胡杨DREB2A和WRKY19基因启动子逆境响应元件及其功能分析
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摘要
胡杨(Populus euphratica Oliv.)是唯一适应沙漠环境的高大乔木树种,抗逆性极强。其抗逆机制对植物抗性育种极为重要,国内外极为关注。本研究以生物信息学和转GUS基因烟草瞬时表达等手段解析胡杨PeDREB2A和PeWRKY19基因启动子序列顺式作用元件。结果表明,两个启动子均有干旱、NaCl、4℃、ABA响应顺式作用元件,CANNTG (N=A/T/C/G)、GRWAAW (R=G/A, W=A/T)、ACGT和CACATG等,增强GUS基因表达。PeDREB2A基因启动子序列1 886 bp,分别含干旱、盐、低温、ABA响应顺式作用元件等9个、11个、9个和5个;-888～-1 271 bp为盐负调控区,-1 271～-1 738 bp为干旱正调控区和ABA负调控区,-1 738～-1 878 bp为盐正调控区和干旱负调控区。PeWRKY19基因启动子序列为1 734 bp,分别含干旱、盐、低温和ABA响应顺式作用元件16个、5个、11个和4个;-176～-417 bp为盐负调控和ABA负调控区,-417～-696 bp为干旱负调控区和低温正调控区,-696～-849 bp为盐正调控区。本研究为阐释胡杨抗逆分子机制提供基础。
Populus euphratica is the onl y arbor species adapted to the desert land, which has extremely strong stress resistance. Its resistance mechanism is quite importance for resistance breeding of plant, and it has attracted much attention at home and abroad. In this study, cis-acting elements of promoter sequences of PeDREB2 A and PeWRKY19 genes in Populus euphratica were analyzed by bioinformatics and transient expression of GUS transgenic tobacco. The results showed that these two promoters both had cis-acting elements CANNTG(N=A/T/C/G),GRWAAW(R=G/A, W=A/T), ACGT and CACATG in responding to drought, NaCl, 4℃, and ABA, respectively.They enhanced the expression of GUS gene. PeDREB2 A promoter sequence was 1 886 bp, containing 9, 11, 9 and5 cis-acting elements in responding to drought, salt, low temperature and ABA, respectively. The region rangeed from-888 to-1 271 bp was negative regulation in responding to salt,-1 271 to-1 738 bp was positive regulation to drought and negative to ABA, and-1 738～-1 878 bp was positive regulation to salt and negative to drought.The promoter sequence of PeWRKY19 was 1 734 bp in full length, containing 16, 5, 11 and 4 cis-acting elements in responding to drought, salt, low temperature and ABA, respectively. Its regions ranged from-176 to-417 bp was negative regulation to salt and ABA,-417 to-696 bp was negative to drought but positive to low temperature,and-696 to-849 bp was positive to salt, respectively. The study could provide the foundation for explaining the molecular mechanism of stress resistance in Populus euphratica.
Populus euphratica is the onl y arbor species adapted to the desert land, which has extremely strong stress resistance. Its resistance mechanism is quite importance for resistance breeding of plant, and it has attracted much attention at home and abroad. In this study, cis-acting elements of promoter sequences of PeDREB2 A and PeWRKY19 genes in Populus euphratica were analyzed by bioinformatics and transient expression of GUS transgenic tobacco. The results showed that these two promoters both had cis-acting elements CANNTG(N=A/T/C/G),GRWAAW(R=G/A, W=A/T), ACGT and CACATG in responding to drought, NaCl, 4℃, and ABA, respectively.They enhanced the expression of GUS gene. PeDREB2 A promoter sequence was 1 886 bp, containing 9, 11, 9 and5 cis-acting elements in responding to drought, salt, low temperature and ABA, respectively. The region rangeed from-888 to-1 271 bp was negative regulation in responding to salt,-1 271 to-1 738 bp was positive regulation to drought and negative to ABA, and-1 738～-1 878 bp was positive regulation to salt and negative to drought.The promoter sequence of PeWRKY19 was 1 734 bp in full length, containing 16, 5, 11 and 4 cis-acting elements in responding to drought, salt, low temperature and ABA, respectively. Its regions ranged from-176 to-417 bp was negative regulation to salt and ABA,-417 to-696 bp was negative to drought but positive to low temperature,and-696 to-849 bp was positive to salt, respectively. The study could provide the foundation for explaining the molecular mechanism of stress resistance in Populus euphratica.
