产黄青霉菌抗稻曲病菌活性物质的鉴定及其作用机制研究

With the large-scale cultivation of hybrid rice, Ustilaginoidea virens has become one of the major hazards affecting rice yield and quality. But so far, active substances from microbial sources against Ustilaginoidea virens and their fungicidal mechanisms have not been reported. Our research group isolated a strain Penicillium chrysogenum KF-25, with high activity against Ustilaginoidea virens. Its active substance may be a small molecule anti-fungal peptide (AFP). AFP can inhibit spore germination and mycelium cell necrosis or apoptosis of the pathogen. This project intends to adopt technologies such as HPLC, MS, NMRI, for the separation and identification of AFP and to clarify its chemical composition and structure; to apply techniques such as proteomics, physiological observation and biochemical detection to study the differences of physiology and protein profile of pathogen after AFP processing; to consider about the functions and three-dimensional structure models of differentially expressed proteins, and the virtual interactions between their structure models and AFP, to screen AFP's targets involved in pathogen growth and pathogenicity; to use techniques of qPCR, GFP tag and X-ray diffraction to resolve the dynamics transcription of targeted genes, as well as cellular localization and crystal structure of targeted proteins. This study not only helps to reveal the action mechanism of AFP against Ustilaginoidea virens, but also provides target references for combating other fungal plant pathogens.

随着杂交稻的大面积种植，稻曲病已成为影响水稻产量和稻米品质的重大危害之一。但到目前为止，微生物来源的抗稻曲病活性物质及其杀菌机制尚无报道。本课题组分离到一株高效抗稻曲病菌的产黄青霉KF-25，其抑菌活性物质可能为一种小分子抗真菌肽(antifungal peptide, AFP)，可抑制病原菌孢子萌发和使菌丝体细胞坏死或凋亡。本课题拟采用HPLC、MS、NMRI技术对AFP进行分离鉴定，阐明其化学组成和结构；采用蛋白质组学和生理生化检测技术，研究AFP作用下稻曲病菌的生理差异和蛋白表达差异；结合病原菌差异蛋白的功能、结构及其与AFP的虚拟互作，筛选AFP靶向的与病原菌生长和致病相关靶标；运用qPCR、GFP标记、X衍射技术，解析若干靶标的基因转录动态、蛋白的细胞定位和晶体结构。本研究不仅有助于揭示AFP抗稻曲病的作用机制，亦为防治其它真菌性植物病害提供靶标层面的有力借鉴。

随着杂交稻的大面积种植，稻曲病已成为影响水稻产量和稻米品质的重大危害之一。但到目前为止，微生物来源的抗稻曲病活性物质尚无报道。本研究开展了稻曲病菌拮抗菌产黄青霉的基因组学和转录组学研究，旨在筛选潜在的抗菌活性物质，为水稻病害的生物安全防治提供理论依据。.产黄青霉(Penicillium chrysogenum)被用于生产青霉素和β-内酰胺类抗生素具有十分悠久的历史。在本实验室前期研究工作中，分离得到了一株产黄青霉菌株P. chrysogenum KF-25，该菌株对稻曲病菌(Ustilaginoidea virens)具有高效拮抗作用。本研究对稻曲病菌的拮抗菌产黄青霉KF-25进行了全基因组测序和分析及不同渗透压下的转录组测序和分析，KF-25是第一株完成全基因组测序的产黄青霉野生型菌株，其基因组大小为29.9Mb，共有9804个编码基因。KF-25的基因组中共有355个特异性基因，这些基因编码的蛋白除了一部分参与转运、代谢、信号传导等功能，大部分是功能未知的基因（232个，占65%）。在KF-25的基因组中预测到33个二级代谢通路，其中，KF25_5480和KF25_1814是KF-25的355个特有基因中显著上调且参与二级代谢通路功能的基因，这些基因可能与抗菌活性代谢产物的生物合成有关，为新的生物活性物质的发掘提供了参考。.在高渗透压条件下，产黄青霉中与二级代谢相关的转录调控因子等基因的转录水平上调，这可能是在高盐条件下，KF-25可以产生更多抗稻曲病菌生物活性物质的原因。转录组学分析发现了在高渗透压条件下转录差异较大且与二级代谢有关的基因FadA和LaeA，在后续的研究中，可以通过构建这些基因缺失的突变株来研究其对活性物质表达调控的差异。对产黄青霉高渗条件下转录组学的研究从转录水平揭示了高渗透压可以提高代谢产物产量的可能的机理，为抗稻曲病菌发酵培养基的优化提供了理论依据。