A manganese superoxide dismutase in blood clam Tegillarca granosa: Molecular cloning, tissue distribution and expression analysis

Comparative Biochemistry and Physiology, Part B 159 (2011) 64–70

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

A manganese superoxide dismutase in blood clam Tegillarca granosa: Molecular cloning, tissue distribution and expression analysis Chenghua Li a,⁎, Jingjing He a, Xiurong Su a, Taiwu Li a,b a b

Faculty of Life Science and Biotechnology, Ningbo University, Fenghua Road, Ningbo, Zhejiang Province 315211, PR China Ningbo City College of Vocational Technology, Ningbo, 315100 PR China

a r t i c l e

i n f o

Article history: Received 20 December 2010 Received in revised form 16 February 2011 Accepted 17 February 2011 Available online 23 February 2011 Keywords: Tegillarca granosa mMn-SOD Heavy metal Western blot

a b s t r a c t Superoxide dismutase (SOD, EC 1.15.1.1) is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by transforming O− 2 into hydrogen peroxide and oxygen. The full-length mitochondrial Mn-SOD cDNA of blood clam Tegillarca granosa (denoted as TgmMnSOD) was identified from haemocytes by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of TgmMnSOD consisted of 1106 bp with a 5′ UTR of 195 bp, a 3′ UTR of 227 bp with a candidate polyadenylation signal sequence ATTAAA and a short polyA tail, and an open reading frame (ORF) of 648 bp encoding a secreted polypeptide of 227 amino acids residues. The deduced amino acid sequence of TgmMnSOD shared significant homology to mMnSODs from other species, indicating that TgmMnSOD should be a novel member of the mMnSOD family. Several highly conserved motifs including three mMnSOD signatures, amino acid residues responsible for coordinating the manganese and the putative active center were almost completely conserved in the deduced amino acid of TgmMnSOD. The mRNA expression of TgmMnSOD in the tissues of mantle, foot, gill, haemocytes and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of TgmMnSOD were mainly detected in hepatopancreas, haemocytes, and gill and weakly detected in the mantle and foot. The temporal expression of TgmMnSOD in haemocytes after heavy metal exposure revealed that TgmMnSOD could be induced by the three pollutants with different response profiles. The polyclonal antibodies generated from the recombinant product of TgmMnSOD could specifically identify not only the recombinant product, but also the native protein from haemocytes. The present results strongly suggested that TgmMnSOD was a cute response protein involved in marine heavy metal contaminants challenge in T. granosa. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Generation of reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) is an unavoidable consequence in most aerobic organisms (Chiu et al., 2007; An et al., 2008). ROS are not only agents of disease and cellular damage, but also second messengers involved in many normal cellular function (Schrek and Baeuerle, 1991). In order to balance the harmful and positive effect of ROS production, organisms evolved to use antioxidant systems to maintain oxygen radicals at fitting concentrations (Arenas-Ríos et al., 2007; Park et al., 2004). Superoxide dismutase (SOD, EC 1.15.1.1) is one member of this antioxidant enzyme family that catalyzes the dismutation reaction of O− 2 and transforming it into hydrogen peroxide and oxygen (Vaughan, 1997). It is originally discovered by McCord and Fridovich (1969) and occurs as different metalloproteins with different cellular distributions. Since then,

⁎ Corresponding author. E-mail address: [email protected] (C. Li). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.02.003

many different types of SODs were reported from a wide range of organisms, mainly including cytosolic Cu/Zn-SOD, Mn-SOD, Fe-SOD and Ni-SOD (Bannister et al., 1987; Kim et al., 1998). Mn-SOD containing manganese ion was found mostly in the mitochondrial matrix (mMn-SOD) (Weisiger and Fridovich, 1973), but some cytosolic Mn-SOD (cMn-SOD) was also reported (Brouwer et al., 2003). Recently, much attention had been paid to Mn-SOD because it was thought to be a major scavenger of damaging ROS metabolites in the mitochondrial matrix. More importantly, the protein was also demonstrated to play an important role in promoting cellular differentiation and tumorigenesis (St Clair et al., 1994), in immune response induced by bacteria (Jung et al., 2005; Cheng et al., 2006), virus infection (Zhang et al., 2007a,b) or toxic chemical exposure (Kim et al., 2007). Several cellular pathologies including cancer (Oberley and Buettner, 1979), ischemia/reperfusion (I/R) injury (Gupta et al., 1997) and cell apoptosis (Keller et al., 1998) had also been shown to correlate with a decrease in Mn-SOD activity. Although mMn-SOD has been identified from some bacteria, trematode (Clonorchis sinensis), abalone (Haliotis discus discus), oyster (Crassostrea gigas), silkworm (Bombyx mori), and Chinese shrimp

