小麦TaUBC基因泛素结合酶活性分析
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摘要
已报道的RNA水平研究表明TaUBC可能是小麦产量相关基因,生物信息学分析显示该基因属于泛素结合酶家族.荧光定量PCR显示TaUBC在小麦根茎叶中均有表达.为了验证TaUBC蛋白是否具有泛素结合酶活性,本文利用原核系统表达野生型TaUBC及三种点突变TaUBC~(C21S)、TaUBC~(C85S)、TaUBC~(C107S)蛋白,并对这些蛋白进行体外泛素化活性分析.结果证实TaUBC是一个有活性的泛素结合酶.另外,点突变C85S使得TaUBC丧失E2活性,C21S和C107S则对活性没有影响,证明其UBC结构域中第85位Cys是结合泛素的关键作用位点.
Reported results of TaUBC gene in RNA level indicated it may be a yield-relevant gene in wheat.Bioinformatic analysis showed that TaUBC was expressed in root,stem and leaf.In order to verify whether TaUBC protein has ubiquitin conjugating enzyme activity,wild type and three different point mutation types of TaUBC protein(TaUBC~(C21S)、TaUBC~(C85S)、TaUBC~(C107S))were expressed in bacteria respectively.Ubiquitin conjugating enzyme activity were then detected by in vitro ubiquitination assay.The results showed that TaUBC was an active ubiquitin conjugating enzyme.Point mutation C85Sresulted in loss of ubiquitin conjugating enzyme activity of TaUBC,while C21Sand C107Shad no influence on the activity,proving that C85in the UBC domain is the crucial catalytic site for ubiquitin binding.
Reported results of TaUBC gene in RNA level indicated it may be a yield-relevant gene in wheat.Bioinformatic analysis showed that TaUBC was expressed in root,stem and leaf.In order to verify whether TaUBC protein has ubiquitin conjugating enzyme activity,wild type and three different point mutation types of TaUBC protein(TaUBC~(C21S)、TaUBC~(C85S)、TaUBC~(C107S))were expressed in bacteria respectively.Ubiquitin conjugating enzyme activity were then detected by in vitro ubiquitination assay.The results showed that TaUBC was an active ubiquitin conjugating enzyme.Point mutation C85Sresulted in loss of ubiquitin conjugating enzyme activity of TaUBC,while C21Sand C107Shad no influence on the activity,proving that C85in the UBC domain is the crucial catalytic site for ubiquitin binding.
引文
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[21] Gu X,Jiang D,Wang Y,et al.Repression of the floral transition via histone H2Bmonoubiquitination[J].Plant J,2009,57:522-533.
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[23] Wang S,Cao L,Wang H.Arabidopsis ubiquitin-conjugating enzyme UBC22is required for female gametophyte development and likely involved in Lys11-linked ubiquitination[J].J Exp Bot,2016,67:3277-3288.
[24] Kao Y T,Bartel B.Elevated growth temperature decreases levels of the PEX5peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4ubiquitin-conjugating enzyme[J].BMC Plant Biol,2015,15:224.
[25] Bahmani R,Kim D,Lee B D,et al.Over-expression of tobacco UBC1encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26Sproteasome and by decreasing Cd accumulation and oxidative stress in tobacco(Nicotiana tabacum)[J].Plant Mol Biol,2017,94:433-451.
[26] Chung E,Cho C W,So H A,et al.Overexpression of VrUBC1,a Mung Bean E2Ubiquitin-Conjugating Enzyme,Enhances Osmotic Stress Tolerance in Arabidopsis[J].PLoS One,2013,8:e66056.
[27] Wan X,Mo A,Liu S,et al.Constitutive expression of a peanut ubiquitin-conjugating enzyme gene in Arabidopsis confers improved water-stress tolerance through regulation of stress-responsive gene expression[J].J Biosci Bioeng,2011,111:478-484.
[28] Zhou G A,Chang R Z,Qiu L J.Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis[J].Plant Mol Biol,2010,72:357-367.
[29] E Z,Zhang Y,Li T,et al.Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments[J].PLoS One,2015,10:e0122621.
