桃PpIAA1与PpERF4协同调控果实成熟软化的分子机制研究

Fruit ripening and quick softening is one of the major problems limiting the development of peach industry. The fruit of melting-flesh peach cultivars produce high concentration of IAA which causes considerable amount of ethylene production, thereby resulting in rapid fruit softening at the late-ripening stage. However, the molecular mechanism is still unclear. In our previous study, PpIAA1, the important factor in auxin signal transduction, as well as ethylene response factor (PpERF4), were closely related to auxin accumulation and ethylene emission during fruit ripening in melting-flesh peaches. To further investigate the molecular mechanism of ripening and softening in peach fruits mediated by PpIAA1 and PpERF4, the current project was proposed to be conducted as follows. First, the gene expression pattern and subcellular localization of PpIAA1 and PpERF4 were investigated. Second, the over-expression manipulation and virus-induced gene silencing (VIGS) were carried out to verify the function of PpIAA1 and PpERF4. Finally, using yeast two hybrid assay (Y2H), electrophoretic mobility shift assay (EMSA), GST Pull-down, co-Immunoprecipitation (CoIP) and tobacco dual-Luciferase reporter assay system to study how PpIAA1 and PpERF4 regulate the transcription of ethylene biosynthetic genes PpACS1 and PpACO1, as well as cell wall modifying gene PpPG1, moreover, the effect of the interaction between PpIAA1 and PpERF4 on the transcription of PpACS1, PpACO1 and PpPG1. The results may advance our understanding of the molecular mechanism of peach fruit ripening and softening mediated by auxin and ethylene, and provide theoretical basis for developing the efficient storage and handling technologies of peach fruits.

果实成熟后迅速软化是长期制约桃产业发展的关键问题。溶质桃果实成熟期产生高浓度的IAA，诱导乙烯的大量释放，导致果实迅速软化，其中的分子机制尚不明确。申请人前期研究发现，桃果实成熟期生长素信号途径关键因子PpIAA1和乙烯响应因子PpERF4的表达与乙烯释放和生长素积累呈显著正相关，且两者之间存在互作。本项目拟从解析PpIAA1和PpERF4的协同调控机制入手，首先明确PpIAA1和PpERF4的诱导表达特性和亚细胞定位情况；随后通过超量表达和病毒介导的基因沉默等手段，鉴定PpIAA1和PpERF4的功能；最后利用酵母双杂交、凝胶阻滞迁移、双荧光素酶瞬时表达等多种互作技术，研究PpIAA1和PpERF4之间的相互作用，以及它们如何调控乙烯合成关键基因和细胞壁相关基因的转录。研究结果将有助于深入解析生长素和乙烯协同调控桃果实成熟软化的分子机理，为开发高效的桃果实贮藏保鲜措施提供理论依据。

果实成熟后迅速软化是长期制约桃产业发展的关键问题。溶质桃果实成熟期产生高浓度的IAA，诱导乙烯的大量释放，导致果实迅速软化，其中的分子机制尚不明确。.本项目主要研究内容包括：.1.阐明乙烯通过ERF转录因子调控桃果实成熟软化的分子机制；以溶质型中油桃13（不耐贮运）和SH型桃中油桃16（耐贮运）为实验材料研究生长素和乙烯调控桃果实成熟软化分子机制。取得以下研究结果： I. 首次发现乙烯可通过调控PpNCED基因表达调节ABA的生物合成；II. ERFs通过调控乙烯合成基因的表达来反馈调节乙烯生物合成；.2. 利用抗体芯片技术筛选桃果实成熟的差异蛋白并构建乙烯合成的生物学模型，构建了首款针对桃果实蛋白组的单克隆抗体库，基于抗体芯片及代谢组等技术开发了生长素和乙烯参与桃果实成熟调控的动力学预测模型 ； .3. 生长素酰胺水解酶参与桃生长素和乙烯的交互作用；发掘了桃果肉生长素氨基酸水解酶PpILR1，亚细胞定位发现该基因定位于细胞核中。功能分析表明ILR1可以水解IAA-Ala或IAA-Leu，释放游离态的IAA。ACS1是乙烯生物合成的限速酶，分子互作实验表明ILR1蛋白可以直接结合并激活ACS1启动子的表达。首次发现生长素氨基酸水解酶PpILR1不仅可以水解结合态生长素至游离态有活性的生长素，同时还参与乙烯的调控；.4.阐明生长素和乙烯通过IAA1和ERF4协同调控桃果实成熟软化的分子机制；1）乙烯通过PpERF4调控脱落酸合成基因PpNCED2/3的表达来调节脱落酸的合成； 2）PpIAA1可直接结合PpACS1、PpNCED2、PpNCED3、PpPG1启动子，并激活基因的表达；3）PpIAA1与PpERF4互作，并能进一步激活PpNCED2、PpNCED3、PpPG1基因的转录活性。.研究结果将有助于深入解析生长素和乙烯协同调控桃果实成熟软化的分子机理，为开发高效的桃果实贮藏保鲜措施提供理论依据。项目执行期间发表论文14篇，其中SCI论文8篇；授权国家发明专利1项，获河南省科技厅科技进步贰等奖1项。