胡杨Ring finger E3连接酶PeRH2提高烟草耐旱机制研究
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摘要
近期研究表明泛素化在植物胁迫响应中扮演一个关键的角色。作为泛素-蛋白酶体系统(UPS)中的关键酶,泛素E3连接酶更是在细胞的新陈代谢中起着重要的作用。本文从胡杨中克隆了一个C端含有Ring finger结构域的蛋白(152个氨基酸)编码基因(Pe RH2),经过序列比对推断该基因编码产物为泛素E3连接酶,属于Ring finger家族中的Ring-H2构型。研究显示,甘露醇胡杨苗木在255 mmol/L甘露醇胁迫下,叶片Pe RH2表达量上调。拟南芥原生质体中瞬时表达融合质粒p Yellow0029-Pe RH2的研究表明,Pe RH2蛋白定位于细胞核。以农杆菌介导法将Pe RH2转入烟草,Sq RT-PCR结果显示,Pe RH2在转基因烟草中获得过表达。在255 mmol/L甘露醇处理下,转基因烟草幼苗耐受渗透胁迫的能力高于野生型和转空载体烟草。干旱处理后,转基因烟草的叶片保水力明显高于野生型和转空载体烟草,而叶片相对电导率、MDA的含量也要低于野生型和转空载体烟草。因此,过表达Pe RH2能够提高烟草的抗旱能力。
Recent studies show that ubiquitination plays a critical role in regulating plant responses to abiotic stresses. Being as a key enzyme of the ubiquitin 26 S proteasome system(UPS), ubiquitin E3 ligases mediates numerous cellular processes in plant cells. In this study, we cloned a putative Pe RH2 gene encoding ubiquitin E3 ligase from populus euphratica leaves. The Pe RH2 gene encodes a protein(152 amino acids) with a Ring finger motif located at the C-terminus. Sequence analysis suggests that Pe RH2 is a member of Ring-H2 type belong to Ring finger family. Pe RH2 was upregulated under 255 mmol/L mannitol in P. euphratica. Transient expression of the gene construct of p Yellow0029-Pe RH2 in Arabidopsis mesophyll protoplast demonstrated the nucleus loclization of the Pe RH2 protein. Through Agrobacterium-mediated method Pe RH2 was transferred into tobacco plants.Sq RT-PCR analysis showed that Pe RH2 gene was overexpressed in transgenic tobacco and two transgenic lines were obtained. Under drought treatment, the leaf water-retaining capacity(WRC) of Pe RH2-transgenic tobacco was higher than that of wild type and vector control plants. The relative electrolytic leakage and MDA contents were much lower in leaves of Pe RH2-transgenic plant. We conclude that overexpression of Pe RH2 enhance drought tolerance in tobacco.
Recent studies show that ubiquitination plays a critical role in regulating plant responses to abiotic stresses. Being as a key enzyme of the ubiquitin 26 S proteasome system(UPS), ubiquitin E3 ligases mediates numerous cellular processes in plant cells. In this study, we cloned a putative Pe RH2 gene encoding ubiquitin E3 ligase from populus euphratica leaves. The Pe RH2 gene encodes a protein(152 amino acids) with a Ring finger motif located at the C-terminus. Sequence analysis suggests that Pe RH2 is a member of Ring-H2 type belong to Ring finger family. Pe RH2 was upregulated under 255 mmol/L mannitol in P. euphratica. Transient expression of the gene construct of p Yellow0029-Pe RH2 in Arabidopsis mesophyll protoplast demonstrated the nucleus loclization of the Pe RH2 protein. Through Agrobacterium-mediated method Pe RH2 was transferred into tobacco plants.Sq RT-PCR analysis showed that Pe RH2 gene was overexpressed in transgenic tobacco and two transgenic lines were obtained. Under drought treatment, the leaf water-retaining capacity(WRC) of Pe RH2-transgenic tobacco was higher than that of wild type and vector control plants. The relative electrolytic leakage and MDA contents were much lower in leaves of Pe RH2-transgenic plant. We conclude that overexpression of Pe RH2 enhance drought tolerance in tobacco.
