Functional Characterization Of FGSG₁0666 Gene In Fusarium Graminearum

Fusarium head blight （Wheat scab） is a kind of fungal disease for wheat and barley, which lead to serious decline in yield and quality. Mycotoxins produced by the pathogen, Fusarium graminearum, include deoxynivalenol （DON） and its derivatives,3-acetyl deoxidization （3-ADON） and 15-acetyl deoxidization （15-ADON）. Cereal products contaminated with mycotoxins cause serious harm to humans, animals and plants. In order to control the occurrence of wheat scab effectively, it is necessary to find pathogenesis-related genes and reveal its pathogenic molecular mechanism.In this study, RNA-Seq was applied to find differential expressed genes between wild type PH-1 and three mutants of Fusarium graminearum. Compared with wild type,541 genes were up regulated and 1116 genes were down regulated in â–³fgcpka; and 1729 genes were up regulated and 2062 genes were down regulated in â–³mgvl; also,503 genes were up regulated and 796 genes were down-regulated in â–³fgac.â–³fgac is the deletion mutant of adenylate cyclase gene, the result of differential expression showed that the FGSG₁0666 gene almost ceased to express after the gene was knocked out. KEGG analysis revealed that it was involved in steroid biosynthesis. In this study, we choose the gene as research object to analyze its function through gene deletion strategy.The Split-marker method was applied to construct the deletion cassette of the FGSG 10666 gene. The primers were designed according the sequence in the Fusarium Genome Database and the PCR products were transformed into the protoplast of wild-type PH-1 by the PEG-mediated transformation method. Transformants were preliminarily selected through medium containing hygromycin and then the knock-out mutants were confirmed by PCR using positive and negative primers.Compared with the wild type, the colony of FGSG 10666 deletion mutant was pinkish and its mycelium was loose. There was no significant difference between mutant and wild-type PH-1 in growth rate, conidium morphology and amount. The result of tomato infection experiment showed that deletion of FGSG₁0666 gene did not reduce in virulence. However, the result of temperature sensitive experiment showed that the mycelium of the mutant were thinner and had the trend of the fracture when cutured whether at 25℃ or 32℃ in TB3 liquid medium.The deletion mutant of FGSG₁0666 gene and the preliminary analysis on the mutant obtained in this research will provide a basis for further study the function and the pathogenicity association of the gene.