苹果转录因子MdWRKY33与MdVQ相互作用抗炭疽叶枯病的分子机理研究

WRKY transcription factors as positive or negative regulators in host-pathogen interaction can regulate plant disease resistance genes through different signaling pathways, and make plant disease resistance broad-spectrum and lasted. In our preliminary study, the expression patterns of apple WRKY transcription factor gene family were examined in response to the treatment with Colletotrichum spp. As a resutl, MdWRKY33 was chosen for further investigation due to its high expression level. Furthermore, it was found that MdWRKY33 interacts with an apple protein MdVQ. Besides, the transgenic apple plantlets of MdWRKY33-GFP have been obtained. In this project, we plan to characterize the function of MdWRKY33 in disease resistance using the transgenic apple plantlets and functional complementation experiments in Arabidopsis wrky33 mutant. In addition, ChIP-Seq, ChIP-PCR and EMSA will be performed to get its target genes and verify the binding of MdWRKY33 to the promoter regions of target genes. Subsequently, it will be examined if MdVQ influences the binding activity of MdWRKY33. The expected results will contributable to get further insight into the disease resistance signal networks mediated by MdWRKYs. Simultaneously, we will obtain some genetically improved apple germplasm with high resistance to Colletotrichum spp..

WRKY转录因子作为寄主-病原物互作中一个重要的调控子，通过不同信号途径正调控或者负调控植物抗病相关基因表达，使植物的抗病能力广谱持久。项目组对炭疽叶枯病菌诱导条件下苹果WRKY家族基因的表达进行了分析，分离了1个病菌诱导强烈表达的转录因子MdWRKY33，且获得了过表达MdWRKY33-GFP转基因苹果植株，酵母双杂交试验表明，MdWRKY33与MdVQ能够相互作用；在此基础上，拟通过对苹果转基因植株进行抗病性鉴定以及拟南芥突变体功能互补试验，确定MdWRKY33的抗病功能，通过ChIP-Seq、ChIP-PCR和EMSA技术证明MdWRKY33对靶基因启动子的结合及MdVQ调控MdWRKY33结合活性。通过本项目的实施，不仅能初步阐明MdWRKY33的抗病功能和作用机制，获得抗苹果炭疽叶枯病的苹果新种质，同时对将来进一步构建苹果中WRKY转录因子参与的抗病信号网络奠定坚实的基础。

WRKY转录因子作为寄主-病原物互作中一个重要的调控子，通过不同信号途径正调控或者负调控植物抗病相关基因表达，使植物的抗病能力广谱持久。但是关于苹果WRKY转录因子和VQ转录调控子在苹果抗病过程中的相关机制还不甚了解。在本研究中，我们对苹果中MdWRKY和MdVQ家族基因进行了全基因组鉴定和分析。利用酵母双杂交和双分子荧光互补技术证明了MdWRKY33与MdVQ之间的相互作用。克隆了WRKY转录因子家族中I组成员MdWRKY33，并通过转化拟南芥wrky33突变体进行了功能互补，通过人工接种Botrytis cinerea对MdWRKY33的抗病功能进行了鉴定。将MdWRKY33在苹果愈伤组织中过量表达，通过对转基因材料进行表达分析，筛选到2个MdWRKY33的候选靶基因。克隆了MdWRKY33候选靶基因MdJAZ9及MdABI5的启动子，利用软件对启动子上可能的结合位点进行了分析，并利用ChIP-qPCR技术对MdWRKY33与启动子的结合进行了验证。通过上述研究，我们系统地对MdWRKY33转录因子的功能及作用机制进行了鉴定，初步阐明了MdWRKY33抗病功能和作用机制，对进一步构建苹果中WRKY转录因子和其靶基因参与的抗病信号网络奠定坚实的基础。目前，已发表SCI论文2篇（已标注），一级学报论文2篇（已标注），培养研究生1名。