Fish Physiol Biochem DOI 10.1007/s10695-015-0085-2

Molecular characterization of heat–shock protein 90 gene and its expression in Gobiocypris rarus juveniles exposed to pentachlorophenol Qiuping Liu . Shuting Huang . Chuan Deng . Li Xiong . Xiang Gao . Yun Chen . Chunqing Niu . Yan Liu

Received: 8 February 2015 / Accepted: 1 June 2015  Springer Science+Business Media Dordrecht 2015

Abstract Heat–shock protein 90 (HSP90) is an abundant and highly conserved molecular chaperone, and it fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity, and proper regulation of a subset of cytosolic proteins. In this study, the full-length 2693-bp cDNA of HSP90 was cloned by rapid amplification of cDNA ends (RACE) technique from the liver of rare minnow (Gobiocypris rarus) for the first time, designated as GrHSP90. The complete coding sequence of GrHSP90 is 2181 bp in length, which encodes a polypeptide of 726 amino acids with a predicted molecular mass of 83.4 kDa and a theoretical isoelectric point of 4.90. Phylogenetic tree analysis indicated that deduced protein GrHSP90 had extensive sequence similarities to other fish HSP90s. Tissue distribution showed that GrHSP90 was constitutively expressed in a wide range of tissues including gill, blood, brain, fin, gonad, heart, intestine, kidney, liver, muscle, spleen, skin, and swim bladder. The highest expression was found in the gonad. Furthermore, significant increase in GrHSP90 mRNA in the liver was observed after exposure to Q. Liu  S. Huang  C. Deng  L. Xiong  X. Gao  Y. Chen  C. Niu  Y. Liu (&) Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, Key Laboratory of Ecoenvironments in Three Gorges Reservoir Region, School of Life Sciences, Southwest University, Chongqing 400715, China e-mail: [email protected]

pentachlorophenol C8 lg/L (p \ 0.05). Our results suggest that GrHSP90 is indeed an ortholog of the HSP90 family and may be act as a biomarker to assess the effect of environmental contaminant. Keywords Gobiocypris rarus  HSP90  RACE  Pentachlorophenol  Expression

Introduction Heat–shock proteins (HSPs) are generally known as stress proteins because they are induced in response to a wide variety of physiological and environmental insults. HSPs are the most abundant soluble intracellular proteins which conserved in all organisms, from prokaryotes to eukaryotes (Bose et al. 1996; Srivastava 2002; Ahn et al. 2003). HSPs have been classified into five families according to their molecular weight: HSP110, HSP90, HSP70, HSP60, and the family of small HSPs (Lindquist and Craig 1988). Among all HSPs, HSP90 is one of the most abundant proteins (Lai et al. 1984), with the abundance at 1–2 % of the total soluble cytosolic protein in most unstressed tissues. HSP90, co-chaperoned with other proteins such as the HSP70 family proteins (Pratt and Toft 2003; Zhang et al. 2013), is a highly conserved and abundant protein. In fish, like in mammals, the HSP90 has been related to cytoprotection, cell survival, and immune responses exerting a protective role (Aneja et al. 2006; Celi et al. 2012). In addition, in

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aquaculture industry, the HSP90 gene expression had even been characterized as a sensitive marker to examine environmental stress (Wang et al. 2011). HSP90 has potential as a new biomarker in environment because it can be triggered by a range of stressful conditions such as high temperature and humidity (Palmisano et al. 2000; Gibbs et al. 2003; Sonoda et al. 2006), hyperosmotic stress (Pan et al. 2000), food deprivation (Cara et al. 2005), oxidative stress (Gophna and Ron 2003), polychlorinated biphenyl (PCB) (Kietz and Fischer 2003), arsenates (Somji et al. 2002), and the presence of heavy metals (Ali et al. 1996; Gao et al. 2007; Liu et al. 2012). While HSP90 was not extensively studied compared to HSP70, to our knowledge, there were no reports on HSP90 expression under pentachlorophenol exposure in G. rarus. Pentachlorophenol (PCP) is a widespread, persistent environmental contaminant, and the International Agency for Research on Cancer (IARC) classified PCP as a group 2B carcinogen, possibly carcinogenic to humans (Jin et al. 2012; Zheng et al. 2012). Actually, since 1980s, the concern about the toxicity of PCP and the potential adverse effects on human being and the environment led to a regulatory action to limit its use (Buono et al. 2012). In China, PCP and its salts were used principally as a molluscicide to control snail-borne schistosomiasis as well as a wood preservative (Jin et al. 2012). PCP could result in many adverse effects and diseases in humans and animals, including liver defects, damage to the immune system, reproductive defects, genetic toxicity, and endocrine disrupting activity (Pavlica et al. 2000; Yin et al. 2006; Zha et al. 2006), and was identified by the US Environmental Protection Agency (USEPA) as an endocrine-disrupting chemical (Zhao et al. 2006; Zhang et al. 2014). Buono showed that the PCP effects were not significant at concentrations below 30 lg/L (p \ 0.05), and the lowest observed effect concentration (LOEC) was 50 lg/L, but increased at higher values reaching 100 % of abnormalities at 300 lg/L, and yielding an EC50 of (147.84 ± 12.30) lg/L (Buono et al. 2012). Rare minnow (Gobiocypris rarus) is a Chinese freshwater cyprinid and now developing to be as an alternative organism in aquatic toxicity tests due to its small size, ease culture, short life cycle, and prolific egg production with high fertilization and hatching rates (Liao et al. 2006; Cao et al. 2012). In

