한국어병학회지 제
22
권 제3
호(2009) J. Fish Pathol., 22(3) ： 353 ~ 366 (2009)
353
Introduction
Many molecular tools are available for character- izing the immune systems of mammals, especially those of human and mouse. In fish, information of immune-related molecules is still limited. Recently, remarkable progress was made in genetic technolo- gy. Rapid expressed sequence tag (EST) analysis allows acquisition of huge number of DNA sequence information in a short time period for many organisms including several species of fish (Zeng and Gong, 2002; Clark et al., 2003; Rise et al., 2004). Large scale EST analysis is also an effi- cient way for identification of genes and for analysis of their expression by means of expression profiling (Franco et al., 1995; Azam et al., 1996; Lee et al., 2000). It offers a rapid and valuable first look at
genes expressed in specific tissue types, under spe- cific physiological conditions, or during specific developmental stages. Currently, the number of fish- related ESTs in the public databases is still small compared with mammalian sequences and there are relatively few tissue-specific cDNA libraries (Ton et al., 2000). ESTs have been shown to be an excellent and proven method of identification and characteriz- ing novel genes.
Rock bream Oplegnathus fasciatus belongs to Oplegnathidae family and inhabits coastal rocky- shores of Korea, Japan, Taiwan and Hawaii (Nakabo, 2002). This species has attracted great interest from Korean fish farmers due to its high market value and consumer demand. The total pro- duction of this species, however, is not satisfactory compared to other commercial important fishes in
Expressed sequence tags analysis of immune-relevant genes in rock bream Oplegnathus fasciatus peripheral
leukocytes stimulated with LPS
Jeong-Ho Lee, Jae Koo Noh, Hyun Chul Kim, Choul-Ji Park, Byung Hwa Min, Sang Jun Choi, Jeong-In Myeong, Hyung-Jun Park
�and Chan-Il Park
��Genetics and Breeding Research Center, National Fisheries Research and Development Institute, Geoje, 656-842, Korea
�
Department of Marine Biology & Aquaculture, Institute of Marine Industry, College of Marine Science, Gyeongsang National University, 455, Tongyeong, 650-160, Korea
We constructed a rock bream Oplegnathus fasciatus leukocyte cDNA library and a total of 795 expressed sequence tag (EST) clones were generated. Gene annotation procedures and homology searches of the sequenced ESTs were locally done by BLASTX for amino acid similarity comparisons. Of the 795 EST clones, 491 different ESTs showed significant homology to previously described genes while 304 ESTs were unidentified, hypothetical, or unnamed proteins. Encoding 121 different sequences were identified as putative bio-defense genes or genes associated with immune response.
Key words: Expressed sequence tags, ESTs, Rock Bream, Oplegnathus fasciatus, Leukocyte
�Corresponding Author : Chan-Il Park, Tel : 82-55-640-3103 FAX : 82-55-642-4509 E-mail : [email protected]
17...
Korea. Since then, RSIV disease has been the major culprit of mass mortality of rock bream in Korea (Sohn et al., 2000; Jung and Oh, 2000).
Despite these interest, relatively a little informa- tion is available about rock bream genes (Cho et al., 2000). In this study, EST sequenced to screen for bio-defence or immune response related genes from rock bream peripheral blood leukocytes (PBL) after the treatment with LPS as an in vitro activation sys- tem.
This lack of knowledge may represent one obsta- cle to the effective use of genetics in aiding both rock bream aquaculture and conservation activities.
Materials and Methods
cDNA library construction
Peripheral blood samples were taken from a rock bream as previously described (Nam et al., 2000).
Isolated leukocytes were adjusted to a concentration of 10
7cells/㎖ and cultured in RPMI1640 contain- ing 10% fetal bovine serum (FBS), 50 ㎍/㎖ LPS at 20℃ for 1, 3, 6, and 12 h. Following each incuba- tion, leucocytes were harvested, washed twice with phosphate buffered saline (PBS) and frozen at -80
℃ until use. mRNA was isolated from the LPS induced leucocytes using a micro mRNA purifica- tion kit (Amersham-Pharmacia, U.S.A.). The puri- fied mRNAs of four different time periods were pooled to ensure complete coverage of expressed genes in the allotted time frame and used to con- struct a cDNA library.
Libraries were constructed by using a modifica- tion of Maruyama and Sugano's method (Maruya- ma and Sugano, 1994). The synthesis of first-strand cDNA from the purified mRNA and cDNA ampli- fication were performed as described by Maruyama and Sugano (1994). The amplified PCR products were then digested with SfiI, and cDNAs longer
than 400 bp were ligated into DraIII-digested pCNS-D2 in an orientation-defined manner. The pCNS-D2 vector contains 5' EcoRI-DraIII-EcoRV- DraIII sites at multi cloning sites, which was achieved by modifying pCNS vector (GenBank Accession no. AF416744). The ligated cDNA was then transformed into E. coli Top10F' (Invitrogen) by electroporation (Gene Pulser II, BioRad).
The 5'-termini base sequencing of selected cDNA clones in phagemid form were performed using the ABI 3100 automatic DNA sequencer (PE Applied Biosystems, CA, U.S.A.) and the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, U.S.A.).
