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Indian Journal of Animal Research

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Full Research Article

CDS Region of Uncoupling Protein 1 (UCP1) Gene in Eothenomys miletus: A Bioinformatics and Selection Pressure Analysis

Lijuan Cao1, Ting Jia1, Shunrong Xu1, Wanlong Zhu1,*, Hao Zhang1
  • 0009-0006-5629-9250, 0000-0001-8261-4089, 0000-0001-8261-4089
1School of Life Sciences, Yunnan Normal University, Kunming, 650500, China.

ABSTRACT

Background: The present study aimed to elucidate the fundamental composition of the CDS region sequence of UCP1 gene in Eothenomys miletus, as well as the physicochemical properties, higher-order structure and structural domains of the UCP1 protein. Additionally, we analyzed the selection pressure on the UCP1 gene in mammals inhabiting different latitudes, providing a data foundation for further exploration of the UCP1 gene and its role in mammalian environmental adaptation.

Methods: The CDS region sequence of UCP1 gene was cloned using PCR and the structure and function of the UCP1 gene were characterized employing a bioinformatics platform. The UCP1 gene of Eothenomys miletus was compared with other species for sequence similarity and phylogenetic tree construction. Furthermore, selection pressure on UCP1 genes from mammals at different latitudes was analyzed.

Result: UCP1 gene encodes a protein comprising 307 amino acids, located in the mitochondria, with a molecular formula of C1498H2402N392O434S16. Threonine (Thr, T) is the most abundant amino acid, accounting for 11.1%. The protein is hydrophobic, positively charged, alkaline in nature and contains one potential N-glycosylation site and five O-glycosylation sites. It lacks a transmembrane region or signal peptide distribution and is a non-GPI-anchored protein. The secondary structure is predominantly composed of α-helices and random coils, featuring three Mito_carr structural domains. Homology comparisons revealed that the highest nucleotide sequence similarity exists between Eothenomys miletus and the species Myodes glareolus, Microtus oregoni and Arvicola amphibius. Selection pressure analysis indicated no positive selection sites for the UCP1 gene in mammals at different latitudes. The UCP1 gene in low-latitude, high-altitude mammals underwent purifying selection, forming a relatively stable function. These findings provided a theoretical basis for subsequent research on the mechanism of fat heat production and energy supply in the Eothenomys miletus UCP1 gene and a data foundation for further exploration of the role of the UCP1 gene in the adaptation of Eothenomys miletus to future environmental changes.

INTRODUCTION

The mechanisms underlying thermogenesis are of critical importance for mammals in their ability to withstand low temperatures (Geng ang Zhu, 2024), which involves not only reducing heat loss through insulation mechanisms, but also enhancing heat production via elevated metabolism (Xia, 2021), primarily through facultative thermogenesis, specifically non-shivering thermogenesis (NST) (Alhamoud et al., 2024; Chen et al., 2021). NST is associated with mitochondria in brown adipose tissue (BAT), which is distributed around the scapulae, neck and aorta (Cannon and Nedergaard, 2004). These thermogenic mechanisms play a regulatory role in body temperature, body weight and body fat content (Wang et al., 2001). Mitochondria contain uncoupling proteins (UCPs), a family of tissue-specific mitochondrial inner membrane proteins belonging to the mitochondrial anion carrier family (MACF) (Luévano-Martínez, 2012). UCPs are distributed across diverse organisms including plants, animals, fish, fungi and protozoa (Adams, 2000).
       
Rodents, as commonly used models for studying cold adaptation and energy metabolism, exhibit physiological adjustments when exposed to prolonged cold environments: Their bodies cease shivering thermogenesis (ST) and elevate NST to maintain a higher metabolic rate (Carlson and Cottle, 1956). In this process, norepinephrine released from sympathetic nerve terminals activates BAT, leading to the upregulated expression of uncoupling protein 1 (UCP1) in mitochondria (Bartness et al., 2010). This mechanism suppresses adenosine triphosphate (ATP) synthesis on BAT mitochondrial membranes redirecting energy from substrate oxidation toward heat generation (Li et al., 2014; Matthias et al., 1999). The produced heat is then distributed to other body regions via blood circulation (Stier et al., 2014), ultimately enhancing survival rates in cold conditions (Ballinger and Andrews, 2018; Janský, 1973; Kang and Chen, 2023). Furthermore, studies have shown that in rodent adipocytes, UCP1 operates in parallel with creatine kinase b (CKB), highlighting a sophisticated regulatory mechanism that underscores the crucial role of UCP1 in body temperature regulation and energy homeostasis (Guan et al., 2022; Rahbani et al., 2024; Simon et al., 1986).
       
