绿僵菌seipin蛋白调控脂滴生成和杀虫毒力的机理研究

Entomopathogenic fungi infect insects via direct penetration of the host cuticle, the accumulation and metabolism of lipid droplets (LDs) are important guarantees for turgor pressure building up in the appressorium. However, it is still unknown that whether the morphology and distribution of LDs affect the utilization of LDs and virulence in Metarhizium. Our previous studies found that deletion of seipin, an endoplasmic reticulum membrane protein that regulate the size of LDs, results in aberrant LD size but also the formation of nuclear LDs in M. robertsii. In this project, we will use seipin deletion and truncated mutants to investigate the domain that regulate the morphology and distribution of LDs, study how seipin affects the distribution of nuclear and cytoplasmic LDs by transmission electron microscopy and labeling of LD proteins, detect lipid content and LD degradation to evaluate the utilization of LDs in Metarhizium, perform bioassays and elucidate the mechanism by which seipin regulates lipid droplet and insecticidal virulence. Results from this project will offer theoretical support for improving the virulence of insecticidal fungi used in ecological control of agricultural pests.

杀虫真菌通过附着胞主动穿透昆虫体壁实现侵染，附着胞内膨压形成所依赖的高浓度甘油来源于脂滴的降解。杀虫真菌胞内脂滴积累与正常代谢是感染昆虫寄主的重要保障，然而脂滴的形态和分布是否影响绿僵菌对脂滴的利用，进而影响杀虫毒力仍然未知。申请人前期研究发现绿僵菌内质网膜蛋白seipin的功能缺失不仅导致脂滴大小不一，而且出现大量核内脂滴。本项目将构建seipin缺失和截短的突变体用于分析seipin蛋白不同区域对脂滴形态和分布的调控；通过透射电镜观察和标记不同的脂滴相关蛋白，分析seipin如何影响脂滴的核内外分布；检测seipin缺失菌株内脂类含量和脂滴降解水平，分析脂滴大小和分布是否影响绿僵菌对脂滴的利用；最后结合生物测定，阐释seipin蛋白如何调控脂滴生成进而影响绿僵菌的致病性。本项目的研究成果将为改造提高杀虫真菌的毒力以应用于农业害虫绿色防治提供理论支撑。

杀虫真菌通过附着胞主动穿透昆虫体壁实现侵染，脂滴的积累与代谢是附着胞内膨压形成的重要保障，但涉及的机理仍不十分清楚。在哺乳动物中，内质网膜蛋白seipin参与脂滴的生成，其功能缺陷导致先天性脂质营养不良症等。本项目研究发现绿僵菌的seipin蛋白调控脂滴大小、含量及其核内外分布，绿僵菌seipin的N端，loop环域、近膜C端与核内脂滴的出现有关。Seipin蛋白的长短型C端在酵母菌中出现分化，绿僵菌的seipin蛋白能行驶酵母中fld1和ldb16的功能，丝状真菌绿僵菌和曲霉的seipin蛋白功能保守。MrSeip缺失导致孢子脂滴、磷脂、甘油脂、总脂肪酸减少，但是胞内脂滴蛋白MPL1增加，且MPL1在核内外脂滴中都存在，但是其在脂滴表面的聚集并不均一。ΔMrSeip中自噬和膜运输等通路上调，脂肪酸代谢和过氧化物酶体等通路下调。MrSeip蛋白缺失导致的脂滴、脂代谢和自噬等通路的异常进一步造成了ΔMrSeip的毒力下降。本研究不仅可以加深了对丝状真菌的脂滴生成以及杀虫真菌致病机制的认知，也为人类seipin和脂滴相关疾病的研究提供理论参考。