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Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8β (C8β) in Nile tilapia (Oreochromis niloticus)
Fish & Shellfish Immunology 35 (2013) 1055e1058
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short sequence report
Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8b (C8b) in Nile tilapia (Oreochromis niloticus) Anyuan He 1, Jie Yang 1, Shoujie Tang, Chenghui Wang* Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 26 June 2013 Accepted 11 July 2013 Available online 19 July 2013
Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8b subunit (C8b), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 50 -untranslated region (UTR) and a 236 bp 30 UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8b cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 50 -UTR and a 170 bp 30 -UTR. The predicted protein of C8b contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Complement components C1INH C8b Tilapia
1. Introduction The complement system is essential to innate and acquired host defenses against microbial infection [1e3]. Complement component 1 inhibitor (C1INH), one of the most important elements of the complement system that controls the classical pathway, has important functions in regulating vascular permeability and suppressing inflammation [4]. Complement component 8b (C8b) is another complement gene and a subunit of C8 comprising one a, b, and g subunits. In the membrane attack complex (MAC) assembly, the lytic complement components, including C6, C7, C8, and C9, aggregate with C5b. MAC formation induces the destruction of bacterial and eukaryotic cells by increasing their membrane permeability [5,6]. A lot of studies of complement genes have focused on mammals, such as humans and mice [7,8]. However, only a few complement genes of teleosts have been described in a
few fishes (e.g. Mi-iuy croaker, zebrafish, common carp and rainbow trout) [9]. Nile tilapia (Oreochromis niloticus) is one of the most important species in freshwater aquaculture and accounts for about 82% of the total production of tilapia [10]. However, high frequent disease outbreaks have annually resulted in large economic losses of tilapia in China since 2008 [11]. Major disease pathogens include Streptococcus shiloi, S. difficile, and S. iniae [11e15]. In this paper, the cloning and characteristics of C1INH and C8b genes of Nile tilapia were reported for the first time. Meanwhile, their expression profiles in different tissues were also described by quantitative real-time PCR (qPCR). The aim of the present study was to provide better understanding of these two molecules and the complement system of Nile tilapia.
2. Materials and methods 2.1. Sample preparation
* Corresponding author. Tel./fax: þ86 21 6190 0439. E-mail address: [email protected] (C. Wang). 1 These authors contributed equally to this paper. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.07.019
Three Nile tilapias with an average body weight of 120 5 g were sampled from the Fishery Genetic Resources Experiment
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Station of Shanghai Ocean University (Shanghai, China). The fishes were maintained in 250 L aquaria at 28 2 C for one week to acclimate laboratory conditions prior to tissue collection and RNA isolation. 2.2. RNA extraction Total RNA was isolated from eight tissues (gill, brain, dorsal muscle, kidney, liver, intestine, heart, and spleen) in the each sample by using an RNAiso Plus Kit (Takara, Japan). The quality and quantity of the total RNA were determined using a NanoDrop 1000 spectrophotometer (Hach, America). 2.3. Gene cloning The total RNA from one liver tissue was used to clone the two genes. For the C1INH gene, a series of degenerate primers (Table 1) were designed based on the C1INH cDNA sequence of Larimichthys crocea and Oncorhynchus mykiss [16,17]. For the C8b gene, primers (Table 1) were designed according to a partial sequence from a cDNA library of Nile tilapia [18]. Some partial encoding sequences of the two genes were isolated by polymerase chain reaction (PCR) under the following PCR conditions: 94 C for 5 min; 30 cycles at 94 C for 30 s, 54 C for 30 s and 72 C for 1 min; and 72 C for 5 min. PCR was performed in a reaction mixture of 50 mL. Amplified products were evaluated by 1.5% agarose gel electrophoresis and purified by BioSpin Gel Extraction Kit (Bioer, China). These purified products were then cloned in pGEM-T vector (Promega, USA) and transformed to competent cells. The cloned PCR fragments were sequenced on an ABI3730 capillary sequencer by using standard sequencing primers (M13). To obtain the complete cDNA sequences of C1INH and C8b genes in Nile tilapia, 50 and 30 rapid amplification of cDNA ends (RACE) techniques were performed using a SMART RACE cDNA amplification kit (Clontech, USA) according to the manufacturer’s
