Functional Analysis Of PKC Gene In Setosphaeria Turcica

Protein kinase C (PKC) located in the middle of Ca2+ signaling pathways. It is a phospholipid and calcium-dependent protein kinase that might play an important role in the processes of conidium germination, appressorium formation and pathogenicity. The functional analysis of PKC gene in Setosphaeria turcica were done by gene-specific inhibitor analysis, constructing PKC gene knock-out mutants based on the gene homologous combination theory and PEG gene transformation system, 5'upstream analysis of PKC gene and the function prediction.It was found by PKC gene-specific inhibitor (chelerythrine) analysis that the color of the colony growing on the PDA containing 10μmol/L inhibitor was still normal, but the mycelial growth was inhibited. There also wasn't any change in the color and shape of appressorium while treated with inhibitor. After inoculating maize seedlings with the spore suspension containing specific inhibitor, the appressorium formed from germinating spore on the leaf could not penetrate directly through the skin of epidermal cells and only to invade leaf tissue from the stoma. It was found that 10μmol/L inhibitor didn't significantly affect the activity of HT-toxin and no lesions appeared on the leaf after three weeks inoculation.High quality and large quantity of protoplasts was one of the key technologies of genetic transformation. The yield of protoplasts was significantly correlated with the age of hyphae, enzyme system, concentration, temperature and treatment time. The optimum condition for preparing protoplasts was that the conidia cultured for 24 h was digested in enzyme mixture of 1% Lywallzyme, 1% Snailase and 1% Drislase for 4 h in 30℃, and about 10 protoplasts could be produced under above conditions (microscope×400).The PKC gene knock-out vector was constructed based on the gene homologous combination theory, then transformed into S. turcica protoplasts and 12 mutants were screened by hygromycin B resistance from 27 transformants. In contrast to the wild strain 01-23, these mutants had smooth colony, slender hyphae and sporulation ability.A 1.5kb gene flank region was obtained by Genome Walking. Bioinformatic analysis showed that it had a lot of cis-acting elements corresponding SP1,AP1 and HSF, but no typical TATA box. It was calculated that PKC gene perhaps played an important role in the regulation of mating type gene expression, host signal identification and hot tolerance.These results indicated that PKC gene played an important role in the processes of conidium germination, appressorium formation and pathogenicity of S. turcica, Which laid a foundation for investigating pathogenic mechanism of S. turcica, molecular interaction between plant and pathogen, and new methods of disease prevention.