百脉根泛素结合酶LjE2的表达及活性鉴定
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摘要
为了研究泛素化作用在豆科植物共生固氮过程中的调控功能和作用机制,利用拟南芥泛素结合酶E2的序列,在百脉根基因组上鉴定得到14个编码E2(LjE2)的基因,从LjE2中克隆到11个基因并构建了融合表达载体pET-LjE2s:His。通过大肠杆菌表达系统,表达并纯化到10个LjE2:His融合蛋白。经体外泛素化及Western blot分析,在10个LjE2中有9个具有泛素结合酶活性,1个(LjE2-5)未检测到泛素结合酶活性。
Fourteen genes encoding E2(LjE2)in Lotus japonicus genome were identified based on the sequence of E2 in Arabidopsis to study the function and mechanism of ubiquitination in the process of symbiotic nitrogen fixation in legumes.Among them,eleven LjE2 swere cloned to construct the fusion expression vectors pET-LjE2 s:His.Ten LjE2 proteins were successfully expressed and purified under the prokaryotic expression system.The results of ubiquitination in vitro and Western blotting showed that nine LjE2 shad ubquitin-conjugation enzyme activity and one had no detectable ubquitin-conjugation enzyme activity.
Fourteen genes encoding E2(LjE2)in Lotus japonicus genome were identified based on the sequence of E2 in Arabidopsis to study the function and mechanism of ubiquitination in the process of symbiotic nitrogen fixation in legumes.Among them,eleven LjE2 swere cloned to construct the fusion expression vectors pET-LjE2 s:His.Ten LjE2 proteins were successfully expressed and purified under the prokaryotic expression system.The results of ubiquitination in vitro and Western blotting showed that nine LjE2 shad ubquitin-conjugation enzyme activity and one had no detectable ubquitin-conjugation enzyme activity.
引文
[1]SCHWARTZ D C,HOCHSTRASSER M.A superfamily of protein tags:ubiquitin,SUMO and related modifiers[J].Trends in biochemical sciences,2003,28(6):321-328.
[2]陈霞,罗良煌.蛋白质翻译后修饰简介[J].生物学教学,2017,42(2):70-72.
[3]MORENO S P,GAMBUS A.Regulation of unperturbed DNAreplication by ubiquitylation[J].Genes,2015,6(3):451-468.
[4]HERRMANN J,LERMAN L O,LERMAN A.Ubiquitin and ubiquitin-like proteins in protein regulation[J].Circulation research,2007,100(9):1276-1291.
[5]SHAID S,BRANDTS C H,SERVE H,et al.Ubiquitination and selective autophagy[J].Cell death and differentiation,2013,20(1):21-30.
[6]ULRICH H D,WALDEN H.Ubiquitin signalling in DNA replication and repair[J].Nature reviews molecular cell biology,2010,11(7):479-489.
[7]王金利,史胜青,贾利强,等.植物泛素结合酶E2功能研究进展[J].生物技术通报,2010(4):7-10.
[8]HERSHKO A,CIECHANOVER A.The ubiquitin system[J].Annual review of biochemistry,1998,67:425-479.
[9]VIERSTRA R D.The ubiquitin-26Sproteasome system at the nexus of plant biology[J].Nature reviews molecular cell biology,2009,10(6):385-397.
[10]SADOWSKI M,SURYADINATA R,LAI X,et al.Molecular basis for lysine specificity in the yeast ubiquitin-conjugating enzyme Cdc34[J].Molecular&cellular biology,2010,30(10):2316-2329.
[11]WU T,MERBL Y,HUO Y,et al.UBE2Sdrives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex[J].PNAS,2010,107(4):1355-1360.
[12]YE Y,RAPE M.Building ubiquitin chains:E2enzymes at work[J].Nat Rev Mol Cell Biol,2009,10(11):755-764.
[13]CHRISTENSEN D E,KLEVIT R E.Dynamic interactions of proteins in complex networks:identifying the complete set of interacting E2sfor functional investigation of E3-dependent protein ubiquitination[J].Febs Journal,2009,276(19):5381-5389.
[14]杨倩倩,刘元,陈大松,等.蒺藜苜蓿中1个聚半乳糖醛酸酶基因突变体的筛选及共生固氮表型的初步鉴定[J].华中农业大学学报,2017,36(4):62-70.
[15]李文清,曹文刚,李豪,等.百脉根4个共生基因在水稻中的共表达及其对水稻转录组的影响[J].华中农业大学学报,2017,36(4):55-61.
[16]汪涛,冯勇,张忠明.百脉根LjSERKs基因家族在根瘤共生中的功能鉴定[J].华中农业大学学报,2018,37(2):9-15.
[17]RYU H,CHO H,CHOI D,et al.Plant hormonal regulation of nitrogen-fixing nodule organogenesis[J].Molecules&cells,2012,34(2):117-126.
[18]YUAN S,ZHU H,GOU H,et al.A Ubiquitin ligase of symbiosis receptor kinase involved in nodule organogenesis[J].Plant physiology,2012,160(1):106-117.
[19]DEN H G,YOSHIDA S,ANTOL NLLOVERA M,et al.Lotus japonicus E3ligase seven in absentia4destabilizes the symbiosis receptor-like kinase SYMRK and negatively regulates rhizobial infection[J].Plant cell,2012,24(4):1691-1707.
[20]VERNI T,CAMUT S,CAMPS C,et al.PUB1interacts with the receptor kinase DMI2and negatively regulates rhizobial and arbuscular mycorrhizal symbioses through its ubiquitination activity in Medicago truncatula[J].Plant physiology,2016,170(4):1694-2015.