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environmental science research area, the fish has been approved to be sensitive to many kinds of organic pollutants (Zhong et al. 2005; Liao et al. 2006, 2009). Thus, the rare minnow was selected as an ideal fish model for the study of the toxicity and molecular mechanism of PCP. In this study, GrHSP90 was cloned and characterized from G. rarus for the first time, and the expression of HSP90 in G. rarus was examined after PCP exposure in order to better understand its potential roles in response to environmental contaminant.

Materials and methods Test fish and culture conditions G. rarus were purchased from Institute of Hydrobiology (IHB) in Wuhan and had been raised in our laboratory for more than 6 years. The fish were kept in flow-through system with dechlorinated tap water and subjected to a 12:12 h of light/dark at 25 ± 1 C. G. rarus juveniles were obtained from fertilized eggs by artificial fertilization method. They were fed newly hatched brine shrimp (Artemia salina) twice a day. Waste and residue were removed daily, while the test equipment and chambers were cleaned once a week. PCP exposure Thirty days after hatching, G. rarus juveniles (36.6 ± 3.7 mm in body length and 0.53 ± 0.15 g in weight; n = 60) were randomly divided into 12 groups (five fish per group). Six groups were exposed to various concentrations (8, 16, 80, 160 lg/L) of PCP, 50 ng/L EE2 as positive control and 0.001 % DMSO (v/v) as solvent control for 7 days, and other six groups were exposed to 80 lg/L PCP for different times (0, 0.5, 1, 3, 5, 7 days), respectively. The exposure solution was renewed once a day. Tissue sampling Before the caudal fin was severed with a pair of fine scissors and blood was carefully drawn from the wound by using a pipet fitted with a heparinized tip (Fatemeh et al. 2013), G. rarus juveniles were anesthetized on ice. Then, the abdomen of each fish was dissected on the left side, and skin, swim bladder,

Fish Physiol Biochem

spleen, liver, gonad, gill, kidney, muscle, brain, heart, intestine, and fin were excised from three untreated healthy fish, resulting in three same tissue samples were pooled for RNA extraction; the livers excised from the fish in each exposure group were pooled for RNA extraction. These samples were flash-frozen in liquid nitrogen and stored at -80 C until RNA extraction. No fish died during the course of the exposure. Total RNA extraction and cDNA preparation Total RNA was extracted from different tissues using RNA simple Total RNA Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. The RNA samples were dissolved in ribonuclease-free water, and the quality was measured by electrophoresis on 1.2 % agarose gel. The RNA samples were stored at -80 C until being subjected to RT-PCR. The first-strand cDNA was synthesized with 1 lg total RNA using a Prime ScriptTM RT reagent kit with a gDNA Eraser (Takara, Dalian, China). The samples were stored at -20 C until analysis. Molecular cloning of GrHSP90 Two degenerated primers, HSP90-F and HSP90-R (Table 1), were designed based on the conserved cDNA sequence regions of Danio rerio (NP-571403), Tanichthys albonubes (ADK27678), Oncorhynchus mykiss (NP-001117703), Paralichthys olivaceus (AA092751), Epinephelus coioides (ACV04938),

