小分子诱导海洋植物内生真菌次级代谢的化学多样性及其调控机理研究

During the long period of co-evolution, a closely mutualistic relationship has formed between the endophytic fungi and its host plant. Plant hormones are small signal molecules produced in plant with extremely low concentrations, which regulate plant growth, development, and adaptations. However, effects of plant hormones on the growth and development of endophytic fungi, especially regulation of secondary metabolism are very rare. During early exploration, the marine alga endophytic Aspergillus wentii EN-48 and the mangrove plant derived Aspergillus nidulans MA-143 could produce diverse secondary metabolites after epigenetic modifications. Based on previous studies, this project aims to use small-molecule elicitation strategies during fermentation, which include plant hormones and fungal signaling molecules. By activation of potential silent metabolic pathways in endophytic fungi, the chemical diversity could be increased with their secondary metabolism potentials untapped. Isolate and identify new natural products; according to chemical analysis and bioactivity tests, and explore their physiological functions, including plant disease, insect, and stress resistances. For these new potential norditerpenoids, over-express the zinc finger activator to get the mutants with high yields. By further screening the target genes and ligand compounds it binds, the regulatory mechanism would be revealed. Trying to analyze the effects of small-molecule elicitation on multi-omics, speculate the signal transduction pathway, and preliminarily discuss the interaction between the marine plant endophytic fungi and its host.

植物内生真菌在与宿主长期的协同进化过程中形成了密切的互利共生关系。植物激素是植物自身合成的微量小分子信号物质，可调控植物的生长发育和适应性。而植物激素对内生真菌的生长发育，尤其是次级代谢的调控研究较少。前期探索发现，海藻内生真菌温特曲霉EN-48和红树林来源构巢曲霉MA-143经表观遗传修饰后可产生多样化的次级代谢产物。本项目拟在已有基础上，通过植物激素、真菌信号物质等小分子诱导发酵策略，激活潜在的沉默代谢途径，深入挖掘其次级代谢潜力，增加内生真菌代谢产物的化学多样性。分离鉴定新天然产物，结合化学分析和生物活性测试，探讨其对植物抗病虫、抗逆性等生理功能。对已获得的潜力化合物四降二萜，高表达其合成基因簇中的激活子锌指蛋白以获得高产菌株；筛选该锌指蛋白识别的靶基因和配体化合物，进而揭示其调控机理。探索结合多组学比较分析，推测小分子诱导的信号传导途径，初步探讨海洋植物内生真菌与宿主的互作关系。

在长期的协同进化过程中，内生真菌与植物宿主形成了密切的互利共生关系。植物激素是植物自身合成的微量小分子化学信号，可以调控植物的生长发育和环境适应性。而植物激素对内生真菌生长发育的影响，尤其是次级代谢的调控研究较少。.本项目在前期研究基础上，通过植物激素、真菌信号等小分子诱导发酵策略，激活曲霉中潜在的沉默代谢途径，深入挖掘其次级代谢潜力，增加了内生真菌代谢产物的化学多样性。从曲霉中分离鉴定新天然产物10个，结合化学分析和生物活性测试，探讨了其对植物抗病虫、抗逆性等生理功能。对降二萜类潜力化合物，筛选获得了高产菌株和诱导发酵优化条件。对曲霉中锌指类调控蛋白结构功能和进化分析，初步探讨了其小分子诱导调控机理。对次级代谢主要调控蛋白家族与小分子配体的协同进化过程进行了结构功能和分子进化分析，探讨了其扮演的调控功能和重要的进化角色。对次级代谢生物合成关键酶（卤化酶和DAase酶）的结构功能和分子进化分析，发现这些同功酶的独立进化历史和多样化的来源途径。.通过比较转录组和代谢组差异分析发现，植物激素脱落酸也可以调控植物内生真菌构巢曲霉生长和代谢。在100nM脱落酸诱导条件下，发现有上调基因3148个和下调基因3160个。这些差异表达基因主要包括：RNA聚合酶及基础转录因子，核糖体合成、蛋白质合成、蛋白酶体及泛素介导的蛋白降解，核酸代谢及三羧酸循环，细胞周期及次级代谢产物的生物合成等。真菌毒素等代谢产物的合成也随着脱落酸的诱导调控而发生变化，这些毒素有不同抗虫或抗菌活性，可能有利于宿主植物的环境适应性。本研究首次揭示了脱落酸调控植物内生真菌的转录差异，这表明脱落酸可能在植物和微生物互作过程中扮演着重要的信号分子角色，进一步探讨了海洋植物内生真菌与宿主的互作关系。