Table 1 Primers used for cloning and expression of C1INH and C8b genes in Nile tilapia. Name of primers
Sequence (50 to 30 )
C1INH gene isolation Sc6F AHKGC CAATG CRCTY TGGTT Sc6R GTAAV TKCCA GCRTC CTCCA T Sd9F RGARC CCATK YTGCA GGART Sd9R AWCSC RCTGA TRCTG ATMGG W Sh14F AGTTT GCTCT SACRG RTRAS A Sh14R TCYAG YTTGA TTWGG GGCA 1 mF AGACC ACGAT CAGAG CATCC T 1 mR AGAGC ATGTT GCCAG AAGGA 2 mF TCCTT CTGGC AACAT GCTCT 2 mR TGCAG CTTTG CCTCT GTGTT tp1F TGAAG CGAAC AGTGG TTCAA tp1R GGAAG ATAAT CCAAA TCCAC A 3RA ATGAC GGACA GGGCT GTGGT T 5RA CATCC TCCAT GCCAG CGTTT AAGAC C C8 beta gene isolation C8F TATGG CACCC ACTAT GTCAC C C8R TTAGT GATCT CGCAG GCTCT T tp2F GGGGA CACAC AAACA TGCTT T tp2R GGAGA TGAAG CATTG TCAAG G 3 KR ACAAT GGAGT GGCTG TTCTG A 5 KR TTAAA CCTGC CTGTA CGCAG TTCTT GTAG qPCR analysis Bt4F GGGTC AGAAA GACAG CTACG TTGGT G Bt4R TCGTT GTAGA AGGTG TGATG CCAGA C1F CACAC GGAAA TTCAA GAGCT ATGGG C1R ACAGG GAAGC TCCAG CGTAG AGTCA 83F TGTCA GAGTA CCTGG CAGAG GCTAG 83R ATTAG TGATC TCGCA GGCTC TTCCA
Utility Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Full-length confirmation RACE RACE Cloning Cloning Full-length confirmation RACE RACE Beta-actin C1INH C8b
protocol. The 50 RACE products were amplified to determine C1INH and C8b genes by using the primers 5RA and 5 KR, respectively (Table 1), and 30 RACE products were similarly amplified to determine C1INH and C8b genes by using the primers 3RA and 3 KR, respectively (Table 1). The generated 50 and 30 PCR products were purified and cloned in pMD 19-T vector (Takara, Japan) for sequencing. The full-length cDNA of the two genes were confirmed by RT-PCR with the two pairs of terminal primers, tp1 and tp2, respectively. 2.4. Sequence analysis Sequence identities of the two genes were determined by aligning against NCBI databases with BLAST. Multiple alignment of nucleotide and amino acid sequences were conducted using ClustalX 2.0 [19]. The protein domain structure was predicted using PROSITE (http://prosite.expasy.org/, available in June 2012). Signal peptide prediction was performed using SignalP 4.0 server (http:// www.cbs.dtu.dk/services/SignalP/, available in June 2012). 2.5. Gene expression analysis cDNAs of the total RNA from eight tissues of the three fishes were individually synthesized using a PrimerScript RT reagent kit (Takara, Japan). Expression patterns of C1INH and C8b genes were evaluated by using a CFX96 real-time PCR system (Bio-Rad, USA) with SYBR Green Premix Ex Taq (Takara, Dalian, China) based on the specific primers (C1F and C1R for the C1INH gene; 83F and 83R for the C8b gene) (Table 1). The expression pattern of b-actin gene (Accession No.: EU887951) generated by the Bt4F and Bt4R primers was used as an internal control. The reaction volume of qPCR was 25 mL containing 2.0 mL of cDNA template, 12.5 mL of SYBR Premix Ex Taq, 2.0 mL of PCR primers (5 mM), and 6.5 mL of nuclease-free water. The reaction conditions used were as follows: 95 C for 40 s; 40 cycles of 95 C for 5 s; and 60 C for 20 s. The melting curves of the amplified products were analyzed at the end of each PCR to confirm that only one PCR product was amplified and detected. The cDNA abundance was estimated using the 2(DDCt) method. The data were subjected to one-way ANOVA in SPSS 17.0 (SPSS, America). 