Comparative Biochemistry and Physiology, Part B 185 (2015) 24–33

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Molecular cloning and tissue expression of uncoupling protein 1, 2 and 3 genes in Chinese perch (Siniperca chuatsi) Zheng-Yong Wen a, Xu-Fang Liang a,⁎, Shan He a, Ling Li a, Dan Shen a, Ya-Xiong Tao b a College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, Hubei 430070, China b Department of Anatomy, Physiology, and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA

a r t i c l e

i n f o

Article history: Received 17 November 2014 Received in revised form 17 March 2015 Accepted 22 March 2015 Available online 28 March 2015 Keywords: Uncoupling protein Gene structures Gene expression Thermogenesis

a b s t r a c t Uncoupling proteins (UCPs) are mitochondrial anion carrier proteins, which play important roles in several physiological processes, including thermogenesis, reactive oxygen species generation, growth, lipid metabolism and insulin secretion. Although the roles of UCPs are well understood in mammals, little is known in fish. To investigate the thermogenesis roles in Chinese perch (Siniperca chuatsi), we cloned the UCP1, 2 and 3. The UCP1 consisted of six exons and five introns, and the UCP2 consisted of eight exons and seven introns. The UCP1 was primarily expressed in liver, UCP2 was ubiquitously expressed, and UCP3 was primarily expressed in muscle. The mRNA levels of UCP1 and UCP2 in liver, and UCP3 in muscle were significantly increased after prolonged cold exposure, but did not change after prolonged heat exposure, suggesting that Chinese perch might have a mechanism of response to cold environment, but not to hot environment. The intestinal UCP1 mRNA level was significantly up-regulated after prolonged heat exposure, while the UCP2 mRNA level was significantly upregulated after prolonged cold exposure, suggesting that the two paralogs might play different roles in intestine of Chinese perch. In addition, the phylogenetic analysis could shed new light on the evolutionary diversification of UCP gene family. © 2015 Published by Elsevier Inc.

1. Introduction The uncoupling proteins (UCPs) are members of the mitochondrial anion carrier family, transporting substrates across mitochondrial inner membrane (Krauss et al., 2005; Mookerjee et al., 2010). Five UCPs have been identified in mammals. UCP1 is highly expressed in brown adipose tissue and well known for its role in adaptive nonshivering thermogenesis, particularly in hibernators and newborns (Cannon and Nedergaard, 2004, 2010). UCP2 and UCP3 have 59% and 57% of amino acid sequence in common with UCP1, and they are widely distributed throughout the organism, suggesting different functions for these UCPs (Echtay, 2007; Diano and Horvath, 2012). Because of its ubiquitous distribution, UCP2 has been suspected to participate in several metabolic processes, such as food intake (Coppola et al., 2007; Andrews et al., 2008), fatty acid metabolism (Brookes et al., 2008), insulin secretion (Pi and Collins, 2010) and immune responses (Tagen et al., 2009; Emre and Nubel, 2010). UCP3 is expressed most abundantly in skeletal muscle and, to a lesser extent, in brown adipose tissue and heart, and might play important roles in attenuation of reactive oxygen species production

⁎ Corresponding author at: College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, P.R.China. Tel.: +86 27 8728 8255; fax: +86 27 8728 2114. E-mail address: [email protected] (X.-F. Liang).

http://dx.doi.org/10.1016/j.cbpb.2015.03.005 1096-4959/© 2015 Published by Elsevier Inc.

