Most bivalves are sedentary filter feeders that live in estuarine or intertidal regions. They experience a multitude of environmental challenges,such as high temperature and salinity,during their life cycles. While adapting to these environmental changes,marine organisms develop defense systems that protect them from environmental hazards. The heat shock proteins(HSPs),a family of molecular chaperones,are one such defense system, and they express in response to a wide range of biotic and abiotic stressors(Iwama et al., 1998). These proteins can be induced by other stressors in addition to heat; thus,they are commonly called “stress proteins”. HSP genes can be classified into several families based on molecular mass and degrees of homology,including HSP100,HSP90,HSP70,HSP60,HSP47, and small HSPs(sHSPs)(Narberhaus,2002; Franck et al., 2004). Although sHSPs are highly diverse in their primary sequences,they share an evolutionarily conserved C-terminal α-crystallin domain of 80–100 amino acid residues. Moreover,this domain has a high similarity to the vertebrate eye lens protein, and it is frequently flanked by a highly variable N-terminal region and a flexible C-terminal tail(MacRae,2000; Haslbeck,2002; Panasenko et al., 2002; Kappé et al., 2003). The α-crystallin domain is crucial to the formation of small molecular heat shock protein dimers. In addition,a number of studies have confirmed that mutations or deletions in the α-crystallin domain sequence can significantly damage the structure and function of sHSPs(Mehlen et al., 1993; Muchowski et al., 1999; Lentze et al., 2003). Under cellular stress conditions,sHSP bind partially denatured proteins,preventing irreversible protein aggregation(Sun and MacRae, 2005). To date,many sHSP cDNA sequences as well as sHSP mRNAs subjected to a variety of stress conditions have been reported in mollusk species such as the hard clam Meretrix meretrix(Li et al., 2013),the disk abalone Haliotis discus discus(Wan et al., 2012),the razor clam Sinonovacula constricta(Zhang et al., 2013),the bloody clam Tegillarca granosa(Bao et al., 2011) and the bay scallop Argopecten irradians(Zhang et al., 2010).

P. textile is an important bivalve species principally distributed throughout the southeastern coastal areas of China and Southeast Asia; however,frequent changes in temperature may cause it to experience mass mortality in aquaculture(Li et al., 2011). To date,little attention has been paid to exploiting an effective strategy that can efficiently monitor the influence of environmental stress on P. textile . Therefore,it is imperative to study the protective mechanisms of P. textile under different environmental stress conditions. Just as the above studies mentioned,HSP20 expression had a close correlation with outside environment such as high temperature or heavy metal exposure(Wan et al., 2012; Li et al., 2013),but no report about HSP20 expression in P. textile to date. In the present study,we cloned the full-length cDNA encoding PtHSP20 and analyzed its expression under the stress of high temperature for the first time in the heart,digestive gl and ,adductor muscle,gonad,gill and mantle. 2 MATERIAL AND METHOD 2.1 P. textile collection

Approximately 300 P. textile individuals with an average shell length of 44 mm were collected from a commercial market in Xiamen,Fujian,China,in November 2012. They were cultured in a tank(7 m 3)containing s and -filtered seawater(temperature: 19±0.5°C,salinity 20,pH 8.1)for a week prior to performing the experiments. They were fed with 0.8% Tetraselmis chuionce every 24 h. Six tissues,including heart,digestive gl and ,adductor muscle,gonad,gill, and mantle,were collected from this untreated P. textile group. 2.2 High temperature shock

One hundred fifty P. textile individuals were then r and omly divided into two groups,designated as case and control, and placed into separate tanks at 19±0.5°C(control) and 32°C(case). We had conducted a preliminary experiment and found that the limiting high temperature for P. textile was 32°C,so 32°C was selected as the experiment temperature in this study. The case group was gradually returned to the temperature of natural sea water,approximately 19±0.5°C,after 2 h of high temperature stress(32°C). Three individuals were r and omly collected from each group(case and control)at 2,6,12,18,24,36 and 48 h after the high temperature began. No P. textile were died during the experiment. The heart,digestive gl and ,adductor muscle,gonad,gill, and mantle were collected from both the control and case groups. All of the samples were instantly preserved in liquid nitrogen until RNA extraction. 2.3 RNA extraction and first str and cDNA synthesis

