Gene 548 (2014) 210–216

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Gene

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Duplication and differentiation of common carp (Cyprinus carpio)myoglobin genes revealed by BAC analysis

Zi-Xia Zhao a, Peng Xu a, Ding-Chen Cao b, You-Yi Kuang b, Hai-Xia Deng a, Yan Zhang a, Li-Ming Xu a,Jiong-Tang Li a, Jian Xu a, Xiao-Wen Sun a,b,⁎a The Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, PR Chinab Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, PR China

Abbreviations: ASR, aquatic surface respiration; ARP, aP0; BAC, bacterial artificial chromosome; CDS, coding seqFISH, fluorescence in situ hybridization; HIF, hypoxia indducible factor-1; hpf, hours post fertilization; HRE, hypoxglobin; mb-1, myoglobin-1; mb-2, myoglobin-2; MEF2MYA, million years ago; NFAT, nuclear factor of activatedNFAT response elements; qRT-PCR, quantitative real timecies; Sp1, specificity Protein 1; TSS, transcription start sitestor;WGD,whole genomeduplication; zv9, zebrafishwholUTRs, 5′ untranslated regions.⁎ Corresponding author at: The Center for Applied Aqua

of Fishery Sciences, Beijing 100141, PR China.E-mail address: sunxw2002@163.com (X.-W. Sun).

http://dx.doi.org/10.1016/j.gene.2014.07.0340378-1119/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 July 2013Received in revised form 9 May 2014Accepted 11 July 2014Available online 12 July 2014

Keywords:Common carpMyoglobinDuplicationDifferentiationHypoxia

Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is ahypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein anal-ysis has shown functional specialization of the two mb transcripts. In this work, analysis formb-containing bac-terial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1)andmyoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed thatmb-1 andmb-2 are locat-ed on separate chromosomes. In both of themb-1 and mb-2 containing BAC clones, gene synteny was well con-servedwith the homologous region on zebrafish chromosome 1, supporting that the common carp specificmb-2geneoriginated from the recentwhole genomeduplication event in cyprinid lineage. Transcription factor bindingsites search indicated that both common carpmb genes lacked specificity Protein 1 (Sp1) andmyocyte enhancerfactor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region ofcommon carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted thehypoxia-inducible, non-muscle expression pattern ofmb-1. In the case ofmb-2, a 10 bp insertion to the bindingsite of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might causethe non-response to hypoxia treatment ofmb-2. The case of common carpmb gene duplication and subsequentdifferentiation in expression pattern and protein function provided an example for adaptive evolution towardaquatic hypoxia tolerance.

cidic ribosomal phosphoproteinuences; DO, dissolved oxygen;ucible factor; HIF1, hypoxia in-ia response element; mb, myo-, myocyte enhancer factor-2;T-cell; Ngb, neuroglobin; NRE,PCR; ROS, reactive oxygen spe-; USF, upstream stimulatory fac-e genomeassemblyversion 9; 5′

tic Genomics, Chinese Academy

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Myoglobin (mb) gene encodes an oxygen-binding hemoprotein,whichmainly serves oxygen supply function by facilitating the deliveryof oxygen from the plasma membrane to the energy-producing mito-chondria. There used to be a consensus that mb gene was expressedexclusively in muscle tissues, such as oxidative skeletal and cardiacmyocytes, where it could be up-regulated via a calcium-dependent

signaling pathway, according to intensive in vivo and in vitro studieson mammals (Berenbrink, 2010; Mirceta et al., 2013; Riggs and Gorr,2006).

