棉花GhbHLH基因的克隆与表达分析
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摘要
bHLH(basic helix-loop-helix,bHLH)家族基因在植物生长发育和非生物胁迫应答中发挥着重要的作用。本研究从前期构建的棉花cDNA文库中发掘得到一个新的GhbHLH基因,该基因开放阅读框全长600 bp,编码199个氨基酸,序列分析表明,该基因具有典型的helix-loop-helix结构域。荧光定量PCR表明,GhbHLH在棉花根、茎、叶和纤维中均有表达,并且能够受高盐、PEG、低温、ABA和Me JA等的诱导表达,亚细胞定位结果表明,GhbHLH在细胞核内表达。推测该基因可能作为调控因子在棉花非生物胁迫应答中发挥作用。
bHLH(basic helix-loop-helix,bHLH) gene family transcription factors play important roles in plant growth and abiotic stress response.In this study,we cloned a new bHLH gene from a normolized cDNA library,which was named GhbHLH.GhbHLH contained an open reading frame(ORF) of 600 bp,which encoded a protein with 199 amino acids,and contained an Helix-Loop-Helix conserved domain.Quantitative RT-PCR analysis showed that GhbHLH expressed in root,stem,leaf and fiber,and was induced by salt,PEG,cold,ABA and Me JA,subcellular localization analysis showed that GhbHLH-GFP fusion protein was distributed specifically in cell nucleus.Our data indicated GhbHLH may play important role in the stress response in cotton.
bHLH(basic helix-loop-helix,bHLH) gene family transcription factors play important roles in plant growth and abiotic stress response.In this study,we cloned a new bHLH gene from a normolized cDNA library,which was named GhbHLH.GhbHLH contained an open reading frame(ORF) of 600 bp,which encoded a protein with 199 amino acids,and contained an Helix-Loop-Helix conserved domain.Quantitative RT-PCR analysis showed that GhbHLH expressed in root,stem,leaf and fiber,and was induced by salt,PEG,cold,ABA and Me JA,subcellular localization analysis showed that GhbHLH-GFP fusion protein was distributed specifically in cell nucleus.Our data indicated GhbHLH may play important role in the stress response in cotton.
引文
Abe H.,Urao T.,Ito T.,Seki M.,Shinozaki K.,and YamaguchiShinozaki K.,2003,Arabidopsis At MYC2(b HLH)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling,The Plant Cell,15(1):63-78
Chen Y.,Li F.,Ma Y.,Chong K.,and Xu Y.Y.,2013,Overexpression of Orb HLH001,a putative helix-loop-helix transcription factor,causes increased expression of AKT1 and maintains ionic balance under salt stress in rice,J.Plant Physiol.,170(1):93-100
Chen Y.Y.,Li M.Y.,Wu X.J.,Huang Y.,Ma J.,and Xiong A.S.,2015,Genome-wide analysis of basic helix-loop-helix family transcription factors and their role in responses to abiotic stress in carrot,Mol.Breeding,35(5):1-12
Cheng Z.W.,Sun L.,Qi T.C.,Zhang B.S.,Peng W.,Liu Y.L.,and Xie D.X.,2011,The b HLH transcription factor MYC3 interacts with the jasmonate ZIM-Domain proteins to mediate jasmonate response in Arabidopsis,Mol.Plant.,4(2):279-288
Chi J.N.,Cai X.,Zhang J.H.,Zhen J.B.,Liu L.L.,Tian H.Y.,Tang L.Y.,Liu C.J.,Cui R.M.,and Zhang X.Y.,2016,Establishment and identification of a normalized full-Length c DNA library of upland cotton Ji228,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(5):813-824(迟吉娜,蔡肖,张建宏,甄军波,刘琳琳,田海燕,唐丽媛,刘存敬,崔瑞敏,张香云,2016,冀228纤维均一化全长c DNA文库的构建与鉴定分析,中国农业科学49(5):813-824)
Chinnusamy V.,Ohta M.,Kanrar S.,Lee B.H.,Hong X.,Agarwal M.,and Zhu J.K.,2003,ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis,Genes Dev.,17(8):1043-1054
Ding W.,Yu Z.M.,Tong Y.L.,Huang W.,Chen H.M.,and Wu P.,2009,A transcription factor with a b HLH domain regulates root hair development in rice,Cell Res.,19(11):1309-1311
