Heat Shock Proteins (HSPs) are synthesized in response to various environmental stresses including heat shock. The major families of HSPs, HSP70 and HSP90 are most abundant and express in a negligible level in the absence of stress (Somero, 1995; Lai et al., 1984). HSP70 is known to assist the folding of nascent polypeptide chains, act as a molecular chaperone and mediate the repair and degradation of altered or denatured proteins (Kregel, 2002), whereas HSP90 supports various components of the cytoskeleton and steroid hormone receptors (Young et al., 2001). HSPs and heat shock response are highly conserved among phylogenetically divergent organisms (Basu et al., 2002), which attests to significant roles these molecules play in cellular function and adaptation.
For poikilothermal animals, temperature regulation of gene expression is certain to be critical for the survival and success in variable thermal environments. HSPs may play a role in physiological adaptation to wide ranges of temperature change, such as seasonal or diurnal shifts. The most common freshwater sunfish specie, bluegill Lepomis macrochirus Rafinesque is native to the middle and eastern regions of North America. It has been introduced to all over North America and other countries and its distribution is now confirmed in 20 countries (Froese and Pauly, 2006; Welcomme, 1992). Their successful invasion is thought to result from their omnivorous feeding habit and wide tolerance to different environments.
In the present study, partial sequences of HSP70 and 90 from bluegills were isolated and sex- and seasonal- specific expression; May (breeding season of bluegill), September and December, was assessed in the tissues of whole organisms to evaluate the role of HSPs in the tolerance to the seasonal environmental changes.
MATERIALS AND METHODS
Collection of experimental fish: Samplings of L. macrochirus were done by fishing on May, September and December 2006 at wetlands of Iwaki city, Fukushima prefecture, Japan. Immediately after captured, internal organs were extracted at collection site and soaked in 500 μL RNA stabilization solution RNAlater (Applied Biosystems, California, USA) in a 1.5 mL microfuge tube.
RNA extraction and cloning of the partial HSP genes: Total RNA was purified
from brain, liver, reproductive organs (testis or ovary), heart, kidney, gill
and muscle by RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription
of the RNA was performed by ThermoScript RT-PCR System (Invitrogen, California,
USA). HSP70 cDNA and HSP90 cDNA were amplified by PCR with Takara Ex Taq Reaction
Kit (Takara Bio, Shiga, Japan). Total PCR reaction volume of 30 μL was
composed of 3.0 μL 10X Ex Taq Buffer, 3.0 μL dNTP mixture, 2.1 pmol
of each primer, 0.8 units Ex Taq and 1.0 μL DNA solution containing 0.15
μg cDNA. Degenerate oligonucleotide primers used in PCR (HSP70-F; 5'- CARGAYTTYTTYAAYGGAAARGA-3',
HSP70-R; 5'-CCCCCAGCACTYTGRTANAGKTT-3', HSP90-F; 5'-ATGCGCCAAGARGARGAGG-3',
HSP90-R; 5'-CWGARAAGTGCTTGACAGCC-3') were based on previous reports (Palmisano
et al., 1999; Deane et al., 2000). The profile of PCR conditions
was as follows: initial denaturation at 95°C for 5 min; 35 or 40 cycles
of denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec, extension
at 72°C for 1 min and a final extension at 72°C for 7 min. The PCR products
of HSP genes were ligated into the pCR 2.1-TOPO vector by the use of TOPO TA
Cloning (Invitrogen). The sequences of HSP genes were confirmed by DNA sequencing
with ABI PRISMTM 3730xl DNA Analyzer (Applied Biosystems). The HSP
gene sequences were compared with all other known gene sequences through a BLAST
search. Similar DNA sequences were downloaded from the DNA Data Bank of Japan
(DDBJ) and aligned with our sequences.
Evaluation of HSP expression: Detection of HSP70 and HSP90 transcripts
were carried out using RT-PCR. At the same time, amplification of β-actin
was performed and the product was used as an internal standard of RT-PCR. HSP
specific oligonucleotide primers used in RT-PCR (RT-HSP70-F; 5'- GCTCAACAAGAGCATCAATCCAGAT
-3', RT-HSP70-R; 5'- CTCCACAGTCGACTTCATGTTGAAA -3', RT-HSP90-F; 5'- CACCTTCTATTCCAACAAAGAGATC
-3', RT-HSP90-R; 5'- TGAACTGGGAGTGCTTCTTGACAAT -3') were designed by sequence
data of L. macrochirus HSPgenes reported in the present study. β-actin
detective primers (Actin-F; 5'-CAATGGATCCGGTATGTGC-3', Actin-R; 5'-CGTTGTAGAAGGTGTGATGCC-3')
were based on previous report (Naito et al., 1998). RNA extraction, cDNA
synthesis and PCR amplification methods were as previously described.
cDNA partial sequence of HSP70 and HSP90: Two newly reported cDNA sequences
were compared with the DDBJ database. Lepomis HSP70 partial sequence
(751 bp, Fig. 1) showed 93.6% HSP70 sequence similarity to
Dicentrarchus labrax Linnaeus (accession No. AY423555), 93.2% to Rhabdosargus
sarba Forsskål (AY436786), 92.5% to Paralichthys olivace Temminck
et Schlegel (AB006814), 88.0% to Danio rerio Hamilton (L77146), respectively.