3. Results and discussion 3.1. Characterization of C1INH and C8b cDNA of Nile tilapia The full-length cDNA of C1INH (GenBank Accession No. JF791810) was 2128 bp with a 101 bp 50 -untranslated region (UTR), a 1791 bp open reading frame, and a 236 bp 30 -UTR. The typical polyadenylation signal AATAAA (14 bp) was found before the poly-A signal. The size of predicted C1INH protein sequence of Nile tilapia (597 amino acids) was higher than those of the reported mammals (approximately 500 amino acids), which may be attributed to the loss of a protein sequence in an Ig-like domain in mammals. In the predicted amino acids of C1INH, a signal peptide was detected between the residues 20 and 21 (signal peptide and cleavage site probabilities were 70.2% and 80.2%, respectively). The specific domain motifs (Fig. 1) were identified, including a typical serpin domain, two Ig-like domain, and glycosyl hydrolase family-9 active site signature 2 (GLYCOSYL_HYDROL_F9_2). These Ig domains were found in many protein families with different functions and may be involved in proteineprotein and proteineligand interactions [17]. The aligned amino acid sequences revealed that this gene in Nile tilapia was homologous to those in other teleost and mammals, with sequence identities of 60%, 51%, 39%, 34%, 33%, and 33% with L. crocea (ACS83543), O. mykiss (NP_001117851), Danio
A. He et al. / Fish & Shellfish Immunology 35 (2013) 1055e1058
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Fig. 1. Multiple amino acid sequence alignment of the C1INH gene in Nile tilapia with homologs from other selected species. The sequences used in this figure are LcC1inh (L. crocea, ACS83543), OmC1inh (O. mykiss, NP_001117851), HsC1inh (Homo sapiens, CAA30314) and OnC1inh (O. niloticus, JF791810). The grey-highlighted letters and period (.) indicates the identical residue among the sequences.
Fig. 2. Multiple amino acid sequence alignment of the C8 beta gene in Nile tilapia with homologs from other selected species. The sequences used in this figure are PoC8b (P. olivaceus, BAA86877), OmC8b (O. mykiss, AF418597), HsC8b (H. sapiens, NP_000057) and OnC8b (O. niloticus, JN998120). The grey-highlighted letters and period (.) indicates the identical residue among the sequences.
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Acknowledgment The authors want to express special thanks to Miss Lihua Xu and Mr. Kangle Li for their assistance in the experiments. This study was supported by the Tilapia Industry Technology System in China (Grant No. CARS-49-04B) and Shanghai University Knowledge Service Platform (ZF1206).
References
Fig. 3. Expression patterns of C1INH and C8 beta genes in eight tissues of Nile tilapia obtained by qPCR. The eight tissues were the gill (Gi), heart (He), brain (Br), kidney (Ki), liver (Li), intestine (In), spleen (Sp), and muscle (Mu).
rerio (NP_001116757), Sus scrofa (BAF36965o), Mus musculus (NP_033906), and Homo sapiens (CAA30314), respectively. The full-length cDNA of C8b (GenBank Accession No. JN998120) was 1946 bp with a 15 bp 50 -UTR, a 1761 bp coding region (encoding 587 amino acids), and a 170 bp 30 -UTR. In C8b, the functional domain analysis revealed the presence of three motifs observed in mammals and other bony fishes, including thrombospondin type-1 (TSP1) repeat, MAC/perforin (MACPF) domain, and LDL-receptor class A (LDLRA) domain [20]. A signal peptide was predicted between the positions 1 and 35, and a cleavage site was found between the positions 35 and 36 (Fig. 2). The deduced amino acid of the C8b in Nile tilapia showed moderate to high sequence identity with Paralichthys olivaceus (79%, BAA86877), O. mykiss (70%, AF418597), D. rerio (58%, NP_001243652), S. scrofa (51%, NP_001090920), Bos taurus (50%, NP_001039606), Oryctolagus cuniculus (50%, NP_001076137), H. sapiens (50%, NP_000057), Rattus norvegicus (49%, NP_001178688), M. musculus (48%, NP_598643), Chimaera phantasma (47%, BAI47541), and Heterocephalus glaber (47%, EHA99005). 3.2. Tissue distribution of C1INH and C8b expressions The expression of the C1INH and C8b genes in the eight tissues (gill, heart, brain, kidney, liver, intestine, spleen, and dorsal muscle) indicated the highest expression level in the liver (P < 0.01; Fig. 3). The expression characteristics of C1INH were consistent with that observed in rainbow trout [21], but were different from large croaker, in which the expression in the liver was less than that in the spleen, kidney, brain, and heart [22]. In Nile tilapia, C8b was expressed in the eight tissues studied using qPCR method, which was different from the finding in rainbow trout using northern blot method, this phenomenon maybe be thanks to the low resolution of northern blot [21,23].
