| The circadian clock is an endogenous timing mechanism evolved in organisms to adapt to the Earth’s periodic diurnal changes. Cryptochrome and Period genes play pivotal roles in regulating the circadian system. They function by forming PER/CRY heterodimer complexes, which translocate from the cytoplasm to the nucleus. Once there, these complexes inhibit the transcription of CLOCK/BMAL1-driven E-box elements, thereby acting as downstream effector genes. These two genes are also influenced by environmental cues, such as external light, which can remodel the periodic rhythm and ultimately facilitate adaptation to environmental rhythmic changes. The boring giant clam Tridacna crocea is a dominant species within the Tridacnidae family, possessing significant economic and ecological importance. However, due to environmental changes, overfishing and other factors, this species is currently classified as a rare and protected animal. A defining characteristic of this species is its symbiotic relationship with zooxanthellae, wherein light plays a fundamental role in the giant clam-zooxanthellae symbiosis. Additionally, light serves as a crucial regulatory factor influencing the circadian clock. Consequently, exploring the influence of circadian rhythms on the expression of core clock genes in T. crocea can provide crucial data to support conservation efforts and breeding programs for this species.
In this study, the SeqMan software was used to assemble sequences obtained through sequencing. The software SignalP 5.0 Server and SMART 4.0 were used to conduct online analysis and prediction of giant clam Cryptochrome and Period functional domains. ExPASy Server online analysis software was used to analyze and predict the physicochemical properties of the sequences, DNAMMAN software was used to conduct multiple sequence comparison of the sequencing results, and BLAST option of NCBI database was used to conduct homology analysis of Cryptochrome and Period sequences. The phylogenetic tree of Cryptochrome and Period was constructed and analyzed using the Neighbor-joining method in Mega 7.0. For homology modeling, I-TASSER was adopted, and Hdock was utilized to investigate the interaction mode between Cryptochrome and Period. The interaction mode of the docking results was analyzed with Pymol 2.3.0. Furthermore, PCR technology was employed to clone and characterize the coding regions of the Cryptochrome and Period from T. crocea. Subsequently, tissue-specific expression analysis of both Cryptochrome and Period was performed, and their expression levels in various tissues were quantified under different photoperiods.
The results showed that: (1) The coding region of Cryptochrome has a base sequence length of 1641 bp, encoding 546 amino acids, with a theoretical isoelectric point of 6.08 and a molecular weight of 62.98 kDa; the coding region of Period has a base sequence length of 4386 bp, encoding 1461 amino acids, with a theoretical isoelectric point of 6.14 and a molecular weight of 164.99 kDa; the Hdock interaction model showed that these two proteins could form heterodimers with binding energy of -279.88 kcal/mol. (2) Tissue expression analysis indicated that both Cryptochrome and Period were expressed across all seven tissues examined, with relatively high expression levels in the outer mantle, inner mantle, gill and adductor muscle (with Cryptochrome also demonstrating a high level of expression in the heart). (3) The genes Cryptochrome and Period exhibited oscillatory expression patterns that varied with the circadian cycle in the outer mantle, inner and outer mantle, gill, and adductor muscle. Under normal lighting conditions, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and the adductor muscle all reached the maximum value at 1 h of light treatment, while the expression level in the gill reached the maximum value at 5 h of dark treatment, and then showed a decreasing trend with the increase of light time. When the illumination was delayed for 2 h, the expression level of Cryptochrome in the outer mantle, inner and outer mantle, and gill all reached the maximum value at 1 h illumination, and the expression level in the adductor muscle reached the maximum value at 7 h darkness treatment, and then all showed a decreasing trend with the increase of illumination time. Under normal light conditions, the expression level of Period in the outer mantle, inner and outer mantle, and gill reached the maximum value at 5 h of darkness treatment, while the expression levels in the adductor muscle reached the maximum value at 1 h of light treatment, and all showed a trend of first decreasing and then increasing with the increase of light time. When the illumination was delayed for 2 h, the expression level of Period in outer mantle and adductor muscle reached the maximum value at 7 h of darkness treatment, while the expression levels in inner and outer mantle, and gill reached the maximum value at 1 h of light treatment, and both showed a decreasing trend with the increase of illumination time. In summary, this study marks the initial successful cloning of two pivotal circadian clock genes, Cryptochrome and Period, from the species T. crocea. Our preliminary validation of the diurnal rhythmic expression patterns of these genes in key tissues provided valuable insights into the behavioral and physiological rhythms of T. crocea, as well as its mechanisms underlying light adaptation. |