(Affourtit et al., 2007), fatty acid metabolism (Himms-Hagen and Harper, 2001) and exporting lipid hydroperoxides (Goglia and Skulachev, 2003). UCPs are not limited only to mammals, but also found in many eukaryotes, such as protozoa, plants and fish, suggest that their evolutionary emergence probably occurred before the divergence of fungal, plant and animal kingdoms (Saito et al., 2008; Hughes et al., 2009; Tseng et al., 2011). Three members of UCP gene family (UCP1, UCP2 and UCP3) have been unequivocally identified in several fish species, including zebrafish (Danio rerio) (Stuart et al., 1999), puffer fish (Takifugu rubripes) (Jastroch et al., 2005), rainbow trout (Oncorhynchus mykiss) (Coulibaly et al., 2006) and gilthead sea bream (Sparus aurata) (Bermejo-Nogales et al., 2010). The discovery of UCP homologues in fish raises the question concerning their primary physiological role in thermoregulation (Jastroch et al., 2005, 2007; Mark et al., 2006; Bermejo-Nogales et al., 2010; Tseng et al., 2011). Mark et al. (2006) reported the UCP-mediated effects on whole-body thermogenesis in Antarctic fish. UCP1 was involved in the brain thermogenesis in carp (Jastroch et al., 2007). However, Bermejo-Nogales (Bermejo-Nogales et al., 2010) indicated that the expression of hepatic UCP1 was highest in summer and autumn and markedly lower during cold-exposure in winter, suggesting that UCP1 in ectothermic fish might not play a thermogenic role of hepatic. The thermogenic role of fish UCP gene remains unclear since investigations dealing with ectothermic UCP genes are limited.

Z.-Y. Wen et al. / Comparative Biochemistry and Physiology, Part B 185 (2015) 24–33 Table 1 PCR primer sequences for cloning, real-time PCR, and SNP screening of Chinese perch UCP1, UCP2 and UCP3 genes. Name of primer

Sequence of primer

UCP101F UCP102R UCP201F UCP202R UCP301F UCP302R UCP1RT UCP1S1 UCP1A1 UCP1S2 UCP1A2 UCP2 5′01R UCP2 5'02R UCP1P1 UCP1P2 UCP2P1 UCP2P2 UCP1I10F UCP1I10R UCP1I20F UCP1I20R UCP1I30F UCP1I30R UCP1I50F UCP1I50R UCP2I10F UCP2I10R UCP2I30F UCP2I30R UCP2I40F UCP2I40R DUCP103F DUCP104R DUCP203F DUCP204R DUCP303F DUCP304R DACT01F DACT02R DTUBα 1-F DTUBα 1-R

5′-TCACCTTTCCACTGGACACCGCA (C/T) AAG (A) GT-3′ 5′-ATAGGTGACAAACATA (C) ACC (T) ACA (G) TTCCA-3′ 5′-TTTCCACTGGACACCGCAAA (G) GT-3′ 5′-GTGACAAACATAACCACA (G) TTCCA-3′ 5′-TTTACAAC (T)GGACTGGTGGC-3′ 5′-TCCCTCA(T)TCTCGGGCAAT-3′ 5′-(P)CTTGATCAGGTCGTA-3′ 5′-TCTGTAACGGATGCCTTC-3′ 5′-AGGCACACTACCCAACATCAC-3′ 5′-GCAGTCTTCTCTCCCTGAAT-3′ 5′-GCTAGTCAACTGCACAGAGC-3′ 5′-CGATACCAACATGATCAGAGC-3′ 5′-GAGACCAATGCGGACAGAG-3′ 5′-CTAGATACATGAACTCACC-3′ 5′-CAGTACAAGAGCGCTATCA-3′ 5′-CTTGGCCAGTACAGCAGTGTG-3′ 5′-GTGCTGCTGCCATGATGAG-3′ 5′-CTGCATAGCTGACATTGTCAC-3′ 5′-TGTCATAGAGGCCGATTCTGA-3′ 5′-TCAGAATCGGCCTCTATGACA-3′ 5′-CCATTCAGGTTCATCTGG-3′ 5′-CCATTCAGGTTCATCTGG-3′ 5′-GGAAGCAATCACCGTGGTA- 3′ 5′-TACCACGGTGATTGCTTCC- 3′ 5′-AACATCACAACATTCCACG-3′ 5′-GGTCACTTGGAGAGGCAT-3′ 5′-CAGGTGGCTTATTAATGTGG-3′ 5′-TCGCTGACCTGCTCACCTT-3′ 5′-GAGACCAATGCGGACAGAG-3′ 5′-CTCTGTCCGCATTGGTCTC- 3′ 5′-CATCACCACGTTCCAGGAG- 3′ 5′-CGATTCCAAGCCCAGATGA-3′ 5′-GCACCAAACGCAGATACAA-3′ 5′-AGCCACCTGTCTACCCAT-3′ 5′-GCCAAACACTCCACGATA-3′ 5′-CTGGACGCCTACAAGACGAT-3′ 5′-CTAAACGCAGCAGTGAAGTG-3′ 5′-CGTGACATCAAGGAGAAGC-3′ 5′-TCTGCTGGAAGGTGGACAG-3′ 5′-CACTGCCACCCAGAAGACA-3′ 5′-AGGGACACGGAAAGCCAT-3′

