한수지 51(2), 163-169, 2018
163
Copyright © 2018 The Korean Society of Fisheries and Aquatic Science pISSN:0374-8111, eISSN:2287-8815 Korean J Fish Aquat Sci 51(2),163-169,2018
Original Article
서 론
Heat shock protein (Hsp)
은모든생물에서계통발생학적으 로잘보존되어있고,
온도뿐만아니라염분,
중금속,
활성산소 및미생물의감염을비롯한여러가지스트레스요인에의해높은발현을보이는스트레스단백질로알려져있다
(Beckmann
et al., 1990). Hsps
는스트레스요인에의해나타나는충격으 로부터자신을보호함으로써항상성(homeostasis)
을유지하는 중요한역할을담당하며,
분자량을기초로하여Hsp90 (85-90 kDa), Hsp70 (68-73 kDa)
및저분자Hsps (16-47 kDa)
등3
개의그룹으로나누어진다(Forsyth et al., 1997; Iwama et al., 1998; Basu et al., 2002).
이중, Hsp70
은Hsp family
중가장 널리알려져있는단백질중하나로,
인간(Homo sapiens)
뿐만 아니라 제브라피쉬(Danio rerio),
무지개송어(Oncorhynchus mykiss), humphead snapper Lutjanus sanguineus
등여러경골어류에서보고되어진바있으며
,
환경조건의변화나미생물의감염으로인해손상된세포나단백질의회복을돕는다
(Hutchi-
son et al., 1994; Graser et al., 1996; Roux et al., 1996; Ojima et al., 2005; Zhang et al., 2011).
구두충문은작은연충모양의장내기생충으로
1,150
종을포 함하고있으며,
어류에있어구두충은뱀장어의장에기생하고 있는것을최초로보고하였고,
이것을Leewenhock
이Acantho- cephalus anguillae
라고명명하였다(Alava and Aguirre, 2005).
구두충의생활사중소형갑각류와절지동물을중간숙주로하 는데
,
이러한중간숙주를먹이로하는어류가감염되는것으로 알려지고있다(Kim et al., 2011).
이는갈고리를가진proboscis
를숙주의장관벽에침투시켜광범위한조직손상을일으키면서 다양한척추동물에게잠재적인폐사를유발하고있으며,
염증 반응을유발하며어류에스트레스로작용하고있다(Nickol and Crompton, 1985; Hatai et al., 1987).
특히,
어류는구두충에감구두충( Longicollum pagrosomi)에 감염된 참돔(Pagrus major)의 Heat Shock Protein 70 (Hsp70) 발현
박형준 ∙ 민병화 1 *
국립수산과학원 양식관리과, 1동해수산연구소 양식산업과
Expression of a Heat Shock Protein 70 (Hsp70) in Red Seabream Pagrus major Infected with Longicollum pagrosomi
Hyung-Jun Park and Byung-Hwa Min
1
*Aquaculture Research Division, National Institute of Fisheries Science, Busan 46083, Korea
1Aquaculture Industry Research Division, East Sea Fisheries Research Institute, Gangneung 25435, Korea
This study examined the expression of heat shock protein 70 (Hsp70) in red seabream Pagrus major infected by the, acanthocephalan parasites Longicollum pagrosomi . We cloned the full-length Hsp70 cDNA from the liver of the red seabream. The full-length cDNA had a 1,950 bp open reading frame (ORF) that encoded a protein of 650 amino acids. The deduced amino acid sequence of Hsp70 contained all of the conserved Hsp70 family signature sequences and an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding motif, including the EEVD (consensus sequence that terminates in Hsp70 family) consensus sequence. The expression of Hsp70 mRNA was upregulated int the fish head-kidney and liver, as determined by quantitative real-time PCR. We quantified the Hsp70 mRNA expres- sion in normal red seabream and fish infected fish by L. pagrosomi . The expression of Hsp70 mRNA was significantly higher in the infected red seabream. These results suggest that Hsp70 play a role of protection against stress and inflammation caused by the parasite and may help maintain homeostasis.
Key words: Pagrus major , RACE PCR, Hsp70, Longicollum pagrosomi, qPCR
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://doi.org/10.5657/KFAS.2018.0163 Korean J Fish Aquat Sci 51(2) 163-169, April 2018
Received 18 January 2018; Revised 8 February 2018; Accepted 19 March 2018
*Corresponding author: Tel: +82. 33. 660. 8542 Fax: +82. 33. 661. 8514
E-mail address: [email protected]
박형준
ㆍ
민병화164
염이되면대부분소화관에만국한되어기생하고있다는것으 로미루어보아
,
구두충은먹이활동중경구를통해감염되고여러조직중에서도소화관이타겟장기로보고되어있다
(Zeng
and Wang, 2007).
