Beruflich Dokumente
Kultur Dokumente
358
other ATP synthase subunit. In situ hybridization analysis was used to reveal the wide-spread distribution
+
of mitochondrial H -ATP synthase in various tissues in Pinctada fucata. This work will help further research
on pearl energy metabolism to increase the output and quality of pearls to more efficiently utilize our rich
pearl oyster resources.
+
Key words: mitochondrial H -ATP synthase; Pinctada fucata; gene cloning
Introduction
Mitochondrial H+-ATP synthase (F1F0 ATPase) is responsible for the production of the majority of the
Received: 2006-09-08
406
1.1
A pair of degenerate oligonucleiotide primers was designed based on the conserved amino acid segments of
the ATP subunit. The primers were ATPF1 [5ATGAAG(T/C)T(G/C)GA(G/A)TT(G/A)GCICAGT-3]
corresponding to (MKLELAQY) and ATPR1 [5-AC
(A/T)(G/C)(C/T)(A/T/G/C)ACTTGTTCTTCAAT-3]
corresponding to the antisense strand of the sequence
encoding (MAIEEQV). Altogether, 2.5 g of mantle
total RNA was used as a template for the RT reaction
with Superscript II and oligio (dT) primers (Invitrogen,
California, USA). The RT reaction and the following
PCR reaction were performed according to the manufacturers instructions on Tgradient Thermocylcer
(Biometra, Gottingen, Germany). PCR cycles were
performed with denaturation at 95 for 5 min,
In situ hybridization
2.1
The nucleotide sequence was submitted to the GeneBank with Accession No. DQ986328. From an evolutionary standpoint, the ATPase subunit is a remarkably well conserved protein. Multiple protein sequence
alignment analysis showed that the pfATPase subunit sequence shared high similarity with those of
ATPase subunits from carp, Xenopus laevis (X. la),
fruit fly, and mammalian (81% identity to fruit fly,
82% to carp, and 83% to human) (Fig. 2a). The
407
408
A: RT-PCR
(164 bp)
5'-RACE (1458 bp)
3'-RACE (558 bp)
Full-length cDNA (1991 bp)
B:
(1344 - 1507 bp )
(1-1458 bp )
(434 -1991 bp )
(1-1991 bp )
ACGCGGGGATATTGGTCGAAACCGCACACATACGACAAGTAGACAAA
ATGCTTTCGGCCAGATTAGCAGCAAGTCTCGTAAGACAGCTTCCCAGGGCTGCACCAAAGGTTTGCCAACATGCC
M L S A R L A A S L V R Q L P R A A P K V C Q H A
CTGGGAGCTGGACTTATTTCTGCCAAAAAGTTTTCCACATCTACACATCACCATACTGCAGGAGCTAGTGCGGAA
L G A G L I S A K K F S T S T H H H T A G A S A E
GTGTCATCCATCTTGGAGGAGAGGATTTTGGGACAGACAACACAAGCTGGATTAGAGGAAACCGGTCGTGTACTA
V S S I L E E R I L G Q T T Q A G L E E T G R V L
TCCATCGGAGACGGTATTGCTCGTGTTTATGGTCTAAAGAATATTCAGGCAGAAGAAATGGTAGAATTCTCCAGT
S I G D G I A R V Y G L K N I Q A E E M V E F S S
GGTTTAAAGGGTATGGCCTTGAACTTGGAGAGAGATAACGTAGGAGTTGTCGTATTTGGTAACGATAAATTAATT
G L K G M A L N L E R D N V G V V V F G N D K L I
AAAGAAGGAGACATCGTAAAAAGAACAGGAGCTATTGTGGATGTCCCCGTAGGAAAGGAATTATTAGGAAGAGTT
K E G D I V K R T G A I V D V P V G K E L L G R V
GTTGATGCTTTAGGAAATCCTATTGATGGAAAGGGACCAATCACATCACCTGACAGACAGAGGGTAGGAGTTAAG
