蓝光对Penicillium expansum的生长发育及棒曲霉素合成的调控机理研究

Penicillium expansum, an important plant fungal pathogen, can infect many fruits, including apple, pear and grape, and cause huge economic losses. Besids, this pathogen is a main producer of the mycotoxin patulin, which is a toxic secondary metabolite of fungal species and is harmful to consumers’ health. Blue light plays important roles in vital activities of fungal cell, and involves in many processes such as growth and development, and secondary metabolism. While the regulation mechanisms of blue light on patulin production have not been documented. In this study, we are using P. expansum to deeply and systemically study the molecular regulation mechanisms of blue light on fungal growth and secondary metabolism. The aims of the study are: 1) to clear the effects of blue light on growth phenotype, hyphal micromorphology and cell structure of P. expansum. 2) to clear the effects of blue light on patulin production and the expression of related genes, and verify the interaction of blue light receptors with the factors involved in patulin biosynthesis. 3) to identify the main factors that participate in patulin production and are regulated by blue light. 4) to illustrate the regulation network of blue light on P. expansum growth and patulin biosynthesis on the level of cell biology, proteomics, transcriptomics and molecular genetics.

Penicillium expansum是引起苹果、梨、桃、葡萄等大宗水果采后青霉病的主要病原物，不仅造成巨大的经济损失，而且会在果实体内产生棒曲霉素，对消费者的安全造成严重威胁。蓝光是细胞生命活动的重要环境参数，参与病原真菌生长发育、次生代谢等诸多过程，然而关于蓝光对棒曲霉素生物合成的调控机制还未见报道。本研究以P. expansum为对象，研究蓝光与P. expansum生长发育和棒曲霉素积累的关系，观察蓝光处理后P. expansum生长表型及菌体形态和微观结构的变化，揭示蓝光对棒曲霉素积累及相关基因表达的影响，验证蓝光受体与棒曲霉素合成相关因子的互作关系，发现新的受蓝光调控参与棒曲霉素合成的关键因子，并揭示它们与毒素合成的关系，从而在细胞生物学、蛋白质组学、转录组学和分子遗传学水平解析P. expansum的生长发育及棒曲霉素合成的蓝光调控机理。

为了解析光照对P. expansum生长发育及棒曲霉素积累等方面的作用,本研究通过基因敲除等遗传学方法对P. expansum蓝光受体基因PeLreA和PeLreB的功能进行研究。结果表明：.1. P. expansum蓝光受体蛋白PeLreA和PeLreB各功能结构域与其它真菌蓝光受体同源性较高。亚细胞定位结果显示，PeLreA和PeLreB主要定位在细胞核中，PeLreB少部分定位在细胞质中。.2. 通过同源重组和农杆菌转化法对P. expansum的PeLreA和PeLreB进行敲除，获得具有潮霉素抗性的敲除转化子，经DNA水平和RNA水平的双重鉴定，得到3株PeLreA基因缺失突变株和3株PeLreB基因缺失突变株。.3.蓝光处理显著抑制了野生型和突变株ΔPeLreA、ΔPeLreB的菌落直径、产孢量、孢子萌发率、芽管长度、生物量和棒曲霉素产量，降低了孢子致病力。与野生型相比，突变株ΔPeLreA和ΔPeLreB在蓝光处理下产孢量显著降低，棒曲霉素合成受到抑制，孢子致病性显著降低，生物量、孢子萌发率和芽管长度也受到显著抑制。.4．PeLaeA基因在蓝光、白光和黑暗处理条件下，在ΔPeLreB中的表达量都较野生型菌株中上调，而在ΔPeLreA中的表达量都较野生型菌株中下调。在蓝光条件下，PePatL、PePatG和PePatN基因的表达量在野生型中较ΔPeLreA和ΔPeLreB高，而PePatE基因的表达量在野生型中最低，PePatK基因在ΔPeLreA中大量表达，在ΔPeLreB中几乎没有表达。.5．转录组分析结果显示，ΔPeLreB和ΔPeLreA差异表达基因主要为琥珀酸脱氢酶、丝氨酸/苏氨酸蛋白激酶、木酮糖激酶以及丙酮酸脱氢酶激酶2/3/4。在蓝光条件下，与野生型相比，ΔPeLreA中下调的基因主要为2-脱氢-3-脱氧-D-葡萄糖酸5-脱氢酶、组蛋白-赖氨酸N-甲基转移酶、C6转录因子和光敏素。ΔPeLreB中下调的基因主要为琥珀酸脱氢酶、丝氨酸/苏氨酸蛋白激酶、2-脱氢-3-脱氧-D-葡萄糖酸5-脱氢酶和组蛋白-赖氨酸N-甲基转移酶。.综上所述，PeLreA和PeLreB对P. expansum的菌落直径、产孢量、孢子萌发率、芽管长度、生物量和棒曲霉素产量具有直接的影响，糖代谢、氨基酸代谢和脂质代谢在其中发挥了重要作用。