.
Chang Y Y,Chiu Y F,Wu J W,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.
Dirks-Mulder A,But?t R,Schaik P van,Wijnands J W P M,van den Berg R,Krol L,Doebar S,van Kooperen K,de Boer H,Kramer E M,Smets E F,Vos R A,Vrijdaghs A,Gravendeel B.2017.Exploring the evolutionary origin of floral organs of Erycina pusilla,an emerging orchid model system.BMC Evolutionary Biology,17(1):89.
Honma T,Goto K.2001.Complexes of MADS-box proteins are sufficient to convert leaves into floral organs.Nature.409(6819):525-529.
Hu Li-xia,Xu Jing,Pang Ji-liang,Wag Li-lin,Wang Hui-zhong,Yu Hong.2011.Cloning and functional research of an AGL6 homologous gene in hybird of Cymbidium goeringii and Cymbidium hybirdium.Acta Horticulture Sinica,38(2):317-326.(in Chinese)胡立霞,徐京,庞基良,王利琳,王慧中,余红.2011.春兰×大花蕙兰杂种AGL6同源基因的克隆及其功能研究.园艺学报,38(2):317-326.
Hu Yue-miao,Sun Chong-bo,Xiang Lin,Hu Feng-rong.2016.Cloning and real-time expression of AGL6-3 gene from Cymbidium goeringii.Acta Botanica Boreali-Occidentalia Sinica,36(2):225-230.(in Chinese)胡月苗,孙崇波,向林,胡凤荣.2016.春兰AGL6-3基因的克隆及实时定量表达分析.西北植物学报,36(2):225-230.
Hsu H F,Huang C H,Chou L T,Yang C H.2003.Ectopic expression of an orchid(Oncidium Gower Ramsey)AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana.Plant Cell Physiol,44(8):783-794.
Hsu H F,Hsu W H,Lee Y I,Mao W T,Yang J Y,Li J Y,Yang C H.2015.Model for perianth formation in orchids.Nat Plants,1(5):15046.
Kim S,Koh J,Yoo M,Kong H,Hu Y,Ma H,Soltis P S,Soltis D E.2005.Expression of floral MADS-box genes in basal angiosperms:implications for the evolution of floral regulators.The Plant Journal,43(5):724-744.
Li Lai-yun.2017.Molecular mechanism of AGL6 homologous genes involving in regulation of floral development in Manglietia patungensis Hu[M.D.Dissertation].Jingzhou:Yangtze University.(in Chinese)李来运.2017.巴东木莲AGL6同源基因参与调控花发育的分子机制[硕士论文].荆州:长江大学.
Losa A,Caporali E,Spada A,Martinelli S,Marziani G.2004.AOM3 and AOM4:two MADS box genes expressed in reproductive structures of Asparagus officinalis.Sex Plant Reprod,16(5):215-221.
Ma H,Yanofsky M F,Meyerowitz E M.1991.AGL1-AGL6,an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes.Genes Dev,5(3):484-95.
Moon Y H,Kang H G,Jung J Y,Jeon J S,Sung S K,An G.1999.Determination of the motif responsible for interaction between the rice APTELA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system.Plant Physiol,120(4):1193-1203
Mouradov A,Glassick TV,Hamdorf B A,Murphy L C,Marla S S,Yang Y,Teasdale R D.1998.Family of MADS-box genes expressed early in male and female reproductive structures of monterey pine.Plant Physiol,117(1):55-62.
Ohmori S,Kimizu M,Sugita M,Miyao A,Hirochika H,Uchida E,Nagato Y,Yoshida H.2009.MOSAIC FLORAL ORGANS1,an AGL6-Like MADS box gene,regulates floral organ identity and meristem fate in rice.The Plant Cell,21(10):3008-3025.
Petersen K,Didion T,Andersen C H,Nielsen K K.2004.MADS-box genes from perennial ryegrass differentially expressed during transition from vegetative to reproductive growth.J Plant Physiol,161(4):439-447.
Purugganan M D,Rounsley S D,Schmidt R J,Yanofsky M F.1995.Molecular evolution of flower development:diversification of the plant mads-box regulatory gene family.Genetics,140(1):345-356.
Qiu A,Liu Z,Li J,Guan D,He S.2016.The ectopic expression of carop1 modulates the response of tobacco plants to Ralstonia solanacearum and aphids.Frontiers in Plant Science,7(e1904).
Reinheimer R,Kellogg E A.2009.Evolution of AGL6-like MADS-box genes in grasses(Poaceae):ovule expression is ancient and palea expression is new.Plant Cell,21(9):2591-2605.
Rijpkema A S,Zethof J,Gerats T,Vandenbussche M.2010.The petunia AGL6 gene has a SEPALLATA-like function in floral patternin.Plant Journal,60(1):1-9.
