cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda.

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.


Introduction
Reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide anion and singlet oxygen, are thought to be involved in cancer, aging and various inflammatory disorders [1]. In addition, these ROSs can kill foreign invaders efficiently and also play an important role in immune signal transduction [2,3]. However, the mass accumulation of ROS in animals will cause serious cell damage, resulting in various diseases [4][5][6]. To protect themselves against damages of ROS, aerobic organisms have developed a set of antioxidant defense systems, including antioxidant enzymes such as superoxide dismutase, catalase and many kinds of peroxidases [7][8][9].
The ridgetail white prawn E. carinicauda is an important economical shrimp species naturally distributed in the coasts of Yellow Sea and Bohai Sea, China [24], which contributes to one-third of the gross outcome of the polyculture ponds in eastern China [25,26]. However, various diseases caused by bacteria and viruses have blossomed within booming E. carinicauda cultures, causing economic losses to commercial shrimp aquaculture [26]. Better understanding of the innate immune abilities and immune defense mechanisms of shrimp will be beneficial to the development of health management and disease control in shrimp aquaculture. The aim of this study was to clone the full-length cDNA of Prx5 from hemocytes of E. carinicauda, compare its sequence with other known Prx5s from other animals, investigate the expression pattern of EcPrx5 in various tissues, and evaluate its expression in E. carinicauda with Vibrio anguillarum and white spot syndrome virus (WSSV) challenge.

Animal materials
Healthy adult E. carinicauda, averaging weight 1.19 ± 0.32 g, were collected from a commercial farm in Qingdao, China. They were cultured in filtered aerated seawater (salinity 20 %, pH 8.2) at 18 ± 0.5°C for 7 days before processing. There were 30 shrimps in each group. The shrimps were fed daily with a ration of 10 % of body weight, and two-thirds of the water in each group was renewed once daily.

RNA extraction and cDNA synthesis
Hemocytes were collected with syringe contained an equal volume of anti-coagulant buffer [27], and centrifuged at 800 g, 4°C for 15 min. Total RNA was extracted from hemocytes using Trizol Reagent (Invitrogen, USA) according to the manufacturer's instruction. The RNA samples were analyzed in 1.0 % agarose electrophoresis and quantitated at 260 nm, all OD 260 /OD 280 were between 1.8 and 2.0. The 3 0 and 5 0 ends RACE cDNA template were synthesized using SMART TM cDNA Kit (Clontech, USA) following the protocol of the manufacturer.
Cloning the full-length cDNA of EcPrx5 An EST sequences was found in large scale EST sequencing from hemocytes cDNA library of the ridgetail white prawn E. carinicauda (GenBank accession no. JK996159), which was constructed using the SMART cDNA library construction kit (Clontech, USA) and have been reported by Duan et al. [24]. Blast analysis showed that they have high similarities with Prx5. According to the EST sequence, a gene specific primer F1 was designed for 3 0 RACE, and R1 was designed for 5 0 RACE (Table 1).
Based on the partial sequence data of Prx5, its 3 0 and 5 0 ends were obtained using SMART RACE cDNA Amplification Kit (Clontech, USA). For 3 0 RACE, the PCR reaction was performed using the primer F1 and the anchor primer UPM ( Table 1). The PCR reaction systems were 50 lL, including RACE cDNA template 2.5 lL, 10 9 Advantage 2 PCR buffer 5 lL, dNTP Mix (10 lmol/L) 1 lL, 50 9 Advantage 2 Polymerase Mix 1 lL, primer UPM (10 lmol/L) 5 lL, primer F1 (10 lmol/L) 1 lL, PCR-Grade water 34.5 lL. The PCR reaction conditions were 5 cycles of 94°C for 30 s, 72°C for 3 min, 5 cycles of 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min, and 25 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 3 min. For 5 0 RACE, the PCR reaction was performed using the primer R1 and the anchor primer UPM ( Table 1). The PCR reaction systems and conditions were the same as those described above.
The PCR fragments were subjected to electrophoresis on 1.5 % agarose gel to determine length differences, and the target band was purified by PCR purification kit (Promega, USA). The purified products were cloned into PMD18-T vector, following the instructions provided by manufacturer (TaKaRa, Japan). Recombinant bacteria were identified by blue/white screening and confirmed by PCR. Plasmids containing the insert were purified (Promega minipreps) and used as a template for DNA sequencing. The nucleotide and deduced amino acid sequences of EcPrx5 cDNA were analyzed and compared using the BLAST search programs (http://www.blast.ncbi.nlm.nih. gov/Blast.cgi). The multiple sequence alignment of Prx5 amino acid sequences was performed using the programs of Vector NTI advance 10.3 (Invitrogen). A phylogenetic NJ tree of Prxs was constructed by the MEGA 4.0 software [28].

