J. of Aquaculture Vol. 19(4) : 315-322, 2006 µ Journal of Aquaculture Korean Aquaculture Society, Acanthopagrus schlegeli HSP90, HSP70 mrna x x cortisol glucose y *, y 1, ½ùù, y, 1 w ww wy w, 1 w ww w Expression of HSP90, HSP70 mrna and Change of Plasma Cortisol and Glucose During Water Temperature Rising in Freshwater Adapted Black Porgy, Acanthopagrus schlegeli Cheol Young Choi*, Byung Hwa Min 1, Na Na Kim, Sung Hwoan Cho and Young Jin Chang 1 Division of Marine Environment & Bioscience, Korea Maritime University, Busan, 606-791 Korea 1 Department of Aquaculture, Pukyong National University, Busan. 608-737 Korea The objective of the present study was to investigate the expression of heat shock protein 90 (HSP90) and 70 (HSP70) mrna as cellular stress responses, the levels of plasma cortisol with glucose as neuro-endocrine stress responses during water temperature rising in freshwater adapted black porgy, Acanthopagrus schlegeli. A cdna fragment of 891 (HSP90) and 465 (HSP70) bp was cloned from black porgy testis by Reverse transcription-polymerase chain reaction (RT-PCR) with primers designed from the conserved regions of other teleost. The PCR product of HSP90 showed very high homology to red seabream (99%), rainbow trout (95%), Atlantic salmon (94%), zebrafish (94%) HSP90, HSP70 of black porgy was also highly similar to those of rainbow trout (96%), silver seabream (95%), zebrafish (95%) HSP70. Water temperature rising (20~30 o C) induced elevation of HSP90 mrna in black porgy gonad, liver, brain, intestine and kidney, whereas it resulted in an induction of the HSP70 mrna expression in gonad only. Plasma cortisol levels increased significantly at 30 o C in the fish compared to those at 20 o C. Glucose levels of the fish showed a tendency of co-increase with cortisol during water temperature rising. These results suggest that increased HSP90 mrna in liver with plasma cortisol following heat shock may be related to increasing glucose for homeostasis in this species. Keywords: Black porgy, Acanthopagrus schlegeli, Water temperature, HSP90, HSP70, Cortisol, Glucose,, s w e w wù, ù y p w kw k w eš. p, w y x (Logue et al., 1995). ƒ ù sü heat shock proteins (HSPs) w wì w yƒ ù (Schlesinger et al., 1992). w HSP, s, y, y w p w *Corresponding author: choic@hhu.ac.kr 315 x p (Beckmann et al., 1990). HSP p w ùkù l yw w(homeostasis) w w w w(sanders, 1993; Forsyth et al., 1997; Iwama et al., 1998; Iwama et al., 1999; Ackerman and Iwama, 2001). HSP» w HSP90 (85-90 kda), HSP70 (68-73 kda) HSP (16-47 kda) 3 ù(basu et al., 2002). ƒ j HSP90 s 1~2% w p s w(pratt, 1997), sü protein kinases, steroid receptor, actin, tubulin, calmodulin, proteasomes ww, s y w(csermely et al., 1998), p glucocorticoid y cortisol s w w w(pratt, 1997).
