甘蓝转录因子BoLH27的克隆与转基因甘蓝的表型分析
|
摘要
bHLH转录因子对植物生长发育、形态调控有重要作用,为了研究BoLH27基因在甘蓝叶片发育及形态建成中的调控功能,本文以甘蓝品种519为材料,克隆出转录因子BoLH27基因。序列分析表明,该基因含有一个795 bp的开放阅读框,编码264个氨基酸,具有一个保守的典型bHLH结构域。以农杆菌介导的遗传转化方法将正义BoLH27基因导入甘蓝品种519中以增强表达,通过PCR筛选出9株T0代转基因单株,结合T2代单株的q RT-PCR分析表明,筛选的转基因植株内BoLH27基因的表达积累量明显高于野生型。试验基地隔离网内种植的转基因甘蓝表型明显,主要表现为莲座期茎叶间距拉长、叶柄伸长以及植株茎和叶柄显示紫色,植株叶片平展,无向上向内卷曲趋势,表明BoLH27基因可能对甘蓝叶片发育有重要的调控作用。
b HLH transcription factor plays an important role in plant growth, development and morphological control. In order to explore BoLH27 gene regulatory function for leaf development and morphologic formation, the BoLH27 gene was cloned from cabbage(Brassica oleracea L.) variety 519. Sequence analysis indicated that the length of BoLH27 gene was 795 bp, which encoded 264 amino acids. The BoLH27 protein contained conservative structure domains of the b HLH family. The sense BoLH27 gene was transformed into cabbage variety 519 by Agrobacterium mediated method, PCR analysis exhibited that BoLH27 was genetically transformed into nine individual plants, q RT-PCR analysis revealed that BoLH27 had higher expression level in T2 transgenic cabbage than in wild type. The phenotype at rosette stage of transgenic cabbage grown in the test base was obvious, showing elongated the stems and leaves, elongated petiole, purple stems and petiole, and flat leaves without upward inward curling trend. It suggested that BoLH27 may play important roles in the control of leaves development.
b HLH transcription factor plays an important role in plant growth, development and morphological control. In order to explore BoLH27 gene regulatory function for leaf development and morphologic formation, the BoLH27 gene was cloned from cabbage(Brassica oleracea L.) variety 519. Sequence analysis indicated that the length of BoLH27 gene was 795 bp, which encoded 264 amino acids. The BoLH27 protein contained conservative structure domains of the b HLH family. The sense BoLH27 gene was transformed into cabbage variety 519 by Agrobacterium mediated method, PCR analysis exhibited that BoLH27 was genetically transformed into nine individual plants, q RT-PCR analysis revealed that BoLH27 had higher expression level in T2 transgenic cabbage than in wild type. The phenotype at rosette stage of transgenic cabbage grown in the test base was obvious, showing elongated the stems and leaves, elongated petiole, purple stems and petiole, and flat leaves without upward inward curling trend. It suggested that BoLH27 may play important roles in the control of leaves development.
