不结球白菜BrCDF1的克隆和蛋白亚细胞定位及其与BrFKF1蛋白的互作验证
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摘要
[目的]本文旨在揭示CDF1基因在不结球白菜(Brassica rapa ssp.chinensis)抽薹开花过程中的调控机制,验证其是否与FKF1蛋白发生互作。[方法]以‘苏州青’为材料,采用同源克隆方法获得BrCDF1基因全长序列,并与其他物种进行氨基酸序列比对;将BrCDF1与报告基因YFP融合构建亚细胞定位载体p Early Gate101-BrCDF1-YFP,采用农杆菌介导的方法将其瞬时表达于本氏烟(Nicotiana benthamiana)叶片中;激光共聚焦显微镜观察,利用酵母双杂交和双分子荧光互补技术验证不结球白菜BrCDF1和BrFKF1蛋白是否存在互作。[结果]BrCDF1基因含有876 bp开放阅读框,编码292个氨基酸。与甘蓝型油菜(Brassica napus)、野甘蓝(原变种)(Brassica oleracea var.oleracea)、萝卜(Raphanus sativus)的氨基酸序列比对结果显示:BrCDF1在进化过程中的保守程度分别为91.11%、88.25%、84.76%,保守性较高。BrCDF1蛋白定位于细胞核中,BrCDF1和BrFKF1之间存在相互作用。[结论]BrCDF1定位于细胞核,并且与BrFKF1互作,在进化过程中保守性较高,并可能参与了不结球白菜的光周期开花途径。
[Objectives]The paper aims to elucidate the flowering regulatory mechanism of transcription factor CDF1 in the flowering process of Brassica rapa ssp. chinensis and verify if CDF1 interacts with the FKF1 protein. [Methods]A full-length sequence of BrCDF1 was isolated by homologous cloning. The subcellular localization vector p Early Gate101-BrCDF1-YFP,which was constructed by fusion of BrCDF1 and YFP,was transiently expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated method and detected by confocal microscopy. The interaction between BrCDF1 and BrFKF1 in Brassica rapa was studied by yeast two-hybrid and bimolecular fluorescence complementation methods. [Results]The BrCDF1 gene contains an open reading frame of876 bp in length encoding a protein of 292 amino acids. The alignment of Brassica napus,Brassica oleracea var. oleracea and Raphanus sativus showed that the conservatism of BrCDF1 was 91. 11%,88. 25% and 84. 76% respectively in the evolutionary process,which was highly conservative comparing with other species. The detection of yellow fluorescence signal by confocal showed that BrCDF1 was located in nucleus. And there was an interaction between BrCDF1 and BrFKF1. [Conclusions]In this study,BrCDF1 is located in nucleus and interacts with BrFKF1. BrCDF1 is highly conserved during evolution. BrCDF1 possibly plays a role in photoperiod pathway of flowering in non-heading Chinese cabbage.
[Objectives]The paper aims to elucidate the flowering regulatory mechanism of transcription factor CDF1 in the flowering process of Brassica rapa ssp. chinensis and verify if CDF1 interacts with the FKF1 protein. [Methods]A full-length sequence of BrCDF1 was isolated by homologous cloning. The subcellular localization vector p Early Gate101-BrCDF1-YFP,which was constructed by fusion of BrCDF1 and YFP,was transiently expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated method and detected by confocal microscopy. The interaction between BrCDF1 and BrFKF1 in Brassica rapa was studied by yeast two-hybrid and bimolecular fluorescence complementation methods. [Results]The BrCDF1 gene contains an open reading frame of876 bp in length encoding a protein of 292 amino acids. The alignment of Brassica napus,Brassica oleracea var. oleracea and Raphanus sativus showed that the conservatism of BrCDF1 was 91. 11%,88. 25% and 84. 76% respectively in the evolutionary process,which was highly conservative comparing with other species. The detection of yellow fluorescence signal by confocal showed that BrCDF1 was located in nucleus. And there was an interaction between BrCDF1 and BrFKF1. [Conclusions]In this study,BrCDF1 is located in nucleus and interacts with BrFKF1. BrCDF1 is highly conserved during evolution. BrCDF1 possibly plays a role in photoperiod pathway of flowering in non-heading Chinese cabbage.
引文
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[16]Imaizumi T,Schultz T F,Harmon F G,et al.FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J].Science,2005,309:293-297.
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[18]Sawa M,Nusinow D A,Kay S A,et al.FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis[J].Science,2007,318:261-265.
[19]Imaizumi T,Kay S A,Schroeder J I.Daily watch on metabolism[J].Science,2007,318:1730-1731.
[20]Halabian R,Fathabad M E,Masroori N,et al.Expression and purification of recombinant human coagulation factorⅦfused to a histidine tag using Gateway technology[J].Blood Transfusion,2009,7(4):305-312.
