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Protoplast Fusion between the Same Mating Type Strains of Y2HGold

【摘要】:There are two kinds of yeast vegetative bodies,haploid and diploid,which are closely related and widely used.In order to avoid the limitations of sexual fusion and find out the application value of asexual fusion between haploid of same mating type,we chose the MatchmakerTM Gold Yeast Two-Hybrid System strain Y2HGold as the material and finished the exploration about protoplast fusion.By using the plasmid,we constructed leucine-deficient and tryptophan-deficient strains of Y2HGold firstly,then transferred and cultured them until they were steady;we acquired protoplast by enzymolysis method,and discussed four important factors with three-level test.As a result,we found that protoplast preparation was 82% and regeneration rates was 37% during the process,the condition we obtained from asexual fusion of Y2HGold were that it was treated in 40 minutes with 35%(w/v) PEG,then it was managed temperature as 25℃ and the pH value was 5.8.We selected some sub-clones and analyzed their genetic stability.This may provide favorable subtractions for the further study on the value of asexual fusion.

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