Functional Analysis Of STK2 Gene In Mitogen-activated Protein Kinase Cascade From Setosphaeria Turcica

Northern Leaf Blight of Corn, caused by Setosphaeria turcica, is one of the most important diseases in corn production area. MAPK, the universal signal tranduction pathway in fungi, is one of the key protein kinases in regulating growth, development and pathogenicity of fungal pathogens. Function analysis of STK2 gene was explored by constructing the gene replacement vector, which was used to create gene mutants. As a result, this gene was involved in conidiation, appressorium, pathogenicity and secondary metabolism, respectively. This work will be helpful to study and understand the function of other conserved genes in signal transduction pathway of S. turcica.A 1 951 bp for the flanking sequences of 5'and 1 062 bp of 3'have been obtained through genome walking strategy. Two CAAT boxes were found in 356 bp and 1 562 bp before start codon through the software analysis of Softberry and NNPP. TATA box was not been found. The corresponding structures and functions of STK2 were predicted by the bioinformatic software.The STK2 gene replacement vector was constructed with the gene homologous combination theory and PEG-mediated gene transformation system. Transformants were screened by hygromycin B and PCR (polymerase chain reaction) with specific primers corresponding to hygromycin phosphotransferase gene and STK2 gene. One STK2 gene-disruption mutant, named asΔstk2, was obtained by Southern blot analysis performed with the DIG-labeled hph gene. MutantΔstk2 showed a higher vegetative growth rate than wild type, the mutantΔstk2 exhibited white colony, dense mycelia, cell lysis in the center colony, evident reduction of HT-toxin activity, sporulation and pathogenicity-decreasing. The mutantΔstk2 fail to form appressoria on onion epidermis. Moreover, the wild type was more tolerant than mutantΔstk2 under the osmotic stress.The ORF of STK2 was amplified by PCR, and then the fragment was inserted into shuttle plasmid pPIC9K, The recombinant plasmid pPIC9K-STK2 was transformed into DH5α, positive clone was screened by PCR and sequencing, and then plasmid pPIC9K-STK2 was extracted and transformed into Pichia pastoris GS115 by electroporation after linearization, recombinant with stable expression was screened by G418. The recombinant was identified by PCR analysis and sequencing, then induced the recombinant by methanol. The expression products were analyzed by SDS-PAGE. This research laid a foundation for further Western Blot analysis and activity detection of the protein.Homologous DNA fragments of STKK2 were obtained by polymerase chain reaction(PCR)amplification from degenerated primer designed on the basis of the conserved amino acid regions of MAPKK from other fungi. Southern blot results showed STKK2 gene had single copy in the genome of S. turcica.