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摘 要 通过筛选辣椒均一化cDNA文库，获得一个具有AP2结构域的辣椒CaERF4全长cDNA序列，含有一个编码250个氨基酸序列的开放阅读框，与拟南芥等其它植物ERF蛋白之间有35%～38%的氨基酸序列相似性。该基因在辣椒不同器官中均有不同程度的组成型表达，在根系中表达量略低。此外，还发现CaERF4的转录受到茉莉酸甲酯（MeJA）和乙烯利（ETH）等外源激素处理以及青枯菌接种的诱导，但水杨酸（SA）处理作用不明显，高温处理也可诱导其转录表达增强。说明CaERF4可能在辣椒应答病原菌和高温等逆境胁迫中起调节作用。
关键词 辣椒；乙烯响应因子；转录活性；组织特异性；诱导表达
中图分类号 S641.3 文献标识码 A
Full Length cDNA Cloning and Expressional
Characterization of CaERF4 of Capsicum annuum
CAI Hanyang1，XIAO Zhuoli1，YAN Yan2，GUAN Deyi2，HE Shuilin2*，WU Yang3*
1 College of Life Science， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002，China
2 College of Crop Science， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002，China
3 College of Life Science， Jinggangshan University， Ji’an， Jiangxi 343009，China
Abstract ERF（Ethylene Responsive Factor） is an important plant specific transcription factor family implicated in plant growth， development and response to environmental stresses. However， the structure and function of the major members in this family remain unknown， especially in non model plants such as Capsicum annuum. In the present study， a full length cDNA of putative ERF containing AP2 conservative domain was identified from randomly sequencing of a normalized cDNA library of pepper. This full length cDNA harbors an open reading frame encoding a protein of 250 aa in length sharing 35%-38% sequence identity to ERFs from other plant species. The putative ERF transcription factor was designated as CaERF4 due to its highest sequence identity to AtCRF4. By RT-PCR analysis， CaERF4 exhibited a constitutive transcriptional expression in different organs in pepper plants， and it was also found to be induced to different degree by exogenously applied ETH and MeJA as well as by inoculation of Ralstonia solanacearum and treatment of high temperature stress，but not by exogenous application of SA. These results implied that CaERF4 may play key roles in pepper response to R. solanacearum inoculation and heat stress treatment.
Key worlds Capsicum annuu；EPF；Transcriptional activity；Tissue specificity；Induced expression
doi 10.3969/j.issn.1000-2561.2014.06.011
植物生长发育和对逆境的应答在很大程度上受到转录水平的调节，各种转录因子在其中发挥着十分重要的调节作用。乙烯响应因子（Ethylene Response Factor，ERF）转录因子属于植物AP2/ERF转录因子家族，AP2/ERF类转录因子是植物特有的转录因子，在拟南芥中有146个成员，在水稻中有136个成员[1]，初步的研究结果表明AP2/ERF家族成员与生长发育和应答环境胁迫等生物学过程密切相关，参与植物的生长，花的发育，果实的成熟，叶片的衰老，以及植物应答病原菌，低温，干旱等过程的调控[2-3]。此外还发现AP2/ERF类转录因子与水杨酸、茉莉酸、乙烯等多种信号通路相关[4-7]。例如，在烟草和番茄中过量表达TERF2/LeERF2可提高转基因植物的抗冷性。拟南芥AtERF1通过结合GCC盒激活PR基因表达，从而提高拟南芥对生物胁迫和非生物胁迫的抗性[8-9]。拟南芥过量表达AtERF14可通过上调ERF1、PDF1和Chi等防御反应相关基因提高拟南芥对镰刀菌（Fusarium oxysporum）侵染的抗性[10]。番茄JERFs的表达与ET和 ABA 信号途径相关，参与番茄耐盐、耐旱、耐冷和抗病调控[11]。这些结果显示出ERF家族成员在植物应答逆境胁迫过程中的重要作用。迄今关于ERF的研究大多数集中在拟南芥、水稻、番茄和烟草等少数作物的部分成员，人类对不同植物ERF家族的大多数成员的结构和功能还所知甚少。
关键词 辣椒；乙烯响应因子；转录活性；组织特异性；诱导表达
中图分类号 S641.3 文献标识码 A
Full Length cDNA Cloning and Expressional
Characterization of CaERF4 of Capsicum annuum
CAI Hanyang1，XIAO Zhuoli1，YAN Yan2，GUAN Deyi2，HE Shuilin2*，WU Yang3*
1 College of Life Science， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002，China
2 College of Crop Science， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002，China
3 College of Life Science， Jinggangshan University， Ji’an， Jiangxi 343009，China
Abstract ERF（Ethylene Responsive Factor） is an important plant specific transcription factor family implicated in plant growth， development and response to environmental stresses. However， the structure and function of the major members in this family remain unknown， especially in non model plants such as Capsicum annuum. In the present study， a full length cDNA of putative ERF containing AP2 conservative domain was identified from randomly sequencing of a normalized cDNA library of pepper. This full length cDNA harbors an open reading frame encoding a protein of 250 aa in length sharing 35%-38% sequence identity to ERFs from other plant species. The putative ERF transcription factor was designated as CaERF4 due to its highest sequence identity to AtCRF4. By RT-PCR analysis， CaERF4 exhibited a constitutive transcriptional expression in different organs in pepper plants， and it was also found to be induced to different degree by exogenously applied ETH and MeJA as well as by inoculation of Ralstonia solanacearum and treatment of high temperature stress，but not by exogenous application of SA. These results implied that CaERF4 may play key roles in pepper response to R. solanacearum inoculation and heat stress treatment.
Key worlds Capsicum annuu；EPF；Transcriptional activity；Tissue specificity；Induced expression
doi 10.3969/j.issn.1000-2561.2014.06.011
植物生长发育和对逆境的应答在很大程度上受到转录水平的调节，各种转录因子在其中发挥着十分重要的调节作用。乙烯响应因子（Ethylene Response Factor，ERF）转录因子属于植物AP2/ERF转录因子家族，AP2/ERF类转录因子是植物特有的转录因子，在拟南芥中有146个成员，在水稻中有136个成员[1]，初步的研究结果表明AP2/ERF家族成员与生长发育和应答环境胁迫等生物学过程密切相关，参与植物的生长，花的发育，果实的成熟，叶片的衰老，以及植物应答病原菌，低温，干旱等过程的调控[2-3]。此外还发现AP2/ERF类转录因子与水杨酸、茉莉酸、乙烯等多种信号通路相关[4-7]。例如，在烟草和番茄中过量表达TERF2/LeERF2可提高转基因植物的抗冷性。拟南芥AtERF1通过结合GCC盒激活PR基因表达，从而提高拟南芥对生物胁迫和非生物胁迫的抗性[8-9]。拟南芥过量表达AtERF14可通过上调ERF1、PDF1和Chi等防御反应相关基因提高拟南芥对镰刀菌（Fusarium oxysporum）侵染的抗性[10]。番茄JERFs的表达与ET和 ABA 信号途径相关，参与番茄耐盐、耐旱、耐冷和抗病调控[11]。这些结果显示出ERF家族成员在植物应答逆境胁迫过程中的重要作用。迄今关于ERF的研究大多数集中在拟南芥、水稻、番茄和烟草等少数作物的部分成员，人类对不同植物ERF家族的大多数成员的结构和功能还所知甚少。