Salmo salar (NP-001117004). cDNA was used as template to amplify the core region of GrHSP90 with the degenerated primers. The PCR program included an initial denaturation at 94 C for 10 min, followed by 35 cycles of denaturation at 94 C for 40 s, annealing at 56 C for 30 s, and elongation at 72 C for 40 s, followed by extension at 72 C for 10 min. The obtained PCR products were separated on 1 % agarose gel and then gel-purified with the QIAquick Gel Extraction Kit (Qiagen, Germany). The purified PCR product was ligated with the pMD-19T vector (Takara, Dalian, China) and transformed into competent cells of Escherichia coli DH5a. Positive clones were checked for inserts of correct size using PCR and were sequenced (Invitrogen, Shanghai, China). The recombinants were identified through colony PCR. Three of the positive clones were sequenced by Invitrogen Corp (Shanghai, China). To extend the GrHSP90 cDNA sequence in the 30 and 50 directions, respectively, four gene-specific primers (sense primers: HSP90-30 F1 and HSP9030 F2; antisense primers: HSP90-50 R1 and HSP9050 R2; Table 1) were designed according to the sequence of the DNA fragment obtained above. The total RNA obtained above was reversely transcribed into the first-strand cDNA according to the protocol for the synthesis of two separate cDNA populations: 50 -RACE-Ready cDNA and 30 -RACE-Ready cDNA in the SMARTTM RACE cDNA Amplification Kit User Manual. The first-strand cDNA obtained was used as a template. The 30 -end RACE PCR was performed with the specific forward primer HSP90-

Table 1 Sequence of primers used in this study Primers

Nucleotide sequences (50 –30 )

Annealing temperature (C)

Use/function

HSP90-F

GAGACNTTTGCNTTYCARGC

56

Partial cDNA amplification

HSP90-R

RTGNGTYTGNGGRTCRTC

HSP90-30 F1 HSP90-30 F2

AGTCGTCTGCTGGCGGCTCTTTCAC AGACCACCTGGCTGTCAAGCATTTC

60 62

30 RACE 30 RACE

HSP90-50 R1

CAGAGTCCACAACACCACGGATAAAGT

60

50 RACE

HSP90-50 R2

ACGGCGAGGAATGAACAGAAGAGCG

63

50 RACE

HSP90-rF

AGGCCAAGTTTGAGAACCTCTG

58

Real-time quantitative PCR

HSP90-rR

CGTAAGTGCTGGTCACGATACAG

b-actin-rF

CAGGGCGTGATGGTGGGGAT

58

Real-time quantitative PCR

b-actin-rR

GGTTGGCTTTGGGGTTGAG

Letter nucleotide code: Y = C/T; R = A/G; N = A/C/G/T

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30 F1 and the universal primer (UPM) with an annealing temperature at 60 C; the nested PCR was carried out with the primer HSP90-30 F2 and UPM with an annealing temperature at 62 C. For the 50 -end, PCR was initially carried out with the tailed cDNA and the primer HSP0-50 R1 and UPM with an annealing temperature at 60 C; the nested PCR was carried out with the specific reverse primer HSP90-50 R2 and UPM with an annealing temperature at 63 C. The PCR products were gel-purified, cloned, and sequenced as described above.

length GrHSP90 cDNA, the HSP90-rF and HSP90rR primers (Table 1) were designed and used to amplify a 120-bp fragment of the GrHSP90 gene. The cycling conditions were as follows: an initial denaturation step at 95 C for 60 s, followed by 40 cycles of 95 C for 15 s, 58 C for 10 s and 72 C for 20 s, and the last cycle for dissociation curve. All samples were amplified in triplicates. The GrHSP90 gene expression was normalized to bactin and expressed as a folded change relative to the expression level in the control according to the 2-DDCT method (Livak and Schmittgen 2001).

Sequence analysis Statistical analyses The full-length sequence of GrHSP90 was assembled by the DNASTAR multiple program package (DNASTAR Inc., USA). The molecular weight, theoretical pI, and amino acid composition were computed using ProtParam tool (http://web.expasy. org/protparam/). Multiple alignments of the GrHSP90 sequence were performed with Clustal X1.83 (Thompson et al. 1997). The SMART program (Letunic et al. 2006) was used to predict the protein motif features and analyze the domain architecture. Conserved Domain Architecture Retrieval Tool (CDART) (Geer et al. 2002) and Conserved Domain Database (CDD) (MarchlerBauer and Bryant 2004; Marchler-Bauer et al. 2009, 2011) in NCBI were used to find the conserved domains of GrHSP90. A phylogenic tree was constructed using the neighbor-joining method by MEGA 5.0 (Tamura et al. 2007) based on the sequences of GrHSP90 and other known HSP90 in a public database. Quantitative real-time PCR To analyze tissue distribution of GrHSP90 gene and effects of PCP on the expression of GrHSP90, quantitative real-time PCR (qRT-PCR) was conducted with b-actin gene (GenBank accession EF597101) as endogenous control. Total RNA was extracted, and 1 lg total RNA was reverse-transcribed. qRT-PCR was performed using the Bio-Rad iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, USA). The qRT-PCR mixture contained 1 lL cDNA, 5 lL SYBR Premix Ex TaqTM II (29, Takara, China), 0.8 lL of each primer, and 2.4 lL deionized water. Based on the obtained full-

