Verification Of Interactors Of Transcription Factor Ss-FoxE2 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is a cosmopolitan necrotrophic fungal pathogen with a broad host range.Sclerotia of S.sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae which can remain active for more than eight years in the soil and resist physical,chemical,and microbial destruction.In the case of suitable conditions,sclerotium can germinate to produce apothecium.The release of the ascospores from the ascospore as a source of infection will lead to a new round of disease.So the Sclerotinia sclerotiorum is a very difficult disease to prevent and control and resulting in huge economic losses to agriculture.Previous studies have shown that induction of sclerotia for sexual development,apothecia were not formed in Ss-Fox E2 knock-out mutant.The result indicate that transcription factors Ss-Fox E2 appears to be necessary for the regulation of sexual reproduction.Nine candidate proteins interacting with Ss-Fox E2 were obtained through screening of the c DNA library of S.sclerotiorum.On the basis of this study,we verify the candidate interaction proteins by Yeast two-hybrid assay and Bimolecular fluorescence complementary assay and try to analysis how transcription factor Ss-Fox E2 regulate the downstream target sequence so as to establish the interaction mechanism of transcription factor Ss-Fox E2.The ORF of candidate gene was constructed on yeast two-hybrid AD vector and the vector with Ss-Fox E2 was co-transformed into the yeast strain.There are six co-transfer plasmids that can activate the downstream reporter gene,and no self-activation occurs.It was shown that six proteins interact with the Forkhead domain of the transcription factor Ss-Fox E2.And then the ORF of 9 candidate genes and the ORF of Ss-Fox E2 were constructed on bimolecular fluorescentcomplementary vector p SAT4-c YFP/p SAT4-n YFP.Under the irradiation of excitation light,four interacting proteins interact with the transcription factor Ss-Fox E2 emit yellow fluorescence.Therefore,four proteins interacting with the transcription factor Ss-Fox E2 were verified by yeast two-hybrid validation and Bi FC experiments.Chromosome immunoprecipitation assay is the main technique involved in the study of the interaction between the transcription factor Ss-Fox E2 and the promoter region.The technology is more difficult,high quality control requirements,so we explored the key control points in the Ch IP assay.Ultrasonic crushing conditions are explored to 20% of the power,the use of 2mm diameter ultrasonic probe,2s work,2s intermittent working state,the total ultrasound time 8-10 minutes,the degree of chromosome DNA break can reach 500-1000 bp.A polyclonal antibody with an ELISA titer of 1: 50 k and capable of specifically binding to the transcription factor Ss-Fox E2 was prepared to lay the foundation for stabilizing experimental conditions,obtaining reliable data and conducting further in-depth research.The above conclusions provide the basis for the further study of the function of Ss-Fox E2 interacting protein and the further research of regulatory network.