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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
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 2008Received in revised form13 April 2009Accepted 26 April 2009Available online 5 May 2009
Keywords:Large yellow croakermACPReal time PCRFibrinogen beta chainDifferentially expressed genes
a b s t r a c t
In this article, we used a modified ACP system (mACP) developed in our laboratory to analyze differ-entially expressed genes in the liver of large yellow croaker, Pseudosciaena crocea (Richardson). By using20 pairs of mACPs, 7 differentially expressed genes were obtained. One of the genes we identifiedencodes for a fibrinogen 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 wascapable of encoding 492 amino acids with an estimated molecular mass of 55.6 kDa, giving it a predictedpI of 5.94. The deduced amino acid sequence included an FGB profile (V238-Y488) and an FGB familysignature (WWYNRCHSANPNG). Multiple sequence alignments indicated that the large yellow croakerFGB showed homology with FGB sequences of other species (45–77% identity). Real time PCR analysisdemonstrated that the expression of FGB in the liver of large yellow croaker injected with Vibrio para-haemolyticus was significantly (P < 0.05) lower than that of the control group at 8 d, which confirmed theexpression patterns of the results of mACP differential display.
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1. Introduction
The mRNA differential display reverse-transcription PCR (DDRT-PCR) [1] worked well in displaying the expected number ofmRNA per primer combination under optimal conditions, but highfalse-positive rates and poor reproducibility were encountered [2],which due to arbitrary primers (10–13 mers) were employed fordifferential display in combination with two-base anchoredprimers. The annealing temperature is critical for insuring thata primer binds only to its perfect complement or to sequences withone or more mismatches. The use of short primers (10–13 mers)usually requires low annealing temperatures about 40–45 �C for allPCR cycles; these low temperatures cause nonspecific primerannealing. By adjusting the annealing temperature, one can alterthe specificity 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 beenintroduced to increase primer annealing specificity [3–5]. The useof longer primers makes it possible to increase the annealing
temperature to 60 �C after 1–4 initial cycles at 40–45 �C [6].However, the additional tail sequences of longer primers areinvolved in nonspecific annealing to the cDNA template during PCRcycles at low annealing temperatures.
We describe here a more accurate and extensive PCR tech-nology, controlled by an annealing control primer (ACP) [7]. TheACP system is based on two characteristics: the unique tripartitestructure of the primers, which have distinct 30- and 50-end regionsthat are separated by a polydeoxyinosine [poly(dI)] linker, and theinteraction of each region during two-stage PCR. In the presentstudy, we used a modified ACP system (mACP) developed in ourlaboratory [8], to analyze differentially expressed genes in liver oflarge yellow croaker Pseudosciaena crocea (Richardson). BothdT-ACP1 and dT-ACP2 were replaced by dT-mACP, and the 50-endregions of dT-mACP and arbitrary mACPs contained the sameuniversal sequence. By using 20 pairs of mACPs, 7 differentiallyexpressed genes were obtained. One of the genes we identified isthe fibrinogen beta chain (FGB). To our knowledge, identification ofdifferentially expressed genes using an ACP system is focused onmammals [9–12]. This article is the first report characterizingdifferentially expressed genes by using an ACP system in fish.
Fibrinogen plays important roles with fibrin in blood clotting,fibrinolysis, cellular and matrix interactions, inflammation, woundhealing, and neoplasia [13]. Fibrinogen occurs as a dimer, whereeach monomer is composed of three non-identical chains, alpha,beta and gamma, linked together by several disulphide bonds. The
1 Present address: Department of Chemistry & Biochemistry, Texas StateUniversity, San Marcos, TX 78666, USA.
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N-terminals of all six chains come together to form the center of themolecule (E domain), from which the monomers extend in oppositedirections as coiled coils, followed by C-terminal globular domains(D domains). Therefore, the domain composition is: D-coil-E-coil-D. At each end, the C-terminal of the alpha chain extends beyondthe D domain as a protuberance that is important for cross-linkingthe molecule.
