根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。

Establishment of the transformation system of Aspergillus japonicus mediated by Agrobacterium tumefaciens

Abstract: Objective]n order to select Aspergillus japonicus mutant strains that express high-yield of glycerol oxidase, we constructed a mutant library of Aspergillus japonicus that mediated by Agrobacterium tumefaciens. Methods]Tri-relative hybridization experiments were performed to transfer binary vector pBI-hphII into Agrobacterium tumefaciens EHA105, which was used as the infective strain and Aspergillus japonicus was used as the host strain. We established the transformation system of Aspergillus japonicus mediated by Agrobacterium tumefaciens and constructed a mutant library. At the same time, we analyzed the factors affecting the transformation efficiency such as the concentration of Agrobacterium tumefaciens, adding and not adding Acetosyringone, co-culture time, co-culture temperature and so on. Results]The results of PCR detection and Southern analysis showed that the T-DNA was integrated into genome of the Aspergillus japonicus. All of the nine randomly selected transformants were quite stable after ten rounds of successive cultures. Conclusion]The transformation system is a good basis for selection of Aspergillus japonicus mutant strains which give a high-yield of glycerol oxidase.