水稻ARF基因家族新成员ADP-Ribosylation Factor-Like Protein 5(OsARFL5)的克隆及表达分析
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摘要
小G蛋白家族中的亚家族ADP-核糖基化因子(ADP-ribosylation factor, ARF),在生物体内起众多的生理作用,参与基因表达、细胞骨架重组装、微管的形成以及囊泡和核孔运输等。本研究已成功克隆得到ARF基因家族中的一个新基因OsARFL5,系统进化分析表明该基因与SiARFC1亲缘关系较近;以该基因和其启动子序列构建了两种遗传转化表达载体(pOsARFL5::GUS, pCaMV35S::OsARFL5:EGFP),并获得了相应遗传载体的转基因植株;GUS染色结果表明,该基因在不同时期、不同组织部位表达;洋葱表皮亚细胞定位结果显示,Os ARFL5基因编码的蛋白定位在细胞核。RT-PCR结果显示,OsARFL5在水稻不同生育期的根、茎、叶、叶鞘和穗子中均有不同程度的表达。本研究初步探明了ARF基因家族新成员OsARFL5在水稻生长发育过程中的表达模式。
ADP-ribosylation factor(ARF) is a sub-family of G protein family and plays important physiological functions in organisms, including gene expression, cytoskeleton recombination, microtubule formation, vesicle and nuclear pore transport. In this study, a novel gene OsARFL5 belonging to ARF gene family in rice was cloned, and phylogenetic analysis indicated that the gene was closely related to SiARFC1. Two transgenic expression vectors(pOsARFL5::GUS, p CaMV35 S::OsARFL5:EGFP) were constructed by using the gene and its promoter sequence,and transgenic plants with corresponding genetic vectors were obtained. The GUS staining results showed that the gene was expressed at different developmental stage and in different tissues. Subcellular localization in onion epidermal cells indicated that the protein encoded by OsARFL5 gene was located in the nucleus. RT-PCR results showed that OsARFL5 was differently expressed in root, stem, leaf, leaf sheath and head of rice at different growth stages. This study preliminarily demonstrated the expression pattern of OsARFL5, a new member of the ARF gene family, during the growth and development of rice.
ADP-ribosylation factor(ARF) is a sub-family of G protein family and plays important physiological functions in organisms, including gene expression, cytoskeleton recombination, microtubule formation, vesicle and nuclear pore transport. In this study, a novel gene OsARFL5 belonging to ARF gene family in rice was cloned, and phylogenetic analysis indicated that the gene was closely related to SiARFC1. Two transgenic expression vectors(pOsARFL5::GUS, p CaMV35 S::OsARFL5:EGFP) were constructed by using the gene and its promoter sequence,and transgenic plants with corresponding genetic vectors were obtained. The GUS staining results showed that the gene was expressed at different developmental stage and in different tissues. Subcellular localization in onion epidermal cells indicated that the protein encoded by OsARFL5 gene was located in the nucleus. RT-PCR results showed that OsARFL5 was differently expressed in root, stem, leaf, leaf sheath and head of rice at different growth stages. This study preliminarily demonstrated the expression pattern of OsARFL5, a new member of the ARF gene family, during the growth and development of rice.
引文
D'Souza-Schorey C.,and Chavrier P.,2006,ARF proteins:roles in membrane traffic and beyond,Nat.Rev.Mol.Cell Biol.,7(5):347-358
D'Souza-Schorey C.,Donselear E.V.,Hsu V.W.,Yang C.Z.,Stahl P.D.,and Peters P.J.,1998,AR F6 targets recycling vesicles to the plasma membrane:insights from an ultrastructural investigation,J.Cell Biol.,140(3):603-616
Huang Y.D.,2013,Functional analysis of Ras GTPase Ps ARF1in Phytophthora sojae,Thesis for M.S.,Nanjing Agricultura University,Supervisor:Wang Y.C.,pp.9-15(黄轶丹,2013大豆疫霉Ras GTPase蛋白Ps ARF1的功能研究,硕士学位论文,南京农业大学,导师:王源超,pp.9-15)
Jefferson R.A.,1987,Assaying chimeric genes in plants:the GUS gene fusion system,Plant Mol.Biol.Rep.,5(4):387-405
