Fan-miR73靶向作用ABI5转录因子调控草莓果实成熟的分子机制

ABA and its signal transduction are very important in regulating non-climacteric fruit ripening. MiRNAs were recently found to regulate fruit ripening by targeting components in ABA signaling pathway, however, transcription factors involved in the downstream of ABA signaling and feed-back regulatory mechanism have not been reported. This project which is based on the previous newly identified Fan-miR73 and its degradation effect on ABI5 mRNA will investigate the transcriptional levels of Fan-miR73 and ABI5 during wild strawberry (Fragaria vesca) fruit ripening, and it will also study the influences of over-expression or knock-out of Fan-miR73 or ABI5 on fruit ripening. We will explore the feed-back effect of ABI5 on Fan-miR73 by using methods of EMSA, yeast one-hybrid and ChIP-PCR. By taking advantages of ChIP-Seq and transcriptome technologies, we will further seek the downstream regulating genes of ABI5 transcription factor. The result may not only provide technical support for developing efficient preservation approaches for postharvest non-climacteric fruits, it will also illustrate the mechanism that how ABA signaling transduction regulates non-climacteric fruit ripening.

ABA及其信号传导调控非跃变型果实成熟是一个重要科学问题，近年来发现miRNA通过与ABA信号通路上元件互补调控参与果实成熟，ABA信号下游转录因子及其反馈调控模式的研究未见报道。本项目在前期新发现Fan-miR73及靶向作用ABI5转录因子的工作基础上，以二倍体草莓为研究对象，探究Fan-miR73和ABI5在草莓果实成熟期间的表达模式，明确Fan-miR73和ABI5过表达或敲除对果实成熟的影响。采用EMSA、酵母单杂和ChIP-PCR等分子生物学手段，解析ABI5转录因子对Fan-miR73的反馈调控机制。利用ChIP-Seq和转录组学技术，挖掘ABI5转录因子下游调控基因。从而揭示Fan-miR73靶向作用ABI5转录因子调控草莓果实成熟中的分子调控机制。研究结果不仅为开发草莓等非跃变型果实采后保鲜方法提供技术支撑，也为揭示ABA信号传导调控非跃变型果实成熟机制提供理论依据。

果实成熟衰老生物学机制是重要的科学问题。ABA信号如何响应采后非生物胁迫并调控果实营养与品质有待进一步研究。本课题以草莓ABA信号转导途径关键靶点FaABI5及其调控因子Fan_miR73为研究对象，开展了ABA信号转导关键靶点鉴定、FaABI5 过表达与下调对草莓果实成熟的影响、ABI5 转录因子下游调控基因研究等研究内容。研究发现非生物胁迫不仅调控ABA生物合成，同时对ABA信号转导产生影响，改变果实对ABA敏感性。利用转录组学删选到ABA信号转导途径中受非生物胁迫调控的65个潜在靶点，包括12个PYL家族基因，21个PP2C家族基因，13个SnAPK家族基因，6个SnRK2家族基因，10个ABF/AREB家族基因，以及3个未注释的novel genes。进一步研究确定了ABI5为其中的关键靶点。采用WGCNA分析鉴定得到FaABI5下游靶标基因FaFLS1，经酵母单杂及双荧光素酶实验明确了FaABI5对FaFLS1的转录激活作用。此外，研究还通过小RNA组学在草莓果实中鉴定了47个保守microRNA，其属于25类microRNA家族，以及28个新microRNA，并通过降解组测序鉴定了上述microRNA的降解靶标。其中Fan_miR73靶向降解FaABI5，在FaABI5响应ABA信号及UV-B、盐胁迫等非生物胁迫中作为关键因子发挥重要作用。通过以上研究系统性构建了“Fan-miR73_FaABI5-FaFLS1”的信号调控模块，揭示了草莓果实中“Abiotic stress→Fan-miR73→FaABI5→FaFLS1→Flavonoids”的信号传导模式。研究成果完善了草莓果实成熟衰老调控网络，为草莓等浆果果实的精准采后保鲜技术研发提供重要的理论依据。本课题研究成果在New Phytologist、Foood Chemistry、Journal of Agricultural and Food Chemistry、等国内外高水平刊物发表论文17篇，授权国家发明专利2件，培养博士研究生5名，硕士研究生1名。