The adaptation of animals to their environment and adaptive evolution have long been hotspots in research. For instance, Wang et al., (2022) revealed that the accelerated evolution of the FSHR gene contributes to the reproductive adaptation of “high-quality parenting’’ in mammals. Kan et al., (2024) discovered that the rapid evolutionary rate of the FOXP2 gene in songbirds is associated with their exceptional vocal learning abilities. Wang et al., (2023) demonstrated that strong purifying selection on the BMPR1B gene in pigs correlates with their high fecundity traits. Studies on the evolutionary system of the cold-related UCP1 gene can effectively and conveniently reveal its potential biological functions and environmental adaptations. Therefore, this study selected the UCP1 gene from mammals distributed in both high- and low-latitude regions to conduct selection pressure analysis, investigate its evolutionary trends and employed the widely accepted maximum likelihood (ML) method to test selection pressure (Wang et al., 2022).
               
Additionally, Wei et al., (2023) cloned and conducted bioinformatics analyses of the UCP1 gene in Nubian goats, tree shrews (Zhu, 2003), Yanbian cattle (Sun et al., 2021) and yaks (Zhao et al., 2021). However, research on UCP1 genes in species inhabiting the Hengduan Mountains-a global biodiversity hotspot-remains limited. Eothenomys miletus belonging to the subfamily Arvicolinae and genus Eothenomys, is an endemic species to the Hengduan Mountains (Gong et al., 2021; Luo et al., 2000). Characterized by its low-latitude, high-altitude environment, the Hengduan region features small annual temperature variations, large diurnal temperature fluctuations, distinct dry and wet seasons and relatively abundant food resources (Gong et al., 2009). These conditions may drive unique physiological and ecological adaptations in Eothenomys miletus. Therefore, this study sequenced the UCP1 gene of Eothenomys miletus, a rodent species endemic to the Hengduan Mountains and further investigated the biological characteristics of its UCP1 gene to gain a more comprehensive and in-depth understanding of it. This reference facilitates the study of the biological functions of the gene. Furthermore, the present study investigated the evolutionary relationship of this gene in mammals to verify its potential association with the latitude-altitude relationship and to provide fundamental data for further research on its UCP1 gene.

MATERIALS AND METHODS

Animal collection and processing
 
In the winter of 2024, field sampling was conducted in the Hengduan Mountains of Dali (100o42'49″E, 24o90'30″N). All experimental subjects were healthy adult Eothenomys miletus captured during non-reproductive periods. After disinfection and flea removal, the voles were transported to the animal facility of the School of Life Sciences, Yunnan Normal University. The voles were euthanized via CO2 anesthesia and BAT from the interscapular and cervical regions was dissected using sterile scissors, while white adipose tissue (WAT) was removed. The BAT samples were weighed, placed in cryotubes and immediately stored at -80oC for further analysis.
 
Cloning of UCP1 gene in Eothenomys miletus
 
The UCP1 gene sequence of the Alexandromys fortis (GenBank accession: XM_050161578.1) was retrieved from the NCBI database. Polymerase Chain Reaction (PCR) primers were designed using Primer 5.0 software, with forward primer F: CCAGAGCCCGACAACT and reverse primer R: CACCAAGAACACGACCTC, targeting an amplified product of approximately 970 bp. Total RNA was extracted from BAT cells using the Trizol method and RNA concentration and purity were measured. RNA integrity was verified via 1.0% agarose gel electrophoresis. cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser and stored at -20oC.PCR amplification was performed using BAT-derived cDNA as the template. The 10 μL reaction mixture included 5 μL 2 ×  Taq PCR MasterMix, 0.5 μL each of forward and reverse primers, 1 μL cDNA and 3 μL RNase-free dd H2O. The PCR protocol comprised: 94oC pre-denaturation for 3 min; 35 cycles of 94oC denaturation (15 s), 62oC annealing (15 s) and 72oC extension (30 s); followed by a final extension at 72oC for 5 min. PCR products were electrophoresed on a 1.5% agarose gel, purified using a gel recovery kit and sequenced by Kunming Shuoyang Technology Co., Ltd.
 
Bioinformatics analysis
 
Probing the physicochemical properties of Eothenomys miletus UCP1 sequence based on various bioinformatics software and further predicting its protein high-level structure (Choudhary et al., 2016) (Table 1).