EST sequencing and analysis
Bioinformatic analysis was conducted to deter- mine gene identities using Genetyx ver. 8.0 soft- ware (SDC software, Japan). Briefly, vector sequences were removed and database search were limited to ESTs >400 bp in length. The sequence of each cDNA was compared with sequences in the peptide sequence databases at the National Center for Biotechnology Information (NCBI) using the BLAST network service. Nucleotide sequence comparisons were carried out using the program BLASTX (Gish and David, 1993).
Results and Discussion
cDNA library construction
A cDNA library of 6 × 10
6clones was construct- ed from the polyadenylated fraction of mRNA of rock bream leukocytes stimulated LPS. The number of clones in the constructed cDNA library was thought to be sufficient to cover the predominantly expressed mRNAs in LPS stimulated rock bream leukocytes.
A total of 795 randomly selected clones were sin-
gle-pass sequenced from the 5' end, resulting in the characterization of cDNA clones that were longer than 400 bp after elimination of vector sequence.
The number of clones sequenced from the cDNA library, the average size of inserts, and the redundan- cy of the obtained sequences, are given in Table 1.
EST sequencing and analysis
Gene annotation procedures and homology searches of the sequenced ESTs have been locally done by BLASTX for amino acid similarity com- parisons. The ESTs with significant similarities to known proteins were evaluated to determine if the significant similarities were caused by simple amino acid matches (Table 2). Of the 795 clones, 491 (61.8%) were identified as orthologues of known genes from other organisms. The remaining 304 (38.2%) clones could not be identified by BLASTX for amino acid similarity comparisons; most of them are expected to derive from hitherto uncharacterized or novel genes, whereas some of them may be due to the fact that the homology is too low to detect in the region that is sequenced. Among the 491 identi- fied EST clones, 121 clones were immune related genes identified as homologous to the previously reported genes from other organisms (Table 2).
The discovery of novel teleost genes related to the immune response has been accelerated by high throughput sequencing techniques combined with searches for homologous sequences on public data- bases. The sequencing of expressed sequence tags (ESTs) is specially useful, since it allows simultane- ously the novel gene discovery and gene expression analysis.
Leukocytes proliferation is an essential process of a specific immune response where activated lym- phocytes, following stimulation, express numerous cytokine genes and immune response-related genes.
However, it is difficult to detect the proliferation of normal lymphocytes in response to specific anti- gens, only a minute proportion of cells will be stimu- lated to divide. The teleost fish leukocyte immune system possesses B and T lymphocytes, similar to that of mammals, although they show different responses to band T-lymphocyte mitogens (Clem et al., 1984; Manning, 1994). The concanavalin A (Con A) activation system has previously been used for the analysis of molecular determinants of fish leukocyte proliferation (Graham and Secombes, 1988; Yin et al., 1999). Furthermore, phorbol myris- tate acetate (PMA) has also been used as a mitogen together with Con A or lipopolysaccharide (LPS)
Table 1. Summary of sequences and clones represented.
Numbers of ESTs (n) 795
Singletons (n) 570
Putatively identified clones (n) 491 (61.8%)
Hypothetical protein and unnamed protein 33
Ribosomal protein 131
Immune related genes (n) 122
Other genes 205
Unidentified clones (n) 304 (38.2%)
Putatively identified different genes (n) 247
Total nucleotides (bp) 550,949
Average of sequenced length (bp) 693
Table 2. List of identified ESTs from LPS stimulated leukocyte cDNA of rock bream.
