蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析
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摘要
bHLH(Basic helix-loop-helix, bHLH)转录因子家族是植物最大的转录因子家族之一,广泛参与植物生长发育和盐胁迫应答机制。该研究利用同源克隆的方法克隆蒺藜苜蓿(Medicago truncatula)的MtbHLH148基因,采用qRT-PCR方法分析MtbHLH148基因在蒺藜苜蓿中的表达特性,构建超表达载体并通过农杆菌侵染法转化拟南芥(Arabidopsis thaliana),对转基因拟南芥的耐盐性相关功能进行分析研究。结果显示:(1)从蒺藜苜蓿中获得MtbHLH148基因,该基因cDNA全长1 343 bp,包含开放阅读框为603 bp,编码201个氨基酸,蛋白分子量22.7 kD,等电点为11.76;蛋白结构分析显示,该蛋白无跨膜结构域,无信号肽,为亲水性蛋白;含有精氨酸/赖氨酸残基的保守结构域和典型的bHLH结构域;二级结构以α-螺旋和无规则卷曲为主。(2)亚细胞定位表明,MtbHLH148蛋白定位在细胞核。(3)进化树分析表明,MtbHLH148与大豆(Glycine max)的亲缘性最近;启动子分析发现,该基因启动子区域含有光响应元件、MYB结合位点以及ABA应答元件ABRE,可能参与非生物胁迫。(4)qRT-PCR分析发现,MtbHLH148基因在蒺藜苜蓿的茎中表达量最高,叶中表达量最低,且MtbHLH148基因受ABA(100μmol/L)诱导并在盐胁迫(200 mmol/L NaCl)处理8 h内表达量上调,而在低温(4℃)处理时表达量明显下调。(5)成功构建超表达载体pCAMBIA3301-MtbHLH148并转化拟南芥获得16个抗性株系,经鉴定有12个过表达株系,其中表达量最高的转基因株系为OE8;对OE8株系耐盐性功能分析发现,转基因拟南芥植株的发芽率明显高于野生型,盐胁迫下转基因拟南芥的根长是野生型的1.5倍,表明其耐盐性得到了增强。研究表明,MtbHLH148基因可能在盐胁迫调节机制中具有一定的调控作用。
The bHLH transcription factor family, one of the largest transcription factor families in plants, has been reported to involve in plant growth and salt stress response. In this study, homologous cloning was used to acquire the MtbHLH148 gene from barrel clover(Medicago truncatula). qRT-PCR was used to analyze the expression pattern of MtbHLH148 gene in barrel clover. The overexpression vector was constructed and transformed into Arabidopsis through agrobacterium infection, so as to analyze the related functions of salt tolerance of transgenic Arabidopsis. The result shows:(1) the full length of MtbHLH148 cDNA was 1 343 bp, Which contained 603 bp open reading frame and encoding 201 amino acids. The theoretical pI of MtbHLH148 protein was 11.76 and whoes molecular weight was 22.7 kD. Protein structure analysis showed that the protein had no transmembrane domain, no signal peptide and it was a hydrophilic protein. This gene contained highly conserved bHLH domains. As expected, the secondary structure was predominatly α-helix and random curl.(2) Subcellular localization results showed that the proteins were locate in the nucleus.(3) Phylogenetic analysis revealed that MtbHLH148 was closely related to Glycine max Analysis of the cis-regulatory element demonstrated that the promoters of MtbHLH148 contained light, hormone and stress response elements, suggesting their involvement in the biological processes.(4) qRT-PCR analysis of the expression pattern of the MtbHLH148 in barrel clover showed that the highest level in stem and the lowest level in leaf. For treatment, the genes were induced by ABA(100 μmol/L), and were repressed by cold(4 ℃) but up-regulated by NaCl(200 mmol/L) within the first 8 h.(5) The pCAMBIA3301-MtbHLH148 overexpression vector was successfully constructed and transformed into Arabidopsis. We obtained 16 resistant lines and identified 12 over-expressed lines. Among these over-expressed lines, the transgenic line OE8 had the highest expression level. Analysis of salt-tolerance function of OE8 showed that a significantly higher germination rate was observed in spite of their indiscernible phenotypic difference from wild type. Statistical analysis showed that the root length of the transgenic seedlings over-expressing MtbHLH148 was about 1.5 times of the non-transgenic plants, suggesting enhanced salt tolerance. Based on these results, we infer that MtbHLH148 may play a regulatory role in plant response to salinity.
