同种接合型酵母菌株Y2HGold的原生质体融合研究  

Protoplast fusion between the same mating type strains of Y2HGold

作　　者：常登龙[1] 洪玉[1] 杨永军[1] 张贺翠[1] 薛丽琰[1] 杨昆[1] 朱利泉[1] 王小佳[1] 

机构地区：[1]西南大学农学与生物科技学院,重庆400715

出　　处：《食品工业科技》2012年第12期233-238,共6页Science and Technology of Food Industry

基　　金：重庆市自然基金重点项目(cstc2012jjB80010);国家自然科学基金项目(30971849);国家大学生创新计划项目(091063509)

摘　　要：为避开有性融合的局限性,挖掘同种接合型酵母菌株无性融合的潜在应用价值,利用质粒构建了酵母双杂交工程菌株Y2HGold的亮氨酸缺陷型和色氨酸缺陷型菌株;接着采用酶解法进行四因素三水平正交实验获得原生质体制备方案:酶解温度30℃、处理时间80min、预处理剂含0.1%(w/v)巯基乙醇和0.1%(w/v)EDTA-2Na,酶解液为1.5%(w/v)蜗牛酶和1.5%(w/v)纤维素酶混合,使得原生质体的制备及再生率分别达82%和37%;最后采用PEG诱导融合的方法,经过同样正交实验得到Y2HGold无性融合条件为:PEG浓度35%、温度25℃、时间40min、pH5.8。筛选得到的无性融合子在遗传上稳定。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.

关 键 词：酵母 原生质体 无性融合 无性融合菌株 

分 类 号：TS201.3[轻工技术与工程—食品科学]