Fernández-Calvo P.,Chini A.,Fernández-Barbero G.,Chico J.M.,Gimenez-Ibanez S.,Geerinck J.,Eeckhout D.,Schweizer F.,Godoy M.,Franco-Zorrilla J.M.,Pauwels L.,Witters E.,Puga M.I.,Paz-Ares J.,Goossens A.,Reymond P.,De Jaeger G.,and Solano R.,2011,The Arabidopsis b HLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses,The Plant Cell,23(2):701-715
Guang Y.Q.,Song G.C.,Zhang J.F.,Wang X.N.,and Tang C.M.,2014,Molecular cloning and expression analysis of Ghb HL-H130,encoding a novel b HLH transcription factor in upland cotton(Gossypium hirsutum L.),Mianhua Xuebao(Cotton Science),26(4):363-370(光杨其,宋桂成,张金凤,王晓楠,唐灿明,2014,1个新棉花b HLH类基因Ghb HLH130的克隆及表达分析,棉花学报,26(4):363-370)
Heang D.,and Sassa H.,2012a,Antagonistic actions of HLH/b HLH proteins are involved in grain length and weight in rice,PLo S One,7(2):e31325
Heang D.,and Sassa H.,2012b,Overexpression of a basic helix-loop-helix gene Antagonist of PGL1(APG)decreases grain length of rice,Plant Biotechnol.,29(1):65-69
Kavas M.,Baloglu M.C.,Atabay E.S.,Ziplar U.T.,Dasgan H.Y.,and Unver T.,2015,Genome-wide characterization and expression analysis of common bean b HLH transcription factors in response to excess salt concentration,Mol.Genet.Genomics,291(1):129-143
Li F.,Guo S.Y.,Zhao Y.,Chen D.Z.,Chong K.,and Xu Y.Y.,2010,Overexpression of a homopeptide repeat-containing b HLH protein gene(Orb HLH001)from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis,Plant Cell Rep.,29(9):977-986
Meng C.M.,Zhang T.Z.,and Guo W.Z.,2009,Molecular cloning and characterization of a novel Gossypium hirsutum L.,b HLH gene in response to ABA and drought stresses,Plant Mol.Biol.Rep.,27(3):381-387
Payne C.T.,Zhang F.,and Lloyd A.M.,2000,GL3 encodes a b HLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1,Genetics,156(3):1349-1362
Seo J.S.,Joo J.S.,Kim M.J.,Kim Y.K.,Nahm B.H.,Song S.I.,Cheong J.J.,Lee J.S.,Kim J.K.,and Choi Y.D.,2011,Osb HLH148,a basic helix-loop-helix protein,interacts with Os JAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice,Plant J.,65(6):907-921
Song X.M.,Huang Z.N.,Duan W.K.,Ren J.,Liu T.K.,Li Y.,and Hou X.L.,2014,Genome-wide analysis of the b HLH transcription factor family in Chinese cabbage(Brassica rapa ssp.pekinensis),Mol.Genet.Genomics,289(1):77-91
Toledo-Ortiz G.,Huq E.,and Quail P.H.,2003,The Arabidopsis basic/helix-loop-helix transcription factor family,The Plant Cell,15(8):1749-1770
Wang Y.J.,Zhang Z.G.,He X.J.,Zhou H.L.,Wen Y.X.,Dai J.X.,Zhang J.S.,and Chen S.Y.,2003,A rice transcription factor Osb HLH1 is involved in cold stress response,Theor.App.Genet.,107(8):1402-1409
Wu H,Ye H.Y.,Yao R.F.,Zhang T.,and Xiong L.Z.,2015,Os JAZ9acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice,Plant Sci.,232:1-12
Yan Q.,Liu H.S.,Yao D.,Li X.,Chen H.,Dou Y.,Wang Y.,Pei Y.,and Xiao Y.H.,2015,The Basic/Helix-Loop-Helix protein family in Gossypium:reference genes and their evolution during tetraploidization,PLo S One,10(5):e0126558
Yoshida T.,Mogami J.,and Yamaguchi-Shinozaki K.,2014,A-BA-dependent and ABA-independent signaling in response to osmotic stress in plants,Curr.Opin.Plant.Biol.,21:133-139
Zhang L.,Chen J.F.,Li Q.,and Chen W.S.,2016,Transcriptome-wide analysis of basic helix-loop-helix transcription factors in Isatis indigotica and their methyl jasmonate responsive expression profiling,Gene,576(1):150-159