The bHLH transcription factor family, one of the largest transcription factor families in plants, has been reported to involve in plant growth and salt stress response. In this study, homologous cloning was used to acquire the MtbHLH148 gene from barrel clover(Medicago truncatula). qRT-PCR was used to analyze the expression pattern of MtbHLH148 gene in barrel clover. The overexpression vector was constructed and transformed into Arabidopsis through agrobacterium infection, so as to analyze the related functions of salt tolerance of transgenic Arabidopsis. The result shows:(1) the full length of MtbHLH148 cDNA was 1 343 bp, Which contained 603 bp open reading frame and encoding 201 amino acids. The theoretical pI of MtbHLH148 protein was 11.76 and whoes molecular weight was 22.7 kD. Protein structure analysis showed that the protein had no transmembrane domain, no signal peptide and it was a hydrophilic protein. This gene contained highly conserved bHLH domains. As expected, the secondary structure was predominatly α-helix and random curl.(2) Subcellular localization results showed that the proteins were locate in the nucleus.(3) Phylogenetic analysis revealed that MtbHLH148 was closely related to Glycine max Analysis of the cis-regulatory element demonstrated that the promoters of MtbHLH148 contained light, hormone and stress response elements, suggesting their involvement in the biological processes.(4) qRT-PCR analysis of the expression pattern of the MtbHLH148 in barrel clover showed that the highest level in stem and the lowest level in leaf. For treatment, the genes were induced by ABA(100 μmol/L), and were repressed by cold(4 ℃) but up-regulated by NaCl(200 mmol/L) within the first 8 h.(5) The pCAMBIA3301-MtbHLH148 overexpression vector was successfully constructed and transformed into Arabidopsis. We obtained 16 resistant lines and identified 12 over-expressed lines. Among these over-expressed lines, the transgenic line OE8 had the highest expression level. Analysis of salt-tolerance function of OE8 showed that a significantly higher germination rate was observed in spite of their indiscernible phenotypic difference from wild type. Statistical analysis showed that the root length of the transgenic seedlings over-expressing MtbHLH148 was about 1.5 times of the non-transgenic plants, suggesting enhanced salt tolerance. Based on these results, we infer that MtbHLH148 may play a regulatory role in plant response to salinity.
引文
[1] 刘晓月,王文生,傅彬英.植物bHLH转录因子家族的功能研究进展[J].生物技术进展,2011,1(6):391-397.LIU X Y,WANG W S,FU B Y .Research progress of plant bHLH transcription factor family [J].Current Biotechnology,2011,1(6):391-397.
[2] JOLMA A,YIN Y M,NITTA K R,et al.DNA-dependent formation of transcription factor pairs alters their binding specificity J].Nature,2015,527(7 578):384-388.
[3] FELLER A,MACHEMER K,BRAUN E L,et al.Evolutionary and comparative analysis of MYB and bHLH plant transcription factors[J].The Plant Journal:for Cell and Molecular Biology,2011,66(1):94-116.
[4] 罗赛男,杨国顺,石雪晖,等.转录因子在植物抗逆性上的应用研究[J].湖南农业大学学报(自然科学版),2005,31(2):219-223.LUO S N,YANG G S,SHI X H,et al.On the application of transcription factor to plant stress resistance[J].Journal of Hunan Agricultural University (Natural Sciences),2005,31(2):219-223.
[5] MA P C,ROULD M A,WEINTRAUB H,et al.Crystal structure of MyoD bHLH domain-DNA complex:Perspectives on DNA recognition and implications for transcriptional activation[J].Cell,1994,77(3):451-459.
[6] NAIR S K,BURLEY S K.Recognizing DNA in the library[J].Nature,2000,404(6 779):715-717,718.
[7] ATCHLEY W R,TERHALLE W,DRESS A.Positional dependence,cliques,and predictive motifs in the bHLH protein domain[J].Journal of Molecular Evolution,1999,48(5):501-516.