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sequences of mitochondrial Mn-SOD. The RNA extraction, cDNA synthesis, PCR amplification and PCR product sequencing were performed according to Li et al. (2010). Two specific primers, sense primers P3: 5′-CCCTTATTTGGCATTGACG-3′, and reverse primers P4 5′-ATCCAGAACCCTGAATAGCAAC-3′, were designed based on the fragment from homology cloning. The 3′ and 5′ RACE were carried out according to Zhang et al. (2007a,b). The PCR products were cloned into the pMD18-T simple vector (TaKaRa) and sequenced bidirectionally with primers M13-47 and RV-M. The sequencing results were verified and subjected to cluster analysis. 2.3. Sequence analysis of TgmMnSOD The TgmMnSOD gene sequence was analyzed using the BLAST algorithm at NCBI web site (http://www.ncbi.nlm.nih.gov/blast), and the deduced amino acid sequence was analyzed with the Expert Protein Analysis System (http://www.expasy.org/). The SignalP 3.0 (http:// www.cbs.dtu.dk/services/SignalP/) was employed to predict the signal sequence of TgmMnSOD. Multiple alignment of TgmMnSOD was performed with the ClustalW Multiple Alignment program (http:// www.ebi.ac.uk/clustalw/) and Multiple Alignment show program (http://www.biosoft.net/sms/index.html). 2.4. Phylogenetic analysis

Fig. 1. The complete cDNA sequence of mMnSOD from Tegillarca granosa and its deduced amino acid sequence. Nucleotides were numbered from the first base at the 5′ end. The asterisk indicated the stop codon. Potential polyadenylation signal sequence is in italics. The predicted signal peptide is underlined. Amino acid residues involved in metal binding are boxed. Three putative manganese superoxide dismutase signatures are underlined (dotted).

(Fenneropenaeus chinensis) (Henkle-Duhrsen et al. 1995; Hunter et al. 1997; Li et al. 2005; Ekanayake et al., 2006; Yue et al. 2006; Zhang et al., 2007a,b; Parka et al., 2009), the studies regarding molecular features and function of mMn-SOD from commercial mollusk Tegillarca granosa were rarely investigated to our knowledge. The main objectives of the present study were: 1) to clone the full-length cDNA of mMn-SOD from T. granosa (denoted as TgmMnSOD); 2) to investigate the tissues expression profile of TgmMnSOD; 3) to examine the temporal expression profile of TgmMnSOD transcript in haemocytes after three heavy metal exposure; and 4) to generate the polyclonal antibody and detect the native protein in haemocytes by western blot. 2. Materials and methods 2.1. Animal and materials T. granosa were collected from Yueqing, Wenzhou city, Zhejiang province, China, and maintained in the laboratory for a week at 20 ± 1 °C before processing. For heavy metal challenge experiment, thirty blood clams cultured in seawater were selected as control group. Ninety blood clams were divided into three tanks and treated by adding 0.5 M stock solutions of CuCl2, CdCl2 and ZnCl2 with final concentration of 10 mM. After 6, 12, 24 and 48 h exposure, the haemocytes were collected from the control and the treated groups for RNA extraction and cDNA synthesis. There were four replicates for each time point. 2.2. Cloning the full-length cDNA of TgmMnSOD Degenerate primers P1: 5′-TTYAAYGGNGGNGGICAYAT-3′ and P2: 5′-GCRTGYTCCCANACRT-3′ were designed based on the conserved

