中国野生毛葡萄特有芪合成酶基因抗白粉病机理研究

Grapevine ranked the top three of the important fruit trees in the world, largely due to its economic benefits from wines, table grapes, grape juice and raisin. Vitis vinifera are main cultivars in grapevine production which have high quality and various utilizations. Resveratrol in grapevine plays important role in human health. However, most of Vitis vinifera species are susceptible to Uncinula necator, and the fungicide cost of preventing powdery mildew was over 3 billion dollars every year in America. Therefore, the key to improve the resistance of these cultivars is to make use of grapevine germplasm resources with high resistance to the disease. Resveratrol, produced by stilbene synthase in grapevines, not only enhances resistance to plant pathogens but also plays important roles in human health, including antioxidant, anti-aging and anti-cancer. In our previous studies, the result of transcriptome sequencing showed that Chinese wild Vitis quinquangularis Danfeng-2 possesses stronger pathogen resistance, and higher accumulation and expression level of stilbene synthase genes, especially, six novel stilbene synthase genes were identified in Vitis quinquangularis. In the present study, based on the analysis of the sequences and conservative structures from the six genes, they will be transfered into the plants without these specific genes, including the model plant Arabidopsis thaliana and the susceptible European grapevines by Agrobacterium mediated transformation system. After inoculation with Uncinula necator, the interacting proteins with the six stilbene synthase genes will be screened by qPCR method, and co-immunoprecipitation combined with mass spectrometry, so as to analyze gene expressions and pathogen resistance. The regulation of the stilbene synthase genes by the transcription factors will be studied by screening the transcription factors interacting with the promoters of these stilbene synthase genes using the methods, yeast one-hybrid and EMSA. Therefore, the expression net of the stilbene synthase genes and activated resistant genes will be explored in transgenic European grapevines, so as to reveal the function and regulation model of stilbene synthase genes against powdery mildew, and provide useful scientific basis for breeding novel grapevine germplasm with pathogen resistance and high resveratrol content. Nine graduate students will participate in the studies, and five of them are doctoral students and four of them are master students. Moreover, 7-8 articles will be organized and published and 1-2 invention patents will be applied. Therefore, the obtained results will be useful for improving the resistance of cultivars and increasing resveratrol content based on Chinese wild grapevines.

葡萄是世界果树生产中栽培面积广、产量高的最重要果树之一。全球葡萄生产主要栽培是欧洲葡萄品种，这些品种具有酿酒、鲜食、制汁及制干等广泛的用途与创造经济效益高的特性，近年的研究指出葡萄及其制品中含有的白藜芦醇对人体的健康有益。但是，欧洲葡萄多数品种不抗葡萄白粉病，仅美国每年用于防治葡萄白粉病的杀菌剂费用超过了30亿美元。从根本解决欧洲葡萄品种不抗病是利用抗葡萄白粉菌的葡萄种质资源进行抗病育种。本项目就是利用抗白粉病且白藜芦醇含量高的中国野生葡萄为材料，前期通过转录组学测序分析发现中国野生毛葡萄丹凤-2不仅抗病强且芪合成酶基因累积与表达强，又比欧洲葡萄品种多拥有6条特有的芪合成酶基因。本项目拟通过对这些特有芪合成酶基因的序列及其保守结构分析，通过农杆菌介导的方法将这些新基因转入到无此类基因的模式植物拟南芥和不抗病的欧洲葡萄品种中，在白粉菌接种诱导下，利用实时荧光定量技术分析基因的时空表达特性;利用免疫共沉淀结合质谱测序技术筛选与芪合成酶基因互作蛋白，并进行基因表达与抗病功能研究；利用酵母单杂交和体外凝胶阻滞实验EMSA筛选与特有芪合成酶基因启动子互作的转录因子，研究这些转录因子基因对特有的芪合成酶基因的调控功能；通过转芪合成酶基基因欧洲葡萄植株功能研究，分析这些基因的表达及其激活的抗病基因的特性，揭示这些基因抗白粉病的调控机理与作用。在项目实施中拟培养研究生5-8人，其中博士研究生3-5人，硕士研究生2-3人；项目研究结果拟发表7-8篇论文，其中SCI论文4-5篇；拟申请发明专利1-2件。本项目对中国野生毛葡萄6条特有的芪合成酶基因功能的研究，评价这些抗病的育种资源，通过对欧洲葡萄品种杂交育种改良抗病性与增加果实中白藜芦醇的含量有直接利作用，研究结果对利用中国野生葡萄种质，提高欧洲葡萄品种抗病性有重要的应用价值和科学意义。

葡萄是世界上古老而经济价值高用途广的果树。欧洲葡萄品种以产量高、品质佳成为主栽品种，但易受病害突出特别是白粉病的危害，造成巨大经济损失。生产上主要采用化学农药防治病害，不仅污染果品与环境，还会使病菌产生耐药性与突变成新的生理小种加重危害生产。因此，利用抗病育种，是从根本上解决葡萄抗病性问题的有效措施与途径。中国是世界葡萄重要的起源地之一，拥有丰富的抗病种质资源。前期研究证明了中国野生葡萄携带的芪合成酶基因具有抗病作用，通过转录组测序白藜芦醇含量高抗白粉病的中国野生毛葡萄丹凤-2，发现丹凤-2中有6条特异芪合成酶转录本，本研究就是克隆获得这些新芪合成酶转录本序列，分析研究基因序列特征，证明其中5条是具有功能的特有芪合成酶基因，提交GenBank，登录号为：OL589477-OL589481；进一步，将新基因VqNSTS2、VqNSTS3、VqNSTS4和VqNSTS6分别遗传转化至欧洲葡萄“无核白”中，研究了这些新成员的表达特性与抗病性功能；发现了正向调控特有芪合成酶基因的5个转录因子基因VqERF062、VqWRKY33、VqAL4、VqGT33和VqGT3，尤其是正向调控特有芪合成酶基因的3个转录因子基因VqAL4、VqGT33和VqGT3，这是葡萄上首次发现调控芪合酶基因的转录因子及其调控机理；将这些特有芪合成酶基因遗传转化至模式植物拟南芥中，证明这些特有芪合成酶基因在接种白粉菌后主动聚集在白粉菌孢子吸器的颈部和周围，阻挡病原菌的入侵。在白粉菌诱导下，发现了糖基水解酶基因VqBGH40a可以水解云杉新苷生成白藜芦醇，发挥抗病性作用。研究了前期遗留的VqWRKY53、VqMYB154、VqERF112、VqERF114和VqERF072等的特殊调控机制。本研究共获得了转基因葡萄新种质47株；本项目在实施过程共计培养博士研究生4人，硕士研究生9人，其中毕业获得学位11人；项目研究结果目前已发表14篇论文，其中5篇SCI论文。本项目成果为葡萄抗病育种提供了重要的种质资源和参考依据，具有重要的科学意义与应用前景。