CYCLOPHILIN A IN HYRIOPSIS CUMINGII: MOLECULAR CHARACTERIZATION AND EXPRESSION ANALYSIS IN RESPONSE TO AEROMONAS HYDROPHILA CHALLENGE

Cyclophilin A (CypA) serves as a principal receptor of Cyclosporin A (CsA) within cells, exhibiting considerable biodiversity and playing an important role in immune system. In this study, the full-length cDNA sequence (GenBank ID: MN121742) of CypA of Hyriopsis cumingii was obtained using PCR and RACE technology. The cDNA sequence spans 1237 bp, comprising a 57 bp 5' noncoding region (UTR), a 688 bp 3' noncoding region, and a 492 bp open reading frame, encoding a total of 164 amino acids. The theoretical isoelectric point of the CypA cDNA sequence is 8.9, and the relative molecular weight is 17.29 kD. A search for homologous sequences in GenBank revealed high similarities between CypA amino acid sequence of Hyriopsis cumingii and those of other shellfish species, including Hyriopsis schlegelii (99.39%), Cyclina sinensis (77.44%), Ruditapes philippinarum (78.05%), Crassostrea ariakensis (78.66%), and Solen grandis (75.05%). Utilizing SWISS-MODEL, it was predicted that CypA of Hyriopsis cumingii contains 8 β-pleated sheets and 2 α helixes, potentially forming the active central region of CypA. A multiple sequence comparative analysis was carried out by using the MEGA 5.1 Software, and a phylogenetic tree of the genetic system was constructed employing the neighbor-joining method. The analysis revealed that CypA of Hyriopsis cumingii clustered with CypA of shellfish from the same genus, particularly exhibiting a close genetic relationship with CypA of Hyriopsis schlegelii. The expressions levels of CypA in different tissues of Hyriopsis cumingii were assessed through qRT-PCR, indicating the highest expression in the gonad, followed by the liver, kidney and gill. Upon exposure to Aeromonas hydrophila, a bacterial infection, expression levels of CypA in gonad, liver, gill, and intestines exhibited an initial increase followed by a subsequent decrease within the first 72h of infection. Specifically, peak expression levels were observed at 6h in gonad and gill, at 12h in liver, and at 24h in intestines. This proves that Aeromonas hydrophila infection significantly induce the expressions of CypA in Hyriopsis cumingii. Expression level of CypA in gonad reaches the peak earlier than that in liver and intestines. Moreover, the relative expression times (14.91) of CypA in gonad at peak is significantly higher than those in liver (9.89), intestines (9.78) and gill (7.53). It is speculated that CypA plays an important role in defending against bacterial infections in gonad, which may not only serve reproductive functions, but also contribute significantly to immune defense.