一个顺式作用元件的载体构建及与一个MYB转录因子的互作分析
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摘要
转录因子通过与下游基因启动子上的顺式作用元件的互作调控目标基因的表达,引起一系列的应答反应,从而增强植物的抗逆能力。在我们前期的研究中,通过酵母单杂交技术发现白桦的BplMYB46转录因子能够与一个核心序列为"TGTCGC"的顺式作用元件结合。在本研究中,"TGTCGC"顺式作用元件的每个碱基分别被突变后构建到表达载体pHIS2中,然后利用酵母单杂交技术与BplMYB46转录因子进行互作分析,结果显示,当"TGTCGC"的核心序列的第二个碱基为G/A、第六个碱基为C/G时,酵母单菌落能在三缺培养基TDO/3AT上生长,表明与BplMYB46转录因子结合的顺式作用元件的特异性序列为T (G/A) TCG(C/G)。本研究为后续通过BplMYB46转录因子与这个顺式作用元件的结合,来分析BplMYB46转录因子与下游基因的调控以及为筛选优良下游基因改良白桦的遗传性状提供数据基础。
The transcription factors r egulates the expressions of the target genes through the interaction with cis-acting elements on the promoters of the downstream genes, causing a series of response reactions, thereby enhancing the resistance to stress in plants. In our previous study, yeast one-hybrid technique showed that the BplMYB46 transcription factor of Betula platyphylla could bind to a cis-acting element with a core sequence of' TGTCGC''. In this study, every base of cis-acting element ' TGTCGC'' was mutated and constructed into pHIS2 vector, and its interaction analysis with BplMYB46 transcription factor was performed by yeast one-hybrid technique. The results showed that the yeast colony could grow on TDO/3 AT medium when the secondary nucleotide of ' TGTCGC'' was G/A and the sixth was C/G, respectively, which indicated that the specific sequence of the cis-acting element bound by BplMYB46 transcription factor was T(G/A) TCG(C/G). This study could provide a data base for further analysis of the regulation of BplMYB46 transcription factors and downstream genes through the combination of BplMYB46 transcription factor with this cis-acting element, and for screening the genetic traits of Betula platyphylla with excellent downstream genes.
The transcription factors r egulates the expressions of the target genes through the interaction with cis-acting elements on the promoters of the downstream genes, causing a series of response reactions, thereby enhancing the resistance to stress in plants. In our previous study, yeast one-hybrid technique showed that the BplMYB46 transcription factor of Betula platyphylla could bind to a cis-acting element with a core sequence of' TGTCGC''. In this study, every base of cis-acting element ' TGTCGC'' was mutated and constructed into pHIS2 vector, and its interaction analysis with BplMYB46 transcription factor was performed by yeast one-hybrid technique. The results showed that the yeast colony could grow on TDO/3 AT medium when the secondary nucleotide of ' TGTCGC'' was G/A and the sixth was C/G, respectively, which indicated that the specific sequence of the cis-acting element bound by BplMYB46 transcription factor was T(G/A) TCG(C/G). This study could provide a data base for further analysis of the regulation of BplMYB46 transcription factors and downstream genes through the combination of BplMYB46 transcription factor with this cis-acting element, and for screening the genetic traits of Betula platyphylla with excellent downstream genes.
引文
Agarwal M.,Hao Y.J.,Kapoor A.,Dong C.H.,Fujii H.,Zheng X.,and Zhu J.K.,2006,A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance,J.Biol.Chem.,281(49):37636-37645
Allen M.D.,Yamasaki K.,Oheme-Takagi M.,Tateno M.,and Suzuki M.,1998,A novel mode of DNA recognition by a sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA,EMBO J.,17(18):5484-5496
Berroeal M.,MolinaA.,and Solano R.,2002,Constitutive expression of ethylene-response factor 1 in Arabidopsis confers resistance to several necrotrophic fungai,Plant J.,29(1):23-32
