昆蟲致病真菌白僵菌聚酮合成基因pks14調控的代謝體分析

Metabolomics analysis of the entomopathogenic fungus Beauveria bassiana regulated by the polyketide synthase gene pks14

雅妮(Kullyanee Panyawicha)

指導教授 ： 楊玉良

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2020年)

https://doi.org/10.6342/NTU202100114

摘要

白僵菌 (Beauveria bassiana) 為一種蟲生病原真菌，目前已做為生物防治劑並用於控制蟲害。此白僵菌以穿透昆蟲宿主外表皮的方式感染宿主，並將其菌絲入侵到昆蟲的血淋巴中。白僵菌可生成許多與昆蟲致病力有關的小分子代謝物。前人研究發現pks14是白僵菌致病過程的關鍵因子之一，因pks14只在白僵菌感染昆蟲時表現，在體外培養的條件下則不表現。在本研究中，我們使用大蠟蛾 (Galleria mellonella) 為昆蟲宿主，探討PKS14在昆蟲致病及調控二次代謝物的功能。結果顯示，相較於野生型及pks14失活突變株，感染pks14過度表現株的幼蟲，在感染二至四天後的致死率較高。利用原子力顯微鏡觀察發現，野生型白僵菌的分生孢子表面具有棒狀束簇，但pks14失活突變及過度表現的菌株則呈現無定形的表面，不具棒狀結構特徵。此現象說明PKS14可能與細胞壁的形成有關。 此外，我們利用LC-MS/MS、分子網路，及影像質譜分析探討pks14失活突變及過度表現菌株在體外及體內模式的代謝物特徵，發現pks14過度表現株會生產較多的cyclodepsipeptides、磷脂質，及脂肪酸，此說明pks14基因表現可能會促進其致病因子的生合成。

關鍵字

白僵菌 (Beauveria bassiana) ； 聚酮合成酶 ； 昆蟲致病性

並列摘要

Entomopathogenic fungus Beauveria bassiana has been used as a biological control agent to manage pest insects. Through physical infected mechanism, B. bassiana infects the insect host by penetration through the external cuticle. The hypha penetrates the insect and invades the hemolymph of the insect. Likewise, B. bassiana produces numerous low molecular weight metabolites that play an important role in determining their virulence. The previous study demonstrated that pks14 only expresses in vivo (infected insect) rather than in vitro cultural condition, which is important for fungal pathogenesis. In this study, we used Galleria mellonella as a model to evaluate the functional role of PKS14 in the insect pathogenesis and the regulation of secondary metabolites. The result showed that the mortality of B. bassiana pks14 overexpression infected larva was higher than the wild type and the pks14 null mutant infected larva at two to four days after inoculation. Atomic force microscopy revealed that the conidia surfaces of wild type possessed the rodlet bundles or fascicles while pks14 null mutant and pks14 overexpression strains were lacking the rodlet and showing amorphous surface, implying that PKS14 might be involved in cell wall formation. Further, we revealed the metabolic profiles of pks14 gene overexpression strain and pks14 null mutant strain in vitro and in vivo using LC-MS/MS, molecular networking, and imaging mass spectrometry analysis. We found that B. bassiana pks14 overexpression strain produced more cyclodepsipeptides, phospholipids, and fatty acids than pks14 null mutant strain in vitro and in vivo, which demonstrated that the pks14 gene expression would enhance the biosynthesis of the fungal virulence factors.

並列關鍵字

Beauveria bassiana ； polyketide synthase ； insect pathogenesis

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