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c02-A08 actin ACR54088.1 Agelena silvatica 5.83E-64 247 100 611 2
04-A09 actin filament associated protein
NP_689619.1| Homo sapiens 2.00E-33 164 57 827 1 1-like 1 isoform 1
05-D12 Actin-related protein 2/3 complex ACQ58595.1 Anoplopoma fimbria 2.98E-98 362 97 820 4 subunit 3
05-A10 adenine nucleotide translocator s6 NP_001037838.1 Takifugu rubripes 2.00E-04 404 91 770 1 04-D04 Adenosylhomocysteinase B NP_001133847.1 Salmo salar 1.00E-108 405 96 814 1 07-C10 Adenylate kinase ACO09564.1 Osmerus mordax 2.00E-54 223 97 812 1 09-H01 ADP-ribosylation factor 6 ACO09998.1 Osmerus mordax 3.86E-31 139 100 792 1 09-E07 AH receptor-interacting protein NP_001133532.1 Salmo salar 5.85E-107 391 82 707 1 03-C07 Aldose reductase ACI67446.1 Salmo salar 2.06E-61 240 88 785 1 03-A01 Alpha-aspartyl dipeptidase ACO09369.1 Osmerus mordax 4.68E-117 424 88 803 1 04-D07 antigen processing
ACN49172.1 Oryzias luzonensis 8.67E-123 443 92 756 1 proteasome-associated protein
11-G02 atlastin GTPase 3 NP_001037706.1 Rattus norvegicus 2.00E-99 418 87 796 1 12-D07 ATP synthase D chain, mitochondrial ACO10059.1 Osmerus mordax 1.64E-70 269 79 646 1 02-A04 ATP synthase F0 subunit 6 YP_001218735.1 Parajulis
5.45E-71 272 69 834 1 poecilepterus
01-G09 B7-H1/DC BAH30159.1 Takifugu rubripes 2.54E-06 57 28 800 1 04-H08 bactericidal/permeability-increasing
ABO32254.1| Larimichthys crocea 8.00E-82 160 57 872 1 protein
14-B12 B-cell translocation gene 1 NP_956314.1 Danio rerio 3.00E-64 256 97 672 1 05-A07 B-cell translocation gene 3 EDM10603.1 Rattus norvegicus 2.00E-32 293 65 839 1 02-H01 B-cell translocation protein 1 ACM41870.1 Epinephelus
7.57E-90 334 92 883 2 coioides
13-A10 beta actin ACP56688.1 Oncorhynchus
1.00E-114 413 100 616 5 tshawytscha
02-E06 beta-2 microglobulin AAW65850.1 Stizostedion vitreum 2.59E-39 166 76 862 7 07-E10 bromodomain containing 4 XP_001914731.1 Equus caballus 0.00012685 49 36 484 1 10-F07 BTB (POZ) domain containing 8,
EAW73104.1 Homo sapiens 0.0043149 44 58 506 1 isoform CRA_c
calcium/calmodulin-dependent protein
12-H02 kinase (CaM kinase) II gamma 2 XP_001332339.1 Danio rerio 1.10E-40 169 95 444 1 isoform 2
10-D05 Calpastatin ACN10964.1 Salmo salar 3.00E-14 104 52 501 1
07-D01 cAMP-regulated phosphoprotein 19 CAJ83731.1 Xenopus tropicalis 5.00E-49 220 93 792 1 05-E04 capping protein (actin filament) muscle
NP_788821.1 Bos taurus 3.25E-107 392 96 785 1 Z-line, beta
01-F07 Carboxymethylenebutenolidase
ACO13373.1 Esox lucius 1.24E-82 310 86 786 1 homolog
13-F07 Casein kinase II subunit alpha ACM08892.1 Salmo salar 5.00E-36 153 87 424 1
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c03-H12 caspace-like protein NP_001117965.1 Oncorhynchus
3.69E-10 69 47 784 1 mykiss
03-A06 Caspase-1 precursor ACI67164.1 Salmo salar 3.92E-14 83 43 880 1 07-A09 cathepsin B
ACO82382.1 Lutjanus
1.21E-86 324 89 834 1 [Lutjanus argentimaculatus] argentimaculatus
12-G02 cathepsin L ACN93991.1 Dicentrarchus
3.67E-133 478 94 815 1 labrax
10-D04 CC chemokine ligand 4 CAM32187.1 Dicentrarchus
4.97E-36 156 73 899 7 labrax
06-F02 CD2 antigen cytoplasmic tail-binding
ACQ58674.1 Anoplopoma fimbria 4.34E-79 299 62 850 1 protein 2
02-G01 CD200 molecule isoform 1 XP_001103685.1 Macaca mulatta 2.00E-18 114 40 862 1 12-G05 CDC-like kinase 4b NP_001013312.1 Danio rerio 4.10E-30 136 76 776 1 10-D01 Cell division cycle 5-like protein ACN10861.1 Salmo salar 3.88E-43 179 64 746 1 09-C10 CHK1 checkpoint homolog
CAJ83813.1 Xenopus tropicalis 4.25E-17 92 98 808 2 (S. pombe)
12-F05 CHK1 checkpoint-like protein ABK29471.1 Helicoverpa armigera 1.42E-16 89 91 455 18 09-B01 Chondroitin 4-sulfotransferase-2 AAI62078.1 Danio rerio 3.30E-37 159 42 759 1 13-F08 Chromobox protein homolog 3 ACQ58808.1 Anoplopoma fimbria 0.001 46.6 100 599 1
CKLF-like MARVEL
04-F08 transmembrane domain-containing ACO08649.1 Oncorhynchus mykiss 2.06E-50 203 61 772 1 protein 7
14-F01 class I helical cytokine receptor
