Cloning Of PKC Gene Regulating Conidium Germination And Appressorium Formation Of Setosphaeria Turcica

The research was completed in the PKC gene cloning,bioinformatic analysis and the study of PKC gene-specific inhibitor,which confirmed primarily the relationships between the PKC gene and conidium germination and appressorium formation of Setosphaeria turcica.Besides,it was studied about four influence factors on conidium germination and appressorium formation of Setosphaeria turcica,such as light condition,conidial concentration,different nutrient resources and pH value of conidial suspension.The optimal pH value for conidium germination and appressorium formation was between 5.0 and 7.0,in which the rate of conidium germination and appressorium formation was 95%and 85%,respectively.The exogenous nutrient sources were not the indispensable factors for conidium germination,but the 5%sugar solutions were more favorable for appressorium formation than the others.There were no significant differences among different light treatments.Conidium concentration of its suspension affected greatly conidium germination and appressorium formation.Low conidia concentration was propitious to conidium germination and appressorium formation,which were inhibited significantly in higher concentration.It was suggested that the phenomenon was due to the self-inhibitor,a kind of lipophilic substance,existing in the site of conidium germination.Full-length genomic DNA(3837 bases)of the protein kinase C gene,was obtained by polymerase chain reaction(PCR)amplification from degenerated primer designed on the basis of the conserved nucleotide regions of protein kinase C domains from other fungi, and Genomic walking technology.The completed cDNA sequence of PKC gene was obtained by PCR with specific primers designed on the full-length genomic DNA.There was one open reading frame composed of 1172 codons and six deduced introns of 50,62,51, 49,52 and 57 nucleotides in the PKC gene,and all introns were accordance with GT-AG rules. The deduced amino acid sequence of the PKC showed highest identity to the amino acid sequence of the protein kinase C from Cochliobolus heterostrophus,to 92%.SignalP soft predicted that there was no signal peptide,and through TMPRED analysis,it was known that there was a transmembrane domian in the deduced amino acid sequence of PKC gene. Southern blotting analysis indicated that there was a single copy of the PKC gene in the genome of Setosphaeria turcica.Through InterProScan analysis,it was known that the deduced protein of PKC gene included six conserved domains,such as Pkinase_c,S_TKc,ATP site,C1,C2 and HR1.The 3D structure of deduced protein of PKC gene of Setosphaeria turcica was the same to that of Cochliobolus heterostrophus,and similar to that of Magnaporthe grisea by Swissmodel soft.A specific inhibitor(chelerythrine)at 8～10μM could completely inhibit the conidium germination and appressorium formation of Setosphaeria turcica.And the forepart time from induced,chelerythrine show significant inhibition on conidium germination and appressorium.It was studied in the PKC gene cloning,bioinformatic analysis and the study of PKC gene-specific inhibitor,and concludied the PKC gene play an important role in conidium germination and appressorium formation of Setosphaeria turcica,which will lay a foundation for investigating molecular mechanism between pathogenic fungi and host and making new fungicides.