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Fish & Shellsh Immunology 27 (2009) 202209

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Fish & Shellsh Immunology

journal homepage: www.elsevier.com/locate/fsi

Identication of immune responsible brinogen beta chain in the liver of large yellow croaker using a modied annealing control primer system
F.J. Xie, Z.P. Zhang**,1, P. Lin, S.H. Wang, Z.H. Zou, Y.L. Wang*
The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen, Fujian 361021, China

a r t i c l e i n f o
Article history: Received 5 December 2008 Received in revised form 13 April 2009 Accepted 26 April 2009 Available online 5 May 2009 Keywords: Large yellow croaker mACP Real time PCR Fibrinogen beta chain Differentially expressed genes

a b s t r a c t
In this article, we used a modied ACP system (mACP) developed in our laboratory to analyze differentially expressed genes in the liver of large yellow croaker, Pseudosciaena crocea (Richardson). By using 20 pairs of mACPs, 7 differentially expressed genes were obtained. One of the genes we identied encodes for a brinogen beta chain (FGB). The full-length cDNA of FGB was 1645 bp, including 5 bp of 50 untranslated region (50 -UTR), 1479 bp of open reading frame (ORF), and 161 bp of 30 -UTR. The ORF was capable of encoding 492 amino acids with an estimated molecular mass of 55.6 kDa, giving it a predicted pI of 5.94. The deduced amino acid sequence included an FGB prole (V238-Y488) and an FGB family signature (WWYNRCHSANPNG). Multiple sequence alignments indicated that the large yellow croaker FGB showed homology with FGB sequences of other species (4577% identity). Real time PCR analysis demonstrated that the expression of FGB in the liver of large yellow croaker injected with Vibrio parahaemolyticus was signicantly (P < 0.05) lower than that of the control group at 8 d, which conrmed the expression patterns of the results of mACP differential display. 2009 Elsevier Ltd. All rights reserved.

1. Introduction The mRNA differential display reverse-transcription PCR (DDRTPCR) [1] worked well in displaying the expected number of mRNA per primer combination under optimal conditions, but high false-positive rates and poor reproducibility were encountered [2], which due to arbitrary primers (1013 mers) were employed for differential display in combination with two-base anchored primers. The annealing temperature is critical for insuring that a primer binds only to its perfect complement or to sequences with one or more mismatches. The use of short primers (1013 mers) usually requires low annealing temperatures about 4045  C for all PCR cycles; these low temperatures cause nonspecic primer annealing. By adjusting the annealing temperature, one can alter the specicity of pairing between the template and primer. Numerous approaches, such as longer primers with universal, homopolymer, or loop sequence tails at their 50 -ends, have been introduced to increase primer annealing specicity [35]. The use of longer primers makes it possible to increase the annealing

* Corresponding author. Tel. 86 592 6182723; fax: 86 592 6181420. ** Corresponding author. Tel.: 1 512 2450358; fax: 1 512 2451922. E-mail addresses: ziping.zhang@txstate.edu (Z.P. Zhang), ylwang@jmu.edu.cn (Y.L. Wang). 1 Present address: Department of Chemistry & Biochemistry, Texas State University, San Marcos, TX 78666, USA. 1050-4648/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.04.002

temperature to 60  C after 14 initial cycles at 4045  C [6]. However, the additional tail sequences of longer primers are involved in nonspecic annealing to the cDNA template during PCR cycles at low annealing temperatures. We describe here a more accurate and extensive PCR technology, controlled by an annealing control primer (ACP) [7]. The ACP system is based on two characteristics: the unique tripartite structure of the primers, which have distinct 30 - and 50 -end regions that are separated by a polydeoxyinosine [poly(dI)] linker, and the interaction of each region during two-stage PCR. In the present study, we used a modied ACP system (mACP) developed in our laboratory [8], to analyze differentially expressed genes in liver of large yellow croaker Pseudosciaena crocea (Richardson). Both dT-ACP1 and dT-ACP2 were replaced by dT-mACP, and the 50 -end regions of dT-mACP and arbitrary mACPs contained the same universal sequence. By using 20 pairs of mACPs, 7 differentially expressed genes were obtained. One of the genes we identied is the brinogen beta chain (FGB). To our knowledge, identication of differentially expressed genes using an ACP system is focused on mammals [912]. This article is the rst report characterizing differentially expressed genes by using an ACP system in sh. Fibrinogen plays important roles with brin in blood clotting, brinolysis, cellular and matrix interactions, inammation, wound healing, and neoplasia [13]. Fibrinogen occurs as a dimer, where each monomer is composed of three non-identical chains, alpha, beta and gamma, linked together by several disulphide bonds. The

