Research On Expression And Partners’ Analysis Of Transcription Factor SsMADS1in Sclerotinia Sclerotiorum

Sclerotinia sclerotiorium (lib.) de Bary is a typical homothallic fungus, belongingto Ascomycota, Pezizomycotina, Helotiales, Sclerotinia. S.sclerotiorium, with broad-spectrum host range, which can infect more than450species of plants, is a much moreimportant plant pathogenic fungus. The sclerotium disease caused by S.sclerotiorium,will cause great economic loss to the agricultural economy in our country each year.Therefore, the related research of S.sclerotiorium caused widespread concern of plantpathologists.As an important of transcription factors in eukaryotic organisms, MADS-boxprotein family genes widely exists in living organisms and are involved in the regulationof multiple cellular functions, including cell identification, cell metabolism, cell cycle.According to our previous studies, the SsMADS1gene, the S.sclerotiorium MADS-boxprotein family gene, is involved in regulating growth and virulence in S.Sclerotiorium.In order to identify clearly the functions of the SsMADS1and its interactionprotein in the growth and the pathogenicity in S.sclerotiorium, this thesis focuses on thestudy of function mechanism and the interacting network system of the SsMADS1. Thepurpose of this thesis is to provide theoretical basis for the further study on thepathogenic mechanism of S.sclerotiorium and scientific basis for its effectiveprevention and treatment by means of identifying the subcellular localization and theinteracting protein research.1. SsMADS1prokaryotic expression and purified. Having gained the completeopen reading frame of SsMADS1by the reverse transcription of RT-PCR, this gene is681bp in size, encoding226amino acids,25kDa in the molecular weight,6.16in theisoelectric point. The construction of the prokaryotic expression vector and induced byIPTG make SsMADS1fusion protein expressed greatly in the expression host. Bymeans of the Ni-NTA Agarose affinity chromatography, a large amount of purified SsMADS1fusion protein is obtained.2. SsMADS1antiserum preparation. With a large number of purified SsMADS1fusion protein as antigen, the immune function through its own experimental animalpromotes them to produce antiserum. According to the results of the Immuno-doublediffusion of agar, indirect ELISA and Western-blot experimental methods for titerdetermination, it shows that the antiserum and the fusion protein of SsMADS1bindsvery well. The antiserum builds a solid foundation for the further sub-cellular locationand future experiments.3. The analysis of the sub-cellular localization of SsMADS1. Predicted bybioinformatics analysis, the gene is localized in the nucleus. The characteristics of theprepared antiserum and protein binding specificity, total protein, S.sclerotioriumcytoplasm total protein, and S.sclerotiorium cell wall protein were extractedrespectively, using Western-blot method to detect, results showed that total protein,S.sclerotiorium cytoplasm total protein were specific. With the cell wall protein,S.sclerotiorium in no specific bands, namely, the protein in the cytoplasm. On the otherhand, through the design of primers and specific PCR amplified SsMADS1gene,combined with subcellular localization are constructed in our lab to build target genecarrier, subcellular localization vector. By using the method of Agrobacterium mediatedtransformation vector, will be constructed into tobacco, observation and locationinformation in a fluorescence confocal microscope found that the gene localization tothe nucleus.4. The screening of the interacting protein of SsMADS1. Using Bioinformatics onthe analysis on Domain interaction prediction and STRING shows that SsMADS1mainly interacts with protein-synthase, serine-synthase, and protease. On the other hand,screening SsMADS1interacting protein by means of His-tag Pull-down, combiningSDS-polyacrylamide gel electrophoresis separation and temperament linkage detectionand identification phase, the thesis further screens and identifies SsMADS1interactingproteins. 5. The construction of yeast two-hybrid GAL4system. To further study theinteraction with SsMADS1protein, we constructed the SsMADS1of yeast two-hybridGAL4system. And we verify that construction of yeast two-hybrid GAL4system withno self activation can proceed to the next step of the experiment by means of thedetermination of toxicity and self activation detection. It provides the basis for furtherscreening of S.sclerotiorium cDNA library and the verification of protein interactionnetworkIn conclusion, in this study we purifies transcription factor SsMADS1, and getsthe Antiserum with good specificity of the protein successfully. We predict and screenthe interaction proteins of SsMADS1with the method of Bioinformatics and thetechnology of Pull-Down. Meanwhile, we construct the yeast two-hybrid GAL4systemin order to further validate the regulatory network of the transcription factors. At thesame time, by using fluorescence sub-cellular localization and Western-bolt, we provethat the gene is localized to the nucleus. The results show that: SsMADS1is localizedin the nucleus. The regulation of network, which is constructed by the interaction ofprotein synthase, serine synthetase, and protease, phosphoinositide regulation, generegulation, and a variety of other transcription factors in many aspects co-participate inthe growth and pathogenicity of the fungus, which build a solid foundation for thefurther study of regulatory network and the growth and pathogenicity of theS.sclerotiorium.