316, y, ½ùù, y, w HSP90 x chinook salmon, Oncorhynchus tshawytscha (Palmisano et al., 2000),, Oncorhynchus mykiss (Sathiyaa et al., 2001) zebrafish, Danio rerio (Murtha and Keller, 2003). HSP70 ƒ wù, (Roux et al., 1994) zebrafish (Graser et al., 1996), silver seabream, Sparus sarba (Deane and Woo, 2005), (Ojima et al., 2005), y y glucocorticoid w w ƒ x p (Hutchison et al., 1994). w, x cortisol p w t t š (Wedemeyer and McLeay, 1981). p, yw y w cortisol x ùkù(holloway and Leatherland, 1997), p w w w, w vw œ w w(ackerman et al., 2000). g, p s qw» w HSP90, HSP70 mrna x w, w x cortisol glucose qw, p cortisol šwš w. x y w y l s³ 14.4±0.2 cm, 48.6±0.6 g 40 w. y w, ü ƒw 10 w 3 k z w g 35 w. x 20±1 o C, Ÿ» 12L:12D w, 2z œ w. y l(js-wbp-170rp, Johnsam Co., Korea) 40 L y ƒ 2 ƒƒ 20 w z 24 g. z 20 o C 30 o C¾ 1 o C g,» œ w. x 20, 25 30 o C ƒƒ 2 3( 6)l x w. x x MS-222 (200 ppm) w z, heparin sodium»(3 ml) w xl w, (4 o C, 10000 rpm, 5)w x y ¾ -80 o C þš w. x z, x w sk z,,,, w þwƒ total RNA ¾ -80 o C w. HSP90 HSP70 cdna -80 o C,,, l Total RNA Extraction kit (Promega, USA) w total RNA w. PCR ww» w primer š k HSP90 HSP70» š w, ü mw primer w(table 1). sww RT-PCR AccuPower RT/PCR PreMix (Bioneer, Seoul, Korea) w w, HSP90 HSP70 ƒƒ 891 bp 465 bp PCR (Table 1). s PCR 1.0% agarose gel»w z, w, pgem-t Easy Vector (Promega, USA) w ³ DH 5α xyw plasmid purification kit (NucleoGen, Seoul, Korea) w plasmid DNA w. plasmid DNA» ABI DNA sequencer (Applied Biosystems, USA) w w.» BLAST GenBank w, cdna w. Table 1. Primers used for the RT-PCR of the black porgy HSP90, HSP70 and ß-actin Gene Primer DNA sequence Product size (bp) Accession number HSP90 HSP70 ß-actin HSP90-F 5'-AAYGACTGGGARGAHCACYTG-3' AY190704 HSP90-R 5'-CATGATBCKCTCCATGTTBGC-3' AB196458 891 RT-HSP90-F 5 -AACGACTGGGAGGATCACCTG-3' AF135117 RT-HSP90-R 5'-CATGATGCGCTCCATGTTCGC-3' BC065359 HSP70-F 5'-CCCTRCCTACTYCAACGATTYA-3 HSP70-R 5'-AAYGAGCCCTRGTGAHGGAG-3' RT-HSP70-F 5'-CCCTGCCTACTTCAACGATTCA-3 RT-HSP70-R 5'-AACGAGCCCTGGTGATGGAG-3 Actin-F 5'-TCGAGCACGGTATTGTGACC-3' Actin-R 5'-ACGGAACCTCTCATTGCCGA-3' 465 AB196461 AY436786 BC045841 557 AY491380
HSP90, HSP70 mrna x cortisol glucose y 317 HSP90 HSP70 mrna s HSP90 HSP70 mrna ü p x w» w, RT-PCR w. Total RNA,,, l w, RT-PCR ww» w HSP90 HSP70 primer Table 1 ùkü. Total RNA 1 µg w RT-PCR SuperScript TM reverse trasncrptase (Invitrogen, Carlsbad, CA, USA) w 42 o C 50 z, 94 o C 2, 94 o C 1, 54 o C (HSP90) 60 o C (HSP70) 45, 72 o C 45 35z wš, 72 o C 5 w. z s PCR 1.0% agarose gel»w, HSP90 HSP70 mrna x Gelpro 3.1(KBT, Korea) ful v w üt ß-actin x w yw w. x cortisol glucose x cortisol cortisol RIA kit (DPC, Los Angeles, U.S.A.) w tw w w w, Hewlett Packard Gamma Counter (Cobra 5010, Packard Co., U.S.A.) dw. Glucose Biochemistry Autoanalyzer (Hitach 7180, Hitach Co., Japan) w. m ƒ x l SPSS-m qj(version 10.0) w t-test one way ANOVA Tukey s multiple range test s³ w (P=0.05). HSP90 HSP70 cdna PCR w s HSP90 cdna r(891 bp) w, k, w. HSP90 (Pagrus major, AY190704) HSP90 99%, (AB196458) HSP90 95%, (Salmo salar, AF135117) HSP90 94%, zebrafish (BC065359) HSP90 94% ùkü(fig. 1). HSP70 cdna r(465 bp) w, Fig. 1. Multiple alignment of black porgy (Acanthopgrus schlegeli) HSP90 with several teleost. The HSP90 sequences used for alignment were black porgy HSP90 (bphsp90, AY929159), red seabream HSP90 (rshsp90, AY190704), rainbow trout HSP90 (rthsp90, AB196458) Atlantic salmon HSP90 (ashsp90, AF135117) and zebrafish HSP90 (zfhsp90, BC065359). Identical amino acids are indicated by shaded box.
