烟草JAZ1基因的克隆和功能分析
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摘要
JAZ(Jasmonate ZIM-domain)蛋白作为E3泛素连接蛋白降解复合体SCFCOI1受体的靶蛋白,是茉莉酸信号转导途径的关键组分。为了明确JAZ基因在烟草中的功能和调控方式,通过同源克隆技术从烟草中克隆到1个JAZ1基因,并结合生物信息学和基因表达模式分析进一步预测其生物学功能。结果表明:NtJAZ1基因cDNA序列长720 bp,具有完整的开放阅读框,编码239个氨基酸。NtJAZ1蛋白含有TIFY和CCT-2(Jas结构域)两个保守结构域,其二级结构由α-螺旋、延伸链和无规则卷曲组成,其三级结构具有TIFY超家族蛋白的结构特征。进化分析表明,NtJAZ1和番茄SlJAZ1同属1个分支,亲缘关系较近。在被黑胫病侵染发病的烟草根部,NtJAZ1的表达量最高,其次是茎和叶,推测NtJAZ1的高表达抑制了JA响应基因的表达,从而引发烟草黑胫病。
JAZ(Jasmonate ZIM-domain) protein, as a target protein of E3 ubiquitin ligase degradation SCFCOI1 complex receptor, is a key component in jasmonic acid signal transduction pathway. To identify the function and regulation mechanism of JAZ gene in tobacco, NtJAZ1 was cloned by homologous cloning strategy, and NtJAZ1 function was analyzed by bioinformatics and gene expression patterns. The results showed that NtJAZ1 encoded 239 amino acids with the cDNA sequence of 720 bp. NtJAZ1 protein contained two conserved domains: TIFY and CCT-2(also known as Jas domain). The secondary structure of NtJAZ1 consisted ofα-helix, extended chain and random coil, and its tertiary structure had the structural characteristics of TIFY superfamily protein. Phylogenetic analysis indicated that NtJAZ1 and SlJAZ1 belonged to the same branch and had a close genetic relationship. The expression level of NtJAZ1 was the highest in root treated by Phytophthora parasitica var. nicotianae, followed by stem and leaf. It was speculated that the high expression of NtJAZ1 inhibited the expression of JA response genes, which resulted in the occurrence of tobacco black shank.
JAZ(Jasmonate ZIM-domain) protein, as a target protein of E3 ubiquitin ligase degradation SCFCOI1 complex receptor, is a key component in jasmonic acid signal transduction pathway. To identify the function and regulation mechanism of JAZ gene in tobacco, NtJAZ1 was cloned by homologous cloning strategy, and NtJAZ1 function was analyzed by bioinformatics and gene expression patterns. The results showed that NtJAZ1 encoded 239 amino acids with the cDNA sequence of 720 bp. NtJAZ1 protein contained two conserved domains: TIFY and CCT-2(also known as Jas domain). The secondary structure of NtJAZ1 consisted ofα-helix, extended chain and random coil, and its tertiary structure had the structural characteristics of TIFY superfamily protein. Phylogenetic analysis indicated that NtJAZ1 and SlJAZ1 belonged to the same branch and had a close genetic relationship. The expression level of NtJAZ1 was the highest in root treated by Phytophthora parasitica var. nicotianae, followed by stem and leaf. It was speculated that the high expression of NtJAZ1 inhibited the expression of JA response genes, which resulted in the occurrence of tobacco black shank.
引文
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[21]姚笛,王启超,曹玉鑫,等.巴西橡胶树JAZ家族基因的克隆和表达分析[J].分子植物育种,2016,14(5):1115-1122.YAO Di,WANG Qichao,CAO Yuxin,et al.Cloning and expression analysis of JAZ gene family of Hevea brasiliensis[J].Molecular Plant Breeding,2016,14(5):1115-1122.
[22]瞿礼嘉,钱前,袁明,等.2011年中国植物科学若干领域重要研究进展[J].植物学报,2012,47(4):309-356.QU Lijia,QIAN Qian,YUAN Ming,et al.Research advances on plant science in China in 2011[J].Chinese Bulletin of Botany,2012,47(4):309-356.
[23]Gangappa S N,Prasad V B R,Chattopadhyay S.Functional interconnection of MYC2 and SPA1 in the photomorphogenic seedling development of Arabidopsis[J].Plant Physiology,2010,154(3):1210-1219.
[24]Gangappa S N,Chattopadhyay S.MYC2,a bHLHtranscription factor,modulates the adult phenotype of SPA1[J].Plant Signaling&Behavior,2010,5(12):1650-1652.
[2]Shan X Y,Wang Z L,Xie D X.Jasmonate signal pathway in Arabidopsis[J].Journal of Integrative Plant Biology,2007,49(1):81-86.
[3]Chehab E W,Kaspi R,Savchenko T,et al.Distinct roles of jasmonates and aldehydes in plant-defense responses[J].PLoS One,2008,3(4):e1904.
[4]Koo A J K,Gao X L,Jones A D,et al.A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[J].The Plant Journal,2009,59(6):974-986.