[1] Rautemaa R, Meri S. Complement-resistance mechanisms of bacteria. Microb Infect 1999;1:785e94. [2] Rooijakkers S, Strijp J v. Bacterial complement evasion. Mol Immunol 2007;44: 23e32. [3] Muller-Eberhard HJ. Molecular organization and function of the complement system. Annu Rev Biochem 1988;57:321e47. [4] Liu D, Lu F, Qin G, Fernandes S. C1 inhibitor-mediated protection from sepsis. J Immunol 2007;179:3966e72. [5] Aybar L, Shin D, Smith S. Molecular characterization of the alpha subunit of complement component C8 (GcC8 alpha) in the nurse shark (Ginglymostoma cirratum). Fish Shellfish Immunol 2009;27:397e406. [6] Kimura A, Nonaka M. Molecular cloning of the terminal complement components C6 and C8 beta of cartilaginous fish. Fish Shellfish Immunol 2009;27: 768e72. [7] Carroll M. The complement system in regulation of adaptive immunity. Nat Immunol 2004;5:981e6. [8] Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science 2010;327. 291e291. [9] Boshra H, Li J, Sunyer J. Recent advances on the complement system of teleost fish. Fish Shellfish Immun 2006;20:239e62. [10] FAO. Fishery and aquaculture statistics, aquaculture production; 2010. [11] Zhang J, Zhou S, Li A. Streptococcus iniae infection in cultured tilapia, sea bass, and barramundi in Guangdong. J Fish Sci China 2012;19:161e6. [12] Eldar A, Bejerano Y, Bercovier H. Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing a meningoencephalitis in fish. Curr Microbiol 1994;28:139e43. [13] Pereraa RP, Johnson SK, Collins MD, Lewis DH. Streptococcus iniae associated with mortality of Tilapia nilotica T. aurea hybrids. J Aquat Anim Health 1994;6:335e40. [14] Weinstein M, Litt M, Kertesz D. Invasive infections due to a fish pathogen, Streptococcus iniae. N Engl J Med 1997;337:589e94. [15] Eldar A, Perl S, Frelier P, Bercovier H. Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection. Dis Aquat Org 1999;36:121e7. [16] Wei W, Xu H, Wang Q, Zhang X, Chang K, Wu C, et al. Identification of differentially expressed genes in large yellow croaker (Pseudosciaena crocea) induced by attenuated live Vibrio anguillarum. Aquaculture 2009;291:124e9. [17] Wang T, Secombes C. Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss. Immunogenetics 2003;55:615e28. [18] Lee B-Y, Howe AE, Conte MA, D'Cotta H, Pepey E, Baroiller J-F, et al. An EST resource for tilapia based on 17 normalized libraries and assembly of 116, 899 sequence tags. BMC Genomics 2010;11:278e88. [19] Larkin M, Blackshields G, Brown N, Chenna R, McGettigan P, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947e8. [20] Steckel EW, York RG, Monahan JB, Sodetz J. The eighth component of human complement. Purification and physicochemical characterization of its unusual subunit structure. J Biol Chem 1980;255:11997e2005. [21] Kazantzi A, Sfyroera G, Holland M. Molecular cloning of the b subunit of complement component eight of rainbow trout. Dev Comp Immunol 2003;27: 167e74. [22] Wei W, Wu H, Xu H, Zhang X, Chu T, Wu C, et al. Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea). Fish Shellfish Immun 2010;28:221e7. [23] Bustin S, Mueller R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci 2005;109:365e79.
Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short sequence report
Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8b (C8b) in Nile tilapia (Oreochromis niloticus) Anyuan He 1, Jie Yang 1, Shoujie Tang, Chenghui Wang* Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2013 Received in revised form 26 June 2013 Accepted 11 July 2013 Available online 19 July 2013
Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8b subunit (C8b), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 50 -untranslated region (UTR) and a 236 bp 30 UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8b cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 50 -UTR and a 170 bp 30 -UTR. The predicted protein of C8b contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Complement components C1INH C8b Tilapia
1. Introduction The complement system is essential to innate and acquired host defenses against microbial infection [1e3]. Complement component 1 inhibitor (C1INH), one of the most important elements of the complement system that controls the classical pathway, has important functions in regulating vascular permeability and suppressing inflammation [4]. Complement component 8b (C8b) is another complement gene and a subunit of C8 comprising one a, b, and g subunits. In the membrane attack complex (MAC) assembly, the lytic complement components, including C6, C7, C8, and C9, aggregate with C5b. MAC formation induces the destruction of bacterial and eukaryotic cells by increasing their membrane permeability [5,6]. A lot of studies of complement genes have focused on mammals, such as humans and mice [7,8]. However, only a few complement genes of teleosts have been described in a
few fishes (e.g. Mi-iuy croaker, zebrafish, common carp and rainbow trout) [9]. Nile tilapia (Oreochromis niloticus) is one of the most important species in freshwater aquaculture and accounts for about 82% of the total production of tilapia [10]. However, high frequent disease outbreaks have annually resulted in large economic losses of tilapia in China since 2008 [11]. Major disease pathogens include Streptococcus shiloi, S. difficile, and S. iniae [11e15]. In this paper, the cloning and characteristics of C1INH and C8b genes of Nile tilapia were reported for the first time. Meanwhile, their expression profiles in different tissues were also described by quantitative real-time PCR (qPCR). The aim of the present study was to provide better understanding of these two molecules and the complement system of Nile tilapia.
2. Materials and methods 2.1. Sample preparation
* Corresponding author. Tel./fax: þ86 21 6190 0439. E-mail address: [email protected] (C. Wang). 1 These authors contributed equally to this paper. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.07.019
Three Nile tilapias with an average body weight of 120 5 g were sampled from the Fishery Genetic Resources Experiment
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Station of Shanghai Ocean University (Shanghai, China). The fishes were maintained in 250 L aquaria at 28 2 C for one week to acclimate laboratory conditions prior to tissue collection and RNA isolation. 2.2. RNA extraction Total RNA was isolated from eight tissues (gill, brain, dorsal muscle, kidney, liver, intestine, heart, and spleen) in the each sample by using an RNAiso Plus Kit (Takara, Japan). The quality and quantity of the total RNA were determined using a NanoDrop 1000 spectrophotometer (Hach, America). 2.3. Gene cloning The total RNA from one liver tissue was used to clone the two genes. For the C1INH gene, a series of degenerate primers (Table 1) were designed based on the C1INH cDNA sequence of Larimichthys crocea and Oncorhynchus mykiss [16,17]. For the C8b gene, primers (Table 1) were designed according to a partial sequence from a cDNA library of Nile tilapia [18]. Some partial encoding sequences of the two genes were isolated by polymerase chain reaction (PCR) under the following PCR conditions: 94 C for 5 min; 30 cycles at 94 C for 30 s, 54 C for 30 s and 72 C for 1 min; and 72 C for 5 min. PCR was performed in a reaction mixture of 50 mL. Amplified products were evaluated by 1.5% agarose gel electrophoresis and purified by BioSpin Gel Extraction Kit (Bioer, China). These purified products were then cloned in pGEM-T vector (Promega, USA) and transformed to competent cells. The cloned PCR fragments were sequenced on an ABI3730 capillary sequencer by using standard sequencing primers (M13). To obtain the complete cDNA sequences of C1INH and C8b genes in Nile tilapia, 50 and 30 rapid amplification of cDNA ends (RACE) techniques were performed using a SMART RACE cDNA amplification kit (Clontech, USA) according to the manufacturer’s
