酿酒酵母和产朊假丝酵母原生质体的形成、再生及属间融合

使用由亚硝基胍诱变所得到的营养缺陷型作为单倍体融合亲株的核基因标记,同时也采用线粒体球红霉素抗性突变株的小菌落形式作为融合亲株的线粒体基因标记。酿酒酵母(Saccharomyces cerevisiae)和产朊假丝酵母(Candida utilis)两亲株原生质体的制备是用对数生长早期的细胞在蜗牛酶和0.7M KCl及β-巯基乙醇或二巯基苏糖醇的作用下完成的。二者的原生质体的形成率在30—60分钟内达到90—99％。原生质体再生率,酿酒酵母最高为29—35％,产朊假丝酵母为7.5％。两亲株的原生质体在35％PEG(M.W.6,000),10mM CaCl_2条件下被诱导融合。在基础培养基上,长出以营养互补为标记的融合菌株。融合频率为10~(-5)—10~(-6)。试验表明,这些融合菌株具有杂种的性质。其中一株杂合子在同化D-木糖、纤维二糖等的能力上比亲株明显增强。

FORMATION, REGENERATION AND INTERGENERIC FUSION OF PROTOPLASTS IN SACCHAROMYCES CEREVISIAE AND CANDIDA UTILIS

The auxotrophs obtained by nitrosoguanidine treatment were used as the markers for nuclear genotype of the haploid parents, and at the same time the petite form with mitochondrial mutation in globorubermycin resistance was used as the marker for mitochondrial genotype of fusion parents. The protoplast formations of S. cerevisiae and C. utilis were carried out by using cells in early logarithmic phase under the action of snail enzyme in 0.7 M KCl with β-mercaptoethanol or with dithiothreitol respectively. The rates of the protoplast formation in two parents were both 90—99% in 30—60 minutes. The rate of the protoplast regeneration, for S. cerevisiae was 29—35%, for C. utilis 7.5%. The fusion of parental protoplasts was induced by using 35% polyethylene glycol (M. W. 6,000) and 10mM CaCl_2. The fusion strains of nutritional complementation were grown on minimal medium. The fusion frequency was 10~(-5)—10~(-6). These fusion products were found to possess the characteristics of the hybrid. One of the hybrids had obviously higher assimilation ability of D-xylose, cellobiose, glucose and galactose in comparison with the parents.