A Study Of Protoplast Fusion Between The Same Mating Type Strains Of Y2HGold

Protoplast fusion is known as somatic cell hybridization or cell fusion, which use artificial method to make two different genetic traits cells protoplast fusion and produce a single core or multicore fusants. Protoplast fusion is also an important content of the "cell engineering"which include induced fusion and spontaneous fusion, The induced fusion is defined as the process of adding inducer or using other methods to induced different parental protoplast fusion after the separation of the protoplas, and the spontaneous fusion is the the process of the natural fusion in cells or plant tissue in the process of protoplast enzyme separation. Somatic hybridization can occur in intraspecific, interspecific, intergeneric, science and even between of the sector the protoplast, it can not only break the reproductive isolation between species, creating distant hybrids, and also allow parents cytoplasmic gene binding together, so as to achieve the purpose of more improved traits.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 was chose as the material, using the plasmid, the leucine-deficient and tryptophan-deficient strains of Y2HGold was successfully constructed; using orthogonal test with four factors and three levels by enzymolysis method, Finally, using PEG-induced fusion method and the same orthogonal test, Sub-clones we selected were genetically stable. Our results showed that:(1)genetic markersThe experiment genetic marker of yeast two-hybrid project strains Y2HGold was based on it is gene structural features. At last, a leucine, tryptophan-deficient strains was successfully constructed.(2) Influencing factors of integrationThe single-factor experiments and orthogonal test results show that the protoplast four key factors:hydrolysis temperature, enzyme treatment time, enzyme concentration and pretreatment agents Preparation of protoplasts and regeneration has an impact. High significant differences were detected in the hydrolysis temperature and enzyme processing time for on the regeneration rate and preparation rate. significant difference was found in the enzyme mixture for protoplast regeneration rate. no significant difference was detected in pretreatment agent protoplast regeneration rate. The key factor in the PEG concentration in the fusion rate have the trend of upward.(3) Protoplast preparation and integration programsThe preparation protocol of deficient-bacteria protoplasts was concluded as: enzymolysis temperature of 30℃,80minutes 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 40minutes and pH 5.8. Sub-clones we selected were genetically stable.(4) the difference of asexual fusion and the sexual integrationWe can found out that the daughter cell of asexual fusant are bigger than the daughter cell of sexual fusant while mother cells of asexual fusant is smaller than sexual fusant’s mother cell, and it indicated that the sub-form clonal integration may be more thorough than sexual fusion on the performance of fusant. asexual fusion can finish in a short period of 40min while the sexual fusion takes 20～24h, but asexual fusant grow slowly in SD/2 medium. All above mentioned indicated that aa and aa show powerful growth advantage compared with aa.