Table 1 Primers used for cloning and expression of C1INH and C8b genes in Nile tilapia. Name of primers
Sequence (50 to 30 )
C1INH gene isolation Sc6F AHKGC CAATG CRCTY TGGTT Sc6R GTAAV TKCCA GCRTC CTCCA T Sd9F RGARC CCATK YTGCA GGART Sd9R AWCSC RCTGA TRCTG ATMGG W Sh14F AGTTT GCTCT SACRG RTRAS A Sh14R TCYAG YTTGA TTWGG GGCA 1 mF AGACC ACGAT CAGAG CATCC T 1 mR AGAGC ATGTT GCCAG AAGGA 2 mF TCCTT CTGGC AACAT GCTCT 2 mR TGCAG CTTTG CCTCT GTGTT tp1F TGAAG CGAAC AGTGG TTCAA tp1R GGAAG ATAAT CCAAA TCCAC A 3RA ATGAC GGACA GGGCT GTGGT T 5RA CATCC TCCAT GCCAG CGTTT AAGAC C C8 beta gene isolation C8F TATGG CACCC ACTAT GTCAC C C8R TTAGT GATCT CGCAG GCTCT T tp2F GGGGA CACAC AAACA TGCTT T tp2R GGAGA TGAAG CATTG TCAAG G 3 KR ACAAT GGAGT GGCTG TTCTG A 5 KR TTAAA CCTGC CTGTA CGCAG TTCTT GTAG qPCR analysis Bt4F GGGTC AGAAA GACAG CTACG TTGGT G Bt4R TCGTT GTAGA AGGTG TGATG CCAGA C1F CACAC GGAAA TTCAA GAGCT ATGGG C1R ACAGG GAAGC TCCAG CGTAG AGTCA 83F TGTCA GAGTA CCTGG CAGAG GCTAG 83R ATTAG TGATC TCGCA GGCTC TTCCA
Utility Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Cloning Full-length confirmation RACE RACE Cloning Cloning Full-length confirmation RACE RACE Beta-actin C1INH C8b
protocol. The 50 RACE products were amplified to determine C1INH and C8b genes by using the primers 5RA and 5 KR, respectively (Table 1), and 30 RACE products were similarly amplified to determine C1INH and C8b genes by using the primers 3RA and 3 KR, respectively (Table 1). The generated 50 and 30 PCR products were purified and cloned in pMD 19-T vector (Takara, Japan) for sequencing. The full-length cDNA of the two genes were confirmed by RT-PCR with the two pairs of terminal primers, tp1 and tp2, respectively. 2.4. Sequence analysis Sequence identities of the two genes were determined by aligning against NCBI databases with BLAST. Multiple alignment of nucleotide and amino acid sequences were conducted using ClustalX 2.0 [19]. The protein domain structure was predicted using PROSITE (http://prosite.expasy.org/, available in June 2012). Signal peptide prediction was performed using SignalP 4.0 server (http:// www.cbs.dtu.dk/services/SignalP/, available in June 2012). 2.5. Gene expression analysis cDNAs of the total RNA from eight tissues of the three fishes were individually synthesized using a PrimerScript RT reagent kit (Takara, Japan). Expression patterns of C1INH and C8b genes were evaluated by using a CFX96 real-time PCR system (Bio-Rad, USA) with SYBR Green Premix Ex Taq (Takara, Dalian, China) based on the specific primers (C1F and C1R for the C1INH gene; 83F and 83R for the C8b gene) (Table 1). The expression pattern of b-actin gene (Accession No.: EU887951) generated by the Bt4F and Bt4R primers was used as an internal control. The reaction volume of qPCR was 25 mL containing 2.0 mL of cDNA template, 12.5 mL of SYBR Premix Ex Taq, 2.0 mL of PCR primers (5 mM), and 6.5 mL of nuclease-free water. The reaction conditions used were as follows: 95 C for 40 s; 40 cycles of 95 C for 5 s; and 60 C for 20 s. The melting curves of the amplified products were analyzed at the end of each PCR to confirm that only one PCR product was amplified and detected. The cDNA abundance was estimated using the 2(DDCt) method. The data were subjected to one-way ANOVA in SPSS 17.0 (SPSS, America). 