Temperature stress response of heat shock protein 90(Hsp90)in the clam Paphia undulata☆

Xiangyang Lin ,Xiangwei Wu ,Xiande Liu

a Key Laboratory of Mariculture for the East China Sea,Ministry of Agriculture,Fisheries College,Jimei University,Xiamen 361021,China

b Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province,Fisheries Research Institute of Fujian,Xiamen 361013,China

c Animal Science and Technology College,Yunnan Agricultural University,Kunming 650201,China

1.Introduction

Many bivalves are sedentary filter feeders,living in estuarine or intertidal regions.Their health is severely affected by temperature or salinity fluctuations,oxygen levels,toxic metals,or bacterial pathogens(Fabbri,Valbonesi,&Franzellitti,2008;Liet al.,2009).In the process of adaptation,bivalves acquired defense systems to provide protection from harmful environments.The Heat shock proteins(Hsps)are part of the defense system and their expression can rapidly increase in response to environmental stresses(Craig,1986;Gao et al.,2008).Generally,they are regarded as molecular chaperones and thus protect proteins and cells from damage when under stressful conditions.Hsps are highly conserved proteins from bacteria to mammals(Craig,1986;Csermely et al.,1998;Gao et al.,2008).According to their molecular size and sequence homology they are grouped in several families:Hsp110,Hsp90,Hsp70,Hsp60,Hsp47,and low molecular mass Hsps(Heikkila,2010;Lindquist&Craig,1988).To date,several studies have focused on Hsp90s and additionally,according to their subcellular locations,they can be classified into four types:cytosolic,endoplasmic reticulum(ER),mitochondria and chloroplast in plants(Picard,2002).

It was reported that Hsp90 are highly conserved and accounts for approximately 1%-2%of the total soluble proteins in some cells(Buchner,1999).When organisms are exposed to a stress,such as an environmental insult or pathogenic infection,expression of Hsp90 can significantly increase in a short period of time to prevent irreversible aggregation of damaged proteins.Therefore,in most of eukaryotes Hsp90 plays an important role in cellular protection against stress(Buchner,1999),tumor repression(Gress et al.,1994;Whitesell et al.,1994),cell cycle control(Aligue,Akhavan-Niak,&Russell,1994),and antigen recognition(Srivastava et al.,1994).Hsp90 and expression patterns in response to various stressors have been reported in several molluscs,including Laternula elliptica(Kim et al.,2009),Crassostrea gigas(Choi,Jo,&Choi,2008),Haliotis tuberculata(Farcy et al.,2007),Haliotis asinine(Gunter&Degnan,2007),Chlamys farreri(Gao et al.,2007),and Argopecten irradians(Gao et al.,2008).

Table 1 Primer sequences and annealing temperatures.

The clam P.undulata is one of the important farmed bivalve species in China and South East Asia and mass mortality causes great losses in P.undulata aquaculture(Leethochavalit et al.,2004).The reasons of clam mortality are currently unknown but it has been associated with harmful environmental conditions,such as acute changes in temperature.Therefore,in order to understand the physiological responses and protective mechanism in harsh environment,we cloned and characterized Pu Hsp90 gene and analyzed its expression in the gonad,heart,adductor muscle,mantle,gills,digestive gland and hemocytes under high temperature stress.Our study provides preliminary information to monitor the physiological status of P.undulata under different environmental conditions.

2.Materials and m ethods

2.1.Animal collection

More than three hundred individuals of P.undulata,with average shell length of 44 mm,were collected from the main distribution areas of P.undulata in Xiamen,Fujian,China in November 2013(temperature 19±0.5°C).Clams were reared in tanks(7 m3)containing aerated sand-filtered seawater(salinity 20,p H 8.1)at 19±0.5°Cfor 7 days,prior to the experiments.Animals were fed with 0.8%Tetraselmis chui once every 24 h.

Table 2 Accession numbers and percentage of amino acid homology of the Hsp90s used for the phylogenetic analysis.

