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Mitochondrial DNAThe Journal of DNA Mapping, Sequencing, andAnalysisPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713640135

Molecular characterization of myostatin in blackseabream, Acanthopagrus schlegeliiLiangyi Xue a; Qiaoyi Yang a; Zhangkui Xiao a; Lu Li aa College of Life Science and Biotechnology, Ningbo University, Ningbo, Zhejiang,People's Republic of China

First Published: June 2008

To cite this Article: Xue, Liangyi, Yang, Qiaoyi, Xiao, Zhangkui and Li, Lu (2008)'Molecular characterization of myostatin in black seabream, Acanthopagrus

schlegelii', Mitochondrial DNA, 19:3, 217 — 223

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FULL LENGTH RESEARCH PAPER

Molecular characterization of myostatin in black seabream,Acanthopagrus schlegelii

LIANGYI XUE, QIAOYI YANG†, ZHANGKUI XIAO‡, & LU LI{

College of Life Science and Biotechnology, Ningbo University, Ningbo, Zhejiang 315211, People’s Republic of China

(Received 16 December 2006; revised 17 May 2007; accepted 19 June 2007)

AbstractMyostatin is a negative regulator of skeletal muscle growth and has a potential application in aquaculture. The black seabreammyostatin gene was cloned and sequenced. It had three exons encoding a protein of 382 amino acids. A 90 bp 50-untranslatedregion (UTR) and a 536 bp 30-UTR were obtained by RACE. Four microsatellite sequences, a (CAG)9, a (TC)12, a (CA)16

repeat and an “imperfect” (CA)25 microsatellite, were found in the myostatin. Two introns were 329 and 742 bp in length,respectively. The deduced amino acid sequence of the myostatin had a putative amino terminal signal sequence, a TGF-bpropeptide domain, a RXXR proteolytic processing site, a TGF-b domain, and 12 conserved cysteine residues. The myostatingene was expressed in four of the examined ten tissues and organs. The expression of myostatin was the strongest in theskeletal muscle and brain, intermediate in the eye, and low in the heart.

Keywords: Black seabream (Acanthopagrus schlegelii), myostatin, RACE, expression, microsatellite

Introduction

Myostatin (MSTN), also known as growth and

differentiation factor-8 (GDF-8), was first identified

in mice (McPherron et al. 1997a). It is a member of

the transforming growth factor-beta (TGF-b) super-

family, which includes proteins that mediate key

events in cell growth and development through signal

transduction. Like all TGF-b superfamily members,

MSTN is translated as a pre-propolypeptide. It

contains a putative signal sequence, conserved

cysteine residues and a RXXR proteolytic processing

site (Rodgers and Weber 2001a).

MSTN is a negative regulator of skeletal muscle

growth in several mammalian species. Mice knockout

for MSTN produce a “double muscling” phenotype

where individual muscle fibers are 2–3 times larger

than those of wild-type mice (McPherron et al.

1997a). Two breeds of cattle, the Belgian Blue and the

Piedmontese, possess a double muscling phenotype

because of the natural mutations of MSTN gene

(Kambadur et al. 1997; McPherron and Lee 1997b).

In addition to mammals, transgenic fish expressing

MSTN prodomain showed a small but significant

increase in the fiber number of skeletal muscle as a

result of MSTN inhibition (Xu et al. 2003). The

microinjection of dsRNA, corresponding to biologi-

cally active C-terminal domain, in early development

stage of zebrafish produced a giant phenotype in

treated fish (Acosta et al. 2005). Because of its role

in muscle growth, MSTN is an extremely interesting

locus for genetic improvement of farmed animals.

Such potential applications in animal husbandry have

prompted the sequencing of MSTN genes from

several vertebrates, including mammalia (Kambadur

et al. 1997; McPherron and Lee 1997b), aves (Yang

et al. 2001), and pisces (Maccatrozzo et al. 2001a,

ISSN 1042-5179 print/ISSN 1029-2365 online q 2008 Informa UK Ltd.

