水稻HD-Zip基因OsHox24的表达与功能分析
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摘要
同源异型-亮氨酸拉链(homeodomain-leucine-zipper, HD-Zip)转录因子参与高等植物形态建成、生长发育和环境中各种逆境的调控。本研究旨在利用融合蛋白表达、RNA干扰载体(p OsHox24:GUS, pCaMV35S:OsHox24-EGFP, pCaMV35S:OsHox24-RNAi)遗传转化等分子生物学方法,解析水稻中同源异型域亮氨酸拉链转录因子OsHox24在水稻生殖生长过程中的时空表达特性及其生物学功能。生物信息学分析表明,Os-Hox24基因在水稻第2号染色体上,具有2个外显子,1个内含子,编码的蛋白质由261个氨基酸组成,在该蛋白第61～121位氨基酸残基位置处有一个典型的HB类转录因子的DNA结合区(DNA-binding domain)。系统进化分析表明该基因与OsHox22同源性很高。GUS组织染色与RT-PCR实验显示OsHox24在水稻根尖、茎节和节间、叶鞘及不同时期的幼穗中均有表达。洋葱内表皮细胞亚细胞定位显示OsHox24在细胞膜和细胞核中表达。与野生型植株相比,RNA干扰植株的结实率显著降低。本研究初步探明由OsHox24形成的转录因子参与了水稻的生殖发育的调控过程,为进一步探索水稻HD-Zip转录因子功能和育种方面的应用研究提供科学依据。
Homeodomain-leucine-zipper(HD-Zip) transcription factors participate in morphogenesis, growth and development, and regulation of various adversities in the environment of advanced plants. This study aimed to analyze the spatial and temporal expression characteristics and biological functions of the homologous leucine zipper transcription factor OsHox24 in rice during reproductive growth by using fusion protein expression and RNA interference vector(p OsHox24:GUS, pCaMV35 S:OsHox24-EGFP, p CaMV35 S:OsHox24-RNAi) transformation. Bioinformatic analysis showed that OsHox24 was on the second chromosome of rice, containing 2 exons and one intron.The encoded protein consisted of 261 amino acids. There was a typical DNA-binding domain of HB transcription factor at the position of 61～121 amino acid residues. Phylogenetic analysis indicated that the gene was highly homologous with OsHox22. GUS staining and RT-PCR revealed that OsHox24 was expressed in root tip, stem node and internode, leaf sheath and young panicle of rice at different stages. Subcellular localization of onion epidermis cells showed that OsHox24 had expression in cell membrane and nucleus. Compared with wild-type plants, the seed setting rate of RNA interference plants decreased significantly. This study preliminarily demonstrated that transcription factors formed by OsHox24 were involved in the regulation of reproductive development in rice,which could provide scientific basis for further exploring the function of HD-Zip transcription factors and their application in rice breeding.
Homeodomain-leucine-zipper(HD-Zip) transcription factors participate in morphogenesis, growth and development, and regulation of various adversities in the environment of advanced plants. This study aimed to analyze the spatial and temporal expression characteristics and biological functions of the homologous leucine zipper transcription factor OsHox24 in rice during reproductive growth by using fusion protein expression and RNA interference vector(p OsHox24:GUS, pCaMV35 S:OsHox24-EGFP, p CaMV35 S:OsHox24-RNAi) transformation. Bioinformatic analysis showed that OsHox24 was on the second chromosome of rice, containing 2 exons and one intron.The encoded protein consisted of 261 amino acids. There was a typical DNA-binding domain of HB transcription factor at the position of 61～121 amino acid residues. Phylogenetic analysis indicated that the gene was highly homologous with OsHox22. GUS staining and RT-PCR revealed that OsHox24 was expressed in root tip, stem node and internode, leaf sheath and young panicle of rice at different stages. Subcellular localization of onion epidermis cells showed that OsHox24 had expression in cell membrane and nucleus. Compared with wild-type plants, the seed setting rate of RNA interference plants decreased significantly. This study preliminarily demonstrated that transcription factors formed by OsHox24 were involved in the regulation of reproductive development in rice,which could provide scientific basis for further exploring the function of HD-Zip transcription factors and their application in rice breeding.
