Construction Of Aspergillus Oryzae Uracil Auxotrophy Strain By Seamless Knockout

Aspergillus oryzae is widely used in the food industry because of its strong capacity of producing enzymes.In China,A.oryzae 3.042 is mainly used as a fermentation strain.Due to its multi-core and polyspore characteristics,research progress in genetic breeding in the past three decades has been difficult.In this study,homologous recombination was used to knock out the pyrG marker gene of A.oryzae to obtain stable inheritance of A.oryzae 3.042 uracil auxotrophic strains,namely strains T4,T25,and T35.It lays the critical foundation for the subsequent molecular biological operation and genetic breeding of A.oryzae 3.042.Due to the multinucleate nature of A.oryzae spores,the mutants screened by traditional methods could not be inherited stably.As a consequence,in this paper,we used A.oryzae 3.042-3 obtained by our group from A.oryzae 3.042 in the past as a transforming recepter,which can produce mononuclear spores stably.In order to achieve the goal of seamless knockout,this thesis first constructed a targeting vector YEplac 195-PU-PD,which seamlessly connected the upstream and downstream fragments of the pyrG gene of A.oryzae 3.042,that enabled homology to the genome to prevent the introduction of exogenous gene fragments.The plasmid YEplac195-PU-PD was double-digested to obtain the fragment PU-PD.A.oryzae 3.042-3 was used to prepare protoplasts,and the resulting fragment PU-PD was introduced.35 transformants were screened with 5-FOA resistant plates at a concentration of 1.4 g/L,and were inoculated on MM medium and SM medium,respectively.Passage uracil dependence verification was performed on them,and the three best performing ones were eventually selected.Transformants 4,25,and 35 were validated for 5-FOA resistance and proved that they can be inherited phenotypically.At the same time,they were verified at the gene molecular level,and the results of PCR proved that the pyrG gene had been successfully knocked out.DAPI staining of the nucleus showed that the three transformants all showed the characteristics of mononuclear.As have discussed above,the A.oryzae 3.042 uracil auxotrophic strains were successfully constructed and named as strains T4,T25,T35.The pyrG gene was amplified from wild-type A.oryzae 3.042,and the complementary plasmid pMD19-T-pyrG was constructed and transformed into three screened strains T4,T25,T35.It could be observed that all three strains returned to wild-type state,that is,recovered from their ability to produce uracil.Therefore,these three strains T4,T25,and T35 can be used as receptors for the transformation system of the pyrG marker gene.