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
Lee W.Y.,Hong J.K.,Kim C.Y.,Chun H.J.,Park H.C.,Kim J.C.Yun D.J.,Chung W.S.,Lee S.H.,Lee S.Y.,Cho M.J.,and Lim C.O.,2003,Over-expressed rice ADP-ribosylation fac tor 1(RARF1)induces pathogenesis-related genes and pathogen resistance in tobacco plants,Physiol.Plantarum,119(4)573-581
Mei F.L.,Hu Y.H.,Zhang X.,Zhang P.C.,Shi N.N.,and Yu Z.M.2016,ADP-ribosylation factor1(ARF1)gene function in plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):740-744(梅凤玲,胡逸华,张弦,章鹏程,施农农,俞志明,2016,植物核糖基化因子ARF1的功能研究进展,基因组学与应用生物学,35(3)740-744)
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nucleic Acids Res.,8(19)4321-4325
Muthamilarasan M.,Mangu V.R.,Zandkarimi H.,Prasad M.,and Baisakh N.,2016,Structure,organization and evolution of ADP-ribosylation factors in rice and foxtail millet,and their expression in rice,Sci.Rep.,6(1):24008
Pu Z.E.,Wang J.R.,Wei Y.M.,and Zheng Y.L.,2009,Sequence analysis of genes for ADP-ribosylation factors in wheat Sichuan Nongye Daxue Xuebao(Journal of Sichuan Agricultural University),27(1):20-25(蒲至恩,王际睿,魏育明郑有良,2009,小麦ADP-核糖基化因子克隆及序列分析四川农业大学学报,27(1):20-25)
Takai Y.,Sasaki T.,and Matozaki T.,2001,Small GTP-binding proteins,Physiological Reviews,81(1):153-208
Ueda T.,Yamaguchi M.,Uchimiya H.,and Nakano A.,2001,Ara6,a plant-unique novel type Rab GTPase,functions in the endocytic pathway of Arabidopsis thaliana,EMBO J.,20(17):4730-4741
Wang L.,Su H.Y.,Wang C.L.,and Mu C.H.,2007,Structure and functional mechanism of the ADP-ribosylation factor,Zhongguo Xibao Shengwuxue Xuebao(Chinses Journal of Cell Biology),29(5):675-681(王磊,宿红艳,王昌留,穆春华,2007,ADP核糖基化因子的结构及其功能机制,中国细胞生物学学报,29(5):675-681)
Wang X.,and Zhong K.,2005,Current progress on the small GT-Pase gene superfamily in plants,Zhiwu Xuebao(Chinese Bulletin of Botany),22(1):1-10(王昕,种康,2005,植物小G蛋白功能的研究进展,植物学报,22(1):1-10)
Wu J.,2007,The analysis of structure MuArf1,a member of small GTPase gene family,and its expression during postharvest ripening in banana fruit,Thesis for M.S.,South China University of Tropical Agriculture,Supervisors:Jin Z.Q.,and Xu B.Y.,pp.1-54(吴静,2007,香蕉小G蛋白基因MuArf1的组织结构分析及其在果实采后成熟中的表达研究,硕士学位论文,华南热带农业大学,导师:金志强,徐璧玉,pp.1-54)
Yuan J.C.,Song J.H.,Ma H.L.,Song X.Q.,Wei H.P.,and Liu Y.H.,2015,Ectopic expression a maize ADP-ribosylation factor gene in Arabidopsis,increase plant size and growth rate,J.Plant Biochem.Biotechnol.,24(2):161-166
Zhang Z.W.,2010,Research on the regulation of small G protein in potato defense reacting system,Dissertation for Ph.D.,Inner Monggolia Agricultural University,Supervisor:Fan M.S.,pp.1-4(张之为,2010,小G蛋白在马铃薯防卫反应体系建立过程中调控机理的研究,博士学位论文,内蒙古农业大学,导师:樊明寿,pp.1-4)
Zhou X.J.,Li J.,Cheng W.,Liu H.,Li M.M.,Zhang Y.,Li W.B.,Han S.C.,and Wang Y.D.,2010,Gene structure analysis of rice ADP-ribosylation factors(OsARFs)and their m RNAexpression in developing rice plants,Plant Mol.Biol.Rep.,28(4):692-703
Zhuang X.L.,2004,Functional study of an rice ArfGAP-Os-AGAP,Dissertation for Ph.D.,Institute of botany,Chinese Academy of Sciences,Supervisors:Xu Z.H.,and Zhong K.,pp.47-99(庄晓蕾,2004,水稻小G蛋白Arf GTPase激活蛋白OsAGAP功能研究,博士学位论文,中国科学院植物研究所,导师:许智宏,种康,pp.47-99)
D'Souza-Schorey C.,Donselear E.V.,Hsu V.W.,Yang C.Z.,Stahl P.D.,and Peters P.J.,1998,AR F6 targets recycling vesicles to the plasma membrane:insights from an ultrastructural investigation,J.Cell Biol.,140(3):603-616
