土槿皮乙酸对植物病原真菌的抑菌作用靶标研究

Pseudolaric acid B (PAB) is a natural diterpenoic acid with unique chemical structure, low toxicity, and strong fungicidal activity, which has been considered an ideal fungicide lead compound. PAB showed strong inhibition against mangoanthracnose (Colletotrichum gloeosporioides) and other plant pathogenic fungi. Previous studies by cytology, physiology, biochemistry and gene transcription indicate that the mechanism of PAB against plant pathogenic fungi was complex. Unfortunately, the fungicidal target of PAB is still unknown. Active based protein profile (PAL-CC-ABPP) analysis using the tag-free clickable photoaffinity probe in combination with Click reaction has been confirmed an efficient and promising strategy for searching taget of small active molecules. In this study, C. gloeosporioides will be chosen as a tested fungus, and carry out the researches in the following: 1) to design, synthesize and screen PAB probe with tag-free photoaffinity; 2) to observe the sub-cellular location of binding protein of PAB in mycelia cell using active PAB probe and further isolate and identify specific binding proteins of PAB to obtain candidate target; 3) construct a revertant mutant with knockouting target protein gene, investigate the change in drug susceptibility of the mutants, and observe the subcellular location of target protein and its distribution. As a result, the target of PAB in plant pathogenic fungi will be ascertained. The results of this study will help us to discover new fungicidal targets, and provide a theoretical basis creating efficient and safe fungicides by utilizing the natural compound PAB.

土槿皮乙酸（pseudolaric acid B，PAB）结构独特、毒性低、活性强、抑菌谱广，是理想的杀菌先导化合物。前期在细胞学、生理生化和基因转录水平对其抑菌机制进行了初步研究，但靶标尚不清楚。利用光亲和标记的多功能探针结合Click反应开展基于活性的蛋白质分析是一项高效新颖的小分子靶标研究策略。本研究拟以芒果炭疽病菌（Colletotrichum gloeosporioides）为靶标生物，（1）设计、合成并筛选PAB多功能探针；（2）利用活性探针研究PAB结合蛋白在菌丝体细胞中的亚细胞定位，分离和鉴定PAB特异性结合蛋白，获得候选靶标；（3）构建候选靶蛋白基因敲除/回复突变体，测定突变体的药敏性变化，研究其在细胞中的定位及分布，确定其抑菌作用靶标。本研究结果将有助于发现新的杀菌靶标，阐明先导化合物PAB抑菌作用的分子机制，为创制以分子靶标为导向的系列新型杀菌剂提供理论依据。

本研究在前期研究的基础上，采用PAL-CC-ABPP策略和反向遗传学思路，本研究通过化学生物学、ABPP、基因组学和计算化学等方法技术明确了天然杀菌化合物土槿皮乙酸（PAB）在胶孢炭疽菌中具有多个杀菌靶标，结合前期研究结果，确定了微管蛋白是土槿皮乙酸的杀菌靶标之一，但PAB与现有的以微管蛋白为靶标的杀菌剂不同，结合于微管蛋白上一个新的活性位点。同时，本研究获得了吲哚2,3双加氧酶等候选杀菌靶标，这些目前尚未见杀菌活性相关报道，进一步研究有望发现更多全新的杀菌剂靶标，可为创制全新机制的杀菌剂提供理论基础。