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et al., 1988; Li, 1991). Chinese perch adapts well to the extremely cold winter of Amur River. In contrast to few disease outbreaks in cultivation areas within its natural distribution, Chinese perch has been transferred to and reared in southern China (especially Guangdong Province) with lots of disease outbreaks in the hot seasons. Examination of UCPs genes in Chinese perch will supply informative clues to the currently nonhealthy culture beyond its natural distribution, and may also allow future development of tools for improving the culture of this species. It could also lead to a better understanding the thermogenesis mechanisms of uncoupling protein genes in Chinese perch, especially the acanthomorphic fish. 2. Materials and methods 2.1. Fish sampling Chinese perch, Siniperca chuatsi, (weight 400–500 g) used in this study were obtained from Guangdong Mandarin Fish Farm (Nanhai, Guangdong Province, China) and transported to the experimental aquarium in the College of Fisheries (Huazhong Agricultural University). Fish were acclimated in 100 L tanks for 3 weeks (20 animals per tank) in constantly running aerated well water (temperature 25.5 ± 0.5 °C, pH 6.8 ± 0.1) under a 12 h:12 h light:dark photoperiod (lights on at 8:00 h). Fish were fed once a day, at 10:00 h, with live prey fish (ratio equivalent to 4% of body mass per day). All experiments were carried out in the morning to avoid possible effects of circadian variations. Following acclimation, fish were randomly assigned to experimental tanks and reared at different temperature (18, 25 or 32 °C) for a month. For cloning and tissue distribution studies, fish were dissected to obtain brain, gill, heart, stomach, intestine, kidney, eye, spleen, liver, muscle and adipose tissue. For gene expression studies, experimental fish were dissected to obtain liver, intestine and muscle tissue. Fish were euthanized with MS-222 (10 mg L−1) before being handled and sampled. Tissues were immediately dissected out, frozen in liquid nitrogen and stored at stored at −80 °C until RNA extractions were performed. 2.2. RNA isolation and reverse transcription

Chinese perch Siniperca chuatsi (Basilewsky) is one of the most valuable food fish in China. It is endemic to the fresh waters between the Yangtze River and the Amur River, and the distribution of this fish is suggested to be mainly determined by environmental temperature (Zhou

Total RNA was isolated from different tissues using SV Total RNA Isolation System (Promega, USA), according to instructions from the manufacturer. Reverse transcription was performed with oligo(dT)18 primer using First Strand cDNA Synthesis Kit (Toyobo, Japan), according to instructions from the manufacturer.

Table 2 Listing of uncoupling protein sequences used in this study. Protein IDs are given to allow access to the protein sequence on Ensembl or GenBank website. Species

Takifugu Tetraodon Stickleback Medaka Chinese perch Zebrafish Cavefish Anole Lizard Frog Chicken Mouse Rat Cow Dog Elephant Human

Protein ID UCP1

UCP2

UCP3

UCP3L

ENSTRUP00000033443 ENSTNIP00000009630 ENSGACP00000022833 ENSORLP00000023151 ACJ09053.1 ENSDARP00000037614 ENSAMXP00000005735

ENSTRUP00000037074 ENSTNIP00000014758 ENSGACP00000026903 ENSORLP00000011390 ACI32422.1 ENSDARP00000063358 ENSAMXP00000007005 ENSACAP00000006358 ENSXETP00000055772

ENSTRUP00000037001 ENSTNIP00000014759 ENSGACP00000026900 ENSORLP00000011396 KP339882 ENSDARP00000105553 ENSDARP0000007013 ENSACAP00000009237

ENSTRUP00000017095 ENSTNIP00000019332 ENSGACP00000014905 ENSORLP00000005419

ENSXETP00000032640 ENSMUSP00000034146 ENSRNOP00000004900 ENSBTAP00000006097 ENSCAFP00000005489 ENSLAFP00000005947 ENSP00000262999