In 2006, Fraser et al. discovered simultaneous expression of two dis-tinct mb transcripts in common carp (Cyprinus carpio) (Fraser et al.,2006). Myoglobin-1 (mb-1) was expressed in the muscle and someother metabolically active tissues, including the liver, kidney, and gills.Myoglobin-2 (mb-2), a unique novel transcript, was expressed only inthe brain, co-existing with neuroglobin (Ngb). Known as a hypoxia-tolerant teleost, common carp was often found living in eutrophic hab-itats with greatly fluctuant dissolved oxygen (DO) levels (Stecyk andFarrell, 2002). In summer, diurnal oxygen tension might range from30.66 kPa in the afternoon to 6.66 kPa during the pre-dawn hours. Inwinter, common carp survived in the ice-covered pondswith constantlydropping oxygen supply. Under laboratory observation, common carpwas able to endure extreme hypoxia treatment of 0.5 mg/L DO expo-sure for 42 days without inducing apoptosis (Poon et al., 2007). The un-precedented expression pattern of common carpmb genes, which wasinferred related to hypoxia adaption, immediately arousedwide refocuson the function and regulation of globins (Riggs and Gorr, 2006).

Fig. 1. Results of PCR amplification for mb-1 (left) and mb-2 (right) in BAC clone(A) CYC001O02, (B) CYC002I05, and (C) CYC041G12.

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mb's non-muscle expression was soon confirmed in other teleosts,including crucian carp (Roesner et al., 2008), zebrafish (Cossins et al.,2009), and medaka (Wawrowski et al., 2011). However, the uniquemb-2, was only found in common carp and its closely related species,crucian carp, which was an extreme hypoxia-tolerant fish (WittenbergandWittenberg, 2012). Recentwork by Helbo et al. indicated functionalspecialization between MB-1 and MB-2 for tissue-specific protect roles,since the MB-1 protein displayed higher oxygen affinity and faster cat-alytic velocity for nitric oxide, while the mb-2 encoded protein provedto be significantly faster at eliminating hydrogen peroxide (Helboet al., 2012).

Gene duplication and alternative splicing isoforms are the twomajorevolutionary mechanisms that increase gene diversification (Jin et al.,2008; Su and Gu, 2012). In human genome, all the three known tran-script variants ofmb shared the same gene locus. In this work, genomicsequences and genome location of common carpmb genes were inves-tigated, suggesting that the two differentmb transcripts originated fromrecent whole genome duplication (WGD) event, rather than alternativesplicing isoforms.

2. Materials and methods

2.1. Screening of mb containing BAC clones

A three-dimension bacterial artificial chromosome (BAC) pool ofcommon carp (Li et al., 2011), containing 18,432 individual clones(1.5× haploid genome coverage), was screened by PCR amplificationfor mb-1 and mb-2. Distinctive primers were obtained from earlierwork of Fraser et al. (mb-1F: ATGGCCGATCACGAACTGGTT; mb-1R:GCCGTGGGCCTTCACCGCTGCGTT; mb-2F: ATGGCTGATTACGAGCGGTTT; mb-2R: ACCGTGGGACGCCACCAACGTGTC) (Fraser et al., 2006).The mb primers were amplified in 88 wells, representing column pool,row pool, and plate pool. Positive wells served as three-dimensional co-ordinates to locate the original mb containing clones. Candidate cloneswere picked up from the BAC library, and then validated by anotherround of PCR.

2.2. Sequence analysis of mb containing BAC clones

Isolated BAC clones, CYC041G12 containing mb-1, CYC001O02 andCYC002I05 containing mb-2, were separately sequenced on 314 chipsof Ion Torrent PGM (Life technologies). More than 200× coverage se-quencing data was obtained for each BAC clone, at quality cutoff Q20.Read assembly was performed with Torrent Suite and Newbler. Poten-tialmb promoter regionswere checked by Sanger sequencing. Gene an-notation was performed via BLASTx against Ensembl peptide databaseof zebrafish whole genome assembly version 9 (zv9). 5′ untranslatedregions (5′ UTRs) were identified by BLASTn searching in sequencingdata of common carp full-length cDNA library. Schematic diagrams ofgene structure and gene synteny were drawn with the online toolfancyGENE v1.4 (Rambaldi and Ciccarelli, 2009). Transcription factorbinding sites were checked using the online TFSEARCH program(Akiyama, 1998) against TRANSFAC databases (Heinemeyer et al.,1998), combined with online search by JASPAR (Bryne et al., 2008),PROMO (Farre et al., 2003; Messeguer et al., 2002), and local searchby CLC Genomics Workbench.

