双亲灭活米曲霉原生质体融合中原生质体制备的研究

Protoplast preparation in fusion of inactivated parental protoplasts of Aspergillus oryzae

对双亲灭活米曲霉原生质体融合育种中原生质体制备的条件进行了研究,将菌丝的预处理与细胞壁的酶解合为一步,并讨论了DTT的加入量。结果表明,原生质体制备的最佳破壁酶为纤维素酶、溶壁酶、蜗牛酶3种酶混合浓度比为5:3:1;菌丝培养15h;酶解时加入3mol/L DTT;酶解2.5h;0.8mol/L NaCl作为渗压稳定剂。所得双亲菌株原生质体的融合率为3.31%。

The conditions for protoplast preparation in the fusion of inactivated parental protoplasts of Aspergillus oryzae were studied in the article. The pretreatment ofmyeelium and the enzymatic hydrolysis of cell wall were combined into one stage. The addition amount of DDT was discussed. The results showed that the optimal enzyme for cell wall broken in protoplast preparation were the mixture of eellulase, lywallzyme and snailase, which was mixed at 5：3：1. The myeelium culture time was 15h. DDT was added at 3 mmol/L during 2.5 h hydrolysis, in which 0.8 mol/L NaCI was used as osmotic stabilizers. The fusion rate of the inactivated parental protoplasts was 3.31%.