The deduced amino acid sequences of TgmMnSOD were used for phylogenic analysis. A NJ tree was constructed with Mega3.1 software package (http://www.megasoftware.net/) (Kumar et al., 2004) and Clustal X (1.81). To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. 2.5. Tissue-specific expression of TgmMnSOD mRNA transcripts The mRNA expression of TgmMnSOD in different tissues of healthy blood clams was measured by quantitative real-time PCR in RotorGeneTM 6000 real-time PCR detection system. Two TgmMnSOD gene specific primers P5: 5′-TCCAAATAGCTTCCGTTTACGTG-3′ and P6: 5′CACTGTTTAGCAGCAGCAAGTC-3′ were designed to amplify a product of 209 bp. For normalizing the TgmMnSOD gene transcripts, T. granosa βactin gene was amplified as an internal control. Based on our preliminary study, two conserved primers, P7: 5′-GCCGCTTCTTCATCCTCAT-3′ and P8: 5′-GTCGGCAATACCTGGGAAC-3′ were used to amplify a 246 bp fragment. Total RNA was isolated from foot, mantle, haemocytes, hepatopancreas and gill. The first-strand cDNA was synthesized based on Promega M-MLV RT Usage information (Promega) using total RNA treated with DNase Ι (Promega) as template. cDNA mix was diluted to 1:50 for subsequent experiment. There were three replicates for each tissue. The data were then subjected to analysis by one-way analysis of variance (ANOVA). Differences were considered significant at P b 0.05 and extremely significant at P b 0.01. 2.6. Temporal expression of TgmMnSOD transcripts after heavy metal exposure Haemocytes were selected to investigate the temporal expression of TgmMnSOD transcripts after heavy metal exposure by qT-PCR. The PCR was performed in a total volume of 20 μL, containing 10 μL of 2× SYBR Green Master Mix (Applied Biosystems), 4 μL of the diluted cDNA mix, 1 μL of each of the primers (P5 and P6 for TgmMnSOD or P7 and P8 for β-actin), and 4 μL of DEPC-water. The thermal profile for qPCR was 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. In a 96-well plate, each sample was run in four replicates along with the internal control gene. Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. To maintain consistency, the baseline

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Shrimp-mMn Crab-mMn Scallop-mMn Clam-mMn 53 Abalone-mMn Snail-mMn 100 Prawns-cMn White-shrimp-cMn 99 100 Tiger-prawns-cMn Clam-Cu/Zn Scallop-Cu/Zn Mussel-Cu/Zn 61 77 Oyster-Cu/Zn 100

was set automatically by the software. The comparative Ct method was used to analyze the relative expression level of TgmMnSOD. The Ct for the target amplified TgmMnSOD and the Ct for the internal control β-actin were determined for each sample. Difference in the Ct for the target and the internal control, called ΔCt, was calculated to normalize the differences in the amount of template and the efficiency of the RT-PCR. The ΔCt value for untreated sample was used as the reference sample, called the calibrator. The ΔCt for each sample was subtracted from the ΔCt of the calibrator; the difference was called ΔΔCt. The expression level of TgmMnSOD could be calculated by 2− ΔΔCt, and the value stood for an n-fold difference relative to the calibrator. All data were given in terms of relative mRNA expression as mean ± SD. The results were subjected to analysis by one-way analysis of variance (ANOVA). 2.7. Expression and purification of recombinant protein of TgmMnSOD The strategy for generation recombinant protein in vitro was according to our previous work (Wang et al., 2010). PCR fragment encoding the mature peptide of TgmMnSOD was amplified with genespecific primers introduced with BamH I and Hind III sites at their 5′ end. The PCR product was cloned into the BamH I/Hind III digested pET-28a(+) vector (Novagen). The recombinant plasmid was transformed into Escherichia coli BL21(DE3) (Novagen) and subjected to DNA sequencing. After sequencing to ensure in-frame insertion, positive clones were incubated in SOB medium (containing 25 mg/L kanamycin) at 37 °C with shaking at 220 rpm. When the culture reached OD600 of 0.6, IPTG with final concentration of 1 mmol/L was added to the culture, and incubated for additional 1 h, 2 h, 3 h, 4 h and 5 h under the same conditions. Cells were harvested by centrifugation at 10,000 g for 2 min, and suspended in 50 mM Tris containing 5 mM EDTA, 50 mM NaCl, and 5% glycerol (pH 7.9). After being sonicated at 4 °C for 60 min, the rTgmMnSOD was purified by HisTrap Chelating Columns (Amersham Biosciences) according to the manufacturer's instruction. The purified protein was subjected to 15% SDS-PAGE according to the method of Laemmli (1970). After washing the PAGE in 3 M KCl solution for 3–5 min, the target protein band was excised from the gel, grinded into small pieces and dissolved in PBS for antibody preparation. 2.8. Preparation of polyclonal antibodies The purified rTgmMnSOD (~100 μg) was injected subcutaneously to immunize Balb/c mice mixed with an equal volume of complete Freund's adjuvant, followed by two booster injections in incomplete Freund's adjuvant. After the last injection, blood samples were taken from the marginal vein of the rabbit ear and centrifuged at 10,000 g for 10 min before clotting at 4 °C overnight. The antisera were stored at − 20 °C. At the same time, the negative control group was immunized with PBS as antigen using the same method. 2.9. Western blot analysis The protein extraction from E. coli BL21 (DE3) strain and clam haemocytes were boiled for 10 min and isolated by 12% SDS-PAGE. The gel was then blotted onto a sheet of nitrocellulose transfer membrane by electrophoresis at 20 V for 14 h. After blotting, the membrane was blocked by incubation in TBST (10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 0.05% (w/v) Tween 20) containing 5% skimmed milk for 4 h at room temperature. Subsequently, the membrane was incubated with above mouse polyclonal antibody by 1:5000 dilutions

