组蛋白乙酰化修饰在采后香蕉果实成熟衰老中的作用及调控机制

Banana, as a climacteric fruit, is very susceptible to postharvest losses due to rapid softening. Thus, the ripening mechanism of banana fruit and its regulation is merited to study and is very important for extending postharvest life of banana fruit. Post-translational modification of histones, including acetylation, play a key role in modulating gene activity. Histone acetylation is modulated through the action of histone acetyltransferase (HATs) and histone deacetylase (HDACs). It has been reported that HDACs plays more important role in the regulation of histone acetylation and is involved in plant development and stress response by regulating expression of ethylene signaling related ERF or interacting with EIN3/EILs protein, however, little attention has been paid to the role of HDACs in fruit ripening, and its relationship with ethylene synthesis, perception and signaling. .In the present project, based on our preliminary results that HDACs might be involved in ripening in banana fruit, we further unravel the role and mechanism of HDACs in regulating fruit ripening, including determining the expression patterns of HDACs in relation to fruit ripening, examining the up-stream genes regulated HDACs and down-stream genes regulated by HDACs, identifying interaction proteins of HDACs, testing the histone acetylase levels of their target genes, moreover, the role and the mechanism of HDACs in fruit ripening were confirmed by over-expressing HDACs in tomato. The results obtained in the project will not only explore the new genes regulating fruit ripening, but also give a novel insight to improve our understanding about the regulation network of fruit ripening.

香蕉果实采后极易成熟、软化并腐烂，探讨果实采后成熟衰老及调控机制对于延长果实货架期及供应期具有重要意义。组蛋白去乙酰化酶（HDACs）是参与组蛋白乙酰化修饰的主要酶类之一，HDACs基因通过调控乙烯信号相关ERF基因表达或与EIN3/EIL蛋白互作，在植物根系发育及抗逆性等过程中发挥重要作用。然而，有关组蛋白乙酰化修饰在果实成熟衰老中的作用及调控机制，目前尚未见报道。.本项目中，我们以主要参与组蛋白乙酰化修饰的HDACs为切入点，在初步明确HDACs基因家族成员参与香蕉果实成熟的前提下，通过筛选、鉴定调控HDACs的上游基因，HDACs调控的下游靶基因及互作蛋白，检测靶基因的乙酰化修饰水平，并且通过在番茄中超表达与香蕉果实成熟相关的HDACs基因，进一步探讨组蛋白乙酰化修饰在采后果实成熟衰老过程中的作用及其作用机制。研究结果可以发掘调控果实成熟的新基因，加深对果实成熟调控网络的认识。

香蕉是呼吸跃变型果实，果实成熟启动后迅速软化，给贮运保鲜带来很大困难。本课题通过凝胶组织迁移实验（EMSA）、蛋白互作技术和染色质免疫共沉淀（ChIP）等多种实验手段，深入研究了组蛋白乙酰化参与调控香蕉果实成熟机制。研究结果拓宽了果实成熟的转录调控网络，尤其是在组蛋白去乙酰化修饰是否以及如何调控果实成熟机制方面加深了人们对果实成熟及其调控的认识。课题获得的主要研究结果如下：.1.从香蕉基因组数据库及NCBI的香蕉基因序列中鉴别出17个MaHDACs基因，其中，MaHDA1-12编码的氨基酸序列包含一个HDAC保守结构域，属于RPD3/HDA1亚家族；MaHDT1-3编码的氨基酸序列在N端都有一个MEFWG保守域, 属于HD2亚家族；而MaSRT1/2编码的氨基酸序列都有一个SIR2保守域，属于SIR2亚家族。研究结果显示，17个MaHDACs在香蕉果实后熟进程中呈现出不同的表达模式，其中， MaHDA6的表达在三个不同处理的香蕉果实中均表现出明显的上调趋势。 .2. MaACO1和MaEXP2/7/8的表达及其组蛋白乙酰化水平与果实成熟密切相关。qRT-PCR分析显示，MaACO1和MaEXP2/7/8基因的表达在香蕉后熟进程中均呈现不同程度的上升趋势，并且成熟的香蕉果实的MaACO1和MaEXP2/7/8基因染色质的组蛋白H3和H4的乙酰化水平高于未成熟的香蕉。.3.分析了MaHDA1参与调控香蕉果实成熟的机制。以MaERF11作为诱饵，分离到与其互作的蛋白MaHDA1，进一步研究表明，MaHDA1具有HDAC保守域和组蛋白去乙酰化酶活性，并且可以增强MaERF11对下游靶基因MaACO1和MaEXP2/7/8的抑制作用。说明MaERF11与MaHDA1可以形成转录抑制复合体，共同调控后熟相关基因MaACO1和MaEXP2/7/8的表达，从而参与调控香蕉果实的后熟衰老过程。 .4.探讨了MaHDA6在乙烯信号转导和果实成熟中的调控机制。研究表明，MaHDA6定位于细胞核内，具有组蛋白去乙酰化酶活性，可以结合MaERF11/15的启动子，并且成熟香蕉果实的MaERF11/15基因染色质上组蛋白乙酰化的水平低于未成熟的香蕉，说明，MaHDA6可能通过去乙酰化作用来调控下游基因MaERF11/15的表达，从而参与调控乙烯信号转导途径和香蕉果实的后熟衰老。