AAR25685.1 Tetraodon nigroviridis 3.00E-46 188 54 623 1 number 22
10-F08 claudin 11a AAT64084.1 Takifugu rubripes 2.25E-89 333 74 893 1 13-A07 coagulation factor II (thrombin)
CAM13186.1 Danio rerio 8.00E-31 200 67 554 1 receptor
10-A10 Cu/Zn superoxide dismutase AAT36615.1 Oplegnathus fasciatus 5.66E-86 321 100 810 1 11-H07 CUGBP1 variant protein dbj|BAE06101.1 Homo sapiens 1.80E-05 54 76 835 1 06-B02 CXC chemokine receptor 4 ABP48751.1 Psetta maxima 2.12E-40 170 71 813 2 04-C02 Cystatin precursor ACO09585.1 Osmerus mordax 6.11E-33 145 50 741 1 03-B12 Cysteine-rich protein 1 ACQ58281.1 Anoplopoma fimbria 7.00E-40 169 92 644 1 05-A08 cytochrome b YP_001974630.1 Oplegnathus fasciatus 4.49E-122 441 89 768 1 06-A02 cytochrome c oxidase I YP_002274334.1 Micropterus dolomieu 2.78E-63 245 85 545 1
Cytochrome c oxidase polypeptide
12-C11 VIIa-liver/heart, mitochondrial ACO09236.1 Osmerus mordax 5.00E-29 149 87 511 1 precursor
10-C03 Cytochrome c oxidase polypeptide
ACN09873.1 Salmo salar 2.00E-23 141 85 477 1 VIIb, mitochondrial precursor
11-A03 cytochrome c oxidase subunit I ACA62816.1 Lutjanus
1.16E-104 383 87 784 2 argentimaculatus
08-C11 cytochrome c oxidase subunit II YP_001218733.1 Parajulis poecilepterus 5.97E-57 190 94 416 1
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c09-D05 cytochrome c oxidase subunit Va
AF255355_1 Scombridae gen. sp. 2.77E-72 275 96 629 1 precursor
01-B02 Cytochrome c oxidase subunit VIb
ACQ58800.1 Anoplopoma fimbria 1.29E-43 180 91 645 1 isoform 1
Cytochrome c oxidase subunit
07-G04 VIIa-related protein, mitochondrial ACO14482.1 Esox lucius 1.00E-48 201 81 665 1 precursor
01-F02 Cytohesin-1 ACO09516.1 Osmerus mordax 6.93E-62 241 87 777 1 10-G01 DEAD (Asp-Glu-Ala-Asp) box
NP_001003876.1 Danio rerio 1.64E-114 416 86 798 1 polypeptide 56
02-H07 DNA polymerase III, alpha subunit YP_002940603.1 Kosmotoga olearia 1.00E-06 138 51 830 1 04-D08 DNA repair protein RAD50 ACE77687.1 Sorex araneus 2.00E-106 470 91 853 1 01-D04 DNA-damage-inducible transcript
ACQ58934.1 Anoplopoma fimbria 1.08E-76 290 74 811 1 4 protein
12-D03 DnaJ (Hsp40) homolog, subfamily
NP_956067.1 Danio rerio 5.00E-78 334 71 870 4 B, member 1
14-D07 DnaJ (Hsp40) homolog, subfamily
NP_001002353.1 Danio rerio 4.00E-20 96.3 69 799 1 B, member 6
03-D11 dual specificity phosphatase 5 NP_997730.1 Danio rerio 3.00E-39 178 68 806 2 04-D02 dynein light chain LC8-type 2 XP_001787488.1 Bos taurus 3.07E-45 185 97 643 3 07-D12 Eef1g protein AAH66518.1 Danio rerio 1.44E-116 423 75 828 1 02-G08 elongation factor 1 beta CAB40840.1 Oryzias latipes 1.00E-37 160 93 321 1 05-F08 elongation factor 1-alpha AAP20169.1 Pagrus major 1.77E-148 529 97 805 2 09-G04 Elongation factor 1-alpha, oocyte
ACI69350.1 Salmo salar 0.0002162 49 71 398 1 form
endothelial differentiation,
13-D06 lysophosphatidic acid NP_001112363.1 Danio rerio 2.00E-07 76.6 48 669 1 G-protein-coupled receptor, 6
04-C09 Engulfment and cell motility
NP_001133480.1 Salmo salar 1.77E-87 326 95 813 1 protein 1
04-A12 erythrocyte membrane protein
NP_036439.2| Homo sapiens 2.00E-84 377 76 776 1 band 4.1-like 3
09-A08 Eukaryotic translation initiation
ACH70927.1 Salmo salar 1.00E-68 274 82 828 2 factor 5A-1
06-D10 FAM32A-like ACQ58612.1 Anoplopoma fimbria 5.44E-34 148 67 582 1 14-A05 F-box and leucine-rich repeat
XP_001915305.1 Equus caballus 1.00E-20 245 50 825 1 protein 10
11-E08 Fermitin family homolog 3 ACI34024.1 Salmo salar 9.99E-63 242 84 480 1 02-C09 ferritin heavy polypeptide ACN80998.1 Dicentrarchus
1.09E-96 357 96 883 2 labrax
07-E12 Ferritin, middle subunit ACQ57875.1 Anoplopoma fimbria 7.44E-91 337 91 794 2 12-E01 friend leukemia integration 1 CAK04699.1 Danio rerio 2.00E-91 353 87 712 1
FUS interacting protein
11-C04 (serine/arginine-rich) 1, isoform EAW95104.1 Homo sapiens 9.67E-17 91 85 814 1 CRA_b