The total RNA was extracted from the gill,heart,gonad,digestive gl and ,mantle, and adductor muscle of P. textile using the TRIZOL Reagent(Invitrogen,USA)in accordance with the manufacturer’ s protocol. The total RNA was treated with RNase-free DNase I(Roche,USA)to eliminate the DNA. First-str and cDNA was synthesized from the total RNA by M-MLV reverse transcriptase(Promega,USA)following the manufacturer’s instructions. 2.4 cDNA cloning and sequencing

In order to obtain the HSP20 cDNA from P. textile,four degenerated primers,HSP20-F1,HSP20-R1,HSP20-F2,HSP20-R2(Table 1),were designed using highly conserved sequences of amino acids from known HSP20 genes(Cyclina sinensis,AET13647; Meretrix meretrix,AFK80359; Bactrocera dorsalis,AEJ88464)in the GenBank database. The total RNA was used for the cDNA synthesis, and then cDNA was used as a PCR template for homologous cloning. The cloning of homologous fragments was achieved using the following steps. First,a first-round PCR was performed using the primers HSP20-F1 and HSP20-R1. The PCR program was set as follows: 94°C for 5 min,followed by 30 cycles at 94°C for 30 s,60°C for 30 s, and 72°C for 40 s,followed by extension at 72°C for 10 min. Next,a nested PCR was implemented with HSP20-F2 and HSP20-R2. The PCR profile was set as described above,but a lower annealing temperature with 58°C was used. PCR was run in 25 μL volume containing 1 μL cDNA(50 ng),10×PCR buffer 2.5 μL(TaKaRa,Dalian,China),10 mmol/L dNTPs 2.0 μL(TaKaRa,Dalian,China),10 μmol/L of primer,1 U of TaKaRa Ex Taq and 17.3 μL double distilled water. The PCR products were cloned into pMD-19T vectors(TakaRa,Japan) and transformed into Escherichia coli DH5αcompetent cells. The recombinants were identified via blue-white color selection on ampicillin-containing LB plates. Three of the positive clones were then sequenced by Invitrogen Corp(Shanghai,China).

Table 1 Information on the primers used in this study

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Based on the sequence of HSP20,the 3' and 5' ends were cloned using a RACE approach with genespecific and adapter primers. The 3' end RACE PCR reaction was carried out with the HSP20-3'F1 genespecific primer and the AOLP adapter primer with an annealing temperature of 54°C. The semi-nested PCR was performed with the HSP20-3'F2 gene-specific primer and the AP adaptor primer with an annealing temperature of 58°C. The mantle mRNA for the 5' end was transcribed by M-MLV reverse transcriptase and the HSP20-5'R1 gene-specific primer. Next,the cDNA was purified with a DNA purification kit(Roche,USA) and tailed with poly C at the 5' end of the cDNA by terminal deoxynucleotidyl transferase(TdT)(Fermentas,USA). The PCR was initially performed with the HSP20-5'R1 primer and the AAP adapter primer using tailed cDNA with an annealing temperature of 51°C. The nested PCR was carried out with the HSP20-5'R2 primer and the AP adapter primer with an annealing temperature of 52°C. The PCR products were gel-purified,cloned, and sequenced as described above. All of the primer sequences are listed in Table 1. 2.5 Sequence analysis and phylogenetic tree construction

The full-length sequence of PtHSP20 was assembled using the DNASTAR multiple program package(DNASTAR Inc.,USA). The deduced amino acid sequence of PtHSP20 was analyzed using ExPASy(http://www.expasy.org). The protein motif features were predicted by the SMART program(Letunic et al., 2006). The potential protein subcellular localization was predicted by the PSORT II prediction system(http://www.psort.org). Multiple alignments of the PtHSP20 sequence were performed with BioEdit version 7.1.3.0(Hall,1999). A phylogenetic tree was constructed based on the amino acid sequences of selected HSPs using the neighborjoining(NJ)method of the MEGA 4.0 program(Tamura et al., 2007). 2.6 mRNA expression of HSP20 by quantitative real-time PCR