Zhang L.Y.,Bai M.Y.,Wu J.X.,Zhu J.Y.,Wang H.,Zhang Z.G.,Wang W.F.,Sun Y.,Zhao J.,Sun X.H.,Yang H.J.,Xu Y.Y.,Kim S.H.,Fujioka S.,Lin W.H.,Chong K.,Lu T.G.,and Wang Z.Y.,2009,Antagonistic HLH/b HLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis,The Plant Cell,21(12):3767-3780
Zhang X.,Zhen J.B.,Li Z.H.,Kang D.M.,Yang Y.M.,Kong J.,and Hua J.P.,2011,Expression profile of early responsive genes under salt stress in upland cotton(Gossypium hirsutum L.),Plant Mol.Biol.Rep.,29(3):626-637
Zhen J.B.,Zhang X.,Wang Y.M.,Zhai Z.X.,Li Z.H.,and Hua J.P.,2011,Isolation and sequence analysis of salt responsive gene Gh CPK5 from Gossypium hirsutum L.,Zhongguo Nongye Daxue Xuebao(Journal of China Agricuitural university),16(3):8-14(甄军波,张曦,王玉美,翟志席,李召虎,华金平,2011,棉花盐胁迫应答基因Gh CPK5的克隆及序列分析,中国农业大学学报,16(3):8-14)
Zhou J.,Li F.,Wang J.L.,Ma Y.,Chong K.,and Xu Y.Y.,2009,Basic helix-loop-helix transcription factor from wild rice(Orb HLH2)improves tolerance to salt-and osmotic stress in Arabidopsis,J.Plant Physiol.,166(12):1296-1306
Chen Y.,Li F.,Ma Y.,Chong K.,and Xu Y.Y.,2013,Overexpression of Orb HLH001,a putative helix-loop-helix transcription factor,causes increased expression of AKT1 and maintains ionic balance under salt stress in rice,J.Plant Physiol.,170(1):93-100
Chen Y.Y.,Li M.Y.,Wu X.J.,Huang Y.,Ma J.,and Xiong A.S.,2015,Genome-wide analysis of basic helix-loop-helix family transcription factors and their role in responses to abiotic stress in carrot,Mol.Breeding,35(5):1-12
Cheng Z.W.,Sun L.,Qi T.C.,Zhang B.S.,Peng W.,Liu Y.L.,and Xie D.X.,2011,The b HLH transcription factor MYC3 interacts with the jasmonate ZIM-Domain proteins to mediate jasmonate response in Arabidopsis,Mol.Plant.,4(2):279-288
Chi J.N.,Cai X.,Zhang J.H.,Zhen J.B.,Liu L.L.,Tian H.Y.,Tang L.Y.,Liu C.J.,Cui R.M.,and Zhang X.Y.,2016,Establishment and identification of a normalized full-Length c DNA library of upland cotton Ji228,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(5):813-824(迟吉娜,蔡肖,张建宏,甄军波,刘琳琳,田海燕,唐丽媛,刘存敬,崔瑞敏,张香云,2016,冀228纤维均一化全长c DNA文库的构建与鉴定分析,中国农业科学49(5):813-824)
Chinnusamy V.,Ohta M.,Kanrar S.,Lee B.H.,Hong X.,Agarwal M.,and Zhu J.K.,2003,ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis,Genes Dev.,17(8):1043-1054
Ding W.,Yu Z.M.,Tong Y.L.,Huang W.,Chen H.M.,and Wu P.,2009,A transcription factor with a b HLH domain regulates root hair development in rice,Cell Res.,19(11):1309-1311
Fernández-Calvo P.,Chini A.,Fernández-Barbero G.,Chico J.M.,Gimenez-Ibanez S.,Geerinck J.,Eeckhout D.,Schweizer F.,Godoy M.,Franco-Zorrilla J.M.,Pauwels L.,Witters E.,Puga M.I.,Paz-Ares J.,Goossens A.,Reymond P.,De Jaeger G.,and Solano R.,2011,The Arabidopsis b HLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses,The Plant Cell,23(2):701-715
Guang Y.Q.,Song G.C.,Zhang J.F.,Wang X.N.,and Tang C.M.,2014,Molecular cloning and expression analysis of Ghb HL-H130,encoding a novel b HLH transcription factor in upland cotton(Gossypium hirsutum L.),Mianhua Xuebao(Cotton Science),26(4):363-370(光杨其,宋桂成,张金凤,王晓楠,唐灿明,2014,1个新棉花b HLH类基因Ghb HLH130的克隆及表达分析,棉花学报,26(4):363-370)
Heang D.,and Sassa H.,2012a,Antagonistic actions of HLH/b HLH proteins are involved in grain length and weight in rice,PLo S One,7(2):e31325
Heang D.,and Sassa H.,2012b,Overexpression of a basic helix-loop-helix gene Antagonist of PGL1(APG)decreases grain length of rice,Plant Biotechnol.,29(1):65-69