[21]盖钧镒,章元明,王建康.QTL混合遗传模型扩展至2对主基因+多基因时的多世代联合分析[J].作物学报,2010,26(4):385-391.Gai J Y,Zhang Y M,Wang J K.A joint analysis of multiple generations for QTL models extended to mixed two major genes plus polygene[J].Acta Agronomica Sinica,2000,26(4):385-391(in Chinese with English abstract).
[22]郁有健.大白菜抽薹开花相关基因SNP分析与晚抽薹开花性状QTL定位[D].哈尔滨:东北农业大学,2010.Yu Y J.Dissertation for the masteral degree in agricultural SNP analysis of bolting and flowering gene and QTL localization of late-bolting and late-flowering in Chinese cabbage[D].Harbin:Northeast Agricultural University,2010(in Chinese with English abstract).
[23]Kim H S,Kim S J,Abbasi N,et al.The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis[J].The Plant Journal,2010,64(3):524-535.
[24]胡娟,王丽鸳,韦康,等.茶树Cs CDF1基因克隆及表达分析[J].茶叶科学,2015,35(5):501-511.Hu J,Wang L Y,Wei K,et al.Cloning and expression analysis of Cs CDF1(Cycling Dof Factor 1)gene in tea plant(Camellia sinensis)[J].Journal of Tea Science,2015,35(5):501-511(in Chinese with English abstract).
[25]Zheng N N,Wang Z W,Wei W Y.Ubiquitination-mediated degradation of cell cycle-related proteins by F-box proteins[J].International Journal of Biochemistry and Cell Biology,2016,73:99-110.
[26]Ho M S,Tsai P I,Chien C T.F-box proteins:the key to protein degradation[J].Journal of Biomedical Science,2006,13:181-191.
[27]Putterill J,Robson F,Lee K,et al.The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors[J].Cell,1995,80:847-857.
[28]彭凌涛.控制拟南芥和水稻开花时间光周期途径的分子机制[J].植物生理学通讯,2006,42(6):1021-1031.Peng L T.Molecular mechanism of flowering time controlling photoperiod pathway in Arabidopsis and rice[J].Plant Physiology Communications,2006,42(6):1021-1031(in Chinese).
[29]Han S H,Yoo S C,Lee B D,et al.Rice FLAVIN-BINDING,KELCH REPEAT,F-BOX 1(Os FKF 1)promotes flowering independent of photoperiod[J].Plant Cell Environment,2015,3:95-107.
[30]Niwa Y,Ito S,Nakamichi N,et al.Genetic linkages of the circadian clock-associated genes,TOC1,CCA1 and LHY,in the photoperiodic control of flowering time in Arabidopsis thaliana[J].Plant and Cell Physiology,2007,48(7):925-937.
[2]张学铭.白菜类作物抽薹开花的遗传分析[D].北京:中国农业科学院,2014.Zhang X M.Genetic analysis of the bolting and flowering time in Brassica rapa[D].Beijing:Chinese Academy of Agricultural Sciences,2014(in Chinese with English abstract).
[3]户秋稳,吕炜,张蜀宁,等.优质晚抽薹四倍体不结球白菜的创制及特性[J].南京农业大学学报,2015,38(5):757-763.DOI:10.7685/j.issn.1000-2030.2015.05.009.Hu Q W,LüW,Zhang S N,et al.The induction and characteristics of high quality and late-bolting tetraploid non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2015,38(5):757-763(in Chinese with English abstract).
[4]Li L,Li X,Liu Y,et al.Flowering responses to light and temperature[J].Science China Life Sciences,2016,59(4):403-408.
[5]Song Y H,Shim J S,Kinmonth-Schultz H A,et al.Photoperiodic flowering:time measurement mechanisms in leaves[J].Annual Review of Plant Biology,2015,66:441-464.
[6]黄和喜,王岩,许玉超,等.不结球白菜雄蕊瓣化相关AP3基因的克隆和表达分析[J].南京农业大学学报,2015,38(5):748-756.DOI:10.7685/j.issn.1000-2030.2015.05.008.Huang H X,Wang Y,Xu Y C,et al.Clone and expression analysis of stamen patelody-associated AP3 genes from non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2015,38(5):748-756(in Chinese with English abstract).
[7]Xu P P,Chen H Y,Ying L,et al.At DOF5.4/OBP4,a DOF transcription factor gene that negatively regulates cell cycle progression and cell expansion in Arabidopsis thaliana[J].Scientific Reports,2016,6:27705.
[8]Yan H,Huang J,Liao B,et al.DOF transcription factors in developing peanut(Arachis hypogaea)seeds[J].American Journal of Molecular Biology,2012,2:60-71.
[9]Cai X F,Zhang Y Y,Zhang C J,et al.Genome-wide analysis of plant-specific Dof transcription factor family in tomato[J].Plant Biology,2013,55(6):552-566.
[10]Cui X H,Xu X F,He Y Y,et al.Overexpression of an F-box protein gene disrupts cotyledon vein patterning in Arabidopsis[J].Plant Physiology and Biochemistry,2016,102:43-52.