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N-terminals of all six chains come together to form the center of the molecule (E domain), from which the monomers extend in opposite directions as coiled coils, followed by C-terminal globular domains (D domains). Therefore, the domain composition is: D-coil-E-coilD. At each end, the C-terminal of the alpha chain extends beyond the D domain as a protuberance that is important for cross-linking the molecule. During clot formation, the N-terminal fragments of the alpha and beta chains (within the E domain) in brinogen are cleaved by thrombin, releasing brinopeptides A and B, respectively, and producing brin [14]. Fibrin forms a soft clot, and is then converted to a hard clot by factor XIIIA, which cross-links brin molecules. Platelet aggregation involves the binding of the platelet protein receptor integrin alpha (IIb)-beta(3) to the C-terminal D domain of brinogen [15]. Fibrin bres interact with platelets to increase the size of the clot, as well as with several different proteins and cells, thereby promoting the inammatory response and concentrating the cells required for wound repair at the site of damage. Research about the function of FGB in sh is unavailable, although bastard halibut Paralichthys olivaceus FGB (GenBank accession no. EF581895) and zebrash Danio rerio FGB (GenBank accession no. NM_212774) have been cloned. Currently, brinogenlike protein genes related to the toxicity in puffersh liver were investigated by comparing mRNA expression patterns in akamefugu Takifugu chrysops and kusafugu Takifugu niphobles having different concentrations of tetrodotoxin (TTX) [16]. Here we report the cloning of FGB and its expression pattern in the liver of large yellow croaker. 2. Materials and methods 2.1. Fish Healthy large yellow croaker (200250 g) were obtained from the Fishery Extension Station of Ningde (Fujian, China) and were kept in net cages at about 25  C and fed with a commercial feed. Before challenging experiments, the sh were maintained for at least two weeks and then were injected intraperitoneally with 1.0 mL of a 0.9% NaCl solution (control group) or Vibrio parahaemolyticus (experimental group) in a titer of 6.6 108 cfu mL1. Liver from six control sh and six experimental sh were rapidly harvested at 1 d, 2 d, 4 d, 8 d, 12 d, 16 d after injection, frozen in liquid nitrogen and stored at 80  C until RNA extraction and analysis. 2.2. RNA isolation Total RNA was isolated from above samples as described in our previous study [17]. Quantity and purity of isolated RNA were determined by absorbance measures at 260 nm and 280 nm, and its integrity was tested by electrophoresis in agarose gels. 2.3. mACP-based differential display 2.3.1. For rst-strand cDNA synthesis 2 mL dT-mACP (50 -AAGCAGTGGTATCAACG CAGAGTTTTTTTTTTTT TTTTTTTTT-30 , 10 mM) primer was added to 3 mg RNA isolated from liver. The RT mix contained 4 mL 5 RT buffer, 2 mL DTT (20 mM), 1 mL dNTP (10 mM), and 200 U of SuperScript II reverse transcriptase (Invitrogen). The mixture was incubated in the tube at 42  C for 90 min, and then heated at 94  C for 2 min to inactivate the reaction. The reaction mixture was chilled on ice for 2 min and spinned briey. First-strand cDNA was diluted by adding 80 mL of ultra puried water.

2.3.2. mACP-based PCR Second-strand cDNA synthesis and subsequent PCR amplication were conducted in a single tube. Second-strand cDNA synthesis was conducted at 50  C during one cycle of rst stage PCR in a nal reaction volume of 20 mL containing 1 mL of the diluted rst-strand cDNA, 2 mL of 10 PCR buffer, 0.4 mL of dNTP (10 mM), 1 mL of dT-mACP (10 mM), and 1 mL of arbitrary mACP (10 mM) preheated to 94  C. The tube containing the reaction mixture was held at 94  C while 1 U of Taq polymerase (TaKaRa) was added to the reaction mixture. The PCR protocol for second-strand synthesis was one cycle at 94  C for 5 min, followed by 50  C for 5 min, and 72  C for 2 min. After second-strand DNA synthesis was completed, 35 amplication cycles were performed. Each cycle involved denaturation at 94  C for 40 s, annealing at 65  C for 40 s, and extension at 72  C for 40 s. A nal extension step of 5 min at 72  C was performed to complete the PCR. The amplied PCR products were separated on 2.0% agarose gels and stained with ethidium bromide. 2.3.3. Cloning and transformation The differentially expressed bands were extracted and cloned into pMD-T (TaKaRa) vector following the manufacturers instructions. In order to verify the identity of insert DNA, isolated plasmids were sequenced. Complete sequences were analyzed by searching for similarities using BLASTX search program at the National Center for Biotechnology Information (NCBI) GenBank. 2.4. 50 -RACE cloning of full-length FGB cDNA Based on the partial sequence of large yellow croaker FGB, specic primers FGB-RACE (GSP1: 50 -TCCTCACACTCCTTACCGGACACCA-30 , GSP2: 50 -GCTGTACTGACCAACCACCCTGTTG-30 ) were designed to 50 end cDNA sequences of large yellow croaker FGB by rapid amplication of cDNA ends (RACE). Total RNA isolated from large yellow croaker liver was used to synthesize the 50 -RACE cDNA templates using the BD SMART RACE cDNA amplication kit (Clontech) according to the manufacturers instructions. 3 mg of RNA was reverse-transcribed to cDNA with 1 mL of 50 CDS primer and 1 mL of SMART II Oligo, then RNase free water was added to a nal volume of 5 mL for each reaction. The RNA was denatured at 70  C for 2 min and cooled on ice for 2 min. The other components in the 10 mL reversetranscription reaction mixture included 2 mL 5 rst-strand buffer, 1 mL DTT (20 mM), 1 mL dNTP (10 mM), 1 mL SuperScript II reverse transcriptase (200 U mL1, Invitrogen). The reaction mixtures were incubated at 42  C for 1.5 h. The rst-strand reaction was terminated and diluted by adding 90 mL of RNase free water and heating at 72  C for 7 min. The primary PCRs were carried out in a 25 mL reaction mixture composed of 1 mL diluted cDNA, 0.5 mL GSP1 (10 mM), 2.5 mL UPM, 2.5 mL 10 PCR-buffer, 0.5 mL dNTP Mix (10 mM), 0.5 mL DNA polymerase (5 U mL1), and 17.5 mL ultra puried water. The PCR conditions were: 5 cycles of 95  C for 30 s, 72  C for 3 min; 5 cycles of 95  C for 30 s; 70  C for 30 s; 72  C for 3 min; 25 cycles of 95  C for 30 s; 68  C for 30 s; 72  C for 3 min. The secondary PCRs were set up and run similar to the primary PCR except the UPM primer was replaced with NUP primer, GSP1 replaced with GSP2, and the primary PCR products 1 mL as templates. The PCR products were separated by electrophoresis on 1% agarose gel. PCR bands of the predicted size were cloned and sequenced. 2.5. Bioinformatic analyses Multiple sequence alignments were performed with T-Coffee (http://www.ch.embnet.org/) [18,19]. A phylogenetic tree was constructed using the Neighbour-Joining method based on the CLUSTAL W alignment [20]. The Interpro prole library [21] was used to identify characteristic domains or patterns. The SignalP

