4.5 Article

Molecular characterization and expression analysis of hypoxia-inducible factor-1 alpha, factor-2 alpha, and factor-3 alpha and physiological response to hypoxia exposure in Amur minnow (Phoxinus lagowskii)

Journal

AQUACULTURE INTERNATIONAL
Volume 30, Issue 2, Pages 607-632

Publisher

SPRINGER
DOI: 10.1007/s10499-021-00826-y

Keywords

Phoxinus lagowskii; Hypoxia-inducible factors; Characterization; Gene expression; Physiological response

Categories

Funding

  1. National Natural Science Foundation of China [32170523]
  2. Guangdong South China Sea Key Laboratory of Aquaculture for Aquatic Economic Animals Guangdong Ocean University [KFKT2019Y08]
  3. Program of Natural Science of Heilongjiang Province of R. P. China [LH2019C040]
  4. Blue granary scientific and technological innovation of China [2019YFD0900405]

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This study investigated the hypoxia tolerance in Phoxinus lagowskii by cloning HIF genes and analyzing their biological function. The findings suggest that P. lagowskii has a high potential for hypoxia tolerance and HIF genes play important roles in modulating circulatory and metabolic responses to hypoxia. Additionally, HIF-3α was found to be crucial for liver homeostasis in hypoxic conditions.
To further enhance our understanding of hypoxia tolerance in the fish Phoxinus lagowskii, hypoxia-inducible-factor (HIF) genes were cloned to analyze their biological function during hypoxia exposure. The complete cDNAs of HIF-1 alpha, HIF-2 alpha, and HIF-3 alpha were 2912, 3444, and 2228 bp in length, encoding 773, 829, and 641 amino acid residues, respectively. The HIF-1 alpha and HIF-2 alpha proteins contain HLH, PAS-A, PAS-B, PAC, N-TAD, and C-TAD domains, while HIF-3 alpha has no C-TAD domain. A high number of serine residues in the oxygen-dependent degradation (ODD) domain are connected with hypoxia tolerance in fish. More than 40 serine residues were found in the ODD domain of HIF-alpha genes in P. lagowskii, suggesting that P. lagowskii has a high potential for hypoxia tolerance. The HIF-alpha genes were highly expressed in the heart, spleen, and liver of P. lagowskii, suggesting an important role for these genes in modulating the circulatory and metabolic responses to hypoxia. Relative long-term hypoxia exposure and recovery resulted in significantly increased transcript abundance for three genes in liver, gill, and spleen, but short-term exposure and recovery did not, which is indicative of a unique characteristic of hypoxia-tolerant fish. In addition, this study found for the first time that P. lagowskii upregulates HIF-3 alpha within 24 h of liver hypoxia, confirming that HIF-3 alpha plays an important role in liver homeostasis. Therefore, we performed fluorescence in situ hybridization on HIF-3 alpha after inducing liver hypoxia. We also constructed an overexpression vector pcDNA3.1-HIF-3a and transfected it into CIK cells. The results showed that overexpression of HIF-3 alpha significantly increased the expression of hypoxia-related genes such as P53, Glut1, VEGF, and VHL. Moreover, our results showed that enzymes of the defense and antioxidant mechanisms showed a noticeable increase in relatively long-term hypoxia to counteract the oxidative stress generated after re-oxygenation.

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