全 文 ：生物技术通报
BIOTECHNOLOGY BULLETIN 2009年第 2期·研究报告·
收稿日期：2008-07-21
基金项目：国家自然科学基金重点项目“鲢、鳙、草鱼和团头鲂遗传资源的变迁”（30630051）
作者简介：杨琴玲（1985-），女，浙江温岭人，硕士，研究方向：水产动物种质资源与种苗工程
通讯作者：李思发，教授，博导，Tel：021-65710333，E-mail：sfli@shou.edu.cn
1 Introduction
Bighead carp （Aristichthys nobilis）is one of the most
important native freshwater species in China. Since the
success of artificial propagation in 1958，bighead carp
had become a wide alien species in Europe，North
America and South Asia. In 2005，the production
reached 2.18 million tons in China and 2.21 million
tons in the world[1]. Associated studies on this species
had been conducted，including traditional biology [2，3]，
cytogenetics[3]，isoenzymes [4~6]and molecular genetics [7~13].
However，the complete mtDNA sequence has not been
determined yet.
Mitochondrial DNA（mtDNA）is a kind of double-strand
circular molecule，approximately 16 ~18 kb in size in
vertebrates. It features small size，maternal inheritance，
and rapid evolutionary rate，lack of recombination，
relatively conserved gene content and organization，all
of which are suited for genetic research，therefore，it
A Report of Complete Nucleotide Sequence of Mitochondrial
Genome of Bighead carp（Aristichthys nobilis）
Yang Qinling Li Sifa Xu Jiawei Chen Qin Wang Chenghui
（Key Laboratory of Aquatic Genetic Resources and Aquacultural Ecosystems，Ministry of Agriculture，Shanghai Ocean
University，Shanghai 200090）
Abstract： The complete mitochondrial DNA sequences from three individuals of bighead carp，Aristichthys nobilis
were determined. The genome is a circular molecule with 16621bp in length，and the overall base composition included
31.6% of Adenine（A），27.1% of cytosine（C），16.0% of Guanine（G），25.3%，of thymine（T），and with 56.9% the A+T
content. The genome contains the typical 37 vertebrates’mitochondri genes，including 13 protein-coding g es，2 rRNA
genes，22 tRNA genes，and also a putative control region（D-loop）. It was confi med that the gene arrangement of the
bighead carp could be used to identify the other Cyprinidae fishes. Meanwhile，12 out of 13 protein coding genes were
observed that was encoded by the heavy strand and only ND6 was encoded by the light strand. The codon usage also
followed the typical vertebrate pattern（ATG）except for an unusual GTG start codon for theCOI gene.
Key words： Bighead carp Mitochondrial genome Complete sequence Gene arrangement
鳙的线粒体基因组核苷酸全序列分析
杨琴玲 李思发 徐嘉伟 陈琴 王成辉
（上海海洋大学农业部水产种质资源与养殖生态重点开放实验室，上海 200090）
摘 要： 对采集自我国长江的鳙的线粒体DNA全序列进行了测定。结果表明，鳙的线粒体DNA全长为166
221bp，其碱基因组成为A=31.6%；C=27.1%；G=16.0%；T=25.3%，A+T含量为56.9%。鳙线粒体基因组的排列、结构和组成
与其它鲤科鱼类相似，包括37个基因，即13个蛋白质编码基因，2个rRNA基因，22个tRNA基因和一个非编码控制区
（D-loop）。在13个蛋白编码基因中，除ND6由轻链编码外，其余12个基因均由重链编码。COI基因的起始密码子为
GTG，而其它12个蛋白编码基因的起始密码子均为ATG。
关键词： 鳙鱼 线粒体基因组 全序列 基因排列
2009年第 2期
has been widely used in the study on phylogenetic
relationship and genetic structure at various taxonomic
levels[14，15].
In this paper，the complete nucleotide sequences of the
mitochondrial DNA of bighead carp were first reported.
The aim was to provide the more sequence information
for studies not only on population genetic structure of
bighead carp，but also on phylogenetic relationships of
the Cyprinidae in future.
2 Materials and methods
2.1 Sample collection and DNA extraction
Three individuals with weight of 400 ~480 g were
collected in the lower stream of the Yangtze River in
2005. A small piece of caudal-fin clips from each
individual was used and stored in 95% ethanol. Whole
genomic DNA including mtDNA and nDNA was
extracted by using a proteinase K and phenol-
chloroform procedure[16]. The quantity and quality of the
extracted DNA were estimated onagarose gels（1%）sta-
ined with ethidium bromide（EB）.