2.3. Chromosome localization of mb containing BAC clones

Twelve-month common carpswere obtained from Songpu hatcheryat Harbin, China. Head kidney was used for mitosis metaphase chromo-some preparation, and fluorescence in situ hybridization (FISH) wasperformed with BAC plasmid probe, by methods described previously(Deng et al., 2013). Using a nick translation kit (Roche), BAC cloneCYC041G12 was labeled by biotin-16-dUTP (Fermentas), while BACclone CYC002I05 was labeled by digoxigenin-16-dUTP (Roche). The

mb-1 containing chromosome region was stained with green fluores-cence, through successive reactions with biotin labeled BAC plasmidprobe, avidin-FITC (Sigma-Aldrich), biotinylated anti-avidin D (Vector),and avidin-FITC. Meanwhile, the mb-2 containing chromosome regionwas stained with red fluorescence, through successive reactions withdigoxigenin labeled BAC plasmid probe, mouse anti-digoxin antibody,anti-mouse IgG-TRITC antibody produced in rabbit, and anti-rabbitIgG-TRITC antibody produced in goat (Sigma-Aldrich). After counter-stained by DAPI (Sigma-Aldrich), chromosome images were observedand captured on a fluorescence microscopy Axio Imager M2 (Zeiss),with AxioVision software system. More than 30 successfully stainedmetaphase spreads from 3 individuals were analyzed.

2.4. Sampling

Full siblings of common carp were obtained from Hulan hatchery atHarbin, China. Healthy individuals, three-month old, with body weightaround 150 gwere selected and kept in 25 °C for 14 days before hypoxiatreatment. Stop feeding for 24 h, and then stop aeration to make DOvalue drop naturally. Tissue samples were collected and flash frozen inliquid nitrogen. Five experimental groups were set: (1) normoxia con-trols, collected before stopping aeration, at DO value around 6.0 mg/L;(2) accelerated breathing group, collected 20 min after stopping aera-tion, at DO value around 3.0 mg/L; (3) aquatic surface respiration(ASR) group, collected 3 h after stopping aeration, at DO value around1.0 mg/L; (4) coma group, collected about 6 h after stopping aeration,when individuals lost balance and had no response to gentle touch, atDO value around 0.3 mg/L; and (5) recovery group, move the fishesinto fully oxygenated water 10 min after they fell into coma, sampleswere collected when individuals recovered to normal swimming, atDO value around 6.0 mg/L. Embryos were collected after timed intervals:0, 3, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, 192, and 240 hours post-fertilization (hpf) at 20 °C and flash frozen in liquid nitrogen.

2.5. Expression analysis of mb-1 and mb-2

Total RNA was extracted using TRIzol reagent (Invitrogen), accordingto the manufacturer's instruction. DNase I (Sigma-Aldrich) digestion wasperformed to avoid contamination by genomic DNA, before reverse tran-scription of mRNA using the First Strand cDNA Synthesis Kit ReverTraAce-α-RT-PCR (TOYOBO). Quantitative real time PCR (qRT-PCR) waspracticed on Applied Biosystems 7500 Real-Time PCR System (LifeTechnologies), using SYBR Green Realtime PCR Master Mix (TOYOBO).mb-1 and mb-2 were amplified with distinctive primers mentioned inSection 2.1. ARP (acidic ribosomal phosphoprotein P0, GenBank accession

Fig. 2. Chromosome localization of common carpmb-1 (green) and mb-2 (red) genes by fluorescence in situ hybridization (FISH) of BAC clones.

