Cloning of catalase gene PpCAT from Pleurotus pulmonata and its response to H2O2 stress

Catalase (CAT) and peroxidase (POD) are key enzymes for scavenging H 2 O 2 in organisms. To investigate their roles in Pleurotus pulmonarius in response to H 2 O 2 stress, two genes, PpCAT (1590 bp) and PpPOD (1515 bp), were mined based on the genomic data of P. pulmonarius from the NCBI database. Bioinformatics analysis indicated that the coding proteins of PpCAT and PpPOD were localized to the peroxisome and extracellular space, respectively, and possessed typical conserved domains of their respective families. Overexpression vectors pYES2-PpCAT and pYES2-PpPOD were constructed and heterologously expressed in yeast INVSC1 along with the empty vector pYES2. Through H 2 O 2 stress experiments with different concentrations, it was found that under 2000 mmol/L H 2 O 2 stress, strains containing pYES2 and pYES2-PpPOD failed to grow, while the strain containing pYES2-PpCAT could still grow. Under 5000 mmol/L H 2 O 2 stress, the strain containing pYES2-PpCAT also failed to grow normally, indicating that PpCAT is a key gene in response to H 2 O 2 stress, but its response has a limit. H 2 O 2 stress experiments over different time courses revealed that the strain containing PpCAT consistently exhibited higher biomass (OD 600 ) and stronger viability. qRT-PCR analysis showed that during H 2 O 2 stress over different times, the CAT gene expression level in the pYES2-PpCAT-containing strain was continuously up-regulated. CAT enzyme activity peaked at 6 h of stress, significantly higher than the control, and effectively maintained intracellular H 2 O 2 content at a low level. Correlation analysis further confirmed that in the pYES2-PpCAT-containing strain, CAT enzyme activity was negatively correlated with H 2 O 2 content ( r = −0.484). In conclusion, the PpCAT gene plays an important role in protecting P. pulmonarius against H 2 O 2 stress by increasing CAT enzyme activity and effectively scavenging intracellular H 2 O 2 .