2.2 Primers design，PCR amplification and sequen-
cing
According to the alignment of the reported complete
mtDNA sequences of common carp[17，18]，crucian carp[19]，
and tinca[20]，18 pairs of primers were designed to ampl-
ify the complete mtDNA sequence of the three
individuals（Table 1）.
The polymerase chain reaction （PCR）was performed in
Forward Sequences (5to 3 ) Reverse Sequences (5to 3)
BH-A0 CATGCCGAGCATTCTTTT BH-B0 GAGACTTGCATGTGTAAG
BH-A1 CAAAGCATAGCACTGAAGATGC BH-B1 TTTTGACAGGGGAGAGTGA
BH-A2 CCAGCCTATATACCGCCGT BH-B2 CTA TCACCAGGTTCGGTAGG
BH-A3 TTAGCCAGTACACCCAAGCA BH-B3 AAAGACAAGTGATTGCGCT
BH-A5 AAGGAACTCGGCAAACACAA BH-B5 TGGTGCTCATAAGGTTATGG
BH-A6 CCATCCACATCATCCCCA BH-B6 ATTGGCGGAGGAGGGACTTT
BH-A7 CCCTACCAATTGCACTAGCA BH-B7 GTTTGTAGGATCGAGGCCTT
BH-A8 ACAACTCACCCTTCTCGCT BH-B8 CGTGAAGGACAATGTCAAGTG
BH-A9 TCCGCAACAATAATTATCGC BH-B9 TGGGACTGCGTCCATTTTTA
BH-A10 CACCCAACGCAACTAGGT(A)TT BH-B19 GGCTTGCAAATTGGTCGAA
BH-A11 CACCCACAACACAACTATCA BH-B11 TGAGCCTCATCAATAGATGG
BH-A12 CTTCACAATTGCAGAT(C)GGG(A)G BH-B12 AGCTG(A)AAATGTACGGGTGTC
BH-A13 AGAATGAGCAGAATAGGGA BH-B13 TTCGTTCATAGGCTGTGTT
BH-A14 GGTGGATACGGAATAATACGA BH-B14 GGCACAGGTGGCTGTAAATA
BH-A15 ATGATGACAT(C)GGACGA(G)GCAG BH-B15 GCGGCTGATTGT(A)CCTAGA(G)GT
BH-A16 GACTTGCCTGAGGAAGCATTA BH-B16 GAACAGCCCGGTTAGGATTT
BH-A17 CTTGCTCAGACTTTAACCGA BH-B17 GTTTAGAATTCTGGCTTTGG
BH-A18 GGTCTTGTAATCCGAAGATC BH-B18 GGGGTTTGACAAGGATAA
Table 1 PCR and sequencing primers for bighead carp
an Eppendorf Thermal Cycler with 50 μl reaction
mixture，which was containing 2 μl DNA polymerase
（Tiangen products，China），5 μl PCR buffer （Tiangen
products，China），2 μl template DNA（50 ng/μl），2 μl
dNTP （0.4 mM），4 μl primers （0.2 μM each），and 35
μl distilled water.
The amplification conditions were 94°C for 5 min. This
was followed by 30 cycles at 94°C for 30 s，54°C for
30 s，and 72° C for 1 min，and a final extension was
done at 72°C for 10 min. The verification of successful
PCR amplification was assessed by agarose gel
electrophoresis.
All amplified products were purified by using 3S Spin
PCR Product Purification Kit （Biocolor Inc.，China）
following the supplier’s instructions. The purified
products then were directly sequenced on an Applied
杨琴玲等：鳙的线粒体基因组核苷酸全序列分析 113
生物技术通报 Biotechnology Bulletin 2009年第 2期
Biosystems ABI 3730 capillary sequencer using the
same PCR primers.
2.3 Sequence assembly and gene identification
DNA sequences were put into BLAST searches on
Genbank，then edited and assembled by BioEdit
software [21]and checked by eyes. Finally，the complete
mtDNA of the bighead carp was obtained. Meanwhile，
the base composition for the mitochondrial genome of
bighead carp was also obtained（Fig.1）.
tRNA genes were identified using tRNAscan-SE 1.21[22]，
using the default search mode and specifying mitochon-
drial/chloroplast DNA as the source and using the
vertebrate mitochondrial genetic code for tRNA structu-
re prediction. Then BioEdit software and MEGA [23]was
used to analyze the protein-coding genes.
