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.