Molecular Characterization and Expression Response Under Hypoxic-Reoxygenation Stress of a Crustacyanin-Like Gene in Procambarus clarkii

Crustacyanin (CRCN), a family of lipocalin proteins specific to crustaceans, was primarily found in the exoskeleton of crustaceans. By binding with astaxanthin to form the astaxanthin-binding protein complex, it resets the proton at the end of astaxanthin and modifies the acidic site in astaxanthin, thereby regulating crustacean shell color. In addition to regulating shell colors, crustacyanin is involved in the transport of small molecule lipids such as steroid hormones and pheromones and has important biological functions in moult growth, gonadal and nervous system development, resistance to heavy metal lipid metabolism, and hypoxic stress. It was first discovered that the pigment in the blue shell of lobster could be extracted with ammonium chloride via a complex combination of organic bases and lipid pigments. It was later proposed that the pigment was a combination of astaxanthin and multimeric protein complex known as α-CRCN. This complex is composed of an octomer of dimeric β-CRCN subunits, with this dimer formed by two types of CRCN A and C in association with two astaxanthin molecules. Natural crustacyanins are all α-CRCN, comprising a total of 16 molecules. Procambarus clarkii, a member of the order Crustacea (Decapoda, Crayfish), is native to North America and is an important freshwater crustacean in China. It had been established that P. clarkii is susceptible to low oxygen stress during the culture process. The low oxygen environment inhibits the metabolic rate of P. clarkii, leading to increased susceptibility to pathogens; therefore, the ability of P. clarkii to tolerate and physiologically regulate stress due to other environmental factors was also affected, which can lead to irreversible damage and even death in severe cases. To understand the role of crustacyanin-like genes in gonadal development and hypoxic-reoxygenation stress in P. clarkii, a cDNA sequence of the PcCRCN-L gene was isolated from the hepatopancreas of this organism. The structural characteristics and evolutionary patterns of the PcCRCN-L gene were analyzed, and the expression characteristics of the PcCRCN-L gene in different tissues and gonad development stages were investigated. The expression response pattern of PcCRCN-L under hypoxic-reoxygenation stress was investigated. The DNA sequence of the PcCRCN-L gene was 6 130 bp long and located on chromosome 12 of the P. clarkii genome. The cDNA sequence was 2 700 bp, and its open reading frame (ORF) length was 1 587 bp. It contained five exons and four introns, encoded 528 amino acid residues, had a theoretical isoelectric point of 5.71 and a relative molecular weight of 55 613.55, and was a hydrophilic protein. The intron/exon splicing pattern was in accordance with the GT-AG rule. The PcCRCN-L protein had a complete lipocalin domain, which included the typical sequences G-X-W of conserved region Ⅰ (SCR1), T-D-Y of conserved region Ⅱ (SCR2), and arginine R of conserved region Ⅲ (SCR3). The results of multiple sequence alignments and phylogenetic analyses showed that PcCRCN-L, as well as the crustacyanin A and crustacyanin C subgroups, were separately clustered into a branch. The expression characteristics of the PcCRCN-L gene in different tissues showed that PcCRCN-L was expressed in all the tissues; however, the highest expression level was found in the hepatopancreas. The expression trend of the PcCRCN-L gene in the ovary and the hepatopancreas was similar in different stages of gonadal development. The expression level of the PcCRCN-L gene was significantly decreased in stages Ⅰ~Ⅱof ovarian development (P < 0.05), but no significant difference was found in stages Ⅱ~Ⅴ (P > 0.05). The expression of the PcCRCN-L gene was significantly decreased under hypoxia stress for 1 h (P < 0.05), but there was no significant difference between hypoxia stress for 1 h and hypoxia stress for 6 h. After reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly up-regulated (P < 0.05). Compared with reoxygenation for 1 h, the expression of the PcCRCN-L gene was significantly increased at 12 h (P < 0.05). The results showed that PcCRCN-L should be closely involved in the regulation of gonadal development and hypoxia-reoxygenation stress in P. clarkii.