3 Results
3.1 Genome organization and composition
The complete mtDNA nucleotide sequences of the three
individuals of bighead carp were all 16 621 bp in
length，containing the same 13 protein-coding genes
（Cytb，ATP6，ATP8，COI-III，ND1-6，ND4L），22 transfer
RNAs，2 ribosomal RNAs genes （12S sRNA and 16S
rRNA）and a putative control region as found in other
Cyprinidae fishes （Table 2）. The overall base compo-
sition of the mitochondrial genome was 31.6% of Aden-
ine （A），27.1% of cytosine （C），16.0% of Guanine
（G），25.3%，of thymine（T），and with 56.9% the A+T
content（Table 3）.
The organization of the mitochondrial genome in the
bighead carp is displayed in Fig.1. The arrangement
order of the bighead carp mtDNA genes was consistent
with other Cyprinidae fishes，such as common carp [17，18]，
crusian carp [19，24]，and tinca [20]. Meanwhile，some nucle-
otides that the space and overlap in adjacent genes
were also observed as the other fishes. There were 14
bases spacer and 9 bases overlap in the bighead carp
（Table 2）. The complete sequences of the bighead
carp mitochondrial genome was deposited in GenBank
（Accession No. EU315941，NC_010156）.
3.2 Protein-coding genes
The mtDNA protein-coding genes of the bighead carp
showed the regular start codon ATG except gene COI，
which starts with GTG （Table 2）. The ends of the open
reading frames （ORFs） for protein-coding genes were
signaled with TAA （COI，ATPase8，ATPase6，COIII，
ND4L，ND5 and ND6），TAG （ND2，ND3，ND4），and
incomplete stop codon T-（ND1，COII，Cytb）. The
incomplete stop codons were common among mtDNA
genes in other fish[24，25]. 12 protein-coding genes were e-
ncoded by the H-strand of the mtDNA，and only ND6
was encoded by the L-strand （Table 2）. 7 nucleotides
overlap were found between the ATPase8 and
ATPase6，and the same phenomenon was seen between
the ND4 （H strand）and ND4L （L strand）genes. One
nucleotide overlap was detected between the ATPase6
and COIII，and 4 nucleotide overlap were detected
between the DN5 and DN6. The total A+T content was
56.8% in the 3 protein-coding genes. The total length
of the 13 protein-coding genes was 11 429 bp，account-
ing for 68.76% of the whole mtDNA length of the
bighead carp. The total A+T content was 56.8% in the
3 protein-coding genes（Table 2）. The A+T contents in
the 13 protein-encoding genes slightly fluctuate within
50%~60% with exception of 62.4% in ATPase8（Table
4）. In the 13 protein-encoding genes，the highest A
content（39.39%）of and the lowest G content （9.70%）
of ATPase8 were observed（Table 4）.
3.3 Non-coding sequence
The major non-coding sequence（D-loop）in the mtDNA
Fig.1 The mitochondrial genome of bighead carp
H-strand was designated on the upside and L-strand on the
downside of the molecule； 12S and 16S.genes of the 12S and
16S ribosomal RNA；ND1-6 and 4L.nicotinamide adenine
dinucleotide dehydrogenase subunits 1-6 and 4L；COI-III.
cytochrome c oxidase subunits I-III；ATP6 and ATP8.ATPase
subunits 6 and 8；cytb.cytochrome b；CR.control region；tRNA
were designated by single-letter amino acid codes except leucine
and serine were labeled as L1 （tRNALeu （UUR）），L2 （tRNALeu
（CUN）），S1（tRNASer（AGN））and S2（tRNASer（UCN））.
