泡盛曲霉抗FOA突变株的筛选与转化

根据泡盛曲霉SG1菌株分生孢子的紫外线致死曲线,选择死亡率为85%～90%的诱变时间诱变分生孢子,然后将其涂布于含FOA(5-flourooroticacid)和尿嘧啶核苷的基本培养基上,选择抗FOA的突变株。经过纯化和回复突变检测后,获得了5株需要尿嘧啶或尿嘧啶核苷才能在基本培养基上生长的稳定突变株。进一步分析鉴定结果表明,这些突变株的URA3基因发生了突变。Northern杂交及RTPCR方法证明这些突变株中URA3基因突变均发生在转录水平上。选择突变株SA5作为受体菌,用含来自黑曲霉的野生型URA3基因的质粒转化该受体菌,结果获得了稳定的转化子。Southern杂交证明野生型URA3基因取代了突变株的ura3基因。

CHARACTERIZATION AND TRANSFORMATION OF ANTI-FOA STRAIN OF ASPERGILLUS AWAMORI

A suspension of freshly harvested spores of Aspergillus awamori SG1 strain was exposed to UV light. The irradiated spores with survival rate of 10%-15% were plated directly on minimal medium (MM) agar plates containing 10 mmol/L uridine and 5-fluoro-orotic acid (FOA 1 mg/mL). Five stable anti-FOA colonies with the reversed rate below 10(-9) were obtained. All anti-FOA strains, can grow on uridine or uricil-containing minimal medium agar plate but not on orotic acid-containing MM plate, indicating that the mutation happened in the URA3 gene. Northern blotting analysis showed that the mutation of URA3 gene could not be transcribed and it was confirmed by RT-PCR. With modified PEG-mediated protoplast transformation method the wild URA3 gene could complement the ura3 mutant strain SA5 to wild type. Southern blotting of the transformant indicated the wild RUA3 gene replaced the mutated ura3 gene.