따라서숙주는소화기능장애를유발하고,
장 기간의기능장애와식욕저하로인하여면역력이감소함으로써 심하게는폐사에이르게한다(Mehlhorn, 2008).
기생충의감염 으로발생하는스트레스로인해어류의생리및생태학적으로영향을미치게되면서세포내
Hsp70
의합성과더불어다양한생리적변화가일어나게된다
(Frank et al., 2013).
참돔
(Pagrus major)
은한국의주요해산양식어류로써고급어 종으로분류되고있으며,
수요가점점증가되고있을뿐만아니 라,
활선어로서수출되어외화획득에도상당한몫을차지하고 있다(Pyen and Jo, 1982).
주로한국의남해안에서해상가두리 양식을통해생산량이증가하고있지만,
참돔양식에있어종종 고수온,
밀식,
외상,
저산소(Hypoxia),
어병세균및바이러스의 감염을통한스트레스로인해심각한폐사를유발하기도하여 양식어가소득에많은영향을미치고있다(Kader et al., 2012).
따라서
,
본연구에서는참돔으로부터분리된Hsp70
의분자생 물학적특성과발현분석을통해질병및스트레스로인한폐사 를막기위한기초적인생체방어메커니즘을조사하고자한다.
재료 및 방법
실험어 및 실험조건
감염어는통영에소재하고있는참돔가두리양식장으로부터 구두충감염이의심되는실험어
100
미를제공받아국립수산과 학원(
부산기장군)
으로옮겨졌다.
이중,
육안적관찰을통해감 염을확인한참돔중10
미(30.7±4.8 cm,
체중697.1±54.8 g)
를선발하여실험어로이용하였다.
대조구인비감염어는국립 수산과학원양식생물연구동사육실내원형수조(2
톤)
에서상업 용사료를급이하며,
사육수온17±0.5℃
광주기10L:14D
조 건하에서사육중인참돔을이용하였다.
이중,
감염이확인되지 않은참돔10
미(
평균전장37.3±2.5 cm,
체중818.6±124.7 g)
를선발하여대조구로이용되었다.
선발된감염어및비감염 어의기타세균및바이러스성질병을확인하기위해신장,
비 장및간을적출하여국립수산과학원병리연구과에질병검사 를의뢰하였다.
세균(
비브리오,
연쇄구균및에드와드균)
및바 이러스(red seabream iridovirus, RSIV)
검사에서모두음성으 로판정되었으며,
감염어에기생하고 있는장내구두충수는299.8±91.7 parasites/fish
로확인되었다.
조직샘플링
선발된감염어와비감염어의
Hsp70 mRNA
발현분석을위 해조직샘플링을실시하였다.
해수가수용된100 L
수조에tric- aine methane sulfonate (MS-222; Sigma, USA)
를150 mg/L
의농도가되도록첨가한다음실험어를옮겨마취시킨후,
샘플링을수행하였다
.
참돔의Hsp70 full-length cDNA
를밝히기위해대조구인참돔의간조직을통해
RACE PCR
에이용되었고
,
건강한참돔의Hsp70 mRNA
조직별발현분석을수행하기 위하여간,
비장,
두신,
후신,
장,
아가미,
뇌하수체및근육을적 출하여액체질소에서급속냉동시킨다음−80℃
에서실험전 까지보관하였다.
또한,
구두충에감염된실험어는두신,
간및 장을적출하여대조구와의Hsp70 mRNA
발현의비교를위해 이용되었다.
Total RNA분리 및 cDNA합성
참돔의
Hsp70
의primer
를제작하기위해앞서선행된연구에 서밝혀진감성돔(Acanthopagrus schlegeli) Hsp70 (GenBank accession no. AAX07833)
의sequence
를토대로합성시켰다.
이primer set
는다음과같다. Hsp70 forward primer (5’-AGA AGG ACA TCA GCG ACA AC-3’), Hsp70 reverse primer (5’-TCA GAC TTG TCA CCA GAC AG-3’).