Chen F.,Yu J.P.,Fang X.H.,and Zhang G.Y.,2006,Characterization of a novel AP2/EREBP transcription factor TSH1specifically binding to GCC elements,Shengwu Huaxue Yu Shengwu Wuli Jinzhan(Progress in Biochemistry and Biophysics),33(7):627-634(陈峰,余军平,方晓红,张贵友,2006,鉴定水稻中一个新的专一结合GCC元件的AP2/EREBP族转录因子,生物化学与生物物理进展,33(7):627-634)
Guo H.,Wang Y.,Wang L.,Hu P.,Wang Y.,Jia Y.,Zhang C.,Zhang Y.,Zhang Y.,Wang C.,and Yang C.,2017,Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla,Plant Biotechnol.J.,15(1):107-121
Guo J.Y.,Zheng X.Y.,Zou C.X.,and Li Q.L.,2011,Research progress of cis-elements of abiotic stress inducible promoters and associated transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):16-30(郭晋艳,郑晓瑜,邹翠霞,李秋莉,2011,植物非生物胁迫诱导启动子顺式元件及转录因子研究进展,生物技术通报,(4):16-30)
Hao D.Y.,Ohme-Takagi M.,and Sarai A.,1998,Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor(ERF domian)in plant,J.Biol.Chem.,273(41):26857-26861
Kitajima S.,Koyama T.,Ohme-Takagi M.,Shinshi H.,and Sato F.,2000,C haracterization of gene expression of Ns ERFs,transcription factor of basic PR genes from Nicotiana sylvestris,Plant Cell Physiol.,41(6):817-824
Kocabek T.,Mishra A.K.,Matousek J.,Patzak J.,Lomnicka A.,Khare M.,and Krofta K.,2018,The R2R3 transcription factor Hl MYB8 and its role in flavonoid biosynthesis in hop(Humulus lupulus L.),Plant Sci.,269:32-46
Qi T.,Huang H.,Song S.S.,and Xie D.X.,2015,Regulation of jasmonate mediated stamen development and seed production by a bHLH-MYB complex in Arabidopsis,Plant Cell,27(6):1620-1633
Sun X.,Gong S.Y.,Nie X.Y.,Li Y.,Li W.,Huang G.Q.,and Li X.B.,2015,A R2R3-MYB transcription factor that is specifically expressed in cotton(Gossypium hirsutum)fibers affects secondary cell wall biosynthesis and deposition in transgenic Arabidopsis,Physiol.Plant,154(3):420-432
Sun X.L.,Li Y.,Cai H.,Bai X.,Ji W.,Ji Z.J.,and Zhu Y.M.,2011,Arabidopsis bZIP1 transcription factor binding to the ABRE Cis-element regulates abscisic acid signal transduction,Zuowu Xuebao(Acta Agronomica Sinica),37(4):612-619(孙晓丽,李勇,才华,柏锡,纪巍,季佐军,朱延明,2011,拟南芥bZIP1转录因子通过与ABRE元件结合调节A-BA信号传导,作物学报,37(4):612-619)
Van der Fits L.,and Memelink J.,2001,The jasmonate-indueible APZ/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element,Plant J.,25(1):43-53
Wang H.Q.,Guo X.R.,Yang X.B.,Su J.,and Cao X.Y.,2016,Screening of the proteins interacting with Sm PAPI of R2R3-MYB transcription factor from Salvia miltiorrhiza bunge by yeast two-hybrid system,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(10):2819-2826(王怀琴,郭晓荣,杨新兵,苏娇,曹晓燕,2016,利用酵母双杂交筛选与丹参R2R3-MYB类转录因子Sm PAP1互作的蛋白,基因组学与应用生物学,35(10):2819-2826)
Wang X.Y.,2010,The analysis of DRE,W and S elements in the transgenic rice,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisor:He S.L.,pp.1-2(王小燕,2010,DRE、W和S盒在转基因水稻中的作用分析,硕士学位论文,福建农林大学,导师:何水林,pp.1-2)
Xu R.,Wang Y.H.,Zheng H.,Lu W.,Wu C.G.,Huang J.G.,Yan K.,Yang G.D.,and Zheng C.C.,2015,Salt-induced transcription factor MYB74 is regulated by the RNA-directed DNA methylation pathway in Arabidopsis,J.Exp.Bot.,66(19):5997-6008
Zhang H.W.,2002,Studies on a jasmonates-responsive cis-acting element of PDFl.2 promoter and its interactions with relevant transcription factors,Dissertation for Ph.D.,Hunan A-gricultural University,Supervisor:Lu X.Y.,pp.4-5(张海文,2002,PDF1.2启动子中茉莉素应答的顺式作用元件及其相关转录因子的研究,博士学位论文,湖南农业大学,导师:卢向阳,pp.4-5)
Allen M.D.,Yamasaki K.,Oheme-Takagi M.,Tateno M.,and Suzuki M.,1998,A novel mode of DNA recognition by a sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA,EMBO J.,17(18):5484-5496
