番茄2个ERF-B1亚族转录因子基因的克隆及其对生物和非生物胁迫响应
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摘要
为进一步研究番茄ERF转录因子调控植物抗逆的分子机理,本研究以番茄浙杂-301为试验材料,分别克隆获得2个乙烯反应元件结合蛋白基因SlERF83和SlERF109,并对其进行序列及表达分析。结果表明,番茄SlERF83和SlERF109基因分别含有678 bp和669 bp的开放阅读框,编码225和222个氨基酸,各含有1个保守的AP2结构域,属于亲水性蛋白。进化分析表明,这2个ERF转录因子均属于AP2/ERF家族中ERF亚族的B1组。SlERF83和SlERF109三级结构都含有1个α-螺旋和3个β-折叠。酵母单杂交及β-半乳糖苷酶活性测定结果证明SlERF83转录因子可以与GCC-box结合。番茄2个ERF蛋白启动子含有多种逆境相关的顺式作用元件。采用荧光定量PCR检测SlERF83和SlERF109在非生物和生物胁迫下的表达,结果表明,高盐和干旱胁迫下,SlERF83和SlERF109基因的表达均被抑制;水杨酸处理能够诱导SlERF83和SlERF109基因的表达;茉莉酸甲酯处理下SlERF83表达水平提高,SlERF109表达量降低;番茄黄化曲叶病毒侵染下,SlERF83和SlERF109基因的表达均被抑制。SlERF83和SlERF109转录因子可能参与了番茄对生物及非生物胁迫的响应过程。本研究结果为进一步深入开展ERF转录因子调控番茄抗逆分子机制研究提供了一定的理论依据。
In order to further understand the molecular mechanism of stress regulation of tomato ERF transcription factor, two ERF genes, SlERF83 and SlERF109, were cloned from tomato cultivar Zheza-301 in this study, their sequence and expression were analyzed. The results indicated that the ORF lengths of SlERF83 and SlERF109 tenes were 678 and 669 bp, which encoded 225 and 222 amino acids, respectively. SlERF83 and SlERF109 were hydrophilic proteins and contained a conserved AP2-domain. Phylogenetic analysis showed that SlERF83 and SlERF109 belonged to B1 group of ERF subfamily of AP2/ERF family. SlERF83 and SlERF109 proteins contained one α-helix and three β-sheets according to the protein three-dimensional structure analysis. Yeast one-hybrid and β-galactosidase activity assays demonstrated that SlERF83 transcription factor could bind to GCC-box. Some stress-related cis-elements existed in the promoter regions of SlERF83 and SlERF109 genes. The expression level of SlERF83 and SlERF109 were detected by RT-qPCR. The results showed that the expression level of SlERF83 and SlERF109 genes were inhibited under salt or drought stress. SA induced the expression level of the two ERF genes. MeJA increased the expression level of SlERF83 and decreased the expression level of SlERF109. The expression level of SlERF83 and SlERF109 were downregulated after tomato yellow leaf curl virus infection. These results indicated that SlERF83 and SlERF109 may involve in response to biotic and abiotic stresses in tomato. The results of this study provides a theoretical basis for revealing the molecular mechanism of ERF transcription factor regulating the stress of tomato.
In order to further understand the molecular mechanism of stress regulation of tomato ERF transcription factor, two ERF genes, SlERF83 and SlERF109, were cloned from tomato cultivar Zheza-301 in this study, their sequence and expression were analyzed. The results indicated that the ORF lengths of SlERF83 and SlERF109 tenes were 678 and 669 bp, which encoded 225 and 222 amino acids, respectively. SlERF83 and SlERF109 were hydrophilic proteins and contained a conserved AP2-domain. Phylogenetic analysis showed that SlERF83 and SlERF109 belonged to B1 group of ERF subfamily of AP2/ERF family. SlERF83 and SlERF109 proteins contained one α-helix and three β-sheets according to the protein three-dimensional structure analysis. Yeast one-hybrid and β-galactosidase activity assays demonstrated that SlERF83 transcription factor could bind to GCC-box. Some stress-related cis-elements existed in the promoter regions of SlERF83 and SlERF109 genes. The expression level of SlERF83 and SlERF109 were detected by RT-qPCR. The results showed that the expression level of SlERF83 and SlERF109 genes were inhibited under salt or drought stress. SA induced the expression level of the two ERF genes. MeJA increased the expression level of SlERF83 and decreased the expression level of SlERF109. The expression level of SlERF83 and SlERF109 were downregulated after tomato yellow leaf curl virus infection. These results indicated that SlERF83 and SlERF109 may involve in response to biotic and abiotic stresses in tomato. The results of this study provides a theoretical basis for revealing the molecular mechanism of ERF transcription factor regulating the stress of tomato.
