【作者】 万丽云；

【导师】 黄荣峰；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 植物激素影响植物的生长发育与对环境胁迫的应答反应。乙烯是植物重要的激素之一,它不仅参与植物种子的萌发、根的生长、叶片及花的形成与发育、果实的成熟乃至植物衰老与凋亡等发育过程,而且还参与植物对低温、干旱、高盐的适应以及抵抗病原菌侵染等生物和非生物胁迫应答过程。植物体内乙烯水平的变化是调控植物生长发育与逆境胁迫应答反应的前提条件之一,研究乙烯生物合成的调控机理对阐明乙烯的生物学功能及其信号传导与调控机理具有重要的意义。研究表明转录和转录后水平调控在乙烯生物合成中具有重要的作用。通过转录水平和转录后水平的协同调控作用,植物便可调节乙烯的生物合成和信号转导途径开闭调节乙烯的功能,协调乙烯信号途径与其它激素信号途径的互作,完成各种生命活动和适应各种胁迫反应。水稻是重要的粮食作物,水稻乙烯合成的调控虽然取得了一定的研究进展,但有关转录调控的分子机理还有待进一步的探讨。本研究通过分析转录组基因表达数据库,发现了一组干旱应答的ERF基因(Rice drought-responsive ERF genes, OsDERF),并对其中的一个新的转录激活子OsDERF1开展了系统的功能分析。它通过激活乙烯应答相关抑制子的表达负调控乙烯合成和干旱胁迫反应。本研究OsDERF1,这个基因的表达受干旱、ABA和ACC诱导。过量表达OsDERF1(OE)降低水稻对干旱胁迫的耐受性,而OsDERF1(RI)增加水稻对干旱胁迫的耐受性。过量表达OsDERF1植株中可溶性糖、脯氨酸和叶绿素的含量比野生型中高,而丙二醛的含量比对野生型低,OsDERF1(RI)中则相反,在干旱胁迫条件下这种差异更加明显。OsDERFl在渗透适应性反应中的负调控也验证了其在干旱胁迫中的负调控的功能。本研究通过Affymetrix GeneChip的方法寻找OsDERF1下游目标基因,以求阐明其在干旱耐受反应中的分子机理,结果发现OsDERF1激活了许多逆境相关的负调控因子,包括ERF抑制子。通过生化和分子的研究方法发现OsDERF1能够直接同ERF抑制子OsERF3和OsAP2-39基因启动子区域的GCC box结合。深入的研究表明乙烯合成相关基因在OsDERF1过量表达植株中下调,而在OsDERF1(RI)植株中升高,这也使得乙烯的释放量发生了相应的变化。过量表达OsERF3/OsAP2-39抑制乙烯合成相关基因的表达和乙烯释放,而ACC处理能够恢复OsERF3过量表达植株对干旱的敏感表型,这证明了乙烯在水稻干旱胁迫应答过程中起到非常重要的作用。本研究认为OsDERF1通过转录激活OsERF3/OsAP2-39的表达抑制了乙烯合成基因的表达,从而负调控乙烯的释放和干旱胁迫反应,以上数据揭示了一个ERF转录调控复合体通过控制乙烯的合成来调控干旱逆境反应,此结果深化了我们对ERF蛋白参与调控乙烯合成相关的干旱胁迫反应方面的理解。此外,为了更深入的了解OsDERF1的功能,通过筛选酵母表达库,得到几个OsDERF1的互作蛋白,其中有一个是C3H2C3环指蛋白将它命名为DEIP1(OsDERF1Interacting Protein1),DEIP1的表达模式和OsDERF1非常类似,它可能参与到OsDERF1的降解。酵母中的实验初步证明了它们之间的相互作用,DEIP1和OsDERF1都定位在细胞核。更多还原

【Abstract】 Phytohormone function as signal molecules regulated a variety of developmental processes and stress responses in plants. Ethylene is one of the important hormones not only involved in plant development, such as seeds germination, root growth, and fruit rippeness, but also participated in plant biotic and abiotic stress response, like low temperature, drought, high salt and pathogen infect. It is important to elucidate the biological function of ethylene and the mechanism of ethylene signal transduction by studying the regulation mechanism of ethylene biosynthesis. Ethylene biosynthesis is modulated by many factors or regulators at both transcriptional and post-transcriptional levels. Combining the regulations at both transcriptional and post-transcriptional levels of ethylene synthesis and transduction, plant can coordinate the ethylene signal pathways and other hormone signaling pathway, keep their life and adapt various stresses. Rice is an important food crop, and studies in rice ethylene synthesis have made great progress in recent years. However, the detail regulatory mechanisms are limited that ethylene production is transcriptionally regulated in rice. In the present research, using genomic transcriptional data in the websites of rice massively parallel signature sequencing,12drought-responsive ERF genes (DERF) were identified. One of the DERF genes, OsDERF1(Os08g35240), is located in rice chromosome8. It is a novel transcriptional activator that modulates the expression of ethylene response factor (ERF) repressors, the ethylene synthesis and the drought tolerance in rice. Through analysis of transcriptional data, one of the drought-responsive ERF genes (DERF), OsDERF1, was identified for its activation in drought, ABA and ethylene precursor ACC. Transgenic plants overexpressing OsDERF1(OE) led to deduced while knockdown the expressing OsDERF1(RI) conferred enhanced tolerance to drought stress in rice seedling and tilling stages. And this regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1using the Affymetrix GeneChip, which activated the expression of cluster stress-related negative regulators including ERF repressors. Biochemical and molecular approaches evidenced that OsDERF1at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3and OsAP2-39. Further investigations showed that the expression of ethylene synthesis genes reduced in OE seedlings while enhanced in RI seedlings, thereby resulting in the changes of ethylene production. Moreover, overexpression of OsERF3/OsAP2-39suppressed the ethylene synthesis. In addition, application of ACC recovered the drought sensitive phenotype in overexpression of OsERF3transgenic lines, evidencing that the ethylene production contributes to the drought response in rice. Thus our data reveal that ERF transcriptional complex modulates drought response through controlling the ethylene synthesis, deepening our understanding of ERF protein regulation in ethylene synthesis-related drought response.In addition, in order to deepen our understanding on OsDERF1, we identified several OsDERF1interacting proteins by screening yeast expression library, one of those was a C3H2C3Ring finger protein DEIP1(OsDERF1-Interacting Protein1), the expression pattern of DEIP1was similar with OsDERF1, and it might participate in the degradation of OsDERF1. Experiments in yeast demonstrated that DEIP1could interact with OsDERF1, DEIP1and OsDERF1were both located in the nucleus.更多还原