转录组揭示禾谷镰刀菌酸碱胁迫条件下的基因表达模式

Transcriptomics reveals gene expression patterns of Fusarium graminearum under pH stress

摘要:

【目的】 研究不同pH胁迫条件下禾谷镰刀菌(Fusarium graminearum)的转录组调控机制，分析基因表达水平及差异，探究在酸性或碱性条件下与禾谷镰刀菌细胞抗胁迫反应相关的代谢通路，揭示禾谷镰刀菌如何主动调节细胞内代谢与合成过程，以适应细胞外pH环境的变化。【方法】 在初始pH为4.5、6.5、8.0的PDB培养基中培养禾谷镰刀菌48 h，提取菌株的总RNA，构建cDNA文库。利用转录组测序技术及生物信息学技术鉴定相关差异表达基因(differentially expressed genes, DEGs)，进一步分析涉及的代谢通路。采用反转录实时定量PCR (reverse transcription-quantitative PCR, RT-qPCR)对基因表达水平进行验证。【结果】 在酸性条件下，共有4 283个DEGs，其中2 032个上调DEGs和2 251个下调DEGs；在碱性条件下，共有498个DEGs，其中269个上调基因和229个下调基因。基因本体论(gene ontology, GO)富集分析结果显示，在酸性条件下上调基因显著富集到的GO terms有211个，下调的有72个；碱性条件下上调的GO terms有33个，下调的有40个。京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)富集分析结果显示，在酸性条件下上调DEGs显著富集到的通路有22个，下调有32个；在碱性条件下上调通路有8个，下调有13个。膜转运蛋白的表达和糖类化合物的水解等相关基因表达上调以及蛋白质代谢相关基因表达下调，协助禾谷镰刀菌细胞适应外界环境的变化。与此同时，禾谷镰刀菌在酸性和碱性条件下分别通过降低次生代谢和降低氨基酸代谢来维持自身细胞的内环境平衡，从而抵抗细胞外pH胁迫。【结论】 在酸性环境中，禾谷镰刀菌通过促进核糖蛋白复合体的产生以及次生代谢来适应细胞外环境的变化；在碱性环境中，禾谷镰刀菌细胞通过氨基酸代谢来响应和感知外界的胁迫。通过对禾谷镰刀菌细胞代谢相关通路分析为禾谷镰刀菌对不同pH环境的响应提供重要的基因表达数据支撑，研究结果有助于理解禾谷镰刀菌的致病机制。

关键词:酸碱胁迫;禾谷镰刀菌;转录组;差异表达基因

Abstract:

[Objective] To study the transcriptome regulation mechanism of Fusarium graminearum under different pH stress conditions, analyze the changes in gene expression levels, explore the metabolic pathways involved in the responses of F. graminearum cells to acidic or alkaline conditions, and reveal how F. graminearum regulates intracellular metabolism and synthesis to adapt to the changes in extracellular pH. [Methods] F. graminearum was cultured in the PDB media with initial pH 4.5, 6.5, and 8.0 for 48 h, and the total RNA of the strains was extracted to construct the cDNA library. Transcriptome sequencing and bioinformatics analysis were used to identify the differentially expressed genes (DEGs), and the metabolic pathways involved were analyzed. The expression levels of target genes were determined by RT-qPCR. [Results] Under acidic conditions, a total of 4 283 DEGs were identified, including 2 032 genes with up-regulated expression and 2 251 genes with down-regulated expression. Under alkaline conditions, a total of 498 DEGs were identified, including 269 genes with up-regulated expression and 229 genes with down-regulated expression. Gene ontology (GO) enrichment analysis revealed 211 GO terms for the up-regulated genes and 72 GO terms for the down-regulated genes under acidic conditions. Under alkaline conditions, GO analysis yielded 33 GO terms for the up-regulated genes and 40 GO terms for the down-regulated genes. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed 22 up-regulated pathways and 32 down-regulated pathways under acidic conditions as well as 8 up-regulated pathways and 13 down-regulated pathways under alkaline conditions. The expression of the genes associated with membrane transporters and hydrolysis of carbohydrates was up-regulated, and that of the genes related to protein metabolism was down-regulated, which assisted F. graminearum cells to adapt to changes in the external environment. At the same time, F. graminearum maintained the internal environment balance by reducing secondary metabolism and amino acid metabolism under acidic and alkaline conditions, respectively, to resist extracellular pH stress. [Conclusion] In the acidic environment, F. graminearum adapts to the changes in the extracellular environment by promoting the production of ribonucleoprotein complexes and secondary metabolism. In an alkaline environment, F. graminearum senses and responds to external stresses via amino acid metabolism. The analysis of the metabolic pathways of F. graminearum cells provides gene expression data for studying the responses of F. graminearum to different pH environments. The findings of this study are helpful to understand the pathogenic mechanism of F. graminearum.

Key words:pH stress;Fusarium graminearum;transcriptome;differentially expressed genes