引文
[1] Zhuang J,Zhang J,Hou X L,Wang F,Xiong A S.Transcriptomic,proteomic,metabolomic and functional genomic approaches for the study of abiotic stress in vegetable crops[J].Critical Reviews in Plant Science,2014,33(2/3):225-237
[2] Deinlein U,Stephan A B,Horie T,Luo W,Xu G,Schroeder J Ⅰ.Plant salt-tolerance mechanisms[J].Trends in Plant Science,2014,19(6):371-379
[3] 陈娜,迟晓元,程果,潘丽娟,陈明娜,王通,王冕,杨珍,禹山林.花生中低温胁迫相关转录因子基因的筛选[J].核农学报,2016,30(1):19-27
[4] 王雅慧,王永鑫,王文丽,吴致君,庄静.茶树CsGIF1基因的克隆及其对非生物胁迫的响应分析[J].西北植物学报,2017,37(12):2363-2369
[5] Licausi F,Ohme-Takagi M,Perata P.APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors:Mediators of stress responses and developmental programs[J].New Phytologist,2013,199(3):639-649
[6] Sakuma Y,Liu Q,Dubouzet J G,Abe H,Shinozaki K,Yamaguchi-Shinozaki K.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression[J].Biochemical & Biophysical Research Communications,2002,290(3):998-1009
[7] Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182
[8] Gu C,Guo Z H,Hao P P,Wang G M,Jin Z M,Zhang S L.Multiple regulatory roles of AP2/ERF transcription factor in angiosperm[J].Botanical Studies,2017,58(1):6
[9] Huang P Y,Catinot J,Zimmerli L.Ethylene response factors in Arabidopsis immunity[J].Journal of Experimental Botany,2016,67(5):1231-1241
[10] Wang F,Wang C,Liu P,Lei C,Hao W,Gao Y,Liu Y G,Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-Targeted mutagenesis of the ERF transcription factor gene OsERF922[J].PLoS One,2016,11(4):e0154027
[11] 庄静,彭日荷,高峰,付晓燕,朱波,金晓芬,Zhang J,熊爱生,姚泉洪.沪油15中两个AP2/ERF-B1亚族转录因子的克隆与分析[J].核农学报,2009,23(3):435-441
[12] Zhang G,Chen M,Chen X,Xu Z,Guan S,Li L C,Li A,Guo J,Mao L,Ma Y.Phylogeny,gene structures,and expression patterns of the ERF gene family in soybean (Glycine max L.)[J].Journal of Experimental Botany,2008,59(15):4095-4107
[13] 杨德鑫,周时荣,权瑞党,黄荣峰.AP2/ERF蛋白调控植物的非生物胁迫应答机制[J].中国农业科技导报,2012,14(6):23-29
[14] Zhang Z,Huang R.Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis[J].Plant Molecular Biology,2010,73(3):241-249
[15] Gao S,Zhang H,Tian Y,Li F,Zhang Z,Lu X,Chen X,Huang R.Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity[J].Plant Cell Reports,2008,27(11):1787-1795
[16] Wu L,Zhang Z,Zhang H,Wang X C,Huang R.Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt,drought and freezing[J].Plant Physiology,2008,148(4):1953-1963
[17] Pan Ⅰ C,Li C W,Su R C,Cheng C P,Lin C S,Chan M T.Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato[J].Planta,2010,17(1):103-112
[18] Huang Y,Zhang B L,Sun S,Xing G M,Wang F,Li M Y,Tian Y S,Xiong A S.AP2/ERF transcription factors involved in response to tomato yellow leaf curly virus in tomato[J].Plant Genome,2016,9(2):8201-8215
[19] 高世超,钟凤林,林义章,赵瑞丽,林俊芳,杨碧云,胡海非.青花菜BrERF2基因的RACE克隆与模拟酸雨胁迫下的表达分析[J].核农学报,2015,29(11):2093-2102
[20] Ken C M,Bruno D,John M M,Peer M S,Cameron I E,Donald J M,Wdf-Rücliger S,Michae L K U,Kemal K.Repressor-and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression[J].Plant Physiology,2005,139(2):949-959
[21] Fischer U,Dr?gelaser W.Overexpression of NtERF5,a new member of the tobacco ethylene response transcription factor family enhances resistance to Tobacco mosaic virus[J].Molecular Plant-Microbe Interactions,2004,17(10):1162-1171
