山葡萄抗灰霉病关键基因发掘及抗病机理研究

European grapevines (Vitis vinifera) are the most widely planted grapes both for fresh consumption and wine production in the word. However, most grape cultivars of V. vinifera are highly susceptible to a fungal disease, grape grey mould, caused by Botrytis cinerea Pers.. Compared to European grapes, Chinese wild Vitis germplasm are resistant to many fungal pathogens, which make them valuable and promising resources for breeding programs of grey mould resistance breeding. Previously, our group found a Chinese wild Vitis, V. amurensis Rpur cv ‘Shuangyou’ was highly resistant to grey mould and we also have the F1 segregating population of this resistant genotype hybrided with V. vinifera cv ‘Red Globe’, which is highly susceptible to B.cinerea. Firstly, we want to use RNA-Seq technology to identify differentially expressed genes (DEGs) in both resistant grape (‘Shuangyou’) and susceptible grape (‘Red Globe’) challenged by B.cinerea in different time points. We will select DEGs that showing different expression patterns in these two genotypes and then narrow down to those DEGs with functional annotations related to disease resistance. In addition, we will also use resistant F1 population and susceptible F1 population to further testify the association of the grey mould resistance and our resistanct gene candidates. At last, we will use transgenic technology to verify the function of these resistance genes. And other methods, such as yeast two-hybrid system and chromatin immunoprecipitation assay, will be also applied to find other interacting genes. We believe that our identified grey mould resistant genes will facilitate studies on the molecular mechanisms of grey mould resistant and contribute to the grape breeding programs.

葡萄灰霉病是危害欧洲葡萄生产最大的真菌病害之一，并随着我国葡萄设施栽培的广泛应用而日趋严重。我国葡萄资源蕴藏着丰富的抗病基因，因而发掘山葡萄抗灰霉病基因具有重要的育种价值。项目组前期以高抗（几乎免疫的）灰霉病的山葡萄双优、高感灰霉病的欧洲葡萄红地球及其杂交群体为材料，对接种灰霉菌后的双优、红地球转录组测序，已筛选出大量在灰霉菌（Botryis cinerea）诱导下特异表达和增强表达的基因片段。在此基础上，本项目拟通过基因结构分析与功能预测，结合差异基因在接种灰霉菌后双优、红地球及其抗、感病杂种后代植株间的差异表达分析，发掘葡萄抗灰霉病关键基因；再利用同源克隆或cDNA末端快速扩增（RACE）技术克隆抗灰霉病关键基因全长，并通过遗传转化验证基因功能，进而通过酵母双杂交和染色质免疫共沉淀筛选其互作调控基因，明晰葡萄抗灰霉病调控路径，阐明抗灰霉病机理，为葡萄抗灰霉病育种提供理论依据。

葡萄灰霉病是危害葡萄产业最大的真菌病害之一，我国葡萄资源蕴藏着丰富的抗病基因，发掘山葡萄抗灰霉病基因具有重要的育种价值。本研究采用灰霉菌接种诱导下的转录组技术、葡萄抗灰霉病的组织细胞学观察及miRNAs转录组分析等揭示了葡萄抗病作用机制，挖掘出葡萄抗灰霉病VaERF11、VaERF99 和ERF20，抗灰霉病WRKY30、VqWRKY10和VqWRKY31，抗灰霉病芪合成酶VaSTS19等7个基因，感灰霉病VqJAZ4、WRKY52、VqSTS36等3个基因。构建了葡萄抗灰霉病ERF20、VaERF11和VaERF99，抗灰霉病WRKY30、VqWRKY10和VqWRKY31，抗灰霉病芪合成酶VaSTS19基因过量表达载体和CRISPR/Cas9 DNA编辑载体，感灰霉病VqJAZ4、WRKY52和VqSTS36过量表达载体转化葡萄及模式植物拟南芥，通过过表达植株、沉默植株及野生型植株抗病性表型分析，观察不同材料活性氧，死细胞积累和病斑直径，定量分析抗病相关基因的相对表达情况等明确抗病功能。为进一步研究葡萄抗灰霉病基因VaERF11的抗病机理和功能，构建灰霉菌诱导下的葡萄酵母文库，经过酵母双杂交mating筛选后，获得互作蛋白基因VaTLP，回复实验证明在酵母中VaERF11与VaTLP互作，将VaTLP过表达转化模式植物拟南芥，过表达植株及野生型植株抗病性表型分析证明了VaTLP抗病功能。结合项目实施，发表SCI论文22篇，获得国家授权发明专利3项，创制一批抗病葡萄新种质材料，为抗病优质葡萄品种选育奠定基础。培养毕业研究生6人，其中博士生4人；毕业博士研究生1人获得校优秀博士论文，在读研究生5人，研究生2人获得国家奖学金。