[8] CASTELAIN M,LE HIR R,BELLINI C.The non-DNA-binding bHLH transcription factor PRE3/bHLH135/ATBS1/TMO7 is involved in the regulation of light signaling pathway in Arabidopsis[J].Physiologia Plantarum,2012,145(3):450-460.
[9] ABE H,URAO T,ITO T,et al.Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling[J].The Plant Cell,2003,15(1):63-78.
[10] FRIEDRICHSEN D M,NEMHAUSER J,MURAMITSU T,et al.Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth[J].Genetics,2002,162(3):1 445-1 456.
[11] PIRES N,DOLAN L.Origin and diversification of basic-helix-loop-helix proteins in plants[J].Molecular Biology and Evolution,2010,27(4):862-874.
[12] 王翠,兰海燕.植物bHLH转录因子在非生物胁迫中的功能研究进展[J].生命科学研究,2016,20(4):358-364.WANG C,LAN H Y.Research progresses on functions of plant bHLH transcription factors involved in abiotic stresses[J].Life Science Research,2016,20(4):358-364.
[13] BAILEY P C,MARTIN C,TOLEDO-ORTIZ G,et al.Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana[J].The Plant Cell,2003,15(11):2 497-2 502.
[14] SUN X,WANG Y,SUI N.Transcriptional regulation of bHLH during plant response to stress[J].Biochemical and Biophysical Research Communications,2018,503(2):397-401.
[15] IKEDA M,MITSUDA N,OHME-TAKAGI M.ATBS1 INTERACTING FACTORs negatively regulate Arabidopsis cell elongation in the triantagonistic bHLH system[J].Plant Signaling & Behavior,2013,8(3):e23448.
[16] ENDO T,FUJII H,SUGIYAMA A,et al.Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1),which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes,modulates carotenoid metabolism in transgenic tomato[J].Plant Science:an International Journal of Experimental Plant Biology,2016,243:35-48.
[17] TOTSUKA A,OKAMOTO E,MIYAHARA T,et al.Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation[J].Breeding Science,2018,68(1):139-143.
[18] HEISLER M G,ATKINSON A,BYLSTRA Y H,et al.SPATULA,a gene that controls development of carpel margin tissues in Arabidopsis,encodes a bHLH protein[J].Development (Cambridge,England),2001,128(7):1 089-1 098.
[19] RAJANI S,SUNDARESAN V.The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence[J].Current Biology:CB,2001,11(24):1 914-1 922.
[20] SORENSEN A M,KR?BER S,UNTE U S,et al.The Arabidopsis ABORTED MICROSPORES(AMS) gene encodes a MYC class transcription factor[J].The Plant Journal,2003,33(2):413-423.
[21] SEO J S,JOO J,KIM M J,et al.OsbHLH148,a basic helix-loop-helix protein,interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice[J].The Plant Journal:for Cell and Molecular Biology,2011,65(6):907-921.
[22] ZHAO Q,XIANG X H,LIU D,et al.Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis[J].Frontiers in Plant Science,2018,9:381.
[23] GUAN Q M,WU J M,YUE X L,et al.A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis[J].PLoS Genetics,2013,9(8):e1003755.
[24] 赵苏州,卢运明,张占路,等.玉米和拟南芥的原生质体制备及瞬时表达体系的研究[J].安徽农业科学,2014,42(12):3 479-3 482.ZHAO S Z,LU Y M,ZHANG Z L,et al.Protoplast preparation and transient expression system in maize and Arabidopsis[J].Journal of Anhui Agricultural Sciences,2014,42(12):3 479-3 482.
[25] 张晓慧,韩榕,等.两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位[J].生物技术通报,2017,33(5):57-62.ZHANG X H,HAN R,et al.Subcellular localization of profilin-1 from Arabidopsis utilizing two transient expression systems[J].Biotechnology Bulletin,2017,33(5):57-62.
[26] ATCHLEY W R,FITCH W M.A natural classification of the basic helix-loop-helix class of transcription factors[J].Proceedings of the National Academy of Sciences,1997,94(10):5 172-5 176.
[27] FERRé-D?AMARé A R,PRENDERGAST G C,ZIFF E B,et al.Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain[J].Nature,1993,363(6 424):38-45.