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All quantitative data were expressed as mean ± SEM. Statistical analysis of variance (ANOVA) was performed using SPSS (version 17.0), and all graphs are made by origin (version 8.0). A probability of p \ 0.05 was considered to be significant, and p \ 0.01 was considered to be statistically extremely significant.

Results Cloning of the GrHSP90 cDNA The core region of GrHSP90 cDNA was amplified with the degenerated primers (Table 1), and a 1995-bp fragment (Fig. 1A) was amplified. Based on the known partial sequence, specific primers (Table 1) were designed to clone the 30 end (Fig. 1B) and the 50 end of GrHSP90 (Fig. 1C) by rapid amplification of cDNA ends (RACE) technique, respectively. Then, a 2693-bp nucleotide sequence representing the complete cDNA sequence of GrHSP90 gene was obtained by sequence assembly of the above sequences using DNASTAR multiple program package (DNASTAR Inc., USA). The complete cDNA sequence of the G. rarus HSP90 gene was deposited in GenBank under accession no. KM201321. Characterization of the full-length GrHSP90 Sequence analysis of GrHSP90 cDNA revealed: (1) an ORF of 2181 bp encoding 726 amino acids, with a calculated molecular mass of 87.1 kDa and an isoelectric point of 4.96; (2) instability index computed to be 41.34, which classifies the protein as

Fish Physiol Biochem

Fig. 1 Electrophoresis of HSP90 from G. rarus amplified by PCR. A: core region of GrHSP90 cDNA, B: amplified fragment of 30 RACE, C: amplified fragment of 50 RACE, M: DNA Marker DL2000

unstable; (3) a 95 bp of 50 -UTR located upstream of a start codon (ATG); and (4) a 417 bp 30 -UTR that ended in a poly (A) tail (Fig. 2). The deduced amino acid sequence of GrHSP90 included one typical HSP90 family motif at residues 32–41 (-YSNKEIFLRE-), and the characteristic cytoplasmic motif (-MEEVD-) of HSP90 was located at residues 722–726 (Fig. 3). Analysis of conserved domains of GrHSP90 found that there was a HATPases-c domain (histidine kinase-like ATPases) and a pfam domain (HSP90 protein) located at residues 38–184 and 190–726, respectively (Fig. 2). Comparison and phylogenetic analysis of GrHSP90 Multiple alignments were performed to compare the GrHSP90 amino acid sequence with known HSP90 sequences from different species (Fig. 3). The result showed that HSP90 family is highly conserved. The phylogenetic tree based on the GrHSP90 and other known HSP90 amino acid sequences showed that all of the sequences were divided into six major groups: fish HSP90, amphibians HSP90, reptiles HSP90, birds HSP90, mammals HSP90, and homo sapiens HSP90 (Fig. 4). GrHSP90 was located in the fish HSP90 group and was most closely related to TaHSP90 (Tanichthys albonubes HSP90). Tissue distribution of GrHSP90 mRNA Using qRT-PCR, the expression of GrHSP90 was investigated in various tissues of G. rarus juveniles. The expression of GrHSP90 mRNA was detected in all

13 tissues with different expression levels. There was a high expression level of GrHSP90 in the gonad, kidney, intestine, and liver, while a low expression level was in blood, fin, skin, and swim bladder (Fig. 5). The highest expression level of GrHSP90 was detected in gonad. Expression of GrHSP90 in response to PCP To study the response to resist the environmental contaminant, the expression of GrHSP90 mRNA in liver after PCP exposure was measured by qRT-PCR. G. rarus juveniles were exposed to 8, 16, 80, 160 lg/L PCP for 7 days, respectively. The expression of GrHSP90 mRNA was found to be extremely significantly up-regulated after 8, 16, 80 lg/L PCP and 50 ng/L EE2 exposure (p \ 0.01), and 7-day exposure to 160 lg/L PCP caused significant increase in GrHSP90 mRNA expression compare to control (p \ 0.05) (Fig. 6A). In the early days after 80 lg/L PCP exposure, mRNA levels of GrHSP90 were not significantly change, as time progressed, and the expressions of GrHSP90 were significantly up-regulated after exposure (p \ 0.05) (Fig. 6B).