3. Results and discussion 3.1. Characterization of C1INH and C8b cDNA of Nile tilapia The full-length cDNA of C1INH (GenBank Accession No. JF791810) was 2128 bp with a 101 bp 50 -untranslated region (UTR), a 1791 bp open reading frame, and a 236 bp 30 -UTR. The typical polyadenylation signal AATAAA (14 bp) was found before the poly-A signal. The size of predicted C1INH protein sequence of Nile tilapia (597 amino acids) was higher than those of the reported mammals (approximately 500 amino acids), which may be attributed to the loss of a protein sequence in an Ig-like domain in mammals. In the predicted amino acids of C1INH, a signal peptide was detected between the residues 20 and 21 (signal peptide and cleavage site probabilities were 70.2% and 80.2%, respectively). The specific domain motifs (Fig. 1) were identified, including a typical serpin domain, two Ig-like domain, and glycosyl hydrolase family-9 active site signature 2 (GLYCOSYL_HYDROL_F9_2). These Ig domains were found in many protein families with different functions and may be involved in proteineprotein and proteineligand interactions [17]. The aligned amino acid sequences revealed that this gene in Nile tilapia was homologous to those in other teleost and mammals, with sequence identities of 60%, 51%, 39%, 34%, 33%, and 33% with L. crocea (ACS83543), O. mykiss (NP_001117851), Danio
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Fig. 1. Multiple amino acid sequence alignment of the C1INH gene in Nile tilapia with homologs from other selected species. The sequences used in this figure are LcC1inh (L. crocea, ACS83543), OmC1inh (O. mykiss, NP_001117851), HsC1inh (Homo sapiens, CAA30314) and OnC1inh (O. niloticus, JF791810). The grey-highlighted letters and period (.) indicates the identical residue among the sequences.
Fig. 2. Multiple amino acid sequence alignment of the C8 beta gene in Nile tilapia with homologs from other selected species. The sequences used in this figure are PoC8b (P. olivaceus, BAA86877), OmC8b (O. mykiss, AF418597), HsC8b (H. sapiens, NP_000057) and OnC8b (O. niloticus, JN998120). The grey-highlighted letters and period (.) indicates the identical residue among the sequences.
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Acknowledgment The authors want to express special thanks to Miss Lihua Xu and Mr. Kangle Li for their assistance in the experiments. This study was supported by the Tilapia Industry Technology System in China (Grant No. CARS-49-04B) and Shanghai University Knowledge Service Platform (ZF1206).
References
Fig. 3. Expression patterns of C1INH and C8 beta genes in eight tissues of Nile tilapia obtained by qPCR. The eight tissues were the gill (Gi), heart (He), brain (Br), kidney (Ki), liver (Li), intestine (In), spleen (Sp), and muscle (Mu).
rerio (NP_001116757), Sus scrofa (BAF36965o), Mus musculus (NP_033906), and Homo sapiens (CAA30314), respectively. The full-length cDNA of C8b (GenBank Accession No. JN998120) was 1946 bp with a 15 bp 50 -UTR, a 1761 bp coding region (encoding 587 amino acids), and a 170 bp 30 -UTR. In C8b, the functional domain analysis revealed the presence of three motifs observed in mammals and other bony fishes, including thrombospondin type-1 (TSP1) repeat, MAC/perforin (MACPF) domain, and LDL-receptor class A (LDLRA) domain [20]. A signal peptide was predicted between the positions 1 and 35, and a cleavage site was found between the positions 35 and 36 (Fig. 2). The deduced amino acid of the C8b in Nile tilapia showed moderate to high sequence identity with Paralichthys olivaceus (79%, BAA86877), O. mykiss (70%, AF418597), D. rerio (58%, NP_001243652), S. scrofa (51%, NP_001090920), Bos taurus (50%, NP_001039606), Oryctolagus cuniculus (50%, NP_001076137), H. sapiens (50%, NP_000057), Rattus norvegicus (49%, NP_001178688), M. musculus (48%, NP_598643), Chimaera phantasma (47%, BAI47541), and Heterocephalus glaber (47%, EHA99005). 3.2. Tissue distribution of C1INH and C8b expressions The expression of the C1INH and C8b genes in the eight tissues (gill, heart, brain, kidney, liver, intestine, spleen, and dorsal muscle) indicated the highest expression level in the liver (P < 0.01; Fig. 3). The expression characteristics of C1INH were consistent with that observed in rainbow trout [21], but were different from large croaker, in which the expression in the liver was less than that in the spleen, kidney, brain, and heart [22]. In Nile tilapia, C8b was expressed in the eight tissues studied using qPCR method, which was different from the finding in rainbow trout using northern blot method, this phenomenon maybe be thanks to the low resolution of northern blot [21,23].
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