DOI: 10.1080/10425170701517564

Correspondence: L. Xue, College of Life Science and Biotechnology, Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang 315211,People’s Republic of China. Tel: 86 574 87600165. Fax: 86 574 87608347. E-mail: xueliangyi@nbu.edu.cn†E-mail: joyiiiii@163.com‡E-mail: hbxfxzk@163.com{E-mail: lu3303@126.com

DNA Sequence, June 2008; 19(3): 217–223

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2002; Rodgers et al. 2001b; Kocabas et al. 2002; Biga

et al. 2004; Terova et al. 2006; Ye et al. 2007).

The black seabream, Acanthopagrus schlegelii, is a

marine teleost belonging to the perciform family

Sparidae. It is an ideal commercial fish because of its

high meat quality and large size, and has been a major

marine-cultured species in China. Reported herein is

the molecular cloning and tissue expression of MSTN

from Acanthopagrus schlegelii.

Materials and methods

Materials

Adult black seabreams were collected at the Ningbo

Bay Breeding Center of Aquatic Animals. Skeletal

muscle tissues were dissected from living black

seabreams which were not anesthetized, rapidly frozen

in liquid nitrogen and stored at 2808C.

Genomic DNA and total RNA extraction

Genomic DNA from skeletal muscle tissue was

extracted according to the procedure (Sambrook

et al.1989). Total RNA was extracted from skeletal

muscle tissue using Trizol Reagent (Sangon, China),

following the manufacturer’s instructions. Total RNA

quality and quantity were checked by ultraviolet

spectrophotometry.

PCR amplification, cloning and DNA sequencing

Two degenerate primers, based on highly conserved

regions of an alignment of reported MSTN genes from

gilthead seabream AF258447, shi drum AF316882,

zebrafish AF019626, striped sea-bass AF290910, and

white perch AF290911, were designed to amplify the

complete coding region of MSTN gene. The two

primers have the following sequences: MygF2 50-

ATGCATC(T/C)GTCTCAGATTGTG-30 and

MygR2 50-TCA(A/C)GAGCA (T/G)CCACAAC-

GGTC-30. PCR components: DNA 200 ng, dNTP

200mmol/l each, Taq 1.5 U, two primers 1.0mmol/l

each, 10 £ PCR buffer 5ml, MgCI2 2.0 mmol/l and

sterile water to 50ml. PCR conditions: 948C, 2 min for

pre-denaturation; followed by 30 cycles (948C, 1 min;

548C, 1.5 min; 728C, 3 min); 728C, 10 min for final

extension. The PCR product was run in 1% agarose gel,

and then purified by using QIAquick Gel Extraction Kit

(QIAGen, USA). The purified PCR product was cloned

according to manufacturer’s protocols (TOPOe TA

Cloning Kit; Invitrogen, USA). Recombination plas-

mids were isolated from transformed TOP10 cells with

QIAGen Plasmid Mini Kit (QIAGen, USA). The

inserted PCR fragment was sequenced on an ABI

PRISM 3700 DNA sequencer with M13 reverse primer.

RT-PCR and RACE

An RT-PCR approach together with the rapid

amplification of cDNA ends (RACE) technique was

used to obtain the complete MSTN cDNA sequence.

RT-PCR was carried out by using TaKaRa RNA LA

PCR Kit (TaKaRa Biotechnology, Dalian, China)

according to the manufacturer’s instructions. The first

strand cDNA was made in a 20ml reverse transcrip-

tase reaction containing: 1mg total RNA, 10 £ RNA

PCR buffer 2ml, MgCI2 (25 mM) 4ml, dNTP mixture

(10 mM each) 2ml, RNase inhibitor (40 Uml) 0.5ml,

AMV Reverse Transcriptase (5 Uml) 1ml, Oligo dT-

Adaptor Primer (2.5 pmolml) 1ml. The reaction was

incubated at 428C for 30 min, and then at 998C for

5 min, at 58C for 5 min. After the first strand cDNA

was synthesized, the obtained cDNA was then used as

template for subsequent PCR reactions. Two different

primer pairs were used (MygF2–MygR3 and

MygF3–MygR2, Figure 1) with the following PCR

reaction condition: 1 cycle at 948C for 2 min; followed

by 40 cycles (948C, 30 s; 548C, 30 s; 728C, 1.5 min);

728C, 10 min for final extension. The products of RT-

PCR were cloned into pCRw-TOPO Vector and

sequenced as described above. The acquired cDNA

sequences were used to define the exon/intron

junction of the gene.