Discussion HSP90s are a group of abundantly expressed and highly conserved molecular chaperones whose exact function is presently undefined. They recognize and regulate the activity of several intracellular substrates and also operate in the absence of stress (Jakob and Buchner 1994).

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In the present study, the complete cDNA sequence of GrHSP90 (GenBank accession no. KM201321) was determined by 50 /30 RACE. The full-length cDNA of GrHSP90 is 2693 bp, with an open reading frame of 2181 bp encoding a polypeptide of 726 amino acids. Amino acid sequence analysis shows that GrHSP90 displayed one signature sequence (-YSNKEIFLRE-), which is well conserved in the eukaryotic HSP90 family. Additionally, a highly conserved motif (-MEEVD-) is found at the C-terminal region, which is believed as an important character of cytosolic HSPs that mediates inter-domain communication and peptide-binding capacity (Gupta 1995). The pentapeptide is essentially existed at all eukaryotic HSP90s C-terminus (Picard 2002), which constitutes the core of the HSP90 interaction surface for the tetratricopeptide repeats (TPRs) of HSP90 co-chaperones (Minami et al. 1994; Meng et al. 1996; Maruya et al. 1999; Ramsey et al. 2000; Scheufler et al. 2000; Richter et al. 2001; Picard 2002). There is a histidine kinase-like ATPases (HATPases-c domain) in the GrHSP90 N-terminal region, which is the N-terminal ATPase domain (Zhao and Houry 2005). HATPases-c domain is found in several ATP-binding proteins, for example: histidine kinase, DNA gyrase B, topoisomerases (Bellon et al. 2004), heat–shock protein 90 (Immormino et al. 2004; Roe et al. 2004; Wright et al. 2004), phytochrome-like ATPases, and DNA mismatch repair proteins. In addition, a pfam domain (HSP90 protein) is also found in the GrHSP90 C-terminal region, which is a protein-binding domain (Zhao and Houry 2005). These conserved molecular structures indicate that GrHSP90 may function as molecular chaperone just like the HSP90s from other organisms. Phylogenetic analysis shows that the HSP90 evolutionary tree is divided into six groups. GrHSP90 is grouped with fish HSP90 and have closer relationship with Tanichthys albonubes (Fig. 4), suggesting that GrHSP90 is homologue of the known fish HSP90s. Under non-stressed condition, GrHSP90 transcripts in all 13 tissues tested. This universal distribution reveals that as an important molecular chaperone molecule, HSP90 plays a housekeeper role in organisms while controlling a variety of protein activities, transportation, and updates (Young 1990). Previous review reported that biomarkers were used as early biological signals to indicate the harmful effects of

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Fig. 2 The cDNA sequence and the deduced amino acid c sequence of GrHSP90. The HATPases-c domain (histidine kinase-like ATPases) is underlined by …, the pfam domain (HSP90 protein) is underlined by —, conserved consensus signature sequence (-YSNKEIFLRE-) is boxed, and conserved motif (-MEEVD-) of HSP90 family is shown in box and shadowed. The stop codon is marked with an asterisk (*)

environmental stress, which could help us to recognize the impact of pollutants on aquatic animals in the early stages and to assess the risk to the aquatic environment (Tom and Auslander 2005). Heat–shock proteins are often used as biomarkers in environmental toxicology (Ryan and Hightower 1996). There are some studies demonstrated the potential utility of HSP90 as a biomarker in aquatic toxicology: HSP90 mRNA expression displayed a clear dose-dependent expression pattern in Cd2?-treated Chlamys farreri (Gao et al. 2007); the expression of HSP90 significantly increased in both heart and brain tissues of Caretta caretta in response to acute heat stress (Tedeschi et al. 2015); food deprivation significantly enhanced HSP90 protein expression in trout larvae, while fed controls had undetectable protein content (Cara et al. 2005); while as a biomarker, the mRNA expression of GDH (glutathione peroxidase) did not display a clear dosedependent expression pattern, and the extremely significant increase was only about 1.8-fold in G. rarus exposed to PCP (Fang et al. 2010). In our study, the expression of GrHSP90 in the liver, at the messenger RNA (mRNA) transcription level, was obtained after G. rarus juveniles subjected to PCP treatment. After different concentrations of PCP exposure for 7 days, the expression of GrHSP90 reached the peak level at 80 lg/L PCP (Fig. 6a), and after 80 lg/L PCP exposure for different times, the expression of GrHSP90 reached the peak level at the 3rd day (Fig. 6b). Under PCP treatment, there was a dosedependent or time-dependent expression change in GrHSP90, and it had a decreasing tendency after the peak expressive level in both concentration and time. Our data are in accordance with the HSP90 mRNA expression in the liver of Tanichthys albonubes subjected to 27 lg/L Cu2? or 2.31 mg/L Cd2? for different times (Liu et al. 2012). In the previous study, there were also some results to indicate low-dose-dependent response or the opposite results between the lower dose and the highest dose (Meucci and Arukwe 2006; Jin

