衔接蛋白复合体调控禾谷镰刀菌囊泡转运和毒素代谢机制的研究

The adaptor protein complexes, as a class of highly conserved eukaryotic protein, play an important role as a bridge between the membrane cargoes and coat proteins during vesicle formation, and they are involved in the regulation of cargo protein sorting and intracellular signal transduction, in addition to the important role they play in endocytosis and secretion. Fusarium graminearum, one of the most devastating plant pathogens, causes severe disease of cereal crops world-wide, yield losses and produces large amount of toxins which threaten food safety. Previous studies have shown that endocytosis and secretion mediated by Rab family (small GTPases) are essential for growth and development, DON metabolite, as well as pathogenicity in F. graminearum. AP2 complexes regulate growth and pathogenesis, and there is a high coefficient of correlation between some subunits of the AP complexes, RAB1 and TRI6 in F. graminearum which suggests that the functions of AP complexes and RAB1 are highly correlated. Based on these results, we proposed to elucidate the functional mechanism of the adaptor complexes involved in regulating pathogenicity and DON synthesis and secretion in F. graminearum. This project intends to study the biological functions of the adaptor complexes and their expression patterns during pathogenesis of F. graminearum, and explore the molecular mechanisms of the regulation of secretion of DON mycotoxin mediated by the adaptor complex and Rab1 through cell biology, biochemistry and genetics technology. The results from this project will advance our current knowledge on the mechanism of pathogenesis of F. graminearum, expand our understanding of the importance of vesicle trafficking in F. graminearum pathogenesis and lay a theoretical foundation for control of wheat scab caused by F. graminearum.

衔接蛋白（AP）复合体是一类高度保守的真核生物蛋白，在网格蛋白和受体配体复合物之间起着桥梁角色，参与调控货物蛋白分拣和胞内信号传导，对胞吞和分泌起重要作用。禾谷镰刀菌是麦类作物赤霉病的重要病原菌，引起产量损失并产生大量毒素引起食品安全问题。我们已有研究表明Rab小GTP酶介导的胞吞和分泌对该菌的生长发育、DON毒素代谢及致病性至关重要，AP-2复合体调控该菌生长和致病性，并与Rab1及毒素合成基因Tri6等共表达，说明他们可能存在极高的功能相关性。为此，我们拟以AP复合体为切入点，通过细胞生物学、生化和遗传学方法深入研究其在禾谷镰刀菌的致病和毒素代谢过程中的作用，阐明AP复合体与Rab1蛋白的关系，及其在囊泡转运和毒素合成与分泌中的作用机制。研究结果将进一步明确囊泡转运、毒素代谢的调控机制及其在禾谷镰刀菌致病机制中的作用，为防治小麦赤霉病提供理论依据。

囊泡转运蛋白分别通过胞吞和分泌调控禾谷镰刀菌的生长发育、毒素产生和致病性。衔接蛋白复合物（Aadaptor Protein, AP）是囊泡运输系统中极为关键环节，在胞内货物分拣过程中发挥重要的调控作用，不同的衔接蛋白也决定货物蛋白的分选途径进而调节不同的生长发育过程。本项目前期发现禾谷镰刀菌中AP2复合体调控该菌生长和致病性，生物信息预测FgAP1α与小GTP蛋白FgRab1及毒素合成基因FgTRI6等共表达，推测他们可能存在功能相关性。在此基础上本项目研究发现，禾谷镰刀菌中共有三种AP蛋白复合物，分别为FgAP1，FgAP2和FgAP3。三种AP蛋白复合物基因在禾谷镰刀菌不同生长发育和侵染小麦不同时段均有表达，特别是FgAP1和FgAP2部分亚基基因上调表达；FgAP1定位于早期内涵体和反式高尔基体，通过影响v-SNARE蛋白FgSnc1的分泌及延缓胞吞调控禾谷镰刀菌生长发育、DON产生及致病性；FgAP2定位于新生菌丝亚尖端颈环及隔膜处，通过影响磷脂翻转酶FgDnfA和FgDnfB极性定位，影响真菌中膜流动性以调控生长发育和致病性；FgAP3复合物主要定位于内涵体与高尔基体，调控禾谷镰刀菌分生孢子的产生，但不参与胞吞过程。FgAP1复合体和FgRab1之间没有直接互作，FgRab1对DON毒素的合成没有显著影响，但它们可能通过不同的途径分别影响分泌蛋白的分泌。在本项目资助下我们还研究了禾谷镰刀菌翻转酶家族蛋白和高尔基体钙镁离子转运蛋白的生物学功能，发现它们在禾谷镰刀菌的生长发育、致病性及DON毒素的合成中起重要作用。. 因此本项目较好地完成了预定目标，较为全面地解析了禾谷镰刀菌衔接蛋白AP复合体、翻转酶家族蛋白和高尔基体钙镁离子调控禾谷镰刀菌生长发育毒素产生和侵染致病的分子机制，为后期开发靶向防治赤霉病和DON毒素危害的新药剂提供了潜在的靶标和理论支撑。

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