During clot formation, the N-terminal fragments of the alphaand beta chains (within the E domain) in fibrinogen are cleaved bythrombin, releasing fibrinopeptides A and B, respectively, andproducing fibrin [14]. Fibrin forms a soft clot, and is then convertedto a hard clot by factor XIIIA, which cross-links fibrin molecules.Platelet aggregation involves the binding of the platelet proteinreceptor integrin alpha (IIb)-beta(3) to the C-terminal D domain offibrinogen [15]. Fibrin fibres interact with platelets to increase thesize of the clot, as well as with several different proteins and cells,thereby promoting the inflammatory response and concentratingthe cells required for wound repair at the site of damage.
Research about the function of FGB in fish is unavailable,although bastard halibut Paralichthys olivaceus FGB (GenBankaccession no. EF581895) and zebrafish Danio rerio FGB (GenBankaccession no. NM_212774) have been cloned. Currently, fibrinogen-like protein genes related to the toxicity in pufferfish liver wereinvestigated by comparing mRNA expression patterns in akame-fugu Takifugu chrysops and kusafugu Takifugu niphobles havingdifferent concentrations of tetrodotoxin (TTX) [16]. Here we reportthe cloning of FGB and its expression pattern in the liver of largeyellow croaker.
2. Materials and methods
2.1. Fish
Healthy large yellow croaker (200–250 g) were obtained fromthe Fishery Extension Station of Ningde (Fujian, China) and werekept in net cages at about 25 �C and fed with a commercial feed.Before challenging experiments, the fish were maintained for atleast two weeks and then were injected intraperitoneally with1.0 mL of a 0.9% NaCl solution (control group) or Vibrio para-haemolyticus (experimental group) in a titer of 6.6 � 108 cfu mL�1.Liver from six control fish and six experimental fish were rapidlyharvested at 1 d, 2 d, 4 d, 8 d, 12 d, 16 d after injection, frozen inliquid nitrogen and stored at �80 �C until RNA extraction andanalysis.
2.2. RNA isolation
Total RNA was isolated from above samples as described in ourprevious study [17]. Quantity and purity of isolated RNA weredetermined by absorbance measures at 260 nm and 280 nm, and itsintegrity was tested by electrophoresis in agarose gels.
2.3. mACP-based differential display
2.3.1. For first-strand cDNA synthesis2 mL dT-mACP (50-AAGCAGTGGTATCAACG CAGAGTTTTTTTTTTTT
TTTTTTTTT-30, 10 mM) primer was added to 3 mg RNA isolated fromliver. 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 transcrip-tase (Invitrogen). The mixture was incubated in the tube at 42 �C for90 min, and then heated at 94 �C for 2 min to inactivate the reac-tion. The reaction mixture was chilled on ice for 2 min and spinnedbriefly. First-strand cDNA was diluted by adding 80 mL of ultrapurified water.
2.3.2. mACP-based PCRSecond-strand cDNA synthesis and subsequent PCR amplification
were conducted in a single tube. Second-strand cDNA synthesis wasconducted at 50 �C during one cycle of first stage PCR in a finalreaction volume of 20 mL containing 1 mL of the diluted first-strandcDNA, 2 mL of 10 � PCR buffer, 0.4 mL of dNTP (10 mM), 1 mL ofdT-mACP (10 mM), and 1 mL of arbitrary mACP (10 mM) preheated to94 �C. The tube containing the reaction mixture was held at 94 �Cwhile 1 U of Taq polymerase (TaKaRa) was added to the reactionmixture. The PCR protocol for second-strand synthesis was one cycleat 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 amplificationcycles were performed. Each cycle involved denaturation at 94 �C for40 s, annealing at 65 �C for 40 s, and extension at 72 �C for 40 s. A finalextension step of 5 min at 72 �C was performed to complete the PCR.The amplified PCR products were separated on 2.0% agarose gels andstained with ethidium bromide.