ENSMUSP00000120967 ENSRNOP00000024156 ENSBTAP00000004810 ENSCAFP00000008312 ENSLAFP00000013302 ENSP00000312029

UCP4

UCP5 ENSTNIT00000010587 ENSGACT00000027594 ENSORLT00000019382

ENSDARP00000062924 ENSAMXT00000010680

ENSDART00000139178 ENSAMXT00000007261 ENSACAT00000008802

ENSXETP00000035487 ENSGALP00000027932 ENSMUSP00000032958 ENSRNOP00000024005 ENSBTAP00000006918 ENSCAFP00000008310 ENSLAFP00000004523 ENSP00000323740

ENSMUSP00000024705 ENSRNOP00000039207 ENSBTAP00000021691 ENSCAFP00000002993 ENSLAFP00000017959 ENSP00000360398

ENSGALT00000032427 ENSMUST00000033431 ENSRNOT00000009637 ENSBTAT00000065224 ENSCAFT00000038395 ENSLAFT00000037448 ENST00000545805

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2.3. PCR cloning and sequencing of partial and full-length Chinese perch UCP1-3 cDNA Degenerate primers were designed to clone partial UCP1-3 cDNA sequences of Chinese perch by PCR (UCP101F and UCP102R for UCP1, UCP201F and UCP202R for UCP2, UCP301F and UCP302R for UCP3) (Table 1). Gene-specific primers were designed for 5′-RACE and 3′-RACE of UCP1 and UCP2 cDNA (Table 1). The expected cDNA fragments were 800 bp and 620 bp, respectively. For UCP1, 5′-RACE was performed with 5′-Full RACE Core Set (TaKaRa, Japan). For UCP2, 5′-

RACE was performed with SMART RACE cDNA Amplification Kit (Clontech, USA). The final PCR products were analyzed by agarose gel electrophoresis, cloned into pGEM-T Easy vector and sequenced. 2.4. Cloning of intronic DNA and 5′-flanking region of Chinese perch UCP1 and UCP2 genes Genomic DNA was isolated from Chinese perch with Blood & Cell Culture DNA Kit (Qiagen, USA) according to the manufacture's recommendations. Universal Genome Walker Kit (Clontech, USA) was used

Fig. 1. The UCP1 gene sequence of Chinese perch. The ATG start codon and TGA translation stop codon are boldfaced. The thicker line under (AATAAA) is polyadenylation signal. The introns are lowercase. The sense strand is displayed from the 5′ to 3′ direction. Putative regulatory elements including CEBP, SP1, AP1, AP2, CAAT box, TATA box, CREB, CREBP and AHR/ARNT identified in the sequence are underlined.

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for cloning the 5′-flanking region of Chinese perch UCP1 and UCP2. The final PCR products were analyzed by agarose gel electrophoresis, cloned into pGEM-T Easy vector and sequenced. Primers for PCR amplification of introns of UCP1 and UCP2 genes are listed in Table 1.

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used to construct the phylogenetic trees with Neighbor-Joining approach using Mega 6.0. The vertebrate tree with midpoint rooting as implemented in Mega. The robustness of the tree topology was assessed by non parametric bootstrap analysis with 1,000 resampling replicates. The predicted protein sequences IDs are shown in Table 2.

2.5. Phylogenetic analysis 2.6. mRNA quantification by real-time PCR The predicted Chinese perch UCP amino acid sequences together with other teleost UCP genes as well as those identified in other vertebrates were aligned by CLUSTAL X2.1. The aligned amino acid data sets were

Extraction of total RNA from fish tissues and first strand cDNA synthesis were performed as described above. Then real-time PCR was

Fig. 2. The UCP2 gene sequence of Chinese perch. The ATG start codon and TAA translation stop codon are boldfaced. The thicker line under (AATAAA) is polyadenylation signal. The introns are lowercase. The sense strand is displayed from the 5′ to 3′ direction. Putative regulatory elements including HNF3, CEBP, CDXA, AP1, USF, XFD1, SRY,, GATA box, CAAT box, TATA box GRE, and EpRE identified in the sequence are underlined.

