Cloning and tissue expression analysis of the calcium/calmodulin dependent protein kinase Ⅳ gene (CaMKⅣ) in Onchidium struma

Calcium/calmodulin-dependent protein kinase Ⅳ (CaMKⅣ) which has multiple physiological functions in organisms, is a regulatory factor widely found in eukaryotes. To investigate the role and molecular mechanism of CaMKⅣ in Onchidium struma in the process of evolution from sea to land, and further explain the physiological function of CaMKⅣ to provide a scientific theoretical basis, a series of experiments have been carried out. In this study, the cDNA sequence of CaMKⅣ gene was cloned by RACE-PCR using ganglion of O. struma, and bioinformatics analysis and qRT-PCR experiment were performed. The results suggested that the full-length cDNA of CaMKⅣ gene consists of 1 603 base pairs (bp), 315 bp 5′ non-coding region (UTR), 256 bp 3′ non-coding region (UTR) and 1 032 bp open reading frame (ORF) which encode a total of 343 amino acids. The atom of the polypeptide chain encoded by this gene was predicted. The CaMKⅣ protein was predicted to contain 5 490 atoms, the molecular weight is about 3.87 ku, the theoretical isoelectric point is 6.12, the molecular formula is C_{1 741}H_{2 768}N₄₅₂O₅₁₄S₁₅, and the N-terminal signal peptide is composed of 1-29 amino acids. The amino acid sequence alignment and phylogenetic tree results suggest that the CaMKⅣ gene was the closest to the CaMKⅣ gene of Aplysia californica and Biomphalaria glabrata, and its phylogenetic analysis was consistent with the traditional morphological classification. The results of qRT-PCR indicated that the CaMKⅣ gene was expressed in all tissues, and the relative expression in the pleopod was the highest, followed by the dorsal skin and mouthparts, and the expression level in the ganglion, albumen gland, intestine, hepatopancreas and other organs were low. It was preliminarily speculated that the gene played an important role in the ganglion of O. struma, or it was the molecular basis for the biological body to perceive external environmental variables. The results of this experiment will provide a theoretical support for further understanding of the regulation of the physiological functions of nervous structure and the function of CaMKⅣ gene, and also provide a reference for exploring the environmental adaptation mechanism of marine animals in the process of evolution from ocean to land.