Analysis of Multiplicity of Hypoxia-Inducible Factors in the Evolution of Triplophysa Fish (Osteichthyes: Nemacheilinae) Reveals Hypoxic Environments Adaptation to Tibetan Plateau.

Juan Chen,Gang Ouyang,Shunping He,Jingliang Kang,Wenqi Lv,Yanjun Shen,Jing Wang,Liandong Yang

doi:10.3389/fgene.2020.00433

Abstract

HIF (Hypoxia-inducible factor) gene family members function as master regulators of cellular and systemic oxygen homeostasis during changes in oxygen availability. Qinghai-Tibet Plateau is a natural laboratory for for long-term hypoxia and cold adaptation. In this context, T. scleroptera that is restricted to >3500 m high-altitude freshwater rivers was selected as the model to compare with a representative species from the plain, P. dabryanus. We cloned different HIF-α and carried out a phylogenetic analysis from invertebrates to vertebrates for identifying HIF-α genes and analyzing their evolutionary history. Intriguingly, the HIF-α has undergone gene duplications might be due to whole-genome duplication (WGD) events during evolution. PAML analysis indicated that HIF-1αA was subjected to positive selection acted on specific sites in Triplophysa lineages. To investigate the relationship between hypoxia adaptation and the regulation of HIF-α stability by pVHL in plateau and plain fish, a series of experiments were carried out. Comparison the luciferase transcriptional activity and protein levels of HIF-αs and the differing interactions of HIF-αs with pVHL, show clear differences between plateau and plain fish. T. scleroptera pVHL could enhance HIF-α transcriptional activity under hypoxia, and functional validation through pVHL protein mutagenesis showed that these mutations increased the stability of HIF-α and its hetero dimerization affinity to ARNT. Our research shows that missense mutations of pVHL induced evolutionary molecular adaptation in Triplophysa fishes living in high altitude hypoxic environments.

Highlights

Tibetan Plateau is commonly referred to as the “Roof of the World” (Zhou et al, 2006), which is the highest and largest plateau on earth
The sequences of the Hypoxia inducible factor (HIF)-α genes (HIF-1αA/B and HIF-2αA/B) and pVHL were obtained here for high -land Triplophysa fish and for fishes living at low altitudes (P. dabryanus)
Bioinformatics analysis showed that all HIF-α/pVHL genes contained complete ORF regions, and the biochemical and physical parameters are listed in Additional file 3: Supplementary Table S3

Summary

INTRODUCTION

Tibetan Plateau is commonly referred to as the “Roof of the World” (Zhou et al, 2006), which is the highest and largest plateau on earth. To better understand the relationship between the adaptation to hypoxia and the regulation of HIF-α stability by pVHL in plateau and plain fish, we cloned and characterized four distinct HIF-α isoforms (HIF-1αA/B and HIF-2αA/B) and pVHL genes to determined their expression levels and impact on physiological regulation in T. scleroptera (highland loach) and P. dabryanus (plain loach), attempted to find differences in the hypoxic signaling pathways between plateau fish and plain fish. This findings substantially advanced our understanding of evolutionary biology and functional adaptation to the hypoxic ecological environment of the plateau

MATERIALS AND METHODS

RESULTS

DISCUSSION

ETHICS STATEMENT