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23 29 29

0.2 Fig. 3. Consensus neighbor-joining tree based on the sequences of different types of SODs. The numbers at the forks indicate the bootstrap. The detail information for the used sequences were as follows: scallop-mMn (ABW98672), abalone-mMn (ABF67504), snail-mMn (AAS83980), shrimp-mMn (ABB05539), crab-mMn (AAF7477), white shrimp-cMn (AAY57407), prawn-cMn (AAY79405), tiger prawncMn (AAW50395), oyster-Cu/Zn (CAD42722), mussel-Cu/Zn (CAE46443), clam-Cu/Zn (ACU83236), and scallop-Cu/Zn (ABD58974).

in the block buffer for 2 h. The membrane was washed extensively before secondary antibody (1:10000) was added and incubated for 1 h at 37 °C. After washing three times, signal was then visualized with BCIP/NBT for 1–3 min and stopped by rinsing strips with distilled water.

3. Results and discussion 3.1. Cloning and analysis of full-length cDNA of TgmMnSOD A product around 300 bp was amplified with degenerated primers P1 and P2. Blastx analysis indicated the fragment was similar to mMnSOD from scallop (BAE78580). Based on the fragment, two genespecific primers (P3 and P4) were designed to clone the full-length cDNA of TgmMnSOD. A 371 bp fragment was produced by 3′ RACE with primer P3 and oligodT. In 5′ RACE reactions, the product was of 643 bp with primer P4 and oligodG. By overlapping the two fragments with the fragment obtained from homology cloning, an 1106 bp nucleotide sequence representing the full-length cDNA of TgmMnSOD was assembled and deposited in GenBank under accession no. GU078656. The complete nucleotide and the deduced amino acid sequence were shown in Fig. 1. The cDNA sequence of TgmMnSOD contained a 684 bp open reading frame (ORF), flanked by a 195 bp 5′-terminal untranslated region (UTR) with two stop codons TAA preceding the initial methionine and a 227 bp 3′ UTR with a shorter polyA tail. Canonical polyadenylation signal sequence AATAAA was instead by ATTAAA in TgmMnSOD (Fig.1). In the deduced amino acid of TgmMnSOD, a putative signal peptide of 19 amino acids was identified by SignalP program. The existence of signal peptide was a strong proof that TgmMnSOD should be located in mitochondrial matrix, which was also validated by mitochondrial targeting sequence analysis with MITOPROT online software (http:// ihg.gsf.de/ihg/mitoprot.html). Some conserved motifs were also identified in TgmMnSOD by multiple alignments (Fig. 2). Three mMnSOD signatures from 92aa to 100aa (FNGGGHINH), from 144aa to 153aa (IQGSGWGWLG), and from 185aa to 192aa (DVWEHAYY) were almost

Fig. 2. Multiple alignments of TgmMnSOD with other known mMnSODs. Amino acid residues that are conserved in at least 80% of sequences are shaded in black, and similar amino acids are shaded in gray. The species and the GenBank accession numbers are as follows: Argopecten irradians (ABW98672), Haliotis discus discus (ABF67504), Biomphalaria glabrata (AAS83980), Macrobrachium rosenbergii (AAZ81617), Fenneropenaeus chinensis (ABB05539), Scylla paramamosain (AAF7477), Drosophila melanogaster (NP_476925), Tatumella ptyseos (AAQ14590), Homo sapiens (P04179), and Danio rerio (NP_956270).