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program [22] was used for identication of signal peptides and prediction of cleavage sites. Analysis of secondary structure was performed using the PHD program [23] at the Protein Predict server (www.embl-heidelberg.de/predictprotein/). The three-dimensional structure of large yellow croaker FGB was modeled using Swissmodel [24] in the rst approach mode accessible via the internet (http://www.expasy.org/swissmod). The co-ordinate les were imported to RASMOL software version 2.7.3.1 for analyzing bond lengths and other conformational features of the molecule.

followed by 40 cycles consisting of 94  C for 15 s, 60  C for 1 min. Thereafter, PCR products were analyzed by generating a melting curve. Since the melting curve of a product is sequence-specic, it can be used to distinguish between nonspecic and specic PCR products. PCR products for FGB and 18S rRNA were ligated into pMD-T vector and transformed in DH5a competent cells. Mini-preps of isolated plasmid DNA were then prepared (Promega) for sequencing to check the sequence of the real time PCR products.

2.6. Real time PCR analysis of FGB expression All the RNA samples, including different exposure groups (V. parahaemolyticus challenge and control) and exposure times (4 d, 8 d, 12 d and 16 d), with four biological replicates were isolated from different individual sh (sh number: 2 4 4 32) for real time PCR. 18S rRNA was used as a normalization factor (endogenous control) to correct for different loading amounts of RNA since this gene is considered to be constitutively expressed. Primers for FGB (forward primer: 50 -GGCGGAGCCTACACTAAACAA-30 ; reverse primer: 50 -TGCTGATGCCTTTGAGTGAATAC-30 ) and 18S rRNA (forward primer: 50 -ACACGGAAAGGATTGACAGATTG-30 ; reverse primer: 50 -CAGACAAA TCGCTCCACCAA-30 ) were designed using Primer Express 3.0 (ABI) and tested to ensure amplication of single discrete bands with no primerdimers. 3 mg of RNA pre-treated with DNase I was used as template for total cDNA synthesis in 20 mL reactions with random hexamers using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). For real time PCR, 1 mL of cDNA, 0.8 mL each of forward and reverse primer (10 mM) and 10 mL SYBR Green Realtime PCR Master Mix (TOYOBO) were brought to a nal volume of 20 mL with ultra-pure water in each reaction and analyzed in the ABI 7500 real time system. The cycling conditions were: 95  C for 10 min,

Fig. 1. Agarose gel photograph indicating products of mACP3 in liver of large yellow croaker obtained by using the modied ACP system. Candidate gene was indicated with arrowhead. The length of the product is about 1500 bp (M: marker, C: control group, V: experimental group after challenge of V. parahaemolyticus).

Fig. 2. Nucleotide and deduced amino acid sequence of large yellow croaker FGB. mACP-based PCR product is dropped shadow and GSP of 50 -RACE are underlined; FGB family signature is represented in bold with italicized letters; the box points to signal peptide; two poly A signal sequences are double underlined.

