MOLECULAR CLONING OF MCCASPASE 3-4 AND ITS FUNCTIONS IN MYTILUS CORUSCUS LARVAL METAMORPHOSIS

As an essential way to remove redundant tissues or cells, apoptosis plays a crucial role in regulating the metamorphosis of vertebrates and insects. However, little is known about the function of apoptosis and its executioner genes caspase-3 in the metamorphosis of marine mollusks. In the present work, a full-length caspase-3 cDNA had 1269 bp full length and 855 bp sequences of Open Reading Frame (ORF), which coded a polypeptide of 1486 amino acids, was cloned from the Mytilus coruscus. It encodes a predicted protein containing conserved caspase p20 and p10 domains and has the caspase family cysteine active site (KPKIFIFQCSRR) in the p20 domains. However, compared with conserved caspase 3 in other species, three amino acids sites of the pentapeptide active motif (QACXG, where X is R, Q, or D) at the end of the cysteine active site is mutated. Multi-sequence alignment and phylogenetic analysis showed that this gene had the highest similarity with the Caspase 3/7-4 gene in Mytilus galloprovincialis and was named McCaspase 3-4. The mRNA expression level of McCaspase 3-4 in different tissues of the adult and larval metamorphosis were analyzed by real-time quantitative PCR. Results showed that McCaspase 3-4 mRNA expression in the mantle and labial palp was significantly higher than that in other tissues and was 9.34 and 7.37 times higher than that in the adductor muscle, which had the lowest expression level, suggesting this gene might be involved in host immune defense or tissue renewal and repair of labial palp. The McCaspase 3-4 mRNA expression also increased at 12 to 24h after the epinephrine inducing and peaked at 24h after inducing when the expression level was 3.0 times higher than that at 0h, indicating the McCaspase 3-4 might play a role in the early stage of larval metamorphosis (larval metamorphosis usually begin at 48 to 72h after epinephrine inducing). Furthermore, after the pediveligers were transfected with the specific McCaspase 3-4 siRNA by electroporation, the metamorphosis rate of pediveligers at 48h induced by epinephrine was decreased to 5%, which were significantly lower than the epinephrine inducing after non-target gene siRNA transfected group (25.7%). These results indicated that although the pentapeptide active motif of McCaspase 3-4 is mutated, this molecular still has the function of metamorphosis regulation and plays a role in the early stage of larval metamorphosis. However, we do not know whether the active site mutation will affect its metamorphosis regulation effect. These results will contribute to understanding the role of apoptosis in mussel larval metamorphosis and the molecular mechanism of marine mollusks’ metamorphosis.