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Relative expression level of TgmMnSOD

45

**

40 35 30 25 20 15

**

**

10 5 0

Foot

Mantle

Gill Tissues

Haemocytes

Hepatopancreas

Fig. 4. Tissue distribution of TgmMnSOD transcripts measured by quantitative real-time PCR. Each symbol and vertical bar represent the mean ± SD (n = 3). Significant differences between other tissues and foot are indicated by an asterisk at P b 0.05 and two asterisks at P b 0.01.

completely conserved in the deduced amino acid of TgmMnSOD. The only mutation occurred in the second signature with synonymous substitution Val by Ile. Four amino acid residues responsible for coordinating the manganese were also identified in the deduced amino acid of TgmMnSOD, although inconsistent results had been reported in a wide range of organisms (Ken et al., 2005; Cheng et al., 2006; Zhang et al., 2010, 2011; Bao et al., 2008). In our study, all the candidate five amino acids potentially involved in manganese binding were totally conserved (His-29, His-57, His-101, Asp-186 and His-190). In addition, the putative active center involving 11 residues in bacteria mMn-SOD (H-27, H-31, Y-35, H-81, F-84, W-85, W-128, Q-146, D-167, W-169, and H-171) was also conserved in TgmMnSOD (Fig. 2) (Ken et al., 2005). Further work should be performed to validate the exact amino acid residues for metal binding. All these conserved characteristics together supported that TgmMnSOD was a novel member of mMnSOD family. 3.2. Phylogenentic analysis The evolutionary tree for different types of SODs was constructed based on protein sequences of some invertebrate SODs (Fig. 3). In the phylogenetic tree, mMn-type, cMn-type and Cu/Zn-type SOD were clustered independently and formed three sister groups. TgmMnSOD was identified in mMn-type SOD group, further indicating that the identity of TgmMnSOD belonged to the mMn-type SOD family. It was highly consistent with the fact that the significant difference between mMn-type and cMn-type was the existence of the mitochondrial transit peptide or not at the sequence level. Among this group, two crustacean mMn-type SODs clustered together firstly, then with other mollusk species. The order of cluster was in agreement with the

Relative expression level of TgmMnSOD

5

phylogeny of organism. According to the phylogenetic tree, mMntype SODs were more closely related to the cMn-type rather than to the Cu/Zn-type SODs. It was suggested that mMn-type and cMn-type might be diverged from a common ancestor, consistent with some researchers' opinions (Zelko et al., 2002).

3.3. Spatial-course expression of TgmMnSOD mRNA in different tissues To examine the tissue distribution profile of TgmMnSOD, total RNA from the tissues of mantle, foot, gill, haemocytes and hepatopancreas was extracted from unchallenged T. granosa. The amplification specificity for TgmMnSOD and β-actin were determined by analyzing the dissociation curves of PCR products. Only one peak presented in the dissociation curves for both TgmMnSOD and β-actin gene, indicating that the amplifications were specific. The result was shown in Fig. 4. The TgmMnSOD transcript could be detectable in all the examined tissues, which was highly consistent with mMn-SOD expression profile in other animals (Cheng et al., 2006; Cho et al., 2006; Zhang et al., 2007a,b; Zhang et al., 2011). Concerning the expression level of different tissues, TgmMnSOD transcript was expressed in the hepatopancreas the highest followed by haemocytes and gill, while there seemed to be a lower expression level in the mantle and foot. However, different tissue-specific expression patterns were also found in other animals. In silver carp, mMn-SOD was differently higher expressed in gill, spleen and liver (Zhang et al., 2011), While in the shrimp, the highest expression level was detected in the hepatopancreas (Zhang et al., 2007a,b). The variable expression level of mMnSOD was speculated to be related with tissue-dependent mitochondrial content and oxidative load, since this antioxidant

Zn2+ Cu2+ Cd2+

4.5 4 3.5 3

**

*

**

2.5

**

2 1.5 1

**

0.5 0h

6h

12 h

** 24 h

48 h

Time elapsed Fig. 5. Time-course expression level of TgmMnSOD transcript in haemocytes after three heavy metal exposures measured at 0, 6, 12, 24, and 48 h. Each symbol and vertical bar represent the mean ± SD (n = 4). Significant differences across control are indicated with an asterisk at P b 0.05 and two asterisks at P b 0.01.

