天然产物柠檬醛对稻瘟病菌细胞壁靶标蛋白的作用机制

Rice blast disease, caused by Magnaporthe grisea, is one of the most severe diseases of rice throughout the world and is recognized as one of the major pathological threats to China food supplies. Currently, the three "R" problems caused by the chemical control of this disease have aroused increasing concern. Efficient and safe prevention and control of rice blast has become an important research topic in plant protection experts. As a natural acyclic monoterpenes, citral is the major component of Litsea cubeba oil which was extracted from its fruits, present at levels. Citral (3,7-dimethyl-2,6-octadienal) is the name given to a natural mixture of two isomeric acyclic monoterpene aldehydes(geranial and neral). Our previous study results showed that citral has a strong inhibitory activity against M. grisea. Because of its particular aroma, substantial antibacterial, antifungal and insecticidal effects, low toxicity and low carcinogenecity, it has been widely used as food additive, fragrance material in cosmetics and agricultural crops insects and diseases. In recent years, several studies reported that citral was capable of preventing plant pathogen, such as Botrytis Cinerea and M. grisea, but the active components of citral and their pharmacological effects are still unclear. However, the effect mechanism of citral against M.grisea. In the present study, therefore, M. grisea is chosen to systemicly study the effect and mechanism of citral against the target protein of M.grisea cell wall using gene cloning and computer aided drug design techniques bansed on the theoretical basis of pesticide pharmacology and pharmaceutical chemistry. The objectives of this work are (i) to definitize the target of citral resistsing M.grisea, (ii) to built the structure-modeling of the interaction between target proteins and citral, and (iii) to study and reveal the effect mechanisms of mycelial morphology and cell ultrastructure under the different concentrations of citral. The aim of the present study is not only to develop a new plant-based fungicide of citral, but also to provide the important scientific basis and clue for the rational design of biological fungicides.

水稻稻瘟病是由水稻稻瘟病菌(Magnaporihe oryzae)引起的真菌病害，世界各国水稻产区均有发生，同时也是影响我国水稻生产安全的严重病害之一。当前该病害化学防治导致的三"R"问题已引起社会高度关注。高效、安全防控水稻稻瘟病已成为植保专家的重要研究课题。笔者前期研究发现柠檬醛对稻瘟病菌具有显著的抑菌活性，但其具体作用机制尚不明确。为此，本项目以水稻稻瘟病菌为研究对象，运用农药药理学和药物化学理论，采用基因克隆手段及计算机辅助药物设计技术，系统研究柠檬醛对稻瘟病菌细胞壁靶标蛋白的影响及机制，以探明柠檬醛对稻瘟病菌靶标蛋白的结合位点；建立靶标蛋白与柠檬醛相互作用的模型；揭示柠檬醛对稻瘟病菌菌丝形态和超微结构的变化，以及对细胞生理生化指标的影响规律。柠檬醛对稻瘟病菌新型靶标蛋白的研究发现，不仅有望开发出防治水稻稻瘟病的天然植物源农药，而且可为杀菌剂的生物合理设计提供重要的科学依据和线索。

水稻稻瘟病是由水稻稻瘟病菌(Magnaporihe oryzae)引起的真菌病害，试验表明，稻瘟病菌菌丝生长抑制率与柠檬醛浓度成正相关；稻瘟病菌孢子萌发率随着柠檬醛浓度的增大而减小，芽管长度亦是类似。其中柠檬醛对稻瘟病菌菌丝生长和孢子萌发的EC50分别为40.28和37.12 μg/mL。柠檬醛可明显提高稻瘟病菌细胞壁中几丁质酶的活性。采用酶活性法测定了柠檬醛对稻瘟病菌几丁质酶活性的作用。相同浓度的柠檬醛在不同时间段对稻瘟病菌几丁质酶基因表达均有上调作用，不同浓度的柠檬醛在一定时间内对稻瘟病菌几丁质酶基因表达均有上调作用。柠檬醛能引起稻瘟病菌细胞壁的消溶，导致细胞膜的损伤，进而引起其生理代谢紊乱，表明柠檬醛对稻瘟病菌的作用部位位于菌丝体的细胞壁上。从稻瘟病菌菌株克隆纯化了稻瘟病菌靶标蛋白，预测蛋白质的三级结构，对接结果表明活性中心形成一个大的疏水空腔，具有氢键、共扼作用。研究结果不仅有望开发出防治水稻稻瘟病的天然植物源农药，而且可为杀菌剂的生物合理设计提供重要的科学依据和线索。