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c10-G05 G protein-coupled receptor 178 NP_001038305.1 Danio rerio 9.00E-73 283 85 811 1 03-F11 G1 to S phase transition 1 NP_942101.1| Danio rerio 5.00E-156 572 93 904 1 11-A08 galectin like protein NP_001117663.1 Oncorhynchus
2.29E-57 226 71 775 1 mykiss
14-E09 Glia maturation factor gamma NP_001134643.1 Salmo salar 2.00E-60 235 82 627 1 07-A10 Glia-derived nexin NP_001133589.1 Salmo salar 7.00E-90 350 80 815 1 02-F10 glucose transporter 3 AAT67456.1 Gadus morhua 6.01E-05 52 53 781 1 06-D04 Glutamate dehydrogenase,
ACI32916.1 Salmo salar 1.81E-118 429 95 781 1 mitochondrial
03-E09 Glycolipid transfer protein ACQ58231.1 Anoplopoma fimbria 3.93E-103 378 88 812 1 12-A05 glycoprotein A-rich protein AAY27078.1 Gasterosteus aculeatus 9.08E-107 390 76 769 1
13-F12 gp39 YP_001456769.1 Corynebacterium
0.066 41.2 31 621 1 phage BFK20
04-B06 guanine nucleotide binding protein,
NP_032165.3 Mus musculus 5.00E-65 249 82 854 1 alpha q polypeptide
11-A01 hCG2007536 EAX09573.1 Homo sapiens 0.00123056 47 39 568 1
09-E10 hCG2036828 EAX04850.1 Homo sapiens 0.0428929 43 87 851 1
03-B07 Heat-shock protein 90 BAF57908.1 Lepomis
6.51E-100 367 92 782 1 macrochirus
13-F03 hematopietic transcription factor
NP_001098355.1 Oryzias latipes 5.00E-93 344 83 745 1 GATA-1
12-C03 Hemoglobin subunit alpha-2 ACQ58312.1 Anoplopoma fimbria 6.00E-07 59 90 416 1 11-E07 Hexokinase-2 ACQ58058.1 Anoplopoma fimbria 1.66E-93 346 88 814 1
High affinity immunoglobulin
12-B04 epsilon receptor subunit gamma ACI66690.1 Salmo salar 0.0001112 50 39 627 1 precursor
12-C05 High mobility group protein B1 ACQ59037.1 Anoplopoma fimbria 1.05E-64 251 81 857 2 09-E03 HN1-like protein DAA01819.1 Takifugu rubripes 7.00E-63 255 60 800 1 08-B01 Homeodomain-only protein NP_001134639.1 Salmo salar 2.39E-25 119 75 690 2 03-D09 Homo sapiens guanine nucleotide
AAP36465.1 synthetic construct 4.25E-26 122 87 654 1 binding protein (G protein), gamma 5
02-D02 ICER protein NP_989718.1 Gallus gallus 3.53E-31 139 68 879 11 06-G06 immunoglobulin heavy chain AF327364_1 Siniperca chuatsi 1.58E-62 242 86 521 3 03-E08 Immunoglobulin lambda-like
ACQ58137.1 Anoplopoma fimbria 9.08E-54 213 87 603 2 polypeptide 1 precursor
02-D07 immunoglobulin light chain AF327366_1 Siniperca chuatsi 5.36E-52 207 85 563 1 inhibitor of kappa light polypeptide
12-E03 gene enhancer in B-cells, kinase NP_001102324.1 Rattus norvegicus 3.00E-99 478 100 813 1 epsilon
14-G02 inositol hexaphosphate kinase 2 NP_958878.1 Danio rerio 3.00E-10 69.3 94 730 1
09-C04 interferon regulatory factor 8 NP_001002622.1 Danio rerio 1.00E-83 324 66 804 1
13-C11 interleukin 2 receptor, beta NP_001121739.1 Danio rerio 2.00E-10 69.7 34 690 1
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c05-D06 Iron-sulfur cluster assembly
ACQ58345.1 Anoplopoma fimbria 4.10E-83 312 94 893 5 enzyme ISCU, mitochondrial
precursor
03-A12 keratin 18 ACE06742.1 Epinephelus coioides 2.92E-136 488 92 845 1 03-C06 keratin 8 ACH73075.1 Epinephelus coioides 9.66E-88 325 74 872 1 11-E06 lin-52 homolog ACI66771.1 Salmo salar 1.27E-57 227 100 751 1 03-F04 low molecular weight heat shock
AAB46594.1 Poeciliopsis lucida 6.08E-87 325 78 890 1 protein Hsp30B
macrophage galactose
03-E02 N-acetyl-galactosamine specific CAM28084.1 Mus musculus 5.00E-12 165 41 830 1 lectin 1
02-G02 major histocompatibility complex II
CAP47207.1 Sparus aurata 9.60E-77 291 72 884 2 gamma chains
02-B07 methyltransferase like 11A CAF96711.1 Danio rerio 1.65E-104 383 84 826 1 11-A09 MHC class II antigen alpha chain ABV48906.1 Larimichthys crocea 3.80E-50 201 75 625 1 09-F05 MHC II invariant chain AAS77256.1 Siniperca chuatsi 6.62E-106 387 81 820 2
03-H06 MIP1alpha BAC10649.1 Paralichthys
1.50E-23 113 68 647 2 olivaceus
11-H01 Myosin regulatory light chain 2,
ACQ58528.1 Anoplopoma fimbria 9.35E-94 347 99 797 3 smooth muscle isoform
05-G04 NADH dehydrogenase 1 beta
ACN09926.1 Salmo salar 1.00E-47 205 78 617 1 subcomplex subunit 7
03-C08 NADH dehydrogenase 1 subunit
ACQ58548.1 Anoplopoma fimbria 3.92E-49 147 92 640 1 C2
