Cloning,prokaryotic expression and expression analysis of StSse19 gene,an effector of Seosphaeria turcica
Northern leaf blight of corn is a fungal disease caused by Seosphaeria turcica.In the primary research of the transcriptome analysis of the early infection stage of maize by S.turcica showed that the effector StSse19 was a significant pathogenic factor.However,its structure and function have not been reported.Analysis of gene expression pattern during infection of maize by the pathogen showed that the expression level of StSse19 peaked after 24 hours of incubation,followed by a decrease at 72 hours.In the present research,we cloned the specific effector StSsel9 using the cDNA of S.turcica wild-type strain 01-23 as the template and analyzed its structure using bioinformatics technology.The prokaryotic expression vector pET28a-StSse19 was constructed and the target gene was induced by IPTG to express in Escherichia coli BL21(DE3).Further,the target protein was confirmed by SDS-PAGE analysis.Moreover,qRT-PCR was employed to investigate the expression level of the StSse19 gene during infection periods.The results revealed that the CDS of the specific effector,StSse19 was consisted of 330 nucleotides,encoding a protein of 109 amino acids.Sequence comparison showed that this protein was unique in S.turcica as it has no homologue compared to other fungus.The results indicated that this gene played a key role in the process of pathogen infection.Furthermore,the StSse19 protein was 28 kD.The purified target protein was verified through purification by Nickel column chromatography and identification by Western blot.This study will not only clarify the structural characteristics of the StSse19 and its expression pattern during the infection stage of the pathogen,but also lay the foundation for further understanding its functions in S.turcica.
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