The mRNA expression of PtHSP20 in the P. textile case and control tissues was measured by quantitative real-time PCR(qRT-PCR)using the ABI 7500 realtime system(Applied Biosystems,USA). A qRT-PCR was performed with the HSP20-rF and HSP20-rR primers β-actin rF and β-actin rR,respectively(Table 1). The PCR reaction was carried out in a total volume of 20 μL consisting of 9 μL of RealMasterMix SYBR Green(TIANGEN,Beijing),0.5 μL of each primer,0.5 μL of 50 × ROX Reference Dye,1 μL of cDNA, and 8.5 μL of deionized water. The qRT-PCR was performed as follows: initial denaturation at 95°C for 2 min,followed by 40 cycles at 95°C for 15 s and 60°C for 25 s, and elongation at 68°C for 20 s. Each sample was measured in triplicate. The qRT-PCR products were separated on 1.0% agarose gels to detect specific b and s. In addition,a dissociation curve analysis was performed at the end of each reaction to confi rm that only one PCR product had been amplified. Expression levels were analyzed using the comparative CT method(Schmittgen and Livak, 2008), and all of the data were analyzed in SPSS 15.0(SPSS Inc.,Chicago,IL,USA). 3 RESULT3.1 Characterization of the full-length cDNA of PtHSP20

The full-length cDNA of HSP20 in P. textile is 1 090 bp long(Fig. 1). It contains a 522-bp ORF that encodes 173 amino acids,a 93 bp 5′UTR,a 475 bp 3′UTR,putative ATTTA instability sequences in the 3′UTR, and a Poly(A)tail. Furthermore,three polyadenylation signals(AATAA)were detected downstream of the stop codon: at 226–230 bp(844 bp from the first nucleotide),522–526 bp(1 037 bp from the first nucleotide), and 536–540 bp(1 054 bp from the first nucleotide). The predicted molecular mass and theoretical isoelectric point were 20.2 kDa and 6.2,respectively. According to the BLASTX results,the deduced amino acid sequence had significant homology to other reported mollusk HSP20s. PtHSP20 included an alpha-crystallin domain(ACD)at the C-terminal region that encodes approximately 95 amino acid residues. According to PSORT II,PtHSP20 is predicted to be located in the cytoplasm with a 56.5% probability.

Fig. 1 The cDNA sequence and deduced amino acid sequence of P. textile HSP20 The bold letters in the box show the start codon(ATG). The bold letters in the box and asterisk indicate the stop codon(TAA). The putative instability sequences(ATTTA)in the 3′UTR are shown in bold letters with single-underlining. The polyadenylation signal sequence(AATAA)is indicated with light gray. The Glx residues are shown in light gray bold letters. The qRT-PCR primer positions for HSP20 are shown in bold letters

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A multiple sequence alignment revealed that PtHSP20 has relatively high similarities to known sHSPs in other mollusk species(Fig. 2). Specifically,it showed the highest similarity to Ruditapesphilippinarum(98.3%),93.6% similarity to Cyclinasinensis,92.5% similarity to M. meretrix, and 64.6% similarity to Mytilus galloprovincialis . However,it showed 40.4% and 41.1% similarity to Chlamysfarreri and A. irradians,respectively. A phylogenic tree was constructed based on the sHSP protein sequences(Fig. 3). The tree was automatically divided into different clades on the basis of phylogenetic taxonomy including bacteria,plants,mollusks,arthropods,cephalochordates,fishes,amphibians,reptiles, and mammals. PtHSP20 first clustered with HSP22 from R. philippinarum and then grouped with C. farreri and A. irradians . In summary,the relationship displayed in the phylogenic tree is in sound agreement with traditional taxonomy.

Fig. 2 Multiple alignments of the deduced amino acid sequence of P. textileHSP20 with other known sHSPs The species and GenBank accession numbers used for the multiple alignments were as follows: Ruditapes philippinarum(ACU83231),Meretrix meretrix(AFK80359),Cyclina sinensis(AET13647),Sinonovacula constricta(AGM14597),Mytilus galloprovincialis(AEP02967),Chlamys farreri(AAR11780), and Argopecten irradians(ACA23998). The conserved amino acid residues are shaded in black. The sequence identities and similarities are shown at the end of the alignments. The N-terminal region,α-crystallin domain, and C-terminal tail alignments are displayed on separate lines.

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Fig. 3 A phylogenetic tree of the sHSP genes The tree was constructed by the amino acid sequences whose GenBank accession numbers are showed in Table 2 .