The 50- and 30-RACE was performed to generate

the 50- and 30-terminal regions of MSTN using the

SMART RACE cDNA Amplification Kit (Clontech,

Palo Alto, CA, USA) according to the manufacturer’s

instructions. Total RNA (1mg) from black seabream

skeletal muscle was converted into first strand cDNA

using SMART II, an oligonucleotide and Power-

Script reverse transcriptase. According to the

sequencing results of PCR and RT-PCR products,

two primers for the 50-RACE, M5gsp1 (50-TCCTC-

CATAACCGCATCCCT-30) and M5gsp2 (50-GTC-

GAGGAGCTGCTGCAGC-30), and two primers for

the 30-RACE, M3gsp1 (50-GAGTGTGAGTACAT-

GCACTTG-30) and M3gsp2 (50-GCTGTACCCC-

CACCAAGATG-30), were designed (Figure 1). For

the 30-RACE, two PCR runs were done. In the first

PCR run, the outer primer M3gsp1 as sense and

poly(dT)18 as antisense primer were used. One

microliter of PCR product from the first run was

used as template in a second PCR run, with the inner

primer M3gsp2 as sense and poly(dT)18 as antisense

primer. For the 50-RACE, First strand cDNA was

subjected to two rounds of PCR as follows: the first

reaction employed an outer adapter primer (AP1)

provided with the kit and gene specific primer

M5gsp1. One microliter of the first PCR mixture

was used as a template for nested PCR using adapter

primer AP2 provided with the kit and a nested

specific primer M5gsp2. PCR conditions were as

follows: an initial denaturation (948C, 2 min),

followed by 35 cycles of amplification (948C, 45 s;

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Figure 1. Complete nucleotide sequence of Acanthopagrus schlegelii myostatin cDNA (1775 bp) (GenBank DQ303480) and sequence of

intron 1 (329 bp) and intron 2 (742 bp) as determined by PCR amplification and sequencing of genomic DNA (GenBank DQ251470). Exons

are shown in capital letters and introns in small letters. The deduced amino acid sequence are shown by single letter code of amino acids below

the exons. Translational termination site is indicated by an asterisk. Name and position of each primer used are indicated. Microsatellites are

underlined. Conserved cysteine residues and putative proteolytic processing site are indicated with gray shading.

Molecular characterization of myostatin in black seabream 219

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558C, 30 s; 728C, 3 min) and a final extension (728C,

7 min). Conditions for the second PCR were the

same as those for the first PCR, with the exception

that amplification was 25 cycles. The products of 50-

and 30- RACE were cloned into pCRw-TOPO Vector

and sequenced as described above.

Expression analysis

Expression analysis by RT-PCR was carried out on

total RNA using the same method described above,

extracted from ten different tissues and organs,

skeletal muscle tissue, liver, kidney, spleen, intestine,

brain, eye, heart, gill and adipose tissue, of adult black

seabreams. The pair of primers used in expression

analysis were as follows: ExpF 50-TCCCTGAAGA-

TCGACGTGAA-30 and ExpR 50-TTTGGGGCAA-

TAATCCAGTC-30. The pair of primers rang partial

exon 2 and 3. In RT-PCR, b-actin gene was served as a

positive control. The primers for b-actin were

designed based on the sequence of black seabream

b-actin gene AY491380. They were upper primer 50-

CCAGATCATGTTCGAGACCTTC-30, and lower

primer 50-GAACCTCTCATTGCCAATGGTG-30.

The RT-PCR products from each tissue were

electrophoresed on 1% agarose gel.