2.3.3. Cloning and transformationThe differentially expressed bands were extracted and cloned
into pMD-T (TaKaRa) vector following the manufacturer’s instruc-tions. In order to verify the identity of insert DNA, isolated plasmidswere sequenced. Complete sequences were analyzed by searchingfor similarities using BLASTX search program at the National Centerfor Biotechnology Information (NCBI) GenBank.
2.4. 50-RACE cloning of full-length FGB cDNA
Based on the partial sequence of large yellow croaker FGB, specificprimers FGB-RACE (GSP1: 50-TCCTCACACTCCTTACCGGACACCA-30,GSP2: 50-GCTGTACTGACCAACCACCCTGTTG-30) were designed to 50
end cDNA sequences of large yellow croaker FGB by rapid amplifi-cation of cDNA ends (RACE). Total RNA isolated from large yellowcroaker liver was used to synthesize the 50-RACE cDNA templatesusing the BD SMART RACE cDNA amplification kit (Clontech)according to the manufacturer’s instructions. 3 mg of RNA wasreverse-transcribed to cDNA with 1 mL of 50CDS primer and 1 mL ofSMART II Oligo, then RNase free water was added to a final volume of5 mL for each reaction. The RNA was denatured at 70 �C for 2 min andcooled on ice for 2 min. The other components in the 10 mL reverse-transcription reaction mixture included 2 mL 5 � first-strand buffer,1 mL DTT (20 mM), 1 mL dNTP (10 mM), 1 mL SuperScript II reversetranscriptase (200 U mL�1, Invitrogen). The reaction mixtures wereincubated at 42 �C for 1.5 h. The first-strand reaction was terminatedand diluted by adding 90 mL of RNase free water and heating at 72 �Cfor 7 min. The primary PCRs were carried out in a 25 mL reactionmixture composed of 1 mL diluted cDNA, 0.5 mL GSP1 (10 mM), 2.5 mLUPM, 2.5 mL 10 � PCR-buffer, 0.5 mL dNTP Mix (10 mM), 0.5 mL DNApolymerase (5 U mL�1), and 17.5 mL ultra purified water. The PCRconditions were: 5 cycles of 95 �C for 30 s, 72 �C for 3 min; 5 cycles of95 �C for 30 s; 70 �C for 30 s; 72 �C for 3 min; 25 cycles of 95 �C for30 s; 68 �C for 30 s; 72 �C for 3 min. The secondary PCRs were set upand run similar to the primary PCR except the UPM primer wasreplaced with NUP primer, GSP1 replaced with GSP2, and theprimary PCR products 1 mL as templates. The PCR products wereseparated by electrophoresis on 1% agarose gel. PCR bands of thepredicted 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 wasconstructed using the Neighbour-Joining method based on theCLUSTAL W alignment [20]. The Interpro profile library [21] wasused to identify characteristic domains or patterns. The SignalP
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program [22] was used for identification of signal peptides andprediction of cleavage sites. Analysis of secondary structure wasperformed using the PHD program [23] at the Protein Predict server(www.embl-heidelberg.de/predictprotein/). The three-dimensionalstructure of large yellow croaker FGB was modeled using Swiss-model [24] in the first approach mode accessible via the internet(http://www.expasy.org/swissmod). The co-ordinate files wereimported to RASMOL software version 2.7.3.1 for analyzing bondlengths and other conformational features of the molecule.
