小麦赤霉病菌CYP51C基因影响DON毒素合成的作用机制研究

The recent expansion of publically available fungal genome sequence data has revealed many ascomycete fungi carry more than one copy of the sterol 14α- demethylase gene (CYP51) encoding the target for azole fungicides. In several Fusarium species, including pathogens of both humans and plants, three paralogous CYP51 genes (FgCYP51A, FgCYP51B, FgCYP51C) have been identified. FgCYP51C is unique to the genus Fusarium. Fusarium graminearum causes Fusarium Head blight (FHB), contaminates wheat grains with DON mycotoxin, and usually controled by azole fungicides. The previous study has demonstrated that FgCYP51C, a Fusarium-specific CYP51 gene, no longer functions as a sterol 14α-demethylase, but rather is specifically required for full virulence on host wheat ears. This is the first example of functional diversification of fungal CYP51 gene. Currently, the exactly functions of FgCYP51C gene and the rationale for their conservation within the Fusaria, is unknown. In this study, combining DON metabolic profile by HPLC/GC-MS and quantitative real-time PCR to reveal the DON pathoway changes induced by CYP51C gene deletion. To determine the protein interacted with FgCYP51C, split-ubiquitin based membrane yeast two-hybrid system will be performed. This study finally explains the mechanism of Fusaria-specific CYP51C impact on DON mycotoxin biosynthesis in Fusarium graminearum. The data will lays the foundation of reduce DON content in wheat grains, as well as provides the theory basis for the evolution of CYP51 and P450 superfamily.

甾醇14α脱甲基酶（CYP51）是甾醇生物合成中的关键酶，也是唑类杀菌剂的靶标。大部分真菌中含有一个或两个CYP51基因（CYP51A和CYP51B），而镰刀菌属真菌中还含有一个特异性CYP51C基因。禾谷镰刀菌引起小麦赤霉病，并产生DON真菌毒素影响小麦品质，田间主要应用唑类药剂进行防治。申请者前期研究表明，禾谷镰刀菌CYP51C基因可间接影响甾醇生物合成，并在小麦上的致病力及DON毒素合成中发挥重要作用，研究首次报道了真菌中CYP51基因的功能分化。本研究拟在前期工作基础上，进一步探究CYP51C基因是否直接影响DON毒素的合成，并通过分离鉴定CYP51C的互作蛋白，分析其参与的代谢合成途径，进而明确CYP51C影响DON毒素合成的作用机制。预期研究结果为小麦赤霉病的防治以及DON真菌毒素的防控奠定理论基础，为丰富真菌中CYP51基因功能多样性以及P450家族基因进化提供理论依据。

细胞色素P450-14α脱甲基酶（CYP51）是真菌细胞膜上甾醇生物合成中的关键酶，也是唑类杀菌剂的靶标。大部分真菌中含有一个或两个CYP51基因（CYP51A和CYP51B），而镰刀菌属真菌中还含有一个特异性CYP51C基因。前期研究结果表明，CYP51C与禾谷镰刀菌对小麦麦穗的致病性相关，且可能是通过影响DON毒素的合成进而影响致病性。为探究禾谷镰刀菌CYP51C影响DON毒素合成的作用机制，本项目通过研究CYP51C基因敲除转化子和野生型菌株PH-1的产生DON毒素的能力，结果表明，CYP51C基因敲除转化子的单位菌丝量产生DON毒素的含量显著下降，明确了CYP51C基因敲除转化子对小麦麦穗的致病性降低主要是由于其产生的DON毒素减少。对DON毒素合成途径中TRI4，TRI5和TRI15基因的表达量研究，虽然TRI5基因表达量增加，但是由于DON毒素合成量降低，因此推测DON合成途径中下游基因的负调控导致DON毒素合成量降低。通过结合转录组学和代谢组学研究，进而探寻CYP51C基因影响DON毒素产生的作用机制，转录组和代谢组学研究结果都表明，基因敲除转化子的嘌呤代谢途径、氨基糖和核苷酸糖代谢途径、生物素代谢途径、核黄素代谢通路、精氨酸和脯氨酸代谢，果糖和甘露糖代谢途径都具有显著的影响。研究结果表明，CYP51C可能通过影响其他生物合成的途径，从而间接影响到DON毒素的合成。该研究结果也为小麦赤霉病的防治以及DON真菌毒素的控制提供依据和思路。