甘蓝型油菜BnTCP7基因的克隆和表达分析
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摘要
该研究克隆获得了甘蓝型油菜预测转录因子TCP7-like同源编码基因,命名为BnTCP7,与甘蓝型油菜TCP7-like NC_027757基因的核苷酸序列相似性为95.68%。BnTCP7基因开放阅读框全长为648bp,编码215个氨基酸。BnTCP7氨基酸序列与十字花科(Brassicaceae)中其他19条TCP7或者TCP7-like氨基酸序列比对发现,BnTCP7与芸薹属和萝卜属等TCP7同源蛋白具有很强的相似性和保守性,尤其在靠近N端的TCP保守结构域,具有典型的螺旋-环-螺旋结构,且BnTCP7属于TCP家族Ⅰ类,为亲水性不稳定蛋白。通过BnTCP7和拟南芥(Arabidopsis thaliana)AtTCP7(NC_003076)蛋白的二级和三级结构在线预测比对分析,进一步证实BnTCP7具有TCP家族典型结构。进化树分析表明,BnTCP7蛋白与甘蓝型油菜TCP7-like NC_027757蛋白聚在同一分支,两者亲缘关系最近。利用转录组数据对不同时期不同器官BnTCP7基因表达模式分析发现,其表达量呈现一定差异性,其中营养器官中的表达量高于生殖器官。非生物胁迫及激素处理对甘蓝型油菜幼苗植株中BnTCP7基因的表达影响分析发现,BnTCP7基因不仅响应了冷、热、损伤等非生物胁迫,而且参与了ABA和GA3激素的信号转导,表明BnTCP7基因在植物维持正常生长发育和逆境胁迫中可能发挥重要作用。
We cloned the homologous which was the predicted gene of Brassica napus transcription factor TCP7-like,named BnTCP7.The nucleotide sequence similarity of BnTCP7 and Brassica napus TCP7-like NC_027757 was 95.68%.The open reading frame was 648 bp,encoding 215 amino acids.The comparison of BnTCP7 with other 19 TCP7 or TCP7-like amino acids in Brassicaceae's,homologous proteins were found of TCP7 in B.napus and Raphanus sativus.And the domains have strong similarity and conservatism,especially near the N end with a typical helix-loop-helix structure.It also showed that BnTCP7 is a hydrophilic unstable protein which belongs to classⅠof TCP family.The online prediction and comparative analysis of secondary and tertiary structures of BnTCP7 and Arabidopsis thaliana AtTCP7(NC_003076)further revealed that BnTCP7 has the typical structure of TCP family.Phylogenetic analysis indicated that BnTCP7 protein was clustered with Brassica napus(rape)NC_027757 in the same branch,and had the closest relationship.The expression patterns of BnTCP7 gene expression in different organs at different stages were analyzed by the transcriptional data which was found that there was a certain difference in the expression level,and the expression in the vegetative organs was higher than that of the reproductive organs.In addition,the effects of abiotic stresses and hormone treatments on the expression ofBnTCP7 gene on the seedling of B.napus were analyzed experimentally.It was found that the BnTCP7 gene not only responds to the abiotic stresses of cold,heat and wound,but also participates in the signals transduction of ABA and GA3 hormones,which indicated that BnTCP7 might play an important role in maintaining normal growth and development and stresses of plants.
We cloned the homologous which was the predicted gene of Brassica napus transcription factor TCP7-like,named BnTCP7.The nucleotide sequence similarity of BnTCP7 and Brassica napus TCP7-like NC_027757 was 95.68%.The open reading frame was 648 bp,encoding 215 amino acids.The comparison of BnTCP7 with other 19 TCP7 or TCP7-like amino acids in Brassicaceae's,homologous proteins were found of TCP7 in B.napus and Raphanus sativus.And the domains have strong similarity and conservatism,especially near the N end with a typical helix-loop-helix structure.It also showed that BnTCP7 is a hydrophilic unstable protein which belongs to classⅠof TCP family.The online prediction and comparative analysis of secondary and tertiary structures of BnTCP7 and Arabidopsis thaliana AtTCP7(NC_003076)further revealed that BnTCP7 has the typical structure of TCP family.Phylogenetic analysis indicated that BnTCP7 protein was clustered with Brassica napus(rape)NC_027757 in the same branch,and had the closest relationship.The expression patterns of BnTCP7 gene expression in different organs at different stages were analyzed by the transcriptional data which was found that there was a certain difference in the expression level,and the expression in the vegetative organs was higher than that of the reproductive organs.In addition,the effects of abiotic stresses and hormone treatments on the expression ofBnTCP7 gene on the seedling of B.napus were analyzed experimentally.It was found that the BnTCP7 gene not only responds to the abiotic stresses of cold,heat and wound,but also participates in the signals transduction of ABA and GA3 hormones,which indicated that BnTCP7 might play an important role in maintaining normal growth and development and stresses of plants.
