Abstract:
Objective Calmodulin-dependent protein kinases (CaMKs) are key components of calcium ion-mediated signaling pathways and may serve as potential drug targets for the treatment of scuticociliatosis. In this study, we attempted to clone the CaMK gene from Pseudocohnilembus persalinus, a scuticociliate pathogen that infects certain maricultured animals, analyze the structure and function of the CaMK protein, and constructed a phylogenetic tree based on its amino acid sequence. In order to elucidate the evolutionary characteristics of CaMK proteins in P. persalinus and provide insights into the phylogenetic relationships among related ciliates.
Method Based on the RNA-seq sequencing results of P. persalinus, the Rapid Amplification of cDNA Ends (RACE) technique was employed to obtain the full-length cDNA sequence of the CaMK gene,which was designated as PpCaMK1. Bioinformatics methods were subsequently employed to analyze its sequence features, gene structure, physicochemical properties, protein domains, protein structure, and phylogenetic relationships.
Result The PpCaMK1 gene contains 9 exons and 8 introns. The full cDNA length is 1737 bp (GenBank accession number: PQ278249), including a 5'-UTR of 56 bp, a 3'-UTR of 307 bp, and an ORF of 1374 bp encoding 457 amino acids residues. The overall hydrophilicity average of PpCaMK1 is -0.802, with an instability index of 54.78, indicating that it was a hydrophilic and unstable protein. Subcellular localization predictions indicated that PpCaMK1 was distributed within the cytoplasm and nucleus. Notably, the protein lacks transmembrane domains and signal peptides. Analysis of the secondary structure composition revealed a significant proportion (50.33%) of random coils. Phylogenetic analysis showed that P. persalinus was closely related to ciliates in the class Oligohymenophorea based on PpCaMK1.
Conclusion In this study, the full-length cDNA of the PpCaMK1 gene was successfully cloned, and its structure was elucidated. A preliminary analysis of the basic physicochemical properties and structural characteristics of the PpCaMK1 protein was also conducted. These findings provide a foundation for further functional studies of PpCaMK1 and offer valuable reference data for investigating the CaMK gene family in P. persalinus.