Fish Physiol Biochem 1

ACATG

6

GGGCTCCTCTGCTTCCCTGAGTATCAGTGTAGGGGAGATCAGCTGTTTCTGTGGCGATTAAACCTTCAAAAATCAACGAAACTAACCAAG

96 ATGCCTGAAGAAATGCGCCAAGATGAGGAGGCCGAGACCTTTGCCTTCCAGGCAGAGATTGCCCAGCTGATGTCTCTTATTATTAACACC M

P

E

E

M

R

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E

A

E

T

F

A

F

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A

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I

A

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L

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186 TTCTACTCCAACAAAGAGATTTTCCTCAGGGAGCTCATCTCCAACGCCTCTGATGCTCTTGACAAAATCAGATATGAAAGTTTGACGGAT F

Y

S

N

K

E

I

F

L

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L

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A

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A

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276 CCCACCAAACTGGACAGTGGCAAGGACCTGAAGATCGACATCATCCCCAATGTTCATGATCGCACTCTTACCATTATTGACACCGGTATT P

T

K

L

D

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G

K

D

L

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366 GGCATGACCAAAGCCGATCTCATCAATAACCTGGGTACCATCGCAAAGTCCGGAACAAAGGCCTTCATGGAGGCTCTGCAGGCTGGTGCT G

M

T

K

A

D

L

I

N

N

L

G

T

I

A

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G

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A

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A

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A

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A

456 GATATCTCCATGATTGGGCAGTTTGGTGTGGGCTTCTACTCTGCCTACCTGGTGGCTGAAAAGGTCACAGTCATCACTAAAAACAATGAT D

I

S

M

I

G

Q

F

G

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G

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Y

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546 GACGAGCAGTACGCCTGGGAGTCGTCTGCTGGCGGCTCTTTCACAGTCAAGGTGGACCATGGTGAGCCCATTGGCCGTGGAACCAGAGTC D

E

Q

Y

A

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636 ATTCTTCACCTGAAAGAAGATCAGACCGAGTACGTCGAGGAGAAGAGGGTGAAGGAAGTGGTCAAGAAACACTCCCAGTTCATCGGATAC I

L

H

L

K

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726 CCAATCACTCTCTTCGTGGAGAAGGAACGCGACAAGGAGATCAGCGACGACGAGGCAGAGGAGGAGAAGGCTGAGAAGGAAGAAAAGGAG P

I

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L

F

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816 GAAGTCGAAGGCGAAGACAAACCCAAGATTGAAGATGTCGGCTCAGATGACGAAGAGGACTCCAAAGACAAGGACAAGAAGAAAAAGAAG E

V

E

G

E

D

K

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906 AAGATCAAGGAGAAGTACATTGACCAGGAGGAGCTGAACAAAACCAAACCCATCTGGACCCGCAACCCCGACGACATCTCCAACGAGGAG K

I

K

E

K

Y

I

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996 TACGGAGAGTTTTACAAGAGCCTGACTAATGATTGGGAAGACCACCTGGCTGTCAAGCATTTCTCCGTGGAGGGTCAGCTGGAGTTTCGC Y

G

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Y

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1086 GCTCTTCTGTTCATTCCTCGCCGTGCCCCCTTCGACCTCTTCGAGAACAAGAAAAAGAAGAATAACATCAAGCTGTACGTCAGAAGGGTC A

L

L

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1176 TTCATCATGGACAGCTGTGAGGAGCTCATCCCAGAATATCTGAACTTTATCCGTGGTGTTGTGGACTCTGAAGATCTGCCCCTCAACATC F

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C

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1266 TCCAGAGAGATGCTTCAACAGAGCAAGATTCTCAAGGTCATCCGCAAGAACATTGTCAAGAAGTGCCTGGAGCCGTTTGCTGAGCTGGCC I