[22] Chen T Z,Lv Y D,Zhao T M,Li N,Yang Y W,Yu W G,He X,Liu T L,Zhang B L.Comparative transcriptome profiling of a resistant vs.susceptible tomato (Solanum lycopersicum) cultivar in response to infection by tomato yellow leaf curl virus[J].PLoS One,2013,8(11):e80816
[23] Lescot M,Dehais P,Thijs G,Marchal K,Moreau Y,Van de Peer Y,Rouze P,Rombauts S.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327
[24] Stothard P.The sequence manipulation suite:JavaScript programs for analyzing and formatting protein and DNA sequences[J].Biotechniques,2000,28(6):1102-1104
[25] Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology Evolution,2011,28(10):2731-2739
[26] Arnold K,Bordoli L,Kopp J,Schwede T.The SWISS-MODEL workspace:A web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201
[27] Chen T Z,Lv Y D,Zhao T M,Li N,Yang Y W,Yu W G,He X,Liu T L,Zhang B L.Comparative transcriptome profiling of a resistant vs.susceptible tomato (Solanum lycopersicum) cultivar in response to infection by tomato yellow leaf curl virus[J].PLoS One,2013,8(11):e80816
[28] Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45
[29] Allen M D,Yamasaki K,Ohme-Takagi M,Tateno M,Suzuki M.A novel mode of DNA recognition by a β-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].EMBO Journal,1998,17(18):5484-5496
[30] Maruyama Y,Yamoto N,Suzuki Y,Chiba Y,Yamazaki K,Sato T,Yamaguchi J.The Arabidopsis transcriptional repressor ERF9 participates inresistance against necrotrophic fungi[J].Plant Science,2013,213(6):79-87
[31] Iwase A,Harashima H,Ikeuchi M,Rymen B,Ohnuma M,Komaki S,Morohashi K,Kurata T,Nakata M,Ohme-Takagi M,Grotewold E,Suqimoto K.WIND1 promotes shoot regeneration through transcriptional activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis[J].Plant Cell,2017,29(1):54-69
[32] Lee S J,Park J H,Lee M H,Yu J H,Kim S Y.Isolation and functional characterization of CE1 binding proteins[J].BMC Plant Biology,2010,10(1):1-13
[33] 莫纪波,李大勇,张慧娟,宋凤鸣.ERF转录因子在植物对生物和非生物胁迫反应中的作用[J].植物生理学报,2011,47(12):1145-1154
[34] Zhu Q,Zhang J,Gao X,Tong J,Xiao L,Li W,Zhang H.The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA,salt and osmotic stress responses[J].Gene,2010,457(1/2):1-12
[35] Zhang G,Chen M,Li L,Xu Z,Chen X,Guo J,Ma Y.Overexpression of the soybean GmERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796
[36] Lorenzo O,Piqueras R,Sanchez-Serrano J J,Solano R.ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[J].Plant Cell,2003,15(1):165-178
[37] Pre M,Atallah M,Champion A,De Ⅴ M,Pieterse C M,Memelink J.The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[J].Plant Physiology,2008,147(3):1347-1357
[38] Huang Y,Li T,Xu Z S,Wang F,Xiong A S.Six NAC transcription factors involved in response to TYLCV infection in resistant and susceptible tomato cultivars[J].Plant Physiology & Biochemistry,2017,120:61-74
[39] Berrocal-Lobo M,Molina A,Solano R.Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi[J].Plant Journal,2002,29(1):23-32
[40] Guo Z J,Chen X J,Wu X L,Ling J Q,Xu P.Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco[J].Plant Molecular Biology,2004,55(4):607-618
