油菜miR1885和miR6030调控菌核病抗性的分子机制研究

The Sclerotinia disease caused by Sclerotinia sclerotiorum is the major reason for reducing the production of Brassica napus. However, the mechanisms underlying B. napus against this pathogen are still poorly understood. Although it has been widely reported that miRNAs play an important role in the interaction between plant and pathogen, their roles in plant defense against S. sclerotiorum remain unknown. In our previous work, we identified two B. napus miRNAs, miR1885 and miR6030, which were downregulated after the infection of S. sclerotiorum. Interestingly, these two miRNAs both targeted resistance (R) genes, and their expression might be regulated by Calmodulin-binding transcription activator 3 (CAMTA3), which play a negative role in plant defense against S. sclerotiorum. In this study, we proposed to focus on these two miRNAs. Firstly, we will study the regulatory function and mechanisms of miRNAs and R genes in the resistance to S. sclerotiorum by transgenic plants. Secondly, ChIP and other technologies will be used to analyze the transcriptional regulation of miRNA genes by CAMTA3. Taken together, the final aim of our project is to reveal the function of CAMTA3-miRNA-R genes signaling pathway in the resistance to S. sclerotiorum. This study has important theoretical and practical significance in revealing the interaction mechanism between B.napus and S. sclerotiorum, as well as for carrying out resistance breeding of B. napus.

菌核病是我国油菜生产上危害最大的病害，然而目前油菜与核盘菌互作机制尚不明确。miRNA广泛参与植物与病原物互作，但其与菌核病抗性相关研究稀缺。我们前期发现油菜miR1885和miR6030响应核盘菌侵染，且下游靶标抗病（R）基因，上游可能受负调控菌核病抗性的转录因子CAMTA3（Calmodulin-binding transcription activator 3）调控。本研究拟围绕这两个miRNA，利用遗传学、分子生物学等手段，构建转基因油菜植株，明确miRNA及其下游R基因对菌核病抗性的调控功能和机制；同时利用ChIP等技术解析上游CAMTA3对miRNA基因的转录调控作用，最终揭示CAMTA3→miRNA→R基因这一途径在调控菌核病抗性中的作用。本研究对于揭示油菜与核盘菌互作机制以及开展油菜抗菌核病分子育种具有重要的理论和实践意义。

由植物病原真菌核盘菌（Sclerotinia sclerotiorum）引起的菌核病是我国油菜生产上危害最大的病害，然而目前油菜与核盘菌互作机制尚不明确。miRNA广泛参与植物与病原物互作，但其与菌核病抗性相关研究稀缺。本项目以重要作物油菜为植物对象，利用遗传学、分子生物学、生物化学、植物病理学等技术，围绕miR1885a、miR1885b和miR6030对核盘菌抗性的调控功能及分子机制开展了系统研究。首先采用转基因油菜植株材料，明确了miR1885a、miR1885b和miR6030均对核盘菌抗性起负调控作用，对应的靶标基因R基因BnRSS1正调控核盘菌抗性；其次，利用荧光素酶报告系统、凝胶阻滞等分析方法，结合CAMTA3转基因油菜植株中miRNA和R基因的表达结果，证明了CAMTA3能够直接结合MIR1885和MIR6030启动子区并负调控其转录；最后，利用CAMTA3和miRNA的转基因油菜植株材料，结合油菜植株中基因瞬时表达技术，分析了CAMTA3与miRNA及靶标R基因在调控菌核病抗性中的遗传关系，明确了CAMTA3→miRNA→R基因模块在调控菌核病抗性中的作用，揭示了CAMTA3通过直接结合MIR1885基因启动子区并负调控其转录，从而抑制miR1885a和miR1885b表达并促进下游靶标基因R基因BnRSS1的表达，进而激活下游防卫反应，最终正调控菌核病抗性。本项目研究结果促进了对miRNA、R基因和CAMTA3的抗病调控机制的认识，为阐明油菜与核盘菌互作机制提供了更多线索，为抗病品种分子育种提供了新资源和新策略。