[11]Ito S,Song Y H,Imaizumi T.LOV domain-containing F-box proteins:light-dependent protein degradation modules in Arabidopsis[J].Molecular Plant,2012,5(3):573-582.
[12]Schwager K M,Calderon-Villalobos L I A,Dohmann E M N,et al.Characterization of the VIER F-BOX PROTEINE genes from Arabidopsis reveals their importance for plant growth and development[J].The Plant Cell,2007,19(4):1163-1178.
[13]Fornara F,Panigrahi K C S,Gissot L,et al.Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response[J].Developmental Cell,2009,17:75-86.
[14]李芳.大豆FKF1和GI基因克隆、表达模式及功能分析[D].北京:中国农业科学院,2012.Li F.Molecular cloning,expression profiles and founctional analysis of FKF1 and GI genes in soybean(Glycine max)[D].Beijing:Chinese Academy of Agricultural Sciences,2012(in Chinese with English abstract).
[15]黄芬娜.龙眼Dl GI和Dl FKF1基因的克隆及功能研究[D].福州:福建农林大学,2016.Huang F N.Cloning and founctional analysis of Dl GI and Dl FKF1 in Longan(Dimocarpus longan Lour.)[D].Fuzhou:Fujian Agriculture and Forestry University,2016(in Chinese with English abstract).
[16]Imaizumi T,Schultz T F,Harmon F G,et al.FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis[J].Science,2005,309:293-297.
[17]吴连成,常丽丽,陈晓,等.CO基因的调控表达与植物光周期反应[J].中国农学通报,2010,26(2):116-121.Wu L C,Chang L L,Chen X,et al.Expression regulation of CONSTANS and plant photoperiod responses[J].Chinese Agricultural Science Bulletin,2010,26(2):116-121(in Chinese with English abstract).
[18]Sawa M,Nusinow D A,Kay S A,et al.FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis[J].Science,2007,318:261-265.
[19]Imaizumi T,Kay S A,Schroeder J I.Daily watch on metabolism[J].Science,2007,318:1730-1731.
[20]Halabian R,Fathabad M E,Masroori N,et al.Expression and purification of recombinant human coagulation factorⅦfused to a histidine tag using Gateway technology[J].Blood Transfusion,2009,7(4):305-312.
[21]盖钧镒,章元明,王建康.QTL混合遗传模型扩展至2对主基因+多基因时的多世代联合分析[J].作物学报,2010,26(4):385-391.Gai J Y,Zhang Y M,Wang J K.A joint analysis of multiple generations for QTL models extended to mixed two major genes plus polygene[J].Acta Agronomica Sinica,2000,26(4):385-391(in Chinese with English abstract).
[22]郁有健.大白菜抽薹开花相关基因SNP分析与晚抽薹开花性状QTL定位[D].哈尔滨:东北农业大学,2010.Yu Y J.Dissertation for the masteral degree in agricultural SNP analysis of bolting and flowering gene and QTL localization of late-bolting and late-flowering in Chinese cabbage[D].Harbin:Northeast Agricultural University,2010(in Chinese with English abstract).
[23]Kim H S,Kim S J,Abbasi N,et al.The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis[J].The Plant Journal,2010,64(3):524-535.
[24]胡娟,王丽鸳,韦康,等.茶树Cs CDF1基因克隆及表达分析[J].茶叶科学,2015,35(5):501-511.Hu J,Wang L Y,Wei K,et al.Cloning and expression analysis of Cs CDF1(Cycling Dof Factor 1)gene in tea plant(Camellia sinensis)[J].Journal of Tea Science,2015,35(5):501-511(in Chinese with English abstract).
[25]Zheng N N,Wang Z W,Wei W Y.Ubiquitination-mediated degradation of cell cycle-related proteins by F-box proteins[J].International Journal of Biochemistry and Cell Biology,2016,73:99-110.
[26]Ho M S,Tsai P I,Chien C T.F-box proteins:the key to protein degradation[J].Journal of Biomedical Science,2006,13:181-191.
[27]Putterill J,Robson F,Lee K,et al.The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors[J].Cell,1995,80:847-857.
[28]彭凌涛.控制拟南芥和水稻开花时间光周期途径的分子机制[J].植物生理学通讯,2006,42(6):1021-1031.Peng L T.Molecular mechanism of flowering time controlling photoperiod pathway in Arabidopsis and rice[J].Plant Physiology Communications,2006,42(6):1021-1031(in Chinese).
[29]Han S H,Yoo S C,Lee B D,et al.Rice FLAVIN-BINDING,KELCH REPEAT,F-BOX 1(Os FKF 1)promotes flowering independent of photoperiod[J].Plant Cell Environment,2015,3:95-107.
[30]Niwa Y,Ito S,Nakamichi N,et al.Genetic linkages of the circadian clock-associated genes,TOC1,CCA1 and LHY,in the photoperiodic control of flowering time in Arabidopsis thaliana[J].Plant and Cell Physiology,2007,48(7):925-937.