Total RNA
는 실험어로부터 적출한 간 및 조직들로부터TRizol Reagent (Gibco/BRL, USA)
를 이용하여 추출하였 다. Total RNA
를주형으로하여oligo-d (T)
18anchor primer
와M-MLV reverse transcriptase (Bioneer, Korea)
를이용하여cDNA
를합성하였다. PCR
은Takara Taq (Takara, Japan)
을이 용하여수행하였으며,
그조건은다음과같다. 95℃
에서2
분동 안initial denaturation
과정을거친후에, 95℃
에서20
초동안denaturation, 56℃
에서40
초동안annealing, 72℃
에서30
초 동안extension
반응을30
주기수행한후에,
마지막주기에서30
초동안extension
반응을시켰다.
증폭된PCR
산물은aga- rose gel
에전기영동하여얻어진DNA
밴드를잘라내고정제 하였다.
준비된DNA
산물을pGEM-T Easy Vector (Promega, USA)
와ligation
시킨후, ligation
시킨반응물은DH5α com- petent cells (RBC Life Sciences, Korea)
를이용하여형질전환 하였다. Plasmid DNA
는LaboPass plasmid DNA purification kit (Cosmo, Korea)
를이용하여분리하였고,
추출된plasmid DNA
를ABI DNA Sequencer (Applied Biosystems, USA)
를 이용하여염기서열을확인하였다.
Full-length Hsp70 cDNA 분리 (3’ 및 5’ RACE)
참돔의Hsp70 full-length cDNA sequence
를얻기위해oligo (dT) anchor primer (5’-CTG TGA ATG CTG CGA CTA CGA T(T)
18-3’)
와CapFishing
TMadaptor (Seegene, Korea)
를 이용하여 합성하였다.
간의RACE-Ready cDNA
를tem- plate
로하여target primer
와gene specific primer
를이용한일 련의RACE-PCR
을진행하였다.
3’ RACE
분리는5 µL
의3’ RACE cDNA, 1 µL
의10 mM 3’
RACE target primer (5’-CTG TGA ATG CTG CGA CTA
CGA T-3’), 1 µL
의10 mM 3’ RACE HSP70-specific primer
(5’-GCC ATC CTG TCT GGT GAC AAG TCT GAG-
3’)
와25µL
의SeeAmpTaq Plus Master Mix
가포함된50 µL
구두충 감염에 따른 참돔의 Hsp70 mRNA 발현
165
PCR
시약을이용하여 분리하였다. PCR
조건은94℃
에서5
분 간denaturation
과정을거친후에, 94℃
에서40
초동안의de- naturation, 62℃
에서40
초동안의annealing, 72℃
에서1
분동 안의extension
반응을40
주기수행한후에,
마지막주기에서5
분동안extension
반응을시켰다.
5’ RACE
분리는5 µL
의5’ RACE cDNA, 1 µL
의10mM 5’
RACE target primer (5’-GTC TAC CAG GCA TTC GCT TCA T-3’), 1 µL
의10mM 5’ RACE HSP70-specific primer (5’-CAG CTC TCT TGT TGT CGC TGA TGT CCT TC-3’)
와25µL
의SeeAmpTaq Plus Master Mix
가포함된50
Hsp70 family signature 1 ATG/GTP binding site motif
Hsp70 family signature 2 Bipartite nuclear localization signal sequence
EEVD consensus sequence
Red seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Black porgy 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMK 88 Humphead snapper 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Goldlined seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Mandarin fish 1:MAPAKGVAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPSNTVFDAKRLIGRKMDDQVVQADMK 90
..**.*.***************************************.*************.**.************..**.***.***
Red seabream 89:HWPFNVINDNTRPKVQVEYKGESKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Black porgy 89:HWPFNVINDNTRPKAQVEYKGESKSFYPEEVSSTVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Humphead snapper 89:HWPFNVINDNTRPKVQVEYKGESKTFYPEEISSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Goldlined seabream 89:HWPFNVINDNTRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Mandarin fish 91:HWPFKVVGDGGKPKIQVEYKGEDKTFYPEEISSMVLVKMNEIAEAYLGQQVSNAVITVPAYFNDSQRQATKDAGVIAGLNVLKIINEPTA 180
****.*..*...**.*******.*.*****.**.**.**.********..*.**********************.*.*****.*******
Red seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Black porgy 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Humphead snapper 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIGEFKRKYKKDISDNKRAVRRLRTACE 268 Goldlined seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Mandarin fish 181:AAIAYGLDKNKTGERNVLIFDLGGGTFDVSILTIEDGIFEVKATAGDTHLGGEKFDNRMVNHFVEEFKRKHKKDISHNKRPLRRLRTACE 270
*********....*****************************.**********.*********..*****.*****.***..********
Red seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGLIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Black porgy 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Humphead snapper 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Goldlined seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Mandarin fish 271:RAKRTLSSSTQASIEIDSLFEGIDFYTSITRARFEELNSELFRGTLDPVEKALQDAKLDKSKIHEVVLVGGSTRIPKIQKLLQDFFNGRE 360