引文
Abe M.,Katsumata H.,Komeda Y.,and Takahashi T.,2003,Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis,Development,130(4):635-643
Agalou A.,Purwantomo S.,Overnas E.,Johannesson H.,Zhu X.,Estiati A.,Kam R.J.,Engstrom P.,Slamet-Loedin I.H.,Zhu Z.,Wang M.,Xiong L.Z.,Meijer A.H.,and Ouwerkerk P.B.,2008,A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members,Plant Mol.Biol.,66(1-2):87-103
Ai L.P.,Shen A.,Gao Z.C.,Li Z.L.,Sun Q.L.,and Luan W.J.,2014,Reverse genetics analysis of the transcription factor oshox9,a member of homeobox family,in rice,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),28(3):223-228(艾丽萍,申奥,高志超,李政龙,孙琼琳,栾维江,2014,水稻同源异型域转录因子OsHox9的反向遗传学分析,中国水稻科学,28(3):223-228)
Ariel F.D.,Manavella P.A.,Dezar C.A.,and Chan R.L.,2007,The true story of the HD-Zip family,Trends Plant Sci.,12(9):419-426
Baima S.,Possenti M.,Matteucci A.,Wisman E.,Altamura M.M.,Ruberti I.,and Morelli G.,2001,The Arabidopsis ATHB-8HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristems,Plant Physiol.,126(2):643-655
Bhattacharjee A.,Khurana J.P.,and Jain M.,2016,Characterization of rice homeobox genes,OsHOX22 and OsHOX24,and over-expression of OsHOX24 in transgenic Arabidopsis suggest their role in abiotic stress response,Front.Plant Sci.,7(1):627
Depègefargeix N.,Javelle M.,Chambrier P.,Frangne N.,Gerentes D.,Perez P.,Rogowsky P.M.,and Vernoud V.,2011,Functional characterization of the HD-ZIPⅣtranscription factor OCL1 from maize,J.Exp.Bot.,62(1):293-305
Dezar C.A.,Giacomelli J.I.,Manavella P.A.,RéD.A.,Alvesferreira M.,Baldwin I.T.,Bonaventure G.,and Chan R.L.,2011,HAHB10,a sunflower HD-ZipⅡtranscription factor,participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress,J.Exp.Bot.,62(3):1061-1076
Emery J.F.,Floyd S.K.,Alvarez J.,Eshed Y.,Hawker N.P.,Izhaki A.,Baum S.F.,and Bowman J.L.,2003,Radial patterning of Arabidopsis shoots by classⅢHD-ZIP and KANADI genes,Curr.Biol.,13(20):1768-1774
Hanson J.,Johannesson H.,and Engstrom P.,2001,Sugar-dependent alterations in cotyledon and leaf development in transgenic plants expressing the HD-Zip gene ATHB13,Plant Mol.Biol.,45(3):247-262
Harris J.C.,Hrmova M.,Lopato S.,and Langridge P.,2011,Modulation of plant growth by HD-Zip classⅠandⅡtranscription factors in response to environmental stimuli,New Phytol.,190(4):823-837
Henriksson E.,Olsson A.S.B.,Johannesson H.,Johansson H.,Hanson J.,Engstrom P.,and Soderman E.,2005,Homeodomain leucine zipper classⅠgenes in Arabidopsis expression patterns and phylogenetic relationships,Plant Physiol.,139(1):509-518
Itoh J.,Hibara K.,Sato Y.,and Nagato Y.,2008,Developmental role and auxin responsiveness of classⅢHD-Zip gene family members in rice,Plant Physiol.,147(4):1960-1975
Izhaki A.,and Bowman J.L.,2007,KANADI and classⅢHD-Zip gene families regulate embryo patterning and modulate auxin flow during embryogenesis in Arabidopsis,Plant Cell.,19(2):495-508
Johannesson H.,Wang Y.,and Engstrom P.,2001,DNA-binding and dimerization preferences of Arabidopsis homeodomainleucine zipper transcription factors in vitro,Plant Mol.Biol.,45(1):63-73
Kim Y.S.,Kim S.G.,Lee M.,Lee I.,Park H.Y.,Seo P.J.,Jung J.H.,Kwon E.J.,Suh S.W.,Paek K.H.,and Park C.M.,2008,HD-ZIPⅢactivity is modulated by competitive inhibitors via a feedback loop in arabidopsis shoot apical meristem development,Plant Cell,20(4):920-933
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.,Tan X.L.,Lu B.R.,Li J.,Gan S.X.,Kang S.G.,Suh H.S.,and Zhu Y.,2013,The bsister MADS gene FST determines ovule patterning and development of the zygotic embryo and endosperm,PLoS One,8(3):e58748