引文
[1]Ludwig S R,Habera L F,Dellaporta S L,Wessler S R.Lc,a member of the maize R gene family responsible for tissuespecific anthocyanin production,encodes a protein similar to transcriptional activators and contains the myc-homology region.Proc Natl Acad Sci USA,1989,86:7092–7096
[2]Ling H Q,Bauer P,Bereczky Z,Keller B,Ganal M.The tomato FER gene encoding a b HLH protein controls iron-uptake responses in roots.Proc Natl Acad Sci USA,2002,99:13938–13943
[3]Kiribuchi K,Jikumaru Y,Kaku H,Minami E,Hasegawa M,Kodama O.Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants.Biosci Biotechnol Biochem,2005,69:1042–1044
[4]Carreteropaulet L,Galstyan A,Roigvillanova I,Martínezgarcía J F,Bilbaocastro J R,Robertson D L.Genome-wide classification and evolutionary analysis of the b HLH family of transcription factors in Arabidopsis,poplar,rice,moss,and algae.Plant Physiol,2010,153:1398–1412
[5]Pires N,Dolan L.Origin and diversification of basic-helixloop-helix proteins in plants.Mol Biol Evol,2010,27:862–874
[6]Grove C A,De M F,Barrasa M I,Newburger D E,Alkema M J,Bulyk M L.A multiparameter network reveals extensive divergence between C.elegans b HLH transcription factors.Cell,2009,138:314–327
[7]Toledo-Ortiz G,Huq E,Quail P H.The Arabidopsis basic-helix-loop-helix transcription factor family.Plant Cell,2003,15:1749–1770
[8]Li X,Duan X,Jiang H,Sun Y,Tang Y,Yuan Z.Genome-wide analysis of basic-helix-loop-helix transcription factor family in rice and Arabidopsis.Plant Physiol,2006,141:1167–1184
[9]Atchley W R,Terhalle W,Dress A.Positional dependence,cliques,and predictive motifs in the b HLH protein domain.J Mol Evol,1999,48:501–516
[10]Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Bailey P C.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Mol Biol Evol,2003,20:735–747
[11]Yin J,Chang X,Kasuga T,Mai B,Reid M S,Jiang C Z.A basic helix-loop-helix transcription factor,Ph FBH4,regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.Hortic Res,2015,2:15059–15068
[12]Ko S S,Li M J,Sun-Ben K M,Ho Y C,Lin Y J,Chuang M H.The b HLH142 transcription factor coordinates with TDR1 to modulate the expression of EAT1 and regulate pollen development in rice.Plant Cell,2014,26:2486–2504
[13]Takahashi Y,Ebisu Y,Kinoshita T,Doi M,Okuma E,Murata Y.b HLH transcription factors that facilitate K?uptake during stomatal opening are repressed by abscisic acid through phosphorylation.Sci Signal,2013,6:ra48
[14]Shen Q,Lu X,Yan T,Fu X,Lv Z,Zhang F.The jasmonate-responsive Aa MYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua.New Phytol,2016,210:1269–1281
[15]Kurbidaeva A,Ezhova T,Novokreshchenova M.Arabidopsis thaliana ICE2 gene:phylogeny,structural evolution and functional diversification from ICE1.Plant Sci,2014,229:10–22
[16]Makkena S,Lamb R S.The b HLH transcription factor SPATULA is a key regulator of organ size in Arabidopsis thaliana.Plant Signal Behav,2013,8:e24140
[17]An R,Liu X,Wang R,Wu H,Liang S,Shao J,Qi Y,An L,Yu F.The over-expression of two transcription factors,ABS5/b HLH30and ABS7/MYB101,leads to upwardly curly leaves.PLo S One,2014,9:e107637
[18]Nath U,Crawford B C,Carpenter R,Coen E.Genetic control of surface curvature.Science,2003,299:1404–1407
[19]Qin G,Gu H,Zhao Y,Ma Z,Shi G,Yang Y.An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development.Plant Cell,2005,17:2693–2704
[20]Sarvepalli K,Nath U.Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation.Plant J,2011,67:595–607
