米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建

米曲霉对潮霉素等多种抗生素不敏感,对其基因改造有一定困难。该研究以米曲霉3.042为出发菌株,建立pyrG筛选标记遗传转化体系。首先,分别运用紫外诱变法和左右臂同源重组法破坏米曲霉自身的pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,最终两种方法分别获得性状稳定的尿苷/尿嘧啶营养缺陷型突变株米曲霉O11和米曲霉g-1。随后以缺陷型突变株为出发菌株,利用农杆菌转化法转入包含pyrG回补标记的质粒,其中米曲霉g-1突变株获得原养型的回补菌株,紫外诱变法获得的缺陷型突变菌株米曲霉O11未能恢复为原养型。研究表明米曲霉3.042 pyrG基因大片段缺失的缺陷型菌株,可直接以自身pyrG基因作为选择标记进行遗传改造。

Construction of Aspergillus oryzae 3.042 Uridine/Uracil Auxotrophy Genetic Transformation System

Since Aspergillus oryzae(A.oryzae)are nonsusceptible to multiple antibiotics such as hygromycin,it is difficult to genetic modification on them.A.oryzae 3.042 was used as the parent strain to establish pyrG selection genetic modification system.Firstly,A.oryzae pyrG gene was destroyed through ultraviolet mutagenesis and homologous recombination method.Stable uridine/uracil auxotrophic mutants A.oryzae O11 and A.oryzae g-1 were obtained on medium containing uridine/uracil and 5-fluoroorotic acid.Then the mutants were used to transform the plasmid containing pyrG selectable marker,so as to obtain prototrophic complemented strains.The results showed that the mutant A.oryzae g-1 obtained from homologous recombination could successfully recover to be prototrophic,while the mutant A.oryzae O11 obtained from mutagenesis didn′t recover to prototrophic strains.This study demonstrated that uridine/uracil auxotrophic strains harvested from pyrG disruption in A.oryzae 3.042 could be directly used to genetically modify with its own pyrG gene as selectable markers.