2.6. Real time PCR analysis of FGB expression
All the RNA samples, including different exposure groups (V. par-ahaemolyticus challenge and control) and exposure times (4 d, 8 d,12 d and 16 d), with four biological replicates were isolated fromdifferent individual fish (fish number: 2 � 4 � 4 ¼ 32) for real timePCR. 18S rRNA was used as a normalization factor (endogenouscontrol) to correct for different loading amounts of RNA since thisgene 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-CAGACAAATCGCTCCACCAA-30) were designed using Primer Express 3.0 (ABI)and tested to ensure amplification of single discrete bands with noprimer–dimers. 3 mg of RNA pre-treated with DNase I was used astemplate for total cDNA synthesis in 20 mL reactions with randomhexamers using the Superscript First-Strand Synthesis System forRT-PCR (Invitrogen). For real time PCR, 1 mL of cDNA, 0.8 mL each offorward and reverse primer (10 mM) and 10 mL SYBR Green RealtimePCR Master Mix (TOYOBO) were brought to a final volume of 20 mLwith ultra-pure water in each reaction and analyzed in the ABI 7500real time system. The cycling conditions were: 95 �C for 10 min,
followed by 40 cycles consisting of 94 �C for 15 s, 60 �C for 1 min.Thereafter, PCR products were analyzed by generating a meltingcurve. Since the melting curve of a product is sequence-specific, it canbe used to distinguish between nonspecific and specific PCR products.PCR products for FGB and 18S rRNA were ligated into pMD-T vectorand transformed in DH5a competent cells. Mini-preps of isolatedplasmid DNA were then prepared (Promega) for sequencing to checkthe sequence of the real time PCR products.
Fig. 1. Agarose gel photograph indicating products of mACP3 in liver of large yellowcroaker obtained by using the modified ACP system. Candidate gene was indicatedwith arrowhead. The length of the product is about 1500 bp (M: marker, C: controlgroup, 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; FGBfamily signature is represented in bold with italicized letters; the box points to signalpeptide; two poly A signal sequences are double underlined.
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The comparative threshold cycle (CT) method was used tocalculate the relative concentrations. This method involvesobtaining CT values for the FGB; normalizing to the housekeepinggene, 18S rRNA; and comparing the relative expression level amongliver samples. The relative quantification of gene expression wasanalyzed by the 2 [-Delta Delta C (T)] method [25]. StatisticsPackage for Social Science (SPSS) 13.0 for windows was used toanalyze the data from all experiments. Data were expressed asthe arithmetic mean � standard deviation (S.D.). Significance ofdifferences was determined by the independent-samples T-test.P values of <0.05 were considered significantly different; P valuesof <0.01 were considered most significantly different.
3. Results
3.1. Isolation of differentially expressed genes in liver oflarge yellow croaker
To identify genes that are expressed in liver of large yellowcroaker in response to the challenge of V. parahaemolyticus, wecompared the RNA expression profiles of control group andexperimental group at 8 d. By using the modified ACP system, sevenproducts with different expression levels were identified andsequenced. Queries of publicly available databases using the BLASTalgorithm showed that one of these products (from mACP3: 50-AAGCAGTGGTATCAACGCAGAGTIIIIICCGGAGGATG-30) (Fig. 1) hadhigh similarity to FGB gene. The length of the product was 1360 bp(Fig. 2). Two gene specific primers, FGB-GSP1 and FGB-GSP2 (Fig. 2)were designed from the product for 50-RACE. The full-length cDNAsequence of FGB was obtained by 50-RACE method. The nucleotidesequence 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 esti-mated molecular mass of 55.6 kDa, giving it a predicted pI of 5.94.Using the SignalP program, a signal peptide (residue numbers 1–16,MRTLLLLYLCVYTAWA, Fig. 2) was predicted at the N-terminus.Interpro analysis indicated that the sequence included an FGBprofile (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 relatednessof FGB between large yellow croaker and other species, we performeda phylogenetic analysis with 11 different members of FGB using theNeighbour-Joining method based on the CLUSTAL W alignment. Thephylogenetic tree was bootstrapped 1000 times. The tree (Fig. 3)indicates that the teleost FGB sequences form a cluster separate fromthe other FGB sequences. The sequence of the large yellow croakerFGB showed homology with FGB sequences of other species (45–77%identity), with sea lamprey Pertromyzon marinus FGB being the lowhomology with 45% identity. In contrast, bastard halibut P. olivaceusFGB sequence showed marked homology (77% identity).