05-H02 NADH dehydrogenase iron-sulfur
ACI66006.1 Salmo salar 4.81E-58 227 84 509 1 protein 6, mitochondrial precursor
13-A12 NADH dehydrogenase subunit 4 YP_002922043.1 Lutjanus sebae 7.00E-54 213 71 605 1 09-G05 Nascent polypeptide-associated
ACQ58397.1 Anoplopoma fimbria 3.83E-61 239 86 824 1 complex subunit alpha
10-G09 natural killer-tumor recognition
AAI21751.1 Danio rerio 2.00E-94 252 87 765 1 sequence
04-H11 NCK-associated protein 1 XP_001254874.2 Bos taurus 2.00E-33 148 97 802 1 08-C02 NEDD8 precursor ACI66839.1 Salmo salar 5.21E-29 131 77 575 1 10-A06 Nfe2 ABS70765.1 Astatotilapia burtoni 1.04E-53 214 68 836 1 10-G02 novel protein (wu:fi34c02) CAN88628.1 Danio rerio 5.08E-18 95 37 705 1 07-A03 novel protein (zgc:101116) CAK05291.1 Danio rerio 0.088188 40 76 565 1 02-H08 novel protein similar to vertebrate
CAN88412.1 Danio rerio 6.00E-73 281 88 732 1 DAB2 interacting protein
06-D07 novel protein similar to vertebrate
CAX12366.1 Danio rerio 4.93E-48 195 52 830 1 hect domain and RLD 3 (HERC3)
06-C09 novel protein similar to vertebrate
CAQ15540.1 Danio rerio 1.64E-64 250 84 713 1 proline rich 6 (PRR6)
12-G01 novel serine protease protein CAQ14751.1 Danio rerio 1.00E-90 425 90 839 1
05-C11 novel zinc finger protein CAQ14792.1 Danio rerio 7.00E-15 157 50 839 1
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c14-B11 Nuclear protein Hcc-1 ACQ58767.1 Anoplopoma fimbria 7.00E-56 219 71 539 1 08-G03 nucleoside diphosphate kinase AAY79301.1 Siniperca chuatsi 7.65E-41 171 96 640 2 05-H12 nucleoside phosphorylase NP_991218.2 Danio rerio 9.00E-88 411 86 742 1 13-D08 oligophrenin 1 CAM13477.1 Danio rerio 1.00E-31 140 62 726 1 01-H05 p53 tumor supressor protein ABQ42582.1 Paralichthys
2.69E-14 83 53 824 2 olivaceus
07-D04 p53-related protein kinase NP_001018386.1 Danio rerio 1.00E-35 155 69 482 1 05-D08 pantothenate kinase 2 NP_001074075.1 Danio rerio 2.00E-60 302 79 810 1 09-A02 Peptide methionine sulfoxide
ACO08867.1 Osmerus mordax 1.53E-64 249 80 610 1 reductase msrB
12-C07 Peroxiredoxin-5, mitochondrial
ACQ58198.1 Anoplopoma fimbria 8.42E-91 337 90 850 1 precursor
13-B10 Peroxiredoxin-6 ACI67008.1 Salmo salar 1.00E-104 382 83 716 1 06-B09 phosducin-like 3-1 ACH70961.1 Salmo salar 8.00E-98 379 81 784 1 12-D04 Pinx1 protein AAH54670.1 Danio rerio 3.02E-51 204 88 435 1 12-H11 pol-like protein dbj|BAC82624.1 Ciona intestinalis 1.94E-06 57 25 699 1
14-A03 polyprotein AAM34208.1 Danio rerio 7.00E-09 63.5 40 513 1
05-D09 Polypyrimidine tract-binding protein 2 ACN10491.1 Salmo salar 6.79E-62 241 64 675 1 10-A01 PolyrC-binding protein 2 NP_001133381.1 Salmo salar 2.62E-110 402 94 824 1 13-C01 polyubiquitin NP_001009202.1 Ovis aries 1.00E-116 421 94 730 1 05-B11 polyubiquitin with 3 Ub domains XP_626192.1 Cryptosporidium
7.50E-124 447 100 849 3 parvum Iowa II
07-G05 PQ-loop repeat-containing protein 2
ACO14120.1 Esox lucius 2.88E-90 335 67 804 1 [Esox lucius]
10-C01 Prmt1 protein AAH44522.1 Danio rerio 2.56E-148 528 92 847 1 02-C06 Probable ATP-dependent RNA
NP_001133649 Salmo salar 4.00E-41 211 68 890 1 helicase DHX58
07-C05 Probable ribosome biogenesis protein
ACQ59000.1 Anoplopoma fimbria 9.24E-84 314 91 819 1 RLP24
10-B02 Prohibitin-2 ACN10295.1 Salmo salar 8.08E-20 100 61 451 1
08-A07 Prostaglandin E synthase 3 ACQ59002.1 Anoplopoma fimbria 1.02E-65 254 91 772 1 06-A10 Proteasome subunit alpha type-3 ACQ58289.1 Anoplopoma fimbria 5.73E-136 487 96 790 1 06-B03 protein phosphatase 3, regulatory
NP_000936.1 Homo sapiens 4.11E-92 342 100 815 1 subunit B, alpha isoform 1
Protein-L-isoaspartate
11-F11 O-methyltransferase ACN11306.1 Salmo salar 3.00E-16 104 54 839 1 domain-containing protein 1
10-D09 putative transient receptor protein 2 AAP20203.1 Pagrus major 1.75E-29 133 89 634 1 11-D06 rCG26050 EDL77776.1 Rattus norvegicus 0.00140713 48 37 881 1 01-H06 receptor for activated protein kinase
AAQ91574.1 Oreochromis
5.33E-126 454 94 787 1
C mossambicus