[30] Jue D,Sang X,Lu S,et al.Genome-Wide Identification,Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize[J].PLoS One,2015,10:e0143488.
[31] Jeon E H,Pak J H,Kim M J,et al.Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice,OgUBC1,confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana[J].Biochem Biophys Res Commun,2012,427:309-314.
[32] Millyard L,Lee J,Zhang C,et al.The ubiquitin conjugating enzyme,TaU4regulates wheat defence against the phytopathogen Zymoseptoria tritici[J].Sci Rep,2016,6:35683.
[33]尹丽娟,陈阳,刘沛,等.小麦泛素结合酶TaE2的表达分析及互作[J].植物资源学报,2014,15:144-149.
[34] Yao Y,Ni Z,Zhang Y,et al.Identification of differentially expressed genes in leaf and root between wheat hybrid and its parental inbreds using PCR-based cDNA subtraction[J].Plant Mol Biol,2005,58:367-384.
[35] Zhao Q,Tian M,Li Q,et al.A plant-specific in vitro ubiquitination analysis system[J].Plant J,2013,74:524-533.
[2] Bachmair A,Novatchkova M,Potuschak T,et al.Ubiquitylation in plants.a post-genomic look at a post-translational modification[J].Trends Plant Sci,2001,6:463-470.
[3] Vierstra R D.The ubiquitin/26Sproteasome pathway,the complex last chapter in the life of many plant proteins[J].Trends Plant Sci,2003,8:135-142.
[4] Stone S L.The role of ubiquitin and the 26Sproteasome in plant abiotic stress signaling[J].Front Plant Sci,2014,5:135.
[5] Wang F,Deng X W.Plant ubiquitin-proteasome pathway and its role in gibberellin signaling[J].Cell Res,2011,21:1286-1294.
[6] Smalle J,Vierstra R D.The ubiquitin 26Sproteasome proteolytic pathway[J].Annu Rev Plant Biol,2004,55:555-590.
[7] Lei L,Bandola-Simon J,Roche P A.Ubiquitin-conjugating enzyme E2D1(Ube2D1)mediates lysine-independent ubiquitination of the E3ubiquitin ligase March-I[J].J Biol Chem,2018,293:3904-3912.
[8] Sanchez-Martin P,Roma-Mateo C,Viana R,et al.Ubiquitin conjugating enzyme E2-N and sequestosome-1(p62)are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex[J].Int J Biochem Cell Biol,2015,69:204-214.
[9] Sheng Y,Hong J H,Doherty R,et al.A human ubiquitin conjugating enzyme(E2)-HECT E3ligase structure-function screen[J].Mol Cell Proteomics,2012,11:329-341.
[10] Soss S E,Yue Y,Dhe-Paganon S,et al.E2conjugating enzyme selectivity and requirements for function of the E3ubiquitin ligase CHIP[J].J Biol Chem,2011,286:21277-21286.
[11] Vanni E,Gatherer D,Tong L,et al.Functional characterization of residues required for the herpes simplex virus 1E3ubiquitin ligase ICP0to interact with the cellular E2ubiquitin-conjugating enzyme UBE2D1(UbcH5a)[J].J Virol,2012,86:6323-6333.
[12] Wang D,Tian Y,Wei D,et al.DNA Damage-Induced Foci of E2Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3Ubiquitin Ligase[J].Biochem Genet,2016,54:147-157.
[13] Ibarra R,Sandoval D,Fredrickson E K,et al.The San1Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1during Protein Quality Control[J].J Biol Chem,2016,291:18778-18790.
[14] Ye Y,Rape M.Building ubiquitin chains.E2enzymes at work[J].Nat Rev Mol Cell Biol,2009,10:755-764.
[15] Li W,Tu D,Brunger A T,et al.A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate[J].Nature,2007,446:333-337.
[16] Hill S,Harrison J S,Lewis S M,et al.Mechanism of Lysine 48Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2Ubiquitin-Conjugating Enzyme[J].Mol Cell Biol,2016,36:1720-1732.