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Fig. 6. SDS-PAGE analysis of recombinant TgmMnSOD (A) and the purified rTgmMnSOD (B). After electrophoresis, the gel was visualized by Coomassie brilliant blue R250 staining. Lane 1: protein molecular standard; lane 2, 3, and 4: negative control for rTgmMnSOD (without induction); lane 5: induced expression for 1 h of rTgmMnSOD; lane 6: induced expression for 2 h of rTgmMnSOD; lane 7: induced expression for 3 h of rTgmMnSOD; lane 8: induced expression for 4 h of rTgmMnSOD; and lane 9: induced expression for 5 h of rTgmMnSOD.

enzyme is a principal scavenger of ROS generated in mitochondrial respiration (Benard et al., 2006; Cho et al., 2009). 3.4. mRNA expression of TgmMnSOD after heavy metal stress As a stress protein, mMn-SOD was expressed in response to a wide range of stressors, among them environmental contaminants like heavy metals were a major one. Heavy metals were responsible for many toxic effects such as generating abnormal or denatured proteins and causing oxidative stress in various living organisms (Gao et al., 2007). In the present study, all the examined heavy metals (Zn2+, Cu2+ and Cd2+) could induce TgmMnSOD expression in haemocytes of T. granosa. The temporal expression of TgmMnSOD transcript after heavy metal exposure was shown in Fig. 5. At the concentration of Zn2+ at 10 mM, the expression level of TgmMnSOD was obviously up-regulated and reached around 2.42-fold compared with that in the control group at 6 h (P b 0.05). As time progressed, the expression of TgmMnSOD mRNA decreased significantly, and the expression level was only 0.55-fold at 12 h (P b 0.01) compared with that in the control group. After the stage, the expression level was recovered to the original level at 24 h and 48 h. Concerning the same concentration of Cu2+, a different expression profile was detected compared to that of the Zn2+ exposure (Fig. 5). The expression level of TgmMnSOD was slightly up-regulated from 12 h (P b 0.01) to 24 h (P b 0.01). The peak expression level was detected at 24 h with 4.00-fold increase compared to the control group. After that, the expression level was sharply recovered and reached the original level at 48 h. Similar expression profile was also found between Zn2+ and Cd2+ exposure. The exclusive difference occurred at 24 h post cadmium exposure. At this time point, the expression level of TgmMnSOD sharply down-regulated and arrive to 0.20-fold compared with that in the control group (P b 0.01). These different expression trends of TgmMnSOD towards the three heavy metal exposures indicated that blood clams had different tolerance to the different heavy metals. Cu2+ might be the most detrimental pollutants compared to Zn2+ and Cd2+. Similar phenomena had also been observed in clams (Li et al., 2010), scallops (Zhang et al., 2010) and shrimps (Lorenzon et al., 2001). For the three heavy metal exposures, up-regulation of TgmMnSOD expression was all detected in the first 6 h indicating that TgmMnSOD should be related to its roles in protecting the host against oxidative damage caused by pollutant stimulation (Li et al., 2007). With time progressed, the host could differentiate their different hazard effects and adopt different strategies to control the ROS at a fitting level by utilization or control antioxidant enzymes, leading to the different expression profiles compared to different heavy metals. The speculation reminded us that TgmMnSOD was an acute response protein involved in

marine heavy metal contaminant challenge in T. granosa. Whether TgmMnSOD was a candidate biomarker for marine heavy metal contaminant monitoring should depend on examining the dosedependent expression of TgmMnSOD towards different heavy metal pollutants. 3.5. Expression and purification of recombinant protein of TgmMnSOD The recombinant plasmid pET-28a-TgmMnSOD was transformed and expressed in E. coli BL21(DE3). After IPTG induction for 1 h, 2 h, 3 h, 4 h and 5 h, the whole cell lysate was analyzed by SDS-PAGE and a distinct band with a molecular weight of 28.90 kDa were detected (Fig. 6 A), which was further purified to homogeneity by HiTrap Chelating Columns (Fig. 6 B). The molecular mass of the purified product was in good agreement with the predicted molecular mass by SMS software. The intensity of the recombinant protein band is increased with increased time. The peak expression level of recombinant protein was observed at 5 h after IPTG was introduced into the culture (Fig.6 A). 3.6. Western blot analysis with polyclonal antibodies With the purified rTgmMnSOD, polyclonal antibodies were generated as described by Guo et al. (2011). The specificity of the polyclonal antibodies was analyzed by western blot (Fig. 7). The results showed that the mouse antisera could specifically identify not only the recombinant protein, but also the native protein from the haemocytes. No signals were detected in other control samples

Fig. 7. Specificity of mMnSOD polyclonal antibody was determined by western blot. Lane1: protein molecular standard; lane 2, and 3: negative control for rTgmMnSOD; lane 4: rTgmMnSOD; lane 5: total protein extraction from haemocytes of T. granosa; and lane 6: negative serum of the mouse.

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