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c04-A03 Regulator of G-protein signaling 13 ACQ58902.1 Anoplopoma fimbria 6.56E-56 182 76 751 1 12-A08 Regulator of G-protein signaling 18 NP_001134783.1 Salmo salar 5.00E-48 206 81 816 1 11-B01 retinoblastoma-binding protein 1 NP_001026838.1 Danio rerio 2.00E-79 325 68 812 1 05-C08 Rho-related GTP-binding protein
ACI69844.1 Salmo salar 4.00E-74 293 87 851 1 RhoF precursor
03-D01 RNA binding motif protein 42 NP_001003850.1 Danio rerio 7.00E-15 108 41 821 1 05-F02 RNA polymerase B transcription
ABJ98669.1 Scophthalmus
5.81E-81 305 92 831 3
factor 3 maximus
11-D07 s100 calcium binding protein CAJ90906.1| Salmo salar 2.00E-17 107 74 357 1 08-E01 Secretagogin ACQ58799.1 Anoplopoma fimbria 4.39E-107 391 93 701 1 09-B04 Serglycin precursor ACM09258.1| Salmo salar 1.00E-03 93 73 760 1 08-H12 Serine/threonine-protein kinase
NP_001134186.1 Salmo salar 3.80E-37 159 88 820 1 SNF1-like kinase 2
Serine/threonine-protein
02-F11 phosphatase PP1-gamma catalytic NP_001135184.1 Salmo salar 4.55E-28 129 86 738 1 subunit
14-C05 Serine--pyruvate aminotransferase ACO09121.1 Osmerus mordax 1.00E-14 55.5 82 453 1 13-A05 sex comb on midleg-like 2 XP_002197012.1 Danio rerio 3.00E-09 75.5 94 844 1 05-E08 SH2 domain-containing protein 1A NP_001134225.1 Salmo salar 3.00E-39 204 89 859 1 13-C04 si:ch211-213a13.3 XP_697912.3 Danio rerio 6.00E-13 78.2 47 685 1 13-A02 Signal peptidase complex catalytic
ACQ57971.1 Anoplopoma fimbria 3.00E-95 351 100 643 1 subunit SEC11A
07-D09 similar to Ac1147 XP_001231536.1 Gallus gallus 2.70E-23 111 88 355 1 13-D07 AP-3 complex beta3A subunit XP_001367655.1 Monodelphis
2.00E-81 306 81 780 1 domestica
03-D12 CD48 antigen precursor XP_700774.3 Danio rerio 7.00E-09 154 41 769 1 05-G07 cellular FLICE-like inhibitory
XP_421935.1 Gallus gallus 5.00E-32 218 60 834 1 protein long form
05-D02 chromosome condensation 1-like XP_001334658.2 Danio rerio 2.81E-90 335 89 791 1 08-C06 DNA mismatch repair protein Msh2 XP_538482.2 Canis familiaris 2.00E-85 320 64 807 1 12-E10 gametogenetin binding protein 2
XP_853479.1 Canis familiaris 1.24E-24 87 81 678 1 isoform 2
09-H02 guanine nucleotide binding protein
XP_002187101.1 Taeniopygia guttata 1.89E-61 238 98 478 1 (G protein), q polypeptide isoform 1
04-C11 interleukin 17 receptor E XP_002187060.1 Taeniopygia guttata 0.0008 47 41 339 1 08-G09 myelin protein zero-like 1 XP_701836.3 Danio rerio 2.00E-23 139 73 647 1 02-G11 NOD3 protein XP_001919724.1 Danio rerio 3.18E-38 150 54 727 1 08-B03 phosphoribosyl pyrophosphate
XP_420181.1 Taeniopygia guttata 5.29E-104 381 97 755 1 synthetase 1 isoform 1
14-C10 plexin A2 XP_689780.3| Danio rerio 7.00E-37 167 89 657 1
09-D09 SPECC1-like XP_692458.2 Danio rerio 2.00E-15 95 95 818 1
Table 2. (Continued)
Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c05-H11 Stromal interaction molecule 2 XP_692356.3 Danio rerio 3.37E-09 66 61 713 1 02-E04 tuberin-like protein 1 isoform 1 XP_001363188.1 Monodelphis
0.0175497 44 26 707 1 domestica
14-G12 vacuolar protein sorting 53 XP_001364702.1 Monodelphis
9.00E-43 176 94 311 1 domestica
05-G05 SNF1-like kinase NP_001119855.1 Danio rerio 6.67E-22 108 33 759 1 04-D10 Sorting nexin-10 ACQ58111.1 Anoplopoma fimbria 9.61E-05 50 93 593 1 09-G11 Sorting nexin-24 ACO09890.1 Osmerus mordax 4.02E-68 262 84 702 2 07-B09 Sorting nexin-9 NP_001133490.1 Salmo salar 3.15E-50 202 93 728 1 07-A02 SRC-like-adapter ACQ58849.1 Anoplopoma fimbria 2.17E-29 133 84 807 1 02-C08 STAT4 AAS10464.1 Takifugu rubripes 5.94E-119 273 88 843 1 14-C02 stress protein HSC70-1 BAG82848.1 Seriola
1.00E-128 462 87 829 2 quinqueradiata
11-B07 Synaptophysin-like protein 1 ACI32929.1 Salmo salar 7.71E-107 391 75 815 2 11-D01 syntaxin 4 ACO35366.1 Lateolabrax japonicus 1.94E-91 339 84 790 1 08-B02 TATA binding protein associated
ABG78598.1 Sander vitreus 8.19E-112 407 88 825 1 factor 9
