禾谷镰刀菌醛脱氢酶(ALDH)基因敲除突变体的转录组分析

Transcriptome analysis of aldehyde dehydrogenase (ALDH)gene knockout mutant in Fusarium graminearum

【目的】对野生型PH-1菌株和醛脱氢酶(ALDH)基因FGSG_04194敲除突变体(ΔFg04194)进行转录组测序,分析禾谷镰刀菌ALDH调控菌丝生长和脱氧雪腐镰刀菌烯醇(DON)生成情况,为深入探究禾谷镰刀菌ALDH的生物学功能、DON生成机制及致病机理提供了理论依据。【方法】接种禾谷镰刀菌野生型PH-1、ΔFg04194突变体和基因回补突变体(ΔFg04194-C)菌株至CM培养基和小麦基质中,分析FGSG_04194基因对菌丝生长和DON生成的影响。利用高通量转录组测序技术对野生型PH-1菌株和ΔFg04194突变菌株在CM培养基中培养72 h的菌丝进行转录组测序,对筛选出的差异表达基因(DEGs)进行GO功能注释和KEGG信号通路富集分析,利用实时荧光定量PCR对转录组测序结果进行验证。【结果】ΔFg04194突变体的菌落直径和生成的DON含量较野生型PH-1和ΔFg04194-C突变体菌株显著减少(P<0.05,下同)。野生型PH-1和ΔFg04194突变体转录组原始数据经过滤后获得34.9 Gb Clean data,共鉴定出329个DEGs,其中263个基因表达显著上调,66个基因表达显著下调。GO功能注释结果显示,DEGs显著富集在质膜成分、碳水化合物代谢和物质转运过程等条目上。KEGG信号通路富集分析显示,DEGs显著富集在碳水化合物代谢通路、氨基酸代谢通路、脂质代谢、能量代谢、生物降解代谢通路、细胞生长和死亡相关途径、信号转导途径等。将FGSG_04194基因调控下有代表性的DEGs分成五大类:几丁质合成相关的基因、跨膜转运蛋白相关的基因、转录因子相关基因、脂质代谢相关基因和能量代谢相关的基因。ΔFg04194突变体的ATP含量显著高于野生型PH-1和ΔFg04194-C突变体。运用实时荧光定量PCR检测7个DEGs的表达情况,其结果与转录组测序结果基本一致。【结论】FGSG_04194影响禾谷镰刀菌的菌丝生长和DON生成,并在ATP生成中起负调控作用,其机制可能与调控转录因子的表达、调控脂类代谢、氨基酸代谢和能量代谢等通路有关。

【Objective】The purpose of the study was to perform transcriptome sequencing of wild-type PH-1 strain and aldehyde dehydrogenase(ALDH)gene FGSG_04194 knockout mutant(ΔFg04194),to analyze the regulation of mycelial growth and deoxynivalenol(DON)production by ALDH in Fusarium graminearum,and to provide a theoretical basis for in-depth study of the biological function of ALDH,DON production mechanism and pathogenesis of F.graminearum.【Method】The wild-type PH-1,ΔFg04194 mutant and gene complementation mutant(ΔFg04194-C)strains of F.graminearum were inoculated into CM medium and wheat grains,respectively.The impact of the FGSG_04194 gene on mycelial growth and DON production was analyzed.By employing high-throughput transcriptome sequencing technology,mycelium from wild-type PH-1 andΔFg04194 mutant strains cultured in CM medium for 72 h were conducted to transcriptome sequencing.The screened differentially expressed genes(DEGs)were analyzed through GO functional annotation and KEGG signaling pathway analysis,and the transcriptome sequencing results were confirmed using real-time fluorescence quantitative PCR.【Result】In comparison to the wild-type PH-1 andΔFg04194-C mutant strains,theΔFg04194 mutant exhibited significantly reduced colony diameter and generated DON content(P<0.05,the same below).The raw data of the wild-type PH-1 andΔFg04194 mutant transcriptome were filtered,resulting in 34.9 Gb of Clean data.A total of 329 DEGs were identified,with 263 genes showing significant up-regulation and 66 genes displaying significant down-regulation in expression.The GO functional annotation results indicated a significant enrichment of DEGs in categories of plasma membrane components,carbohydrate metabolism and substance transport processes.KEGG metabolic pathway analysis demonstrated a significant enrichment of DEGs in carbohydrate metabolism pathway,amino acid metabolism pathway,lipid metabolism pathway,energy metabolism pathway,biodegradation metabolism pathway,pathways associated with cell growth and death and signaling pathways.Representative and significant DEGs regulated by FGSG_04194 gene were classified into five main groups:genes associated with chitin synthesis,genes associated with transmembrane transporter proteins,genes associated with transcription factors,genes associated with lipid metabolism,and genes associated with energy metabolism.ATP content of theΔFg04194 mutant was significantly higher than that of the wild-type PH-1 andΔFg04194-C mutants.Expression levels of the 7 DEGs were analyzed using real-time fluorescence quantitative PCR,and the results were generally consistent with the transcriptome sequencing results.【Conclusion】The FGSG_04194 influences both the mycelial growth and DON production in F.graminearum,and plays a negative regulatory role in ATP production.Its mechanism may be associated with the regulation of transcription factors as well as metabolism pathways such as lipid metabolism,amino acid metabolism and energy metabolism.