新三糖酯MTE拮抗采后柑桔青霉病（Penicillium italicum）的作用机制研究

Citrus blue and green mold caused by of Penicillium pathogenic fungi is one of the most common and serious citrus diseases during postharvest storage and transportation. The challenge of drug-resistance and public concern of pesticide residue requires an immediate development of antifungal agents with novel structure, novel mechanism of action, high efficiency, low toxicity and low residue. Historical records of fighting against pathogenic fungi suggest the need for effective development in novel antifungal agents with new targets and new mechanism of action to address the challenges of fungal drug-resistance. .In previous research, applicant has identified eight novel 6-O-β-L-mannopyranosyltrehalose esters, MTE-I~VIII, from an endophytic fungi, Pezicula sp. SC1337. Structures of these compounds are simple but unique, with carbon scaffolds completely different from existing antifungal drugs. In vitro and in vivo experiments showed that they had significant antagonistic activity against three main pathogenic fungi of citrus, citrus blue mold, citrus green mold, and citrus sour rot, caused by P. italicum, P. digitatum and Geotrichum citri-aurantii respectively. These compounds display similar or even improved antifungal activities to the existing pesticide antifungal agents. This project aims to unravel the underlying molecular mechanisms of action for these novel antifungal compounds, and to find the potential targets of MTE against P. itallicum by using micro technology, proteomics and molecular biology methods. Accomplishment of this project will clearly aid in the development of structure optimization of MTE and its analogues, and will help with the development of novel biological antifungal oligosaccharide ester agents against Penicillium.

意大利青霉（Penicillium itallicum，PI）是导致柑桔采后损失的最重要致病真菌之一，当前针对柑橘青霉病的防腐保鲜剂因耐药性、安全性、防腐效果等问题在使用逐渐受到限制，从生物源中寻找安全、高效、低毒和低残留的新型抑菌成分并解析其作用机理是柑桔采后保鲜领域的重要发展方向。前期研究中，项目申请者从落羽杉内生真菌代谢产物中发现了一类能高效抑制柑橘青绿霉病的新三糖酯（6-O-β-L-甘露糖基海藻糖酯，MTE)，它们骨架新，易降解。本项目拟采用植物化学、生物化学和分子生物学方法，主要从细胞壁和细胞膜代谢调控角度研究MTE抑制PI的机理。主要研究内容包括：MTE对PI抑菌效果和安全性评价；MTE抑制PI的形态学基础；抑制PI生长的调控网络解析；抑制PI细胞壁合成和细胞膜合成的作用机制。研究结果可为新型寡糖酯抗青霉药物研发和MTE后续结构优化提供理论依据，具有重要的科学意义和应用价值。

新三糖酯MTE虽结构简单，但骨架新、易降解，对柑橘青霉等的拮抗活性好，其相关的制备、生物活性和作用机制研究意义重大，因此在本项目的资助下，项目组开展了与MTE的来源、制备工艺、生物安全性、抗真菌效果和作用机制相关的研究。共获得了以下结果：1）初步阐明了MTE产生菌株Pezicula neosporulosa与裸子植物协同进化与生态适应性机制，发现该菌具有生产类型丰富的代谢产物的潜力；2）通过重离子束辐照诱变获得了多个突变株，并比较了它们的抗菌活性及代谢产物差异，且发现MTE产量高于野生型的突变株；3）通过优化发酵条件和放大制备，共获得18个MTE同系物，并阐明了它们的抗真菌构效关系；发现了13个地衣酸衍生物等其它类型化合物；4）评价了MTE的抗真菌活性及抗菌谱，证实MTE不仅抗菌活性好，且抗菌谱宽；5）评价了MTE对柑橘、葡萄、木瓜和香蕉等水果采后主要致病真菌的体外活性和水果防腐保鲜效果，证实MTE可以有效抑制各种水果致病真菌侵染和病情发展，具有明显的保鲜效果。6）发现MTE可以有效抑制肺癌A549、肝癌HepG2、乳腺癌MCF-7、宫颈癌HeLa等癌细胞的体外增殖，并可以显著抑制荷瘤小鼠体内的肿瘤生长，但不影响小鼠体重的正常增长；7）证实MTE在小鼠体内代谢快、口服最大耐受量大于5g/kg体重，是安全性极高天然产物；8）研究了MTE对柑橘青霉、柑橘白地霉和稻绿核病菌的抑菌效果和作用机制，发现MTE可以通过破坏柑橘青霉、柑橘白地霉的细胞膜、细胞壁，影响活性氧代谢水平，糖、脂质和氨基酸代谢等基础代谢，调控相关证据的毒力相关基因或蛋白，从而有效抑制真菌的生长，高效抑制柑橘采后保鲜过程中的真菌侵染和发病率。研究结果在一定程度上突破了MTE作为新型抗真菌剂产业化相关的生产成本、保鲜效果和生物安全等多个关键技术节点，初步阐明了MTE抗真菌的作用机制，具有明显的学术创新和应用价值。