【作者】 李丛；

【导师】 于静娟；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 干旱胁迫严重影响植物生长,是限制农作物产量的主要环境因素之一。植物进化出了多种分子机制来适应和抵抗干旱胁迫。在植物中,响应干旱胁迫的信号途径主要有两个：一个是脱落酸(abscisic acid, ABA)依赖信号途径,另一个是非ABA依赖信号途径。两条信号通路之间并不是孤立的,它们彼此相互联系,形成了一个复杂的调控网络。谷子是原产于我国的一种古老的粮食作物,具有营养丰富和抵抗干旱环境等特点,是我国重要的战略粮食作物。目前对谷子抗旱的分子机理还知之甚少。在本研究中,我们以干旱响应元件(dehydration responsive element, DRE)为诱饵,通过酵母单杂交技术从谷子cDNA文库中克隆到一个响应ABA信号的DRE元件结合蛋白基因。我们将该基因命名为SiARDP (Setaria italica ABA-responsive DRE-binding protein)。 Quantitative real-time PCR (qRT-PCR)和Northern blot结果显示SiARDP受干旱、高盐、ABA和低温诱导表达。SiARDP在谷子的根、茎、叶和花序中都有表达,并且在叶中表达量较高。谷子原生质体亚细胞定位显示SiARDP蛋白定位于细胞核；凝胶阻滞(electrophoretic mobility shift assay, EMSA)实验表明原核表达的SiARDP-His融合蛋白能与DRE元件结合；酵母转录激活实验证明SiARDP蛋白具有转录激活活性。在拟南芥中异源表达SiARDP能够提高植物对干旱和高盐的耐受力,干旱胁迫下转基因植株比野生型植株积累更多的脯氨酸,高盐胁迫下转基因植株的离子渗透率低于野生型植株。在谷子中超表达SiARDP提高了转基因谷子的耐旱性,并且干旱胁迫下转基因谷子比野生型谷子积累了更多的脯氨酸。超表达转基因谷子中多个启动子区存在DRE元件与干旱胁迫相关基因的表达量升高,说明超表达谷子植株中SiARDP通过调控干旱胁迫相关基因的表达来提高植株耐旱能力。分析显示SiARDP启动子区存在两个ABA响应元件(ABA-responsive element, ABRE)。我们从谷子中克隆了两个ABRE结合蛋白(ABRE-binding protein, AREB)基因分别命名为SiAREBl和SIAREB2。 SiAREBl和SiAREB2受干旱、高盐和ABA诱导,但不受低温胁迫诱导。谷子原生质体亚细胞定位显示SiAREB1和SiAREB2定位于细胞核,酵母转录激活实验证明SiAREB1和SiAREB2具有转录激活活性。EMSA,酵母单杂和ChIP分析证明SiAREB1和SiAREB2在体内和体外都能够与SiARDP启动子区的ABRE元件直接结合。在拟南芥中异源表达SiAREBl和SiAREB2同样能够提高转基因拟南芥对干旱和高盐的耐受力。点突变和蛋白磷酸化实验表明SiAREB2蛋白活性受磷酸化修饰调控。综上所述,SiARDP作为DREB类转录因子参与谷子非生物胁迫应答,在干旱和高盐胁迫下受AREB转录因子调控,参与ABA信号通路,在低温胁迫下可能受其它因子调控参与非ABA信号通路。更多还原

【Abstract】 Drought stress greatly impacts growth, and is one of the major environmental factors that limit crop production. Plants have evolved many molecular mechanisms to adapt and resist to drought stress. There are two main transcriptional signal pathways to response to drought stress:abscisic acid (ABA)-dependent and-independent signal pathway. Drought stress responsive signals are complex, and some cross-talks exist between two signal pathways.Foxtail millet, an ancient crop in China, provides rich nutrient elements and has excellent drought tolerance, and is an important strategic food crops in China. However the molecular mechanisms of drought stress in foxtail millet remain unknown. In this study, an ABA-responsive dehydration responsive element (DRE) binding protein gene, named SiARDP, was cloned from foxtail millet using a yeast one-hybrid screening assay. Northern blot and qRT-PCR assay showed that SiARDP induced by drought, high salt, low temperature stress and ABA treatment. The qRT-PCR assay showed that the transcriptional levels of SiARDP in leaf were higher than in root, stem and inflorescence. The SiARDP was located in the nucleus of foxtail millet protoplasts. Electrophoretic mobility shift assay (EMSA) showed that the fusion protein SiARDP-His could bind to the DRE core element, and the SiARDP had the ability of transcriptional activity in yeast. Heterologous expression of SiARDP in Arabidposis enhanced the tolerance to drought and high salt stresses. Under drought stress, transgenic Arabidposis accumulated more proline than the wild type plants, and under high salt stress, the electrolyte leakage of transgenic plants was much lower than that of wild type plants. Overexpression of SiARDP in foxtail millet enhanced tolerance to drought stress, and accumulated more proline. The qRT-PCR assay showed that the transcriptional levels of many stress-relevant genes with several DRE core elements in their promoter regions increased in transgenic foxtail millet. The data suggested that SiARDP responds to drought stress and regulates some functional genes to improve tolerance.Further analyses showed that two ABA-responsive element (ABRE) exist in the promoter region of SiARDP. We cloned two ABRE binding protein (AREB) genes from foxtail millet, named SiAREBl and SiAREB2. The qRT-PCR showed that SiAREB1and SiAREB2induced by drought, high salt stresses and ABA treatment. SiAREB1and SiAREB2located in nucleus of foxtail millet protoplasts and had the ability of transcriptional activation in yeast. The EMSA, yeast one hybird and ChIP assays shown that SiAREB1and SiAREB2could bind to the ABRE core element in SiARDP promoter region in vitro and vivo. Heterologous expression of SiAREB1and SiAREB2in Arabidopsis enhanced the tolerance to drought and high salt stresses and activated the transcription of the target genes. The point mutant and protein phosphorylation assay demonstrated that the ability of transcriptional activation of SiAREB2was regulated by phosphorylation.Taken together, SiARDP was DREB-type transcriptional factor and responsive to abiotic stress. SiAREB transcriptional factors regulated the expression of SiARDP under drought and salt stress in ABA-dependent pathway, but under low temperature other regulatory factors regulated SiARDP in ABA-independent pathway.更多还原