Maimaiti Y.S.J.,2012,Expression of rice HD-ZIP transcription factor Oshox4,Oshox6 and IPT gene in the rice and drought resistance analyse of these transgenic plants,Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Qiu D.W.,pp.50-51(玉山江·麦麦提,2012,水稻HD-ZIP转录因子Oshox4,Oshox6和IPT基因在水稻中的表达及其抗旱功能研究,博士学位论文,中国农业科学院研究生院,导师:邱德文,pp.50-51)
Manavella P.A.,Dezar C.A.,Bonaventure G.,Baldwin I.T.,and Chan R.L.,2008,HAHB4,a sunflower HD-Zip protein,integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses,Plant J.,56(3):376-388
McConnell J.R.,Emery J.,Eshed Y.,Bao N.,Bowman J.,and Barton M.K.,2001,Role of PHABULOSA and PHAVOLU-TA in determining radial patterning in shoots,Nature,411(6838):709-713
Ohashiito K.,Kubo M.,Demura T.,and Fukuda H.,2005,ClassⅢhomeodomain leucine-zipper proteins regulate xylem cell differentiation,Plant Cell Physiol.,46(10):1646-1656
Sawa S.,Ohgishi M.,Goda H.,Higuchi K.,Shimada Y.,Yoshida S.,and Koshiba T.,2002,The HAT2 gene,a member of the HD-Zip gene family,isolated as an auxin inducible gene by DNA microarray screening,affects auxin response in Arabidopsis,Plant J.,32(6):1011-1022
Scarpella E.,Boot K.J.,Rueb S.,and Meijer A.H.,2002,The procambium specifcation gene OsHOXl promotes polar auxin transport capacity and reduces its sensitivity toward inhibition,Plant Physiol.,130(3):1349-1360
Stamm P.,and Kumar P.P.,2010,The phytohormone signal network regulating elongation growth during shade avoidance,J.Exp.Bot.,61(11):2889-2903
Wenkel S.,Emery J.,Hou B.H.,Evans M.M.,and Barton M.K.,2007,A feedback regulatory module formed by LITTLEZIPPER and HD-ZIPⅢgenes,Plant Cell,19(11):3379-3390
Yao P.F.,Zhao X.R.,Li M.F.,Wang A.H.,and Wu Q.,2016,Cloning and expression analysis of transcription factor gene FtbHLH3 from Fagopyrum tataricum under abiotic stress,Jiy inzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):429-435(姚攀锋,赵学荣,李茂菲,王安虎,吴琦,2016,苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,36(2):429-435)
Agalou A.,Purwantomo S.,Overnas E.,Johannesson H.,Zhu X.,Estiati A.,Kam R.J.,Engstrom P.,Slamet-Loedin I.H.,Zhu Z.,Wang M.,Xiong L.Z.,Meijer A.H.,and Ouwerkerk P.B.,2008,A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members,Plant Mol.Biol.,66(1-2):87-103
Ai L.P.,Shen A.,Gao Z.C.,Li Z.L.,Sun Q.L.,and Luan W.J.,2014,Reverse genetics analysis of the transcription factor oshox9,a member of homeobox family,in rice,Zhongguo Shuidao Kexue(Chinese Journal of Rice Science),28(3):223-228(艾丽萍,申奥,高志超,李政龙,孙琼琳,栾维江,2014,水稻同源异型域转录因子OsHox9的反向遗传学分析,中国水稻科学,28(3):223-228)
Ariel F.D.,Manavella P.A.,Dezar C.A.,and Chan R.L.,2007,The true story of the HD-Zip family,Trends Plant Sci.,12(9):419-426
Baima S.,Possenti M.,Matteucci A.,Wisman E.,Altamura M.M.,Ruberti I.,and Morelli G.,2001,The Arabidopsis ATHB-8HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristems,Plant Physiol.,126(2):643-655
Bhattacharjee A.,Khurana J.P.,and Jain M.,2016,Characterization of rice homeobox genes,OsHOX22 and OsHOX24,and over-expression of OsHOX24 in transgenic Arabidopsis suggest their role in abiotic stress response,Front.Plant Sci.,7(1):627
Depègefargeix N.,Javelle M.,Chambrier P.,Frangne N.,Gerentes D.,Perez P.,Rogowsky P.M.,and Vernoud V.,2011,Functional characterization of the HD-ZIPⅣtranscription factor OCL1 from maize,J.Exp.Bot.,62(1):293-305
Dezar C.A.,Giacomelli J.I.,Manavella P.A.,RéD.A.,Alvesferreira M.,Baldwin I.T.,Bonaventure G.,and Chan R.L.,2011,HAHB10,a sunflower HD-ZipⅡtranscription factor,participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress,J.Exp.Bot.,62(3):1061-1076
Emery J.F.,Floyd S.K.,Alvarez J.,Eshed Y.,Hawker N.P.,Izhaki A.,Baum S.F.,and Bowman J.L.,2003,Radial patterning of Arabidopsis shoots by classⅢHD-ZIP and KANADI genes,Curr.Biol.,13(20):1768-1774