Sequence analysis

Similarity searches of the sequenced DNA fragment and

deduced amino acid sequence were done by BLAST

(Altschul et al. 1997). A multiple sequence alignment

was performed using CLUSTAL W (Thompson et al.

1994). The species in the sequence alignment represent

Mammalia (human AF019627, mouse U84005, rat

AF019624, hare AY169410, bovine AF019620, hama-

dryas baboon AF019619, sheep AF019622, pig

AF019623), Aves (chick AF346559, turkey

AF019625) and Pisces (shi drum AF316882, gilthead

seabream AF258447, striped sea-bass AF290910,

white perch AF290911, tilapia AF197193, white bass

AF197194, Atlantic salmon AJ316006, croceine croa-

ker AY842934, brook trout AF247650, white seabass

AY966401, European seabass AY839106, orange-

spotted grouper AY856860, arctic char AJ829532,

rainbow trout isoform I AF273035, rainbow trout

isoform II AF273036, zebrafish AF019626, sea perch

AY965685, red seabream AY965686 and channel

catfish AF396747). Signal peptide was predicted by

SignalP 3.0 Server (http://www.cbs.dtu.dk/services/

SignalP). ScanProsite were performed to analyze

functional sites of the deduced amino acid sequence

(http://www.expasy.org/prosite). The Neighbor-joining

method in MEGA version 3 was used to construct the

phylogenetic tree based on Poisson-corrected pairwise

distances between protein sequences, and node robust-

ness was assessed using a bootstrap approach (Kumar

et al. 2004).

Results

Molecular characterization of myostatin gene

PCR amplification of genomic DNA from Acanthopa-

grus schlegelii produced a band of 2220 bp with a pair of

primers MygF2 and MygR2, which was subsequently

TA-cloned and sequenced. The resulting sequence

was submitted to the GenBank database, and the

accession number was DQ251470. The sequence

covered the region from the initiator codon to the

stop codon. Compared with its cDNA sequence, the

boundary between exon and intron was determined.

Exon 1–3 are comprised of 397, 371 and 381 bp,

respectively. The two intron sequences are 329 and

742 bp. Outside of the protein coding region, 90 bp 50-

UTR and 536 bp 30-UTR were obtained by RACE.

The complete mRNA was 1775 bp in length, and the

accession number was DQ303480. The open reading

frame (OFR) was 1149 bp, which encoded a peptide

sequence of 382 amino acids. Four simple sequence

repeats were found in black seabream MSTN. One

(CAG)9 repeat was in the first exon encoding a

polyglutamine tract, and (TC)12 in the first intron.

A (CA)16 and an “imperfect” (CA)25 microsatellite

existed in the second intron and 30-UTR, respectively

(Figure 1).

Blastn and Blastp searches showed that the cloned

nucleotide sequence from black seabream and putative

amino acid sequence had over 90% identities with

MSTNs from gilthead seabream, red seabream and

other several fishes. Two conserved domains, TGF-b

domain (residues 288–382) and TGF-b propeptide

(residues 47–262), have also been detected in the

submitted amino acid sequence by Blastp.

The multiple sequence alignment showed that the

TGF-b active domain, which was situated between

the RXXR motif (residues 270–273, RARR, match-

ing the RXXR consensus site) and the carboxy

terminal, was highly conserved from fish to human.

Twelve conserved cysteine residues (positions 47, 50,

145, 146, 279, 288, 289, 316, 320, 347, 379, 381)

were found in black seabream MSTN (Figure 1). In

the carboxy terminal region, 8 conserved cysteine

residues were shared among all vertebrate MSTNs.

A signal peptide was predicted in the amino

terminal. The cleavage site to remove the signal

peptide was most likely located between position 22

and 23. ScanProsite results showed that there existed

many functional domains/sites in the predicted

protein sequence, such as the glutamine-rich region

profile (residues 24–35), the TGF-beta family

signature sequence (residues 305–320), Casein kinase

II phosphorylation site (residues 22–25, 39–42, 40–

43, 131–134, 247–250, 275–278), protein kinase C

phosphorylation site (residues 58–60, 151–153,

198–200), N-glycosylation site (residues 79–82),

N-myristoylation site (residues 208–213, 231–236,

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241–246), and cAMP- and cGMP-dependent protein

kinase phosphorylation site (residues 272–275).