114
2009年第 2期
Table 2 Organization and profile of the mitochondrial DNA of bighead carp
Position number Codon
Feature
Start Stop
Size
(bp) Start Stop
Intergenic
nucleotide
Strand
tRNA 1 69 69 H
12SrRNA 71 1029 959 1 H
tRNA 1030 1101 72 0 H
16S rRNA 1102 2792 1691 0 H
tRNA
2793 2868 76 0 H
ND1 2870 3844 975 ATG T++ 1 H
tRNA
3849 3920 72 4 H
tRNA 3919 3989 71 -2 L
tRNA 3991 4059 69 1 H
ND2 4060 5106 1047 ATG TAG 0 H
tRNA 5105 5175 71 -2 H
tRNA 5177 5245 69 1 L
tRNA 5247 5319 73 1 L
tRNA 5352 5419 68 32 L
tRNA 5421 5491 71 1 L
CO 5493 7043 1551 GTG TAA 1 H
tRNA
7044 7114 71 0 L
tRNA 7118 7191 74 3 H
CO 7205 7895 691 ATG T++ 13 H
tRNA 7896 7971 76 0 H
ATPase8 7973 8137 165 ATG TAA 1 H
ATPase6 8131 8814 684 ATG TAA -7 H
CO 8814 9599 786 ATG TAA -1 H
tRNA 9599 9671 73 -1 H
ND3 9672 10022 351 ATG TAG 0 H
tRNA 10021 10090 70 -2 H
ND4L 10091 10387 297 ATG TAA 0 H
ND4 10381 11763 1383 ATG TAG -7 H
tRNA 11763 11831 69 -1 H
tRNA 11832 11900 69 0 H
tRNA
11902 11974 73 1 H
ND5 11975 13810 1836 ATG TAA 0 H
ND6 13807 14328 522 ATG TAA -4 L
tRNA 14329 14397 69 0 L
Cytb 14402 15542 1141 ATG T++ 4 H
tRNA 15543 15614 72 0 H
tRNA 15614 15683 70 -1 L
D-loop 15684 16621 938 0
Gene/Fragment
Protein coding
A C G T A+T
1 28.7 26.8 23.9 20.6 49.3
2 20.1 27.5 13.7 38.8 58.9
3 43.6 31.6 6.1 18.7 62.3
Total 30.8 28.6 14.5 26.0 56.8
tRNA 30.3 24.3 19.9 25.5 55.8
sRNA 34.8 24.2 21.1 19.9 54.7
CR 35.4 20.6 12.0 32.0 67.4
Overall 31.6 27.1 16.0 25.3 56.9
Table 3 Base composition (%) for the mitochondrial genome of bighead carp
杨琴玲等：鳙的线粒体基因组核苷酸全序列分析 115
生物技术通报 Biotechnology Bulletin 2009年第 2期
of the bighead carp was 938 bp long（Table 2）. The D-
loop of the bighead carp locates between tRANPro and
tRANPhe. Meanwhile，compared with corresponding
regions of other fish[26]，the D-loop of the bighead carp can
be divided into three domains. Domain I which was
hypervariable domain in mtDNA，consisted of a termin-
ation associated sequence （TAS：TACAT AAT GTA
CTA ATA CCT ATA TATGTATTAT）. The conserved
motif TACAT and its complement ATGTA could form a
thermostable “hairpin” which would be involved in the
regulation of mitochondrial gene replication. Domain II
was the central conserved region with 375 ~575 bp.
Domain III consisted of three conserved blocks （CSB-
1，CSB-2，CSB-3）. Furthermore，a TA-dinucleotide
micr-osatellite repeat was observed in the domain III，
and the number of TA core sequence is all 8 repeats in
the three individuals.
A small non-coding region，a putative origin of light
strand replication （OL），in the bighead carp was
located in a cluster of tRANTrp - tRANAla - tRANAsn
- tRANCys - tRANTyr region （WANCY region）and
comprised with 42 nucleotides in length. This region
has the potential to fold into a stable stem-loop
secondary structure with 19 bp in the stem and 11 bp
in the loop.
3.4 Ribosomal and transfer RNA genes
As other mitochondrial genome in vertebrates，the
bighead carp mtDNA contained two submits of rRNA，a
small one （12S）and a large one （16S）. The start
position number of 12S rRNA was at 71 bp while the
stop position number was at 1 029 bp with the length
of 959bp. Meanwhile，the start position number of 16S
rRNA was at 1 102 bp while the stop position number
was at 2 792 bp with the length of 1 691 bp. These two
submits were separated by tRNAVal，and are both on
the H-strand.
The secondary structure of 22 tRNA genes were
completed presented as typical clover-leaf structure by
using the tRNAscan-SE 1.21 serves （Fig.2）. The
length of 22 tRNA genes of the bighead carp were
ranged from 68 to 76 bp. Meanwhile，some nucleotides
overlaps were found in some adjacent tRNA genes. The
base composition of the 22 tRNA genes was A=30.3%；
C=24.3%；G=19.9%；T=25.5%，and A+T content was
55.8%（Table 2）. The average A+T content was higher
than that of the rRNA and lower than that of the
protein-encoding genes.
4 Acknowledgement
The authors want to express thanks to Mr. Minghu Tang
and Lixia Fu for their kind help in collecting samples.
