| The filamentous fungus Setosphaeria turcica is the causal agent of Northern Leaf Blight of Corn disease and its appearance and overspread frequently cause serious economic losses. The cAMP signal tranduction pathway plays an important role in regulating the growth, development and pathogenicity of fungal pathogens. Encoding cAMP-dependent protein kinase A gene (PKA), which is a key component in the downstream of cAMP transduction pathway. In this research, the function of PKA was studyed by PKA gene-specific inhibitor, the full length sequence of cAMP-dependent protein kinase A regulatory subunit gene (PKA-R) and the homologous fragment of cAMP-dependent protein kinase A catalytic subunit gene (PKA-C) from S. turcica 01-23 genomic DNA were cloned by candidate gene approach and Genome Walking technique. Then the research was completed in bioinformatic analysis and the mutant of gene deletion, all of which laid a foundation for investigating the role of cAMP pathway between pathogenic fungi and host.A specific inhibitor (H89) of PKA could inhibit the conidium germination and appressorium formation of S. turcica. As the increasing of H89 concentration, the inhibition bacame stronger. At the same concentration, appressorium formation was more sensitive to H89 compared to conidium germination.Degenerate oligonucleotide primers were designed based on conserved nucleotide sequences of the known PKA-R gene and PKA-C gene in plant pathogenic fungi. We amplified their homologous fragments from S. turcica 01-23 genomic DNA. Then its flanking regions were obtained by Genome Walking. A whole PKA-R gene which is 2688 bp in length and a DNA homologous fragments of PKA-C which is 1382 bp in length were got. The specific probes of PKA-C and PKA-R were prepared to apply for Southern blotting. The results showed that PKA-C gene and PKA-R gene only had single copy in genomic DNA of S. turcica, respectively.The sequence information by bioinformatic analysis indicated that the deduced amino acid sequence of the PKA-C showed 69%~85% identity with other fungal PKA-C gene including Alternaria alternata, Phaeosphaeria nodorum, Pyrenophora tritici-repentis, and so on. Both sequences of DNA and cDNA of PKA-R gene are 1398 bp and there is no intron in this sequence through alignment. It has a completely open reading frame composed of 465 amino acid residues, the molecular weight is about 50.437 kD, and pI 5.68. The deduced amino acid sequence of the PKA-R showed 58%~88% identity with other fungal PKA-R gene including P. tritici-repentis, P. Nodorum, Botryotinia fuckeliana, and so on. Furthermore, we analyzed the sequence of PKA-R gene, the results showed that, the PKA-R protein was hydrophilic cytoplasmic protein, and there were no signal peptide and transmembrance domain. The secondary structure had been predicted that there were 34.84% alpha helix, 13.55% extended strand, 4.52% beta turn and 47.10% random coil.To study the gene function deeply, the vector of double-cross homologous recombination of PKA-R gene and the RNA interference vector of PKA-C gene were constructed. To PKA-C gene silencing, the protoplasts of S. turcica were transformed through PEG-mediated. 11 transformants were obtained after scanning with hygromycin B. It is determined that the transformant m5-1 was mutant through Southern blotting. In contrast to the wild strain 01-23, the PKA-C mutant m5-1 showed different phenotypes at the same growth conditions. On PDA medium, filamentous growth of PKA-C mutants was impaired. The vertical axis of mycelium cells became shorter than the normal type, and conidia were not found. |
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