野生番茄SpIDD1基因的表达分析及其VIGS载体构建
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摘要
为鉴定野生番茄的SpIDD1基因并探究其功能,以野生醋栗番茄‘PI365967’为试验材料,利用RT-PCR克隆得到SpIDD1基因的CDS序列,全长1 020 bp,编码339个氨基酸,与普通番茄参考基因组序列相比,SpIDD1只存在一个核苷酸位点的差异。蛋白序列比对和结构分析表明,Sp IDD1含有1个核定位信号和4个保守锌指结构域(C2H2·C2H2·C2HC·C2HC),是一个典型的IDD蛋白。系统进化分析表明,SpIDD1与马铃薯和辣椒的IDD蛋白亲缘关系最近。荧光定量PCR显示,SpIDD1在叶、顶芽和果实中的表达较高,并受干旱胁迫的显著诱导。利用TRV-VIGS系统构建了Sp IDD1的基因沉默载体,并成功侵染番茄。研究结果表明,SpIDD1作为一个典型的IDD基因,很可能参与了番茄的干旱胁迫。本研究为进一步研究该基因的功能及作用机理提供理论依据。
To identify the SpIDD1 gene and explore its function in wild tomato, this study took Solanum pimpinellifolium 'PI365967' as a test materials, cloned the CDS sequence of SpIDD1 gene with 1 020 bp full length and encoded for 339 amino acids. There was only one nucleotide divergence compared with the reference genome sequence of the cultivated tomato Solanum lycopersicum. Sequence alignment and structure analysis demonstrated that SpIDD1 contained a nuclear localization signal(NLS) and four conserved zinc finger domain(C2 H2·C2 H2·C2 HC·C2 HC). It was a typical IDD protein. Phylogeny analysis showed that SpIDD1 might have the closest relationship with the IDD of potato and pepper. Quantitative real-time PCR analysis illustrated that SpIDD1 expressed highest in leaf, terminal bud and fruit. It was significantly induced by drought stress. Using the Tobacco rattle virus(TRV)-based VIGS system, we constructed the pTRV2-Sp IDD1 vector and infected the cotyledon of Solanum pimpinellifolium 'PI365967' successfully. The results suggested that SpIDD1, as a typical IDD gene, might participate in the drought stress response in tomato. It might provid the foundation for further study the function of SpIDD1.
To identify the SpIDD1 gene and explore its function in wild tomato, this study took Solanum pimpinellifolium 'PI365967' as a test materials, cloned the CDS sequence of SpIDD1 gene with 1 020 bp full length and encoded for 339 amino acids. There was only one nucleotide divergence compared with the reference genome sequence of the cultivated tomato Solanum lycopersicum. Sequence alignment and structure analysis demonstrated that SpIDD1 contained a nuclear localization signal(NLS) and four conserved zinc finger domain(C2 H2·C2 H2·C2 HC·C2 HC). It was a typical IDD protein. Phylogeny analysis showed that SpIDD1 might have the closest relationship with the IDD of potato and pepper. Quantitative real-time PCR analysis illustrated that SpIDD1 expressed highest in leaf, terminal bud and fruit. It was significantly induced by drought stress. Using the Tobacco rattle virus(TRV)-based VIGS system, we constructed the pTRV2-Sp IDD1 vector and infected the cotyledon of Solanum pimpinellifolium 'PI365967' successfully. The results suggested that SpIDD1, as a typical IDD gene, might participate in the drought stress response in tomato. It might provid the foundation for further study the function of SpIDD1.
