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β-1, 6-glucan synthase is a key enzyme of β-1, 6-glucan synthesis, which plays a vital role in the cell wall cross-linking of fungi. However, the role of the β-1, 6-glucan synthase gene in the development of the fruiting body and the stress response of macrofungi is largely unknown. In this study, four overexpression transformants of the β-1, 6-glucan synthase gene (FfGS6) were successfully obtained, and gene function was studied in Flammulina filiformis. The overexpression of FfGS6 can increase the width of mycelium cells and improve the tolerance ability under mechanical injury and oxidative stress. Moreover, FfGS6 gene expression fluctuated in up-regulation during the recovery process of mycelium injury but showed a negative correlation with H2O2 concentration. Fruiting body phenotype tests showed that mycelia's recovery ability after scratching improved when the FfGS6 gene was overexpressed. However, primordia formation and the stipe elongation ability were significantly inhibited. Our findings indicate that FfGS6 is involved in regulating mycelial cell morphology, the mycelial stress response, and fruit body development in F. filiformis. [ABSTRACT FROM AUTHOR]