Kavas M.,Baloglu M.C.,Atabay E.S.,Ziplar U.T.,Dasgan H.Y.,and Unver T.,2015,Genome-wide characterization and expression analysis of common bean b HLH transcription factors in response to excess salt concentration,Mol.Genet.Genomics,291(1):129-143
Li F.,Guo S.Y.,Zhao Y.,Chen D.Z.,Chong K.,and Xu Y.Y.,2010,Overexpression of a homopeptide repeat-containing b HLH protein gene(Orb HLH001)from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis,Plant Cell Rep.,29(9):977-986
Meng C.M.,Zhang T.Z.,and Guo W.Z.,2009,Molecular cloning and characterization of a novel Gossypium hirsutum L.,b HLH gene in response to ABA and drought stresses,Plant Mol.Biol.Rep.,27(3):381-387
Payne C.T.,Zhang F.,and Lloyd A.M.,2000,GL3 encodes a b HLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1,Genetics,156(3):1349-1362
Seo J.S.,Joo J.S.,Kim M.J.,Kim Y.K.,Nahm B.H.,Song S.I.,Cheong J.J.,Lee J.S.,Kim J.K.,and Choi Y.D.,2011,Osb HLH148,a basic helix-loop-helix protein,interacts with Os JAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice,Plant J.,65(6):907-921
Song X.M.,Huang Z.N.,Duan W.K.,Ren J.,Liu T.K.,Li Y.,and Hou X.L.,2014,Genome-wide analysis of the b HLH transcription factor family in Chinese cabbage(Brassica rapa ssp.pekinensis),Mol.Genet.Genomics,289(1):77-91
Toledo-Ortiz G.,Huq E.,and Quail P.H.,2003,The Arabidopsis basic/helix-loop-helix transcription factor family,The Plant Cell,15(8):1749-1770
Wang Y.J.,Zhang Z.G.,He X.J.,Zhou H.L.,Wen Y.X.,Dai J.X.,Zhang J.S.,and Chen S.Y.,2003,A rice transcription factor Osb HLH1 is involved in cold stress response,Theor.App.Genet.,107(8):1402-1409
Wu H,Ye H.Y.,Yao R.F.,Zhang T.,and Xiong L.Z.,2015,Os JAZ9acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice,Plant Sci.,232:1-12
Yan Q.,Liu H.S.,Yao D.,Li X.,Chen H.,Dou Y.,Wang Y.,Pei Y.,and Xiao Y.H.,2015,The Basic/Helix-Loop-Helix protein family in Gossypium:reference genes and their evolution during tetraploidization,PLo S One,10(5):e0126558
Yoshida T.,Mogami J.,and Yamaguchi-Shinozaki K.,2014,A-BA-dependent and ABA-independent signaling in response to osmotic stress in plants,Curr.Opin.Plant.Biol.,21:133-139
Zhang L.,Chen J.F.,Li Q.,and Chen W.S.,2016,Transcriptome-wide analysis of basic helix-loop-helix transcription factors in Isatis indigotica and their methyl jasmonate responsive expression profiling,Gene,576(1):150-159
Zhang L.Y.,Bai M.Y.,Wu J.X.,Zhu J.Y.,Wang H.,Zhang Z.G.,Wang W.F.,Sun Y.,Zhao J.,Sun X.H.,Yang H.J.,Xu Y.Y.,Kim S.H.,Fujioka S.,Lin W.H.,Chong K.,Lu T.G.,and Wang Z.Y.,2009,Antagonistic HLH/b HLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis,The Plant Cell,21(12):3767-3780
Zhang X.,Zhen J.B.,Li Z.H.,Kang D.M.,Yang Y.M.,Kong J.,and Hua J.P.,2011,Expression profile of early responsive genes under salt stress in upland cotton(Gossypium hirsutum L.),Plant Mol.Biol.Rep.,29(3):626-637
Zhen J.B.,Zhang X.,Wang Y.M.,Zhai Z.X.,Li Z.H.,and Hua J.P.,2011,Isolation and sequence analysis of salt responsive gene Gh CPK5 from Gossypium hirsutum L.,Zhongguo Nongye Daxue Xuebao(Journal of China Agricuitural university),16(3):8-14(甄军波,张曦,王玉美,翟志席,李召虎,华金平,2011,棉花盐胁迫应答基因Gh CPK5的克隆及序列分析,中国农业大学学报,16(3):8-14)
Zhou J.,Li F.,Wang J.L.,Ma Y.,Chong K.,and Xu Y.Y.,2009,Basic helix-loop-helix transcription factor from wild rice(Orb HLH2)improves tolerance to salt-and osmotic stress in Arabidopsis,J.Plant Physiol.,166(12):1296-1306