Table 1: Bioinformatics software.


       
The UCP1 gene sequence of Eothenomys miletus was subjected to sequence similarity analysis using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) available in the NCBI database. Following this, a phylogenetic tree was reconstructed using the ML method implemented in MEGA X software, incorporating the following species: Myodes glareolus (XM_048447499.1), Microtus oregoni (XM_041663724.1), Arvicola amphibius (XM_038313207.1), Peromyscus eremicus (XM_059262645.1), Meriones unguiculatus (XM_021632264.2), Mus musculus (NM_009463.3), Ochotona curzoniae (XM_040967137.1) and Bos mutus (XM_005895812.2). Nucleotide sequences were aligned using default parameters and bootstrap analysis (1,000 replicates) was performed to assess node support. The resulting tree topology was visualized and annotated to elucidate evolutionary relationships among taxa.
               
To investigate the adaptive evolution of the UCP1 gene in mammals inhabiting distinct latitudinal and altitudinal environments, we retrieved 18 mammalian UCP1 gene sequences from the NCBI database (Table 2). A phylogenetic tree was first reconstructed using the maximum likelihood (ML) method in MEGA X, followed by selection pressure analysis implemented in PAML 4.9a (codeml module) using site and branch models (Wu et al., 2021). The selection coefficient ω (nonsynonymous/synonymous substitution rate ratio) was calculated to quantify adaptive evolutionary trends at codon levels. We employed the following nested models to detect codon-level positive selection: M0 (single-ratio model: uniform ω across all sites), M1 (nearly neutral model: two site classes-conserved sites (0 < ω < 1) and neutral sites (ω = 1), M2 (positive selection model: adds a third class of sites with ω >1), M3 (discrete model: ω varies across sites under a discrete distribution), M7 (β-distribution model: ω varies between 0 and 1), M8 (β+ω > 1 model: extends M7 by allowing a class of positively selected sites). Likelihood ratio tests (LRTs) were performed to compare nested models: M1 vs. M2, M0 vs. M3 and M7 vs. M8. Sites with posterior probabilities >0.95 under Bayesian empirical Bayes (BEB) analysis were considered under positive selection. To detect lineage-specific accelerated evolution, branch models were applied under two scenarios: Low-latitude, high-altitude mammals labeled as foreground branches. High-latitude, low-altitude mammals labeled as foreground branches. For each scenario, models M0 (single ω for all branches) and M2 (separate ω for foreground vs. background branches) were compared using LRTs. A significantly higher ω in foreground branches (p<0.05) indicated accelerated evolution driven by positive selection.

Table. 2: Mammalian UCP1 gene information.

RESULTS AND DISCUSSION

Homology comparison of UCP1 in Eothenomys miletus with other species
 
The nucleotide sequences of UCP1 in Eothenomys miletus showed the highest similarity with those of Myodes glareolus, Microtus oregoni and Arvicola amphibius, while lower similarity was observed with Ochotona curzoniae (81.696%) and Bos mutus (80.022%) (Table 3). Phylogenetic tree analysis aligned with sequence comparisons (Fig 1). Consequently, it is hypothesized that the UCP1 gene is relatively conserved in rodents, yet it also exhibits specificity among different species (Liu et al., 2016 and Fu et al., 2006). This finding is analogous to the results of Gao, (2014) analysis of partial sequences of the UCP1 gene in the Chinese-Myanmar tree shrew and also supports the view of Beáta et al., (2024) that the UCP1 gene exhibits both conservation and species specificity between humans and rodents.

Fig 1: Phylogenetic tree of UCP1 gene.



Table 3: BLAST comparison of species information.