引文
An G.,1987,Binary ti vectors for plant transformation and promoter analysis,In Methods in Enzymology,153:292-305
Chen C.W.,Yang Y.W.,Lur H.S.,Tsai Y.G.,and Chang,M.C.,2006,A novel function of abscisic acid in the regulation ofrice(Oryza sativa L.)root growth and development,Plant&Cell Physiology,47(1):1-13
Cheng M.C.,Hsieh E.J.,Chen J.H.,Chen H.Y.,and Lin T.P.,2012,Arabidopsis RGLG2,functioning as a RING E3 ligase,interacts with AtE RF53 and negatively regulates theplant drought stress response,Plant Physiology,158(1):363-375
Cutler A.J.,and Krochko J.E.,1999,Formation and breakdownof ABA,Trends in Plant Science,4(12):472-478
Cutler J.M.,Shahan K.W.,and Steponkus P.L.,1980,Influenceof water deficits and osmotic adjustment on leaf elongationin rice,Crop Sci.,20(3):314-318
Dong C.H.,Agarwal M.,Zhang Y.Y.,Xie Q.,and Zhu J.K.,2006,The negative regulator of plant cold responses,HOS1,is a RING E3 ligase that mediates the ubiquitination anddegradation of ICE1,Proc.Natl.Acad.Sci.USA,103(21):8281-8286
Du Z.,Zhou X.,Li L.,and Su Z.,2009,PlantsU PS:a database ofplants'Ubiquitin Proteasome System,BMC Genomics,10:227
Finkelstein R.R.,Gampala S.S.,and Rock C.D.,2002,Abscisicacid signaling in seeds and seedlings,The Plant Cell,14:S15-S45
Freemont P.S.,2000,RING for destruction?Current Biology,10(1):R84-R87
Freemont P.S.,1993,The RING finger a novel protein sequencemotif related to the zinc finger,Annals of the New York Academy of Sciences,684(1):174-192
Han Y.S.,Wang W.,Sun J.,Ding M.Q.,Zhao R.,Deng S.R.,WangF.F.,Hu Y.,Wang Y.,Lu Y.J.,Du L.P.,Hu Z.M.,DiekmannH.,Shen X.,Polle A.,and Chen S.L.,2013,Populus euphraticaXTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants,Journal of Experimental Botany,64(14):4225-4238
Heath R.L.,and Packer L.,1968,Photoperoxidation in isolatedchloroplasts:玉.Kinetics and stoichiometry of fatty acidperoxidation,Archives of Biochemistry and Biophysics,125(1):189-198
Horsch R.B.,Fry J.E.,and Hoffmann N.L.,1985,A simple andgeneral method for transferring genes into plants,Science,227(4691):1229-1231
Hu X.L.,Yang H.R.,and Li C.H.,2009,Regulating mechanismof drought in maize with ABA,Xibei Zhiwu Xuebao(ActaBot.Boreal.-Occident.Sin.),29(11):2345-2351(胡秀丽,杨海荣,李潮海,2009,ABA对玉米响应干旱胁迫的调控机制,西北植物学报,29(11):2345-2351)
Khadri M.,Tejera N.A.,and Lluch C.,2006,Alleviation of saltstress in common bean(Phaseolus vulgaris)by exogenousabscisic acid supply,J.Plant Growth Regul.,25(2):110-119
Khadri M.,Tejera N.A.,and Lluch C.,2007,Sodium chlorideABA interaction in two common bean(Phaseolus vulgaris)cultivars differing in salinity tolerance,Environmental andExperimental Botany,60(2):211-218
Ko J.H.,Yang S.H.,and Han K.H.,2006,Upregulation of anArabidopsis RING-H2 gene,XERICO,confers drought tolerance through increased abscisic acid biosynthesis,ThePlant Journal,47(3):343-355
Kraft E.,Stone S.L.,Ma L.G.,Su N.,Gao Y.,Lau O.S.,Deng X.W.,and Callis J.,2005,Genome analysis and functionalcharacterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis,Plant Physiology,139(4):1597-1611
Lee H.K.,Cho S.K.,Son O.,Xu Z.Y.,Hwang I.,and Kim W.T.,2009,Drought stress-induced Rma1H1,a RING membrane-anchor E3 ubiquitin ligase homolog,regulates aquaporin levels via ubiquitination in transgenic Arabidopsisplants,The Plant Cell,21(2):622-641