[21]Efroni I,Blum E,Goldshmidt A,Eshed Y.A protracted and dynamic maturation schedule underlies Arabidopsis leaf development.Plant Cell,2008,20:2293–2306
[22]莫晓婷.玉米逆境相关转录因子的克隆与初步分析.中国农业科学院硕士学位论文.北京,2013Mo X T.Cloning and Functional Characterization of Stress-related Transcription Factors in Maize.MS Thesis of Chinese Academy of Agricultural Sciences,Beijing,China,2013(in Chinese with English abstract)
[23]何绍敏,李春雨,兰彩耘,邹敏,任雪松,司军,李成琼,宋洪元.转MLPK反义基因对甘蓝自交不亲和性的影响.园艺学报,2015,42:252–262He S M,Li C Y,Lan C Y,Zou M,Ren X S,Si J,Li C Q,Song H Y.Effect of antisense RNA of the MLPK gene on self-incompatibility in cabbage.Acta Hortic Sin,2015,42:252–262(in Chinese with English abstract)
[24]Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Bailey P C.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Mol Biol Evol,2003,20:735–747
[25]Grotewold E,Sainz M B,Tagliani L,Hernandez J M,Bowen B,Chandler V L.Identification of the residues in the Myb domain of maize C1 that specify the interaction with the b HLH cofactor R.Proc Natl Acad Sci USA,2000,97:13579–13584
[26]Bernhardt C,Lee M M,Gonzalez A,Zhang F,Lloyd A,Schiefelbein J.The b HLH genes GLABRA3(GL3)and ENHANCER OF GLABRA3(EGL3)specify epidermal cell fate in the Arabidopsis root.Development,2003,130:6431–6439
[27]Zhang L Y,Bai M Y,Wu J,Zhu J Y,Wang H,Zhang Z.Antagonistic HLH/b HLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis.Plant Cell,2009,21:3767–3780
[28]Meng Y,Li H,Wang Q,Liu B,Lin C.Blue light-dependent interaction between cryptochrome 2 and CIB1 regulates transcription and leaf senescence in soybean.Plant Cell,2013,25:4405–4420
[29]Husbands A,Bell E M,Shuai B,Smith H M S,Springer P S.LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific b HLH proteins.Nucl Acids Res,2007,35:6663–6671
[30]Shuai B,Reynagape?a C G,Springer P S.The lateral organ boundaries gene defines a novel,plant-specific gene family.Plant Physiol,2002,129:747–761
[31]Aguilar-Martínez J A,Sinha N.Analysis of the role of Arabidopsis class I TCP genes At TCP7,At TCP8,At TCP22,and At TCP23in leaf development.Front Plant Sci,2013,8:e24140
[32]Guo Z,Fujioka S,Blancaflor E B,Miao S,Gou X,Li J.TCP1modulates brassinosteroid biosynthesis by regulating the expression of the key biosynthetic gene DWARF4 in Arabidopsis thaliana.Plant Cell,2010,22:1161–1173
[33]Palatnik J F,Allen E,Wu X,Schommer C,Schwab R,Carrington J C.Control of leaf morphogenesis by micro RNAs.Nature,2003,425:257–263
[34]Chandler V L,Radicella J P,Robbins T P,Chen J,Turks D.Two regulatory genes of the maize anthocyanin pathway are homologous:isolation of B utilizing R genomic sequences.Plant Cell,1989,1:1175–1183
[35]de Vetten N,Quattrocchio F,Mol J,Koes R.The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast,plants,and animals.Genes Dev,1997,11:1422–1434
[36]Baudry A,Caboche M,Lepiniec L.TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and b HLH factors,allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana.Plant Mol Biol,2006,46:768–779
[37]Zhang F,Gonzalez A,Zhao M,Payne C T,Lloyd A.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis.Development,2003,130:4859–4869
[38]Liu X F,Yin X R,Allan A C,Wang K L,Shi Y N,Huang Y J.The role of Mrb HLH1,and Mr MYB1,in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry(Myrica rubra)during anthocyanin biosynthesis.Plant Cell Tissue Org,2013,115:285–298
[39]Chiu L W,Zhou X,Burke S,Wu X,Prior R L,Li L.The purple cauliflower arises from activation of a MYB transcription factor.Plant Physiol,2010,154:1470–1480