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number: KF572122) was utilized as the internal control gene for hypoxiatreatment analysis (Roesner et al., 2006; Simpson et al., 2000),while beta-actinwas utilized as the internal control gene for embryonic developmentand tissue distribution analysis (Casadei et al., 2011), with primers ARP-F:GGCTTTGGGAATCACCACCA; ARP-R: GCTGTAGACGCTGCCGTTAT; actin-F:TGCAAAGCCGGATTCGCTGG; and actin-R: AGTTGGTGACAATACCGTGC.For each sample, 5 biological replicates with 3 experimental replicateswere examined. The comparative CT (2−ΔΔCT) method (Schmittgenand Livak, 2008) was applied to analyze the expression ofmb genes.

3. Results

3.1. Screening of mb containing BAC clones

BLAST search against EST data from a 2.0 × 105 CFU full-length cDNAlibrary and the transcriptome data of four common carp stains (Xu et al.,2012)was performed to searchpotential unknownmb transcripts.mb-1andmb-2were the only transcripts found. Different PCR results formb-1and mb-2 specific primers were found in the BAC screening pool, sug-gesting different genome loci for the two transcripts. Positive amplifica-tion for mb-1 was found in plate 41, row 7, and column 12, indicatingthat the BAC clone CYC041G12 might contain mb-1 gene. For mb-2,PCR amplification in plate 1, plate 2, row 9, row 15, column 2, and

Fig. 3. Gene synteny ofmb containing genom

column5all yieldedpositive signals. A second roundof PCR in 8 candidateBAC clones verified that clone CYC001O02 and CYC002I05 containedmb-2 gene. As shown in Fig. 1, mb-1 and mb-2 existed in different BACclones.

3.2. Genome localization of mb-1 and mb-2

By metaphase chromosome FISH of BAC clones, mb-1 and mb-2genes were localized on separate chromosomes (Fig. 2). Among the100 chromosomes of common carp, a pair of submetacentric chromo-somes was found bearing mb-1 gene (green), while mb-2 gene (red)was located on short arms of a pair of acrocentric chromosomes.

3.3. Conserved synteny of flanked genes near mb loci

BAC clones containing mb-1 and mb-2 were sequenced and assem-bled, separately. Over 100 kb high-quality genomic sequences for eachclone were obtained. BAC clone CYC001O02 and CYC002I05, whichboth contained mb-2 gene, were found overlapped. Fig. 3 indicated thegene synteny with homologous region on zebrafish chromosome 1,which was the sole locus of mb in zebrafish genome. The available7 genes adjacent to mb kept in the same order with zebrafish inboth of the common carp BAC clones. The coding sequences (CDS)

ic regions of zebrafish and common carp.

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were conserved, with nucleotide similarity of 86%. Meanwhile, sizesof introns and intergenic regions varied. Most of the introns andintergenic regions of common carp were shorter than their counter-parts of zebrafish.

3.4. Comparison of mb gene structures

Structure of mb genes of common carp, zebrafish, and human wascompared in Fig. 4. Exactly the same genome locus, identical CDS and3′ UTR were shared by the three human mb transcript variants (mb-1:NM_005368.2, mb-2: NM_203377.1, mb-3: NM_203378.1), which dif-fered in the 5′ UTR due to alternative transcription initiation. No tran-script variant of zebrafish mb was reported up to now. Common carpmb-1 (KC342292.1), mb-2 (DQ338464.1), and zebrafish mb (NM_200586.1) had similar gene structure to a recently reported mb tran-script, human mb-2, which was discovered in breast cancer cells withhypoxia-inducible non-muscle expression (Kristiansen et al., 2011).Compared with the well-known human mb-1, the three teleost mbgenes possessed an additional intron in 5′ UTR.

