| Volvariella volvacea was a saprophytic fungus grown in the tropics and subtropics area with nutritional and medicinal value. V. volvacea could not grow and can not be stored under cold stress. When the ambient temperature was lower than 5℃, Low temperature could induce the degradation of protein in V. volvacea cells, resulting in the autolysis of the mycelia. Fruit bodies would soften, colour and decay. The characteristic of V. volvacea had an adverse effect on its cultivation, storage and transportation. Therefore, the study of responsive mechanism of V.volvacea under cold stress means a lot for screening cold-tolerance strains.The mycelia of two V. volvacea strains differing in cold resistance, V23 (cold-sensitive) and VH3 (cold-tolerant) were exposed to cold stress (0℃) for 0,2,4, 6, and 8h, respectively. The mycelia were collected for different experiments. (1) Indicators of cell damage which include conductivity, MDA, growth recovery time of mycelium and mycelium length were measured to determine the difference of two strains. (2) Cold stress could cause the increase of reactive oxygen in V. volvacea and high level of ROS could bring severe damage to V. volvacea cell. The expression of some key enzymes’ gene including Cu/Zn-SOD, Mn-SOD, CAT and GR were analyzed by RT-PCR. (3) Phylogenetic analysis of key antioxidant enzymes (Cu/ Zn-SOD, Mn-SOD, CAT, GR) were analyzed by using bioinformatics software. The activity of these antioxidant enzymes including SOD, CAT, APX and GR were measured.The results showed that:1 The trend of conductivity changes was consistent in two strains of V. volvacea. The electrical conductivity increased sharply from Oh to 2h, and then stayed stable in 6-8h, then increased from 6h to 8h. The corresponding conductivity value of VH3 less than strain V23 indicated that VH3 could better maintain the integrity of the cell membrane under cold stress.2 The longer the low temperature stress, the longer the recovery time needed for growth of V. volvacea mycelia, the slower growth rate of recovery. The mycelium of strain V23 requiring recovery time was longer than VH3 and the growth rate of the mycelium of V23 was slower than VH3. The difference was more obvious for recovery time and length of mycelia between these two strains when cold stress time was much longer.3 The content of MDA in strain VH3 did not change significantly but MDA content of strain V23 increased during 0-2h.However, MDA content in strain VH3 increased rapidly in 2-4h during cold stress. No difference of MDA content in two strains almost was observed during 4-8h.4 The amino acid sequence of antioxidant enzymes (Cu/Zn-SOD, Mn-SOD, CAT, GR) was analyzed with other fungal corresponding enzymes by sequence alignment and phylogenetic tree analysis. The results confirmed that these four enzymes had high homology with other fungal corresponding enzymes. It also ensured that the obtained nucleotide sequence encoding antioxidant enzymes as well as gene quantification was validity.5 The relative expression of Cu/Zn-SOD gene increased at first and then decreased in two strains during cold stress. Although small difference was found in two strains, relative expression in VH3 was higher than that in V23.Relative expression of Mn-SOD genes in strains VH3 increased at first and then fell. Relative expression of Mn-SOD genes in strains V23 declined and then increased.The expression level Mn-SOD gene in strain VH3 is much higher than that in strain V23. SOD activity increased and then decreased during low temperature treatment. SOD activity in strain VH3 was higher than strain V23 from Oh to 4h, but SOD activity decreased rapidly later in VH3 strains. It suggested that the expression of SOD might have to do with cold tolerance, especially with Mn-SOD.6 The relative expression of catalase gene in two strains decreased first and then rised, but the relative expression in strain VH3 was much higher than V23.The trend of CAT activity showed an upward during cold stress. Besides, CAT activity in VH3 increased higher than the V23 strain. It meant that CAT in strain VH3 could remove H2O2 more effectively.7 Ascorbate peroxidase was an important enzyme for clearing H2O2.The activity of APX in two strains increased first and then decreased during cold stress. No difference found in the activity of APX suggested that APX was not the reason for low temperature tolerance between two strains.8 During cold stress, the relative expression of GR gene showed a rise after the first fall, and finally a downward trend. The level of gene transcription of GR in strain VH3 was lower than strain V23. GR activity of two strains showed the same trend during cold stress. It rose at first, then remained stable, then dropped, and finally on the rise. GR activity in V23 was much higher than that in VH3. It suggested that V. volvacea could adjust the expression of GR in response to low temperature stress. V. volvacea tended to use antioxidant enzyme system(SOD,CAT et al) to eliminate ROS in the process of cold stress. |
PDF Full Text Request