ABA介导的miRNA及其靶基因调控草莓成熟的分子机理研究

The regulatory role of ABA on non-climacteric fruit ripening is an important scientific issue. In recent years, it is found that the miRNA can regulate the expression of target genes at the post-transcriptional level, which may provide innovative ideas to the study of ABA’s regulatory mechanism on non-climacteric fruit ripening. On the basis of our achievements, strawberry fruit, a typical non-climacteric fruit is selected as the subject of this research. With the application of genomic technologies (transcriptome, miRNA sequencing and degradome sequencing) and other techniques, this project aims to select the key miRNAs that specifically regulated by ABA, screen the target genes, analyze their biological functions, and explore the bidirectional interactions between ABA and the key miRNA. Meanwhile, it is supposed to construct key miRNA over-expressed vector and target gene transient expression and RNAi silenced genetic vectors, in order to further clarify the regulation of strawberry ripening through the expression of key miRNA and target genes in ABA signaling pathways, thus revealing the molecular mechanism of ABA-mediated miRNA and its target genes in the regulation of strawberry ripening. The results of this study can be conducive to reveal the molecular mechanism in ABA’s regulation of strawberry ripening, providing reference for further miRNA functional studies on other non-climacteric fruit. This research not only provides a theoretical basis for revealing non-climacteric fruit ripening mechanism, but also offers technical support for the development of effective preservation methods in post-harvest non-climacteric fruit.

ABA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可在转录后水平调节靶基因表达，这为研究ABA调控非跃变型果实成熟机制创新了思路。本项目以非跃变型果实模式材料草莓为代表，在已有成果基础上，利用转录组学、miRNA测序和降解组测序等技术手段，挖掘受ABA特异性调控的关键miRNA，筛选鉴定靶基因，分析其生物学功能，明确ABA与关键miRNA双向互作调控模式；构建关键miRNA过表达和靶基因瞬态表达及RNAi沉默遗传载体，明确ABA信号路径中关键miRNA和靶基因的表达操控对草莓成熟的调控，从而揭示ABA介导的miRNA及其靶基因在调控草莓成熟中的分子机制。研究结果不仅有利于揭示ABA在草莓果实成熟中的分子调控机制，而且对其他非跃变型果实开展miRNA功能研究也具有借鉴作用。这不仅为揭示非跃变型果实成熟机制提供理论依据，也为开发有效的非跃变型果实采后保鲜方法提供技术支撑。

果实的成熟机制是个重要的科学问题，ABA调控非跃变型果实成熟的机制尤其是信号下游和转录后调控机制还有待深入开展。本课题以草莓成熟过程中ABA 信号调控的miRNA 及其靶基因为核心内容，开展了ABA 调控草莓成熟过程中对Fan-miR73表达水平的影响、草莓成熟模型中调控ABA 信号传导的关键Fan-miR73的挖掘与鉴定；ABA 信号路径中Fan-miR73和转录因子ABI5的表达模式、ABA 信号路径中Fan-miR73和转录因子ABI5表达操控对草莓成熟过程影响等研究内容。转录组学分析发现ABA处理或者NDGA处理能够使4164个基因的表达水平产生显著差异。通过对转录组学差异表达基因的代谢通路富集分析发现，ABA下调了细胞分裂素受体基因AHK2/3/4和下游重要基因CYCD3的表达，ABA抑制了生长素信号基因IAA27和SAUR基因的表达，这表明ABA在草莓果实成熟过程中对其他激素的信号通路起到了调节作用。ABA对乙烯合成基因ACO3和关键信号元件CTR1表达产生影响，暗示乙烯在草莓果实成熟过程中可能发挥了某些作用。转录因子与差异表达基因共表达分析发现，WRKY和HSF家族转录因子是ABA诱导的基因高表达的重要调控因子，而SPL18和KNOX家族转录因子在ABA抑制基因表达起到了调节作用。研究发现了草莓果实成熟过程中47个保守miRNA和28个新miRNA，其中20个保守miRNA和6个新miRNA受到ABA差异调控，降解组分析发现Fa-novel6能够靶向HERCULES1基因。发现了Fan-miR73能够靶向ABA信号下游转录因子ABI5，外源ABA处理可以显著下调Fan-miR73的表达，从而促进ABI5基因的表达。UV-B辐照和盐胁迫均能下调Fan-miR73的表达，而上调ABI5的表达水平。Fan-miR73和ABI5表达水平之间高度的负相关性暗示了Fan-miR73可能在草莓果实成熟和非生物胁迫过程中起到了对ABA信号下游转录因子ABI5表达水平的调节作用。这些研究结果有利于了解ABA 在草莓等非跃变型果实成熟中的作用机理，也为开发有效的非跃变型果实采后保鲜技术提供理论支撑。本课题发表论文18篇，授权国家发明专利2件；培养博士生3名，硕士研究生3名。