稻瘟病菌过氧化物酶体前体产生关键基因MoPEX16的功能及调控机理研究

Magnaporthe oryzae causes rice blast, the most devastating disease on rice world widely. During the infection of the rice blast fungus, lipids in its conidia are rapidly degraded to provide material and energy for the invasion of the fungus. Peroxisomes are the main organelles involved in lipid metabolism, and play vital roles in fungal pathogenicity. However, the mechanism of peroxisome biogenesis, especially of the occurrence of pre-peroxisome, is still obscure. PEX16 is a key factor regulating the presence of pre-peroxisomes in mammals and plants, but interestingly, is absent in most of the yeast species. Filamentous fungi contain the PEX16 orthologues, however, the characterization of PEX16 in filamentous fungi was quite limited so far, and its relationship with fungal pathogenicity was undetermined. We have previously generated the MoPEX16 deleted mutants by using gene knockout procedures. In the present proposal, we are going to perform the phenotypic analysis of the mutants, as well yeast complementation, protein localization and gene expression, to find out: 1. Whether Pex16 distributed to peroxisomes or ER; 2. The expression and roles of PEX16 in peroxisomal membrane protein import and metabolisms; 3. The roles of the gene in fungal developments.and infection of the fungus; 4. The regulation networks of the gene. The findings in these investigations will benefit the researches on peroxisomal biogenesis, fungal pathogenesis and rice blast control, and may also provide targets for disease control and fungicide screening.

稻瘟病菌是水稻上重要的致病菌，侵染时孢子内脂肪迅速降解，为侵入提供物质和能量。过氧化物酶体是脂肪代谢的主要细胞器，对侵染至关重要，但目前其发生机制尤其是前体过氧化物酶体的产生还不清楚。动植物细胞中PEX16是调控过氧化物酶体前体产生的关键因子，有趣的是该基因在多数酵母中不存在。丝状真菌含有PEX16，但功能不明确，而植物病原真菌中尚无报道。通过基因敲除我们获得了稻瘟病菌PEX16突变体并进行了初步的表型分析。本项目拟在上述基础上，明确PEX16：1.是否定位于过氧化物酶体或内质网；2. 在过氧化物酶体形成、代谢过程中的表达动态及作用；3.在病菌生长发育和致病过程中的作用；4.蛋白互作与调控网络。从而明确PEX16在过氧化物酶体形成过程中的作用及机制，揭示其在病菌生长发育和致病过程中的生物学功能。本研究对病原真菌致病机理与过氧化物酶体产生机制的研究具有重要意义，为病害防治提供可能的药物靶点。

我们克隆并分析了稻瘟病菌过氧化物酶体前体形成相关基因 MoPEX16，通过启动子与 GFP 融合表达发现该基因在稻瘟病菌菌丝、孢子、芽管及附着胞中均表达，以孢子为最高。通过同源置换获得了这两个基因的突变体，表型分析表明MoPEX16 在过氧化物酶体生物合成及真菌致病过程中起着重要作用。发现过氧化物酶体膜蛋白 PEX16（MGG_09011.7）与 YlPEX16最为相似，氨基酸相似度为 29.4%。MGG_09011.7 开放阅读框 ORF全长 1320bp，该基因包含有 1 个内含子和 2 个外显子，编码一个由439 个氨基酸组成的多肽。MGPEX16 是含有过氧化物酶体定位信号 1 和定位信号 2 蛋白定位所必须的，同时参与了 mPTS定位信号的转运。MoPEX16 基因敲除突变体脂肪酸利用率降低、活性氧（ROS）耐受性下降且细胞壁的完整性收到影响。此外，Δmopex16 突变体分生孢子产量下降、附着胞形成时间推迟且侵染寄主植物的能力下降，这些缺陷最终导致突变体致病能力丧失。这些数据表明过氧化物酶体前提形成相关蛋白Mopex16 在真菌过氧化物酶体生物合成、生长发育及致病过程中起着重要作用。