Comparison of known FGB sequences revealed that many of thesignature residues are conserved in large yellow croaker FGB (Fig. 4).Most of the sequence identities are confined to the C-terminaldomain, as reported for other species. The C-terminal domaincontains four conserved cysteines involved in two disulfide bonds.Analysis of secondary structure using the PHD program at theProtein Predict server (www.embl-heidelberg.de/predictprotein/)suggested that large yellow croaker FGB possesses 11 alpha helixesand 14 beta-pleated sheets, which was similar to human FGB. Thus,the overall predicted structure of large yellow croaker FGB wascompared with the 3D structure of human FGB (Fig. 5). Using thismodel, we could predict similar biological functions from largeyellow croaker FGB similar to that of human FGB.
3.3. Confirmation of expression of large yellow croakerFGB by real time PCR
To validate the results of mACP differential display of FGB and todetermine the relative abundance of target sequences in liver oflarge yellow croaker after challenge of V. parahaemolyticus, realtime PCR was performed. The results (Fig. 6) demonstrated that theexpression of FGB in liver of large yellow croaker injected withV. parahaemolyticus was significantly (P < 0.05) lower than that ofthe control group at 8 d, and there was no significant differencebetween other control group and experimental group (P > 0.05).This analysis revealed that our target transcripts which showed theexpression patterns in agreement with the results of mACP differ-ential display.
Pseudosciaena crocea
Paralichthys olivaceus
Danio rerio
1000
Xenopus tropicalis
Xenopus laevis
Gallus gallus
Rattus norvegicus
Equus caballus
Canis familiaris
Homo sapiens
758
611
1000
1000
752
1000
994
Petromyzon marinus0.02
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, Speciesnames are represent: Pseudosciaena crocea: large yellow croaker (GenBank accession no. ABJ98546); Paralichthys olivaceus: bastard halibut (GenBank accession no. ABQ41317);Danio rerio: zebrafish (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); Equuscaballus: 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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4. Discussion
The specificity and sensitivity with which a primer anneals to itstarget sequence are the most critical factors in determining thesuccess of PCR amplification. In this study, we employed a newdifferential display RT-PCR technique [7,9] to compare the geneexpression in liver of large yellow croaker. The ACP-based RT-PCRmethod involves an ACP that has a unique tripartite structure in thatits distinct 30- and 50-end portions are separated by a regulator. ThisACP-based RT-PCR system is easy and accurate and lacks false posi-tives, the PCR products can be detected on standard ethidiumbromide-stained agarose gels, and hence, obviating the use of PAGEgels with the attendant issues in handling and use. The 50-end regionsof dT-mACP and mACP primers contain the same universal sequence,which result in the synthesis of the second-strand cDNA containingthe inverted repeat sequence at both ends. Through the technique ofstep-out PCR, suppression PCR inverted repeat elements are incor-porated next to all the second-strand cDNA sequences. During PCR,these inverted repeats of short cDNA fragments easily anneal to eachother intramolecularly. This rapid first-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 beamplified. So the modified ACP system which we used could generatea wide range of PCR products ranging from 300 bp to 2.0 kb, which notonly increases the chances of identifying differentially expressedgenes, but also provides more significant sequence information for theprediction of gene function. With this technique, we identified 7differentially expressed genes that are specifically or more promi-nently expressed in liver of control group to experimental group at 8 d.And it was adapted to identify genes differentially expressed in ovaryand testis at different developmental stages in penaeid shrimpMarsupenaeus japonicus in our laboratory [26]. By using 20 pairs ofmACPs, 8 differentially expressed genes were obtained. One of thesegenes is ubiquitin-conjugating enzyme E2r (UBE2r), which has animportant role in oogenesis and spermatogenesis.
