The Transcriptional Activity And Regulation Mechanism Of Regulated Gene StMR1 In Melanin Biosynthesis Pathway Of Setosphaeria Turcica

Northern Leaf Blight of Corn(NLBC) is a serious leaf disease of maize, which directly impact on corn production, causing severe economic losses. Setosphaeria turcica, the pathogenic fungus to induce NLBC, can produce some vital pathogenic factors including toxins, enzymes and melanin. As a result, researching on biosynthetic regulation mechanism of melanin is of significance to identify pathogenic mechanism of S.turcica. In our previous work, a transcription factor St MR1 that regulates the biosynthetic pathway of melanin was isolated and identified. And in this paper, 1) its transcriptional activity and toxicity on yeast cells are analyzed via yeast self-activated experiments; 2) by constructing GFP labeled over-expression vector, subcellular location was investigated; 3)Chromatin Immunoprecipitation was utilized to analyze its molecular mechanism in regulating melanin synthetases. The results are shown as follows:1. The bait vector of St MR1 gene expression was constructed, and high transcriptional activation and little toxicity to yeast cells were showed through transcriptional activation analysis;2. Over expression vector of St MRl gene was constructed based on GFP labeled p BARKS1 plasmid and by PEG mediating method, S. turcica strain 01-23 was transformed.; Finally, an over-expression mutants were obtained and laid foundation for the next function research;3. By observing the fluorescence images of express site of GFP labeled St MRl in the over-expression mutant, it was confirmed that the subcellular was located in nucleus which accorded with the characteristics St MR1 gene as a transcription factor to regulate target gene expression by acting on its promoter region;4. Investigating the conditions of chromatin immunoprecipitation of S. turcica.With conditions controlled as: 1% formaldehyde, crosslinking time 30 min, 80 W 5 s ultrasonic/10 s interval for 2-3 min. Under this condition, chromatin pieces were broken into 200~1000 bp, suitable for downstream test and laid foundation for researching transcription factors;5. Upstream promoters about 2000 bp base sequences of melanin biosynthesis enzyme genes were analyzed by NNPP and softberry. Transcription factor binding siteswere predicted in them;6. Chromatin immunoprecipitation was used to analyze the St MR1 genes for six melanin biosynthesis enzyme genes regulating role, it indicated that St MR1gene-for-gene regulation should be existed.