Bioinformatics analysis of UCP1 gene sequence
 
The UCP1 gene in Eothenomys miletus encodes 307 amino acids (Table 4), with a molecular weight of 33 kDa, theoretical isoelectric point (pI) of 9.23, molecular formula C1498H2402N392O434S16, total atom count of 4,742, instability index of 32.61, extinction coefficient of 24,785, grand average of hydropathicity (GRAVY) of 0.157 and a half-life of 30 hours. The protein is alkaline, with threonine (Thr, T) being the most abundant amino acid (11.1%) (Fig 2). It contains 19 negatively charged and 28 positively charged residues. Post-translational modifications (PTMs) include 33 phosphorylation sites (15 serine [S], 17 threonine [T] and 1 tyrosine [Y]) (Fig 3), 1 potential N-glycosylation site at position 188 (Fig 4) and 5 O-glycosylation sites at positions 3, 5, 6, 7 and 12. Eothenomys miletus is an endemic species of the Hengduan Mountains in China (Luo et al., 2000). In order to adapt to environmental changes at different altitudes in this region, the species enhances its thermogenic capacity by upregulating UCP1 expression in brown adipose tissue, thereby better coping with cold environments (Han et al., 2022). Furthermore, post-translational modifications (PTMs), including phosphorylation and glycosylation, have been demonstrated to play a pivotal regulatory role in the process of mRNA translation into proteins and are implicated in a variety of biological processes (Wang and Zhang, 2019). Therefore, the present study conducted a bioinformatics analysis of the UCP1 protein in Eothenomys miletus, systematically elucidating its basic characteristics, including amino acid composition, molecular weight, hydrophilic/hydrophobic amino acid ratio and phosphorylation and glycosylation sites. This study provides important data for further exploration of the functional role of the UCP1 gene.

Table 4: Amino acid composition of UCP1 protein.



Fig 2: Amino acid proportion of UCP1 protein.



Fig 3: Prediction of UCP1 protein phosphorylation site in Eothenomys miletus.



Fig 4: Prediction of N-glycosylation site of UCP1 protein in Eothenomys miletus.


 
Subcellular localization and structural features
 
UCP1 is localized to mitochondria, lacks a GPI anchor or transmembrane domains and contains ~11.20 amino acids embedded in transmembrane regions (Fig 5). The first 60 amino acids include 5.52 transmembrane helices, with a 21.93% probability of cytoplasmic orientation. The protein exhibits strong hydrophobicity (peak value: 2.311) and weak hydrophilicity (minimum: -2.033), with dominant hydrophobic regions at positions 4-7 (Fig 6). Signal peptide analysis (D=0.214; cutoff=0.450) confirmed UCP1 as a non-secretory protein (Fig 7). Secondary structure comprises 51.79% α-helices, 37.13% random coils and 11.07% extended strands (Fig 8). Three Mito_carr domains span residues 10–107, 109–206 and 209–300 (Fig 9). Tertiary modeling (SWISS-MODEL) yielded a high-quality structure (GMQE=0.87, 82.27% sequence identity) (Fig 10). The first identification of UCP1 was in the fat mitochondria of hamsters, rats and guinea pigs in the mid-1970s, with particularly high activity in BAT (Rodríguez-Cuenca et al., 2010). Subsequent studies revealed that UCP1 is also expressed in the thymus of mice (Adams et al., 2008). As a pivotal mitochondrial inner membrane protein, UCP1 exerts a pivotal role in temperature regulation, energy metabolism and obesity control in animals (Zhou et al., 2022). This study shows that the UCP1 protein in E. miletus is localized in mitochondria. The secondary and tertiary structure prediction results of the UCP1 protein are consistent, indicating significant structural stability and functionality. This provides a foundation of data for a better under-standing of UCP1.

Fig 5: Prediction of UCP1 protein transmembrane region in Eothenomys miletus.



Fig 6: Prediction of hydrophilicity/hydrophobicity of UCP1 protein in Eothenomys miletus.



Fig 7: UCP1 protein signal peptide prediction in Eothenomys miletus.



Fig 8: Prediction of UCP1 protein secondary structure in Eothenomys miletus.



Fig 9: Prediction of UCP1 protein domain in Eothenomys miletus.



Fig 10: Prediction of tertiary structure of UCP1 protein in Eothenomys miletus.


 
Selection pressure analysis
 
In this study, the ML method in MEGA X was used to construct the phylogenetic tree of the UCP1 gene (Fig 11). The topology of the phylogenetic tree constructed by MEGA X was basically consistent with the traditional classification and the bootstrap values were also basically consistent, with most above 90 and a few below 70. It is inferred that the evolutionary rate of the UCP1 gene is moderate and has basically formed a relatively stable function.

Fig 11: Phylogenetic tree based on mammalian UCP1 gene.


       
Likelihood ratio tests (2ΔlnL) revealed significant differences between models M0 and M3 (2ΔlnL = 92.3228, p<0.05) but not between M7 and M8 (p = 0.99985601). M3 indicated three site classes (p0=0.02029, p1=0.02341, p2=0.58008; ω=0, 0, 0.05405), while M1 showed two classes (p0=0.82334, p1=0.17666; ω=0.11383, 1.00000). No positive selection signals were detected. During the course of a long-term evolutionary process, selective pressure does not invariably act on the entirety of a gene; rather, it acts on specific functional sites, such as particular sites within the coding region of a gene (Kamath and Getz, 2011). In this study, no positive selection sites were identified in the UCP1 gene across the selected species, indicating that UCP1 is a relatively conserved gene.
       