引文
[1]CUBAS P,LAUTER N,DOEBLEY J,et al.The TCP domain:a motif found in proteins regulating plant growth and development[J].Plant Journal,1999,18(2):215-222.
[2]谢磊,王姗珊,胡博文,等.基于CRISPR/Cas9技术的月季TCP9基因转化拟南芥[J].分子植物育种,2017,(3):928-933.XIE L,WANG S S,HU B W,et al.Transformation of rose TCP9gene in Arabidopsis thaliana based on CRISPR/Cas9technology[J].Molecular Plant Breeding,2017,(3):928-933.
[3]刘丽娟,高辉.TCP家族基因研究进展[J].生物技术通报,2016,32(9):14-22.LIU L J,GAO H.Research progress on the family of TCP genes[J].Biotechnology Bulletin,2016,32(9):14-22.
[4]刘春浩,梁楠松,于磊,等.水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析[J].北京林业大学学报,2017,39(6):22-31.LIU C H,LIANG N S,YU L,et al.Cloning,analysing and homologous expression of TCP4transcription factor under abiotic stress and hormone signal in Fraxinus mandschurica[J].Journal of Beijing Forestry University,2017,39(6):22-31.
[5]MA J,WANG Q,SUN R,et al.Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii[J].Scientific Reports,2014,4(7 522):6 645.
[6]杨洋.白桦BpTCP7基因的功能研究[D].哈尔滨:东北林业大学,2016.
[7]DANISMAN S.TCP transcription factors at the interface between environmental challenges and the plant’s growth responses[J].Frontiers in Plant Science,2016,7(406):1 930-1 943.
[8]ILHAN E,BUYUK I,INAL B.Transcriptome-Scale characterization of salt responsive bean TCP transcription factors[J].Gene,2018,642:64-73.
[9]DU J,HU S,YU Q,et al.Genome-wide identification and characterization of BrrTCP transcription factors in Brassica rapa ssp.rapa[J].Frontiers in Plant Science,2017,8:1 588.
[10]王晓珠,孙杨,肖仁坚,等.甘蓝型油菜BnABCG8基因的克隆及表达分析[J].分子植物育种,2018,16(1):49-56.WANG X Z,SUN Y,XIAO R J,et al.Cloning and expression analysis of BnABCG8gene in Brassica napus[J].Molecular Plant Breeding,2018,16(1):49-56.
[11]GASTEIGER E,HOOGLAND C,GATTIKER A,et al.Protein Identification and Analysis Tools on the ExPASy Server[M].//John.M.Walker.The Proteomics Protocols Handbook.Humana Press,2005:571-607.
[12]GEOURJON C,DELEAGE G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Computer Applications in the Biosciences:CABIOS,1995,11(6):681-684.
[13]GAO Y,ZHANG D,JIA L.TCP1 modulates DWF4expression via directly interacting with the GGNCCC motifs in the promoter region of DWF4in Arabidopsis thaliana[J].Journal of Genetics and Genomics,2015,42(7):383-392.
[14]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[15]NARUYA S,MASATOSHI N.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[16]JOSEPH F.Confidence limits on phylogenies:an approach using the bootstrap[J].Evolution,1985,39(4):783-791.
[17]ZUCKERKANDL E,PAULING L.Evolutionary divergence and convergence in proteins[J].Evolving Genes&Proteins,1965,97:97-166.
[18]陆奇丰,黄至欢.芸薹属物种(B.napus,B.rapa,B.oleracea)TCP基因家族全基因组鉴定和分析[J].分子植物育种,2016,(12):3 289-3 298.LU Q F,HUANG Z H.Genome-wide identification and analysis of TCP gene family in Brassica species(B.napus,B.rapa,B.oleracea)[J].Molecular Plant Breeding,2016,(12):3 289-3 298.
[19]RIECHMANN J L,HEARD J,MARTIN G,et al.Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes[J].Science,2000,290(5 499):2 105-2 110.
[20]NICOLAS M,CUBAS P.TCP factors:new kids on the signaling block[J].Current Opinion in Plant Biology,2016,33:33-41.
[21]LI Z,WOO H R,GUO H.Genetic redundancy of senescence-associated transcription factors in Arabidopsis[J].Journal of Experimental Botany,2018,69(4):811-823.
[22]YIN D,LIU X,et al.An AT-hook protein DEPRESSED PALEA1physically interacts with the TCP family transcription factor RETARDED PALEA1in rice[J].Biochemical&Biophysical Research Communications,2018,495(1):487-492.
[23]STEINER E,YANAI O,EFRONI I,et al.Class I TCPs modulate cytokinin-induced branching and meristematic activity in tomato[J].Plant Signaling&Behavior,2012,7(7):807-810.
[24]胡立勇,傅廷栋,吴江生,等.油菜生长发育期间内源激素含量的变化[J].植物生理与分子生物学学报,2003,29(3):239-244.HU L Y,FU T D,WU J S,et al.Changes in endogenous hormone content of Brassica napus during growth and development[J].Journal of Plant Physiology and Molecular Biology,2003,29(3):239-244.