2.2.Heat shock stress

In this study,one hundred and fifty P.undulata were divided into two groups,i.e.,stressed and control.The control group was cultured at 19±0.5°C while the stressed group was heat shock at 32°Cfor 2 h and allowed to gradually recover to 19±0.5°C.Three samples were collected from different tissues(gonad,heart,adductor muscle,mantle,gill,digestive gland,and hemocytes)before(0,1,and 2 h)and after the recovery period(2,6,12,18,24,36,and 48 h),immediately snap-frozen in liquid nitrogen and stored at 80°C until the total RNA extraction.No mortality was observed during the experimental procedure.

2.3.RNA extraction and cDNA synthesis

Total RNA was extracted using Trizol reagent(Invitrogen,USA)following the manufacturer's instructions.The total RNA was treated with RNase-free DNase I(Roche,USA)to remove possibly genomic DNA contamination.First-strand cDNA was synthesized from the total RNA using M-MLV reverse transcriptase(Promega,USA)according to the manufacturer's instructions.

Fig.1.Complete cDNA sequence of the PuHsp90 and predicted amino acid sequence.The start codon(ATG),stop codon(TAA)and the canonical polyadenylation signal sequence AATAAA are within boxes.The five Hsp90 family signatures and conserved motif MEEVD are shaded.The ATP binding domain between amino acids 35-189 is underlined,the conserved motif Gxx Gx G is in italics and the methyl-Cp G binding domain is underlined by a dash line.

2.4.Cloning

Four degenerated primers,Hsp90-F1,Hsp90-R1,Hsp90-F2 and Hsp90-R2(Table 1),were designed based on the conserved nucleotide sequence regions of available Hsp90 amino acid sequences of other molluscs retrieved from the NCBI database.A partial PuHsp90 c DNA fragment was isolated by polymerase chain reaction(PCR)using cDNAfrom the mantle as template.First-round PCR was performed with the primers Hsp90-F1 and Hsp90-R1,according to the cycle:initial denaturation at 94°C for 2 min followed by 27 cycles of denaturation at 94°Cfor 30 s,annealing at 53°C for 45 s,and elongation at 72°C for 60 s,and finally by extension at 72°Cfor 8 min.Subsequently,a nested PCR was performed with primers Hsp90-F2 and Hsp90-R2 according to the same cycle but with annealing at 55°C.The PCRproduct obtained was ligated in the p MD-19Tvector(Takara,Japan)and transformed into Escherichia coli DH5a competent cells.The recombinant clones were identified by blue-white color selection on ampicillin containing LB plates,and 3 clones were selected for sequencing(Invitrogen Corp.,Shanghai,China).

Based on the obtained partial PuHsp90 sequence,the 3′and 5′ends were cloned using RACE with gene-specific primers and adapter primers(Table 1).The 3′-end RACE PCR was performed with primer Hsp90-3′F1 and the adapter AOLP with annealing at 53°C;the semi-nested PCRwas then carried out with Hsp90-3′F2 and an adaptor primer AP with annealing at 56°C.For the 5′-end,mantle m RNA was transcribed by M-MLV reverse transcriptase using the gene-specific primer Hsp90-5′R1.Next,cDNA was purified using a DNApurification kit(Roche,USA)and then tailed poly C by terminal deoxynucleotidy1 transferase(Td T)(Fermentas,USA).PCR was initially carried out with the tailed cDNA,primer Hsp90-5′R1 with the primer AAP with annealing at 51°C;the semi-nested PCR was subsequently performed with Hsp90-5′R2 and the adapter primer AP with annealing at 53°C.

Fig.2.Multiple sequence alignment of PuHsp90 with Hsp90 from other molluscs.Family signatures and the consensus sequence MEEVD localized at the C-terminus are shaded.

