Cyclophilin A in <em>Megalobrama amblycephala</em>: Molecular characterization and expression analysis in response to <em>Aeromonas hydrophila</em> challenge

Abstract

Cyclophilin A (CypA) is a cell’s primary receptor of Cyclosporin A (CsA). It is essential for biodiversity and plays an important role in the immune system. In this study, the full-length cDNA sequence (GenBank ID: PV256473) of Megalobrama amblycephala CypA was acquired by PCR and RACE technology. The complete cDNA sequence of M. amblycephala CypA is 901 bp. The lengths of the 5′ untranslated region (UTR), the 3′ untranslated region, and the open reading frame (ORF) are 77 bp, 329 bp, and 495 bp, respectively. It encodes a total of 164 amino acids. The theoretical isoelectric point of M. amblycephala CypA is 8.6, and the relative molecular weight is 17.43 KDa. According to homologous sequence retrieval in GenBank, the similarity between CypA amino acid sequence of M. amblycephala and other fish was Cyprinus carpio (94.51%), Ctenopharynodon idellus (92.07%), Carassius gibelio (92.07%), Labeo rohita (92.07%), Danio rerio (91.46%), and Onychostoma macrolepis (91.46%). ESyPred3D predicted that the CypA of M. amblycephala contained 8 β-pleated sheets and double α-helixes, which might form the active central region of CypA. MEGA 5.1 software was used for multiple sequence comparison analysis, and the adjacency method constructed the genetic phylogenetic tree. It was found that the CypA gene of M. amblycephala was clustered into one branch of Cyprinidae fish CypA, and CypA of M. amblycephala was closely related to CypA of Cyprinus carpio. Expressions of CypA in different tissues of M. amblycephala were detected through qPCR; it was found that the expression level of the CypA gene was highest in the head kidney, followed by liver, kidney, and spleen. The M. amblycephala was immersed in Aeromonas hydrophila for infection; it was found that expression levels of CypA in head kidney, liver, gills, and intestines first increased, then decreased in the first 72 h of infection. Specifically, the expression levels reached a peak at 6h in the head kidney, at 12 h in the liver and gills, and at 24h in the intestines. It indicated that the expression of M. amblycephala CypA gene could be significantly induced after A. hydrophila infection—expression levels of CypA in the head kidney peak earlier than in the liver and intestines. Moreover, the relative expression level of CypA in the head kidney at peak (15.02-fold) is significantly higher than that in the liver (6.89-fold), intestines (9.92-fold), and gills (7.42-fold). It is suggested that CypA might play an essential role in the head kidney of M. amblycephala in defense against bacterial infection.