3.5. Expression divergence of mb-1 and mb-2

QRT-PCR analysis indicated thatmb-1 andmb-2 had different expres-sion patterns, as shown in Fig. 5(a) for representative stages during em-bryonic development, and Fig. 5(b) for tissue distribution in adultcommon carp (3 month old). No significative expression of both mbgenes could be observed during the first 12 hours post fertilization(hpf), corresponding to the end of gastrulation by embryos at tail budstage. Until 18 hpf, significant expression was detected for both mb-1andmb-2. The expression ofmb-1 gradually increased during segmenta-tion (12–48 hpf), pharyngula (48–72 hpf), and hatching (72–96 hpf),then experienced a sharp increase in the larval period after hatching, ob-served from 96 to 240 hpf. Meanwhile, mb-2 exhibited significantlylower expression level all along the examined periods, with no morethan 1/10 of mb-1 mRNA expression quantity at 240 hpf. And no greatfluctuation of mb-2 expression was observed during the developmentalperiods from 18 to 240 hpf. In adult common carp, expression of mb-1

Fig. 4. Comparison ofmb gene structures for human (mb-1: NM_005368.2,mb-2: NM_203377.1mb-2: DQ338464.1).

was detected in the skin, muscle, gill, liver, kidney, and slightly in thebrain, while mb-2 was uniquely expressed in the brain.

3.6. Hypoxia response of mb-1 and mb-2

As shown in Fig. 6,mb-1 expression in themuscle and gill significantlyfluctuated under severe hypoxia, butmb-2 seemed unaffected by varioushypoxia conditions. mb-1 expression was not affected under temporaryslight hypoxia, as shown in the accelerated breathing group. While com-mon carp suffered from severe hypoxia, the fish moved to water surfaceto increase oxygen supply, and mb-1 was two-fold up-regulated. ThenDO level continued dropping, the fish fell into a coma status with min-imized vital movements, accompanied with decreasing mb-1 expres-sion. Unlike many hypoxia-sensitive teleosts, common carp was ableto recover from hypoxia induced coma, once re-oxygenated. The fishquickly returned to normal behavior before the hypoxia conditions ininternal tissueswere relieved, suggested by highermusclemb-1 expres-sion in the recovery group.

3.7. Analysis of transcription factor binding sites in mb-1 and mb-2

Potential transcription factor binding siteswere searched in 1 kb up-stream regions of mb-1 and mb-2 transcription start sites (TSS). Classicmb promoter consisted of an A/T motif for myocyte enhancer factor-2(MEF2) binding, a CCAC box for Specificity Protein 1 (Sp1) binding,and 2 NFAT response elements (NRE) for nuclear factor of activatedT-cell (NFAT) binding (Kanatous and Mammen, 2010; Kanatous et al.,2009). Neither Sp1 nor MEF2 binding site was found in common carpmb-1 or mb-2, while no less than 3 NREs appeared in both mb genes.Hypoxia response elements (HREs), which were commonly considerednot existing in mb gene, were found in both mb-1 andmb-2. Althoughmb-2 was expressed exclusively in the brain, no neuron-specific tran-scription factor binding sites was found. Near TATA box, there was anupstream stimulatory factor (USF) binding site in mb-1, while thehomologous site inmb-2was interrupted by a 10 bp insertion. Compar-ison for mb-1 and mb-2 regulatory regions was shown in Fig. 7. In the

,mb-3: NM_203378.1), zebrafish (NM_200586.1), and common carp (mb-1: KC342292.1,

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selected 599 bp region, only 75% sequence similarity was observed,which was much lower than that of the CDS region (86%).

