Bioinformatics characterization and tissue-specific expression of MARCKS and MLP genes in the regenerating salamander...

Myristoylated alanine-rich C-kinase substrate (MARCKS) and MARCKS-like protein (MLP) play a key role in initial axolotl limb regeneration. However, the basic bioinformatics characteristics and expression profiles of the two genes in newts remain unclear. To investigate the important biological characteristics of newt MARCKS and MLP, the open reading frame (ORF) sequences of MARCKS and MLP were obtained from Cynops orientalis (C. orientalis) and subjected to comprehensive bioinformatics and tissue expression analyses for the two genes. The results showed that the complete ORF sequences of Co-MARCKS and Co-MLP were 837 bp (encoding 278 amino acids) and 684 bp (encoding 227 amino acids) respectively. Both Co-MARCKS and Co-MLP were dominated by random coils, and neither protein contained a signal peptide. Both Co-MARCKS and Co-MLP contain three highly conserved domains, including the N-terminal myristoylation motif, the MARCKS homology domain 2, and the effector domain. Furthermore, Co-MARCKS and Co-MLP were predicted to contain eight and five potential phosphorylation sites respectively. Notably, the phosphorylation sites at Ser¹³¹ of Co-MARCKS and at Ser⁹⁵ of Co-MLP were located in their respective effector domains, implying critical roles in protein function. Phylogenetic analysis revealed that Co-MARCKS and Co-MLP shared the closest evolutionary relationship with those of Pleurodeles waltl. Expressions of the two genes were detected in the liver, spleen, stomach and intestine of C. orientalis. Importantly, the expression levels of Co-MLP in the liver and stomach were each significantly higher than those in the spleen and intestine (P < 0.05). In conclusion, this study indicated that the dynamic structures and phosphorylation of newt MARCKS and MLP play a crucial role in their function, providing a molecular foundation for future functional studies of these genes in newt limb regeneration.