Hanson J.,Johannesson H.,and Engstrom P.,2001,Sugar-dependent alterations in cotyledon and leaf development in transgenic plants expressing the HD-Zip gene ATHB13,Plant Mol.Biol.,45(3):247-262
Harris J.C.,Hrmova M.,Lopato S.,and Langridge P.,2011,Modulation of plant growth by HD-Zip classⅠandⅡtranscription factors in response to environmental stimuli,New Phytol.,190(4):823-837
Henriksson E.,Olsson A.S.B.,Johannesson H.,Johansson H.,Hanson J.,Engstrom P.,and Soderman E.,2005,Homeodomain leucine zipper classⅠgenes in Arabidopsis expression patterns and phylogenetic relationships,Plant Physiol.,139(1):509-518
Itoh J.,Hibara K.,Sato Y.,and Nagato Y.,2008,Developmental role and auxin responsiveness of classⅢHD-Zip gene family members in rice,Plant Physiol.,147(4):1960-1975
Izhaki A.,and Bowman J.L.,2007,KANADI and classⅢHD-Zip gene families regulate embryo patterning and modulate auxin flow during embryogenesis in Arabidopsis,Plant Cell.,19(2):495-508
Johannesson H.,Wang Y.,and Engstrom P.,2001,DNA-binding and dimerization preferences of Arabidopsis homeodomainleucine zipper transcription factors in vitro,Plant Mol.Biol.,45(1):63-73
Kim Y.S.,Kim S.G.,Lee M.,Lee I.,Park H.Y.,Seo P.J.,Jung J.H.,Kwon E.J.,Suh S.W.,Paek K.H.,and Park C.M.,2008,HD-ZIPⅢactivity is modulated by competitive inhibitors via a feedback loop in arabidopsis shoot apical meristem development,Plant Cell,20(4):920-933
Lee D.S.,Chen L.J.,Li C.Y.,Liu Y.,Tan X.L.,Lu B.R.,Li J.,Gan S.X.,Kang S.G.,Suh H.S.,and Zhu Y.,2013,The bsister MADS gene FST determines ovule patterning and development of the zygotic embryo and endosperm,PLoS One,8(3):e58748
Maimaiti Y.S.J.,2012,Expression of rice HD-ZIP transcription factor Oshox4,Oshox6 and IPT gene in the rice and drought resistance analyse of these transgenic plants,Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Qiu D.W.,pp.50-51(玉山江·麦麦提,2012,水稻HD-ZIP转录因子Oshox4,Oshox6和IPT基因在水稻中的表达及其抗旱功能研究,博士学位论文,中国农业科学院研究生院,导师:邱德文,pp.50-51)
Manavella P.A.,Dezar C.A.,Bonaventure G.,Baldwin I.T.,and Chan R.L.,2008,HAHB4,a sunflower HD-Zip protein,integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses,Plant J.,56(3):376-388
McConnell J.R.,Emery J.,Eshed Y.,Bao N.,Bowman J.,and Barton M.K.,2001,Role of PHABULOSA and PHAVOLU-TA in determining radial patterning in shoots,Nature,411(6838):709-713
Ohashiito K.,Kubo M.,Demura T.,and Fukuda H.,2005,ClassⅢhomeodomain leucine-zipper proteins regulate xylem cell differentiation,Plant Cell Physiol.,46(10):1646-1656
Sawa S.,Ohgishi M.,Goda H.,Higuchi K.,Shimada Y.,Yoshida S.,and Koshiba T.,2002,The HAT2 gene,a member of the HD-Zip gene family,isolated as an auxin inducible gene by DNA microarray screening,affects auxin response in Arabidopsis,Plant J.,32(6):1011-1022
Scarpella E.,Boot K.J.,Rueb S.,and Meijer A.H.,2002,The procambium specifcation gene OsHOXl promotes polar auxin transport capacity and reduces its sensitivity toward inhibition,Plant Physiol.,130(3):1349-1360
Stamm P.,and Kumar P.P.,2010,The phytohormone signal network regulating elongation growth during shade avoidance,J.Exp.Bot.,61(11):2889-2903
Wenkel S.,Emery J.,Hou B.H.,Evans M.M.,and Barton M.K.,2007,A feedback regulatory module formed by LITTLEZIPPER and HD-ZIPⅢgenes,Plant Cell,19(11):3379-3390
Yao P.F.,Zhao X.R.,Li M.F.,Wang A.H.,and Wu Q.,2016,Cloning and expression analysis of transcription factor gene FtbHLH3 from Fagopyrum tataricum under abiotic stress,Jiy inzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(2):429-435(姚攀锋,赵学荣,李茂菲,王安虎,吴琦,2016,苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,36(2):429-435)