A comparison of the high-latitude, low-altitude (foreground branch) model (M0 vs. M2: 2ΔlnL = 0.23402, p>0.05) with the low-latitude, high-altitude model (2ΔlnL = 7.17748, p<0.05, dN/dS = 0.15496) indicates that UCP1 in low-latitude, high-altitude rodents tends toward purifying selection (Table 5). This evolutionary phenomenon can be inferred from the environmental adaptation mechanisms of Eothenomys miletus, a species endemic to the Hengduan Mountains. The low-latitude, high-altitude regions of the Hengduan Mountains possess unique environmental characteristics, including small annual temperature fluctuations and relatively abundant food resources (Gong et al., 2001). This environmental stability may have reduced the selective pressure on the UCP1 gene. Mu, (2015) observed that this species employs metabolic, regulatory and hormonal mechanisms to mitigate the effects of environmental fluctuations on the organism. Research has demonstrated that regulatory processes, encompassing metabolic and hormonal regulation, have been shown to provide adaptive contributions in response to environmental stress (Scherbarth et al., 2010; Kuzmenko et al., 2024; Kuti et al., 2022). Consequently, these regulatory adaptive mechanisms can be employed to mitigate selection pressure on the UCP1 gene. In relatively stable environments, natural selection tends to preserve the existing functional conservatism of the UCP1 gene rather than promote new adaptive mutations. Animals have been shown to optimize the thermogenic efficiency of brown adipose tissue by maintaining the stability of the UCP1 gene. This gene may confer evolutionary advantages more effectively than developing new metabolic pathways (Kamath and Getz, 2011).

Table 5: Results of the likelihood ratio test and germline selection pressure of the branch model.


               
From a functional perspective, the UCP1 gene primarily participates in energy metabolism and body temperature regulation processes, which are crucial for the survival and reproduction of the species. During the course of long-term evolution, it is imperative for this gene to preserve relatively stable functions, thereby ensuring the normal physiological activities of the organism. For instance, in cold environments, the UCP1 protein converts chemical energy into thermal energy through uncoupling oxidative phosphorylation to maintain body temperature (Dieckmann et al., 2022). This fundamental and critical function subjects the gene to strong purifying selection, continuously eliminating harmful mutations and making it difficult for adaptive mutations represented by positive selection sites to accumulate. Consequently, this process maintains functional conservation. Furthermore, UCP1 demonstrates adaptive differentiation in thermal environments: high-latitude mammals exhibit higher evolutionary rates (Su et al., 2025), while low-latitude species, such as Heteroce- phalus glaber, exhibit loss-of-function mutations (Kim et al., 2011). The UCP1 gene, located in low-latitude, high-altitude regions, functions as an evolutionary "hub," balancing ancestral thermogenesis (cold adaptation) with regulatory flexibility (environmental variability). The coexistence of thermal stress and hypoxia in high-altitude regions may drive a trade-off between thermogenesis efficiency and energy conservation, providing a natural model for studying metabolic adaptation thresholds in cross-latitude dispersal. However, further research is needed in subsequent studies to elucidate the precise function of the UCP1 gene and its regulatory mechanisms.

CONCLUSION

In conclusion, the UCP1 gene of Eothenomys miletus encodes a protein comprising 307 amino acids, characterized as hydrophobic and predominantly composed of α-helices, random coils and extended chains. Homology analysis revealed high sequence similarity between the UCP1 gene of Eothenomys miletus and that of the Myodes glareolus. Selective pressure analysis further indicates that the UCP1 gene is highly conserved in mammals. However, the UCP1 gene in mammals inhabiting low-latitude, high-altitude environments exhibits a propensity for purifying selection. Collectively, these findings contribute to a more comprehensive understanding of the functional data of the Eothenomys miletus UCP1 gene and provide a valuable foundation for future studies on its physiological functions and adaptive significance.

ACKNOWLEDGEMENT

This work was supported by the National Natural Scientific Foundation of China (No. 32160254), Yunnan Provincial Department of Education Science Research Fund Project (2025Y0308), Student Research and Training Fund Project of Yunnan Normal University (KX2024105).
 
Ethical approval
 
All animal procedures were within the rules of Animals Care and Use Committee of School of Life Sciences, Yunnan Normal University. This study was approved by the committee (13-0901-011).

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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