Lepomis HSP90 partial sequence (910 bp, Fig. 2) showed
94.8% HSP90 sequence similarity to Dicentrarchus labrax (AY395632), 91.4%
to Paralichthys olivace (AY214170), 87.6% to Oncorhynchus mykiss
Walbaum (AB196457), 87.0% to Salmo salar Linnaeus (AF135117), respectively.
The cDNA sequence data reported in this study submitted to the DDBJ/EMBL/Gen-Bank
database and were assigned the accession numbers AB296301 to HSP70 and AB296302
Evaluation of HSP mRNA expression: RT-PCR revealed that Lepomis
HSP70 and 90 mRNA were widely distributed in different organs (Fig.
3). HSP70 and 90 mRNA were found in brain, liver and gill regardless of
sex or seasonality.
macrochirus HSP70 cDNA partial sequence together with its deduced
amino acid translation. GenBank DDBJ/EMBL/Gen-Bank database Accession
macrochirus HSP90 cDNA partial sequence together with its deduced
amino acid translation. GenBank DDBJ/EMBL/Gen-Bank database Accession
analysis in different organs at different season. RT-PCR amplification
products for HSP70 and HSP90 fragments and the constitutive control of
Although there were slight variations, they were also detected in gonad, heart,
kidney and muscle and it seemed that there were neither sex- nor seasonal-specific
expression. There was no apparent difference of tissue distribution between
HSP70 and 90.
The present study reports the isolation and sequencing of cDNA partial clone corresponding to the Lepomis HSP70 and HSP90 that have highly sequence homology with other teleost species. And the comparison of the distribution of mRNA revealed that bluegill HSP70 and 90 mRNA were widely abundant in the tissues regardless of sex or seasonality including breeding season.
Numerous studies have been documented about HSPs mRNA expression induced by
heat shock (Arai et al., 1995; Molina et al., 2000; Basu et
al., 2002; Ojima et al., 2005). However, many of these were carried
out using in vitro cell culture. Few studies were reported about the
induction of HSP in the tissues of whole organisms in response to elevated temperature
by the effect of naturally occurring variations in environmental temperature.
For example, the threshold induction temperature of HSP90 protein had a seasonal
variation in the gobies Gillichthys mirabilis Cooper and G. seta
Ginsburg (Dietz and Somero, 1992). Four marine teleost that habited in 10°C
water showed tissue-specific variation in a specie and the wide variation in
induction temperature among the species in the level of both HSP70 and HSP90
proteins (Dietz and Somero, 1993). As for mRNA expression, tissue-specific expression
induced by heat shock was observed in embryonic zebrafish HSP70 and 90 mRNA
(Krone et al., 1997) and semi-quantitative PCR revealed that HSP70 mRNA
was detected in several organs of female tilapia Oreochromis mossambicus
Peters by heat stress, while no signal was observed in the control fish (Molina
et al., 2000). Furthermore, it was demonstrated that HSP expression was
absent in an antarctic fish Trematomus bernacchii Boulenger (Hofmann
et al., 2000). These results indicated that the expression manner of
HSPs might be extremely variable among teleost species.
Steady expression of Lepomis HSP70 and 90 mRNA regardless of sex and seasonality as seen in the present study suggests that certain level of HSP expression might be advantageous to survive in labile environment, namely, bluegill could rapidly and well adapt the change of the environment. In fact, bluegill is tolerant to wide range of different water temperature; it generally prefers temperate water, but could adapt higher temperature (O'Hara, 1968) and sustain water temperature at 2.5°C (Petrosky and Magnuson, 1973). However, only qualitative evaluation, i.e., RT-PCR, was conducted in the present study, further quantitative investigation was necessary to reveal detailed HSPs functions in the thermotolerance of bluegill.
The result in the present study suggested that HSPs play some roles in environmental adaptation in poikilothermal animals and the heat shock response may have a significant variation among teleost species according to their capacity of adaptation.
The authors are thankful to the Iwaki Public Office of the Fukushima Prefecture and the Agricultural Land Division of Iwaki City for permission to use the agricultural water reservoir in Fukushima Prefectural Iwaki Park as the sampling site for bluegill.