利用根癌农杆菌(Agrobacterium tum efaciens)介导的转化方法,以紫杉醇产生菌———树状多节孢HQD33的分生孢子为受体材料,分析影响转化效率的主要因素.获得高效的遗传转化体系,转化率最高可达1.3×10-4.PCR检测表明,T-DNA已整合进树状多节孢HQD33的基因组中,而且大多数都是以单拷贝的形式整合,转化子都能够稳定遗传.结果表明,根癌农杆菌菌株类型、菌株生长状态、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要影响.通过对以上因素的分析,为建立紫杉醇产生菌———树状多节孢HQD33稳定的遗传转化体系奠定了基础.
To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%―44.72% higher than that of the parent strains.
ZHAO Kai, PING WenXiang, ZHANG LiNa, LIU Jun, LIN Yan, JIN Tao & ZHOU DongPo Laboratory of Microbiology, College of Life Science, Heilongjiang University, Harbin 150080, China
