siRNA silencing and hypoxia challenge indicate that the function of common carp hif-1[alpha]b genes are tightly linked to hif-1[alpha]a and hif-3[alpha] genes

Fishes are susceptible to hypoxia stress, while the common carp is known for its high tolerance to hypoxia. The hypoxia-inducible factor (HIF) pathway directly regulates the cell's response to hypoxia. Still, it is currently unknown which members of the hif-[alpha] genes are present in common carp and their specific functions. In this study, we found that the hif-1[alpha], hif-2[alpha], and hif-3[alpha] genes of common carp all contained twice the number of copies of their orthologs in zebrafish. Common carp has four copies of the hif-1[alpha] gene, of which the two hif-1[alpha]a genes were expressed at low levels in the vast majority of tissues, while the two hif-1[alpha]b genes were expressed at high levels in multiple tissues. We silenced the two hif-1[alpha]b genes using chitosan nanoparticles (CSNPs) carrying siRNA and subjected two groups to hypoxic stress. Transcriptome sequencing results show that whether under normoxia or hypoxia, the number of differentially expressed genes (DEGs) caused by silencing the hif-1[alpha]b genes in the heart exceeds 1,000, far more than the number of DEGs in the gills or brain. GO enrichment and KEGG enrichment showed that DEGs in the heart were mainly related to immune function and myocardial contraction. DEGs in the gills and brain also enriched many immune-related terms, and some DEGs in the gills were related to iron metabolism and erythropoiesis. Among the paralogs, the two hif-1[alpha]a genes were most obviously up-regulated under normoxia, while the hif-3[alpha] genes were most obviously up-regulated under hypoxia. We did not find any downstream genes of the HIF pathway that were specifically regulated by the hif-1[alpha]b genes. The main effect site of the common carp hif-1[alpha]b genes is the heart, and their main functions are to regulate immune response and myocardial contraction. Their functions are partially redundant with the hif-1[alpha]a genes and hif-3[alpha] genes. When their expressions are inhibited, the expression of hif-1[alpha]a genes or hif-3[alpha] genes would be up-regulated in specific contexts, thereby compensating for their loss of function. The downstream genes of the HIF pathway in common carp may be generally regulated by multiple hif-[alpha] genes.