Fig.3.Multiple sequence alignment of Hsp90 and homologues from several representative species.Shaded regions indicate the C-terminus sequences for the cytosolic(cyt),ER(e),mitochondria(mit),and chloroplast(chl)Hsp90.No consensus sequence was identified at the C-terminus of the bacteria Hsp90.Abbreviations used and Gen Bank accession numbers are reported in Table 2 are as follows:Human(cyt),Homo sapiens,NP_005339;Rat(cyt),Rattus norvegicus,NP_786937;Pig(cyt),Sus scrofa,NP_999138;Chicken(cyt),Gallus gallus,NP_001103255;Frog(cyt),Xenopus laevis,AAV41061;Zebra fish(cyt),Danio rerio,NP_571385;Crab(cyt),Eriocheir sinensis,ADE60732;Shrimp(cyt),Fenneropenaeus chinensis,ABM 92446.1;Human(e),Homo sapiens,AAH66656;Rat(e),Mus musculus,AAH11439;Chicken(e),Gallus gallus,P08110;Dog(e),Canis lupus familiaris,AAA17708;Pig(e),Sus scrofa,CAA70347;Frog(e),Xenopus tropicalis,NP_001039228;Zebra fish(e),Danio rerio,AAH63951;Oyster(e),Crassostrea gigas,BAF63637.1;Sea urchin(e),Strongylocentrotus purpuratus,AAO21341;Barley(e),Hordeum vulgare,CAA48143;Pine(e),Xerophyta viscosa,AAN34791;Human(mit),Homo sapiens,NP_057376;Rat(mit),Rattus norvegicus,NP_001034090;Silkworm(mit),Bombyx mori,AFG30049.1;Fruit fly(mit),Drosophila melanogaster,AAD29307;Arabidopsis(chl),Arabidopsis thaliana,NP_178487;Rye(chl),Secale cereale,CAA82945;Kp Htp G,Klebsiella pneumoniae,CCI78437;DaHtp G,Desulfobacterium autotrophicum,ACN17860.1;PfHtp G,Pseudomonas fluorescens,AEV64226.1;BsHtp G,Bacillus subtilis,NP_391861;CaHtp G,Clostridium acetobutylicum,NP_349907;BaHtp G,Buchnera aphidicola,AHG61639.1.

The PCR program included an initial denaturation at 94°C for 2 min followed by 30 cycles of denaturation at 94°C for 30 s,annealing at the specific temperature for 30 s,and elongation at 72°C for 90 s,followed by extension at 72°C for 8 min.All PCR products obtained were gel-purified,cloned and sequenced to confirm identity.

2.5.Sequence analysis and phylogenetic tree construction

The consensus complete sequence of the PuHsp90 was obtained by assembling all the sequence fragments using the DNASTAR multiple program package(DNASTARInc.,USA).Multiple sequence alignments were performed with Clustal X 1.83 software(Thompson et al.,1997).The predicted PuHsp90 amino acid sequence was deduced using Ex PASy(http://www.expasy.org)and the SMART program(Letunic et al.,2006)was used to predict the protein motif features.To explore the evolutionary relationship of PuHsp90 a phylogenic tree was constructed using the neighborjoining(NJ)method with 1000 bootstrap replicates by MEGA 4.0(Tamura et al.,2007)including other metazoan Hsp90 sequences retrieved from the nr(non-redundant protein database,NCBI)database.To predict the intracellular location of PuHsp90,multiple sequence alignment of the PuHsp90 C-terminus with other cytosolic,ER,mitochondria and chloroplast Hsp90 sequences was performed.

Fig.4.Multiple sequence alignment of the bivalve and vertebrate Hsp90 N-terminus.Two consensus sequences:one is QTQDQ motif,a glutamine-rich motif,in the vertebrate Hsp90a N-terminus is highlighted in light gray,and the other is V(H/Q)HG motif in the vertebrate Hsp90βN-terminus is marked in dark gray.Gen Bank accession numbers are described in Table 2 and Human1,NP_005339;Rat1,NP_786937;pig1,NP_999138;monkey1,XP_003902333;Chicken1,NP_001103255;Human2,NP_031381;Rat2,XP_217339;pig2,NP_001231362;monkey2,AFJ71824.1;Chicken2,CAA49704.

Fig.5.Phylogenetic NJtree of the Hsp90 sequences.Bootstrap confidence values(1000 replications)are indicated at the nodes.Abbreviations of the species names and Gen Bank accession numbers are listed in Table 2,Figs.3,and 4.PuHsp90 is denoted by a black diamond.