318, y, ½ùù, y, Fig. 2. Multiple alignment of black porgy (Acanthopgrus schlegeli) HSP70 with several teleost. The HSP70 sequences used for alignment were black porgy HSP70 (bphsp70, AY929160), rainbow trout HSP70 (rthsp70, AB196461) silver seabream HSP70 (sshsp70, AY436786) and zebrafish HSP70 (zfhsp70, BC045841). Identical amino acids are indicated by shaded box. w, (AB196461) HSP70 96%, silver seabream (Sparus sarba, AY436786) HSP70 95%, zebrafish (BC045841) HSP70 95% HSP90 ƒ yw (Fig. 2). HSP90 HSP70 mrna x HSP90 HSP70 mrna x w, HSP90 mrna xw ùkû. ù 30 o C g HSP90 mrna x 20 o C x w 7~9 ùkû(p<0.05) (Fig. 3A). 20 o C x HSP70 mrna x w ù, 30 o C x x w ùkû(p<0.05) (Fig. 3B). x cortisol glucose x cortisol 20 o C x 6.5±0.7 ng/ml 25 o C x 32.3±1.1 ng/ml w Fig. 3. Tissue-specific expression of HSP90 (A) and HSP70 (B) mrnas in five different tissues from black porgy (Acanthopagrus schlegeli) by RT-PCR. Amplification of ß-actin gene was used as an internal control at 20 o C. B: brain, L: liver, G: gonad, K: kidney, I: intestine. Value represents a mean±s.e. (n=3). Asterisks indicate difference between 20 o C and 30 o C (P<0.05).
HSP90, HSP70 mrna x cortisol glucose y 319 Fig. 4. Plasma cortisol and glucose in black porgy (Acanthopagrus schlegeli) during water temperature rising. Value represents a mean ±S.D. (n=6). Different letter indicate significantly different (P<0.05). ƒw(p<0.05), 30 o C x 106.6±29.9 ng/ ml 20 o C x w 16 ùkû(p<0.05) (Fig. 4). Glucose 20 o C x 49.5±6.5 mg/dl, 25 o C x ¾ w ù, 30 o C x 91.0±6.2 mg/dl w ƒw (P<0.05) (Fig. 4). š w w p ü s p j, w n y, w, w(ackerman et al., 2000). w w k w» w w vw (glucose) v w, ü w j(munck et al., 1984), s p w ww (Gamperl et al., 1994). p ü s w, y šw. w y, w, sz sw w p j w, 1 w-w- y, w cortisol x (Perry and Reid, 1993; Wendelaar Bonga, 1997; Chang and Hur, 1999), 2 - ³x,, ƒ ƒ, x glucose ùkù(tomasso et al., 1980; Eddy, 1981; Carmichael et al., 1984; McDonald and Milligan, 1997). w ü x t š. g, x cortisol glucose ƒ w ƒw, e(paralichthys olivaceus: Chang et al., 2002), sunshine bass (Morone chrysops Morone saxatilis: Davis, 2004) š. w p w s p, ü HSP, WTA65 (warm temperature acclimationassociated 65-KDa protein), cytochrome P450 monooxygenases, etallothioneins p w š(stegeman et al., 1992). p p w 2 l s yw, ù p e (Ciavarra and Simeone, 1990; Hightower, 1991). 20 o C 30 o C g, p HSPs mrna x RT-PCR mw (,,, ) w. 20~25 o C(Min et al., 2006), k x cortisol ƒ 35 ng/ml w (Chang et al., 2002; Min et al., 2003) x 20 o C x x cortisol ƒ s³ 9 ng/ml, 20 o C x p š k. 20 o C x HSP90 mrnaƒ w xw, HSP90 mrna p w ƒš w. w, HSP90 p k ü s 1~2% wš š(lai et al., 1984) e wš. 30 o C x HSP90 mrna x 20 o C x w w ƒw, x cortisol w 30 o C x ƒw. p w HSP90 x x cortisol w ƒw. HSP90 gpg y wš, gpg sww l y s( w 3, v»)» w vwš (Pratt et al., 1996; Pratt and Toft, 1997). w gpg z s w gpg w (Munck et al.,