Rounsley S D,Ditta G S,Yanofsky M F.1995.Diverse roles for MADS box genes in Arabidopsis development.Plant Cell,7(8):1259-1269.
Shindo S,Ito M,Ueda K,Kato M,Hasebe M.1999.Characterization of MADS genes in the gymnosperm Gnetum parvifolium and its implication on the evolution of reproductive organs in seed plants.Evolution&Development,1(3):180-190.
Sun Chong-bo,Xiang Lin,Shi Ji-sen,Hu Feng-rong,Guo Fang-qi,Qin De-hui.2010.Cloning and real-time expression analysis of AGL6 gene from Cymbidium goeringii.Molecular Plant Beeeding,8(5):939-944.(in Chinese)孙崇波,向林,施季森,胡凤荣,郭方其,秦德辉.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.
Sun W,Huang W,Li Z,Song C,Liu D,Liu Y,Hayward A,Liu Y,Huang H,Wang Y.2014.Functional and evolutionary analysis of the AP1/SEP/AGL6 superclade of MADS-box genes in the basal eudicot Epimedium sagittatum.Ann Bot,113(4):653-668.
Tang Wen-long,Zhao Peng-fei,Li Yong-hua,He Dan,Lu Jiu-xing.2018.Cloning and expression analysis of transcription factor gene PsAGL6 in tree peony(Paeonia suffruticosa L.).Acta Botanica Boreali Occidentalia Sinica,37(3):439-444.(in Chinese)唐文龙,赵鹏飞,李永华,贺丹,逯久幸.2018.牡丹PsAGL6基因克隆与表达分析.西北植物学报,37(3):439-444.
Theissen G,Becker A,Di Rosa A,Kanno A,Kim J T,Münster T,Winter K U,Saedler H.2000.A short history of MADS-box genes in plants.Plant Mol Biol,42(1):115-149.
Thompson B E,Bartling L,Whipple C,Hall D H,Sakai H,Schmidt R,Hake S.2009.Bearded-ear encodes a MADS-box transcription factor critical for maize floral development.Plant Cell,21(9):2578-2590.
Tsuchimoto S,Mayama T,Van Der Krol A,Ohtsubo E.2000.The whorl-specific action of a petunia class B floral homeotic gene.Genes to Cells,5(2):89-99.
Viaene T,Vekemans D,Becker A,Melzer S,Geuten K.2010.Exspression divergence of the AGL6 MADS domain transcription factor lineage after a core eudicot duplication suggests functional diversification.BMC Plant Biology,10:148.
Wang P P,Liao H,Kong H Z.2015.Flexibility in the structure of spiral flowers and its underlying mechanisms.Nature Plants,15188.
Wu Jing-hua,Wu Shao-hua,Yang Chao,Zhang Zhi-zhong.2013.Cloning and expression analysis on AP1 homologous gene from Cymbidium ensifolium and interaction analysis between AP1 and MADS-box transcription factors.Acta Horticulturae Sinica,40(10):1935-1942.(in Chinese)吴菁华,吴少华,杨超,张志忠.2013.建兰AP1基因的克隆、表达及其与MADS-box转录因子相互作用的分析.园艺学报,40(10):1935-1942.
Xiang Lin,ChenYue,Chen Li-ping,Sun Chong-bo.2018.Expression analysis of B,C and E class MADS-box genes in Cymbidium ensifolium.Acta Horticulturae Sinica,45(8):1595-1604.(in Chinese)向林,陈跃,陈丽萍,孙崇波.2018.建兰花发育相关B、C和E类MADS-box基因的表达分析.园艺学报,45(8):1595-1604.
Yim Y S,Moak P,Sanchez-Villeda H,Musket T A,Close P,Klein P E,Mullet J E,McMullen M D,Fang Z,Schaeffer M L,Gardiner J M,Coe E H Jr,Davis G L.2007.A BAC pooling strategy combined with PCR-based screenings in a large,highly repetitive genome enables integration of the maize genetic and physical maps.BMC Genomics,8(1):47.
Yockteng R,Almeida AM,Morioka K,Alvarez-Buylla E R,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.Mol Biol Evol,30(11):2401-2422.
Yu X,Chen G,Guo X,Lu Y,Zhang J,Hu J T,Tian S B,Hu Z L.2017.Silencing SlAGL6,a tomato AGAMOUS-LIKE 6 lineage gene,generates fused sepal and green petal.Plant Cell Reports,36(6):1-11.
Zahn L M,Kong H,Leebens-Mack J H,Kim S,Soltis P S,Landherr L L,Soltis D E,Depamphilis C W,Ma H.2005.The evolution of the SEPALLATA subfamily of MADS-Box genes:a preangiosperm origin with multiple duplications throughout angiosperm history.Genetics,169(4):2209-2223.