This research was funded and supported by the
National Natural Science Foundation of China （Grant
No.30630051）and the Shanghai Leading Academic
Discipline Project（Grant No. Y1101）.
Gene A C G T A+T Total number
ND1 30.56 28.51 14.97 25.95 56.51 975
ND2 34.19 31.61 11.65 22.54 56.73 1047
CO 27.21 25.40 17.60 29.79 57 1551
CO 29.52 27.35 17.51 25.62 55.14 691
ATPase8 39.39 27.88 9.70 23.03 62.42 165
ATPase6 28.95 28.80 13.60 28.65 57.6 684
CO 27.93 27.68 16.84 27.55 55.48 786
ND3 27.64 32.19 15.10 25.07 52.71 351
ND4L 26.60 30.98 15.49 26.94 53.54 297
ND4 30.51 28.20 14.82 26.46 56.98 1383
ND5 32.95 29.14 12.20 25.71 58.66 1836
ND6 41.38 31.03 12.64 14.94 56.32 522
Cytb 29.45 28.75 14.46 27.34 56.79 1141
Table 4 Base composition (%) of 13 protein-coding genes in the mitochondrial genome of bighead carp
116
2009年第 2期
References
1 FAO. Fishery Statisitcs Yearbook-Aquaculture Production，2005.
2 Li SF，Zhou BY，Lu GQ，Zhao JL，Yao DX，Shen WK. Journal of
Fisheries of China，1997，21（2）：143～151.
3 Li SF，Genetical Characterization of Major Freshwater Culture Fishes
in China. Shanghai：Shanghai Scientific & Technical Publishers，
1998.
4 Xia DQ，Yan H，Wu TT，Dong ZJ，Jian JC，Cao Y，Zhang YS.
Journal of Fishery Science of China，1996，3（4）：11～18.
5 Zhao JL，Li SF. Journal of Fisheries of China，1996，20（2）：104～
110.
6 Jiang JG，Xiong QW，Yao RH. Hereditas（Beijing），1998，20（2）：
19～22.
7 Fan LC，Cui JX，Yu QX. Journal of Wuhan University（Natural Sci-
ence Edition），1994，40（1）：121～125.
8 Jian JC，Xia DQ. Journal of Fishery Science of China，1999，6（4）：
18～20.
9 Zhang XY，Yang JQ，Zhang DC，et al. Bio-phys，1999，26（5）：
469～472.
10 Zhang DC，Zhang XY，Yang DS，et al. Journal of Wuhan Universi-
ty（Natural Science Edition），1999，45（6）：857～860.
11 Zhang DC. J of China Three Gorge Univ（Natural Sciences），2002，
24（1）：379～381.
12 Lu CY，Sun XW，Lian LQ. Journal of Fishery Science of China，
2005，12（2）：192～196.
13 Shan Q，Dong S，Wu HF，Taniguchi N.Journal of Fishery Science
of China，2006，13（2）：174～180.
14 Avise JC，Molecular Markers，Natural History and Evolution. New
York：Chapman & Hall，1994.
15 Jondeung A，Sangthong P，Zardoya R. Gene，2007，387：49～57.
16 Sambrook J，Russell DW，Molecular Cloning：A Laboratory M ann-
ual（3rd edn）. New York：Cold Spring Harbor Laboratory Press，
2001，463~471.
17 Chang YS，Huang FL，Lo TB. J Mol Evol，1994，38（2）：138~155.
18 Mabuchi K，Miya M，Senou H，et al. Aquaculture，2006，257：
68～77.
19 Murakami M，Yamashita Y，Fujitani H. Zool Sci，1998，15：335～
337.
20 Saitoh K，Sado T，Mayden RL，et al. J Mol Evol，2006，63（6）：
826～841.
21 Hall TA. Nucl Acid Symp Ser，1998，41：95～98.
22 Lowe TM，Eddy SR. Nucl Acids Res，1997，25：955～964.
23 Kumar S，Tamura K，Nei M.Bioinformatics，2004，5：150～16
24 Guo X，Liu S，Liu Y. DNA Seq，2007，18（4）：247~256.
25 Peng Z，Wang J，He X. Gene，2006，376：290~297.
26 Kartavtsev YP，Jung SO，Lee YM，et al. Gene，2007，396：13~27.
Fig.2 The putative secondary structure of 22 tRNAs in the mitochondrial genome of bighead carp
杨琴玲等：鳙的线粒体基因组核苷酸全序列分析 117