[21]BACHMAIR A,NOVATCHKOVA M,POTUSCHAK T,et al.Ubiquitylation in plants:apost-genomic look at a posttranslational modification[J].Trends in plant science,2001,6(10):463-470.
[22]ZHAO Q,TIAN M,LI Q,et al.A plant-specific in vitro ubiquitination analysis system[J].Plant journal for cell&molecular biology,2013,74(3):524-533.
[23]MARKSON G,KIEL C,HYDE R,et al.Analysis of the human E2ubiquitin conjugating enzyme protein interaction network[J].Genome research,2009,19(10):1905-1911.
[24]WIJK S J L V,VRIES S J D,KEMMEREN P,et al.A comprehensive framework of E2-RING E3interactions of the human ubiquitin-proteasome system[J].Molecular systems biology,2009,5(1):295.doi:10.1038/msb.2009.55.
[25]DESHAIES R J,JOAZEIRO C A P,RING Domain E3ubiquitin ligases[J].Annual review of biochemistry,2009,78(1):399-434.
[2]陈霞,罗良煌.蛋白质翻译后修饰简介[J].生物学教学,2017,42(2):70-72.
[3]MORENO S P,GAMBUS A.Regulation of unperturbed DNAreplication by ubiquitylation[J].Genes,2015,6(3):451-468.
[4]HERRMANN J,LERMAN L O,LERMAN A.Ubiquitin and ubiquitin-like proteins in protein regulation[J].Circulation research,2007,100(9):1276-1291.
[5]SHAID S,BRANDTS C H,SERVE H,et al.Ubiquitination and selective autophagy[J].Cell death and differentiation,2013,20(1):21-30.
[6]ULRICH H D,WALDEN H.Ubiquitin signalling in DNA replication and repair[J].Nature reviews molecular cell biology,2010,11(7):479-489.
[7]王金利,史胜青,贾利强,等.植物泛素结合酶E2功能研究进展[J].生物技术通报,2010(4):7-10.
[8]HERSHKO A,CIECHANOVER A.The ubiquitin system[J].Annual review of biochemistry,1998,67:425-479.
[9]VIERSTRA R D.The ubiquitin-26Sproteasome system at the nexus of plant biology[J].Nature reviews molecular cell biology,2009,10(6):385-397.
[10]SADOWSKI M,SURYADINATA R,LAI X,et al.Molecular basis for lysine specificity in the yeast ubiquitin-conjugating enzyme Cdc34[J].Molecular&cellular biology,2010,30(10):2316-2329.
[11]WU T,MERBL Y,HUO Y,et al.UBE2Sdrives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex[J].PNAS,2010,107(4):1355-1360.
[12]YE Y,RAPE M.Building ubiquitin chains:E2enzymes at work[J].Nat Rev Mol Cell Biol,2009,10(11):755-764.
[13]CHRISTENSEN D E,KLEVIT R E.Dynamic interactions of proteins in complex networks:identifying the complete set of interacting E2sfor functional investigation of E3-dependent protein ubiquitination[J].Febs Journal,2009,276(19):5381-5389.
[14]杨倩倩,刘元,陈大松,等.蒺藜苜蓿中1个聚半乳糖醛酸酶基因突变体的筛选及共生固氮表型的初步鉴定[J].华中农业大学学报,2017,36(4):62-70.
[15]李文清,曹文刚,李豪,等.百脉根4个共生基因在水稻中的共表达及其对水稻转录组的影响[J].华中农业大学学报,2017,36(4):55-61.
[16]汪涛,冯勇,张忠明.百脉根LjSERKs基因家族在根瘤共生中的功能鉴定[J].华中农业大学学报,2018,37(2):9-15.
[17]RYU H,CHO H,CHOI D,et al.Plant hormonal regulation of nitrogen-fixing nodule organogenesis[J].Molecules&cells,2012,34(2):117-126.
[18]YUAN S,ZHU H,GOU H,et al.A Ubiquitin ligase of symbiosis receptor kinase involved in nodule organogenesis[J].Plant physiology,2012,160(1):106-117.
[19]DEN H G,YOSHIDA S,ANTOL NLLOVERA M,et al.Lotus japonicus E3ligase seven in absentia4destabilizes the symbiosis receptor-like kinase SYMRK and negatively regulates rhizobial infection[J].Plant cell,2012,24(4):1691-1707.
[20]VERNI T,CAMUT S,CAMPS C,et al.PUB1interacts with the receptor kinase DMI2and negatively regulates rhizobial and arbuscular mycorrhizal symbioses through its ubiquitination activity in Medicago truncatula[J].Plant physiology,2016,170(4):1694-2015.
[21]BACHMAIR A,NOVATCHKOVA M,POTUSCHAK T,et al.Ubiquitylation in plants:apost-genomic look at a posttranslational modification[J].Trends in plant science,2001,6(10):463-470.
[22]ZHAO Q,TIAN M,LI Q,et al.A plant-specific in vitro ubiquitination analysis system[J].Plant journal for cell&molecular biology,2013,74(3):524-533.
[23]MARKSON G,KIEL C,HYDE R,et al.Analysis of the human E2ubiquitin conjugating enzyme protein interaction network[J].Genome research,2009,19(10):1905-1911.
[24]WIJK S J L V,VRIES S J D,KEMMEREN P,et al.A comprehensive framework of E2-RING E3interactions of the human ubiquitin-proteasome system[J].Molecular systems biology,2009,5(1):295.doi:10.1038/msb.2009.55.
[25]DESHAIES R J,JOAZEIRO C A P,RING Domain E3ubiquitin ligases[J].Annual review of biochemistry,2009,78(1):399-434.