Tissue expression of EcPrx5
Hemocytes, gill, hepatopancreas, muscle, ovary, eyestalk, stomach and bowel were dissected from unchallenged E. carinicauda. The mRNA expressions of EcPrx5 in different tissues were determined by quantitative real-time RT-PCR. Total RNA was extracted as described above.
The RNA samples were analyzed in 1.0 % agarose electrophoresis and quantitated at 260 nm, all OD 260 /OD 280 were between 1.8 and 2.0. Total RNA (5 lg) was reverse transcribed using the PrimeScript TM Real time PCR Kit (TaKaRa, Japan) for real-time quantitative RT-PCR analysis.

Experimental design of V. anguillarum and WSSV challenge
The experiments were divided into the bacterial challenged group, the virus challenged group and the control group. V. anguillarum strains was obtained from Germplasm Resources and Genetic Breeding Laboratory, Yellow Sea Fisheries Research Institute, activating on marine agar 2611E. WSSV crude extract were obtained from 10 grams of WSSV-infected tissue from Litopenaeus vannamei, which provided from Mariculture Disease Control and Pathogenic Molecular Biology Laboratory, Yellow Sea Fisheries Research Institute, the methods referred to Li et al. [29]. In the experiment, the challenged groups were injected individually with 20 lL live V. anguillarum suspended in 0.9 % normal saline (2 9 10 8 CFU/mL) or 20 lL WSSV crude extract, the control group received individually an injection of 20 lL sterile 0.9 % saline solution. Hemocytes and hepatopancreas of six shrimps from each treatment (the challenged group and the control group) were randomly sampled at 0, 3, 6, 12, 24, 48 and 72 h post-injection respectively, then the samples were snap-frozen in liquid nitrogen. There were three replicates for each time point. Total RNA was extracted and the first strand cDNA was synthesized as described above. RT-PCR data from three replicate samples were analyzed with the ABI 7300 system SDS Software (Applied Biosystems, USA), for estimating transcript copy numbers for each sample. The comparative C T method was to analyze the relative expression level of EcPrx5. The C T for the target amplified products of EcPrx5 and internal control 18S were determined for each sample. Difference in the C T between the target and the internal control, called 4C T , was calculated to normalize the differences in the amount of template and the efficiency of the RT-PCR. In the same challenge time, the 4C T of the control group was used as the calibrator, and the difference between the 4C T of the challenged group and the control group was called 44C T . The expression level of EcPrx5 was calculated by the 2 ÀMM C T comparative C T method [30].
Statistical analysis was performed using SPSS software (Ver 11.0). Statistical significance was determined using one-way ANOVA [20] and post hoc Duncan multiple range tests. Significance was set at P \ 0.05.

Sequence characterization of EcPrx5
The full-length EcPrx5 cDNA of E. carinicauda was obtained by RACE, and the results are shown in Fig. 1   Peroxiredoxins signatures VPGAFTPGCSKTHLPG and DGTGLTCSL were marked by frame and the two highly conserved amino acids were indicated by solid lines (53 %), Bos taurus (52 %), Homo sapiens (51 %), and so on.
Multiple sequence alignment revealed that the cysteine residue (Cys 83 and Cys 184 ) was conserved in all the analyzed Prx5 s. In addition, Cys 83 and Cys 184 , positioned within VPGAFTPGCSKTHLPG and DGTGLTCSL respectively, were deduced to form the intramolecular disulfide bond. No signal peptide was identified by the signalP program (Fig. 2).
A molecular phylogenetic tree was constructed to further analyze the evolutionary relationships among animal Prx sequences (Fig. 3). Base on MEGA 4.0 analysis, the Prxs were identified with three distinct clades, 1-Cys, 2-Cys, and atypical 2-Cys. All the Prx5s formed the atypical 2-Cys subgroup, and all the Prx6s were clustered together and formed a sister group with Prx5 s. Prx1, Prx2, Prx3 and Prx4 clustered into a branch and constituted to 2-Cys subgroup. EcPrx5 was clustered into atypical 2-Cys subgroup. In the Prx5s subgroup, the sequences from vertebrate diverged from invertebrate, and EcPrx5 was placed in the invertebrate branch with other arthropods.

