3.2 AaLaeA is involved in the metabolism of antitumor substances and regulates the expression of metabolism-related genes
To investigate the material basis of the loss of antitumor activity ofAaLaeA OE26, LC/MS nontargeted metabolomics analysis of the endophytic fungus A. alstroemeria WT andAaLaeA OE26 was performed. Principal component analysis (PCA) showed that there was significant separation between the WT and AaLaeA OE26 samples, with PC1 and PC2 accounting for 95.14% and 4.44% of the total variation, respectively (Fig. 2A). Hierarchical cluster analysis and Plots from partial least squares discriminant analyses (PLS-DA) likewise showed significant separation of WT and AaLaeA OE26 metabolites (Fig.S2); Further analysis of the differing substances revealed noteworthy metabolic differences between the two groups of samples (Fig. 2B). Screening for metabolites(log2 FC > 0.5、P value < 0.05 and VIP > 1) with significant differences revealed that the flavonoid epicatechin gallate [25] was the most significantly down-regulated of the top 10 live compounds (Table 1) in terms of anti-tumour compounds; Phenolic and indole derivatives were also included in the down-regulated antitumour activators (Fig. 2C). Through KEGG pathway enrichment, it was found that differential metabolites were enriched into 20 metabolic pathways, including fatty acid synthesis, amino acid synthesis and indole alkaloid synthesis (Fig. 2D). The above results suggest that significant down-regulation of several major classes of antitumour compounds in theAaLaeA OE26 strain might be responsible for the loss of antitumour activity in AaLaeA OE26.
To clarify the molecular basis of the loss of antitumor activity ofAaLaeA OE26, the transcriptome patterns of WT and AaLaeA OE26 were analyzed comparatively at the same time points as the above metabolome sampling. Analysis of the depth of sequencing and the clean reads obtained showed that the samples all reached 5.90 Gb of Clean Data and over 19,755,288 Clean reads (Table 2), all meeting transcriptome quality standards. A total of 3795 differential genes (|log2FoldChange| > 1, FDR < 0.05) were detected, of which 1893 were up-regulated and 1902 were down-regulated (Fig. 3A). GO functional analysis showed that the differential genes were mainly involved in substance metabolic processes, catalytic enzyme activity and binding activity (Fig. 3B). Metabolic pathway clustering analysis showed that diverse genes were mainly involved in four pathways: metabolism, genetic information processing, cellular processing and environmental information processing (Fig. 3C). This suggests that AaLaeA is involved in the metabolism ofA. alstraoemeria and may be involved in the regulation of catalase genes in the metabolic process.