NaCl胁迫下哈茨木霉ACCC32524的转录组和代谢组分析

Transcriptome-metabolome analysis of Trichoderma harzianum ACCC32524 under NaCl stress

摘要:

[目的] 通过分析NaCl胁迫下哈茨木霉（Trichoderma harzianum）ACCC32524转录组和代谢组数据，研究差异表达基因及次级代谢产物的变化情况，初步探索响应NaCl胁迫的分子机制。[方法] 利用Illumina HiSeq XTen高通量测序平台完成0、0.4、0.6 mol/L NaCl浓度胁迫培养下哈茨木霉ACCC32524的转录组测序，GC-TOF-MS技术完成对0 mol/L和0.6 mol/L NaCl胁迫培养下的差异次级代谢产物检测，利用相关软件及数据库对差异表达基因（DEGs）和次级代谢产物的注释、筛选和分类，并进行RT-qPCR验证。[结果] 本研究分别得到0.4 mol/L和0.6 mol/L NaCl胁迫下417和733条差异表达基因；GO富集分析显示，分别有318和582条差异表达基因注释到生物学过程、分子功能和细胞组分3个一级分类和40个二级分类；COG分类结果表明分别有232和414条转录本为20个类别，涉及差异表达基因最多的分别为氨基酸的转运和代谢、一般功能预测、碳水化合物的转运和代谢；KEGG代谢途径分析结果表明，分别有75和96条基因归到25个代谢通路中（P ≤ 0.05），其中涉及差异基因最多的是氨基酸的生物合成和2-氧代羧酸代谢通路。从转录组数据中共筛选出与渗透调节、离子转运、活性氧清除等22个耐盐相关基因。0 mol/L和0.6 mol/L NaCl胁迫下的代谢组数据中共筛选出101个差异次级代谢产物，包括8种积累量上调和93种下调物质，其中36个得到定性，分属于糖类、有机酸和氨基酸等9个分类中。RT-qPCR验证挑选的差异表达基因的表达量变化，均与RNA-seq分析结果一致。[结论] NaCl胁迫下引起哈茨木霉ACCC32524基因及次级代谢产物发生明显变化，细胞代谢途径发生明显偏移，这些进程共同作用减少NaCl对细胞的毒害作用，为木霉菌的耐盐机理研究提供重要信息。

Abstract:

[Objective] To identify the candidate genes and second metabolites involved in NaCl stress response of Trichoderma harzianum ACCC32524, thereby exploring the mechanism of NaCl stress adaptation.[Methods] The transcriptomes of ACCC32524 with the treatment of 0, 0.4, 0.6 mol/L NaCl were compared using Illumina HiSeq XTen high-throughput sequencing and the metabolomes with the treatment of 0, 0.6 mol/L were detected by GC-TOF-MS. The annotation, screening and classification of differentially expressed genes (DEGs) and secondary metabolites were completed by using related softwares and databases. Validation of DEGs using RT-qPCR.[Results] A total of 417 and 733 DEGs were found of ACCC32524 with the treatment of 0.4 or 0.6 mol/L NaCl respectively. GO analysis suggested that total of 318 and 582 DEGs were categorized into three functional classifications (biological process, molecular function, cellular component) and forty sub-categories; COG classification results showed that 232 and 414 transcripts were assigned to the same 20 categories and these transcripts were significantly enriched in various known amino acid transport and metabolism, general function prediction only and carbohydrate transport and metabolism; KEGG pathway analysis revealed that filtered 79 and 96 DEGs were enriched in 25 individual pathways, and those genes were significantly enriched in biosynthesis of amino acids and 2-oxocarboxylic acid metabolism pathways. A total of 22 genes were screened from transcriptome data related with osmotic regulation, ion transport and ROS scavenging. A total of 101 differential secondary metabolites were screened from the metabolome data under 0.6 mol/L NaCl stress, including 8 upregulated and 93 downregulated substances, 36 of which were qualitatively identified and distributed in 9 classifications, such as carbohydrates, organic acids and amino acids. The expression level of selected DEGs was tested with RT-qPCR, and they were consistent with the result of RNA-seq analysis.[Conclusion] Under NaCl stress, a large number of genes and secondary metabolites of Trichoderma harzianum ACCC32524 were changed and the metabolic pathway was significantly shifted. These processes together reduced the toxicity of salt ions to cells and enhanced the tolerance of strains to salt stress. This study further provided the gene information for the research of salt tolerance mechanism of Trichoderma spp..