V D A L G N P I D G K G P I T S P D R Q R V G V K
GCCCCTGGTATCATCCCCCGTATCTCAGTAAAGGAACCTATGCAGACCGGTATTAAGACTGTAGATAGCTTAGTA
A P G I I P R I S V K E P M Q T G I K T V D S L V
CCTATTGGTAGAGGTCAGAGAGAACTCATCATCGGTGACAGGCAGACTGGCAAAACCGCTATTGCAATTGACACA
P I G R G Q R E L I I G D R Q T G K T A I A I D T
ATTATTAACCAGAAGAGGTTCAACGATGGAACAAATGAGAAGGCCAAGCTTTATTGTATCTATGTTGCTATTGGT
I I N Q K R F N D G T N E K A K L Y C I Y V A I G
CAGAAGAGATCCACAGTGGCTCAGATTGTAAAGAGGCTTACTGATGCAGATGCCATGAAGTACACTGTGATTGTC
Q K R S T V A Q I V K R L T D A D A M K Y T V I V
AGTGCTACAGCTTCTGATGCTGCTCCCTTACAGTATCTGGCTCCTTACTCGGGATGTGCCATGGGAGAATTCTTC
S A T A S D A A P L Q Y L A P Y S G C A M G E F F
AGAGATAACGGCATGCACGCCCTCATCATCTACGACGATCTGTCAAAACAGGCTGTAGCTTACCGTCAAATGTCA
R D N G M H A L I I Y D D L S K Q A V A Y R Q M S
CTACTTTTGAGACGTCCCCCCGGTCGTGAAGCCTACCCTGGTGATGTGTTCTACCTCCATTCACGTTTGCTGGAG
L L L R R P P G R E A Y P G D V F Y L H S R L L E
CGTGCTGCCAAGATGAATGACGATAACGGTGGTGGATCCCTCACTGCTCTCCCTGTCATCGAGACCCAGGCTGGT
R A A K M N D D N G G G S L T A L P V I E T Q A G
GATGTGTCAGCCTACATCCCCACTAACGTCATCTCTATCACAGACGGACAGATCTTCTTGGAGACTGAGTTGTTC
D V S A Y I P T N V I S I T D G Q I F L E T E L F
TACAAGGGTGTACGTCCAGCAATTAATGTAGGATTATCTGTCAGTAGGGTAGGATCGGCAGCCCAAACAAAGGCC
Y K G V R P A I N V G L S V S R V G S A A Q T K A
ATGAAACAGGTAGCAGGCTCCATGAAGTTGGAGTTGGCCCAGTACAGAGAAGTGGCAGCTTTCGCTCAGTTTGGA
M K Q V A G S M K L E L A Q Y R E V A A F A Q F G
TCTGATTTGGATCAGGCTACACAGAATTTACTGAACAGGGGAGTTCGTCTGACCGAGTTACTCAAACAGGCTCAG
S D L D Q A T Q N L L N R G V R L T E L L K Q A Q
TATGTACCAATGGCCATTGAAGAACAAGTAGCAGTTATCTATGCTGGTGTCAAGGGACGTTTAGACAAAGTTGAT
Y V P M A I E E Q V A V I Y A G V K G R L D K V D
CCATCTAGAATCACAGAATTTGAAGCAGCTTTTGTATCACACATCAGAGGAAGTCAGCAAGAATTATTAGGTCAG
P S R I T E F E A A F V S H I R G S Q Q E L L G Q
ATCAGGAAAGACGGTCAAATCACAGAAGCCTCCGACGCTAAATTAAAGGAGGTTGTCACACAGTTCTTGTCCACC
I R K D G Q I T E A S D A K L K E V V T Q F L S T
TTCGAAGCATAAATTCATGTGACATTAAACATTTTCTCAAACGAATGGTGATATAGACAGTGCGGTTTTCCTTGT
F E A *
GACACTTATTGACTTAGAGTCTATTTATATCAGTGCTAACTTTGAAAGTATGTTGAATTGATTTGTACATGTAAT
GTCCCTTGTTTAAAAGATCTTTATAAACATTACATGGCAGTGACTGTTTTACATTGTTTTTGATGAATTCAGACA
ACTGTTGATGTATAGATGACATTATTAAAGGGATATTATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
-47
75
25
150
50
225
75
300
100
375
125
450
150
525
175
600
200
675
225
750
250
825
275
900
300
975
325
1050
350
1125
375
1200
400
1275
425
1350
450
1425
475
1500
500
1575
525
1650
550
1725
1800
1875
1944
Fig. 1 Oyster pfATPase subunit cDNA and deduced amino acid sequence. The conserved amino acid sequence residues used to deduce the degenerate primers are in bold and the corresponding nucleotides are shaded. The 3-RACE
primer is underlined. The 5-RACE primer is in the box. The stop codon is indicated with an asterisk.
