黑曲霉原生质体电融合研究

Electrofusion of Protoplasts from Aspergillus niger

以NTG和UV分别对生淀粉糖化酶菌株——野生型黑曲霉N-11,N-16(Aspergillus niger)进行诱变处理获Lys^-和Arg^-两类营养缺陷型。采用纤维素酶和蜗牛酶复合酶处理其菌丝并探讨了酶浓及菌龄等因素对原生质体形成和再生的影响。以BAEKON-2000型基因转移仪将上述营养缺陷型进行原生质体电融合处理。产生高融合率的最佳参数为:脉冲幅度4.5kV,脉冲数32,脉宽62.5μs,电激时间0.2s,距离3mm,周期数10。融合率可达60%。经连续传代7次获得4株稳定的融合子,经孢子体积,核DNA含量,生长速度测定证明上述融合子是种内杂合二倍体。以PFA处理得稳定的单倍体分离子。

Wild-type strains of Aspergillus niger N-11 and N-16 (Raw-starchdigesting glucoamylase producers) were mutagenized by UV and NTG respectively. A. niger auxotrophs No.2 (lys^-) and No. 3 and No.5(Arg^-) were obtained and used as direct parents. The auxotrophs were treated by a combined enzyme system containing cellulases and snailases. The effect of factors, including the concentration of enzymes, age of myselium on the formation and regeneration of protoplasts in these auxotrophs werer studied. This work systematically investigated the condition of electrofusion on the protoplasts of the mutants using BAEKON-2000 Advanced Gene Transfer system, an optimal yield of hybrids able to form colonies on MM/CM was found at A=4.5kV/cm, Np=32, Tp=62.5μs, Tb=0.2s, Cy=10, D=3mm and Ff (fusion freqency)=60% of the fusants selected statistically were stable and most of them show the increse in the active unit of enzyme.