| Flammulina velutipes (Fr.) Sing has a long cultural history in our country, which cultivation technology research is relatively perfect. Until now there is no lack studied about cultivation techniques and cultivation mode, But its physiological mechanisms cultivation process and in-depth study should be further explored. After the fungus mycelium reached physiological age fruiting body can differentiation and growth under cold strss. It is not clear molecular mechanism of cold stimulation, leading to deep research and production technology innovation basic theory behind. Flammulina velutipes is an ideal material study fungi under low temperature in molecular level.In this study, a comparison of fruting body and a genetic diversity analysis were used to select F. Velutipes starting strain. To obtain non-fruiting monokaryon mycelia and fruiting dikaryon mycelia of F. Velutipes, this study used single spore isolation and monokaryon-monokaryon crossing. iTRAQ coupled 2D LC-MS/MS was applied to investigate the proteomes of non-fruiting monokaryon mycelia and fruiting dikaryon mycelia of F. Velutipes. To validate the accuracy of iTRAQ coupled 2D LC-MS/MS, real-time quantitative PCR was used to analyze the expression level of the differential genes. The chief results are showed as follows:1. By comparing the characteristics of fruit body and analyzing the genetic diversity, the idear F. Velutipes was chosen as the experimental material.2.Three highly pure and undegraded protein samples were obtained by methanol-ammonium sulfate extraction method and SDS-PAGE analysis method.3.The three protein samples were analyzed by iTRAQ coupled 2D LC-MS/MS which consisted of three technical replicates.1198 proteins were identified by two and three replicates. Results of analyzing the principal component, expression level, distribution range of expression, correlation coefficient, hierarchy clustering and K-means clustering showed that iTRAQ coupled 2D LC-MS/MS was a excellent method to isolated and identified proteins groups of three samples.4. Using the standard of more than 2 times difference screened differential expression proteins from the 1198 identification proteins. The results show that 63(0.95%) differential expression proteins are screened in the three protein samples.3 differential expression proteins all up-regulated in two stages,6 differential expression proteins all down-regulated in two stages,8 differential expression proteins all up-regulated in first stages and down-regulated in the second stages,7 differential expression proteins all down-regulated in first stages and up-regulated in the second stages,15 differential expression proteins are up-regulated and 7 are down-regulated only in the first stages,11 differential expression proteins are up-regulated and 6 are down-regulated only in the second.5. Gene Ontology (GO) analysis show that there is a strong correlation between the protein and the mycelia cell proliferation, biosynthesis, energy metabolism. KEEG pathways analysis show that the total of the 63 differential expression proteins are involved in 22 unique metabolism pathways. The synthesis and metabolism of protein, nucleic acid and carbohydrate are main pathways. The result provides a good protein expression profile data platform which is used for screening proteins associated cold stress mechanism.6. Real-time quantitative PCR analyzed the expression level of the differential expression genes. The results show that 3 differential expression proteins all up-regulated in two stages,14 differential expression proteins all down-regulated in two stages,1 differential expression proteins are up-regulated in first stages and down-regulated in the second stages, 14 differential expression proteins are down-regulated in first stages and up-regulated in the second stages.The results are consistent with the label free quantitative proteomic data. Thus, the iTRAQ coupled 2D LC-MS/MS quantitative proteomic data are highly accurate and reliable. |
PDF Full Text Request