引文
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BroschéM.,Vinocur B.,Alatalo E.R.,Lamminm覿ki A.,Teichmann T.,Ottow E.A.,Djilianov D.,Afif D.,Bogeat-Triboulot M.B.,Altman A.,Polle A.,Dreyer E.,Rudd S.,Paulin L.,Auvinen P.,and Kangasj覿rvi J.,2005,Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert,Genome Biol.,6(12):101
Buchel A.S.,Brederode F.T.,Bol J.F.,and Linthorst H.J.,1999,Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo,Plant Mol.Biol.,40(3):387-396
Chen H.,Lai Z.B.,Shi J.W.,Xiao Y.,Chen Z.X.,and Xu X.P.,2010,Roles of Arabidopsis WRKY18,WRKY40 and WRK-Y60 transcription factors in plant responses to abscisic acid and abiotic stress,BMC Plant Biol.,10:281-296
Chen Y.,Chen S.,Chen F.,Li P.,Chen L.,Guan Z.,and Chang Q.,2012,Functional characterization of a Chrysanthemum dichrum stress-related promoter,Mol.Biotechnol.,52(2):161-169
Cui M.,Zhang W.,Zhang Q.,Xu Z.,Zhu Z.,Duan F.,and Wu R.,2011,Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice,Plant Physiol.Biochem.,49(12):1384-1391
Higo K.,Ugawa Y.,Iwamoto M.,and Korenaga T.,1999,Plant cis-acting regulatory DNA elements(PLACE)database:1999,Nucleic Acids Res.,27(1):297-300
Huda K.M.,Banu M.S.,Pathi K.M.,and Tuteja N.,2013,Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants,PLo S One,8(3):e57803
Jefferson R.A.,Burgess S.M.,and Hirsh D.,1986,β-glucuronidase from Escherichia coli as a gene-fusion marker,Proc.Natl.Acad.Sci.USA,83(22):8447-8451
Li T.,Sun J.K.,and Liu J.T.,2015,Research advances on plant promoter,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(2):18-25(李田,孙景宽,刘景涛,2015,植物启动子研究进展,生物技术通报,31(2):18-25)
Liu J.L.,Wang F.T.,Yu G.,Zhang X.H.,Jia C.G.,Qin J.C.,and Pan H.Y.,2015,Functional analysis of the maize C-repeat/DRE motif-binding transcription factor CBF3 promoter in response to abiotic stress,Int.J.Mol.Sci.,16(6):12131-12146
Srivastava V.K.,Raikwar S.,and Tuteja N.,2014,Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay,Plant Signal.Behav.,9:e29626
Sun X.,Dong J.H.,Chen M.,Xu Z.S.,Ye X.G.,Li L.C.,Qu Y.Y.,and Ma Y.Z.,2008,Isolation and regulative region analysis of promoter of stress-related gene GmDREB3 from soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(8):1475-1479(孙啸,董建辉,陈明,徐兆师,叶兴国,李连城,曲延英,马有志,2008,大豆抗逆基因GmDREB3启动子的克隆及调控区段分析,作物学报,34(8):1475-1479)
Wang S.J.,1996,The status conservation and recovery of global resources of Populus euphradica,Shijie Linye Yanjiu(World Forestry Research),9(6):37-44(王世绩,1996,全球胡杨林的现状及保护和恢复对策,世界林业研究,9(6):37-44)
Xie Z.,Zhang Z.L.,Zou X.,Huang J.,Ruas P.,Thompson D.,and Shen Q.J.,2005,Annotations and functional analyses of the rice WRKY gene super family reveal positive and negative regulators of abscisic acid signaling in aleurone cells,Plant Physiol.,137(1):176-189
Yang Y.Y.,Ren Y.P.,Sun Y.M.,Chen Q.J.,Zhang B.,and Zhang H.,2012,Cloning and analysis of two promoters of stress-related genes in Medicago varia Xinmu-1,Caoye Kexue(Pratacultural Science),29(12):1887-1893(杨云尧,任燕萍,苏豫梅,陈全家,张博,张桦,2012,新牧1号苜蓿两个抗逆相关基因启动子的克隆及分析,草业科学,29(12):1887-1893)
Yazaki J.S.,Shimatani Z.,Hashimoto A.,Nagata Y.,Fujii F.,Kojima K.,Suzuki K.,Taya T.,Tonouchi M.,Nelson C.,Nakagawa A.,Otomo Y.,Murakami K.,Matsubara K.,Kawai J.,Carninci P.,Hayashizaki Y.,and Kikuchi S.,2004,Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice:phenoltyping and comparative analysis between rice and Arabidopsis,Physiol.Genomics,17(2):87-100