[2]谢磊,王姗珊,胡博文,等.基于CRISPR/Cas9技术的月季TCP9基因转化拟南芥[J].分子植物育种,2017,(3):928-933.XIE L,WANG S S,HU B W,et al.Transformation of rose TCP9gene in Arabidopsis thaliana based on CRISPR/Cas9technology[J].Molecular Plant Breeding,2017,(3):928-933.
[3]刘丽娟,高辉.TCP家族基因研究进展[J].生物技术通报,2016,32(9):14-22.LIU L J,GAO H.Research progress on the family of TCP genes[J].Biotechnology Bulletin,2016,32(9):14-22.
[4]刘春浩,梁楠松,于磊,等.水曲柳TCP4转录因子克隆及胁迫和激素下的表达分析[J].北京林业大学学报,2017,39(6):22-31.LIU C H,LIANG N S,YU L,et al.Cloning,analysing and homologous expression of TCP4transcription factor under abiotic stress and hormone signal in Fraxinus mandschurica[J].Journal of Beijing Forestry University,2017,39(6):22-31.
[5]MA J,WANG Q,SUN R,et al.Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii[J].Scientific Reports,2014,4(7 522):6 645.
[6]杨洋.白桦BpTCP7基因的功能研究[D].哈尔滨:东北林业大学,2016.
[7]DANISMAN S.TCP transcription factors at the interface between environmental challenges and the plant’s growth responses[J].Frontiers in Plant Science,2016,7(406):1 930-1 943.
[8]ILHAN E,BUYUK I,INAL B.Transcriptome-Scale characterization of salt responsive bean TCP transcription factors[J].Gene,2018,642:64-73.
[9]DU J,HU S,YU Q,et al.Genome-wide identification and characterization of BrrTCP transcription factors in Brassica rapa ssp.rapa[J].Frontiers in Plant Science,2017,8:1 588.
[10]王晓珠,孙杨,肖仁坚,等.甘蓝型油菜BnABCG8基因的克隆及表达分析[J].分子植物育种,2018,16(1):49-56.WANG X Z,SUN Y,XIAO R J,et al.Cloning and expression analysis of BnABCG8gene in Brassica napus[J].Molecular Plant Breeding,2018,16(1):49-56.
[11]GASTEIGER E,HOOGLAND C,GATTIKER A,et al.Protein Identification and Analysis Tools on the ExPASy Server[M].//John.M.Walker.The Proteomics Protocols Handbook.Humana Press,2005:571-607.
[12]GEOURJON C,DELEAGE G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Computer Applications in the Biosciences:CABIOS,1995,11(6):681-684.
[13]GAO Y,ZHANG D,JIA L.TCP1 modulates DWF4expression via directly interacting with the GGNCCC motifs in the promoter region of DWF4in Arabidopsis thaliana[J].Journal of Genetics and Genomics,2015,42(7):383-392.
[14]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[15]NARUYA S,MASATOSHI N.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[16]JOSEPH F.Confidence limits on phylogenies:an approach using the bootstrap[J].Evolution,1985,39(4):783-791.
[17]ZUCKERKANDL E,PAULING L.Evolutionary divergence and convergence in proteins[J].Evolving Genes&Proteins,1965,97:97-166.
[18]陆奇丰,黄至欢.芸薹属物种(B.napus,B.rapa,B.oleracea)TCP基因家族全基因组鉴定和分析[J].分子植物育种,2016,(12):3 289-3 298.LU Q F,HUANG Z H.Genome-wide identification and analysis of TCP gene family in Brassica species(B.napus,B.rapa,B.oleracea)[J].Molecular Plant Breeding,2016,(12):3 289-3 298.
[19]RIECHMANN J L,HEARD J,MARTIN G,et al.Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes[J].Science,2000,290(5 499):2 105-2 110.
[20]NICOLAS M,CUBAS P.TCP factors:new kids on the signaling block[J].Current Opinion in Plant Biology,2016,33:33-41.
[21]LI Z,WOO H R,GUO H.Genetic redundancy of senescence-associated transcription factors in Arabidopsis[J].Journal of Experimental Botany,2018,69(4):811-823.
[22]YIN D,LIU X,et al.An AT-hook protein DEPRESSED PALEA1physically interacts with the TCP family transcription factor RETARDED PALEA1in rice[J].Biochemical&Biophysical Research Communications,2018,495(1):487-492.
[23]STEINER E,YANAI O,EFRONI I,et al.Class I TCPs modulate cytokinin-induced branching and meristematic activity in tomato[J].Plant Signaling&Behavior,2012,7(7):807-810.
[24]胡立勇,傅廷栋,吴江生,等.油菜生长发育期间内源激素含量的变化[J].植物生理与分子生物学学报,2003,29(3):239-244.HU L Y,FU T D,WU J S,et al.Changes in endogenous hormone content of Brassica napus during growth and development[J].Journal of Plant Physiology and Molecular Biology,2003,29(3):239-244.