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used to detecte the mRNA expression of UCP1-3 with CFX ConnectTM Real-Time system (Bio-Rad). Reverse transcription product was used for real-time PCR in a final volume of 10 μL. The end products of PCR were verified with the melting curves that showing a single peak specific for the target gene. Values are expressed as means ± SD (N = 5). The amplification efficiencies of reference and target genes are given in Table 1. A set of five housekeeping genes (β-actin, GAPDH, EF1α, Tubα 1) were selected from the literature (Mc Curley and Callard, 2008; Zhao et al., 2011) in order to test their transcription stability. By using geNorm software (Vandesompele et al., 2002), two genes with the most stable expression across the experimental conditions were β-actin and Tubα 1. The relative expression levels were calculated using the Pfaffl method (Pfaffl, 2001) when normalized to the geometric mean of the best combination of two genes (β-actin and Tubα 1) as suggested by geNorm.

2.7. Statistical analysis Statistical analysis was performed with SPSS19.0 software. Significant differences were found using one-way analysis of variance (ANOVA), followed by the post hoc test (least significant difference test and Duncan's multiple range test), after confirming for data normality and homogeneity of variances. Differences were considered to be significant if P b 0.05.

3. Results 3.1. Cloning and sequence analysis of the Chinese perch UCP1, 2 and 3 cDNAs The UCP1 cDNA was 1519 bp in length, containing a 116 bp 5′untranslated region (5′UTR), a 461 bp 3′-untranslated region (3′UTR) and a 942 bp open reading frame (ORF) encoding 313 amino acids (Fig. 1). A consensus polyadenylation signal (AATAAA) was located at 17 bp upstream of the poly(A). The UCP2 cDNA was 1882 bp in length, containing a 342 bp 5′UTR, a 604 bp 3′UTR and a 936 bp ORF encoding 312 amino acids (Fig. 2). A consensus polyadenylation signal (ATTAAA) was located at 16 bp upstream of the poly(A). The UCP3 cDNA was 1542 bp in length, containing a 205 bp 5′UTR, a 362 bp 3′UTR and a 930 bp ORF encoding 309 amino acids,the gene include 5 introns and the predicted protein include 6 transmembrane domains (Fig. 3). As shown in Figs. 4–6, the predicted amino acid sequences of Chinese perch UCP1, UCP2 and UCP3 contained three mitochondrial carrier protein motifs, six transmembrane α-helix domains, the UCP signature motifs, and the putative purine-nucleotide binding domain.

3.2. Characterization of 5′-flanking region of Chinese perch UCP1 and UCP2 genes Analysis of a 1333 bp promoter region of Chinese perch UCP1 gene revealed typical TATA box, CAAT box and motifs for SP1, AP1 and AP2 (Fig. 1). Many potential regulatory elements were identified, including binding sites for CEBP, cAMP response element binding protein, peroxisome proliferator activated receptor, aryl hydrocarbon receptor (AHR) and its dimerization partner, the AHR nuclear translocator (ARNT) (Fig. 1). Analysis of an 1800 bp promoter region of Chinese perch UCP2 gene revealed typical TATA box, CAAT box and motifs for AP1 and GATA (Fig. 2). Many potential regulatory elements were also identified, including binding sites for CEBP, CREB, hepatocyte-enriched nuclear transcription factor 3 (HNF3), myeloid zinc finger protein 1 (MZF1), upstream stimulatory transcription factor (USF), and sexdetermining region Y gene product (SRY) as well as glucocorticoid response element (GRE) and electrophile response element (EpRE) (Fig. 2).

Fig. 3. Complete CDS sequences and predicted amino acids of Chinese perch uncoupling protein 3 (UCP3). Putative transmembrane conserved domains are indicated by boxes. The CDS sequences are indicated by lowercase, and the amino acids sequences are indicated by uppercase. Letters in lower case represent the 5′ and 3′ untranslated regions. Intron positions are indicated by arrows. Stop codon is indicated by (*).

