Cloning and characterization of two glutathione S-transferases cDNAs in Foc4 and expression under exogenous oxidative stress

Abstract

In order to identify two putative glutathione S-transferase (GSTs) genes in Fusarium oxysporum f. sp. cubense race 4 (Foc4), cDNA sequences of the entire coding regions of the two genes were cloned from Foc4 using RT-PCR method. Subsequently, the two genes were named Fogst1 and Fogst2 respectively. The length of open reading frame of Fogst1 was 609 bp and encoded a protein including 202 amino acid residues, Fogst2 possessed an open reading frame with 693 bp which encoded a 230-amino acid protein. Phylogenetic analysis showed that Fogst1 belonged to sigma (σ) subtype members of the GSTs superfamily, and Fogst2 was a new member of an unknown subfamily in the GSTs superfamily. To verify the expression of Fogst1 and Fogst2, the recombinant prokaryotic expression vector pET28a-Fogst1 and pET28a-Fogst2 were constructed and transformed into Escherichia coli expression strain BL21(DE3). The soluble recombinant proteins Fogst1 and Fogst2 were obtained after being induced by IPTG. GSTs activity assays showed that both of the two recombinant proteins had specific activity with CDNB. For real time RT- PCR analysis, the mycelium samples of Foc4 were collected after treatment by H₂O₂ for 1, 5, 12, 24 hours. The results showed that the expression of Fogst1 and Fogst2 were significantly up-regulated in the first 5 hours, and then decreased and returned to normal level. These results suggested that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.