Molecular Cloning and Functional Characteristics Reveal the Relationship between <i>Caspase-10</i> and <i>Caspase-3</i> Gene in Strongylocentrotus Intermedius and Their Potential Roles in Apoptosis

Cysteine-aspartic acid protease (caspases) is a key enzyme during apoptosis. In this study, we cloned the full-length sequence of the Caspase-10 and Caspase-3 gene, analyzed their expression profile in different tissues, and during different developmental stages of Strongylocentrotus intermedius,and finally analyzed their functions. The SiCaspase-10 and SiCaspase-3 in S. intermedius were respectively 3107bp and 1642bp in length, which accordingly possessed open reading frames (ORF) of 2064 and 960bp, encoding 687 and 319 amino acids. A 5’UTR (untranslated region) of 620bp and a 3’UTR of 423bp were cloned in SiCaspase-10, and a 5’UTR of 365bp and a 3’UTR of 317bp in Caspase-3. The predicted molecular weights of Caspase-10 and SiCaspase-3 were 254.7 and 35.34 kDa, and their theoretical pI values were 4.85 and 4.84, respectively. The SiCapsase-10 has conserved features, such as a cysyein as partate-specific catalytic domains, and the SiCaspase-3 also has partate-specific catalytic domains. Our findings demonstrated the expression of SiCaspase-10 and SiCaspase-3 in all the six tissues via tissue-specific expression assays with the significant expression in coelomocyte. According to time-course expression assessments at 9 distinct developmental stages, SiCaspase-10 and SiCaspase-3 has the maximum expression in the 8-cell stage. SiCaspase-3 expression was drastically reduced in the intestines and coelomocyte tissue 48 hours after SiCaspase-10 was knocked down by particular small interfering RNA (siRNA), demonstrating that SiCaspase-3 is Caspase-10's downstream target gene. TUNEL staining analysis of coelomic cells after Sicaspase-3 inhibition showed that the apoptosis rate decreased significantly, which proved that Sicaspase-3 was involved in the apoptotic response. The findings contribute to a better knowledge of the SiCaspase-10 and SiCaspase-3 mediated immune response in S. intermedius, as well as a theoretical experimental foundation for investigating the mechanism of action of SiCaspase-10 and SiCaspase-3 genes and the identification of disease-resistant sea urchin families.