4. Discussion

4.1. Origin of common carp specific mb-2 gene

Unlike human mb transcript variants, the common carp mb-2 geneappeared to originate from gene duplication, rather than alternativetranscription or splicing, since BAC library screening (Fig. 1) and chro-mosome localization (Fig. 2) revealed distinct genome loci for mb-1and mb-2. Compared with zebrafish (2n = 50), a typical memberand model organism in cyprinid lineage, the chromosome numberof common carp (2n = 100) doubled. It's estimated that 5.6 to11.3 million years ago (MYA), whole genome duplication (WGD) oc-curred to the ancient cyprinid(s), resulting in the current common carpspeciation (Wang et al., 2012). Crucian carp (2n = 100 and 3n = 150)might experienced the same duplication event, which also possessedthe mb-2 gene copy. Identical syntenic flank genes with the mb regionin zebrafish genome (Fig. 3) confirmed that the specificmb-2 gene copywas derived from large scaled genome duplication, instead of randomtranslocation.

Fig. 5. Expression analysis of common carp mb-1 and mb-2 genes during early develop-mental stages (a) and in adult tissues (b), revealed by qRT-PCR. Error bars representedstandard error of the mean (n = 5).

4.2. Ancestral form of mb gene transcripts

The CCAC box in well studied human mb-1 promoter, related tomuscle-specific expression (Kanatous andMammen, 2010), was absentin common carp mb genes. So did the A/T rich motif, responsive to thecalcineurin/MEF2 signaling pathway (Kanatous and Mammen, 2010).Existence of HREs and NREs suggested that common carp mb might beregulated by hypoxia inducible factor-1 (HIF1) and calcineurin/NFATsignaling pathway. This HIF-inducible, non-muscle expression patternwas also observed in human mb-2, which had a similar three-introngene structure to the teleostmb genes. Thus, humanmb-2, although re-cently reported (Kristiansen et al., 2011) and not universally expressed,might be close to the primitive transcript of mb gene in the aquatic an-cient ancestors of mammals and modern teleosts. After landing onearth, steady oxygen level in the atmosphere relieved the stress of oxy-gen supply, and then the newmb transcripts evolved and became dom-inant in terrestrial animals, such as humanmb-1, whichwas not directlyregulated by HIF, and solely expressed in metabolically active muscles.On the other hand, modern teleosts conserved the ancestral form ofmb gene transcript, butwith shortened intronsduring additional roundsof WGD.

4.3. Differentiation of common carp mb-1 and mb-2

As previously reported (Helbo et al., 2012; Wittenberg andWittenberg, 2012), MB-1 and MB-2 shared 73% similarity in aminoacid sequences, and MB-2 protein was evolutionarily optimized fornew function or new environment, since it exhibited higher efficiencyin eliminating reactive oxygen species (ROS).

Fig. 6. Hypoxia expression of common carpmb-1 (a) and mb-2 (b) genes, determined byqRT-PCR. Brain, gill, and muscle tissue samples were examined for (1) normoxia controls,(2) slight hypoxiawith accelerated breathing, (3) severe hypoxiawith aquatic surface res-piration (ASR), (4) hypoxic coma, and (5) recovery groups. Error bars represented stan-dard error of the mean (n = 5). Statistical differences relative to normoxia controlswere calculated by using Student's t test (*, P b 0.01).