[2]Ling H Q,Bauer P,Bereczky Z,Keller B,Ganal M.The tomato FER gene encoding a b HLH protein controls iron-uptake responses in roots.Proc Natl Acad Sci USA,2002,99:13938–13943
[3]Kiribuchi K,Jikumaru Y,Kaku H,Minami E,Hasegawa M,Kodama O.Involvement of the basic helix-loop-helix transcription factor RERJ1 in wounding and drought stress responses in rice plants.Biosci Biotechnol Biochem,2005,69:1042–1044
[4]Carreteropaulet L,Galstyan A,Roigvillanova I,Martínezgarcía J F,Bilbaocastro J R,Robertson D L.Genome-wide classification and evolutionary analysis of the b HLH family of transcription factors in Arabidopsis,poplar,rice,moss,and algae.Plant Physiol,2010,153:1398–1412
[5]Pires N,Dolan L.Origin and diversification of basic-helixloop-helix proteins in plants.Mol Biol Evol,2010,27:862–874
[6]Grove C A,De M F,Barrasa M I,Newburger D E,Alkema M J,Bulyk M L.A multiparameter network reveals extensive divergence between C.elegans b HLH transcription factors.Cell,2009,138:314–327
[7]Toledo-Ortiz G,Huq E,Quail P H.The Arabidopsis basic-helix-loop-helix transcription factor family.Plant Cell,2003,15:1749–1770
[8]Li X,Duan X,Jiang H,Sun Y,Tang Y,Yuan Z.Genome-wide analysis of basic-helix-loop-helix transcription factor family in rice and Arabidopsis.Plant Physiol,2006,141:1167–1184
[9]Atchley W R,Terhalle W,Dress A.Positional dependence,cliques,and predictive motifs in the b HLH protein domain.J Mol Evol,1999,48:501–516
[10]Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Bailey P C.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Mol Biol Evol,2003,20:735–747
[11]Yin J,Chang X,Kasuga T,Mai B,Reid M S,Jiang C Z.A basic helix-loop-helix transcription factor,Ph FBH4,regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.Hortic Res,2015,2:15059–15068
[12]Ko S S,Li M J,Sun-Ben K M,Ho Y C,Lin Y J,Chuang M H.The b HLH142 transcription factor coordinates with TDR1 to modulate the expression of EAT1 and regulate pollen development in rice.Plant Cell,2014,26:2486–2504
[13]Takahashi Y,Ebisu Y,Kinoshita T,Doi M,Okuma E,Murata Y.b HLH transcription factors that facilitate K?uptake during stomatal opening are repressed by abscisic acid through phosphorylation.Sci Signal,2013,6:ra48
[14]Shen Q,Lu X,Yan T,Fu X,Lv Z,Zhang F.The jasmonate-responsive Aa MYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua.New Phytol,2016,210:1269–1281
[15]Kurbidaeva A,Ezhova T,Novokreshchenova M.Arabidopsis thaliana ICE2 gene:phylogeny,structural evolution and functional diversification from ICE1.Plant Sci,2014,229:10–22
[16]Makkena S,Lamb R S.The b HLH transcription factor SPATULA is a key regulator of organ size in Arabidopsis thaliana.Plant Signal Behav,2013,8:e24140
[17]An R,Liu X,Wang R,Wu H,Liang S,Shao J,Qi Y,An L,Yu F.The over-expression of two transcription factors,ABS5/b HLH30and ABS7/MYB101,leads to upwardly curly leaves.PLo S One,2014,9:e107637
[18]Nath U,Crawford B C,Carpenter R,Coen E.Genetic control of surface curvature.Science,2003,299:1404–1407
[19]Qin G,Gu H,Zhao Y,Ma Z,Shi G,Yang Y.An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development.Plant Cell,2005,17:2693–2704
[20]Sarvepalli K,Nath U.Hyper-activation of the TCP4 transcription factor in Arabidopsis thaliana accelerates multiple aspects of plant maturation.Plant J,2011,67:595–607
