Analyses of the differential expression of cloned Caspase-3 and Caspae-9 genes, of Onchidium reevesii, exposed to extreme hot and cold conditions

Abstract

During low tide intertidal organisms face severe temperature fluctuations. This situation may be more pronounced in the context of climate change. To investigate the molecular response mechanism of Onchidium reevesii when stressed by hot and cold temperatures, we cloned its Caspase-3 and Caspase-9 genes and detected their expression characteristics using RT-qPCR. The results showed that the full-length of the cDNA of Caspase-3 consist of 1831 bp, and included an open reading frame (ORF) of 894 bp, a 5′UTR of 155 bp and a 3′UTR of 300 bp, and encoding for 297 amino acids. The full-length of the cDNA of Caspase-9 consisted of 2425 bp, and included an ORF of 1373 bp, a 5′UTR of 290 bp, and a 3′UTR of 759 bp, and encoding for 457 amino acids. During the evolution of species the two genes have been greatly conserved. A homology analysis and phylogenetic tree reconstruction revealed that O. reevesii is most closely related to Aplysia californica. The RT-qPCR showed that both genes were expressed in all of the tissues and that expression was highest in the hepatopancreas. It is speculated that these genes play an essential role in the immunity of O. reevesii to certain diseases. Conditions of temperature stress (40 °C and 10 °C) showed that both heat and cold stresses could induce significant changes in both genes.Compared to the control group, under cold stress conditions the levels of expression of Caspase-3 and Caspase-9 were significantly upregulated (P < 0.05) in each experimental group. When exposed to a constant temperature of 10 °C, their expression peaked after 1 h (4.32-fold, P < 0.05; and 5.58-fold, P < 0.05, respectively) and was at its lowest after 12 h (1.93-fold, P < 0.05; and 2.35-fold, P < 0.05, respectively). Their expression was upregulated after 24 h (2.68-fold, P < 0.05; and 4.21-fold, P < 0.05, respectively). The expression of both genes showed similar trends. Under heat stress, the expressions of the two genes showed a wave pattern. When exposed to a constant temperature of 40 °C, their expression was up-regulated after 1, 6, and 24 h, and down-regulated after 3 and 12 h. Except at 24 h, the other experimental groups did not differ significantly (P > 0.05) from the control group. When subjected to various stress conditions the two genes displayed different expression patterns, indicating that the species has different strategies for coping with temperature changes. It further indicates that the effect of temperature on the species may play a role through the Caspase-dependent pathway. These may help to better understand the response, at the molecular level, of O. reevesii to conditions of hot and cold stress.