3.3. Characterization of gene organization of Chinese perch UCP1 and UCP2 The total gene size of the Chinese perch UCP1 and UCP2 were 3146 bp and 2890 bp, respectively, and both were much smaller than the mammalian orthologues. Chinese perch UCP1 gene consisted of six exons and five introns, whereas UCP2 gene consisted of eight exons and seven introns (Fig. 7). The exon numbers of Chinese perch UCP1 and UCP2 were identical to the orthologs of other vertebrates although exon lengths were observed to be variable in untranslated regions. Mutations apparently occurred at a slower rate in the coding region than in the untranslated region of Chinese perch UCP1 and UCP2. All the introns in Chinese perch UCP1

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Fig. 4. Alignment of the UCP1 amino acid sequence of Chinese perch with those of other fishes and mammals. The dashes in amino acid sequence indicate the amino acid gaps that are necessary to align these sequences. Asterisks indicate the amino acids conserved among all members of the proteins. Six transmembrane α-helix domains are underlined and labelled I - VI. Three mitochondrial carrier protein motifs are boxed. Purine-nucleotide binding domain is lightly shaded. UCP signature motifs are shown in white letters on black. The protein sequences IDs are shown in Table 2.

and UCP2 started as GT and ended as AG, and appeared at similar positions as in their mammalian orthologs (Fig. 7).

3.4. Phylogenetic analysis of UCPs Phylogenetic inference definitely positioned Chinese perch UCP2 within the corresponding vertebrate orthologues. However, the phylogenetic relationship of Chinese perch UCP1 to their corresponding orthologues of mammals could not be resolved (Fig. 8). Alignment of amino acid sequence indicated that the predicted Chinese perch UCP1 had higher identities to the orthologs of cyprinid fishes (82%) than mammals (55.7–57.3%), whereas Chinese perch UCP2 had similar identities to cyprinid fishes (81%) and mammals (76.9–78.5%), and Chinese perch UCP3 had higher identities to the orthologs of Perciformes fishes (94%) than mammals (66.5–71.2%).

3.5. Analysis of the tissue distribution of Chinese perch UCP1, 2 and 3 Expression of UCPs mRNA was evaluated by real time PCR in different Chinese perch tissues (Fig. 9). The UCP1 mRNA was primarily expressed in liver, and lower level of expression was detected in other tissues (Fig. 9A). UCP2 was ubiquitously expressed in Chinese perch, with high expression in liver, stomach and eye, and lower expression in brain, intestine, kidney, heart, gill, muscle and adipose, and lowest in spleen (Fig. 9B). Regarding to UCP3, it was mainly expressed in muscle, and lower expression in heart and other tissues (Fig. 9C). 3.6. Chinese perch UCPs mRNA expression response to different temperature The mRNA expression levels of UCP1, 2 and 3 of Chinese perch reared in different temperatures (18, 25 or 32 °C) were measured. In contrast

Fig. 5. Alignment of the UCP2 amino acid sequences of Chinese perch with those of other fishes, mammals and amphibian. Dashes indicate the amino acid gaps that are necessary to align these sequences. Asterisks indicate the amino acids conserved among all members of the proteins. Six transmembrane α-helix domains are underlined and labelled I - VI. Three mitochondrial carrier protein motifs are in boxes. Purine-nucleotide binding domain are lightly shaded. UCP signature motifs are shown in white letters on black. The protein sequences IDs are shown in Table 2.

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Fig. 6. Alignment of the UCP3 amino acid sequences of Chinese perch with those of other fishes, mammals and reptile. Dashes indicate the amino acid gaps that are necessary to align these sequences. Asterisks indicate the amino acids conserved among all members of the proteins. Six transmembrane α-helix domains are underlined and labelled I - VI. Three mitochondrial carrier protein motifs are in boxes. Purine-nucleotide binding domain are lightly shaded. UCP signature motifs are shown in white letters on black. The protein sequences IDs are shown in Table 2.

