【作者】 王鹏程；

【导师】 宋纯鹏；

【作者基本信息】 河南大学 ， 植物学， 2010， 博士

【摘要】 在高等植物中,活性氧作为重要的信号分子介导了防御反应、程序性死亡、气孔关闭、细胞生长和植物发育调控等许多重要的生理过程。活性氧能够广泛地调控许多基因的表达,暗示了在植物细胞中存在复杂的活性氧应答的转录调控网络,但目前对于这一复杂调控网络中的许多细节仍不清楚。本研究通过基因芯片技术在基因组水平上系统研究检测了ABA以及H202对拟南芥幼苗基因表达的调控。发现外源H202处理使459个基因的转录水平明显增加,同时使221个基因的表达显著减少,显示H202也是一种重要的转录调节物质,广泛地调控了许多基因的表达。而且ABA和H202对基因表达的调控存在相当大的重叠,它们可能通过一种协同模式调控下游基因的表达。通过生物信息学检索,本研究从活性氧特异应答基因的启动子序列中发现8个潜在的氧化胁迫应答的顺式作用元件。通过基因枪介导的瞬时表达系统检测发现4个保守序列元件在H202、MV等不同种类的活性氧处理下能够诱导下游萤光素酶基因的表达,它们可能作为氧化胁迫应答的元件(ROS responsive element)参与活性氧应答的转录调控。在上述研究的基础上,发现AP2/EREBP家族转录因子ERF6能够特异地与ROE8结合,共转化ERF6能够显著增强ROE8的转录活性,而且H202处理能够促进ERF6对ROE8的激活效应。进一步的研究发现ERF6能够与MPK3／6发生直接的相互作用,MPK6能够磷酸化ERF6蛋白的Ser266和Ser269残基。ERF6磷酸化位点的突变会引起ERF6亚细胞定位的改变,ERF6DD定位于细胞核中,而非活化状态的ERF6AA主要存在于细胞质中。35S-ERF6WT、35S-ERF6DD超表达转基因植物在萌发和生长早期对H202更加敏感,一些活性氧应答基因的表达也明显增加。免疫共沉淀结果还显示H202和磷酸化能增加ERF6和MPK6复合体的积累。上述结果显示ERF6作为活性氧应答的转录因子,接受活性氧激活的MPK6的调控,介导了活性氧对于基因表达和幼苗生长发育的调控过程。本研究工作系统比较分析了H202对基因表达的调控作用,通过生物信息学和遗传学分析检测了在活性氧应答基因启动子中存在的氧化胁迫应答元件,并鉴定了其中一个顺式作用元件ROE8的结合蛋白ERF6的功能,发现ROS-MPK6-ERF6途径部分地介导了活性氧对于基因表达的调控过程。这一研究为阐明活性氧应答的转录调控机制提供了重要的基因组学和遗传学线索。更多还原

【Abstract】 Reactive oxygen species (ROS) has been characterized as an important signal molecule that mediates many developmental and physiological processes in plants, which include biotic and abiotic stress responses, stomotal movement, programmed cell death, growth and development. ROS also plays a global role in gene transcription. So far, details of the ROS responsive transcriptional network are still largely unknown.To gain insight into the coordination of gene expression profiles under abscisic acid (ABA) and H2O2 applications, global changes in gene expression in response to ABA and H2O2 in Arabidopsis seedlings were investigated by microarray analysis.459 transcripts were found to be significantly increased, whereas another 221 decreased following H2O2 treatment compared with control. These results suggest the global role of H2O2 in gene transcription. The transcriptional responses induced by the application of exogenous ABA and H2O2 overlapped substantially. Promoters of ROS up-regulated genes were selected for a bioinformatics search to identify the potential transcription factor binding sites responsible for ROS induced gene expression, and four of eight potential ROS responsive cis-elements (ROEs) are sufficient to confer a rapid increase of luciferase under H2O2 and MV treatment indicated their roles in ROS responsive gene transcription.We also found that an AP2/EREBP transcription factor ERF6 could bind to the ROE8 specially. Co-expression of ERF6 effector enhanced the expression of ROE8 driver luciferase gene, we also found that ERF6 interacted physically with MPK6 in vitro and in vivo, and also served as a substrate of MPK6. Two Ser residues, Ser 266 and Ser 269 were the putative phosphorylation site of ERF6. After application of H2O2, phosphorylation by MPK6, ERF6 relocalizes from cytoplasm to the nucleus. Expression of ROS responsive genes is strongly up-regulated in 35S-ERF6WT and 35S-ERF6DD transgenic plants. Furthermore, growths of 35S-ERF6WT and 35S-ERF6DD seedlings are more sensitive to H2O2 treatment than WT or 35S-ERF6AA transgenic plants. Immunoprecipitation results indicated that H2O2 treatment and SS to DD mutation also increased the stability of ERF6-MPK6 complex. Based on those results, the MPK6-ERF6 pathway has established that it mediated the ROS responsive transcription and seedling growth regulation.In this work, H2O2 responsive gene transcription was compared at genomic ranges and potential ROS responsive cis-elements were identified. The function of ROE8 binding protein ERF6 in controlling the ROS responsive gene expression was also characterized. We found that H2O2 responsive gene expression partly dependent on ROS-MPK6-ERF6 pathway. These data may provide new insight into ROS responsive gene regulation mechanism.更多还原