Molecular Characterization and Response of Prolyl Hydroxylase Domain (PHD) Genes to Hypoxia Stress in Hypophthalmichthys molitrix

SIMPLE SUMMARY: Hypoxia is a common challenge for aquatic organisms, and prolyl hydroxylase domain (PHD) proteins play important roles in hypoxic adaptation by regulating the stability of the hypoxia-inducible factor 1 alpha subunit (HIF-1α). In this study, the full-length cDNAs of three PHD genes were obtained from Hypophthalmichthys molitrix, which is an important freshwater fish and sensitive to low oxygen tension. The amino acid sequence analysis and phylogenetic analysis of PHDs were performed among various species. Furthermore, the expression patterns and the transcriptional responses of H. molitrix PHD genes to acute hypoxia, continued hypoxia, and reoxygenation were explored in different tissues. Our study preliminarily explored the physiological regulation functions of PHD genes at the transcriptional level when addressing the hypoxic challenge and provided a foundation for future systematic explorations of the molecular mechanisms underlying hypoxia adaptation in silver carp. ABSTRACT: As an economically and ecologically important freshwater fish, silver carp (Hypophthalmichthys molitrix) is sensitive to low oxygen tension. Prolyl hydroxylase domain (PHD) proteins are critical regulators of adaptive responses to hypoxia for their function of regulating the hypoxia inducible factor-1 alpha subunit (HIF-1α) stability via hydroxylation reaction. In the present study, three PHD genes were cloned from H. molitrix by rapid amplification of cDNA ends (RACE). The total length of HmPHD1, HmPHD2, and HmPHD3 were 2981, 1954, and 1847 base pair (bp), and contained 1449, 1080, and 738 bp open reading frames (ORFs) that encoded 482, 359, and 245 amino acids (aa), respectively. Amino acid sequence analysis showed that HmPHD1, HmPHD2, and HmPHD3 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at their C-termini. Meanwhile, the evaluation of phylogeny revealed PHD2 and PHD3 of H. molitrix were more closely related as they belonged to sister clades, whereas the clade of PHD1 was relatively distant from these two. The transcripts of PHD genes are ubiquitously distributed in H. molitrix tissues, with the highest expressional level of HmPHD1 and HmPHD3 in liver, and HmPHD2 in muscle. After acute hypoxic treatment for 0.5 h, PHD genes of H. molitrix were induced mainly in liver and brain, and different from HmPHD1 and HmPHD2, the expression of HmPHD3 showed no overt tissue specificity. Furthermore, under continued hypoxic condition, PHD genes exhibited an obviously rapid but gradually attenuated response from 3 h to 24 h, and upon reoxygenation, the transcriptional expression of PHD genes showed a decreasing trend in most of the tissues. These results indicate that the PHD genes of H. molitrix are involved in the early response to hypoxic stress, and they show tissue-specific transcript expression when performing physiological regulation functions. This study is of great relevance for advancing our understanding of how PHD genes are regulated when addressing the hypoxic challenge and provides a reference for the subsequent research of the molecular mechanisms underlying hypoxia adaptation in silver carp.