Lutts S.,Kinet J.M.,and Bouharmont J.,1996,NaC l-inducedsenescence in leaves of rice(Oryza sativa L.)cultivars differing in salinity resistance,Annals of Botany,78(3):389-398
Muhetaer Z.,Qiman Y.,and Norkazu Y.,2010,Effect of exogenous ABA and silicon on leaf water potential and protectiveenzyme activities of Exogenous angustifolia L.seedlings under drought stress,Zhiwu Yanjiu(Bulletin of Botanical Research),30(4):468-472(木合塔尔·扎热,齐曼·尤努斯,山中典和,2010,干旱胁迫下外源脱落酸和硅对沙枣幼苗叶片水势及保护酶活性的影响,植物研究,30(4):468-472)
Pauls K.P.,and Thompson J.E.,1980,In vitro simulation ofsenescence-related membrane damage by ozone-inducedlipid peroxidation,Nature,283(5746):504-506
Qin F.,Sakuma Y.,Tran L.S.P.,Maruyama K.,Kidokoro S.,FujitaY.,Fujita M.,Umezawa T.,Sawano Y.,Miyazono K.,Tanokura M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2008,Arabidopsis DREB2A-interacting proteins function asRING E3 ligases and negatively regulate plant droughtstress-responsive gene expression,The Plant Cell,20(6):1693-1707
Ryu M.Y.,Cho S.K.,and Kim W.T.,2010.,The ArabidopsisC3H2C3-type RING E3 ubiquitin ligase AtA IRP1 is a positive regulator of an abscisic acid-dependent response todrought stress,Plant Physiology,154(4):1983-1997
Tang R.S.,Huang Y.H.,Tang X.H.,Zhong Y.,Yu Y.Z.,and Tong H.Y.,2008,Effect of microorganism-sourced ABA on chillingresistance of pepper seedling,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),24(4):467-470(汤日圣,黄益洪,唐现洪,钟雨,余永柱,童红玉,2008,微生物源脱落酸(ABA)对辣椒苗耐冷性的影响,江苏农业学报,24(4):467-470)
Taylor I.B.,Burbidge A.,and Thompson A.J.,2000,Control ofabscisic acid synthesis,Journal of Experimental Botany,51(350):1563-1574
Xiao C.W.,Chen F.L.,Yu X.H.,Lin C.T.,and Fu Y.F.,2009,Over-expression of an AT-hook gene,AHL22,delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana,Plant Molecular Biology,71(1-2):39-50
Yan H.,Xu B.B.,Zhao F.Y.,He Y.,Yao X.D.,and Tian Z.H.,2008,Effects of abscisic acid and salicylic acid on physiological characteristics of sesame seedl ings under droughtstress,Ganhan Diqu Nongye Yanjiu(Agricultural Researchin the Arid Areas),26(6):163-166(严寒,许本波,赵福永,何勇,姚晓鼎,田志宏,2008,脱落酸和水杨酸对干旱胁迫下芝麻幼苗生理特性的影响,干旱地区农业研究,26(6):163-166)
Yao M.S.,Yang X.H.,and Guo P.Y.,2005,Effects of abscisicacid on water relationship and defensive enzymes activitiesin cotton seedling under water stress,Mianhua Xuebao(Cotton Science),17(3):141-145(姚满生,杨小环,郭平毅,2005,脱落酸与水分胁迫下棉花幼苗水分关系及保护酶活性的影响,棉花学报,17(3):141-145)
Yoo S.D.,Cho Y.H.,and Sheen J.,2007,Arabidopsis mesophyllprotoplasts:a versatile cell system for transient gene expression analysis,Nature Protocols,2(7):1565-1572
Yu R.,Hu Y.,Huang X.X.,Shen Z.D.,Han Y.S.,Deng S.R.,Zhao R.,Shen X.,and Chen S.L.,2013,Overexpression ofKandelia candel C2H2zinc-finger protein gene Kc ZFP enhanced salinity tolerance in tobacco plants,Jiyinzuxue YuYingyong Shengwuxue(Genomics and Applied Biology),32(2):149-158(于瑞,胡月,黄旭新,申泽丹,韩彦莎,邓澍荣,赵瑞,沈昕,陈少良,2013,过表达秋茄C2H2型锌指蛋白基因KcZ FP提高烟草耐盐性,基因组学与应用生物学,32(2):149-158)
Zhang Y.Y.,Yang C.W.,Li Y.,Zheng N.Y.,Chen H.,Zhao Q.Z.,Gao T.,Guo H.S.,and Xie Q.,2007,SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis,The Plant Cell,19(6):1912-1929
