氟吗啉和丁吡吗啉胁迫下辣椒疫霉细胞壁形成相关基因的表达和调控

Flumorph and pyrimorph are original fungicides used to control oomycete pathogens by China. Since 2006, genes responsible for flumorph-resistance in P. capsici were investigated. Point mutations on cesA3 gene were detected to confer resistance in P. capsici. However, the resistance level of transformants was 30~50% recovery by function verification. Resistance genetic analysis revealed that flumorph-resistance in P. capsici was controlled by two dominant genes. It was supposed that there could be other ressitance mechanism except the point mutations on cesA3 gene. Meanwhile, studies on pyrimorph indicated that pyrimorph disrupted the cell wall, and pyrimorph-resistance mutants showed cross-resistance with flumorph. However, there was no mutation responsible for resistance on cesA3 gene in these mutants, suggesting that resistance mechanism to pyrimorph might be the same to the unknown resistance mechanism to flumorph. Based on these, zoospores (without cell wall) and mycelium (with cell wall) were chosen for studies on regulation and expression of genes involved in cell wall formation under the pressure of flumorph and pyrimorph by transcriptome and proteomics methods. This project aims to (i) Screen and identify the genes involved in the biosynthesis of cell wall; (ii) investigate resistance mechanism except cesA3 gene; (iii) identify the function of resistance gene in cell wall biosynthesis; (iv) study the interaction between fungicides and targets. The results would be helpful for understanding the biosynthesis of cell wall and the mode of action of pyrimorph and flumorph. These genes and proteins responsible for cell wall formation would be potential targets for new fungicides development.

氟吗啉和丁吡吗啉是我国自主创制的卵菌抑制剂。自2006年以来申请者研究了疫霉中氟吗啉抗性相关基因，在cesA3基因上发现与抗药性相关的点突变，转化验证表明其转化子只能恢复30-50%抗性；抗性遗传研究显示辣椒疫霉对氟吗啉的抗性是由不完全显性双基因控制，推测可能存在其它未知机制。同期研究表明，丁吡吗啉抑制细胞壁的形成，其抗性突变体对氟吗啉表现抗性，但在cesA3基因上并未发现与抗药性相关的突变，推测丁吡吗啉的抗性机制与氟吗啉未知的抗性机制一致。本课题拟采用转录组和蛋白组学的研究方法，筛选辣椒疫霉细胞壁形成相关的基因，进而构建药剂选择压下抗感菌株的基因转录和蛋白表达差异图谱，筛选和比较细胞壁形成过程中与氟吗啉、丁吡吗啉抗药性相关的候选基因和蛋白，并研究其与药剂的互作。该研究将对阐明供试药剂的作用靶标和抗药性机制具有重要意义；细胞壁形成相关蛋白和调控途径的研究结果可为新药剂靶标的开发提供参考。

辣椒疫霉是一种世界性分布的重要病原卵菌，可以侵染茄科、豆科和葫芦科等多种农作物。每年由辣椒疫霉引起的病害可造成严重的经济损失。氟吗啉和丁吡吗啉是我国自主创制的卵菌抑制剂。本研究采用蛋白组学方法，分析获得了辣椒疫霉细胞壁形成相关的关键基因PcCesA1，并采用异源表达方法，明确了其生物学功能。同时构建药剂选择压下辣椒疫霉抗感菌株的蛋白表达差异图谱，获得了与氟吗啉、丁吡吗啉选择压相关的候选蛋白基因，结合分子生物学和生物化学方法，初步明确了药剂作用机制和辣椒疫霉产生抗性的机制。采用酵母异源表达方法，首次成功地表达出有活性的辣椒疫霉纤维素合酶1（PcCesA1）蛋白，单个PcCesA1蛋白只能催化尿苷二磷酸D-葡萄糖（UDPG）生成纤维二糖。结合免疫共沉淀和辣椒疫霉菌丝膜蛋白催化活性研究结果，发现多个纤维素合酶蛋白相互作用在一起，共同参与纤维素生物合成。该研究为阐明新型药剂的作用靶标和抗性机制提供了新思路，为新药剂靶标的开发提供了参考。