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The comparative threshold cycle (CT) method was used to calculate the relative concentrations. This method involves obtaining CT values for the FGB; normalizing to the housekeeping gene, 18S rRNA; and comparing the relative expression level among liver samples. The relative quantication of gene expression was analyzed by the 2 [-Delta Delta C (T)] method [25]. Statistics Package for Social Science (SPSS) 13.0 for windows was used to analyze the data from all experiments. Data were expressed as the arithmetic mean standard deviation (S.D.). Signicance of differences was determined by the independent-samples T-test. P values of <0.05 were considered signicantly different; P values of <0.01 were considered most signicantly different. 3. Results 3.1. Isolation of differentially expressed genes in liver of large yellow croaker To identify genes that are expressed in liver of large yellow croaker in response to the challenge of V. parahaemolyticus, we compared the RNA expression proles of control group and experimental group at 8 d. By using the modied ACP system, seven products with different expression levels were identied and sequenced. Queries of publicly available databases using the BLAST algorithm showed that one of these products (from mACP3: 50 AAGCAGTGGTATCAACGCAGAGTIIIIICCGGAGGATG-30 ) (Fig. 1) had high similarity to FGB gene. The length of the product was 1360 bp (Fig. 2). Two gene specic primers, FGB-GSP1 and FGB-GSP2 (Fig. 2) were designed from the product for 50 -RACE. The full-length cDNA sequence of FGB was obtained by 50 -RACE method. The nucleotide sequence obtained was 1645 bp in length (GenBank accession no. EF014896), including 5 bp of 50 untranslated region (50 -UTR), 1479 bp of open reading frame (ORF), and 161 bp of 30 -UTR (Fig. 2). The ORF was capable of encoding 492 amino acids with an estimated molecular mass of 55.6 kDa, giving it a predicted pI of 5.94. Using the SignalP program, a signal peptide (residue numbers 116, MRTLLLLYLCVYTAWA, Fig. 2) was predicted at the N-terminus. Interpro analysis indicated that the sequence included an FGB prole (V238-Y488) and an FGB family signature W-W-[LIVMFYW]x(2)-C-x(2)-[GSA]-x(2)-N-G (between residues 433 and 445, WWYNRCHSANPNG, Fig. 2).

3.2. Homology to FGB proteins from other species To analyze more rigorously the degree of evolutionary relatedness of FGB between large yellow croaker and other species, we performed a phylogenetic analysis with 11 different members of FGB using the Neighbour-Joining method based on the CLUSTAL W alignment. The phylogenetic tree was bootstrapped 1000 times. The tree (Fig. 3) indicates that the teleost FGB sequences form a cluster separate from the other FGB sequences. The sequence of the large yellow croaker FGB showed homology with FGB sequences of other species (4577% identity), with sea lamprey Pertromyzon marinus FGB being the low homology with 45% identity. In contrast, bastard halibut P. olivaceus FGB sequence showed marked homology (77% identity). Comparison of known FGB sequences revealed that many of the signature residues are conserved in large yellow croaker FGB (Fig. 4). Most of the sequence identities are conned to the C-terminal domain, as reported for other species. The C-terminal domain contains four conserved cysteines involved in two disulde bonds. Analysis of secondary structure using the PHD program at the Protein Predict server (www.embl-heidelberg.de/predictprotein/) suggested that large yellow croaker FGB possesses 11 alpha helixes and 14 beta-pleated sheets, which was similar to human FGB. Thus, the overall predicted structure of large yellow croaker FGB was compared with the 3D structure of human FGB (Fig. 5). Using this model, we could predict similar biological functions from large yellow croaker FGB similar to that of human FGB. 3.3. Conrmation of expression of large yellow croaker FGB by real time PCR To validate the results of mACP differential display of FGB and to determine the relative abundance of target sequences in liver of large yellow croaker after challenge of V. parahaemolyticus, real time PCR was performed. The results (Fig. 6) demonstrated that the expression of FGB in liver of large yellow croaker injected with V. parahaemolyticus was signicantly (P < 0.05) lower than that of the control group at 8 d, and there was no signicant difference between other control group and experimental group (P > 0.05). This analysis revealed that our target transcripts which showed the expression patterns in agreement with the results of mACP differential display.
0.02 Petromyzon marinus Homo sapiens

611 Canis familiaris 1000 752 994 758 Equus caballus Rattus norvegicus Gallus gallus Xenopus laevis 1000 Xenopus tropicalis Danio rerio 1000 1000 Paralichthys olivaceus Pseudosciaena crocea

Fig. 3. Phylogenetic analysis of large yellow croaker FGB. A phylogenetic tree was constructed using the Neighbour-Joining method based on the CLUSTAL W alignment, Species names are represent: Pseudosciaena crocea: large yellow croaker (GenBank accession no. ABJ98546); Paralichthys olivaceus: bastard halibut (GenBank accession no. ABQ41317); Danio rerio: zebrash (GenBank accession no. NP_997939); Xenopus tropicalis: western clawed frog (GenBank accession no. NP_001107962); Xenopus laevis: African clawed frog (GenBank accession no. NP_001081418); Gallus gallus: chicken (GenBank accession no. XP_420369); Rattus norvegicus: Norway rat (GenBank accession no. AAA64866); Equus caballus: horse (GenBank accession no. XP_001501005); Canis familiaris: dog (GenBank accession no. XP_853738); Homo sapiens: human (GenBank accession no. AAI07767); Petromyzon marinus: sea lamprey (GenBank accession no. P02678).