S

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1356 GAGGACAAGGAAAACTACAAGAAGTTCTACGACGCTTTCTCCAAGAATCTTAAACTGGGCATCCACGAGGACTCTCAGAACCGCAAGAAG E

D

K

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N

Y

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1446 CTGTCAGAACTGTTGCGTTATCAGAGCTCGCAGTCCGGCGACGAGATGACCTCCCTCACAGAATATGTCAGCCGTATGAAGGAAAACCAG L

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1536 AAATCCATCTACTACATCACTGGTGAGAGCAAAGACCAGGTCGCCCACTCCGCCTTTGTGGAGCGCGTGTGCAAGAGAGGTTTCGAGGTG K

S

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Y

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1626 CTGTACATGACCGAGCCCATCGACGAGTACTGCGTCCAGCAGCTTAAGGACTTCGACGGCAAGTCTCTGGTGTCCGTCACCAAAGAGGGA L

Y

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1716 CTGGAGCTGCCTGAGGATGAAGACGAGAAGAAAAAGATGGAGGAGGACAAGGCCAAGTTTGAGAACCTCTGCAAGCTCATGAAGGAGATT L

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1806 CTGGACAAGAAAGTAGAGAAGGTCACCGTATCAAACAGGCTGGTTTCTTCCCCCTGCTGTATCGTGACCAGCACTTACGGTTGGACGGCC L

D

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1896 AACATGGAGAGGATTATGAAGGCCCAGGCCCTGAGGGACAACTCCACCATGGGCTACATGATGGCCAAGAAACACCTGGAGATCAACCCT N

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123

Fish Physiol Biochem 1986 GATCATCCCATCATGGAGACCCTTAGGCAGAAGGCGGACGCAGATAAAAACGACAAAGCTGTGAAGGATCTGGTCATCCTCCTGTTCGAG D

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2076 ACTGCACTGCTTTCCTCTGGATTCTCATTGGACGATCCTCAGACACACTCAAACCGCATTTACAGAATGATCAAGCTGGGTTTAGGTATT T

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2166 GATGAAGATGAAGACGTGCCTGTGGAAGAGCCAACCGCTGCTCCCGCTCCAGAAGAGATCCCACCTCTGGAAGGAGACGATGACGCCTCT D

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2256 CGCATGGAGGAAGTTGATTAA R

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2277 ATCCATCTTGTCTGTAACTCCCCAGTCAATTTTCCGTAGTCATTCCGTAACATCTTATCGTTCTCTTTGGCCCGTAGAAGGTTAACTGAT 2367 TCTCCGTAATGATTCCTTAAAGTATCAAAATGTGTAACTAAAGCATACTGTCCACTTTTGCTCTGTTTTGAGCCGTTATGTACTTTTGTT 2457 TTTTTTACATATGTTCATGGATCAATGGGTTGCTGCAGTATTGTCTGAGCACAATAAGCACTGTGTTTCACTGATACTGAGTCTGCTGGC 2547 TGTGCTGGATTGAGTCAAGCATGATTTCATTCTGCATGGAAGGGGAAAGACCTTTTGCCTCAGTGCAGGCATGTATTGTCTTGGTTTTCA 2637 GAAATTAAAACACGGTGAAACCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Fig. 2 continued

et al. 2009; Wang et al. 2011). These may be related to two aspects as we inferred: first, the 7-day time is far too long, and in some cases, the gene expression changes occur within a few days after exposure; second, GrHSP90 expression in a dose-dependent manner regulated by PCP may be within the limits of the PCP concentration. It may have an endocrine-disrupting function and acute toxicity on G. rarus at PCP concentration \80 and [80 lg/L, respectively. Some findings together with the observation that acute toxicity of PCP to fish occurred after 96 h within a concentration range of 0.19–1.28 lM depending on the species (Dwyer et al. 2005) lead to the conclusion that fish are very sensitive to PCP (Pietsch et al. 2014). The results have been shown that the expression of GrHSP90 in experiment groups is obviously higher than the control group. The increased amount of HSP90 expression is regarded as one of the protective approaches of organisms from additional toxicity. Previous study reported that HSP90 was likely to function as an essential chaperone involved in immune response by hydrolyzing ATP (Pearl and Prodromou 2006). These data would be helpful to understand the significance of HSP90 to immune defense in G. rarus. In addition, our results suggested that GrHSP90 was very sensitive to PCP exposure, and the results confirmed that