引文
Bao H.,Chen X.Y.,LüS.L.,Jiang P.,Feng J.J.,Fan P.X.,Nie L.L.,and Li Y.X.,2015,Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato byγ-aminobutyric acid metabolic pathway,Plant Cell Environ.,38(3):600-613
Colasanti J.,Yuan Z.,and Sundaresan V.,1998,The indeterminate gene encodes a zinc finger protein and regulates a leaf-generated signal required for the transition to flowering in maize,Cell,93(4):593-603
Cui D.Y.,Zhao J.B.,Jing Y.J.,Fan M.Z.,Liu J.,Wang Z.C.,Xin W.,and Hu Y.,2013,The Arabidopsis IDD14,IDD15,and IDD16 cooperatively regulate lateral organ morphogenesis and gravitropism by promoting auxin biosynthesis and transport,PLo S Genet.,9(9):e1003759
Fan S.,Zhang D.,Xing L.B.,Qi S.Y.,Du L.S.,Wu H.Q.,Shao H.X.,Li Y.M.,Ma J.J.,and Han M.Y.,2017,Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus,Mol.Genet.Genomics,292(4):755-771
Feurtado J.A.,Huang D.,Wicki-Stordeur L.,Hemstock L.E.,Potentier M.S.,Tsang E.W.,and Cutler A.J.,2011,The Arabidopsis C2H2zinc finger INDETERMINATE DOMAIN1/ENHYDROUS promotes the transition to germination by regulating light and hormonal signaling during seed maturation,Plant Cell,23(5):1772-1794
Ingkasuwan P.,Netrphan S.,Prasitwattanaseree S.,Tanticharoen M.,Bhumiratana S.,Meechai A.,Chaijaruwanich J.,Takahashi H.,and Cheevadhanarak S.,2012,Inferring transcriptional gene regulation network of starch metabolism in Arabidopsis thaliana leaves using graphical gaussian model,BMC Syst.Biol.,6:100
Kandoth P.K.,Ranf S.,Pancholi S.S.,Jayanty S.,Walla M.D.,Miller W.,Howe G.A.,Lincoln D.E.,and Stratmann J.W.,2007,Tomato MAPKs LeMPK1,LeMPK2,and LeMPK3 function in the systemin-mediated defense response against herbivorous insects,Proc.Natl.Acad.Sci.USA,104(29):12205-12210
Kay R.,Chan A.,Daly M.,and McPherson J.,1987,Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes,Science,236(4806):1299-1302
Levesque M.P.,Vernoux T.,Busch W.,Cui H.,Wang J.Y.,Blilou I.,Hassan H.,Nakajima K.,Matsumoto N.,Lohmann J.U.,Scheres B.,and Benfey P.N.,2006,Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis,PLoS Biol.,4(5):e143
Morita M.T.,Sakaguchi K.,Kiyose S.,Taira K.,Kato T.,Nakamura M.,and Tasaka M.,2006,A C2H2-type zinc finger protein,SGR5,is involved in early events of gravitropism in Arabidopsis inflorescence stems,Plant J.,47(4):619-628
Murashige T.,and Skoog F.,1962,A revised medium for rapid growth and bioassays with tobacco tissue cultures,Physiol.Plant,15(3):473-497
Seo P.J.,Kim M.J.,Ryu J.Y.,Jeong E.Y.,and Park C.M.,2011a,Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism,Nat.Commun.,2:303
Seo P.J.,Ryu J.,Kang S.K.,and Park C.M.,2011b,Modulation of sugar metabolism by an INDETERMINATE DOMAINtranscription factor contributes to photoperiodic flowering in Arabidopsis,Plant J.,65(3):418-429
Tanimoto M.,Tremblay R.,and Colasanti J.,2008,Altered gravitropic response,amyloplast sedimentation and circumnuta tion in the Arabidopsis shoot gravitropism 5 mutant are associated with reduced starch levels,Plant Mol.Biol.,67(1-2):57-69
Welch D.,Hassan H.,Blilou I.,Immink R.,Heidstra R.,and Scheres B.,2007,Arabidopsis JACKDAW and MAGPIEzinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action,Genes Dev.,21(17):2196-2204