2.6.Quantitative expression of PuHsp90

Real-time quantitative RT-PCRwas performed to determine the levels of PuHsp90 m RNA in different tissues.The PCR reaction mixture contained 9μL RealMaster Mix SYBR Green(TIANGEN,Beijing),0.5μLof each primer(10μmol/L)(Table 1),0.5μL 50×ROX Reference Dye,1μL cDNA,and 8.5μL deionized water.A PCR product of 96 bp in size was amplified using the primers HSP90-r F and HSP90-r R.β-actin gene(Gen Bank accession no.JX885712)was used as an internal control and was amplified with the primersβactin r Fandβ-actin r R(Table 1).The RT-PCRprogram consisted of an initial denaturation step at 95°Cfor 2 min,followed by 40 cycles of denaturation at 95°C for 15 s,annealing at 60°C for 25 s,and elongation at 68°Cfor 20 sand was performed in the ABI7500 realtime system(Applied Biosystems,USA).Each sample was tested in triplicate.Specificity of the reaction for PuHsp90 andβ-actin was determined by analyzing the dissociation curves.The obtained data was analyzed using the comparative CTmethod(Schmittgen&Livak,2008)and statistical significant differences were calculated using one-way ANOVA in SPSS 15.0(SPSS Inc.,Chicago,IL,USA).Duncan's test was conducted using SAS9.1 software(SASInstitute,Inc.,Cary NC)to compare the means between the individual treatments,and P＜0.05 was considered statistically significant.

3.Results

3.1.Characterization of the PuHsp90

The isolated full-length c DNA of PuHsp90(Genbank accession no.JX885710)was 2 766 bp in length with an open reading frame(ORF)of 2 181 bp encoding a putative 726 amino acid protein with a 83.5 k Da and 4.86 isoelectric point(Fig.1).The predicted 5′-terminal untranslated region(UTR)was 106 bp and the 3′-terminal UTRwas 474 bp.The putative polyadenylation signal(AATAAA)was found at 445 bp downstream the stop codon(TAA).

In PuHsp90 sequence 5 conserved signatures motifs characteristic of the Hsp90 family were found at residues 35-55(NKEIFLRELISNSSDALDKIR), 102-110 (LGTIAKSGT), 126-141(IGQFGVGFYSAYLVAD),354-363(IKLYVRRVFI),and 380-394(GIVDSEDLPLNISRE)(Fig.2).The conserved motif,Gxx Gx G(x represents any residue)was also observed at the N-terminus(Fig.1;Fig.2).Sequence analysis using SMARTprogram identified a typical histidine kinase-like ATPase domain at residues 35-189 that is ubiquitous in Hsp90 family and a methyl-Cp G binding domain at residues 431-495(Fig.1).

Multiple sequence alignment of the Hsp90 C-terminus showed that they contained various C-terminal consensus sequences,depending on their intracellular locations(Fig.3).The PuHsp90 possessed the MEEVD motif on its C-terminus that was also found in other known cytosolic Hsp90s,suggesting that PuHsp90 is a cytosolic homolog(Fig.3).In contrast,PuHsp90 and also other known mollusk Hsp90s lacked the consensus motifs QTQDQ and V(H/Q)HGat the N-terminus(Fig.4)which are characteristic of the vertebrate Hsp90a and Hsp90βN-terminus,respectively.

3.2.Sequence comparison and phylogenetic analysis of PuHsp90

According to the phylogenetic tree,the various Hsp90s cluster in different groups according to their intracellular locations(Fig.5).The PuHsp90 grouped with other known cytosolic Hsp90s that belonged to one big group,and it was further separated into four monophyletic sub-groups,including mollusks,arthropods,vertebrates,and plants,with high bootstrap support that are in agreement with the evolution of the species.The mollusc cytosolic shared common ancestry with the Hsp90s from vertebrate and arthropods.In vertebrates,the cytosolic Hsp90a and Hsp90β grouped into closely related clusters.The mitochondria Hsp90s are the most divergent protein group and the phylogenetic tree suggest that they diverged prior to the other Hsp90s groups.

3.3.Tissues distribution of PuHsp90

PuHsp90 was found to be expressed in all the tissues analyzed,including mantle,digestive gland,adductor muscle,gonad,hemocytes,gills and heart.The gonad presented the highest m RNA expression level and was followed by the digestive gland,gills and heart.In the mantle,adductor muscle and hemocytes expression levels were relatively low(Fig.6).

