Molecular Evolution Of HIF-1α And GH-IGFs And Its Expression In Naked Carp (Gymnocypris Przewalskii) From The Qinghai Lake Of China

Przewalski's naked carp (Gymnocypris przewalskii, Family Cyprinidae) resides in the alkaline (pH 9.1-9.5) and saline waters (9 ppt) of Lake Qinghai, on the Tibetan plateau of Qinghai-province in western China. Unlike most stenohaline cyprinids, naked carp undergo an annual spawning migration between the saline lake water and freshwater rivers. Between March and July, fish leave the lake and migrate upstream into fresh water river where they spawn. Then they migrate back to the lake for the rest of the year. The growth of these cyprinids appear to be very slow, with estimates of 7-10 years to reach typical reproductive size of 300- 500 g.Large variations in environmental oxygen levels have played a significant role in the evolution of fishes. The transcriptional regulator hypoxia-inducible factor 1 (HIF-1) is an essential mediator of O₂ homeostasis. The expression and function of HIF-1 alpha have not been studied in naked carp. Using RT-PCR and rapid amplification of cDNA ends (RACE), we have cloned the cDNA (2702 base pairs, complete CDS) of the HIF-1 alpha from naked carp. The predicted HIF-1 alpha protein sequence (776 amino acids) of naked carp has a 57% identity to human and rat HIF-1α, about a 62% identity to rainbow trout HIF-1α, and a 89% identity to crucian carp HIF-1α. The results of sequence analysis, Western blot and EMSA indicates the main funtional amino acid, the C-terminal of TAD-C domain, and bHLH domain are higly conserved in the HIF-1αof naked carp, implying similar regulation mechanisms of expression and transactivity with other animals.In this study, maximum-likelihood (ML) models of codon substitutions were used to investigate Darwinian adaptive evolution of the HIF-1αgenes in vertebrates. The results indicate that overall evolution rate of teleosts HIF-1αis higher compared with mammals, and the evolution rate in lineages leading to hypoxia-tolerant carps, just like naked carp and crucian carp, is greater than that in lineages leading to other teleosts. It seems that the evolutionary rate variation seen for HIF-1αs between mammalian and fish is primarily due to a relaxation of purifying selection. The proportion of neutral sites for naked carp(34%), crucian carp(32%) and Antarctic eelpout(38.8) is much higher than for other teleosts(about 22%), which maybe is a combined result of genome duplication and their hypoxia-tolerant ability.Growth in fish, as in all vertebrates, is regulated primarily by the growth hormone (GH)/insulinlike growth factor-I (IGF-I) axis. Besides its growth-promoting action, GH/IGF-I have been implicated as regulator of SW adaptation in fish. Investigation of the GH/IGF-I axis in naked carp is inviting for their slow growth rate and spawning migration. The cDNA sequence corresponding to the coding region of ncGH is 633 bp and codes for a 210-amino-acid protein; The cDNA sequence corresponding to the coding region of IGF-I is 537 bp and codes for a 178-amino-acid protein; The coding region of naked carp IGF-II is 639 base pairs long, with an open reading frame of 212 amino acids. The ncGH showing identities of 94%, 91%, 63% and 32% in comparison with common carp, zebrafish, Cherry salmon and human GH, respectively. Compared to common carp and goldfish, evolution analyses showed that genome duplication has lesser influence on the relaxation of purifying selection in evolution of ncGH.In this study, Naked carp were assigned randomly to 1 of 4 groups: RW (riverwater), RW+Na (NaCl treatment in RW), RW+Mg (MgCl₂ treatment in RW) and LW(lakewater), the concentration of Na⁺ and Mg²⁺ in RW+Na and RW+Mg are equal to iron concentration in LW. After 2 days, the plasma IGF-I level of group RW+Na and LW were significantly higher than control group(RW); The plasma GH levels of LW group were also significantly higher than RW group; RW+ Na rearing significantly increased SS levels in hypothalamus. By contrast, after 5 days these hormones levels did not vary significantly among the groups. To our knowledge, this is the firtst report of salinity-induced increases of GH-IGF-I circulating levels in Cypriniformes fish.In conclusion, high-altitude and salty enviroment of Qinghai lake produced a different influence on the evolution and funciton of the genes related to hypoxia adaption, growth promotion and osmoregulation in naked carp. Although the main functional domains are higly conserved in the HIF-1αof naked carp,the evolution rate in lineages leading to hypoxia-tolerant carps, just like naked carp and crucian carp, is greater than that in lineages leading to other vertebrates, which maybe is a combined result of genome duplication and their hypoxia-tolerant ability. Compared to common carp and goldfish, evolution analyses showed that genome duplication has lesser influence on the relaxation of purifying selection in evolution of ncGH. We observed the phenomena of salinity-induced increases of GH-IGF-I circulating levels in naked carp. To our knowledge, this is the firtst report in Cypriniformes fish. The IGF-I and IGF-II genes of naked carp are higly conserved with other Cypriniformes fish.