409
Carp
Fruit fly
Gallus
X. la
Cow
Homo
Mus
Rattus
100
82
81
82
83
83
83
83
83
80
88
90
90
91
91
90
100
79
81
81
80
80
80
100
Gallus
100
X. la
Cow
93
92
92
92
92
100
92
92
92
93
97
98
07
100
97
96
100
98
100
Homo
Mus
Rattus
100
(a) Matrix indicating the percentage identities of the aligned pfATPase
(b) Phylogenetic tree showing the evolutionary relationship of the oyster pfATPase subunit protein sequence. (The N-J tree was generated
using the method N-J of program Clustal X 1.81.)
Fig. 2
Phylogenetic analysis of pfATPase subunit in Pincatada fucata and their homologies with other species
P. fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la
--MLSARLAASLVRQLPRAA-PKVCQHALGAGLISAKKFSTSTHHHTAGASAEVSSILEERILGQTTQAGLEETGRVLSI
--MLSGRVAAALARTLPRRA-GFVSKNVAAAACVGAKNLHTARPWLQKTGTAEVSSILEEKILGADTSAALEETGRVLSI
--MLSVRVAAAVARALPRRA-GLVSKNALGSSFIAARNLHASNSRLQKTGTAEVSSILEERILGADTSVDLEETGRVLSI
MSIFSARLASSVARNLPKAANQVACKAAYPAASLAARKLHVAST--QR--SAEISNILEERILGVAPKADLEETGRVLSI
--MLSVRVAAVFARSLPRQA-GLVSRNALGAAFVATRNIHASKMRFQKTGTAEVSSILEERILGADTSAELEETGRVLSI
--MLSVRVAAAVVRALPRRA-GLVSRNALGSSFIAARNFHASNTHLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRVAAAVARALPRRA-GLVSKNALGSSFVGARNLHASNTRLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRIAAAVARALPRRA-GLVSKNALGSSFVGTRNLHASNTRLQKTGTAEMSSILEERILGADTSVDLEETGRVLSI
--MLSVRVAAALARALPRQS-GLVSKKALGAAFVATRNIHASGAWLQKSGTAEVSSILEERILGADISTDLEETGRVLSI
77
77
77
76
77
77
77
77
77
P. fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la
GDGIARVYGLKNIQAEEMVEFSSGLKGMALNLERDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGKELLGRVVDALGNP
GDGIARVYGLRNVQAEEMVEFSSGLKGMSLKLEPENVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVYGLNNIQADEMVEFSSGLKGMALNLEPDNVGVVVFGNDKLIKQGDIVKRTGAIVDVPVGDELLGRVVDALGNA
GDGIARVYGLRNVQAEEMVEFFFGLKGMSLNLEPDNVGVVVFGNDRLIKEGDVVKRTGAIVDVPVGEELLGRVVVALGNP
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDVVKRTGAIVDVPVGEELLGRVVDALGNA
GDGIARVHGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGDELLGRVVDALGNA
GDGIARVYGLRNVQAEEMVEFSSGLKGMSLNLEPDNVGVVVFGNDKLIKEGDIVKRTGAIVDVPVGDELLGRVVDALGNA
157
157
157
156
157
157
157
157
157
P.fucata
Carp
Cow
Fruit fly
Gallus
Homo
Mus
Rattus
X. la
IDGKGPITSPDRQRVGVKAPGIIPRISVKEPMQTGIKTVDSLVPIGRGQRELIIGDRQTGKTAIAIDTIINQKRFNDGTN
IDGKGPLGSKQRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTAIAIDTIINQKRFNDGTE
IDGKGPIGSKARRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGAINTKDRFRVGIKAPGIIPRVSVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTALAIDTIINQKRFNEAQD
IDGKGPITSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPIGSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGSD
IDGKGPIGSKTRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPVGSKIRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVPIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNDGTD
IDGKGPLTSKIRRRVGLKAPGIIPRISVREPMQTGIKAVDSLVSIGRGQRELIIGDRQTGKTSIAIDTIINQKRFNEGTD
237
237
237
236
237
237
237
237
237
410
P. fucata
EKAKLYCIYVAIGQKRSTVAQIVKRLTDADAMKYTVIVSATASDAAPLQYLAPYSGCAMGEFFRDNGMHALIIYDDLSKQ 317
Carp
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGGYFRDNGKHALIIYDDLSKQ 317
Cow
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
Fruit fly
ESKKLYCIYVAIGQKRSTVAQIVKRLTDSGAMGYSVIVSATASDAAPLQYLAPYSGCAMGEYFRDKGKHALIIYDDLSKQ 316
Gallus
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
Homo
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
Mus
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
Rattus
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
X. la
EKKKLYCIYVAIGQKRSTVAQLVKRLTDADAMKYTIVVSATASDAAPLQYLAPYSGCSMGEYFRDNGKHALIIYDDLSKQ 317
(b) Nucleotides binding domain257-303 aa
P. fucata