to 25 °C group, hepatic UCP1 and UCP2 mRNA levels were significantly up-regulated in 18 °C group but have no significant change in 32 °C group, as well as UCP3 expression in muscle (Fig. 10A, C, E). However, intestinal UCP1 mRNA level was dramatically up-regulated in 32 °C group but was not different from the 18 °C group, intestinal UCP2 mRNA level was dramatically up-regulated in 18 °C group but dramatically down-regulated in 32 °C group (Fig. 10B, D). 4. Discussion In this study, UCP1, 2 and 3 were cloned and sequenced in Chinese perch. Chinese perch UCP1 shared high amino acid identity (N 80%) with cyprinid fishes, but only 55.7-57.3% identity with mouse and human UCP1. However, Chinese perch UCP2 shared similar identities with cyprinid fishes (81%) and mammals (76.9-78.5%), which is consistent with results observed in other species (Stuart et al., 1999; Jastroch et al., 2005; Tine et al., 2012; Bermejo-Nogales et al., 2013). In addition, Chinese perch UCP3 shared higher identities with the orthologs of

Perciformes fishes (94%) than those of mammals (66.5–71.2%). The UCPs with different identities suggested a functional diversion of vertebrate UCPs in the evolutionary process. Five UCP homologues have been discovered in vertebrates, beginning with canonical UCP1, which is an important regulator of adaptive thermogenesis in mammals (Cannon and Nedergaard, 2004, 2010). With approximately 60% homology with UCP1, the orthologs UCP2 (Affourtit et al., 2007) and UCP3 (Echtay, 2007; Jastroch et al., 2010) are more closely related to UCP1 than to UCP4 (Azzu et al., 2010) or UCP5 (also named BMCP1) (Jia et al., 2009). However, Nedergaard and Cannon (Nedergaard and Cannon, 2003) suggested that these carriers (UCP4 and UCP5) are wrongly annotated as UCP and do not belong to the uncoupling protein family. In the present study, the phylogenetic tree of vertebrate UCPs showed that UCP1, UCP2 and UCP3 were grouped into the same clade, but UCP4 and UCP5 did not belong the core UCP family. The results were consistent with several previous studies (Hughes and Criscuolo, 2008; Hughes et al., 2009; Tine et al., 2012), and suggested that UCPs family members

Fig. 7. Schematic representation of the exon-intron structures of Chinese perch UCP1 (a) and UCP2 (b) genes. Black box indicate UTR and white box represent extrons, the introns are indicated by line. The size of exon 1-6 of Chinese perch UCP1 gene was 242, 199, 198, 102, 181 and 557 bp, respectively, and intron 1-5 was 323, 734, 93, 329 and 148 bp respectively. The size of exon 1-8 of Chinese perch UCP2 gene was 105, 155, 205, 217, 198, 102, 181 and 716 bp, respectively, and intron 1-7, 89, 294, 230, 95, 95, 92 and 106 bp, respectively.

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Fig. 8. Neighbor-Joining tree depicting the phylogenetic relationship of vertebrate UCP proteins. The phylogenetic tree was conducted with 67 amino acid sequences using MEGA 6.0 program. The values at the nodes represent bootstrap percentages from 1000 replicates. The tree was rooted with midpoint rooting. The protein sequences ID in each species are shown in Table 2.

should be reconsidered. In addition, a novel member named UCP3-like is found only in teleost fish lineage (Jastroch et al., 2005; BermejoNogales et al., 2010). We also found that UCP3-like was grouped in the same clade with UCP3, suggesting that UCP3L might be a fish-

specific gene derived from the whole genome duplication (Tine et al., 2012). The UCP1 gene consisted of six exons and five introns, whereas the UCP2 gene consisted of eight exons and seven introns. Chinese

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Fig. 9. The tissue distribution of Chinese perch uncoupling protein genes by real-time PCR. The tissue expression of UCP1 gene in Chinese perch (A). The tissue expression of UCP2 gene in Chinese perch (B). The tissue expression of UCP3 gene in Chinese perch (C). The results are expressed as mean ± SEM.