[2] Deinlein U,Stephan A B,Horie T,Luo W,Xu G,Schroeder J Ⅰ.Plant salt-tolerance mechanisms[J].Trends in Plant Science,2014,19(6):371-379
[3] 陈娜,迟晓元,程果,潘丽娟,陈明娜,王通,王冕,杨珍,禹山林.花生中低温胁迫相关转录因子基因的筛选[J].核农学报,2016,30(1):19-27
[4] 王雅慧,王永鑫,王文丽,吴致君,庄静.茶树CsGIF1基因的克隆及其对非生物胁迫的响应分析[J].西北植物学报,2017,37(12):2363-2369
[5] Licausi F,Ohme-Takagi M,Perata P.APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors:Mediators of stress responses and developmental programs[J].New Phytologist,2013,199(3):639-649
[6] Sakuma Y,Liu Q,Dubouzet J G,Abe H,Shinozaki K,Yamaguchi-Shinozaki K.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration-and cold-inducible gene expression[J].Biochemical & Biophysical Research Communications,2002,290(3):998-1009
[7] Ohme-Takagi M,Shinshi H.Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[J].Plant Cell,1995,7(2):173-182
[8] Gu C,Guo Z H,Hao P P,Wang G M,Jin Z M,Zhang S L.Multiple regulatory roles of AP2/ERF transcription factor in angiosperm[J].Botanical Studies,2017,58(1):6
[9] Huang P Y,Catinot J,Zimmerli L.Ethylene response factors in Arabidopsis immunity[J].Journal of Experimental Botany,2016,67(5):1231-1241
[10] Wang F,Wang C,Liu P,Lei C,Hao W,Gao Y,Liu Y G,Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-Targeted mutagenesis of the ERF transcription factor gene OsERF922[J].PLoS One,2016,11(4):e0154027
[11] 庄静,彭日荷,高峰,付晓燕,朱波,金晓芬,Zhang J,熊爱生,姚泉洪.沪油15中两个AP2/ERF-B1亚族转录因子的克隆与分析[J].核农学报,2009,23(3):435-441
[12] Zhang G,Chen M,Chen X,Xu Z,Guan S,Li L C,Li A,Guo J,Mao L,Ma Y.Phylogeny,gene structures,and expression patterns of the ERF gene family in soybean (Glycine max L.)[J].Journal of Experimental Botany,2008,59(15):4095-4107
[13] 杨德鑫,周时荣,权瑞党,黄荣峰.AP2/ERF蛋白调控植物的非生物胁迫应答机制[J].中国农业科技导报,2012,14(6):23-29
[14] Zhang Z,Huang R.Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis[J].Plant Molecular Biology,2010,73(3):241-249
[15] Gao S,Zhang H,Tian Y,Li F,Zhang Z,Lu X,Chen X,Huang R.Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity[J].Plant Cell Reports,2008,27(11):1787-1795
[16] Wu L,Zhang Z,Zhang H,Wang X C,Huang R.Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt,drought and freezing[J].Plant Physiology,2008,148(4):1953-1963
[17] Pan Ⅰ C,Li C W,Su R C,Cheng C P,Lin C S,Chan M T.Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato[J].Planta,2010,17(1):103-112
[18] Huang Y,Zhang B L,Sun S,Xing G M,Wang F,Li M Y,Tian Y S,Xiong A S.AP2/ERF transcription factors involved in response to tomato yellow leaf curly virus in tomato[J].Plant Genome,2016,9(2):8201-8215
[19] 高世超,钟凤林,林义章,赵瑞丽,林俊芳,杨碧云,胡海非.青花菜BrERF2基因的RACE克隆与模拟酸雨胁迫下的表达分析[J].核农学报,2015,29(11):2093-2102
[20] Ken C M,Bruno D,John M M,Peer M S,Cameron I E,Donald J M,Wdf-Rücliger S,Michae L K U,Kemal K.Repressor-and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression[J].Plant Physiology,2005,139(2):949-959
[21] Fischer U,Dr?gelaser W.Overexpression of NtERF5,a new member of the tobacco ethylene response transcription factor family enhances resistance to Tobacco mosaic virus[J].Molecular Plant-Microbe Interactions,2004,17(10):1162-1171