BroschéM.,Vinocur B.,Alatalo E.R.,Lamminm覿ki A.,Teichmann T.,Ottow E.A.,Djilianov D.,Afif D.,Bogeat-Triboulot M.B.,Altman A.,Polle A.,Dreyer E.,Rudd S.,Paulin L.,Auvinen P.,and Kangasj覿rvi J.,2005,Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert,Genome Biol.,6(12):101
Buchel A.S.,Brederode F.T.,Bol J.F.,and Linthorst H.J.,1999,Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo,Plant Mol.Biol.,40(3):387-396
Chen H.,Lai Z.B.,Shi J.W.,Xiao Y.,Chen Z.X.,and Xu X.P.,2010,Roles of Arabidopsis WRKY18,WRKY40 and WRK-Y60 transcription factors in plant responses to abscisic acid and abiotic stress,BMC Plant Biol.,10:281-296
Chen Y.,Chen S.,Chen F.,Li P.,Chen L.,Guan Z.,and Chang Q.,2012,Functional characterization of a Chrysanthemum dichrum stress-related promoter,Mol.Biotechnol.,52(2):161-169
Cui M.,Zhang W.,Zhang Q.,Xu Z.,Zhu Z.,Duan F.,and Wu R.,2011,Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice,Plant Physiol.Biochem.,49(12):1384-1391
Higo K.,Ugawa Y.,Iwamoto M.,and Korenaga T.,1999,Plant cis-acting regulatory DNA elements(PLACE)database:1999,Nucleic Acids Res.,27(1):297-300
Huda K.M.,Banu M.S.,Pathi K.M.,and Tuteja N.,2013,Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants,PLo S One,8(3):e57803
Jefferson R.A.,Burgess S.M.,and Hirsh D.,1986,β-glucuronidase from Escherichia coli as a gene-fusion marker,Proc.Natl.Acad.Sci.USA,83(22):8447-8451
Li T.,Sun J.K.,and Liu J.T.,2015,Research advances on plant promoter,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(2):18-25(李田,孙景宽,刘景涛,2015,植物启动子研究进展,生物技术通报,31(2):18-25)
Liu J.L.,Wang F.T.,Yu G.,Zhang X.H.,Jia C.G.,Qin J.C.,and Pan H.Y.,2015,Functional analysis of the maize C-repeat/DRE motif-binding transcription factor CBF3 promoter in response to abiotic stress,Int.J.Mol.Sci.,16(6):12131-12146
Srivastava V.K.,Raikwar S.,and Tuteja N.,2014,Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay,Plant Signal.Behav.,9:e29626
Sun X.,Dong J.H.,Chen M.,Xu Z.S.,Ye X.G.,Li L.C.,Qu Y.Y.,and Ma Y.Z.,2008,Isolation and regulative region analysis of promoter of stress-related gene GmDREB3 from soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(8):1475-1479(孙啸,董建辉,陈明,徐兆师,叶兴国,李连城,曲延英,马有志,2008,大豆抗逆基因GmDREB3启动子的克隆及调控区段分析,作物学报,34(8):1475-1479)
Wang S.J.,1996,The status conservation and recovery of global resources of Populus euphradica,Shijie Linye Yanjiu(World Forestry Research),9(6):37-44(王世绩,1996,全球胡杨林的现状及保护和恢复对策,世界林业研究,9(6):37-44)
Xie Z.,Zhang Z.L.,Zou X.,Huang J.,Ruas P.,Thompson D.,and Shen Q.J.,2005,Annotations and functional analyses of the rice WRKY gene super family reveal positive and negative regulators of abscisic acid signaling in aleurone cells,Plant Physiol.,137(1):176-189
Yang Y.Y.,Ren Y.P.,Sun Y.M.,Chen Q.J.,Zhang B.,and Zhang H.,2012,Cloning and analysis of two promoters of stress-related genes in Medicago varia Xinmu-1,Caoye Kexue(Pratacultural Science),29(12):1887-1893(杨云尧,任燕萍,苏豫梅,陈全家,张博,张桦,2012,新牧1号苜蓿两个抗逆相关基因启动子的克隆及分析,草业科学,29(12):1887-1893)
Yazaki J.S.,Shimatani Z.,Hashimoto A.,Nagata Y.,Fujii F.,Kojima K.,Suzuki K.,Taya T.,Tonouchi M.,Nelson C.,Nakagawa A.,Otomo Y.,Murakami K.,Matsubara K.,Kawai J.,Carninci P.,Hayashizaki Y.,and Kikuchi S.,2004,Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice:phenoltyping and comparative analysis between rice and Arabidopsis,Physiol.Genomics,17(2):87-100