MAPK调控采后香蕉果实成熟的分子机理研究

The regulation of fruit ripening by transcription factors (TFs) is a focused issue currently. The researches that TFs regulate fruit ripening focus mainly on the transcriptional levels, but little information is available regarding the post-translational levels. Among various post-translational modifications, protein phosphorylation mediated by mitogen-activated protein kinase (MAPK) is one of the important mechanisms underlying the response of cell to various stimuli. Therefore, attention should be paid to the regulation of fruit ripening through the phosphorylation of ripening-related TFs by MAPK. Banana is one of the most popular tropical fruit with great important economical value in China. However, the fruit is very prone to ripen and senesce at ambient temperature, which leads to reduced shelf life and serious postharvest losses. In this project, on the basis of the isolation of ripening-related MAPK genes from banana fruit, the interactions between MAPK and ripening-related TFs, such as ERF, DREB and NAC, are examined. Moreover, the direct target genes of the interacted TFs are also screened and confirmed. Finally, the effect of MAPK phosporylating ripening-related TFs on the regulation of target genes is studied. The results of our project will provide some novel information on the regulation of ripening by post-translational modification of TFs, and expand our understanding of transcriptional regulatory network of ripening, as well as pay a theoretical basis for the improvement of postharvest technology in controlling fruit ripening.

转录因子调控果实成熟是当前的研究热点，目前研究主要集中在转录水平上，而在翻译后水平上调控果实成熟则未见报道。促分裂原活化蛋白激酶(MAPK)介导的蛋白质磷酸化修饰是调控细胞响应外源信号的重要机制之一，因此，MAPK介导转录因子的磷酸化修饰调控果实成熟及其机制值得关注。香蕉是我国主要热带水果，具有重要的经济价值；然而，香蕉果实在常温下容易成熟衰老，导致货架期短，采后损失严重。本项目以香蕉果实为材料，在获得成熟相关的MAPK的基础上，分析MAPK与ERF、DREB、NAC等重要成熟相关转录因子的互作，鉴定互作转录因子的下游靶基因，并进一步研究MAPK磷酸化互作转录因子对其调控下游靶基因的影响，以期阐明MAPK介导的成熟相关转录因子的磷酸化修饰在采后果实成熟中的作用及机制。研究结果可获得翻译后修饰调控果实成熟的新知识，拓展对果实成熟调控网络的认识，并为改进采后果实成熟控制技术奠定理论基础。

香蕉是我国主要热带水果，具有重要的经济价值。但香蕉果实在常温下容易成熟衰老，导致货架期短，采后损失严重。本项目采用蛋白质-蛋白质互作、染色体免疫共沉淀（ChIP）、凝胶阻滞迁移（EMSA）、双荧光素酶报告基因（DLR）等多种技术手段，深入研究了MAPK调控香蕉果实成熟的分子机制。研究结果可获得翻译后修饰调控果实成熟的新知识，拓展对果实成熟调控网络的认识，并为改进采后果实成熟控制技术奠定理论基础。本项目的主要研究结果如下：.1）从香蕉基因组数据库中分离获得23个MAPK基因，并分析这23个MAPK基因在三种不同成熟进程（包括自然成熟、乙烯催熟、1-MCP抑制成熟）中的表达。结果显示，这23个MAPK基因在果实成熟过程中差异表达，其中MaMPK3-2、MaMPK4、MaMPK6-1、MaMPK6-2、MaMPK6-3、MaMPK9-3、MaMPK9-4、MaMPK20-1、MaMPK20-2、MaMPK20-3和MaMPK20-4随着果实成熟进程呈明显上调的趋势。.2）酵母双杂交和双荧光分子互补（BiFC）实验表明，MaMPK9-3和MaMPK20-2可与转录因子MaDREB2相互作用，并且相互作用的部位在细胞核内。亚细胞定位显示MaMPK9-3和MaMPK20-2主要定位于细胞核和细胞质。.3）染色体免疫共沉淀测序法（ChIP-Seq）显示MaDREB2可以调控697个靶基因；应用MEME-ChIP分析发现MaDREB2的主要结合元件为(A/G)CC(G/C)AC，与DREB转录因子的特异结合元件DRE/CRT即(A/G)CCGAC相似；将MaDREB2的ChIP-Seq数据与香蕉果实成熟的RNA-Seq数据进行整合，发现在697个靶基因中，115个基因是下调，81个基因是上调，并且这些靶基因主要分成四大类：果实成熟相关基因、逆境反应/防御基因、转录因子、翻译后修饰相关基因（包括MAPK）等；MaDREB2可以结合自己启动子。.4）烟草瞬时表达发现，MaMPK9-3、MaMPK20-2分别与MaDREB2共转时，MaDREB2对其中一个靶基因MaADH1启动子的激活能力削弱，推测MaMPK9-3、MaMPK20-2可能促使MaDREB2的磷酸化修饰，而MaDREB2磷酸化后有可能被E3泛素化酶识别并通过泛素化途径降解，进而导致激活能力削弱。