乙烯和活性氧协同调控月季花朵衰老的分子机理解析

Rose is one of the most important commercial flowers worldwide, and ethylene is an important factor that affects the senescence of rose flowers. Reactive oxygen species (ROS) is widely involved in transmitting the senescence signals, and also plays a key role in flower senescence. However, the signal transmission between ethylene and ROS is still not very clear. In order to understand the molecular mechanism underlying ethylene-regulated flower senescence in roses, it would be of great importance to screen and identify the ethylene-responsive transcription factors that regulate ROS levels. Based on the microarray analysis of ethylene-responsive genes in rose petals which has been done by former researchers, we have finished data analysis and quantitive RT-PCR test, and have selected 4 ethylene-responsive transcription factor genes, whose expression is obviously changed during flower senescence. Here, the function of the candidate genes during flower senescence will be studied by over-expression in tobacco and silencing in rose flowers. Through RNA-seq analysis in silenced rose petals, the antioxidase genes whose expression levels were significantly changed will be selected. Further, the transcriptional regulation of the selected antioxidase genes by the ethylene-responsive transcription factors will be studied by transient transcriptional activation and EMSA analysis. The work aims to study the signal transduction between ethylene and ROS during flower senescence of roses, which will be helpful to understand the molecular mechanism underlying ethylene-regulated flower senescence in roses, and provide useful evidence for molecular breeding of roses to improve its quality.

月季是世界第一大切花，乙烯是影响花朵衰老的重要因素。活性氧广泛参与衰老信号传递，同样在花朵衰老过程中起到关键作用。然而，乙烯到活性氧之间的信号传递途径尚不明确。筛选和鉴定响应乙烯并调控活性氧水平的转录因子，成为解析乙烯调控月季花朵衰老分子机理的一个重要课题。在前期工作中，申请者在前人的月季花瓣乙烯响应基因表达谱的基础上，通过芯片数据分析和荧光定量PCR检测，已经筛选出响应乙烯并在月季花朵衰老过程中表达变化明显的基因4个。本课题将通过烟草中过表达以及月季花瓣中的沉默，研究候选基因对花朵衰老的调节。选取沉默月季花瓣，进行RNA-seq，筛选表达变化明显的抗氧化酶基因。通过瞬时转录激活和EMSA等方法，研究目标转录因子对抗氧化酶基因的调节。本研究旨在了解月季花朵衰老过程中，乙烯和活性氧之间的信号传递途径，为进一步解析乙烯调控月季花朵衰老的分子机理奠定基础，进而为月季品质改良分子育种提供理论依据。

月季是世界第一大切花，乙烯是影响花朵衰老的重要因素。活性氧广泛参与衰老信号传递，同样在花朵衰老过程中起到关键作用。然而，乙烯到活性氧之间的信号传递途径尚不明确。本研究在前人的月季花瓣乙烯响应基因表达谱的基础上，通过芯片数据分析和荧光定量PCR检测，筛选出响应乙烯并在月季花朵衰老过程中表达变化明显的转录因子基因4个和功能未知基因3个。通过烟草中过表达对这几个基因进行了功能初步验证，表明一个ERF基因RU03393和一个bHLH基因RU03429在植物衰老过程中可能发挥了一定作用。进一步通过月季花瓣中基因沉默和过表达验证了RU03393和RU03429的功能，表明RU03393和RU03429沉默后明显延缓月季花瓣的衰老，过表达后促进花瓣的衰老。对RU03393和RU03429过表达样本进行RNA-seq分析，发现抗氧化酶、蛋白核酸脂类降解酶和植物激素代谢等相关基因在RU03393和RU03429过表达后呈现显著差异，进一步通过qRT-PCR对这些基因的表达进行了验证，初步确定6个抗氧化酶和蛋白核酸脂类降解酶基因作为可能的下游基因，并克隆了该基因启动子。通过烟草叶片瞬时转录激活实验，表明RU03393强烈诱导一个核酸酶RU21924的启动子活性。接下来将通过EMSA和ChIP实验，进一步验证RU03393对RU21924等基因的调控。本研究对于了解乙烯调控月季花朵衰老的分子机理提供了一些最新证据，为月季品质改良分子育种提供了一些理论依据。

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