06-C04 T-cell receptor alpha dbj|BAB82528.1 Paralichthys olivaceus 1.07E-32 121 64 846 1 13-C03 T-complex protein 1 subunit epsilon ACN11333.1 Salmo salar 4.00E-27 124 91 411 1 09-D04 TGF-beta-inducible nuclear protein 1 ACQ58100.1 Anoplopoma fimbria 8.94E-85 317 100 735 1 03-C03 Thioredoxin ACQ58191.1 Anoplopoma fimbria 1.39E-38 163 72 675 2
12-B06 THO complex 2 ACM47731.1 Paralichthys
8.68E-91 337 85 753 1 olivaceus
05-G02 Thymosin beta-12 P33248.2 Lateolabrax
6.21E-14 82 98 840 8 japonicus
07-C07 TNF superfamily member 14 NP_001118039.1| Oncorhynchus
4.00E-12 167 56 821 1 mykiss
05-E01 topoisomerase I beta CAA23016.1 Takifugu rubripes 9.71E-14 80 74 558 1 09-G02 Transcobalamin-2 precursor ACN10392.1 Salmo salar 2.00E-45 193 67 819 1 13-B02 transcription factor B1, mitochondrial CAJ83142.1 Xenopus tropicalis 6.00E-12 77.8 88 745 1 10-D06 transforming growth factor, beta
CAQ15472.1 Danio rerio 1.00E-26 123 88 566 1 receptor I
13-C05 transgelin NP_001038932.1 Danio rerio 9.00E-90 362 88 825 1 14-D11 translationally-controlled tumor
ACO82289.1 Oryzias latipes 5.00E-57 224 70 595 1 protein
09-D06 Translocon-associated protein subunit
ACM09789.1 Salmo salar 2.00E-79 317 95 815 1 delta precursor
12-F02 tRNA selenocysteine associated
NP_001025137.2 Danio rerio 1.04E-36 156 92 508 1 protein 1, like
12-A01 tropomodulin 4 NP_001133067.1 Salmo salar 4.00E-89 346 84 713 1
08-G04 Tubulin alpha-1 chain ACO09494.1 Osmerus mordax 6.09E-130 467 95 783 1
Table 2. (Continued)
(Johnson et al., 1987; Takeshita et al., 1988; Murphy and Norton, 1993).
In conclusion, this study reports an expressed sequence tag (EST) based gene identification analy- sis of 795 ESTs derived from rock bream leukocyte cDNA library. These EST analysis will be useful for construction of cDNA microarray and recombinant proteins. In particular, the application of cDNA microarrays may facilitate research attempting to answer questions concerning immune responses and other protective responses of rock bream upon infec- tion of pathogens.
Acknowledgement
This research was supported by National Fish- eries Research and Development Institute (RP- 2009-00-000) grant.
References
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Cho, Y.S., Choi, B.N., Kim, K.H., Kim, S.K., Kim D.S., Bang, I.C., Nam, Y.K.: Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream (Oplegnathus fasciatus). Aqua- culture, 253:667-679, 2006.
Clark, M.S., Edwards, Y.J., Peterson, D., Clifton, S.W., Thompson, A.J., Sasaki, M., Suzuki, Y., Kikuchi, K., Watabe, S., Kawakami, K., Sugano, S., Elgar, G. and Johnson, S.L.:
Fugu ESTs: new resources for transcription Clone No. Putative identification Accession No. Closest species E-value I
as
bbp F
c07-G11 Tubulin beta-1 chain ACQ58513.1 Anoplopoma fimbria 2.00E-131 472 92 811 3 02-C12 tumor necrosis factor receptor
NP_777099.1 Bos taurus 0.31 38.1 100 499 1 superfamily, member 1A
10-F03 tyrosine kinase jak2b AAO47335.1 Tetraodon fluviatilis 7.94E-44 179 84 339 1 03-C02 Tyrosine-protein kinase BTK NP_001133410.1 Salmo salar 2.27E-15 86 86 618 1 01-B05 Tyrosine-protein kinase SYK ACN10928.1 Salmo salar 1.13E-129 466 85 824 1 09-F09 U2 small nuclear ribonucleoprotein B NP_001139849.1 Salmo salar 3.00E-97 371 85 794 1
06-A01 Ubiquitin ACM09817.1 Salmo salar 3.46E-87 325 100 664 2
05-G10 ubiquitin C variant BAD93019.1 Homo sapiens 2.00E-79 301 83 652 1 09-D07 Ubiquitin-conjugating enzyme E2 A ACQ58139.1 Anoplopoma fimbria 5.71E-95 351 98 746 1 06-C07 Ubiquitin-conjugating enzyme E2 L3
ACO51728.1 Rana catesbeiana 4.22E-07 58 71 573 1 [Rana catesbeiana]
12-H01 vesicle-associated membrane protein,
NP_997812.1 Danio rerio 1.00E-67 263 85 899 1 associated protein B and C
02-B11 v-ets erythroblastosis virus E26
NP_001017558 Danio rerio 1.00E-96 408 94 814 1 oncogene homolog 1
14-G06 WD repeat domain 40A NP_001071048.1 Danio rerio 2.00E-25 118 98 369 1 08-F03 Zinc finger protein 364 ACO09370.1 Osmerus mordax 4.83E-15 86 69 775 1 Table 2. (Continued)
a