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P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis

1 1 1 1 1 1 1 1 1 1 1 1 35 34 29 25 24 34 35 38 35 34 26 49 76 76 70 66 64 73 73 76 76 75 65 97 124 124 117 113 110 119 119 122 123 122

--------------MRTLLLLYLCVYTAWAQDNLDYDDYDTDSKSKPA --------------MKTLLLLCLCLGVTWAQD-FDFDEYDLDSTSSPA --------------MKLVLLLCLCAVGALAQD--DYDDYGEGK----K --------------MKLLLLLLLCIPAIKPQASVEYD----------N --------------MRHLWLLLLSVSLVQTQAAT-----------TDS ---MVSWNFQKFKTMKLLFLLLLCVSVVRSQALD-YD-------------MVSWNFKNFKSMKNLLLLLLCVFIVKSQAHYYDD----------MKRMVSWSFHKLKTMKHLLLLLLCVFLVKSQGVNDNE------------------------MRVLLLFALCVSAVWCSSDYDEDEGDEGAVISKS --------------MRVLLLFALCVSAVWCSSDYDEDDVDDAAVI-KS ----------------------EDLSLVGQP-ENDYDTGDDBTAADPD

34 33 28 24 23 33 34 37 34 33 25 48

E-NATEVGARGHRPLTRGTDRYSPNRYVPPP-----ISGG-SRYRGRP V-NESGVNARSHRPLSRGRDGYTRNRYVQPP-----ISRDSSRYRGRP E-AKEVVDPRGHRPVSRGRETYSPGPVSQPP-----ISGG-TRYRGRP EEDSPQIDARAHRPLDKRQEAAPTLRPVAPP-----ISGTG--YQPRP DKVDLSI-ARGHRPVDRRKEEPPSLRPAPPP-----ISGGG--YRARP --HEVTFDARSHRPLDKKREEPLSLKPAPPP-----ISGPG--YRPRP ---TSTVDARGHRPLDKKREEAPSLRPAPPP-----ISGGG--YRARP ---EGFFSARGHRPLDKKREEAPSLRPAPPP-----ISGGG--YRARP ENASATVDARGHRPVSRGREPTPTQKPAPPP-----ISGGS--YRGRP DNATASVDARGHRPVSRGREPVPTQRPAPPP-----ISGGS--YRGRP S-NNTAAALDVRRPLPSG------TRVRRPPLRHRRLAPG-A-VMSRD :**: ** :: *

75 75 69 65 63 72 72 75 75 74 64 96 123 123 116 112 109 118 118 121 122 121 110 144 171 171 164 160 157 166 166 169 170 169

APAPTGQPQEEEKVTQPDAGGCTHASEEMGVLCPNGCELKTTLLKQEK TPPPVRGAQLQEEEVQPDAGGCLHASESMGVLCPNGCDLKTTLLKQER TAAPV-GKAVQEKEEQPESGGCNHMSEKMGVLCPTGCELKKALIKQER PKQDK-QAMKKGPIIYPDAGGCKHPLDELGVLCPTGCELQTTLLKQEK AKVDA--GQKKVERKPPDAGGCVHGDGDMGVLCATGCELRQTLLNHER AKVAA--NKKKVERKAPDAGGCVHADPDLGVLCPTGCQLQDTLVKQER VKPV--ASQKKLERKAPDAGGCLHADPDLGVLCPTGCQLQDTLVKQER AKAAA--TQKKVERKAPDAGGCLHADPDLGVLCPTGCQLQEALLQQER TKAPA-KAQKKEPTEYPDAGGCKHAYEELGTLCPTGCELRTTLLKQER TKAPV-KGQKKEATEYPDAGGCKHAYEELGTLCPTGCELRTTLLKQER PPASP--RPQEAQKAIRDEGGCMLPESDLGVLCPTGCELREELLKQRD : : *** .:*.**..**:*: *:::.

NVKMSIGELKPQVDELSRASSNVYNYVNSMSNSLRERQRVINDNNRVV NVKTSINELKPLVDDLSRSSNTIYNYVNSMSNSLRERQKVVNDNTRVA NVKPTVEQLKRAVDDLTQSTNSIHGYVLDMTAEVAQRQKVSEGNGLVV TVKPVLRDLKDRVAKFSDTSTTMYQYVNMIDNKLVKTQKQRKDNDIIL PIKNSIAELNSNINSVYETSSVTFQYLTLLKDMWKKKQAQVKDNENVI SVRNSVNELSHNVESISQSSSSNFQYITALKEMWKKREKQLKDNENVV PIRKSIDELNNNVESVSQSSSSTFQYITLLKDMWKNRQKQIRDNENAI PIRNSVDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNENVV NVKTGISDVRGRVESLSQLTNNIYRYTNVLSQKMKETQQQALDNQNVI NVKTAINDVRGRVETLAQSANNVYRYTTVLGQKIKENQQQTLDNQNVV

Fig. 4. Comparison of the large yellow croaker FGB sequence with those of other FGB homologues. Species names see Fig. 3. Sequence alignment was performed using the T-Coffee at http://www.ch.embnet.org/. Consistency scores are shown by color as: BAD AVG GOOD

