The Construction Of Genetic Transformation System Of Coprinus Chinensis

Coprinopsis cinerea(Schaeff.ex Fr.)S.F.Gray is a model organism commonly used to study developmental processes in the homobasidiomycetous fungi.Although it is of limited edible value,but studies of C.cinerea may be used to understand the development of numerous edible basidiomycetes which fail to grow and/or produce fruiting bodies in the laboratory,it also has important theoretical and practical significance in increase the production of the edible and medicinal mushroom.Previously,we have used the extension instrument of plant cell wall,established a research platform to study the stipe elongation of the mushroom C.cinerea,which is suitable as a model material for wall extension studies.Using a combined chromatography method,we got simultaneously three purified protein fractions,with P-1,3-glucanase activity from extraction of autohydrolyzing pilei of C.cinerea fruiting bodies.These protein fractions can collaborate with each other to hydrolyze the ?-1,3-glucan from C.cinerea's cell walls,these studies can help us initially know about hydrolysis mechanism of basidiomycetes cell wall.But if we intend to explore this phenomenon deeply,we must establish a transformation system of C.cinerea.In this paper,on the basis of the existing theory and experiments,we initially established the transformation method in C.cinerea,and harvested the transformants.In this paper,we explored the collection conditions of C.cinerea auxotrophic strain AmutBmut's oidias and protoplasts firstly.Under optimized conditions,each plate can be harvested about 109-1010cells,and the oidia germination rate can up to 60%-80%.During the preparation of protoplasts with oidias,the preparation rate of protoplasts can up to approximately 70%and the regeneration rate can up to approximately 15%.We constructed a plasmid named pEASY-zero-pabl containing PABA synthase gene(pab1)of C.cinerea,and then transformed into C.cinerea strain AmutBmut by PEG/CaC12-mediated transformation.We obtain a bunch of transformations and confirmed that the foreign gene has been successfully integrated into the genome of C.cinerea by PCR amplification and dot ohart analysis.On the basis of this system,we constructed the expression vectors named pCCGFP-Xpro-1 containing the promoter from different fungus and egfp gene,and then co-transformed with pEASY-zero-pabl into C.cinerea strain AmutBmut by PEG-CaC12-mediated transformation.The results demonstrated that about 30%of transformants were co-transformats.Three of promoters(C.cinerea trpl and bgl,A.bisporus gpdll)were tested in promoting GFP expression in C.cinerea,and the promoter of A.bisporus gpdII is the best.