Expression of the myostatin gene

To further characterize the black seabream MSTN

gene, RT-PCR was conducted to determine its

expression in various tissues. The expected

335 bp fragments were amplified in 4 of 10 tested

tissues. However, its expression levels varied among

tissues. The strongest expression was in the skeletal

muscle and brain; intermediate in the eye; low in the

heart. No band was present in the spleen, gill, kidney,

adipose tissue, liver and intestine (Figure 2). As a

positive control, a b-actin fragment of 406 bp was

successfully amplified in all ten samples by means of

RT-PCR, thereby excluding that the negative result is

due to technical problems.

Evolutionary relationships of myostatins

To evaluate the evolutionary relationships of black

seabream MSTN with other vertebrate MSTNs, we

constructed a phylogenetic tree by using MEGA

version 3 (Figure 3). All analyzed vertebrate MSTNs

were divided into two subgroups. Teleost MSTNs

formed a subgroup, while mammalian and avian

MSTNs formed the other subgroup. The black

seabream and gilthead seabream cluster first, and

then form a branch with red seabream with strong

bootstrap support (100%) in teleost subgroup, and all

of them belong to family Sparidae.

Discussion

Blast searches indicated that the obtained sequence is

most similar to fish MSTN, with more than 90%

identities to those of some fishes such as gilthead

seabream, red seabream, shi drum and white seabass.

Blastp search and ScanProsite analysis showed that

the acquired gene belonged to TGF-b superfamily.

These results confirmed that the cloned PCR

fragment was MSTN gene. The position of black

seabream MSTN in the phylogenetic tree also

confirmed that the obtained sequence is orthologous

to the other vertebrate MSTN genes. Vertebrate

MSTN is very conserved from the initiator codon to

the stop codon, but introns are changeable. TGF-b

domain is located in exon III, which is the most

conserved. The conservation was displayed not only

in the sequence identity, but also in the structure and

organization of the gene. To date, all vertebrate

MSTN genes, whose genomic sequences are publicly

available, had three exons and two introns. The

boundaries of exons and introns are also conserved

and obeys “GT . . .AG” rule.

Four microsatellites were found in the black

seabream MSTN. One (CAG)9 repeat was in the

first exon encoding a polyglutamine tract, and (TC)12

in the first intron. A (CA)16 and an “imperfect”

(CA)25 microsatellite existed in the second intron and

30-UTR, respectively. Microsatellites also existed in

other teleost MSTNs, such as AC repeats in the

second intron and the 30-UTR of gilthead seabream

MSTN (Maccatrozzo et al. 2001a), one AC repeat in

the 30-UTR of shi drum MSTN (Maccatrozzo et al.

2002) and an imperfect CA repeat in the 30-UTR of

croceine croaker (Xue et al. 2006). In channel catfish,

MSTN gene contained multiple microsatellites, one

CAG repeat in the first exon, three microsatellite

sequences, GTTT, TG and TA repeats, in the first

intron, and one AC repeat in the 30-UTR (Kocabas

et al. 2002). These studies indicated microsatellites

were common in teleost MSTNs.

In the aligned MSTNs from 29 vertebrate species,

only four teleosts, channel catfish and three sparid

species, gilthead seabream, red seabream and black

seabream, have a polyglutamine tract. It seems a

characteristic of MSTNs from sparid species. The

consecutive glutamine number is 6 in the channel

catfish, 9 in the black seabream, 11 in the red

seabream and 12 in the gilthead seabream. The three

sparid species have more consecutive glutamine

number than the channel catfish.

MSTN is a member of TGF-b superfamily. Like

other TGF-b superfamily members, MSTN is

synthesized as a precursor protein that contains a

signal sequence, an N-terminal propeptide domain

and a C-terminal domain that is the active ligand.

