关键词: 草菇, 甘露醇, 菌种复壮, 活性氧, 抗氧化酶, 线粒体

Abstract:

【Objective】The aim of this study was to investigate the effects of mannitol on production characteristics and reactive oxygen species (ROS) scavenging ability of Volvariella volvacea subcultured strains, and to explore a simple and effective method for the rejuvenation of V. volvacea degenerated strains. 【Method】The tissue isolation subcultured strains T6, T12 and T19 were obtained by previous study of our research group, and T6 was obtained after 6 successive subculture, while T12 and T19 was obtained after 12 and 19 successive subculture, respectively. The original strain (T0), referred to as V844, was a strain used in commercial agricultural cultivation. The glucose in the traditional potato dextrose agar (PDA) was replaced by mannitol of the same mass, then physiological traits were determined in mycelia. The agronomic characters of fruiting body were measured by adding mannitol to the culture medium. ROS accumulation was reflected using nitrotetrazolium blue chloride (NBT) staining of V. volvacea mycelia, superoxide anion ($\mathrm{O}_{2}^{\bar{.}}$) and hydrogen peroxide (H₂O₂) content. The expression levels of antioxidant enzyme genes were detected by real-time quantitative PCR (RT-qPCR). The activity of antioxidant enzymes was measured by the kit. The number of nuclei and mitochondrial membrane potential were determined by mycelium staining. The energy indexes were determined by high performance liquid chromatography (HPLC). 【Result】Mannitol treatment had no significant effect on non-degraded strains T0 and T6, but could effectively restore the production characteristics and ROS scavenging ability of degraded strains T12 and T19. After mannitol treatment, the mycelial growth rate of T12 and T19 was increased by 31.46% and 20.99%, respectively, and the mycelial biomass was increased by 97.33% and 76.36%, respectively. The mannitol treatment shortened the production cycle of T12 by 12.24% and increased the biological efficiency by 17.97%, thus restoring it to T0 level. In addition, the mannitol treatment caused T19 to regrow its fruiting body, which had been severely degraded and lost its ability to produce fruiting bodies. Meanwhile, mannitol treatment increased the relative expression of Cu/Zn superoxide dismutase (Cu/Zn-sod) gene in T12 and T19 by 24.64% and 61.54%, respectively, and the relative expression of Mn-sod2 gene by 19.76% and 267.09%, respectively. Similarly, the relative expression of glutathione peroxidase (gpx) gene was up-regulated by 25.67% and 55.82%, respectively. More importantly, the activity of SOD in T12 and T19 increased by 10.79% and 72.32%, and the activity of GPX increased by 16.98% and 103.85%, respectively. The accumulation of ROS in T12 and T19 was significantly reduced by mannitol treatment, in which the $\mathrm{O}_{2}^{\bar{.}}$ content in T12 and T19 decreased by 35.96% and 41.62%, while the H₂O₂ content decreased by 14.44% and 18.26%, respectively. Furthermore, the mannitol treatment significantly increased the number of nuclei and mitochondrial membrane potential in T12 and T19. Mannitol treatment could increase ATP content in T12 and T19 by 17.08% and 14.55%, and EC value by 4.52% and 0.92%, respectively. 【Conclusion】Mannitol treatment could significantly improve the antioxidant capacity and mitochondrial function of the degenerated strains T12 and T19, and effectively restore their production traits.

Key words: Volvariella volvacea, mannitol, strain rejuvenation, reactive oxygen species, antioxidase, mitochondria