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The results of qRT-PCR revealed that PtHSP20 mRNA was expressed in all six of the tested tissues,including heart,digestive gl and ,adductor muscle,gonad,gill, and mantle. PtHSP20 mRNA amount was highest in the gonad,followed by the mantle and the heart, and was low in the gill,digestive gl and , and adductor muscle(Fig. 4).

Fig. 4 The tissue distribution of PtHSP20 mRNA was determined by qRT-PCR in the heart,digestive gl and ,adductor muscle,gonad,gill and mantle The bars represent the mean±the st and ard error(SE)for n=3 replicates with 3 individuals per replicate, and the statistical significance is indicated with asterisks(**P<0.01).

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The expression of PtHSP20 mRNA was investigated by qRT-PCR in the heart,digestive gl and ,adductor muscle,gonad,gill and mantle after being subjected to high temperature shock, and the results are shown in Fig. 5. Overall,PtHSP20 mRNA was down-regulated in most of the tissues except the adductor muscle and gonad(P<0.05).

Fig. 5 Temporal expression of PtHSP20 mRNA relative to β-actin analyzed by qRT-PCR in the heart(a),digestive gl and (b),adductor muscle(c),gonad(d),gill(e) and mantle(f)under the stress of high temperature The bars represent the mean±SE for n=3 replicates with 3 individuals per replicate. The statistical significance using the control group is indicated with asterisks(* Temporal expression of PtHSP20 mRNA relative to β-actin analyzed by qRT-PCR in the heart(a),digestive gl and (b),05,** Temporal expression of PtHSP20 mRNA relative to β-actin analyzed by qRT-PCR in the heart(a),digestive gl and (b),01).

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In this study,three polyadenylation signals(AATAA)were found in PtHSP20. This is not consistent with HSP22 in A. irradians(Zhang et al., 2010)where only one polyadenylation signal AATAA was found. PtHSP20 contains 19 glutamine and glutamic acid(Glx)residues,accounting for 10.9% of its total amino acids. These Glx residues could provide additional electrostatic forces contributing to the stability of the protein at elevated temperatures(Jacobsen and Shaw, 1989). Amino acid sequence alignment showed that PtHSP20 shared the highest similarity(98.3%)in amino acid sequence with the HSP22 of R. philippinarum . Yet,it showed 40.4% and 41.1% similarity to the Chlamys farreri and A.irradians,respectively. The results were in agreement with the HSP20 of M. meretrix(Li et al., 2013).

Table 2 The amino acid sequences used for phylogenetic analysis in this study

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HSP20 is a ubiquitous protein participating in many stress response processes. In this study,the expression of PtHSP20 mRNA could be detected in all of the examined tissues under non-stressed conditions. The expression of PtHSP20 mRNA washighest in the gonad. Similar results were also found in M. galloprovincialis,where the HSP24.1 transcript was predominantly expressed in the gonad(You et al., 2014). In a prior study,human sHSPs were found to be specifically expressed in the testes,where they play a crucial role in sperm development(Kappé et al., 2003). Therefore,it is hypothesized that PtHSP20 is likely to play an important role in the processes of gonad development and differentiation.

Water temperature is one of the most decisive factors affecting an organism’s ability to conduct its normal physiological functions. When cells are exposed to heat shock,many proteins tend to aggregate and lose their biological activity,resulting in a series of adverse cellular events(Laszlo,1992). Under stress conditions,the expression of HSP20 may be significant up-regulation or significant downregulation. Ju et al.(2015)identified that the expression of HSP20 in CRC tissues was significant reduced. The results of Wan et al.(2012) and Yang et al.(2012)revealed that the expression of HSP20 and HSP27 were up-regulated under heat stress. In our study,the expression of PtHSP20 mRNAs was only up-regulated in the adductor muscle and gonad and was significantly down-regulated(P<0.05)in other tissues. Gene expression is a very complex course and it influenced by many factors including itself and the outside world. In this study,32°C maybe exceed the adapted capacity to high temperature and led to express disorder. 5 CONCLUSION

In this study,the full-length cDNA of HSP20 in P. textilewas cloned and its expression patterns were characterized under high temperature shock. Our results provide a first step towards studying the mechanisms of P. textile ’s response to high temperature stress.