Tissue expression of EcPrx5
Quantitative real-time RT-PCR was employed to investigate the distribution of EcPrx5 mRNA in different tissues. The mRNA transcripts of EcPrx5 could be detected in all the examined tissues with different expression levels including hemocytes, gill, hepatopancreas, muscle, ovary, intestine, stomach and eyestalk (Fig. 4). The highest expression was found in hepatopancreas, and the lowest was in eyestalk.

EcPrx5 gene expression after V. anguillarum and WSSV challenge
The mRNA expression levels of EcPrx5 in hemocytes and hepatopancreas of E. carinicauda after V. anguillarum and WSSV challenge were quantified by real-time RT-PCR with 18S gene as internal control. For both EcPrx5 and 18S genes, there were only one peak at the corresponding melting temperature in the dissociation curve analysis, indicating that the PCR was specifically amplified.
The expression levels of EcPrx5 in ridgetail white prawn hemocytes after V. anguillarum and WSSV challenge were shown in Fig. 5. Compared to the control, the expression of EcPrx5 in V. anguillarum and WSSV challenged group increased significantly and reached to the maximum at the first 12 and 6 h after challenge respectively, which was 1.78 and 1.52-fold respectively of that in the control group (P \ 0.05). Afterwards, the expression of EcPrx5 in the two groups decreased gradually, and dropped to the lowest level at 48 h after challenge, which was only 0.22 and 0.08-fold respectively of that in the control group (P \ 0.05), then recovered to 0.72 and 0.42-fold respectively of the control at 72 h after challenge. Expression profiles of EcPrx5 in hepatopancreas after V. anguillarum and WSSV challenge was shown in Fig. 6. Compared to the control, the EcPrx5 mRNA expression levels of V. anguillarum and WSSV challenged groups increased significantly and reached the highest at 6 h after challenge, which was 1.71 and 1.51-fold respectively of that in the control group (P \ 0.05). After 6 h challenge, EcPrx5 mRNA expression levels in V. anguillarum challenged group decreased gradually and reached to a low level at 12 h after challenge (0.54-fold of that in control group, P \ 0.05), then recovered to 0.71-fold of the control at 72 h after challenge. However, the EcPrx5 mRNA expression levels of WSSV challenged group decreased and reached the lowest at 48 h after challenge (0.63-fold of that in control group, P \ 0.05), then rised gradually and recovered to 1.14-fold of the control at 72 h after challenge.