[21]Efroni I,Blum E,Goldshmidt A,Eshed Y.A protracted and dynamic maturation schedule underlies Arabidopsis leaf development.Plant Cell,2008,20:2293–2306
[22]莫晓婷.玉米逆境相关转录因子的克隆与初步分析.中国农业科学院硕士学位论文.北京,2013Mo X T.Cloning and Functional Characterization of Stress-related Transcription Factors in Maize.MS Thesis of Chinese Academy of Agricultural Sciences,Beijing,China,2013(in Chinese with English abstract)
[23]何绍敏,李春雨,兰彩耘,邹敏,任雪松,司军,李成琼,宋洪元.转MLPK反义基因对甘蓝自交不亲和性的影响.园艺学报,2015,42:252–262He S M,Li C Y,Lan C Y,Zou M,Ren X S,Si J,Li C Q,Song H Y.Effect of antisense RNA of the MLPK gene on self-incompatibility in cabbage.Acta Hortic Sin,2015,42:252–262(in Chinese with English abstract)
[24]Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Bailey P C.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Mol Biol Evol,2003,20:735–747
[25]Grotewold E,Sainz M B,Tagliani L,Hernandez J M,Bowen B,Chandler V L.Identification of the residues in the Myb domain of maize C1 that specify the interaction with the b HLH cofactor R.Proc Natl Acad Sci USA,2000,97:13579–13584
[26]Bernhardt C,Lee M M,Gonzalez A,Zhang F,Lloyd A,Schiefelbein J.The b HLH genes GLABRA3(GL3)and ENHANCER OF GLABRA3(EGL3)specify epidermal cell fate in the Arabidopsis root.Development,2003,130:6431–6439
[27]Zhang L Y,Bai M Y,Wu J,Zhu J Y,Wang H,Zhang Z.Antagonistic HLH/b HLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis.Plant Cell,2009,21:3767–3780
[28]Meng Y,Li H,Wang Q,Liu B,Lin C.Blue light-dependent interaction between cryptochrome 2 and CIB1 regulates transcription and leaf senescence in soybean.Plant Cell,2013,25:4405–4420
[29]Husbands A,Bell E M,Shuai B,Smith H M S,Springer P S.LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific b HLH proteins.Nucl Acids Res,2007,35:6663–6671
[30]Shuai B,Reynagape?a C G,Springer P S.The lateral organ boundaries gene defines a novel,plant-specific gene family.Plant Physiol,2002,129:747–761
[31]Aguilar-Martínez J A,Sinha N.Analysis of the role of Arabidopsis class I TCP genes At TCP7,At TCP8,At TCP22,and At TCP23in leaf development.Front Plant Sci,2013,8:e24140
[32]Guo Z,Fujioka S,Blancaflor E B,Miao S,Gou X,Li J.TCP1modulates brassinosteroid biosynthesis by regulating the expression of the key biosynthetic gene DWARF4 in Arabidopsis thaliana.Plant Cell,2010,22:1161–1173
[33]Palatnik J F,Allen E,Wu X,Schommer C,Schwab R,Carrington J C.Control of leaf morphogenesis by micro RNAs.Nature,2003,425:257–263
[34]Chandler V L,Radicella J P,Robbins T P,Chen J,Turks D.Two regulatory genes of the maize anthocyanin pathway are homologous:isolation of B utilizing R genomic sequences.Plant Cell,1989,1:1175–1183
[35]de Vetten N,Quattrocchio F,Mol J,Koes R.The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast,plants,and animals.Genes Dev,1997,11:1422–1434
[36]Baudry A,Caboche M,Lepiniec L.TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and b HLH factors,allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana.Plant Mol Biol,2006,46:768–779
[37]Zhang F,Gonzalez A,Zhao M,Payne C T,Lloyd A.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis.Development,2003,130:4859–4869
[38]Liu X F,Yin X R,Allan A C,Wang K L,Shi Y N,Huang Y J.The role of Mrb HLH1,and Mr MYB1,in regulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry(Myrica rubra)during anthocyanin biosynthesis.Plant Cell Tissue Org,2013,115:285–298
[39]Chiu L W,Zhou X,Burke S,Wu X,Prior R L,Li L.The purple cauliflower arises from activation of a MYB transcription factor.Plant Physiol,2010,154:1470–1480