3.4.mRNA expression of PuHsp90 under a high temperature stress

Fig.6.Tissue distribution of PuHsp90 m RNA determined by qRT-PCR in the mantle(Mn),digestive gland(Dg),adductor muscle(Ms),gonad(Gn),hemocytes(Hm),gills(Gi),and heart(Ha).Bars represent the mean±S.E(N=3 replicates,and 3 individuals per replicate).Different letters denote statistical significantly differences(P＜0.05;Duncan's test).

Under a heat shock stress,the expressed levels of PuHsp90 were gradually up-regulated after the stress and continuously increased to the maximum levels(P＜0.05)after 2 h of post-stress in the mantle(3.6-fold),adductor muscle(9.1-fold),hemocytes(12.3-fold)and the gills(7.2-fold).Subsequently,transcript expression was down-regulated and were almost recovery to the pretreatments(P＞0.05)after 48 h of post-stress(Fig.7).However,in the digestive gland,PuHsp90 level was down-regulation throughout all the examined period with significant differences(P＜0.05).In contrast,PuHsp90 levels were slight up-regulated(P＞0.05)in the gonad and heart at the beginning of the stress,and subsequently down-regulated at 6 h in the gonad and at 12 h in the heart.

4.Discussion

The PuHsp90 C-terminal displays greater sequence similarity with the cytosolic Hsp90,suggesting that PuHsp90 is probably a cytosolic Hsp90 homolog.The motif MEEVD is characteristic of the cytosolic metazoan Hsp90 proteins,that constitutes the Hsp90 center of interaction surface for the tetratricopeptide repeats(TPRs)of Hsp90 co-chaperones(Scheufler et al.,2000).Therefore,this structure,existing in PuHsp90 suggests that it may have similar functions than the known cytosolic Hsp90 from other species.In vertebrates,cytosolic Hsp90 can be divided into two different types the Hsp90a and Hsp90β,determined by a glutamine-rich motif(QTQDQ)at the N-terminus that is absent from Hsp90β(Krone&Sass,1994;Lees-Miller&Anderson,1989).The bivalve Hsp90s display a greater sequence similarity for the vertebrate Hsp90βand PuHsp90 and other bivalve sequences lack the typical N-terminus motif.Phylogenetic analysis supported that PuHsp90 grouped with the other mollusk Hsp90s and they shared common origin and diverged prior to the vertebrate a anβHsp90s which subsequently emerged by a gene duplication event(Gupta,1995).

The PuHsp90 m RNA showed highest expression in the gonad,which is consistent with the studied on the homolog transcripts in C.Hongkongensis,the black tiger shrimp Penaeus monodon and marine crab Portunus trituberculatus(Jiang et al.,2009;Zhang et al.,2009).Hsp90 usually plays a role of activating the mitogenactivated protein kinase pathway,which is necessary for oocyte maturation(Fisher,Mandart,&Dor′ee,2000).During spermatogenesis,Hsp90 expression was largely up-regulated in rat testis(Krawczyk,Szymik,&Wi′sniewski,1987)and thus it can be speculated that PuHsp90 is probably involved in gonadal cellular development in the clam P.undulata.

Fig.7.Temporal expression of PuHsp90 in the different tssues(mantle,digestive gland,adductor muscle,gonad,hemocytes,gills and heart)under heat shock stress obtained by qRT-PCR.Transcript relative expression was calculated in relation to the expression ofβ-actin.The values represent the mean±S.E(N=3 replicates,and 3 individuals per replicate).Different letters denote statistical significantly differences between the samples(P＜0.05;Duncan's test).S0 h,S1 h,and S2 h represent 0,1,and 2 h after the heat stress.R 2 h,R 6 h,R 12 h,R 18 h,R 24 h,R 36 h and R 48 h represent 2,6,12,18,24,36,and 48 h of the recovery period.

