Expression And Interactors Screening Of Transcription Factor Ss-FoxE2 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.) de Bary is an important filamentous pathogenic fungus. It can annually cause substantial losses to crop production because of its broad host range and complex life cycle. The scientists around the word are focusing on controlling the disease causing by S.sclerotiorum. The lifecycle of S.sclerotiorum contains asexual and sexual developments. The fungus develops as hyphae and sclerotium in asexual stage. Sexual development in this homothallic fungus is initiated within the sclerotium where multiple apothecia primordia form. Large quantities of ascospores which play an important role in disease epidemiology are released by apothecia during its sexual developmental stage.Our lab found that gene Ss-Ss Fox E2 encoding a transcriotion factor belonging to Forkhead superfamily was essential for apothecia formation. According to this our study was focusing on the gene Ss-Fox E2 expression and screening of its interactors. The experiment results are as following: We found 4 members of Forkhead transcription factor family in S.sclerotiorum genomic DNA database by bioinformatics, they are Ss-Fox1, Ss-Fox E2, Ss-Fox3 and Ss-Fkh1. Multiple sequences alignment and phylogenetic tree analysis showed the ralationship among them and other Forkhead proteins; Ss-Fox E2 encoding 449 amino acids was cloned into p MD18-T vector and it contained an intron; Expression level of Ss-Fox E2 in different stages of S.sclerotiorum was detected by Real-time PCR(q RT-PCR). It indicated that the expression level of Ss-Fox E2 in apothecia is much higer than that in other stages. It would be an envidence that Ss-Fox E2 involved in apothecia formation; To confirm the subcellular localization of the Ss-Fox E2, the co-localization of Ss-Fox E2 and enhanced green fluorescent proteins(e GFP) were analyzed. Ss-Fox E2 was shown to target in the nucleus; Expression vector p ET-28a::Ss-Fox E2 was introduced into E.coli Rosetta(DE3)to express the target protein. The result showed that most of the target protein was insoluble. But some purified protein satisfied with further study can be obtained by Ni-NTA affinity purification; The bait vector p GBKT7::Ss-Fox E2 was constructed successufully. The autoactivation of bait Ss-Fox E2 indicated that it has transcriptional activation ability; 6 candidate proteins interacting with Ss-Fox E2 were obtained through screening of the c DNA library of S.sclerotiorum using Forkhead domain of Ss-Fox E2.