猕猴桃采后果实软化过程中HDAC的作用及其调控机制研究

During the storage of kiwifruit after harvest, the fruit is prone to ripening and softening, seriously affecting the commercial value. Kiwifruit softening is a complex process, which is closely related to ethylene, cell wall metabolism and starch degradation, and it is regulated by many genes and proteins. Histone deacetylase (HDAC) plays an important role in the regulation of fruit development, ripening and senescence of tomato, longan and banana. Whether HDAC mediated epigenetic regulation is involved in the softening process of kiwifruit, as well as the specific regulatory mechanism have not been reported. Based on the confirmation of the changes in histone acetylation levels of structural genes associated with ethylene synthesis, cell wall metabolism and starch degradation during the softening process of kiwifruit, this project will select the fruit softening-related HDAC, ERF, Dof and so on. Further screening of transcription factors such as ERF and Dof that interact with HDAC, and identifying the downstream genes of ERF and Dof, will be investigated. The effects of HADC on the transcriptional regulation of the target genes of ERF and Dof will be determined, and the mechanism of HDAC involved in the ripening and softening of kiwifruit will be clarified. The research results of the project will deepen the understanding of the epigenetic regulation mechanism of fruit ripening and softening, and provide novel ideas and theoretical basis for the development of quality control technology for harvested kiwifruit.

采后猕猴桃贮藏过程中易后熟软化，严重影响果实的商品价值。猕猴桃果实软化是一个复杂的过程，与乙烯、细胞壁代谢和淀粉降解等密切相关，受多个基因和蛋白调控。组蛋白去乙酰化酶（HDAC）在番茄、龙眼和香蕉等果实发育及后熟衰老调控方面发挥着重要作用，而HDAC介导的表观遗传调控是否参与猕猴桃果实后熟软化过程，其具体作用机制是什么，未见相关报道。本项目在明确猕猴桃果实软化中参与乙烯合成、细胞壁代谢和淀粉降解等过程的结构基因组蛋白乙酰化水平变化基础上，筛选与果实软化相关的HDAC以及ERF和Dof等转录因子；进一步筛选与HDAC互作的ERF和Dof等转录因子；确定ERF和Dof等转录因子的下游靶基因，分析HADC对其转录调控效应的影响，从而阐明HDAC在猕猴桃果实后熟软化中的作用机制。项目的研究结果将加深对果实成熟软化表观遗传调控机制的认识，为研发采后猕猴桃品质调控技术提供新思路和理论依据。

采后猕猴桃贮藏过程中，果实易发生成熟软化，严重影响品质。目前，采后猕猴桃的基础研究主要集中在营养功能品质、细胞壁代谢、基因鉴定和表达分析等方面，而猕猴桃成熟软化的分子调控机理并未深入展开，如组蛋白去乙酰化酶（HDAC）参与的表观遗传调控。项目以猕猴桃为试材，研究了不同处理在猕猴桃保鲜中的作用。通过ChIP-qPCR检测，发现AdACO1、AdACO4、AdXTH10、AdXTH12、Adβgal-1、Adβgal-2的启动子的组蛋白乙酰化水平，随着猕猴桃后熟而不断升高。筛选了采后猕猴桃差异表达的3个组蛋白去乙酰化酶基因、16个转录因子（8个NACs、5个ERFs和3个Dofs）。通过蛋白质-DNA互作技术，发现AdNAC5可以绑定AdACO4、AdXTH10、AdXTH12和Adβgal-1启动子，并激活其活性，从而参与调控猕猴桃果实的后熟软化。利用蛋白质-蛋白质互作和蛋白质-DNA互作技术，发现了AdHDA2可以减弱AdNAC5对AdACO4、AdXTH10、AdXTH12和Adβgal-1启动子的激活作用，从而影响下游靶基因的表达，进而参与果实后熟软化。项目从分子生物学角度，阐明了HDAC在猕猴桃采后软化过程中的调控机制，将为调控果实软化和延长贮藏期提供理论支撑。