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Cloning and expression analysis of hif-1α and downstream genes during hypoxic stress in cobia (Rachycentron canadum)

Aquaculture International

April 2022
30(9)

DOI:10.1007/s10499-021-00820-4

Authors:

Eric Amenyogbe

University of Environment and Sustainable Development

Show all 7 authorsHide

Hypoxia is a lack of required oxygen to meet the metabolic demands of living organisms. Cellular hypoxia occurs when the molecular oxygen, essential to maintain sufficient adenosine triphosphate (ATP) levels for normal physiological function, surpasses the vascular supply. Tissue hypoxia can arise during a range of diseases. As molecular oxygen is a crucial metabolic energy source for all living organisms, animals manage the intracellular oxygen levels to sustain homeostasis, with the upregulation of genes that improve tissue perfusion and anaerobic ATP creation via glycolysis. This is facilitated by the hypoxia-inducible factors (HIFs). Hypoxia-inducible factor 1α (hif-1a) is the core regulator of the hypoxia response and plays a crucial role in the cellular/molecular response to hypoxic stress by regulating the transcription of target genes. In the present study, hif-1a cDNA was identified and cloned from cobia (Rachycentron canadum), using rapid amplification of cDNA ends (RACE). The hif-1a and downstream mRNA expression levels in various tissues were then determined. The full length of hif-1a cDNA is 3642 bp, with a 2292 bp open reading frame (ORF), a 5′ non-coding region (5′-UTR) of 293 bp, 3′ non-coding region (3′-UTR) of 1057 bp, and encoding 764 amino acids. The encoded protein contains the basic helix-loop-helix domain (amino acid 22–77), PER-Arnt-SIM domain (amino acid 88–154 and 230–296), and the PAS-associated C-terminal domain (amino acid 302–345). hif-1a mRNA expression was detected in nine tissues, with the highest expression observed in the liver, and the lowest expression in the intestine and spleen. hif-1a, erythropoietin (epo), and vascular endothelial growth factor (vegf) gene expressions were analyzed in the gill, intestine, liver, and muscle under hypoxic stress. In the gills, hif-1a expression was significantly increased at all hypoxia time points as well as in the liver. Erythropoietin (epo) and vascular endothelial growth factor (vegf) showed similar trends, with a significant decrease followed by a significant increase. In the muscle, the expression of all three genes was higher than the control group after hypoxic stress. These results indicate that the expression patterns of hif-1a and related genes after hypoxic stress are tissue-specific and play an essential role in cobia’s response to hypoxia.

The cDNA and amino acid sequence of hif-1a gene of cobia. “ ” indicate the initiation codon; “ ” indicate the termination codon; “ ” indicate the bHLH domain; “ ” indicate the PAS domain; “ ” PAC domain

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Analysis of hif-1a amino acid sequences of cobia by multiple sequence alignment with identified HIF-1α sequences in other species. The predicted cobia HIF-1α sequence was aligned with Danio rerio (AAQ91619.1), Sander lucioperca (ABO26718.1), Larimichthys crocea (XP_027130453.1), Siniperca chuatsi (QDA76729.1), Seriola lalandi dorsalis (XP_023256826.1), Lates calcarifer (XP_018545560.1), Channa argus (KAF3701198.1), Echeneis naucrates (XP_029349501.1), Xenopus laevis (NP_001165655.1), Homo sapiens (AAF20149.1), Gallus gallus (NP_989628.1), and Mus musculus (AAC53461.1) with their accession numbers. The dark row column height indicates the conservation degree of corresponding amino acid sequences. The blue arrow indicates the bHLH domain. The purple accent four arrow indicates the PAS-A domain. The orange accent six elbow arrow indicates the PAS-B domain. The red accent two line indicates the PAC domain

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Cluster analysis of hypoxia-inducible factor α (hif-1a) amino acid sequences of cobia. Systematic evolutionary tree of hif-1a protein from 13 species reconstructed using the neighbor-joining (N-J) method. The digits at each branch indicate the percentage of bootstrap values on 1000 duplicates. The phylogenetic distance is 0.1, as presented in the scale bar. All sequences are obtained from the National Center of Biotechnology Information (NCBI) Protein Database to analyze the phylogenetic tree. The sequence names and the accession numbers are the same as those used for multiple sequence alignment

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Relative expression of hypoxia-inducible factor α (hif-1a) gene in different tissues of cobia. Different letters (a, b, c, d, cd, e, and f) indicate significant differences in expression between different tissues (P < 0.05)

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Relative expression of hypoxia-inducible factor α (hif-1a) in the (a) gill, (b) intestine, (c) liver, and (d) muscle of juvenile cobia after hypoxia. Single asterisk indicates significant difference (P < 0.05), and double asterisks indicate highly significant difference (P < 0.01)

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https://doi.org/10.1007/s10499-021-00820-4

1 3

Cloning andexpression analysis ofhif‑1α anddownstream

genes duringhypoxic stress incobia (Rachycentron canadum)

Jian‑shengHuang1,2,3· EricAmenyogbe1 · Lin‑tongYang1· Zhong‑liangWang1·

GangChen1,2,3· Wei‑zhengWang1· Jian‑dongZhang1

Received: 28 June 2021 / Accepted: 7 December 2021 /

Abstract

Hypoxia is a lack of required oxygen to meet the metabolic demands of living organisms.

