Studies On The Karyotype-related Genes Of Conidia Of Filamentous Fungi And Construction Of Genetic Markers For Aspergillus Oryzae

Aspergillus oryzae is an important production strain in the food fermentation industry and has a long history of safe use.It is also an important strain for industrial production.It has good protein secretion and production capabilities and is used to produce commercial enzymes and secondary metabolites.The spores of A.oryzae have different karyotypes,most of them are multinuclear,and a few are mononuclear,which makes it difficult for strain modification and industrial breeding,because the multinuclear characteristics will dilute the phenotypic effects of genetic manipulation,and heteronuclear transformants can reduce the expression level of recombinant proteins.In previous researches,our laboratory has established a separation technology to isolate and enrich its mononuclear and multinuclear populations using A.oryzae FS1125.And 45 differentially expressed proteins between uninuclear conidia and multinuclear conidia were screened by two-dimensional electrophoresis.Then these proteins are identified by using protein spectrum technology and preliminary analysis of the bioinformatics.Based on the above background,this study intends to explore the biochemical mechanism and genetic background of the karyotype change in Aspergillus oryzae.As a model strain of filamentous fungi,the study results of Neurospora crassa can be applied to other filamentous fungi,and conidia of Neurospora crassa also have a variety of karyotypes.In this study,defective strains of the corresponding genotype of Neurospora crassa were used to screen the genes encoding these differentially expressed proteins.Fluorescence microscopy and blood cell count were used to analyze the spore karyotype and number of each deficient and wild strain under the same culture conditions.The results showed that four defective strains with significant differences from the wild strain karyotype were obtained,namely F187,F188,F192,and F193.Therefore,the corresponding genes of these four deficient strains were subsequently selected for further compensatory verification in Neurospora crassa.Neurospora crassa has different mating types,F187 and F188 are different mating types of the same gene(npc)knockout strain,F192 and F193 are also different mating types of the other same gene(ftt)knockout strain.To verify the function of the two genes,complementary verification is required.In this study,the upstream and downstream flanks of the csr-1 gene were connected on both sides of the expression box of the corresponding gene,and the replacement vectors for the npc gene and the ftt gene were constructed respectively.The target gene was inserted into the csr-1 site by gene recombination.After replacing the csr-1 gene with the target gene,the complemented strains was conferred resistance to cyclosporine,and the complemented strain was screened successfully by cyclosporine.After gene replenishment,the growth rate and the cellulase activity of filter paper were measured and compared among the wild strains,replenishing strains and defective strains.The results showed that the growth rate and cellulase activity of the defective strains were significantly different from those of the supplemented strains and wild strain,while there was no significant difference between the supplemented strains and wild strain,indicating the growth rate and the cellulase activity of the supplemented strains were phenotypic functional recovery.These genes need to be knocked out in Aspergillus oryzae at a later stage to verify whether these gene have a significant effect on the karyotype distribution of Aspergillus oryzae..Hence,it is necessary to develop a genetic transformation system suitable for Aspergillus oryzae,and construct screening genetic markers suitable for Aspergillus oryzae.Studies have shown that overexpression of erg1 gene,a key enzyme encoding ergosterol synthesis,can develop resistance to the antifungal drug terbinafine.In this study,the resistance of Aspergillus oryzae to terbinafine was tested,and the minimum inhibitory concentration of terbinafine against Aspergillus oryzae was determined to be 0.8?g/m L.The constitutive promoter pgkA was used to drive the erg1 gene overexpression to form a selectable marker,and the upstream and downstream homologous sequences of pyrG were ligated on both sides of erg1 gene expression box,and finally linked to the skeleton to construct a plasmid.Using this plasmid as a template,two split fragments were constructed according to the principle of targeted knockout technology and transform to Aspergillus oryzae,and the doublescreening markers strain of terbinafine resistance and uracil auxotrophy was obtained.The above results lay the foundation for subsequent genetic manipulation in Aspergillus oryzae.