[28] 张红梅,刘晓庆,陈华涛,等.大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析[J].华北农学报,2018,33(2):49-57.ZHANG H M,LIU X Q,CHEN H T,et al.Subcellular localization and expression analysis of a soybean transcription factor GmWRKY58 in response to abiotic stresses[J].Acta Agriculturae Boreali-Sinica,2018,33(2):49-57.
[29] 陈婷婷,杨青川,丁旺,等.紫花苜蓿WRKY转录因子基因的克隆与亚细胞定位[J].草业学报,2012,21(4):159-167.CHEN T T,YANG Q C,DING W,et al.Cloning and subcellular localization of a WRKY transcription factor gene of Medicago sativa[J].Acta Prataculturae Sinica,2012,21(4):159-167.
[30] SONG X M,HUANG Z N,DUAN W K,et al.Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp.pekinensis)[J].Molecular Genetics and Genomics,2014,289(1):77-91.
[31] RAGHAVENDRA A S,GONUGUNTA V K,CHRISTMANN A,et al.ABA perception and signalling[J].Trends in Plant Science,2010,15(7):395-401.
[32] BUSK P K,PAGèS M.Regulation of abscisic acid-induced transcription[J].Plant Molecular Biology,1998,37(3):425-435.
[33] BHATTACHARJEE S,SAHA A K.Plant water-stress response mechanisms[M]//BHATTACHARJEE S,SAHA A K.eds.Approaches to plant stress and their management.New Delhi:Springer India,2013:149-172.DOI:10.1007/978-81-322-1620-9_8
[2] JOLMA A,YIN Y M,NITTA K R,et al.DNA-dependent formation of transcription factor pairs alters their binding specificity J].Nature,2015,527(7 578):384-388.
[3] FELLER A,MACHEMER K,BRAUN E L,et al.Evolutionary and comparative analysis of MYB and bHLH plant transcription factors[J].The Plant Journal:for Cell and Molecular Biology,2011,66(1):94-116.
[4] 罗赛男,杨国顺,石雪晖,等.转录因子在植物抗逆性上的应用研究[J].湖南农业大学学报(自然科学版),2005,31(2):219-223.LUO S N,YANG G S,SHI X H,et al.On the application of transcription factor to plant stress resistance[J].Journal of Hunan Agricultural University (Natural Sciences),2005,31(2):219-223.
[5] MA P C,ROULD M A,WEINTRAUB H,et al.Crystal structure of MyoD bHLH domain-DNA complex:Perspectives on DNA recognition and implications for transcriptional activation[J].Cell,1994,77(3):451-459.
[6] NAIR S K,BURLEY S K.Recognizing DNA in the library[J].Nature,2000,404(6 779):715-717,718.
[7] ATCHLEY W R,TERHALLE W,DRESS A.Positional dependence,cliques,and predictive motifs in the bHLH protein domain[J].Journal of Molecular Evolution,1999,48(5):501-516.
[8] CASTELAIN M,LE HIR R,BELLINI C.The non-DNA-binding bHLH transcription factor PRE3/bHLH135/ATBS1/TMO7 is involved in the regulation of light signaling pathway in Arabidopsis[J].Physiologia Plantarum,2012,145(3):450-460.
[9] ABE H,URAO T,ITO T,et al.Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling[J].The Plant Cell,2003,15(1):63-78.
[10] FRIEDRICHSEN D M,NEMHAUSER J,MURAMITSU T,et al.Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth[J].Genetics,2002,162(3):1 445-1 456.
[11] PIRES N,DOLAN L.Origin and diversification of basic-helix-loop-helix proteins in plants[J].Molecular Biology and Evolution,2010,27(4):862-874.
[12] 王翠,兰海燕.植物bHLH转录因子在非生物胁迫中的功能研究进展[J].生命科学研究,2016,20(4):358-364.WANG C,LAN H Y.Research progresses on functions of plant bHLH transcription factors involved in abiotic stresses[J].Life Science Research,2016,20(4):358-364.
[13] BAILEY P C,MARTIN C,TOLEDO-ORTIZ G,et al.Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana[J].The Plant Cell,2003,15(11):2 497-2 502.
[14] SUN X,WANG Y,SUI N.Transcriptional regulation of bHLH during plant response to stress[J].Biochemical and Biophysical Research Communications,2018,503(2):397-401.