tissue-specificity. UCPs tissue mRNA expressions were detected in Chinese perch. UCP1 was primarily expressed in the liver, which is in strong agreement with previous studies in other fishes (Liang et al., 2003; Jastroch et al., 2005; Liao et al., 2006; Bermejo-Nogales et al., 2010; Tseng et al., 2011). Additionally, UCP2 was found to be ubiquitously expressed in Chinese perch, which was also in line with other studies (Liang et al., 2003; Jastroch et al., 2005; Coulibaly et al., 2006; Mark et al., 2006; Tseng et al., 2011). UCP3 was mainly expressed in muscle, and lower expressed in heart. The different gene structures and tissue expression patterns of UCPs might contribute to different functions in fish (Stuart et al., 1999; Dos Santos et al., 2013). In order to better understand the UCPs thermogenesis roles in Chinese perch, the UCPs mRNA expression in response to thermal accumulation were detected. Results showed that the hepatic UCP1 and UCP2 expression were significantly up-regulated after prolonged cold exposure, but did not change after prolonged heat exposure. The result was consistent with the previous study that the hepatic UCP2 of polar fish (Z. Viviparus) was up-regulated both in mRNA and protein levels after prolonged cold exposure (Mark et al., 2006). However, hepatic UCP1 of common carp was highest in summer, and hepatic UCP1 of gilthead sea bream was down-regulated after cold accumulation (Jastroch et al., 2005; Bermejo-Nogales et al., 2010). Moreover, UCP3 expression in muscle of Chinese perch was up-regulated after a long time exposure to low temperatures, which was consistent with other fishes, including carp (Gracey et al., 2004), gilthead sea bream (Bermejo-Nogales et al., 2011) and goldfish (Dos Santos et al., 2012). High levels of UCPs could be the result of an overall increase in mitochondrial capacity, frequently found during cold acclimation (Guderley and St-Pierre, 2002; Portner, 2002; Mark et al., 2006). As the depressed metabolic activity and increased energy demand in low temperature environment, UCP1, 2 and 3 might play an important role in energy metabolism with thermogenic functions at cold acclimation in fish. The increased expression of UCPs in Chinese perch after cold exposure and no changes after warm accumulation, suggest that Chinese perch might have a mechanism of response to cold environment, but not to hot environment. This could lead to increase illness outbreak of Chinese perch in the south of China, where higher water temperature is present all year round. The intestinal UCP1 mRNA level was significantly up-regulated after prolonged heat exposure, while the UCP2 mRNA level was significantly up-regulated after prolonged cold exposure. The results suggested the two paralogs might play different roles in intestine of Chinese perch (Stuart et al., 1999; Dos Santos et al., 2013). Furthermore, we also observed similar thermogenic changes of mRNA expression of UCP1 in liver, UCP2 in liver and intestine, and UCP3 in muscle. It is suggested that all of the three UCPs might have a similar thermal response to cold environment and play a similar role in energy metabolism. In summary, UCP1, 2 and 3 genes were identified from Chinese perch. The different gene structures and tissue expression patterns might contribute to varying physiological functions. The mRNA levels of UCPs genes were increased after prolonged cold exposure, suggesting their thermogenesis roles for meeting the energy and metabolism demands at cold environment. In addition, the phylogenetic analysis of vertebrate UCPs could shed new light on the evolutionary diversification of UCP gene family. Acknowledgements

perch UCP1 and UCP2 genes had a shorter introns compared with other fish or mammals, suggesting that Chinese perch UCP1 and UCP2 could be evolutionarily young and have yet to acquire an indispensable biological function (Gorlova et al., 2014). Furthermore, more putative transcription factor regulatory elements transcription factors were found in UCP1 5′-flanking region than in UCP2, suggesting that UCP1 might be involved in more physiological processes than UCP2, and the genes express differential regulation conferring

We wish to express our thanks to Dr. Jeffrey T. Silverstein for his helpful review of the manuscript. This work was financially supported by the National Natural Science Foundation of China (31272641 and 31172420), the National Basic Research Program of China (2014CB138601), the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAD25B04) and the Fundamental Research Funds for the Central Universities (2011PY030, 2013PY072).

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Fig. 10. Quantification of mRNA expression of uncoupling protein genes of Chinese perch reared at different temperatures using real-time PCR. The relative UCP1 mRNA expression in liver (A). The relative UCP1 mRNA expression in intestine (B). The relative UCP2 mRNA expression in liver (C). The relative UCP2 mRNA expression in intestine (D). The relative UCP3 mRNA expression in muscle (E). The results are expressed as mean ± SEM. Different letters mean significantly different from each other (P b 0.05).

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