番茄EIN3-binding F-box蛋白2超表达诱导单性结实和果实成熟异常的机制研究

Parthenocarpy is the transition of a quiescent ovary into a rapidly growing young fruit, so-called fruit set, without pollination and/or fertilization, and produce seedless fruit. Therefore, an understanding of the mechanisms that induce parthenocarpic fruit formation can facilitate to unravel the molecular regulation system of fruit set and provides a basis for molecular breeding. Tomato (Solanum lycopersicum) is the model system for studying the biological bases of fleshy fruit initiation, development and ripening. Over the years, it has been well established that tomato fruit set depends on the activation of the auxin and gibberellin signaling pathways, although the exact role of each of these two hormones is still poorly understood, and only few of the signaling components involved have been identified so far. However, the biological function of ethylene in regulating tomato fruit set is rarely known. In Arabidopsis, EBF1 and EBF2 (EIN3-binding F-box protein 1 and 2) are critical elements in regulating ethylene signaling pathway through mediating the degradation of EIN3/EIL proteins by ubiquitin/26S proteasome pathway. It is still not clear whether EBF2 regulates other targets besides EIN3/EIL family. Our earlier research found that SlEBF2 overexpression induced parthenocarpy, abnormal fruit ripening and insensitivity to ethylene treatment in tomato, suggesting that the ethylene signaling pathway is involved in regulating tomato fruit set. While the molecular mechanism on tomato fruit set regulated by SlEBF2 protein is completely unknown. In this project, characterization of SlEBF2 overexpression tomato lines, identification of target proteins of SlEBF2 and analysis of their biological functions, and research on whether SlEBF2 mediates the interaction between ethylene and auxin or gibberellin will be carried out. Based on these data, it will be uncovered that SlEBF2 overexpression induces parthenocarpy and abnormal fruit ripening through regulating ethylene signaling pathway or other pathway. If ethylene is critical in this process, we will discover that ethylene regulates directly tomato fruit set or controls indirectly fruit initiation through cross-talk with auxin and/or gibberellin. Finally, the molecular mechanism will be unraveled that SlEBF2 regulates tomato fruit set and fruit ripening. The results is not only important for revealing the biological functions and molecular mechanism of ethylene in regulating fruit set, but also valuable for understanding the process and molecular mechanism of fruit set.

单性结实是子房不经授粉/授精发育成无籽果实，研究单性结实有助于阐明座果过程的调控机制。番茄座果受生长素和赤霉素调控为人们所熟知，而迄今对乙烯在座果过程中的调控功能了解甚少。拟南芥中，EBF1/2通过控制EIN3/EIL蛋白来调控乙烯信号途径。我们前期发现，番茄SlEBF2超表达诱导单性结实和果实成熟异常，预示着乙烯信号途径可能参与调控座果过程，但SlEBF2调控座果和果实成熟的机制尚不清楚。因此，本项目拟通过对SlEBF2超表达株系进行鉴定、SlEBF2靶蛋白分离及其生物学功能分析、以及SlEBF2与乙烯、生长素和赤霉素之间的关联性分析等研究，深入阐明SlEBF2超表达诱导单性结实和果实成熟异常的机制，以及乙烯是否直接或间接通过与生长素或赤霉素之间互作调控番茄座果。研究结果对揭示乙烯在座果过程中的生物学功能及其调控机制具有重要意义，同时也对阐明番茄座果过程的分子调控机理具有重要促进作用。

拟南芥中，EBF1/2通过泛素化途径介导EIN3/EIL蛋白降解来调控乙烯信号转导。前期研究发现，番茄SlEBF2超表达诱导果实发育和成熟异常，本研究采用酵母双杂交系统筛选SlEBF2的互作蛋白，并对其互作蛋白在果实发育和成熟过程中的生物学功能进行分析。结果显示，EIN3/EIL蛋白家族中的SlEIL1和SlEIL3与SlEBF2具有较强的互作作用，并且获得几个非EIN3/EIL家族的潜在互作蛋白，其中包括编码细胞分裂素合成关键酶的基因SlIPT4。通过RNAi介导的基因沉默技术分析发现，无论是SlEIL1和SlEIL3的单基因沉默还是双基因共沉默，与野生型对照植株相比，转基因番茄株系都没有呈现出果实发育和成熟的差异。SlIPT4在番茄座果过程中存在动态表达变化特征，且随着果实的成熟其表达水平持续增强。SlIPT4沉默加速番茄叶片衰老，抑制果实成熟过程中番茄红素的合成，导致永不转红的橙色果实，与SlEBF2超量表达果实具有类似的表型特征。分子水平分析揭示，SlIPT4可能通过控制番茄红素合成酶基因ZISO的表达水平来调控番茄红素合成，且导致果实中ABA水平的下降，而低水平的ABA负反馈调控番茄红素合成途径的第一个酶基因PSY的表达。这些结果表明，SlIPT4可能参与SlEBF2对果实成熟的调控。然而，尽管SlIPT4调控细胞分裂素合成，但SlIPT4沉默并没有引起番茄果实发育过程的改变。随着番茄基因组数据的完善，我们发现番茄中还存在一个与SlEBF2高度相似的新EBF基因，两者之间的氨基酸序列相似度高达74.57%。蛋白功能域和进化树分析表明，该新EBF蛋白属于植物F-box蛋白家族的EBF亚家族，命名为SlEBF2-like。SlEBF2-like的表达受乙烯正调控和生长素的负调控，在子房中表达较强，座果后表达下调，并在果实发育过程中保持温和的表达水平，但随着果实成熟启动，其表达水平迅速增强，果实成熟后表达下调。SlEBF2-like沉默对番茄果实发育和成熟过程没有影响，然而SlEBF2-like超量表达引起番茄果型改变，产生长型果，且果实成熟过程中的色泽变化异常，色泽转换从果尖逐步向果蒂方向延展。统计分析表明，SlEBF2-like超表达极显著地延缓了番茄果实的发育和成熟进程，表明SlEBF2-like参与番茄果实发育和成熟过程调控。