酵母嗜杀质粒融合转移──Ⅰ．融合转移系统的建立

TRANSFER OF DILLER PLASMID BY PROTOPTLAST FUSION IN YEAST- Ⅰ. ESTABLISHMENT OF TRANSFER SYSTEM

以嗜杀酵母ＥＲＲ１和啤酒生产酵母ＡＳ２４２０出发菌株，建立了供体菌ＭＫ２－３：Ｋ＋Ｒ＋Ｌｅｕ－ρ＋（ｎ）和受体菌ＡＳ２４２０－１：Ｋ－Ｒ－ｌｅｕ＋ρ°（２ｎ）．对原生质体制备再生条件的研究结果表明两株菌的原生质体制备条件不尽相同；同一菌株原生质体形成与再生的最佳条件也不相同．有利于融合再生的原生质体制备条件是ＭＫ２－３：菌龄３０ｈ，蜗牛酶解量３０ｇ／Ｌ酶解时间３０ｍｉｎ，此时原生质体形成率为８０％，而再生率达１７．１％，这些条件对嗜杀活性无影响，再生菌落的嗜杀活性率达１００％；而ＡＳ２４２０－１；菌龄２４ｈ，蜗牛酶量２０ｇ／Ｌ，酶解时间３０ｍｉｎ，原生质体形成率为８１％，再生率为１８．１％．对四种渗透压稳定剂的再生效果实验表明甘露醇效果最佳，氯化钾次之，考虑到廉价，氯化钾是很好的代用品，在３０％ＰＥＧ６０００及Ｃａ２＋条件下进行原生质体融合，融合率可达８．９×１０－６，对其中的融合子ＭＡＲ４的遗传性状及嗜杀活性的稳定性检测，ＤＮＡ含量及细胞大小测定，ｄｓＲＮＡ质粒的提取及电泳结果分析和发酵性能测定，结果表明融合子ＭＡＲ４具有较高的嗜杀活性并可以稳定遗传，有与供体菌ＭＫ２－３嗜杀质粒ｄｓＲＮＡ相同的电泳行为，?

The donor MK2-3: K+ R+leu- ρ°(n)and the recipient AS2420-1: K-R-len+ρ° were established from killer yeast ERRI and beer-producing yeast AS2420 respectively. The recipient had no any chromosomal marks and their polypoid cells were used directly. The results shoWed that the optimum conditions of protoplast preparation were not completely same for the two strains. And for the same strain the optimum conditions were quite different between protoplast preparation and regeneration, without affecting the killing ability. All of the regenerative colonies had the killing ability. In the peresence of PEG(6000) and Ca2+,two kinds of protoplast were fused. The fusion frequency was 9. 8×10-6. By testing the stabitity of genetic character and killing ability,contents of DNA per cell and average size of cells, extraction and electrophoretic detection of dsRNAs killer plasmid and properties of fermentation, it was found that the fusant MAR. had a stable high killing ability,its dsRNAs electrophoretic result was the same as that of donor MK2-3,and the properties of fermentation were close even superior to those of the recipient AS2420-1.