123

Fig. 3 Multiple alignment of GrHSP90 with other species in c GenBank. Highly conserved amino acids are highlighted in dark gray and consensus residues in light gray background. Conserved consensus signature sequence (-YSNKEIFLRE-) and conserved motif (-MEEVD-) of HSP90 family are shown in red boxes and underlined. The GenBank accession numbers of the HSP90s are as follows: GrHSP90 (Gobiocypris rarus; KM201321), TaHSP90 (Tanichthys albonubes; ADK27678), MaHSP90 (Megalobrama amblycephala; AGI97008), DrHSP90 (Danio rerio; NP_571385), LoHSP90 (Lepisosteus oculatus; XP_006625986); SsHSP90 (Salmo salar; NP_001117004), DlHSP90 (Dicentrarchus labrax; AAQ95586). (Color figure online)

HSP90 could act as a sensitive biomarker to examine environmental pollution. In conclusion, the complete cDNA sequence of G. rarus HSP90 gene, GrHSP90, was isolated from the liver for the first time (GenBank accession no. KM201321), and its expression under PCP exposure was analyzed. Further studies are required to clarify GrHSP90 as a biomarker in relation to some other stressful conditions, and it is also needed to collect protein expression data to clarify the transient changes in the transcript levels of the GrHSP90 protein. Moreover, a better knowledge of their regulation by hormonal factors both at transcriptional and translational levels would be helpful for developing G. rarus as a potential experimental animal in aquatic toxicology research.

Fish Physiol Biochem

123

Fish Physiol Biochem 88

Dicentrarchus labrax Paralichthys olivaceus Scophthalmus maximus Oncorhynchus mykiss Salmo salar

89 57 41

100

Danio rerio *Gobiocypris rarus Tanichthys albonubes Oreochromis niloticus Xenopus laevis Bufo gargarizans

91 100 100 96

99 61 96 100

Fig. 4 Phylogenetic analysis of HSP90 sequences. A neighborjoining tree was generated using HSP90 amino acids of 17 species from fish, amphibians, reptiles, birds, mammals, and homo sapiens. The percentage bootstrap values obtained from 1000 re-samplings were shown at the nodes. HSP90 amino acids were from the following species with their accession numbers: Tanichthys albonubes (ADK27678), *Gobiocypris rarus (KM201321), Danio rerio (AAC21566), Oncorhynchus mykiss (NP_001117703), Anolis carolinensis (XP_003215948),

Relative HSP90 mRNA expression

14

Reptiles Birds Mammals Homo sapiens

Macaca fascicularis (BAE87589), Xenopus laevis (NP_001086624), Alligator mississippiensis (BAF94147), Salmo salar (NP_001117004), Gallus gallus (NP_996842), Bufo gargarizans (ABD75383), Oreochromis niloticus (XP_003446339), Dicentrarchus labrax (AAQ95586), Scophthalmus maximus (ABU50778), Paralichthys olivaceus (AAO92751), Mus musculus (BAE27553), Homo sapiens (NP_031381)

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Fig. 5 Expression of GrHSP90 mRNA analyzed by real-time PCR in different tissues of G. rarus juveniles. The expression level was analyzed by 2-DDCT method, and the expression of GrHSP90 in gill was taken as reference for the expression in the other tissues. The b-actin gene was explored as an internal

123

Amphibians

Anolis carolinensis Gallus gallus Alligator mississippiensis Macaca fascicularis Homo sapiens Mus musculus

97

Fish

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control. Bars represent the mean ± SEM of three experiments for each condition. Error bars represent the SEM for the average fold changes. *p \ 0.05 and **p \ 0.01 indicate significant differences between tissues and the control tissue

Fish Physiol Biochem

GrHSP90 mRNA expression (relative to control)

3

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Control

8µ g/L

16 µg/L

80 µg/L

160 µg/L 50 ng/L EE2

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GrHSP90 mRNA expression (relative to control)

B

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time (day)

Fig. 6 Effects of PCP on the expression of GrHSP90 mRNA in G. rarus juveniles. The 30-dpf G. rarus juveniles were exposed to (A) different concentrations of PCP for 7 days or (B) 80 lg/L PCP for different days. Bars represent the mean ± SEM for

each experiment and control groups. Error bars represent the SEM for the average fold changes. *p \ 0.05 and **p \ 0.01 indicate significant differences between exposure groups and the control group

Acknowledgments The authors express their sincere thanks for the financial support received from the National Natural Science Foundation, China (21147002), and the National Program on Key Basic Research Project (973 Program; 2012CB723205) for this study.

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