Under a high temperature stress,PuHsp90 levels significantly increased in external organs such as the mantle,adductor muscle,gills and hemocytes,which is consistent with previous reports from other mollusks(Farcy et al.,2007;Kim et al.,2009)but were down regulation in the internal organs,including the digestive gland,gonad and heart suggesting that it may be part of the defense mechanism against environmental challenges(Feder and Hofmann,1999).Therefore,it may be concluded that in P.undulata external organs are more sensitive to thermal stress than the internal organs.However,this is opposite to that was described in the sea cucumber Apostichopus japonicus(Ji,Dong,&Dong,2008).The external organs often connect with outside environment,being sensibility to outside stress.In addition the unbalance energy distribution in the different organs may be another reason for the observed changes in the expression levels of Pu Hsp90 in the different organs.In general,energy metabolism is quick in the adductor muscle,mantle,and gill to meet their physiological functions under environmental stresses.When some events that require energy consumption occur,such as the activation of Hsp90 transcription and Hsp90 synthesis,the energy can be quickly supplied(Somero,2002).Consequently,amounts of PuHsp90 increase in a short time in response to high temperature stress.

5.Conclusion

In conclusion,PuHsp90 is a putative cytosolic Hsp90 and transcript expression level is affected by temperature stress.PuHsp90 displayed tissue specific pattern and this is probably due to natural selective pressures in order to respond effectively to environmental stress factors.Therefore,for the healthy P.undulata farming,the effective ways,e.g.,exchanging water and adding freshwater,should be taken in consideration to avoid dramatic temperature changes.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the Open Program of Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province(2015fjscq05)and the New Century Excellent Talents of Fujian Province University(No.JA14167).

Aligue,R.,Akhavan-Niak,H.,&Russell,P.(1994).A role for Hsp90 in cell cycle control:Wee 1 tyrosine kinase activity requires interaction with Hsp90.The EMBO Journal,13(24),6099-6106.

Buchner,J.(1999).Hsp90&Co.-a holding for folding.Trends in Biochemical Sciences,24(4),136-141.

Choi,Y.K.,Jo,P.G.,&Choi,C.Y.(2008).Cadmium affects the expression of heat shock protein 90 and metallothionein m RNA in the Pacific oyster.Crassostrea gigas.Comp.Biochem.Physiol.C,147(3),286-292.

Craig,E.A.(1986).The heat-shock response.Annual Review of Biochemistry,55(1),1151-1191.

Csermely,P.,Schnaider,T.,S″oti,C.,et al.(1998).The 90-k Da molecular chaperone family:Structure,function,and clinical applications.A comprehensive review.Pharmacology&Therapeutics,79(2),129-168.

Fabbri,E.,Valbonesi,P.,&Franzellitti,S.(2008).HSP expression in bivalves.ISJ,5(135),135-161.

Farcy,E.,Serpentini,A.,Fi′evet,B.,et al.(2007).Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata:Transcriptional induction in response to thermal stress in hemocyte primary culture.Comparative Biochemistry and Physiology B,146(4),540-550.

Feder,M.E.,&Hofmann,G.E.(1999).Heat-shock proteins,molecular chaperones,and the stress response:evolutionary and ecological physiology.Annu.Rev.Physiol.,61,243-282.

Fisher,D.L.,Mandart,E.,&Dor′ee,M.(2000).Hsp90 is required for c-Mos activation and biphasic MAPkinase activation in Xenopus oocytes.The EMBOJournal,19(7),1516-1524.

Gao,Q.,Song,L.S.,Ni,D.J.,et al.(2007).cDNA cloning and mRNA expression of heat shock protein 90 gene in the hemocytes of Zhikong scallop Chlamys farreri.Comparative Biochemistry and Physiology B,147(4),704-715.

Gao,Q.,Zhao,J.M.,Song,L.S.,et al.(2008).Molecular cloning,characterization and expression of heat shock protein 90 gene in the hemocytes of bay scallop.Argopecten irradians.Fish Shell fish Immunol.,24(4),379-385.

Gress,T.M.,Müller-Pillasch,F.,Lerch,M.M.,et al.(1994).Balance of expression of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in chronic pancreatitis.Zeitschrift für Gastroenterologie,32(4),221-225.

Gunter,H.M.,&Degnan,B.M.(2007).Developmental expression of Hsp90,Hsp70 and HSF during morphogenesis in the vetigastropod Haliotis asinina.Dev.Genes Evol.,217(8),603-612.

Gupta,R.S.(1995).Phylogenetic analysis of the 90 k D heat shock family of protein sequences and an examination of the relationship among animals,plants,and fungi species.Molecular Biology and Evolution,12(6),1063-1073.