4. Discussion The specicity and sensitivity with which a primer anneals to its target sequence are the most critical factors in determining the success of PCR amplication. In this study, we employed a new differential display RT-PCR technique [7,9] to compare the gene expression in liver of large yellow croaker. The ACP-based RT-PCR method involves an ACP that has a unique tripartite structure in that its distinct 30 - and 50 -end portions are separated by a regulator. This ACP-based RT-PCR system is easy and accurate and lacks false positives, the PCR products can be detected on standard ethidium bromide-stained agarose gels, and hence, obviating the use of PAGE gels with the attendant issues in handling and use. The 50 -end regions of dT-mACP and mACP primers contain the same universal sequence, which result in the synthesis of the second-strand cDNA containing the inverted repeat sequence at both ends. Through the technique of step-out PCR, suppression PCR inverted repeat elements are incorporated next to all the second-strand cDNA sequences. During PCR, these inverted repeats of short cDNA fragments easily anneal to each other intramolecularly. This rapid rst-order reaction out-competes

the second-order binding of the primers to the short cDNA fragments. As a result, panhandle-like structures are formed, which cannot be amplied. So the modied ACP system which we used could generate a wide range of PCR products ranging from 300 bp to 2.0 kb, which not only increases the chances of identifying differentially expressed genes, but also provides more signicant sequence information for the prediction of gene function. With this technique, we identied 7 differentially expressed genes that are specically or more prominently expressed in liver of control group to experimental group at 8 d. And it was adapted to identify genes differentially expressed in ovary and testis at different developmental stages in penaeid shrimp Marsupenaeus japonicus in our laboratory [26]. By using 20 pairs of mACPs, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r), which has an important role in oogenesis and spermatogenesis. Using the BLASTX showed that one of these products (from mACP3) (Fig. 1) had high similarity to FGB gene. More rigorously bioinformatic analyses indicated that the deduced amino acid sequence included an FGB prole and an FGB family signature (Fig. 2), and the sequence of the large yellow croaker FGB showed

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P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus

111 PVRYKISMLKQNLTYFINSFDRMASDSNTLKQNVQTLRRRLNSRSSTH 145 172 172 165 161 158 167 167 170 171 170 159 193 220 220 213 209 206 215 215 218 219 218 207 :: : : : . : . ..

158 192 219 219 212 208 205 214 214 217 218 217 206 240 267 267 260 256 253 262 262 265 266 265 254 288 314 314 307 303 301 310 310 313 313 312 301 336 362 362 355 351 349 358 358 361 361 360 349 384 410 410 403 399 397 406

GQYSDNVEEQHAYIKETVDTVFPSNIRVLQGVLEKIKLKIQKLEKAIH SQYTDQVEEQHAYIKETVDTVFPSNIRVLQGVLDKVRLKIQKLEKSIL DQYTDSLETQHAYIKDTVDVTFPQNIKVLQGVLDKIREKIQRLEKAIT SEYNTEMELHYNYIKDNLDNNIPSSLRVLRAVIDSLHKKIQKLENAIA NEYSSILEDQKLYIDETVNDNIPLNLRVLRSILEDLRSKMQKLESDIS GEYSSELEKHQLYIDETVNSNVPTNIRVLRSILENLRSKIQKLESDVS NEYSSELEKHQLYIEETVTSNIPTNLRVLRAILENLRSKIQKLESDVS NEYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESDVS NEYNLEVEEQYTFIKENIDNKIPSNIRTLRQVLENVRSKIQKLEIAIA NEYNLELEEQYTFIKDNIDTKIPSNIRILRQVLENLRSKIQKLETAIA VNAQKEIENRYKEVKIRIESTVAGSLRSMKSVLEHLRAKMQRMEEAIK : :* : :. : .. .:: :: ::: :: *:*::* :

AQSEVCKEPCKTKCPIPVVSGKECEDIFRRGGTDSQMYMVHPDTFYPP SQRDLCREPCKTTCPIPVVSGKECEDIYRRGGRDSQMYMIQPDAFFPP TQRAKCQAPCKVTCPIPVVSGKECEDIIRKGGEDSQMYIIRPDPLGTP TQTDYCRSPCVASCNIPVVSGRECEDIYRKGGETSEMYIIQPDPFTTP AQTEYCHTPCTVNCNIPVVSGKECEEIIRKGGETSEMYLIQPDTSSKP AQMEYCRNPCTVSCNIPVVSGRECEEVIRNGGETSEMYLIQPDRSAKP AQMEYCRTPCTVSCNIPVVSGKECEEIIRNGGETSEMYLIQPHSSITP AQMEYCRTPCTVSCNIPVVSGKECEEITRKGGETSEMYLIQPDSSVKP TQVENCRSPCVTNCPIPVVSGKECEEIYRKGGETSEMYLIQPDSFFRP TQVENCRSPCVTTCPIPVVSGKECEEIYRKGGETSEMYLIQPDSFFRP TQKELCSAPCTVNCRVPVVSGMHCEDIYRNGGRTSEAYYIQPDLFSEP * ** ..* :***** .**:: *.** *: * ::*. *