[15] IKEDA M,MITSUDA N,OHME-TAKAGI M.ATBS1 INTERACTING FACTORs negatively regulate Arabidopsis cell elongation in the triantagonistic bHLH system[J].Plant Signaling & Behavior,2013,8(3):e23448.
[16] ENDO T,FUJII H,SUGIYAMA A,et al.Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1),which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes,modulates carotenoid metabolism in transgenic tomato[J].Plant Science:an International Journal of Experimental Plant Biology,2016,243:35-48.
[17] TOTSUKA A,OKAMOTO E,MIYAHARA T,et al.Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation[J].Breeding Science,2018,68(1):139-143.
[18] HEISLER M G,ATKINSON A,BYLSTRA Y H,et al.SPATULA,a gene that controls development of carpel margin tissues in Arabidopsis,encodes a bHLH protein[J].Development (Cambridge,England),2001,128(7):1 089-1 098.
[19] RAJANI S,SUNDARESAN V.The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence[J].Current Biology:CB,2001,11(24):1 914-1 922.
[20] SORENSEN A M,KR?BER S,UNTE U S,et al.The Arabidopsis ABORTED MICROSPORES(AMS) gene encodes a MYC class transcription factor[J].The Plant Journal,2003,33(2):413-423.
[21] SEO J S,JOO J,KIM M J,et al.OsbHLH148,a basic helix-loop-helix protein,interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice[J].The Plant Journal:for Cell and Molecular Biology,2011,65(6):907-921.
[22] ZHAO Q,XIANG X H,LIU D,et al.Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis[J].Frontiers in Plant Science,2018,9:381.
[23] GUAN Q M,WU J M,YUE X L,et al.A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis[J].PLoS Genetics,2013,9(8):e1003755.
[24] 赵苏州,卢运明,张占路,等.玉米和拟南芥的原生质体制备及瞬时表达体系的研究[J].安徽农业科学,2014,42(12):3 479-3 482.ZHAO S Z,LU Y M,ZHANG Z L,et al.Protoplast preparation and transient expression system in maize and Arabidopsis[J].Journal of Anhui Agricultural Sciences,2014,42(12):3 479-3 482.
[25] 张晓慧,韩榕,等.两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位[J].生物技术通报,2017,33(5):57-62.ZHANG X H,HAN R,et al.Subcellular localization of profilin-1 from Arabidopsis utilizing two transient expression systems[J].Biotechnology Bulletin,2017,33(5):57-62.
[26] ATCHLEY W R,FITCH W M.A natural classification of the basic helix-loop-helix class of transcription factors[J].Proceedings of the National Academy of Sciences,1997,94(10):5 172-5 176.
[27] FERRé-D?AMARé A R,PRENDERGAST G C,ZIFF E B,et al.Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain[J].Nature,1993,363(6 424):38-45.
[28] 张红梅,刘晓庆,陈华涛,等.大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析[J].华北农学报,2018,33(2):49-57.ZHANG H M,LIU X Q,CHEN H T,et al.Subcellular localization and expression analysis of a soybean transcription factor GmWRKY58 in response to abiotic stresses[J].Acta Agriculturae Boreali-Sinica,2018,33(2):49-57.
[29] 陈婷婷,杨青川,丁旺,等.紫花苜蓿WRKY转录因子基因的克隆与亚细胞定位[J].草业学报,2012,21(4):159-167.CHEN T T,YANG Q C,DING W,et al.Cloning and subcellular localization of a WRKY transcription factor gene of Medicago sativa[J].Acta Prataculturae Sinica,2012,21(4):159-167.
[30] SONG X M,HUANG Z N,DUAN W K,et al.Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp.pekinensis)[J].Molecular Genetics and Genomics,2014,289(1):77-91.
[31] RAGHAVENDRA A S,GONUGUNTA V K,CHRISTMANN A,et al.ABA perception and signalling[J].Trends in Plant Science,2010,15(7):395-401.
[32] BUSK P K,PAGèS M.Regulation of abscisic acid-induced transcription[J].Plant Molecular Biology,1998,37(3):425-435.
[33] BHATTACHARJEE S,SAHA A K.Plant water-stress response mechanisms[M]//BHATTACHARJEE S,SAHA A K.eds.Approaches to plant stress and their management.New Delhi:Springer India,2013:149-172.DOI:10.1007/978-81-322-1620-9_8
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