According to multiple sequence alignment and protein

structure analysis, MSTN of black seabream had a

putative amino terminal signal sequence with 22

residues, a TGF-b propeptide domain (residues 47–

262), a RXXR proteolytic processing site (RARR,

residues 270–273, matching the RXXR consensus

site), and a TGF-b domain (residues 288–382). The

signal sequence, which direct the protein to the cell

membrane, is cleaved during the secretion. The amino

terminal propeptide is cleaved from the carboxy

terminal mature protein. It is believed that the

precursor protein forms a homodimer before proteo-

lytic processing (Thies et al. 2001). The cleaved

propeptide molecules remain non-covalently bound to

the mature domain dimmer, forming a latent complex

Figure 2. (A) RT-PCR analysis of myostatin expression in different

tissues. Lanes correspond to: spleen (1), brain (2), eye (3), heart (4),

gill (5), kidney (6), adipose tissue (7), liver (8), intestine (9), muscle

(10) and marker (M). (B) As a positive control, a b-actin fragment of

406 bp was successfully amplified in all samples by means of RT-

PCR.

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and inhibiting its biological activity. To interact with

its receptor, the MSTN ligand must be released from

the latent complex (Lee and McPherron 2001). The

most conserved region (residues 288–382), the TGF-

b domain, was the mature bioactive domain of MSTN

protein in black seabream.

The high conservation of sequences among all

known MSTN orthologues suggests the importance

and conservation of its function in vertebrates. MSTN

was initially characterized as a negative regulator of

skeletal muscle growth in mammals (Grobet et al.

1997; McPherron et al. 1997a; Wiener et al. 2002).

Recent studies demonstrate that MSTN also plays

an inhibitory role in hyperplastic muscle growth in

zebrafish (Xu et al. 2003; Acosta et al. 2005). In mice,

MSTN is expressed exclusively in skeletal muscle

(McPherron et al. 1997a). It also expressed in bovine

cardiomyocytes and Purkinje fibers (Sharma et al.

1999) and in porcine mammary gland (Ji et al. 1998),

in addition to the skeletal muscle of these animals.

However, some reports indicated its wide tissue

distribution in several fish, including skeletal muscle,

eyes, gill filaments, brain, kidney, gut and gonads

(Roberts and Goetz 2001; Rodgers et al. 2001b;

Figure 3. Neighbor-joining tree of MSTN s based on Poisson-corrected protein distances. Numbers at tree nodes refer to percentage

bootstrap values after 1000 replicates. The scale bar presents to a phylogenetic distance of 0.05 amino acid substitutions per site. sb, striped

sea-bass; wp, white perch; wb, white bass; cc, croceine croaker; sd, shi drum; gs, gilthead seabream; sp, sea perch; mt, Mozambique tilapia; rt,

ranbow trout; as, Atlantic salmon; bt, brook trout; ac, arctic char; zf, zebrafish; cf, channel catfish; hb, hamadryas baboon; rs, red seabream;

bs, black seabream; ws, white seabass; es, European seabass; og, orange-spotted grouper.

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Maccatrozzo et al. 2001b; Acosta et al. 2005; Terova

et al. 2006; Ye et al. 2007). In our study, the myostatin

gene was expressed in skeletal muscle, brain, eye and

heart in adult black seabream although the expression

level was various. These data suggest that biological

actions of MSTN in lower vertebrates may be more

complex, and not be strictly restricted to skeletal

muscles. MSTN may also help to regulate the

development and growth of other tissues as well in

teleost. Therefore, it is important to understand the

physiological roles of MSTN in fish and the molecular

mechanisms of its actions for eventually manipulating

its functions to stimulate fish muscle growth.

Acknowledgements

Thisworkwas supportedbyZhejiangProvincialNatural

Science Foundation (306295) and Ningbo Municipal

Natural Science Foundation (2006A610083). This

article is sponsored by K.C. Wang Magna Fund in

Ningbo University.

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