320, y, ½ùù, y, 1984; Adcock, 2000), ü d cortisol ƒ, s d HSP90 mrna ƒ xw š., HSP90 mrna cortisol y ey cortisol sw w dw. HSP90 mrna x w,,, ùkû. w (43 o C, 30), HSP90,, w, x(huang al., 1999), w p l y w x Vamvakopoulos (1993) w ew š. HSP70 y mw p. HSP family ƒ ƒ Ÿ w, (Kothary et al., 1984), medaka, Oryzias latipes (Arai et al., 1995), zebrafish (Lele et al., 1997), pv, Oreochromis mossambicus (Molina et al., 2000),, Fugu rubripes (Lim and Brenner, 1999) w l j. w, HSP70 y p wš š (Feder and Hofmann, 1999). s w HSP70 p w w apoptosis (s)l s ywš (Mosser et al., 1997; Mallouk et al., 1999). HSP70 x s apoptosis w(dix et al., 1996), w s (Allen et al., 1988; Matsumoto and Fujimoto, 1990) y ny (Khanna et al., 1995). HSP70 w w w d. (aryl hydrocarbon receptor: AHR, ƒ sw) white sucker, Ictalurus punctatus HSP70 mrna x ùs apoptosis ƒ (Janz et al., 1997, 2001). p w HSP70 mrna x, p w ü s apoptosisƒ y ƒ ƒ d w, x ù w» w HSP70 xw. HSP70 ƒ p, glucocorticoid w w HSP90 ƒ x p (Hutchison et al., 1994),»w p w» w w vw, w cortisol ùkù. cortisol w w HSP70 mrnaƒ w. 30 o C x HSP70 mrnaƒ x»w ù, x yw. ù Wegele et al. (2005) š w HSP90 mrnaƒ cortisol yw HSP70 mrnaƒ x cortisol yy w, HSP90 mrnaƒ HSP70 mrna xw w vw cortisol yw, w HSP70 mrna x ù kù d. p w cortisol-cortisol -HSP70 HSP90-HSP70 y w ƒ vw. g, s p d HSP90 HSP70 mrna x, -ü p d x cortisol glucose w. RT-PCR w l HSP90 (891 bp) HSP70 (465 bp) cdna r j w, k w, HSP90 HSP90 99%, HSP90 95%, HSP90 94%, zebrafish HSP90 94% ùkû, HSP70 HSP70 96%, silver seabream HSP70 95%, zebrafish HSP70 95% ùkü. 30 C g o, HSP90 mrna x ƒ 20 o C x w 7~9 ù, HSP70 mrna xw ùkû. x cortisol glucose 20 o C x w 30 o C x w ƒw ùkû. w w x (y: F01-2006-000-10066-0) w,. šx Ackerman, P. A. and G. K. Iwama, 2001. Physiological and cellular stress responses of juvenile rainbow trout to Vibriosis. J. Aquat. Anim. Health, 13, 173 180. Ackerman, P. A., R. B. Forsyth, C. F. Mazur and G. K. Iwama, 2000. Stress hormones and the cellular stress response in salmonids. Fish Physiol. Biochem., 23, 327 336. Adcock, I. M., 2000. Molecular mechanisms of glucocorticosteroid actions. Pulm. Pharmacol. Ther., 13, 115 126. Allen, R. L., D. A. O'Brien, and E. M. Eddy, 1988. A novel hsp70-like protein (P70) is present in mouse spermatogenic cells. Mol. Cell. Biol., 8, 828 832.
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