葡萄响应赤霉素microRNA的鉴定及其参与果实发育生物学通路的识别

Gibberellin (GA) is a necessary plant hormone for the growth and development of grape berry, involving in regulation of various biological processes, and thus it has been utilized popularly in grape industry. However, the molecular machnism of GA manipulating fruit development is unclear. In recent years, it has been revealed that some microRNAs (miRNAs) can regulate plant development through involving in GA signal transduction and/or responding GA, which has not been studied and reported in grapes even other fruit crops. This project is anticipated to use miRNA as an entrance point to analyze the role of miRNAs responsive to GA during grape berry development, in which the libraries of small RNA and mRNA in GA-treated grape berry and control ones will be constructed, and the candidate miRNAs responsive GA and their target genes will be screened and tested by Solexa and Degradome sequencing, respectively. The temporal and spatial expression of the candidate miRNAs and their target genes will be analyzed by Northern Blotting, qRT-PCR and the array of identification of biological function, and their biological regulatory pathways will be identifid by GO and KEGG pathway analysis together with bioinformatics. Based on the analysis above, we can make an interpretation on the molecular mechanism of grape miRNA responsive to GA duing berry development. The accomplishment of this project can broaden the knowledge about the molecular mechanism of GA's regulating berry development, and provide important referential theory for further studies on the regulation of fruit development even in other fruit crops.

赤霉素（GA）是葡萄果实生长所必需的植物激素，能够调控葡萄果实发育的多种生理过程。因而，在生产中也被广泛应用。但是，目前赤霉素调控果实发育的分子机理并不清楚。近期研究发现一些miRNAs通过参与GA信号转导响应GA作用在植物生长发育中起着重要调控作用，而葡萄乃至果树方面尚无相关报道。本项目拟以miRNA为切入点，构建赤霉素处理与对照葡萄小RNA与mRNA文库，通过Solexa和降解组分析、克隆与Northern杂交验证、GO与KEGG途径识别、qRT-PCR表达分析、生物学功能验证等手段鉴定葡萄响应赤霉素miRNA及其靶基因，识别其参与果实发育生物学通路，分析其表达特征，验证响应赤霉素miRNA的生物学功能，阐释其调控果实发育的分子机制。本研究可从表观遗传学方面拓宽对赤霉素调控果实发育机制的认识，为有效利用基因进行葡萄果实发育调控提供科学依据，也为其它果树果实发育调控机制的研究提供参考。

赤霉素(GA)是葡萄果实生长所必需的植物激素，能够调控葡萄果实发育的多种生理过程。但是，目前GA调控葡萄果实发育的分子机理尚不甚清楚。本项目阐释了miRNAs响应GA信号调控葡萄果实生长发育的分子机制。通过Solexa测序总共鉴定了137个响应GA的miRNAs，其中58条miRNAs的表达被GA下调，另51条被上调，还有21条被抑制，另7条被诱导；运用PsTargetmRNA软件预测了响应GA的miRNAs的靶基因，并注释了其潜在的功能；GO与KEGG途径识别的结果显示，这些miRNAs可能参与了激素、糖及钙离子信号转导，DNA、组蛋白的甲基化与脱甲基化，脯胺酸、酚类、类固醇、萜类物质代谢，木质素、纤维素代谢，芳香物质、色素合成、碳水化合物、有机酸代谢等代谢途径，并可能调控果实细胞的增殖与分化及果实大小、种子、胚及果核的发育、木质部与韧皮部的形成、果实风味及色泽的形成等生物学过程；同时，利用qRT-PCR技术检测了28条miRNAs在葡萄浆果不同发育阶段应答GA的不同表达图谱，并重点分析了响应GA的miR058、miR061在葡萄果实不同组织或不同发育时期应答GA的模式； 通过RLM-RACE验证了一批响应赤霉素miRNAs的靶基因，并检测了其在葡萄果实发育过程中裂解产物的时空变化特性，初步认识响应GA的miRNAs在葡萄果实发育过程中调控作用；利用转基因技术验证了响应GA的miR156b及其靶基因的生物学功能，通过在番茄中过表达葡萄miR156b，发现其能够调控花青苷合成，过表达则可促进着色和成熟。本研究从miRNAose组学角度阐释了GA对葡萄果实不同生理过程的分子调控机制。本项目的完成可从表观遗传学方面拓宽对赤霉素调控果实发育机制的认识，为有效利用基因进行葡萄果实发育调控提供科学依据，也为其它果树果实发育调控机制的研究提供参考。