Molecular Analysis of the Genes Responsible for Catalysing Intracellular Steps of Aurofusarin Biosynthesis in Fusarium culmorum

Abstract

Abstract Fusarium culmorum produces polyketide-structured aurofusarin and its precursor metabolites. Aurofusarin biosynthetic pathway is catalysed by the products of eleven genes located in gene cluster. In this study, the effects of the expression levels of Pks12, Gip6, and Gip7, responsible genes for the intracellular steps of the pathway, on pigmentation were investigated in an F. culmorum field isolate. Presence of three genes were determined via PCR and sequencing. Their expressions were investigated by qPCR at 48th, 72nd, 120th and 168th hours. Aurofusarin and rubrofusarin production were verified by HPLC at the same time periods. Mycelial pigmentation, which was initially white, turned yellow, then carmine red during the seven-day culture. Pks12, Gip6, Gip7 genes with 1282, 449 and 1278 bps had sequence similarities (86,6%, 99,4% and 80,5% respectively) with the genes of reference (FcUK99). The ΣCp values of all genes ranged from 29th to 32nd cycles, whereas the Σ∆∆Ct values were calculated between 9,59 E-02 and 1,30 E-02. Relative quantification revealed these genes were controlled by down-regulation compared to the β-tubulin expression. Aurofusarin quantities ranged from 2,355 ppm to 27,350 ppm between the 72nd and 168th hours whereas the yield of rubrofusarin decreased from 0,098 ppm to 0,063 ppm. (4) Conclusion: This study becomes first report for both investigating the expression levels of the genes responsible for the catalysis of intracellular steps of aurofusarin biosynthesis in F. culmorum, and for examining the relationship between gene expression and mycelium pigmentation.

Keywords

• Fusarium culmorum 1
• aurofusarin 2
• mycelial pigmentation 3
• qPCR 4
• HPLC-DAD 5.