【作者】 吴娟娟；

【导师】 武维华； 陈益芳；

【作者基本信息】 中国农业大学 ， 植物学， 2014， 博士

【摘要】 植物遭受干旱胁迫时,许多基因的表达受到调控,表明转录因子在植物响应干旱胁迫信号通路中发挥重要作用。拟南芥bZIP转录因子家族有75个成员,根据序列保守性分为10个亚族,其D亚族成员TGAs主要参与植物防御病害及生长发育两个过程。本论文工作主要研究拟南芥bZIP转录因子TGA7参与调控植物响应干旱胁迫的机制。土壤干旱实验结果表明,与野生型相比,拟南芥TGA7的T-DNA插入突变体tga7表现出较野生型较晚出现叶片萎蔫的干旱耐受表型,性状互补实验表明tga7突变体的干旱耐受表型是由于TGA7基因功能缺失引起的。过量表达TGA7的转基因株系对干旱胁迫的耐受性显著降低,同时离体叶片失水实验表明TGA7过量表达株系失水量明显高于野生型,而tga7突变体的失水量明显比野生型慢,说明TGA7参与植物响应干旱胁迫。组织表达分析显示TGA7在气孔中有较强的表达,气孔开度实验结果表明,ABA处理不能诱导TGA7过量表达株系的气孔关闭,而tga7突变体气孔的关闭程度明显高于野生型,推测TGA7可能通过调控气孔运动来响应植物干旱胁迫反应。离子通道和ABA在气孔运动中发挥重要作用,对tga7突变体和TGA7过量表达株系中离子通道基因、ABA响应基因及ABA合成代谢途径相关酶类基因表达进行检测,qRT-PCR检测结果表明AtBGI的表达量在TGA7过量表达株系中有明显下调,进一步的烟草瞬时转化实验也说明TGA7能够直接抑制AtBG1的表达。TGA7能识别核心序列为ACGT回文结构的顺式作用元件,对AtBG1启动子区的分析结果表明AtBG1的启动子区含有多个TGA7结合的作用元件,凝胶阻滞实验(EMSA)和染色质免疫共沉淀实验(ChIP)从体外和体内两个方面证明TGA7能够直接结合AtBG1的启动子区。土壤干旱实验中atbg1突变体表现出与TGA7过量表达株系类似的干旱敏感表型,气孔开度实验显示atbg1突变体气孔对ABA处理敏感性降低。AtBG1表达受干旱诱导升高,而在TGA7过量表达株系中这种诱导水平被明显抑制。综上所述,论文实验结果表明TGA7可能通过直接负调控AtBGl的表达来响应植物干旱胁迫反应。更多还原

【Abstract】 When plants suffer from drought stress, the expression of many genes is regulated, indicating that transcription factors play important roles in plant response to drought stress. There are75members of the bZIP family in Arabidopsis, which are subdivided into ten groups using common domains, and the D group proteins TGAs function in plant defense against pathogens and development. This research work focused on the functional characterization and regulation of AtTGA7in response to drought stress.In our research work, we found the TGA7T-DNA insertion mutant tga7showed a much more tolerant phenotype under drought stress compared with wild-type plant. Complemental experiment showed that tga7mutant complementation line had similar phenotype to the wild-type plant, indicating the function of TGA7in response to drought stress. In contrast, the TGA7-overexpression lines were more susceptible to drought stress compared with wild-type plant. The assay of water loss from detached leaves showed that the tga7mutant lost less water than the wild-type plant and tga7mutant complementation line, while the TGA7-overexpression lines lost much more water than wild-type plant, demonstrated that TGA7may play roles in drought stress. The GUS-staining assay showed that TGA7was preferentially expressed in stomatal guard cell, further stomatal experiments showed that induction of stomatal closure and inhibition of stomatal opening by ABA were impaired in the TGA7-overexpressing lines, indicating that TGA7play roles in plant responses to drought stress by regulating stomatal movement.To find the target genes of TGA7, the expression of ion channel genes, ABA-responsive genes and ABA biosynthesis enzyme genes were tested in the tga7mutant and TGA7-overexpression lines, which were proposed to mediate stomatal movements. qRT-PCR results showed that the expression of AtBG1was strongly repressed in the TGA7-overexpressing lines, further transient expression assay in Nicotiama benthamiana leaves also demonstrated the suppression of AtBGl expression by TGA7.TGA7binds to DNA sequence with an ACGT core (ACE motif), and there are three ACE motifs within the AtBGl promoter. EMSA and ChIP assay demonstrated that TGA7could bind to the AtBG1promoter in vitro and in vivo. Similar to the TGA7overexpression lines, loss-of-function mutant of AtBG1showed drought-sensitive phenotype and was less sensitive to ABA in regulation of stomatal movements. AtBGl expression was markedly induced under drought stress, whereas this induction was restained in the TGA7-overexpression lines.Taken together, our data indicate that TGA7is involved in plant responses to drought stress by negatively regulating AtBGl expression.更多还原