与香蕉果实成熟相关且受乙烯诱导的14-3-3互作蛋白的筛选及其生理功能分析

The banana (Musa acuminate L. AAA group) fruit undergoes a postharvest ripening process, which plays an important role in improving the quality and extending the shelf life of bananas. To manipulate postharvest banana ripening, a better understanding of the mechanism of postharvest ripening is necessary. In a previous study, four novel 14-3-3 protein genes were isolated from banana cDNA library and their expression pattern at different fruit postharvest ripening stages was analyzed. The four genes presented differential expression during banana fruit postharvest ripening, and three of them were significantly induced by exogenous ethylene treatment. The findings suggest that 14-3-3 proteins could be involved in regulatory processes during fruit ripening in banana. Based on the results, the aim of this proposal will carry out a comprehensive identification of 14-3-3 interactome present in the preclimacteric banana fruits through a yeast two-hybrid screen using the isolated banana 14-3-3 proteins as baits. We will confirm their interaction for further bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation test analysis. At the last, we will perform physiological and molecular biological methods to investigate 14-3-3 proteins and their positive targets in regulating ethylene biosynthesis relating to fruit ripening and in regulating substance metabolism process during fruit postharvest ripening. The results will have the potential interesting findings to elucidate the mechanism of banana fruit postharvest ripening and will be of great agronomical interest to control banana fruit postharvest ripening.

香蕉采后成熟对于香蕉的品质和货架寿命具有重要作用，只有充分地了解果实采后成熟机理，才能更好地去调控果实成熟。我们前期研究发现，香蕉的三个14-3-3蛋白基因在果实采后成熟过程中受乙烯的强烈诱导，与果实成熟密切相关。本项目拟采用酵母双杂交技术对与果实成熟相关且受乙烯诱导的14-3-3蛋白对果实采后成熟的调控机制进行深入研究。以与成熟相关的14-3-3蛋白为诱饵筛选在果实成熟跃变前与14-3-3有相互作用的蛋白。进一步采用双分子荧光互补和免疫共沉淀技术验证它们之间的相互作用。采用生理学和分子生物学的方法系统分析14-3-3蛋白及其互作蛋白在香蕉果实采后成熟过程中的生理功能。目的是阐明14-3-3蛋白及其互作蛋白在诱导果实成熟乙烯生物合成中的作用，在果实成熟跃变的物质代谢变化中的作用。研究结果将为香蕉果实成熟的基础理论研究提供新的创新点，为调控香蕉果实采后成熟提供理论依据。

运用转录组学结合生物信息学的方法对香蕉中的25个14-3-3蛋白基因家族成员在果实发育、成熟的不同阶段及各种逆境胁迫（渗透、冷和盐胁迫）转录水平进行了系统的分析，结果发现香蕉中的大部分14-3-3蛋白基因家族成员的转录水平在果实发育的早期显著高于其它时期，同时发现香蕉中的某些基因家族成员在果实成熟、各种逆境胁迫（渗透、冷和盐胁迫）转录水平有显著的变化。 据此，我们推测香蕉的14-3-3蛋白对香蕉果实的早期发育、果实成熟以及逆境胁迫起着重要的调控作用。运用酵母双杂交技术筛选了成熟香蕉果实（80-DAF）的cDNA文库，共获得36个可能与14-3-3蛋白互作的目标靶蛋白，这些蛋白包括物质代谢类的相关蛋白（酶），物质跨膜运输，能量代谢，转录调控，信号转导过程中的激酶以及受体蛋白、激素信号转导过程中的相关蛋白与酶。经生物信息学分析，选择与乙烯生物合成相关的ACO1、与乙烯信号转导相关的蛋白Erf1、乙烯受体蛋白等目标靶蛋白，分别构建了酵母双杂的诱饵载体和表达载体，并在酵母中初步验证了ACO1、乙烯受体蛋白与Ma1-4-3-3e、Ma1-4-3-3c和Ma1-4-3-3a蛋白的相互作用。综上所述，本研究初步证实了14-3-3蛋白及其调控蛋白对果实成熟过程中乙烯生物合成及信号转导的调控，丰富了香蕉果实采后成熟理论知识。本项目资助发表SCI 论文四篇，待发表两篇。项目投入经费25.0万元，支出24.3752万元。各项支出基本与预算相符。剩余经费0.6248万元。剩余经费计划用于本项目的后续研究。