Cellular hypoxia occurs when the molecular oxygen, essential to maintain suﬃcient adeno-

sine triphosphate (ATP) levels for normal physiological function, surpasses the vascular

supply. Tissue hypoxia can arise during a range of diseases. As molecular oxygen is a cru-

cial metabolic energy source for all living organisms, animals manage the intracellular oxy-

gen levels to sustain homeostasis, with the upregulation of genes that improve tissue perfu-

sion and anaerobic ATP creation via glycolysis. This is facilitated by the hypoxia-inducible

factors (HIFs). Hypoxia-inducible factor 1α (hif-1a) is the core regulator of the hypoxia

response and plays a crucial role in the cellular/molecular response to hypoxic stress by

regulating the transcription of target genes. In the present study, hif-1a cDNA was identi-

ﬁed and cloned from cobia (Rachycentron canadum), using rapid ampliﬁcation of cDNA

ends (RACE). The hif-1a and downstream mRNA expression levels in various tissues were

then determined. The full length of hif-1a cDNA is 3642bp, with a 2292bp open read-

ing frame (ORF), a 5′ non-coding region (5′-UTR) of 293bp, 3′ non-coding region (3′-

UTR) of 1057bp, and encoding 764 amino acids. The encoded protein contains the basic

helix-loop-helix domain (amino acid 22–77), PER-Arnt-SIM domain (amino acid 88–154

and 230–296), and the PAS-associated C-terminal domain (amino acid 302–345). hif-1a

mRNA expression was detected in nine tissues, with the highest expression observed in

the liver, and the lowest expression in the intestine and spleen. hif-1a, erythropoietin (epo),

and vascular endothelial growth factor (vegf) gene expressions were analyzed in the gill,

intestine, liver, and muscle under hypoxic stress. In the gills, hif-1a expression was signiﬁ-

cantly increased at all hypoxia time points as well as in the liver. Erythropoietin (epo) and

vascular endothelial growth factor (vegf) showed similar trends, with a signiﬁcant decrease

followed by a signiﬁcant increase. In the muscle, the expression of all three genes was

higher than the control group after hypoxic stress. These results indicate that the expres-

sion patterns of hif-1a and related genes after hypoxic stress are tissue-speciﬁc and play an

essential role in cobia’s response to hypoxia.

Keywords Cobia· Hypoxia· Gene cloning· Hif-1a gene

Handling Editor: Gavin Burnell

Extended author information available on the last page of the article

Published online: 19 January 2022

Aquaculture International (2022) 30:803–824

Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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Cobia (Rachycentron canadum) is an important cultured marine fish species in southern China. It is characterized by fast growth. Due to the effects of climate change on water oxygen levels and seawater temperatures, the understanding of the influence of environmental changes on cobia culture has become important. In this study, to explore the stress and adaptability of cobia under hypoxia-reoxygenation conditions, the fish were exposed to an oxygen-deficient environment with dissolved oxygen (DO) level of 2.64 ± 0.25 mg/L. Serum liver function target, lipid metabolism-related enzymes and the serum antioxidant capability were detected in cobia under acute hypoxic stress after which fish returned to normal DO level (6.34 ± 0.15 mg/L) at 8, 24 and 48 h. Conventional paraffin sectioning and hematoxylin and eosin (H-E) staining were used to observe gill and liver tissues of cobia by electron microscopy. After hypoxic stress, the activities of alkaline phosphatase (ALP), total protein (TP), albumin (ALB), triglyceride (TG), total cholesterol (TC) and superoxide dismutase (SOD) in cobia decreased. Under acute hypoxic stress, the gill patches of the fish were contracted and slightly curved. Liver disorder and vacuoles appeared between liver tissues. After reoxygenation, SOD and catalase (CAT) were recovered to the level of the control group with time, and all peaks appeared. The gill and liver of fish were also recovered with time. The results showed that hypoxic stress could cause oxidative damage to cobia.

Identification, characterization, and expressions profile analysis of growth hormone receptors (GHR1 and GHR2) in Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂)

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May 2019

Growth hormone is an essential hormone that plays essential roles in growth, metabolism, cellular differentiation, immunity and reproduction in fish, by means of the growth hormone receptors. The encoding cDNA growth hormone receptors (GHR1 and GHR2) were cloned and characterized from Hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus polyphekadion♂). Sequence analysis of the cloned GHR1 was observed as containing 2176, which comprised an ORF of 1842 bp, 5 UTR of 6 bp and 3 UTR of 328 bp, with 612 amino acids encoding proteins, while GHR2 was observed as containing 1824 bp that encompassed an ORF of 708 bp, 5 UTR of 48 bp and 3 UTR of 1068 bp with 235 amino acids encoding proteins. Relative mRNA expression of GHR1 and GHR2 in the liver and muscle was found to be highest respectively. Our findings provide vital statistics of GHRs likely to play a significant role in the growth of the fish.

Cloning and expression analysis of hif-1α and downstream genes during hypoxic stress in cobia (Rachycentron canadum)

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