禾谷镰孢菌BCAAs合成途径上关键基因的生物学功能研究

Branched-chain amino acids (BCAAs) biosynthetic pathway is uniquely present in bacteria, fungi and plants, but not mammals, which makes enzymes involved in this pathway attractive targets for novel and safe antimicrobials and herbicides development. Currently, several different herbicides that inhibit the first common enzyme in the pathway are wildly used in agriculture, some are effective against bacteria and yeast. In yeast, several BCAAs biosynthetic genes are essential for fungal development and virulence. To date, very little information is available about functional characterization of BCAAs biosynthetic genes in filamentous fungi except for Aspergillus fumigatus. Our previous studies on functional analysis of BCAAs biosynthetic genes in Fusarium graminearum (causal agent of wheat head blight) showed that deletion of FgILV5, FgILV2 or FgILV6 resulted in a significant decrease in aerial hyphae formation, conidiation and DON production. Deletion mutants also showed different pigment accumulation on PDA plates comparing to wild type strain. Besides the growth defects, deletion mutants also showed BCAAs auxotroph and can not grow on FGA medium containing no amino acids. Exogenous supplementation of different combinations of the three BCAAs can rescue the growth defects of the deletion mutants. Plant infection assays revealed that FgILV5, FgILV2 or FgILV6 deletion mutants also showed reduced virulence on flowering wheat heads. Taken together, our results indicate essential roles of BCAAs biosynthetic genes in fungal development, DON biosynthesis and pathogenicty in F. graminearum. Based on current research, this project will further identify essential genes involved in BCAAs biosynthetic pathway and analyze the effects of ILV gene deletion on various life activities in F. graminearum and elucidate the regulating mechanisms of ILV gene in fungal development, DON biosynthesis and pathogenicity. Results of this project will provide molecular targets for novel fungicides development and scientific basis for strategy of controlling wheat head blight and of reducing mycotoxin contamination.

由ILV基因编码的支链氨基酸（BCAAs）合成酶仅存在于细菌、真菌和植物中，哺乳动物体内不存在，因此，该类酶是新型安全药剂研发的潜在药靶。目前靶向该途径的除草剂在生产上广泛应用，某些还对细菌和酵母表现抑菌活性。酵母中该途径多个基因与生长、致病相关；丝状真菌中相关研究甚少，仅在烟曲霉中见过报道。课题组前期研究发现，禾谷镰孢菌FgILV5、FgILV2和FgILV6敲除突变体菌丝稀疏、菌落变黄、不产红色素、表现特定的BCAAs缺陷型、产孢和产毒量下降且致病力减弱，表明，该途径可能对禾谷镰孢菌生长发育、毒素合成和致病起重要作用。在此基础上，本项目将进一步鉴定禾谷镰孢菌BCAAs合成的关键基因，研究ILV缺失对禾谷镰孢菌各项生理代谢活动的影响，并阐明其在BCAAs合成、生长发育、毒素合成和致病过程中的作用机制。本研究将为新型安全药剂的研发提供分子靶标，为赤霉病的持续防控和毒素污染治理提供科学依据。

禾谷镰孢菌复合种是引起麦类赤霉病和镰刀菌毒素污染的重要致病真菌，由ILV基因编码的支链氨基酸（BCAAs）合成酶仅存在于细菌、真菌和植物中，哺乳动物体内不存在，因此，该类酶是新型安全药剂研发的潜在药靶。本项目对参与支链氨基酸合成的17个ILV基因进行了功能研究，利用靶向基因敲除和互补技术对获得了相关基因的缺失和互补突变体，重点分析了其中FgILV5、FgILV1、FgILV2、FgILV6、FgLEU2A/2B的生物学功能，并通过一系列的表型测定阐明其在病原菌生长发育和产毒致病过程中所起的作用。相关的研究成果为开发靶向于支链氨基酸合成途径的新型安全抑制剂提供分子靶标，为提出赤霉病防控策略、减少毒素污染提供科学依据。