Discussion
The peroxiredoxin family is an evolutionarily conserved group of antioxidants that protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides [31]. In the present study, a novel Prx gene (EcPrx5) was cloned from E. carinicauda. Prior to this study, no full-length cDNA of Prx5 has been isolated in E. carinicauda, and our study is the first report on cloning of the full-length cDNA of Prx5 in E. carinicauda. Homology analysis revealed that the deduced amino acid sequence of EcPrx5 had more than 50 % similarity with Prx5 of other animals (89 % with M. rosenbergii, 68 % with M. rotundata, 66 % with H. saltator, 65 % with A. echinatior, 53 % with D. rerio and 51 % with H. sapiens). Phylogenetic analysis showed a closer relationship of Ec-Prx5 with other animals, indicating that the EcPrx5 gene belonged to the atypical 2-Cys Prx subgroup. Multiple sequence alignment analysis revealed that two cysteines (Cys 83 and Cys 184 ) are highly conserved in all of the Prx5 investigated, which located in the Prx signature motifs, and critical for enzyme function serving as a catalytic site and a resolving residue [20]. Compared to Prx5 s of other animals, the Prx signature motifs VPGAFTPGCSKTHLPG appeared to be well conserved, while the motifs DGTGLTCSL had obvious difference. At position I, threonine was replaced by lysine in E. carinicauda, and  Quantitative real-time RT-PCR revealed that EcPrx5 was expressed in all the tested tissues, and the highest expression level occurred in the hepatopancreas. As reported by Maningas et al. [32], the differential expression of Prx in various shrimp tissues shows that it is an important molecule that could effectively be involved in a number of physiological activities. Therefore, the EcPrx5 expressed in different tissues of E. carinicauda indicated that it could potentially be involved in different physiological process, such as ROS clearance [33,34], cell differentiation [35,36], proliferation [37], apoptosis [38], signal transduction [39], immune response [20,21], and so on.
Various diseases, which mostly caused by bacteria and viruses, have affected the commercial shrimp aquaculture. Prx have been proposed to play a part in the physiological oxidative stress response to bacterial and viral infections in arthropods. Information about the expression profile of Prx5 after bacterial and viral challenge would be helpful in understanding its biological function. V. anguillarum and WSSV are both the extremely virulent pathogen prevalent causing mass mortalities and economic losses in shrimp aquaculture [40][41][42]. When pathogens enter into the body of the shrimp, they will encounter the innate immune systems [43] and ROS are released by oxidative stress in response to them. In the present study, live V. anguillarum and WSSV were chosen for challenging the shrimp, so that the shrimp health condition could be affected severely by the production of V. anguillarum and WSSV. The level of Prx5 transcripts in hemocytes of Argopecten irradians was up-regulated and appeared to be time-dependent after V. anguillarum challenged, which indicated that Prx5 is a inducible protein that plays an important role in the immune response against bacterial infection [20]. It reported that in addition to the general antioxidant role of Prx, it may also be associated with immune responses, where Prx could serve to remove ROS [44]. So, the provoked increase of Prx5 expression level was found as one of protection approaches of organisms from further damage.
In our study, EcPrx5 expression in E. carinicauda hemocytes and hepatopancreas was up-regulated at earlier time and then decreased gradually with V. anguillarum and WSSV challenge. The results showed that EcPrx5 might be involved in a transient systemic immune response to the V. anguillarum and WSSV stimulation. Prx5 gene expression was significantly up-regulated until 12 h p.i in gills and then down-regulated in the following p.i. time points at 24 and 48 h in IHHNV infected M. rosenbergii [16], which were consistent with our results. After V. anguillarum and WSSV challenged 6 h, the transcripts level of EcPrx5 in hemocytes and hepatopancreas of both the challenged group were significant higher than that in the control group, it can be deduced that the challenged shrimps would generate a mass of ROS which need to be eliminated by extra Prx translated from more Prx transcripts. As time progressed, the expression of EcPrx5 in hemocytes dropped to a low level at 24 and 48 h in both V. anguillarum and WSSV challenged group, which might because of the infection progress brought more bacterias and virus, and destroyed severely to the normal function of shrimp's cells and finally caused that the expression of EcPrx5 in the challenged group decreased gradually [7]. EcPrx5 showed a different expression profile in hemocytes and hepatopancreas after V. anguillarum and WSSV challenge, this might be caused by the different function of hemocytes and hepatopancreas in the immune defense system. It has been proved that hemocytes are key cells for invertebrate's innate defense reactions [45,46] and play an important role in the host immune functions when the organism is attacked by bacterias or viruses [47,48]. This study showed that EcPrx5 may serve to decrease the cellular damage caused by V. anguillarum and WSSV, and the knowledge on this gene expression studies can provide useful tools in understanding and quantifying how these organisms respond to various biotic environmental stress.
In conclusion, a novel Prx cDNA (EcPrx5) was cloned from E. carinicauda, and it constitutively expressed in the tissues of hemocytes, gill, hepatopancreas, mucle, ovary, intestine, stomach and eyestalk. The rapid and dynamic expression profiles in hemocytes and hepatopancreas challenged with V. anguillarum and WSSV indicated that EcPrx5 was perhaps involved in the immune response against bacterial and viral infection. However, further work is required to better understand the regulation of antioxidant enzymes under oxidative stresses.