Yamauchi Y.,Fukaki H.,Fujisawa H.,and Tasaka M.,1997,Mutations in the SGR4,SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism,Plant Cell Physiol.,38(5):530-535
Colasanti J.,Yuan Z.,and Sundaresan V.,1998,The indeterminate gene encodes a zinc finger protein and regulates a leaf-generated signal required for the transition to flowering in maize,Cell,93(4):593-603
Cui D.Y.,Zhao J.B.,Jing Y.J.,Fan M.Z.,Liu J.,Wang Z.C.,Xin W.,and Hu Y.,2013,The Arabidopsis IDD14,IDD15,and IDD16 cooperatively regulate lateral organ morphogenesis and gravitropism by promoting auxin biosynthesis and transport,PLo S Genet.,9(9):e1003759
Fan S.,Zhang D.,Xing L.B.,Qi S.Y.,Du L.S.,Wu H.Q.,Shao H.X.,Li Y.M.,Ma J.J.,and Han M.Y.,2017,Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus,Mol.Genet.Genomics,292(4):755-771
Feurtado J.A.,Huang D.,Wicki-Stordeur L.,Hemstock L.E.,Potentier M.S.,Tsang E.W.,and Cutler A.J.,2011,The Arabidopsis C2H2zinc finger INDETERMINATE DOMAIN1/ENHYDROUS promotes the transition to germination by regulating light and hormonal signaling during seed maturation,Plant Cell,23(5):1772-1794
Ingkasuwan P.,Netrphan S.,Prasitwattanaseree S.,Tanticharoen M.,Bhumiratana S.,Meechai A.,Chaijaruwanich J.,Takahashi H.,and Cheevadhanarak S.,2012,Inferring transcriptional gene regulation network of starch metabolism in Arabidopsis thaliana leaves using graphical gaussian model,BMC Syst.Biol.,6:100
Kandoth P.K.,Ranf S.,Pancholi S.S.,Jayanty S.,Walla M.D.,Miller W.,Howe G.A.,Lincoln D.E.,and Stratmann J.W.,2007,Tomato MAPKs LeMPK1,LeMPK2,and LeMPK3 function in the systemin-mediated defense response against herbivorous insects,Proc.Natl.Acad.Sci.USA,104(29):12205-12210
Kay R.,Chan A.,Daly M.,and McPherson J.,1987,Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes,Science,236(4806):1299-1302
Levesque M.P.,Vernoux T.,Busch W.,Cui H.,Wang J.Y.,Blilou I.,Hassan H.,Nakajima K.,Matsumoto N.,Lohmann J.U.,Scheres B.,and Benfey P.N.,2006,Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis,PLoS Biol.,4(5):e143
Morita M.T.,Sakaguchi K.,Kiyose S.,Taira K.,Kato T.,Nakamura M.,and Tasaka M.,2006,A C2H2-type zinc finger protein,SGR5,is involved in early events of gravitropism in Arabidopsis inflorescence stems,Plant J.,47(4):619-628
Murashige T.,and Skoog F.,1962,A revised medium for rapid growth and bioassays with tobacco tissue cultures,Physiol.Plant,15(3):473-497
Seo P.J.,Kim M.J.,Ryu J.Y.,Jeong E.Y.,and Park C.M.,2011a,Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism,Nat.Commun.,2:303
Seo P.J.,Ryu J.,Kang S.K.,and Park C.M.,2011b,Modulation of sugar metabolism by an INDETERMINATE DOMAINtranscription factor contributes to photoperiodic flowering in Arabidopsis,Plant J.,65(3):418-429
Tanimoto M.,Tremblay R.,and Colasanti J.,2008,Altered gravitropic response,amyloplast sedimentation and circumnuta tion in the Arabidopsis shoot gravitropism 5 mutant are associated with reduced starch levels,Plant Mol.Biol.,67(1-2):57-69
Welch D.,Hassan H.,Blilou I.,Immink R.,Heidstra R.,and Scheres B.,2007,Arabidopsis JACKDAW and MAGPIEzinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action,Genes Dev.,21(17):2196-2204
Yamauchi Y.,Fukaki H.,Fujisawa H.,and Tasaka M.,1997,Mutations in the SGR4,SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism,Plant Cell Physiol.,38(5):530-535