Selection Of Medium For Aspergillus Ochraceus And Roles Of AopacC Gene Involved In Regulation Of Growth And Toxin Biosynthesis

Ochratoxin A(OTA) is a secondary metabolites mainly produced by Penicillium spp. and Aspergillus spp, and widely distributed in grain, fruits, feedstuff, coffee and animal products. OTA is harmful to human health and food safety, and it was listed as potential carcinogen(class 2B) by IARC. Better understanding of the regulation of OTA biosynthesis is the key to effective prevention and control of OTA contamination in agricultural and horticultural commodities. As a key factor in p H regulatory pathway,Pac C participate in a variety of biological processes in addition to the regulation of mycotoxin biosynthesis. However, little is known about Pac C regulatory pathway in Aspergillus ochraceus. In this work, growth characteristics of Aspergillus ochraceus in different medium and the biological functions of Aopac C gene involved in p H pathway were investigated. The main results were as follows:(1) The OTA production and mycelium growth in Aspergillus ochraceus is quite different when grown in solid medium, liquid medium and the natural medium. YES medium is the optimal toxigenic-producing and developed mycelium medium in the solid medium, and the OTA content was 36.09 ng/mm2 medium. YES medium is suitable for research of physiological characteristics of Aspergillus ochraceus. The highest toxin-producing medium in the natural medium is corn grit medium with OTA content of 251.67 μg/g. The corn medium was suitable for research of toxicology and toxin-producing feature. Methanol extraction was suitable for solid medium because of its convenient and high recovery. Chloroform was selected for extraction solvent in liquid medium.(2) We obtain the Aopac C mutants with the method of homologous recombination and protoplast transformation mediated by PEG. Three Aopac C mutants: ⊿Aopac C22-1,⊿Aopac C-3, ⊿Aopac C-5 were obtained by transformation of target fragment in protoplast and selection of hygromycin resistance. Three Aopac C mutant were un-ectopic mutants by PCR identified and these can be used for function analysis of Aopac C gene.(3) Compared with the wild type, ⊿Aopac C showed some phenotypic defects ingrowth, toxin production and pathogenicity. We predicted that Aopac C involved in the regulation of growth, toxin production and pathogenicity, and this regulation may involve in p H-dependent. The relative expression of global regulation factor, the synthesis genes and regulation genes in OTA biosynthesis gene cluster were studied.Compared with the wild type, the synthesis genes and regulation genes were inhibited in⊿Aopac C. However, the expression analysis of the global regulatory factors showed some differences in different p H. This article presume that the regulation function of Aopac C on OTA is realized by the regulation of related genes. In addition, p H had no significant effect on the spores production and mycelium, but it had effects on growth,OTA production and gene expression. The best growth and maximum OTA production of Aspergillus ochraceus achieved at p H6.0.