Chen C.W.,Yang Y.W.,Lur H.S.,Tsai Y.G.,and Chang,M.C.,2006,A novel function of abscisic acid in the regulation ofrice(Oryza sativa L.)root growth and development,Plant&Cell Physiology,47(1):1-13
Cheng M.C.,Hsieh E.J.,Chen J.H.,Chen H.Y.,and Lin T.P.,2012,Arabidopsis RGLG2,functioning as a RING E3 ligase,interacts with AtE RF53 and negatively regulates theplant drought stress response,Plant Physiology,158(1):363-375
Cutler A.J.,and Krochko J.E.,1999,Formation and breakdownof ABA,Trends in Plant Science,4(12):472-478
Cutler J.M.,Shahan K.W.,and Steponkus P.L.,1980,Influenceof water deficits and osmotic adjustment on leaf elongationin rice,Crop Sci.,20(3):314-318
Dong C.H.,Agarwal M.,Zhang Y.Y.,Xie Q.,and Zhu J.K.,2006,The negative regulator of plant cold responses,HOS1,is a RING E3 ligase that mediates the ubiquitination anddegradation of ICE1,Proc.Natl.Acad.Sci.USA,103(21):8281-8286
Du Z.,Zhou X.,Li L.,and Su Z.,2009,PlantsU PS:a database ofplants'Ubiquitin Proteasome System,BMC Genomics,10:227
Finkelstein R.R.,Gampala S.S.,and Rock C.D.,2002,Abscisicacid signaling in seeds and seedlings,The Plant Cell,14:S15-S45
Freemont P.S.,2000,RING for destruction?Current Biology,10(1):R84-R87
Freemont P.S.,1993,The RING finger a novel protein sequencemotif related to the zinc finger,Annals of the New York Academy of Sciences,684(1):174-192
Han Y.S.,Wang W.,Sun J.,Ding M.Q.,Zhao R.,Deng S.R.,WangF.F.,Hu Y.,Wang Y.,Lu Y.J.,Du L.P.,Hu Z.M.,DiekmannH.,Shen X.,Polle A.,and Chen S.L.,2013,Populus euphraticaXTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants,Journal of Experimental Botany,64(14):4225-4238
Heath R.L.,and Packer L.,1968,Photoperoxidation in isolatedchloroplasts:玉.Kinetics and stoichiometry of fatty acidperoxidation,Archives of Biochemistry and Biophysics,125(1):189-198
Horsch R.B.,Fry J.E.,and Hoffmann N.L.,1985,A simple andgeneral method for transferring genes into plants,Science,227(4691):1229-1231
Hu X.L.,Yang H.R.,and Li C.H.,2009,Regulating mechanismof drought in maize with ABA,Xibei Zhiwu Xuebao(ActaBot.Boreal.-Occident.Sin.),29(11):2345-2351(胡秀丽,杨海荣,李潮海,2009,ABA对玉米响应干旱胁迫的调控机制,西北植物学报,29(11):2345-2351)
Khadri M.,Tejera N.A.,and Lluch C.,2006,Alleviation of saltstress in common bean(Phaseolus vulgaris)by exogenousabscisic acid supply,J.Plant Growth Regul.,25(2):110-119
Khadri M.,Tejera N.A.,and Lluch C.,2007,Sodium chlorideABA interaction in two common bean(Phaseolus vulgaris)cultivars differing in salinity tolerance,Environmental andExperimental Botany,60(2):211-218
Ko J.H.,Yang S.H.,and Han K.H.,2006,Upregulation of anArabidopsis RING-H2 gene,XERICO,confers drought tolerance through increased abscisic acid biosynthesis,ThePlant Journal,47(3):343-355
Kraft E.,Stone S.L.,Ma L.G.,Su N.,Gao Y.,Lau O.S.,Deng X.W.,and Callis J.,2005,Genome analysis and functionalcharacterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis,Plant Physiology,139(4):1597-1611
Lee H.K.,Cho S.K.,Son O.,Xu Z.Y.,Hwang I.,and Kim W.T.,2009,Drought stress-induced Rma1H1,a RING membrane-anchor E3 ubiquitin ligase homolog,regulates aquaporin levels via ubiquitination in transgenic Arabidopsisplants,The Plant Cell,21(2):622-641