Using the BLASTX showed that one of these products (frommACP3) (Fig. 1) had high similarity to FGB gene. More rigorouslybioinformatic analyses indicated that the deduced amino acidsequence included an FGB profile and an FGB family signature(Fig. 2), and the sequence of the large yellow croaker FGB showed
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-Coffeeat http://www.ch.embnet.org/. Consistency scores are shown by color as: BAD AVG GOOD
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homology with FGB sequences of other species (45–77% identity,Fig. 4). Analysis of secondary structure suggested that large yellowcroaker FGB possesses 11 alpha helixes and 14 beta-pleated sheets,which was similar to human FGB. The molecular modeling studiesalso predicted similar biological functions of large yellow croakerFGB to human FGB (Fig. 5). Based on the analysis above, we canconfirm that the large yellow croaker FGB belongs to the FGB family.
Fibrinogen is a soluble plasma glycoprotein that is synthesized inthe liver by hepatocytes and megakaryocytes. Fibrinogen is an acutephase reactant, meaning that fibrinogen concentrations may rise
sharply in any condition that causes inflammation or tissue damage,but dysfunction or disease of the liver can lead to a decrease infibrinogen production or the production of abnormal fibrinogenmolecules with reduced activity (dysfibrinogenaemia)[14]. A study[27] on the V. parahaemolyticus of marine cage-cultured large yellowcroaker showed that incidence of this disease was high and epidemicand occurred during summer. The infection rate varied from 30% to50%, and artificial infection showed that the bacterium isolatedcould cause dysfunction of liver of large yellow croaker. The real timePCR analysis (Fig. 6) confirmed that the expression of FGB in the liver
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of large yellow croaker injected with V. parahaemolyticus was lowerthan that of the control group, and at 8 d it was significantly lower.
Fibrinogen levels are a reflection of clotting ability and activityin the body. Excessive generation of fibrin due to activation of thecoagulation cascade leads to thrombosis, while ineffective gener-ation predisposes to hemorrhage. Reduced concentrations offibrinogen may impair the body’s ability to form a stable blood clot.Chronically low levels may be related to decreased production dueto an inherited condition such as afibrinogenemia or hypofi-brinogenemia or to an acquired condition such as end-stage liverdisease or severe malnutrition [28]. Based on data currently avail-able, several studies revealed fibrinogen containing molecules
functioning as innate-type defence factors not only in vertebratesbut also in invertebrates [29–32]. The horseshoe crab Tachypleustridentatushas has fibrinogen-related molecules in hemolymphplasma and they function as nonself-recognizing lectins [29].Fibrinogen-related proteins are found in the hemolymph of thefreshwater snail Biomphalaria glabrata, are up-regulated followingexposure to digenetic trematode parasites, and bind to trematodelarval surfaces, suggesting a role in internal defence [32]. Based onthis result, fibrinogen levels may be used in diagnosis of relativediseases in large yellow croaker. For example, as an inflammatorymarker: a level above the normal range suggests some degree ofsystemic inflammatory 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-terminalchain 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-terminalglobular domains (D domains).
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In conclusion, we have used a modified ACP system developedin our laboratory to analyze differentially expressed genes in liverof large yellow croaker after challenge of V. parahaemolyticus. Thegene of fibrinogen beta chain which identified here will provideinsights into mechanisms of inflammation or tissue damage, anddysfunction or disease of the liver in fish. Future studies will aim todissect the pathway by using selective gene inactivation tech-niques, such as RNA interference.
Acknowledgement
This project was supported by the program of the Science andTechnology Department of Fujian Province (2005N041, 2008N0121)and the InnovationTeam foundation of Jimei University (2008A001).We thank Miss Lyndsey Kirk of the Department of Chemistry andBiochemistry, Texas State University for critical reading of themanuscript.
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0.0
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4d 8d 12d 16d
Time after challenge
Exp
ress
ion
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o re
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o 18
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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 (þS.D.) of four fish per time point.*Indicate significantly difference expression (P < 0.05).
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