T CCTGTGT T T GT T TGCACAC GTGT CGGTCA T TGT T T TGT T T T CCTGAGCC

T CCTGTGT T T GT T TGCAGAT GTGT CAGT CA T TGT T T TGT T T T CCTGAACT

GAACT CT CAT GTAAAGCCT T GAACGT T CAT CCAT T CACCC CCAAAT CAAA

- - - - - - - - - - - - - - - - - - - - - - - - - T T CAT CCAT T CACCC C- AAAT CAAA

CT CGGAGACG CCT CAT CTGT T T T T T CCAC- - - T ACATGAA CT CGCAGAAG

CT CAGAGATG CT T CAT CTGT T T T CCCACTA TATACGTGAA CT TAAAGAAG

AGCATGACAA AGTGGGA- CT GATGATGCTG TGAAAGCAG- - - - - - AAAAA

AGCATGAAAA AGTGGAAAT T GAAAATGCTA GAAAAGCACT TAT CTAAAAA

CTAGAAAACA AT CATATGGC AC- AT CT CAA AATGACAT T T T TAAAGTGAA

CAAGAAAACA AACAT CTGGC ATGACCT CAA AATGAC- TGT T TAAAGTAAA

TATAGGTGTG CAGATAGGCA T CTGTGTGCA GCCATGGAAC GGCTGTGGAA

T CTAGCTGTG TGT - - - - - - - - - - - T GTGCA GTAATGCAAC TGT TGTGGAA

TAATACAGTG A - - - - - - - - - - GCAT CT CAA ATGGCT T CTG T TGCAGAT CT

TAAAACAGCG ATGGAAT CAG AGCACCT CAA ATGGCT T CGG T CACAGAGCC

GCT CTACGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG CTGGTGG- GC

AGTGT - - GTG TGT T TGT T T T TGT T T T TGTG CGTGTGTGTG CTGGTGGAGC

AAACAGAGGG GTGTGGT TAA CAT T T CAAGA CAAGGAGAGG GACAGTGTGA

AAACGGAGGG GTGGGGT TAA CAT T T CAAGA CACCAAGAGG GACAGTGTGA

T TGACAGGT - - - - - - - - - - G ACAT CACAAG GACGCTGTAT AAAAGGTGAA

T TGACAGGTA AGGGGCAGTG ACAT CACAAG GATACTGTAT AAAAGATGAA

AGTGGGGAA- - - - - - - - - T C TGAGCT CAT T TGGGGAGT T T T CCAAACCTA

CGTGGGGAAA AGTGATGAT C TGAGCT CAT T TGAGGAGT T T T CCAAACCCA

CAT CCAGT CA CACGT T CT T C AGAGAGGACA CCGATAT CGG TAAGGAGT T T

CAACCAGT CA CACGGTCT T C AGAGAGGACG CTGATAT CGG TAAGGAGCT T

GACTGGACT T T CTAATGCT T TGTGCAAAAG T TAAGACGCA

GACTGGACT T T CT T - TGCT T TGTGAAAAAG T TAAGATGCA

Fig. 7. Alignment of common carpmb-1 andmb-2 gene upstream sequences. Hypoxia response elements (HRE), NFAT response elements (NRE), upstream stimulatory factor (USF) bind-ing site, and TATA boxes were indicated.

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The common carpmb genes differentiated not only in protein func-tions, but also in expression patterns, as shown in Figs. 5 and 6, whichmight be caused by the apparent differences in the promoter regionand other transcription factor binding sites. Expression of mb-1 wasup-regulated by hypoxia treatment and greatly fluctuated under differ-ent hypoxia conditions, while expression of mb-2 seemed not affectedby hypoxia stress. Promoter analysis indicated that the common carpmb genes differed at a USF binding site 20 bp upstream to TATA box,where a 10 bp insertion caused mutation of the binding site in mb-2.USF itself was an oxygen independent transcription factor, however,USF sites were often adjacent to HREs and interacted with hypoxia in-ducible factor (HIF) binding (Borley and Sidell, 2011; Dimova andKietzmann, 2006; Hankeln et al., 2005; Hu et al., 2011; Kajimura et al.,2006; Small et al., 1998, 2003). The mutation at the USF binding sitemight lead to the different hypoxia responses ofmb-1 and mb-2.

Genome duplication was an important source for biodiversity andenvironmental adaption, especially for teleosts (Santini et al., 2009;Vandepoele et al., 2004). Duplication of ancestral cyprinid mb genemade the survival stress shared and provided opportunities to evolu-tionary specialization for individual paralogs. As a result, subsequentdifferentiation in expression patterns and protein functions of theduplicated mb genes arose, contributing to the common carp hypoxiatolerance.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The financial supports from the National High-tech R&D Program ofChina (863 Program, 2011AA100401), and the Special Scientific Re-search Funds for Central Non-profit Institutes, Chinese Academy of Fish-ery Sciences (2012C014, 2014C010) are well acknowledged.

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