Heikkila,J.J.(2010).Heat shock protein gene expression and function in amphibian model systems.Comparative Biochemistry and Physiology a,156(1),1531-4332.

Jiang,S.G.,Qiu,L.H.,Zhou,F.L.,et al.(2009).Molecular cloning and expression analysis of a heat shock protein(Hsp90)gene from black tiger shrimp(Penaeus monodon).Molecular Biology Reports,36(1),127-134.

Ji,T.T.,Dong,Y.W.,&Dong,S.L.(2008).Growth and physiological responses in the sea cucumber,Apostichopus japonicus Selenka:Aestivation and temperature.Aquaculture,283(1),180-187.

Kim,M.,Ahn,I.Y.,Kim,H.,et al.(2009).Molecular characterization and induction of heat shock protein 90 in the Antarctic bivalve Laternula elliptica.Cell Stress&Chaperones,14(4),363-370.

Krawczyk,Z.,Szymik,N.,&Wi′sniewski,J.(1987).Expression of hsp70-related gene in developing and degenerating rat testis.Molecular Biology Reports,12(1),35-41.

Krone,P.H.,&Sass,J.B.(1994).Hsp 90a and Hsp 90βgenes are present in the zebra fish and are differentially regulated in developing embryos.Biochemical and Biophysical Research Communications,204(2),746-752.

Lees-Miller,S.P.,&Anderson,C.W.(1989).The human double-stranded DNA-activated protein kinase phosphorylates the 90-k Da heat-shock protein,hsp90 alpha at two NH2-terminal threonine residues.Journal of Biological Chemistry,264(29),17275-17280.

Leethochavalit,S.,Chalermwat,K.,Upatham,E.S.,et al.(2004).Occurrence of Perkinsus sp.in undulata surf clams Paphia undulata from the Gulf of Thailand.Diseases of Aquatic Organisms,60(2),165-171.

Letunic,I.,Copley,R.,Pils,B.,et al.(2006).SMART 5:Domains in the context of genomes and networks.Nucleic Acids Research,34(suppl 1),257-260.

Li,F.H.,Luan,W.,Zhang,C.S.,et al.(2009).Cloning of cytoplasmic heat shock protein 90(FcHSP90)from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia.Cell Stress&Chaperones,14(2),161-172.

Lindquist,S.,&Craig,E.A.(1988).The heat-shock proteins.Annual Review of Genetics,22(1),631-677.

Picard,D.(2002).Heat-shock protein 90,a chaperone for folding and regulation.Cellular and Molecular Life Sciences,59(10),1640-1648.

Scheu fler,C.,Brinker,A.,Bourenkov,G.,et al.(2000).Structure of TPR domainpeptide complexes:Critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine.Cell,101(2),199-210.

Schmittgen,T.D.,&Livak,K.J.(2008).Analyzing real-time PCR data by the comparative CTmethod.Nature Protocols,3(6),1101-1108.

Somero,G.N.(2002).Thermal physiology and vertical zonation of intertidal animals:Optima,limits,and cost of living.Integrative and Comparative Biology,42(4),780-789.

Srivastava,P.K.,Udono,H.,Blachere,N.E.,et al.(1994).Heat shock proteins transfer peptides during antigen processing and CTL priming.Immunogenetics,39(2),93-98.

Tamura,K.,Dudley,J.,Nei,M.,et al.(2007).MEGA4:Molecular evolutionary genetics analysis(MEGA)software version 4.0.Molecular Biology and Evolution,24(8),1596-1599.

Thompson,J.D.,Gibson,T.J.,Plewniak,F.,et al.(1997).The CLUSTAL_X windows interface,fl exible strategies for multiple sequence alignment aided by quality analysis tools.Nucleic Acids Research,25(24),4876-4882.

Whitesell,L.,Mimnaugh,E.G.,De-Costa,B.,et al.(1994).Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins:Essential role for stress proteins in oncogenic transformation.Proc.Nat.Acad.Sci.USA.,91(18),8324-8328.

Zhang,X.Y.,Zhang,M.Z.,Zheng,C.J.,et al.(2009).Identification of two hsp90 genes from the marine crab,Portunus trituberculatus and their specific expression pro files under different environmental conditions.Comparative Biochemistry and Physiology-Part C,150(4),465-473.