Protoplast fusion between the same mating type strains of Y2HGold

In order to avoid the limitations of sexual fusion and find out the potential value of asexual fusion between yeast strains of same mating type,the MatchmakerTM Gold Yeast Two-Hybrid System strain Y2HGold were chosen as the material,using the plasmid,the leucine-deficient and tryptophan-deficient strains of Y2HGold were successfully constructed;using orthogonal test with four factors and three levels by enzymolysis method,the preparation protocol of deficient-bacteria protoplasts was concluded as:enzymolysis temperature of 30℃,80min processing time,pre-treatment agent containing 0.1%(w/v)mercaptoethanol and 0.1%(w/v)EDTA-2Na,hydrolyzate a mixture of 1.5%(w/v)and 1.5% snail enzyme(w/v)cellulase,rendering the protoplast preparation and regeneration rate 82% and 37% respectively.Finally,using PEG-induced fusion method and the same orthogonal test,we obtained the requirements about asexual fusion of Y2HGold:35% PEG concentration,temperature as 25℃ and 40min and pH5.8.Sub-clones we selected were genetically stable.