Huang Y.D.,2013,Functional analysis of Ras GTPase Ps ARF1in Phytophthora sojae,Thesis for M.S.,Nanjing Agricultura University,Supervisor:Wang Y.C.,pp.9-15(黄轶丹,2013大豆疫霉Ras GTPase蛋白Ps ARF1的功能研究,硕士学位论文,南京农业大学,导师:王源超,pp.9-15)
Jefferson R.A.,1987,Assaying chimeric genes in plants:the GUS gene fusion system,Plant Mol.Biol.Rep.,5(4):387-405
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
Lee W.Y.,Hong J.K.,Kim C.Y.,Chun H.J.,Park H.C.,Kim J.C.Yun D.J.,Chung W.S.,Lee S.H.,Lee S.Y.,Cho M.J.,and Lim C.O.,2003,Over-expressed rice ADP-ribosylation fac tor 1(RARF1)induces pathogenesis-related genes and pathogen resistance in tobacco plants,Physiol.Plantarum,119(4)573-581
Mei F.L.,Hu Y.H.,Zhang X.,Zhang P.C.,Shi N.N.,and Yu Z.M.2016,ADP-ribosylation factor1(ARF1)gene function in plants,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):740-744(梅凤玲,胡逸华,张弦,章鹏程,施农农,俞志明,2016,植物核糖基化因子ARF1的功能研究进展,基因组学与应用生物学,35(3)740-744)
Murray M.G.,and Thompson W.F.,1980,Rapid isolation of high molecular weight plant DNA,Nucleic Acids Res.,8(19)4321-4325
Muthamilarasan M.,Mangu V.R.,Zandkarimi H.,Prasad M.,and Baisakh N.,2016,Structure,organization and evolution of ADP-ribosylation factors in rice and foxtail millet,and their expression in rice,Sci.Rep.,6(1):24008
Pu Z.E.,Wang J.R.,Wei Y.M.,and Zheng Y.L.,2009,Sequence analysis of genes for ADP-ribosylation factors in wheat Sichuan Nongye Daxue Xuebao(Journal of Sichuan Agricultural University),27(1):20-25(蒲至恩,王际睿,魏育明郑有良,2009,小麦ADP-核糖基化因子克隆及序列分析四川农业大学学报,27(1):20-25)
Takai Y.,Sasaki T.,and Matozaki T.,2001,Small GTP-binding proteins,Physiological Reviews,81(1):153-208
Ueda T.,Yamaguchi M.,Uchimiya H.,and Nakano A.,2001,Ara6,a plant-unique novel type Rab GTPase,functions in the endocytic pathway of Arabidopsis thaliana,EMBO J.,20(17):4730-4741
Wang L.,Su H.Y.,Wang C.L.,and Mu C.H.,2007,Structure and functional mechanism of the ADP-ribosylation factor,Zhongguo Xibao Shengwuxue Xuebao(Chinses Journal of Cell Biology),29(5):675-681(王磊,宿红艳,王昌留,穆春华,2007,ADP核糖基化因子的结构及其功能机制,中国细胞生物学学报,29(5):675-681)
Wang X.,and Zhong K.,2005,Current progress on the small GT-Pase gene superfamily in plants,Zhiwu Xuebao(Chinese Bulletin of Botany),22(1):1-10(王昕,种康,2005,植物小G蛋白功能的研究进展,植物学报,22(1):1-10)
Wu J.,2007,The analysis of structure MuArf1,a member of small GTPase gene family,and its expression during postharvest ripening in banana fruit,Thesis for M.S.,South China University of Tropical Agriculture,Supervisors:Jin Z.Q.,and Xu B.Y.,pp.1-54(吴静,2007,香蕉小G蛋白基因MuArf1的组织结构分析及其在果实采后成熟中的表达研究,硕士学位论文,华南热带农业大学,导师:金志强,徐璧玉,pp.1-54)
Yuan J.C.,Song J.H.,Ma H.L.,Song X.Q.,Wei H.P.,and Liu Y.H.,2015,Ectopic expression a maize ADP-ribosylation factor gene in Arabidopsis,increase plant size and growth rate,J.Plant Biochem.Biotechnol.,24(2):161-166
Zhang Z.W.,2010,Research on the regulation of small G protein in potato defense reacting system,Dissertation for Ph.D.,Inner Monggolia Agricultural University,Supervisor:Fan M.S.,pp.1-4(张之为,2010,小G蛋白在马铃薯防卫反应体系建立过程中调控机理的研究,博士学位论文,内蒙古农业大学,导师:樊明寿,pp.1-4)
Zhou X.J.,Li J.,Cheng W.,Liu H.,Li M.M.,Zhang Y.,Li W.B.,Han S.C.,and Wang Y.D.,2010,Gene structure analysis of rice ADP-ribosylation factors(OsARFs)and their m RNAexpression in developing rice plants,Plant Mol.Biol.Rep.,28(4):692-703
Zhuang X.L.,2004,Functional study of an rice ArfGAP-Os-AGAP,Dissertation for Ph.D.,Institute of botany,Chinese Academy of Sciences,Supervisors:Xu Z.H.,and Zhong K.,pp.47-99(庄晓蕾,2004,水稻小G蛋白Arf GTPase激活蛋白OsAGAP功能研究,博士学位论文,中国科学院植物研究所,导师:许智宏,种康,pp.47-99)