[17] Ryan P E,Sivadasan-Nair N,Nau M M,et al.The N terminus of Cbl-c regulates ubiquitin ligase activity by modulating affinity for the ubiquitin-conjugating enzyme[J].J Biol Chem,2010,285:23687-23698.
[18] Nguyen L,Plafker K S,Starnes A,et al.The ubiquitin-conjugating enzyme,UbcM2,is restricted to monoubiquitylation by a twofold mechanism that involves backside residues of E2and Lys48of ubiquitin[J].Biochemistry,2014,53:4004-4014.
[19] van Wijk S J,Timmers H T.The family of ubiquitin-conjugating enzymes(E2s):deciding between life and death of proteins[J].FASEB J,2010,24:981-993.
[20] Michelle C,Vourc'h P,Mignon L,et al.What was the set of ubiquitin and ubiquitin-like conjugating enzymes in the eukaryote common ancestor[J].J Mol Evol,2009,68:616-628.
[21] Gu X,Jiang D,Wang Y,et al.Repression of the floral transition via histone H2Bmonoubiquitination[J].Plant J,2009,57:522-533.
[22] Xu L,Menard R,Berr A,et al.The E2ubiquitin-conjugating enzymes,AtUBC1and AtUBC2,play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana[J].Plant J,2009,57:279-288.
[23] Wang S,Cao L,Wang H.Arabidopsis ubiquitin-conjugating enzyme UBC22is required for female gametophyte development and likely involved in Lys11-linked ubiquitination[J].J Exp Bot,2016,67:3277-3288.
[24] Kao Y T,Bartel B.Elevated growth temperature decreases levels of the PEX5peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4ubiquitin-conjugating enzyme[J].BMC Plant Biol,2015,15:224.
[25] Bahmani R,Kim D,Lee B D,et al.Over-expression of tobacco UBC1encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26Sproteasome and by decreasing Cd accumulation and oxidative stress in tobacco(Nicotiana tabacum)[J].Plant Mol Biol,2017,94:433-451.
[26] Chung E,Cho C W,So H A,et al.Overexpression of VrUBC1,a Mung Bean E2Ubiquitin-Conjugating Enzyme,Enhances Osmotic Stress Tolerance in Arabidopsis[J].PLoS One,2013,8:e66056.
[27] Wan X,Mo A,Liu S,et al.Constitutive expression of a peanut ubiquitin-conjugating enzyme gene in Arabidopsis confers improved water-stress tolerance through regulation of stress-responsive gene expression[J].J Biosci Bioeng,2011,111:478-484.
[28] Zhou G A,Chang R Z,Qiu L J.Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis[J].Plant Mol Biol,2010,72:357-367.
[29] E Z,Zhang Y,Li T,et al.Characterization of the Ubiquitin-Conjugating Enzyme Gene Family in Rice and Evaluation of Expression Profiles under Abiotic Stresses and Hormone Treatments[J].PLoS One,2015,10:e0122621.
[30] Jue D,Sang X,Lu S,et al.Genome-Wide Identification,Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize[J].PLoS One,2015,10:e0143488.
[31] Jeon E H,Pak J H,Kim M J,et al.Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice,OgUBC1,confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana[J].Biochem Biophys Res Commun,2012,427:309-314.
[32] Millyard L,Lee J,Zhang C,et al.The ubiquitin conjugating enzyme,TaU4regulates wheat defence against the phytopathogen Zymoseptoria tritici[J].Sci Rep,2016,6:35683.
[33]尹丽娟,陈阳,刘沛,等.小麦泛素结合酶TaE2的表达分析及互作[J].植物资源学报,2014,15:144-149.
[34] Yao Y,Ni Z,Zhang Y,et al.Identification of differentially expressed genes in leaf and root between wheat hybrid and its parental inbreds using PCR-based cDNA subtraction[J].Plant Mol Biol,2005,58:367-384.
[35] Zhao Q,Tian M,Li Q,et al.A plant-specific in vitro ubiquitination analysis system[J].Plant J,2013,74:524-533.
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