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDDNGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
Carp
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDNFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
Cow
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDAFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
Fruit fly
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMSPAMGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 396
Gallus
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPAIETQAGDVSAYIPTNVISITDGQIFLET 397
Homo
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDAFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
Mus
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
Rattus
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDSFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
X. la
AVAYRQMSLLLRRPPGREAYPGDVFYLHSRLLERAAKMNDHFGGGSLTALPVIETQAGDVSAYIPTNVISITDGQIFLET 397
P. fucata
ELFYKGVRPAINVGLSVSRVGSAAQTKAMKQVAGSMKLELAQYREVAAFAQFGSDLDQATQNLLNRGVRLTELLKQAQYV 477
Carp
ELFYKGIRPAINVGLSVSRVGSAAQTRGMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYS 477
Cow
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477
Fruit fly
ELFYKGIRPAINVGLSVSRVGSAAQTKAMKQVAGSMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 476
Gallus
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 477
Homo
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477
Mus
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477
Rattus
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLSRGVRLTELLKQGQYS 477
X. la
ELFYKGIRPAINVGLSVSRVGSAAQTRAMKQVAGTMKLELAQYREVAAFAQFGSDLDAATQQLLNRGVRLTELLKQGQYV 477
P. fucata
PMAIEEQVAVIYAGVKGRLDKVDPSRITEFEAAFVSHIRGSQQELLGQIRKDGQITEASDAKLKEVVTQFLSTFEA 553
Carp
PMAIEEQVAVINAGVRGHLDKMDPSKITKFEKAFLQHVISQQTDLFAAIRTDGKISEASDAKLKEVVLNFLASFE- 552
Cow
PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENAFLSHVISQHQALLSKIRTDGKISEESDAKLKEIVTNFLAGFEA 553
Fruit fly
PMAIEDQVAVIYCGVRGHLDKMDPAKITKFEKEFLQHIKTSEQALLDTIAKDGAISEASDAKLKDIVAKFMSTFQG 552
Gallus
PMAIEEQVAVIYAGVKSHLDKLEPSKITKFESAFLAHVLSQHQALLSTIRTEGKISDQTEAKLKEIFTNFLSTFEA 553
Homo
PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENDFLSHVVSQHQALLGTIRAEGKISEQSDAKLKEIVTNFLAGFEA 553
Mus
PMAIEEQVAVIYAGVRGYLDKLEPSKITKFENAFLSHVISQHQSLLGNIRSDGKISEQSDAKLKEIVTNFLAGFEP 553
Rattus
PMAIEEQVAVIYAGVRGYLDKLEPSKITKFESAFLSHVVSQHQSLLGNIRSDGKISEQSDAKLKEIVTNFLAGFEP 553
X. la
PMAIEEQVTVIYAGVRGHLDKMEPSKITKFESAFLAHVKSQHQELLATIRTDGKISEQADAKLKEIVLNFLSTFEA 553
(c) / signal transduction domain (322-388 aa)
Fig. 3 Alignment of the oyster pfATPase subunit sequence with other ATP synthase subunits from invertebrates to
mammalian. The amino acid domains are indicated by overlining. The 358th and 359th amino acids, which differ from
other species are Asp (D) and Asn (N), respectively.
411
Fig. 4 In situ hybridization of oyster pfATPase subunit mRNA in various tissues of P. fucata. Among these tissues, hybridization signals (arrows) were observed in the mantle epithelial cells (a), mantle gland (b), digestive gland (c), spermatogenic duct (d), and gill (e).
Conclusions
[3]
Pedersen P L. Frontiers in ATP synthase research: Understanding the relationship between subunit movements and
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[4]
[5]
[6]
Senior A E, Nadanaciva S, Weber J. The molecular mechanism of ATP synthesis by F1F0-ATP synthase. Biochim.
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[7]
Huang W, Yao B, Sun L, Pu R, Wang L, Zhang R. Immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (GnRH) and its receptor in
rat digestive tract. Life Sci., 2001, 68(15): 1727-1734.
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