241 :* 268 268 261 257 254 263 263 266 267 266 255

YKVFCDQTTQNGGWLLIQNRLDGSVDFGRRWDGYRRGFGNVAFDS-GK YKVFCDQSTQNGGWLLIQNRLDGSVNFGRRWDEYRRGFGNIAFDV-GK YKVFCDQTSKNGGWVLIQNRMDGSVDFGRRWDDYRRGFGNIAFDV-GK YRVYCDMETDNGGWTLIQNRQDGSVNFGRAWDEYKRGFGNIAKS-GGK YRVYCDMKTENGGWTVIQNRQDGSVDFGRKWDPYKKGFGNIATNEDTK YRVYCDMTTESGGWTVIQNRQDGSVDFGRKWDPYKQGFGNIATNADGK YRVYCDMTTDSGGWTVIQNRQDGSVDFGRTWDPYKQGFGNIATSADGK YRVYCDMNTENGGWTVIQNRQDGSVDFGRKWDPYKQGFGNVATNTDGK FKVYCDMATHDGGWTVIQNRQDGSVNFGRTWDSYKSGFGNIAAN-GGK FKVYCDMATHDGGWTVIQNRQDGSVGFGRTWDSYKSGFGNIAAN-GGK YKVFCDMESHGGGWTVVQNRVDGSSNFARDWNTYKAEFGNIAFGN-GK :..*** ::*** *** .*.* *: *: ***:* . *

289 ::*:** 315 315 308 304 302 311 311 314 314 313 302 337 363 363 356 352 350 359

GHCETPSEYWLGNDRISYLTKMGPTEVLIEMEDWSGAKVYAQYQQFTI GHCETPGEYWLGNDHISQVTNMGPTEVLIEMQDWTGDKVHAQYSQFTI GHCQTPGEYWLGNDRISQLSKMGATELLVEMEDWSGSKVYAQYEQFSM KYCDTPGEYWLGNDKISQLTKIGPTKVLIEMEDWNGDKVSALYGGFTI KYCGLPGEYWLGNDKISQLTRIGPTELLIEMEDWKGDKVKAHYGGFTV KYCGLPGEYWLGNDKISQLTMMGPTVLLIEMEDWRGDKVKALYEGFTV KYCGLPGEYWLGNDKISQLTNMGPTELLIEMEDWKGDKVKALYGGFTM NYCGLPGEYWLGNDKISQLTRMGPTELLIEMEDWKGDKVKAHYGGFTV GICDMPGEYWLGNEKISQLTNLGATEALIEMEDWDGAKVTAQYTGFTV GICDMPGEFWLGNEKISQLTNLGATEALFEMEDWDGAKVTAQYTGFTV SICNIPGEYWLGTKTVHQLTKQHTQQVLFDMSDWEGSSVYAQYASFRP * *.*:***.. : :: . *.:*.** * .* * * *

QSDASNFVLAVDGYSGNAGNSFMEGSSELFGENRTMTIHNGMMFSTYD QSETSNYVMAVNGYSGNAGNCFLEGSLQLFGENRTMTIHNGMMFSTYD QGEASNYILGVGRYSGTAGNTFLEGATELFGENRTMTIHNGMMFSTYD HNEGNKYQLSVSNYKGNAGNALMEGASQLYGENRTMTIHNGMYFSTYD QTEANKYQVSVNKYKGTAGNALMEGASQLVGENRTMTIHNGMFFSTYD KGEGDKYQLSVGKYKGTAGNALVDGASQLVGENRTMTIHNGMYFSTYD

Fig. 4. (continued).

homology with FGB sequences of other species (4577% identity, Fig. 4). Analysis of secondary structure suggested that large yellow croaker FGB possesses 11 alpha helixes and 14 beta-pleated sheets, which was similar to human FGB. The molecular modeling studies also predicted similar biological functions of large yellow croaker FGB to human FGB (Fig. 5). Based on the analysis above, we can conrm that the large yellow croaker FGB belongs to the FGB family. Fibrinogen is a soluble plasma glycoprotein that is synthesized in the liver by hepatocytes and megakaryocytes. Fibrinogen is an acute phase reactant, meaning that brinogen concentrations may rise

sharply in any condition that causes inammation or tissue damage, but dysfunction or disease of the liver can lead to a decrease in brinogen production or the production of abnormal brinogen molecules with reduced activity (dysbrinogenaemia)[14]. A study [27] on the V. parahaemolyticus of marine cage-cultured large yellow croaker showed that incidence of this disease was high and epidemic and occurred during summer. The infection rate varied from 30% to 50%, and articial infection showed that the bacterium isolated could cause dysfunction of liver of large yellow croaker. The real time PCR analysis (Fig. 6) conrmed that the expression of FGB in the liver

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F.J. Xie et al. / Fish & Shellsh Immunology 27 (2009) 202209

C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons P.crocea P.olivaceus D.rerio G.gallus R.norvegicus E.caballus C.familiaris H.sapiens X.tropicalis X.laevis P.marinus cons

359 362 362 361 350

QNEANKYQLSVSKYKGTAGNALIEGASQLFGENQTMTIHNGMYFSTYD QNEANKYQISVNKYRGTAGNALMDGASQLMGENRTMTIHNGMFFSTYD QNEANKYQLSVSGYKGTAGNALMEGASQLKGENRTMTIHNGMFFSTFD QNEANKYQLSVSGYKGTAGNALMDGASQLKGENRTMTIHNGMFFSTFD ENEAQGYRLWVEDYSGNAGNALLEGATQLMGDNRTMTIHNGMQFSTFD * *.*** :::*: :* *:*:******** ***:*

