1 State Key Laboratory of Crop Improvement and Regulation in North China, Baoding 071001, China; 2 College of Life Sciences/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Hebei Agricultural University, Baoding 071001, China; 3 College of Plant Protection, Hebei Agricultural University, Baoding 071001, China
Abstract：Protein kinase A (PKA) is the core element of cAMP signal transduction pathway in eukaryotie, which regulates the growth and development of filamentous fungus through phosphorylation of a variety of active proteins. In order to clarify the possible interaction protein of PKA in Setosphaeria turcica, and further analyze the molecular mechanism of its acts, in this study, the complete coding region of StPKA-C1/2 was amplified from the total complementary DNA of S. turcica to construct the expression vector pGST-StPKA-C1/2, which was introduced into Eschaeria coli. The fusion protein GST-StPKA-C1/C2 was induced in E. coli by isopropyl β-D-thiogalactopyranoside and then purfied by affinity chromatography. The GST pull-down strategy was used to screen the interacting proteins of StPKA-C1/2. The interacting proteins were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that 69 StPKA-C1 interacting proteins and 70 StPKA-C2 interacting proteins were identified. The functional annotations of proteins included catalytically active protein, enzyme regulating active protein, transport electron carrier protein, molecular chaperone protein, structural molecular protein, metabolic enzyme, etc. Venn analysis found that StPKA-C1 shared 52 interacting proteins with StPKA-C2, with only 17 proteins interacting with StPKA-C1, and only 18 proteins interacting with StPKA-C2. The target protein SETTUDRAFT_ 168881, as a member of the elongation factor G (EFG) family, may regulate hyphal growth. The target protein SETTUDRAF_37808 belonged to the Fimbrin protein family of microfilament-binding proteins and may be involved in the regulation of the actin cytoskeleton, oxidative stress response, and morphogenesis. It was suggested that StPKA-C1/2 might play important biological functions by interacting with these target proteins.The above results preliminarily clarified the potential interaction protein of StPKA-C1/2. This study provides a theoretical basis for further analyzing the molecular mechanism of PKA.
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