中国野葡萄抗黑痘病分子机理的研究

Anthracnose, caused by the biotrophic fungus Elsinoe ampelina (de Bary) Shear, is one of the most devastating disease of grapevine (Vitis vinifera L.) prevalent in warm and humid regions of the world. European grapvine is one of the finest grapes grown in the world both for wine and table purposes, but the prevalence of anthracnose is a major risk to grpae production. As Chinese wild Vitis species are more anthracnose tolerant than European cultivars and the inheritance of the resistance to anthracnose to offspring is very strong, it turns out to be the important germplasm resources for developing resistant cultivars. Thus, cloning genes related to E. ampelina-resistance and studying the mechanisms of anthracnose resistance are of important breeding value. Based on our previous studies, Chinese wild V. quinquangularis clone 'Shang-24' which is highly resistant to E. ampelina, and V. vinifera cv 'Red Globe' which is susceptible to anthracnose, and the interspecific hybrid F1 generation, will be used as materials in this study. RNA-Seq, qRT-PCR, Affymetrix microarry data bioinformatic analysis and genetic transformation techniques, will be used to study the transcriptomics and molecular mechanism during Chinese wild Vitis species-Elsinoe ampelina interaction. We aim to isolate target genes related to anthracnose resistance, further identify the resistance relevant metabolic pathways.The full length cDNA sequence of new genes resistant to anthracnose will be isolated by rapid amplification of cDNA ends (RACE)or RT-PCR technique. The over-expression vecator and RNA interference (RNAi) vecator of new genes will be constructed and will be successfully transferred into Arabidopsis thaliana and Chinese wild V. quinquangularis Shang-24 via Agrobacterium tumefaciens mediated transformation method.Phynotype investigation and anthracnose disease resistance identification of different transgenic plants will carried out in field conditions.Information obtained from this study will enhance the understanding of the resistance mechanisms and resistant breeding in grapevine plants to E. ampelina infection.

葡萄黑痘病是危害欧洲葡萄重要的真菌病害之一。中国野葡萄不仅抗病，而且和欧洲葡萄杂交亲和性好，是葡萄抗病育种重要的种质材料。因而，克隆中国野葡萄抗病基因，进行抗病分子机理研究，具有重要的育种价值。本项目在前期研究基础上，以抗病中国野葡萄商-24、感病的红地球及其种间杂交F1代为研究对象，进行商-24与红地球在黑痘菌侵染后不同时期的转录组研究，筛选出黑痘菌诱导下特异表达和增强表达基因片段，明确抗病过程中参与的代谢途径；采用qRT-PCR进行商-24、红地球及其杂种后代抗病、感病植株在黑痘菌及信号分子处理下的抗病基因表达分析，并结合Affymetrix芯片数据表达分析，获得抗黑痘病新基因；构建抗黑痘病新基因过表达载体和RNAi干扰载体，转化商-24和拟南芥，通过过表达、RNAi干扰及未转化葡萄或拟南芥的抗病性表现及基因表达分析，研究基因功能，分析抗黑痘病分子机理，为葡萄抗病育种提供理论依据。

以抗葡萄黑痘病的毛葡萄商-24株系和感黑痘病的欧亚种葡萄红地球叶片为材料，对接种葡萄黑痘病菌不同时间时间叶片及清水处理叶片转录组测序，共获得差异表达基因3414个，来自抗病毛葡萄商-24株系的有2246个，来自感病葡萄红地球的有2013个，且上调表达基因较下调表达基因更多；对差异表达基因参与的代谢通路富集性分析表明，黑痘病菌在抗病和感病葡萄中诱导的差异表达基因和显著性富集的代谢通路大多相同，但是在抗病毛葡萄商-24株系中，抗性相关基因出现差异表达的时间和代谢通路发生富集的时间较感病葡萄红地球的更早，揭示抗病毛葡萄在响应黑痘菌胁迫中表现出一定的抗性特异性；获得了葡萄WRKY家族基因59个，生物胁迫处理有38个WRKY差异表达基因，干旱和盐胁迫处理下55个差异表达基因，表明了葡萄WRKY基因与葡萄非生物和生物胁迫及信号调控存在密切而又复杂的关系；共鉴定葡萄126个bHLH转录因子，在激素和不同胁迫处理下表达分析显示，葡萄bHLH转录因子在不同组织中的表达模式存在差异，多数bHLH基因参与对ABA和MeJA处理的响应，部分bHLH基因对高盐或者干旱胁迫处理有响应，少数bHLH基因参与对病原菌侵染的响应；从抗病毛葡萄商-24中克隆得到VqWRKY52基因及其启动子，在拟南芥中异源表达VqWRKY52基因，对T3代株系接菌处理发现，过量表达株系表现抗性。通过对死细胞与活性氧的染色发现，过量表达株系在接种后，活性氧积累与死细胞数量比野生型明显增多。VqWRKY52的过量表达增强了抗病性，并且推测VqWRKY52可能是在SA途径中起作用；克隆了葡萄VpCN、VpTNL1、VqLecRK 、VqBAK1、AP17、AP13、VqbZIP39、VqbZIP36及VvMBF1等9个基因cDNA全长序列，过量表达分析明晰了其抗病（逆）功能；邀请国内外专家7人次来校学术交流；参加国内外学术会议3次；发表SCI论文14篇；培养学术带头人1人；培养研究生8人，其中博士生5人；获授权国家发明专利3项，申请国家发明专利2项。