Berroeal M.,MolinaA.,and Solano R.,2002,Constitutive expression of ethylene-response factor 1 in Arabidopsis confers resistance to several necrotrophic fungai,Plant J.,29(1):23-32
Chen F.,Yu J.P.,Fang X.H.,and Zhang G.Y.,2006,Characterization of a novel AP2/EREBP transcription factor TSH1specifically binding to GCC elements,Shengwu Huaxue Yu Shengwu Wuli Jinzhan(Progress in Biochemistry and Biophysics),33(7):627-634(陈峰,余军平,方晓红,张贵友,2006,鉴定水稻中一个新的专一结合GCC元件的AP2/EREBP族转录因子,生物化学与生物物理进展,33(7):627-634)
Guo H.,Wang Y.,Wang L.,Hu P.,Wang Y.,Jia Y.,Zhang C.,Zhang Y.,Zhang Y.,Wang C.,and Yang C.,2017,Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla,Plant Biotechnol.J.,15(1):107-121
Guo J.Y.,Zheng X.Y.,Zou C.X.,and Li Q.L.,2011,Research progress of cis-elements of abiotic stress inducible promoters and associated transcription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),(4):16-30(郭晋艳,郑晓瑜,邹翠霞,李秋莉,2011,植物非生物胁迫诱导启动子顺式元件及转录因子研究进展,生物技术通报,(4):16-30)
Hao D.Y.,Ohme-Takagi M.,and Sarai A.,1998,Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor(ERF domian)in plant,J.Biol.Chem.,273(41):26857-26861
Kitajima S.,Koyama T.,Ohme-Takagi M.,Shinshi H.,and Sato F.,2000,C haracterization of gene expression of Ns ERFs,transcription factor of basic PR genes from Nicotiana sylvestris,Plant Cell Physiol.,41(6):817-824
Kocabek T.,Mishra A.K.,Matousek J.,Patzak J.,Lomnicka A.,Khare M.,and Krofta K.,2018,The R2R3 transcription factor Hl MYB8 and its role in flavonoid biosynthesis in hop(Humulus lupulus L.),Plant Sci.,269:32-46
Qi T.,Huang H.,Song S.S.,and Xie D.X.,2015,Regulation of jasmonate mediated stamen development and seed production by a bHLH-MYB complex in Arabidopsis,Plant Cell,27(6):1620-1633
Sun X.,Gong S.Y.,Nie X.Y.,Li Y.,Li W.,Huang G.Q.,and Li X.B.,2015,A R2R3-MYB transcription factor that is specifically expressed in cotton(Gossypium hirsutum)fibers affects secondary cell wall biosynthesis and deposition in transgenic Arabidopsis,Physiol.Plant,154(3):420-432
Sun X.L.,Li Y.,Cai H.,Bai X.,Ji W.,Ji Z.J.,and Zhu Y.M.,2011,Arabidopsis bZIP1 transcription factor binding to the ABRE Cis-element regulates abscisic acid signal transduction,Zuowu Xuebao(Acta Agronomica Sinica),37(4):612-619(孙晓丽,李勇,才华,柏锡,纪巍,季佐军,朱延明,2011,拟南芥bZIP1转录因子通过与ABRE元件结合调节A-BA信号传导,作物学报,37(4):612-619)
Van der Fits L.,and Memelink J.,2001,The jasmonate-indueible APZ/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element,Plant J.,25(1):43-53
Wang H.Q.,Guo X.R.,Yang X.B.,Su J.,and Cao X.Y.,2016,Screening of the proteins interacting with Sm PAPI of R2R3-MYB transcription factor from Salvia miltiorrhiza bunge by yeast two-hybrid system,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(10):2819-2826(王怀琴,郭晓荣,杨新兵,苏娇,曹晓燕,2016,利用酵母双杂交筛选与丹参R2R3-MYB类转录因子Sm PAP1互作的蛋白,基因组学与应用生物学,35(10):2819-2826)
Wang X.Y.,2010,The analysis of DRE,W and S elements in the transgenic rice,Thesis for M.S.,Fujian Agriculture and Forestry University,Supervisor:He S.L.,pp.1-2(王小燕,2010,DRE、W和S盒在转基因水稻中的作用分析,硕士学位论文,福建农林大学,导师:何水林,pp.1-2)
Xu R.,Wang Y.H.,Zheng H.,Lu W.,Wu C.G.,Huang J.G.,Yan K.,Yang G.D.,and Zheng C.C.,2015,Salt-induced transcription factor MYB74 is regulated by the RNA-directed DNA methylation pathway in Arabidopsis,J.Exp.Bot.,66(19):5997-6008
Zhang H.W.,2002,Studies on a jasmonates-responsive cis-acting element of PDFl.2 promoter and its interactions with relevant transcription factors,Dissertation for Ph.D.,Hunan A-gricultural University,Supervisor:Lu X.Y.,pp.4-5(张海文,2002,PDF1.2启动子中茉莉素应答的顺式作用元件及其相关转录因子的研究,博士学位论文,湖南农业大学,导师:卢向阳,pp.4-5)