[22] Chen T Z,Lv Y D,Zhao T M,Li N,Yang Y W,Yu W G,He X,Liu T L,Zhang B L.Comparative transcriptome profiling of a resistant vs.susceptible tomato (Solanum lycopersicum) cultivar in response to infection by tomato yellow leaf curl virus[J].PLoS One,2013,8(11):e80816
[23] Lescot M,Dehais P,Thijs G,Marchal K,Moreau Y,Van de Peer Y,Rouze P,Rombauts S.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327
[24] Stothard P.The sequence manipulation suite:JavaScript programs for analyzing and formatting protein and DNA sequences[J].Biotechniques,2000,28(6):1102-1104
[25] Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology Evolution,2011,28(10):2731-2739
[26] Arnold K,Bordoli L,Kopp J,Schwede T.The SWISS-MODEL workspace:A web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201
[27] Chen T Z,Lv Y D,Zhao T M,Li N,Yang Y W,Yu W G,He X,Liu T L,Zhang B L.Comparative transcriptome profiling of a resistant vs.susceptible tomato (Solanum lycopersicum) cultivar in response to infection by tomato yellow leaf curl virus[J].PLoS One,2013,8(11):e80816
[28] Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):e45
[29] Allen M D,Yamasaki K,Ohme-Takagi M,Tateno M,Suzuki M.A novel mode of DNA recognition by a β-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].EMBO Journal,1998,17(18):5484-5496
[30] Maruyama Y,Yamoto N,Suzuki Y,Chiba Y,Yamazaki K,Sato T,Yamaguchi J.The Arabidopsis transcriptional repressor ERF9 participates inresistance against necrotrophic fungi[J].Plant Science,2013,213(6):79-87
[31] Iwase A,Harashima H,Ikeuchi M,Rymen B,Ohnuma M,Komaki S,Morohashi K,Kurata T,Nakata M,Ohme-Takagi M,Grotewold E,Suqimoto K.WIND1 promotes shoot regeneration through transcriptional activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis[J].Plant Cell,2017,29(1):54-69
[32] Lee S J,Park J H,Lee M H,Yu J H,Kim S Y.Isolation and functional characterization of CE1 binding proteins[J].BMC Plant Biology,2010,10(1):1-13
[33] 莫纪波,李大勇,张慧娟,宋凤鸣.ERF转录因子在植物对生物和非生物胁迫反应中的作用[J].植物生理学报,2011,47(12):1145-1154
[34] Zhu Q,Zhang J,Gao X,Tong J,Xiao L,Li W,Zhang H.The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA,salt and osmotic stress responses[J].Gene,2010,457(1/2):1-12
[35] Zhang G,Chen M,Li L,Xu Z,Chen X,Guo J,Ma Y.Overexpression of the soybean GmERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796
[36] Lorenzo O,Piqueras R,Sanchez-Serrano J J,Solano R.ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense[J].Plant Cell,2003,15(1):165-178
[37] Pre M,Atallah M,Champion A,De Ⅴ M,Pieterse C M,Memelink J.The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense[J].Plant Physiology,2008,147(3):1347-1357
[38] Huang Y,Li T,Xu Z S,Wang F,Xiong A S.Six NAC transcription factors involved in response to TYLCV infection in resistant and susceptible tomato cultivars[J].Plant Physiology & Biochemistry,2017,120:61-74
[39] Berrocal-Lobo M,Molina A,Solano R.Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi[J].Plant Journal,2002,29(1):23-32
[40] Guo Z J,Chen X J,Wu X L,Ling J Q,Xu P.Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco[J].Plant Molecular Biology,2004,55(4):607-618
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