Differentially Expressed Proteins And Genes Analysis Among The Different Developmental Stages Of Tremella Fuciformis

Tremella fuciformis, a typical dimorphic fungus with medicinal and nutritional value, is rich in polysaccharides, proteins, vitamins and inorganic salts, known as "Champion in Mushroom". Protein is the product of gene expression and the executor of physiological function. Studying gene function after cloning gene from the protein level can be more comprehensive and perspective to clarify the actual function that gene plays, which can also better understand the mechanism and regular pattern of life activities. In this study, the total protein was extracted by Tris-saturated phenol method from T. fuciformis yeast-like conidia and mycelia, then to find and identify differentially expressed proteins from the two different developmental stages by 2-DE and MALDI-TOF MS/MS technique, and to clone their differentially expressed genes by designing degenerate primers based on their amino acid sequences of two differentially expressed proteins. This study laid the foundation for the research about regulatory mechanism in different developmental stages of T. fuciformis. The results are as follows:1. In order to compare the total protein extraction effect from T. fuciformis yeast-like conidia by Tris-saturated phenol method, TCA/acetone precipitation method and Tris-saturated phenol combined with TCA/acetone precipitation method, cells were disrupted by ultrasonic wave and high pressure homogenization on the basis of liquid nitrogen grinding, the effect of cell disruption was observed by microscope, protein content was determined by the Bradford method, and SDS-PAGE analysis was used. The obtained results showed that the effect of cell disruption by high pressure homogenization is better than that by ultrasonic wave. The protein contents obtained by the three extraction methods were 2.33 mg/mL, 1.26 mg/m L and 1.02 mg/m L, respectively. And the electrophoresis band brightness and resolution of Tris-saturated phenol method were better than the other two methods. Furthermore, the extraction of total protein from mycelia by Tris-saturated phenol also achieved a good result. So an effective method to extract total protein from T. fuciformis yeast-like conidia and mycelia was established.2. 53 differentially expressed protein spots were selected by 2-DE analysis after the total proteins were extracted by Tris-saturated phenol method from T. fuciformis yeast-like conidia and mycelia. These differentially expressed protein spots were analyzed by MALDI-TOF MS/MS, and 11 protein spots were identified through BLAST in the database. Most of the 11 proteins were related to the metabolic pathway TCA cycle after analyzing their functions and properties, which can speculate that the expression differences of protein in T. fuciformis yeast-like conidia and mycelia is closely related to the differences in metabolic pathways.3. The degenerate primers were designed based on the amino acid sequences of protein-S-isoprenylcysteine O-methyltransferase and cytochrome-c oxidase subunit among the differentially expressed proteins.The corresponding differentially expressed genes were cloned by RT-PCR, PCR amplification and other techniques after extracting the total RNA of T. fuciformis yeast-like conidia.