Cloning Of Fih-1 Gene And Analysis Of Correlation Between SNPs And Hypoxia Traits In Megalobrama Amblycephala

Megalobrama amblycephala(Wuchang bream) is one of the important herbivorous freshwater fish species native to China. It has been widely cultured for its economic traits such as rapid growth and strong disease resistance, therefore, it is regarded as a principal fish species in the polyculture system of freshwater in China. While, it often suffers from hypoxic stress, encumbering the development of M. amblycephala aquaculture. Fih-1 is an upstream regulation gene of Hif-1α, an important transcription factor in response to hypoxia, and controls the transcriptional activity of Hif-1α. Under normoxic conditions, Fih-1 inhibits the transcriptional activity of Hif-1α, thus inhibiting the transcription of Hif-1α-mediated gene. Whereas, under hypoxic conditions, the inhibition is abrogated, allowing Hif-1α to recruit CBP/p300, thus promoting the expression of target genes. In this study, we described the molecular cloning, sequences analysis, and the expression of M. amblycephala fih-1 under hypoxic conditions. Furthermore, we also detected fih-1 SNPs and performed association analysis between SNPs and the hypoxia trait. These results will provide helps for fish genetic breeding with hypoxia-tolerant strains or breeds. Add itionally, the promoter of M. amblycephala fih-1 has been cloned, and the hypoxia response element(HRE) has been verified. The main results are as follows.1) Molecular cloning and sequence analysis of M. amblycephala fih-1In this study, the full-length c DNA(2253 bp) sequence of M. amblycephala fih-1 was obtained, with 5’ UTR of 273 bp, 3’ UTR of 909 bp and ORF of 1071 bp, encoding 356 amino acid residues. SMART analysis revealed that Fih-1 was a Fe(II) and 2-oxoglutarate-dependent dioxygenase enzyme which had a typical Jmj C domain, a Cupin-8 domain, and metal coordination sites for Fe(II)-binding. Phylogenetic tree analysis showed that the deduced amino acid sequence of M. amblycephala Fih-1 had high homology with that of other cyprinid fish, with the highest homology with Aspius aspius(96%).2) The expression pattern of M. amblycephala fih-1 during embryogenesis and in hypoxic conditionThe real-time PCR results showed that fih-1 m RNA was ubiquitously expressed in all detected tissues and developmental stages. During embryogenesis, the fih-1 m RNA was highly expressed at the beginning of embryogenesis, with the highest expression in 0.5 hpf(hours post fertilization, 2-cell stage). The expression was very low from 19.5 hpf(the mid-gastrul stage) to 36.3 hpf(the heartbeat beginning stage), then dramatically increased in 43.5 hpf(hatching) and kept a high level after hatching. Under normoxia condition, fih-1 was widely expressed in all the detected tissues, while the expression was very low in spleen. During hypoxia treatment, the fih-1 expression decreased at 2 h, and then increased at 4 h after hypoxia treatment in different tissues of M. amblycephala, except for spleen where it remained increasing.3) The identification of SNPs and association analysis between SNPs and hypoxia traitThree SNPs(-402T/A,-106G/T and +1557C/T) in the fih-1 gene were detected by amplification, sequencing and alignment. Based on PCR-RFLP and PCR-HRM analysis, the frequencies of alleles and genotypes were calculated in hypoxia-sensitive group and hypoxia-tolerant group. The correlation between SNPs and hypoxia trait was analyzed, and revealed that all of the three SNPs in fih-1 had a significant correlation with hypoxia trait(p < 0.05). Furthermore, the-106G/T SNP showed highly significant correlation with hypoxia trait(p < 0.01).4) Molecular cloning of the promoter of M. amblycephala fih-1 and verification of the HREThermal asymmetric interlaced(TAIL) PCR was used to isolate the promoter sequence(891bp) of the fih-1 from M. amblycephala. Bioinformatics analysis showed,many cis-acting elements including HRE、HIF-1beta、TFAP2A and STAT were predicted on the promoter. Hif-1α binding element(HRE) on the fih-1 promoter was found, and luciferase activity of 293 cells transfected by recombinant vector containing HRE was higher than PGL-3basic which verified the HRE on the promoter of fih-1.