Lutts S.,Kinet J.M.,and Bouharmont J.,1996,NaC l-inducedsenescence in leaves of rice(Oryza sativa L.)cultivars differing in salinity resistance,Annals of Botany,78(3):389-398
Muhetaer Z.,Qiman Y.,and Norkazu Y.,2010,Effect of exogenous ABA and silicon on leaf water potential and protectiveenzyme activities of Exogenous angustifolia L.seedlings under drought stress,Zhiwu Yanjiu(Bulletin of Botanical Research),30(4):468-472(木合塔尔·扎热,齐曼·尤努斯,山中典和,2010,干旱胁迫下外源脱落酸和硅对沙枣幼苗叶片水势及保护酶活性的影响,植物研究,30(4):468-472)
Pauls K.P.,and Thompson J.E.,1980,In vitro simulation ofsenescence-related membrane damage by ozone-inducedlipid peroxidation,Nature,283(5746):504-506
Qin F.,Sakuma Y.,Tran L.S.P.,Maruyama K.,Kidokoro S.,FujitaY.,Fujita M.,Umezawa T.,Sawano Y.,Miyazono K.,Tanokura M.,Shinozaki K.,and Yamaguchi-Shinozaki K.,2008,Arabidopsis DREB2A-interacting proteins function asRING E3 ligases and negatively regulate plant droughtstress-responsive gene expression,The Plant Cell,20(6):1693-1707
Ryu M.Y.,Cho S.K.,and Kim W.T.,2010.,The ArabidopsisC3H2C3-type RING E3 ubiquitin ligase AtA IRP1 is a positive regulator of an abscisic acid-dependent response todrought stress,Plant Physiology,154(4):1983-1997
Tang R.S.,Huang Y.H.,Tang X.H.,Zhong Y.,Yu Y.Z.,and Tong H.Y.,2008,Effect of microorganism-sourced ABA on chillingresistance of pepper seedling,Jiangsu Nongye Xuebao(Jiangsu Journal of Agricultural Sciences),24(4):467-470(汤日圣,黄益洪,唐现洪,钟雨,余永柱,童红玉,2008,微生物源脱落酸(ABA)对辣椒苗耐冷性的影响,江苏农业学报,24(4):467-470)
Taylor I.B.,Burbidge A.,and Thompson A.J.,2000,Control ofabscisic acid synthesis,Journal of Experimental Botany,51(350):1563-1574
Xiao C.W.,Chen F.L.,Yu X.H.,Lin C.T.,and Fu Y.F.,2009,Over-expression of an AT-hook gene,AHL22,delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana,Plant Molecular Biology,71(1-2):39-50
Yan H.,Xu B.B.,Zhao F.Y.,He Y.,Yao X.D.,and Tian Z.H.,2008,Effects of abscisic acid and salicylic acid on physiological characteristics of sesame seedl ings under droughtstress,Ganhan Diqu Nongye Yanjiu(Agricultural Researchin the Arid Areas),26(6):163-166(严寒,许本波,赵福永,何勇,姚晓鼎,田志宏,2008,脱落酸和水杨酸对干旱胁迫下芝麻幼苗生理特性的影响,干旱地区农业研究,26(6):163-166)
Yao M.S.,Yang X.H.,and Guo P.Y.,2005,Effects of abscisicacid on water relationship and defensive enzymes activitiesin cotton seedling under water stress,Mianhua Xuebao(Cotton Science),17(3):141-145(姚满生,杨小环,郭平毅,2005,脱落酸与水分胁迫下棉花幼苗水分关系及保护酶活性的影响,棉花学报,17(3):141-145)
Yoo S.D.,Cho Y.H.,and Sheen J.,2007,Arabidopsis mesophyllprotoplasts:a versatile cell system for transient gene expression analysis,Nature Protocols,2(7):1565-1572
Yu R.,Hu Y.,Huang X.X.,Shen Z.D.,Han Y.S.,Deng S.R.,Zhao R.,Shen X.,and Chen S.L.,2013,Overexpression ofKandelia candel C2H2zinc-finger protein gene Kc ZFP enhanced salinity tolerance in tobacco plants,Jiyinzuxue YuYingyong Shengwuxue(Genomics and Applied Biology),32(2):149-158(于瑞,胡月,黄旭新,申泽丹,韩彦莎,邓澍荣,赵瑞,沈昕,陈少良,2013,过表达秋茄C2H2型锌指蛋白基因KcZ FP提高烟草耐盐性,基因组学与应用生物学,32(2):149-158)
Zhang Y.Y.,Yang C.W.,Li Y.,Zheng N.Y.,Chen H.,Zhao Q.Z.,Gao T.,Guo H.S.,and Xie Q.,2007,SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis,The Plant Cell,19(6):1912-1929