[5]Chini A,Fonseca S,Fernández G,et al.The JAZfamily of repressors is the missing link in jasmonate signalling[J].Nature,2007,448(7154):666-671.
[6]Thines B,Katsir L,Melotto M,et al.JAZ repressor proteins are targets of the SCFCOI1complex during jasmonate signalling[J].Nature,2007,448(7154):661-665.
[7]Yan Y X,Stolz S,Chételat A,et al.A downstream mediator in the growth repression limb of the jasmonate pathway[J].The Plant Cell,2007,19(8):2470-2483.
[8]Pauwels L,Barbero G F,Geerinck J,et al.NINJAconnects the co-repressor TOPLESS to jasmonate signalling[J].Nature,2010,464(7289):788-791.
[9]Katsir L,Chung H S,Koo A J,et al.Jasmonate signaling:a conserved mechanism of hormone sensing[J].Current Opinion in Plant Biology,2008,11(4):428-435.
[10]Kazan K,Manners J M.JAZ repressors and the orchestration of phytohormone crosstalk[J].Trends in Plant Science,2012,17(1):22-31.
[11]段龙飞,慕小倩,李文燕.茉莉酸信号途径中转录抑制因子JAZ蛋白家族的分子进化分析[J].植物学报,2013,48(6):623-634.DUAN Longfei,MU Xiaoqian,LI Wenyan.Molecular evolution of transcriptional repressor JAZ protein family in jasmonic acid signaling pathway[J].Chinese Bulletin of Botany,2013,48(6):623-634.
[12]晋鑫鑫.SmJAZ3基因的克隆及JAZ调控丹参次生代谢的功能研究[D].西安:陕西师范大学,2015.JIN Xinxin.Cloning of SmJAZ3 gene and function of JAZ in regulating secondary metabolism of Salvia miltiorrhiza Bunge[D].Xi.an:Shaanxi Normal University,2015.
[13]孙程,周晓今,陈茹梅,等.植物JAZ蛋白的功能概述[J].生物技术通报,2014,32(6):1-8.SUN Cheng,ZHOU Xiaojin,CHEN Rumei,et al.Comprehensive overview of JAZ proteins in plants[J].Biotechnology Bulletin,2014,32(6):1-8.
[14]武明珠,李锋,王燃,等.烟草转录因子b HLH93基因的克隆及表达分析[J].烟草科技,2015,48(3):1-7.WU Mingzhu,LI Feng,WANG Ran,et al.Cloning and expression of transcription factor gene bHLH93 from Nicotiana tabacum[J].Tobacco Science&Technology,2015,48(3):1-7.
[15]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod[J].Methods,2001,25(4):402-408.
[16]范成明,李成云,赵明富,等.根癌土壤杆菌C58Cereon中分泌蛋白信号肽分析[J].微生物学报,2005,45(4):561-566.FAN Chengming,LI Chengyun,ZHAO Mingfu,et al.Analysis of signal peptides of the secreted proteins in Agrobacterium tumefaciens C58[J].Acta Microbiologica Sinica,2005,45(4):561-566.
[17]Bai Y H,Meng Y J,Huang D L,et al.Origin and evolutionary analysis of the plant-specific TIFYtranscription factor family[J].Genomics,2011,98(2):128-136.
[18]Melotto M,Mecey C,Niu Y J,et al.A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine-and jasmonoyl isoleucine-dependent interactions with the COI1 F-box protein[J].The Plant Journal,2008,55(6):979-988.
[19]Chung H S,Howe G A.A critical role for the TIFYmotif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATEZIM-domain protein JAZ10 in Arabidopsis[J].The Plant Cell,2009,21(1):131-145.
[20]WANG Yiping,LIU Sen,XIA Mu,et al.Research progress on tobacco black shank disease[J].Modern Agricultural Science and Technology,2013,22:129-131.
[21]姚笛,王启超,曹玉鑫,等.巴西橡胶树JAZ家族基因的克隆和表达分析[J].分子植物育种,2016,14(5):1115-1122.YAO Di,WANG Qichao,CAO Yuxin,et al.Cloning and expression analysis of JAZ gene family of Hevea brasiliensis[J].Molecular Plant Breeding,2016,14(5):1115-1122.
[22]瞿礼嘉,钱前,袁明,等.2011年中国植物科学若干领域重要研究进展[J].植物学报,2012,47(4):309-356.QU Lijia,QIAN Qian,YUAN Ming,et al.Research advances on plant science in China in 2011[J].Chinese Bulletin of Botany,2012,47(4):309-356.
[23]Gangappa S N,Prasad V B R,Chattopadhyay S.Functional interconnection of MYC2 and SPA1 in the photomorphogenic seedling development of Arabidopsis[J].Plant Physiology,2010,154(3):1210-1219.
[24]Gangappa S N,Chattopadhyay S.MYC2,a bHLHtranscription factor,modulates the adult phenotype of SPA1[J].Plant Signaling&Behavior,2010,5(12):1650-1652.