中国野生葡萄芪合成酶基因表达调控与功能研究

Resveratrol enriched in grape berries and its products, not only enhances the resistance to fungal pathogens but also plays important roles in human health, including anti-cancer, antioxidant, and reduction of cardiovascular disease. Stilbene synthase (STS) is a key enzyme in the biosynthesis of resveratrol. This project is based on our previous research, which has verified that the expression of stilbene synthase gene in the berry was significantly higher than other organs/tissues from most Chinese wild Vitis species. The main projects are carried out as following:.1) Transcriptome analysis of high expression of stilbene synthase genes and their transcription factors was carried out in the organs of Chinese wild grapevine induced by Uncinula necator. .2) Explore regulations and expressions of selecting high expression of stilbene synthase genes and their transcription factors from the organs. .3) Localizations both of organization and subcellular and functional analysis of these favorable genes in transgenic plants and protoplasts. .4) Dynamic observation of the synthesis, transport, accumulation and degradation of STS and their expression resveratrol under stress by laser scanning confocal microscope to better understand the function of stilbene synthase genes;.5) Agrobacterium-mediated transformation and over-expression of the interesting genes from Chinese wild grape species into Vitis vinifera through somatic embryogenesis system to generate the highly accumulation of resveratrol and novel disease-resistance grapevine germplasm..The research focuses on the regulatory mechanism of stilbene synthase genes in response to biotic and abiotic stress as well as their function to defense powdery mildew. Additonnally, this study also provides gene resource for disease-resistance breeding and a base for future scientific research.

葡萄芪合成酶基因的表达产物白藜芦醇不仅增强植物抗病性，而且存在于葡萄浆果及其制品中，对人体具有抗氧化、抗衰老和防癌的作用。本项目是在前期获得中国野生葡萄高表达的芪合成酶基因及其在成熟果实中表达高于其他器官组织的基础上，拟开展：1）白粉菌胁迫诱导高表达的芪合成酶基因及其转录因子表达，进行高表达芪合成酶基因葡萄组织的转录组测序研究；2）遴选获得高表达芪合成酶基因基因与转录因子基因，探究芪合成酶基因调控机理；3）对这些基因在葡萄器官组织和亚细胞水平定位与功能表达研究；4）进一步研究胁迫条件下芪合成酶及其产物白藜芦醇在合成、转运、积累、作用和降解的动态变化规律，阐明高表达的芪合成酶基因功能；5）利用转基因技术，向欧洲葡萄品种中转入所获基因，创造葡萄新种质。本研究将为弄清芪合成酶基因功能与作用机制，创造抗病优质葡萄新种质提供基因资源与科学依据。

葡萄是跨越南北半球的世界性重要的果树之一。目前生产上主栽的欧洲葡萄品种在酿酒和鲜食等方面拥有优质高效的优点，但突出缺点是抗病性差，常造成葡萄产量的巨大损失，降低品质，生产上主要使用农药减少病害损失。因此，利用抗病种质资源进行葡萄抗病育种是有效途径。然而，抗病性极强的圆叶葡萄（2n=40）与欧洲葡萄品种杂交亲和性极差，抗病性强的美洲葡萄拥有特殊的麝香味限制了其利用。因此，利用抗病性强且不携带麝香味的中国野生葡萄与欧洲葡萄品种杂交，进行抗病育种是改良葡萄品种抗病性的有效措施之一。本项目选择抗病性强且白藜芦醇含量高的中国野生葡萄华东葡萄白河35-1与毛葡萄的丹凤-2为材料，分离筛选出抗病相关的芪合酶基因，分析基因序列与功能，将这些基因转入欧洲葡萄品种中，创造芪合酶基因突变体，研究了在自然状态下芪合成酶存在于油体，在人工接种白粉菌胁迫下芪合成酶转运到液泡，在紫外光胁迫条件下芪合成酶转运到叶绿体的转运机制；研究了芪合酶基因表达与增强抗病功能作用；理清了芪合成酶及其产物与葡萄白粉菌的互作关系；揭示了芪合酶基因表达对增强抗病性的作用及机理，具体是VpSTS29的过量表达引起转基因株系中35个内源芪合成酶基因的协同表达，激活了水杨酸信号通路，引起MYB-WRKY转录因子以及抗病相关基因的高量表达，刺激侵染部位的芪类物质含量迅速增加，产生更多活性的芪类植保素如ε-葡萄素，激发抗病基因参与的共表达调控网络，从而提高葡萄植株的抗病性。本项目在实施过程共计培养博士研究生5人，硕士研究生3人，其中毕业获得学位7人；克隆获得的基因序列25条已登录GenBank；已发表9篇论文，其中6篇SCI论文。本项目研究获得的芪合成酶基因表达与增强葡萄的抗病性，表明中国野生葡萄是今后改良欧洲葡萄品种抗病性的重要基因资源与葡萄抗病育种的重要种质资源；利用中国野生葡萄种质资源进行抗病育种具有重要的应用价值和科学意义。