406 409 409 408 397 432 458 458 451 447 445 454 454 457 457 456 445 480

385 . : . : : * 411 411 404 400 398 407 407 410 410 409 398

EDNDNWNPGDPYTQCSREDGGGWWYNRCHSANPNGRYYIGGAYTKQMA RDNDNWLPGDPSKQCSKEDGGGWWYNRCHSANPNGRYYIGGAYTKQMA RDNDKWIPGDPSKQCSKEDGGGWWYNRCHSCNPNGRYYWGGAYTKYMA RDNDGWLTTDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGTYSWDMA RDNDGWVTTDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGLYSWDMS RDNDGWTTTDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGQYSWDMA RDNDGWITTDPKKQCAREDGGGWWYNRCHAANPNGRYYWGGHYSWDMA RDNDGWLTSDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGQYTWDMA RDNDGWQHADPNKQCSKEDGGGWWYNRCHAANPNGRYYWGGYYTWDMA RDNDGWQHSDPNKQCSKEDGGGWWYNRCHAANPNGRYYWGGYYTWDMA RDNDNWNPGDPTKHCSREDAGGWWYNRCHAANPNGRYYWGGIYTKEQA ** .:*::**.*********:.******* ** *: 492 492 485 480 479 488 488 491 490 488 477 514 :

433 .*** * 459 459 452 448 446 455 455 458 458 457 446

KHGTDDGIVWMNWKGSWYSLKGISMKIRPYFASR KHGTDDGVVWMNWKGSWYSLKAISMKIRPFYPAS KHGTDDGIVWMNWKGSWYSLKTISMKIRPYFKQK KHGTDDGIVWMNWKGSWYSMKKMSMKIKPYFP-D KHGTDDGVVWMNWKGSWYSMRRMSMKIRPVFPQQ KHGTDDGVVWMNWKGSWYSMKKISMKIRPYFPQQ KHGTDDGVVWMNWKGSWYSMKKMSMKIRPFFPRQ KHGTDDGVVWMNWKGSWYSMRKMSMKIRPFFPQQ KHGTDDGVVWMNWKDSWYSMKNMCIKIRPYFN-K KHGTDDGVVWMNWKDSWYSMKKMSIKIRPYFN-DYGTDDGVVWMNWKGSWYSMRQMAMKLRPKWP--

481 .:*****:******.****:: :.:*::* :

Fig. 4. (continued).

of large yellow croaker injected with V. parahaemolyticus was lower than that of the control group, and at 8 d it was signicantly lower. Fibrinogen levels are a reection of clotting ability and activity in the body. Excessive generation of brin due to activation of the coagulation cascade leads to thrombosis, while ineffective generation predisposes to hemorrhage. Reduced concentrations of brinogen may impair the bodys ability to form a stable blood clot. Chronically low levels may be related to decreased production due to an inherited condition such as abrinogenemia or hypobrinogenemia or to an acquired condition such as end-stage liver disease or severe malnutrition [28]. Based on data currently available, several studies revealed brinogen containing molecules

functioning as innate-type defence factors not only in vertebrates but also in invertebrates [2932]. The horseshoe crab Tachypleus tridentatushas has brinogen-related molecules in hemolymph plasma and they function as nonself-recognizing lectins [29]. Fibrinogen-related proteins are found in the hemolymph of the freshwater snail Biomphalaria glabrata, are up-regulated following exposure to digenetic trematode parasites, and bind to trematode larval surfaces, suggesting a role in internal defence [32]. Based on this result, brinogen levels may be used in diagnosis of relative diseases in large yellow croaker. For example, as an inammatory marker: a level above the normal range suggests some degree of systemic inammatory response.

Fig. 5. 3D structure compared the large yellow croaker FGB model (up) with human FGB (down) model which was predicted by Swiss-model and Rasmol software. The N-terminal chain can come together with other chains to form the center of the molecule (E domain), from which the chain extends in opposite direction as coiled coils, followed by C-terminal globular domains (D domains).

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2.5

Expression ratio relative to 18S rRNA

control v.para 2.0

1.5

1.0

0.5

0.0

4d

8d

12d

16d

Time after challenge

Fig. 6. Real time PCR expression of FGB in liver of large yellow croaker. The data are presented relative to 18S rRNA. All bars represent mean value (S.D.) of four sh per time point. *Indicate signicantly difference expression (P < 0.05).

In conclusion, we have used a modied ACP system developed in our laboratory to analyze differentially expressed genes in liver of large yellow croaker after challenge of V. parahaemolyticus. The gene of brinogen beta chain which identied here will provide insights into mechanisms of inammation or tissue damage, and dysfunction or disease of the liver in sh. Future studies will aim to dissect the pathway by using selective gene inactivation techniques, such as RNA interference. Acknowledgement This project was supported by the program of the Science and Technology Department of Fujian Province (2005N041, 2008N0121) and the Innovation Team foundation of Jimei University (2008A001). We thank Miss Lyndsey Kirk of the Department of Chemistry and Biochemistry, Texas State University for critical reading of the manuscript. References
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