Establishment of stable heterologous gene expression platform by mating strategies in Flammulina velutipes

Mushroom molecular pharming, a novel biotechnology application using mushrooms as bioreactors to produce pharmaceutical proteins or oral vaccines, exhibits advantages such as less genetic pollution, simple operation and lower costs etc. In our laboratory, we have established Agropbacterium-mediated transformantion (AMT) in mushrooms and have tried to enhance heterologous gene expression by various molecular biology strategies. However, the previous studies show that the expression of heterologous proteins varies a lot between subcultures, indicating the mycelia are a mixture of transformants and wild type. Futhermore, it appears AMT does not ensure delivering heterologous genes to the two nuclei of dikaryotic mycelia and lead to transformants with unstable heterologous expression. Therefore, it’s critical to acquire the pure transformants which all nuclei contain heterologous genes for further studies. In order to establish the pure transformants of Flammulina velutipes in our study, we used Fluorescence-Activated Cell Sorter (FACS) to obtain the green fluorescent spores with high intensity from Figpd-d3-2 transformants and mated the different haploid mycelia germinating from spores. The result shows we have good chances to obtain the pure and stable transformants expressing heterologous proteins with higher intensity of enhanced green fluorescent protein (eGFP) expression than Figpd-d3-2 transformants. This study is the first report regarding the establishment of selecting pure transformants. In the future, the platform of selecting pure transformants can be applied in oral vaccines of enterovirus 71 or other infectious diseases. We also constructed the plasmid which contains glyceraldehyde-3-phosphate dehydrogenase promoter d1 (gpd-d1) , VP1 as target gene, egfp as report gene, and 2A peptide which can cleave in translation to ensure transformants expressing eGFP and VP1 simultaneously. The selection of pure and stable VP1 transformants is undergoing.