*******************.**.***************..***********.*.***.**..**..**********************.*
Red seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Black porgy 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Humphead snapper 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Goldlined seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Mandarin fish 361:LNKSINPDEAVAYGAAVQAAILMGDTSENVQDLLLLDVAPLSLGIETAGGVMTPLIKRNTTIPSKQTQIFSTYSDNQPGVLIQVYEGERA 450
**********************.**.************.**************.*********.****.*.*******************
Red seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Black porgy 449:MTRDNSLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKIQITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Humphead snapper 449:MTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Goldlined seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Mandarin fish 451:MTKDNNLLGKFELTGIPPAPRGVPQVEVTFDIDANGVLNVSAVDKSTGKENKITITNDKGRLSKEEIERMVQDSEKYKAEDDMQREKITA 540
**.**.*******************.**********..***************.***********.******..********.**.*..*
Red seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVIGWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Black porgy 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDGYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Humphead snapper 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAEKDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMGGG 628 Goldlined seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMPGG 628 Mandarin fish 541:KNSLESYAYNMKSSVEDENMKGKISEEDKKMVIDKCNQTISWLENNQLAGKEEYQHGQKELEKVCQPIISGLYQ--GGAPAGSCGEQAGS 628
**.*****.****.****...****..**....****..*.**..**.*....*.*.********.***..***..**.*.*...*.
Red seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Black porgy 629:FPGAGGAAPGGGSSGPTIEEVD 650
Humphead snapper 629:FPGAGGAAPGGGSSGPTIEEVD 650
Goldlined seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Mandarin fish 629:GSQGPTMEEVD--- 639
...
Hsp70 family signature 3
Austrofundulus limnaeus XP_013888455 Nothobranchius furzeri XP_015816161 Fundulus heteroclitus XP_012712369 Poecilia formosa XP_007541686 Poecilia reticulata XP_008400612 Maylandia zebra XP_004574972
Pagrusmajor AKS36893
Ctenopharyngodon idella ACJ03596 Hypophthalmichthys molitrix ACJ03595 Rattus norvegicus CAA54422
Capra hircus AEX55800 AEX55800 Ovis aries AEX55801 AEX55801
Pelophylax lessonae ACY69994 Xenopus laevis NP_001121147 Genlisea aurea EPS60056
Medicago sativa AAV98051 Camellia sinensis ACD93209
Arabidopsis thaliana CAA05547 Paeonia lactiflora AEK70336 Paeonia suffruticosa AFA51946 100
92 46 51 82 100 99 90 100
99 96 100 63 99 97 95
0.2
Fish
Mammalia Reptilia
Plant
0 20 40 60 80 100
Muscle Intestine Pituitary Spleen Gill Trunk-kidney Liver Head-kidney
Relative expression
Tissues
a ab
b
ab ab
c
d
e
0 5 10 15 20
Head-kidney Liver Intestine
Relative expression
Tissues
Control Infection
a a a
b
b
b
Fig. 1. Comparison of Hsp70 amino acid sequence of red seabream Pagrus major Hsp70, black porgy Acanthopagrus schlegeli (AAX07834), humphead snapper Lutjanus sanguineus (ADO32584), goldlined seabream Rhabdosargus sarba (AAR97293) and mandarin fish Siniperca chuatsi (AHK25484) optimally aligned to match identical residues. The sequences were taken from the GenBank/EMBL/DDBJ sequence database. The Hsp70 family signature are grey shaded. The ATP-GTP binding site motif and bipartite nuclear localization signal sequence are underlined. The EEVD consensus sequence is bolded. Hsp70, heat shock protein 70; EMBL, European molecular biology labolatory;
DDBJ, DNA data bank of Japan; ATP, adenosine triphosphate; GTP, guanosine triphosphate; EEVD, consensus sequence that terminates in Hsp70).
박형준
ㆍ
민병화166
µL PCR
시약을이용하여분리하였다. PCR
조건은94℃
에서5
분동안initial denaturation
과정을거친후에, 94℃
에서40
초 동안denaturation, 62℃
에서40
초동안annealing, 72℃
에서1
분간extension
반응을40
주기수행한후에,
마지막주기에서5
분동안extension
반응을시켰다.
증폭된
PCR
산물은1% agarose gel
을이용한전기영동을통 해분리하였으며,
그다음과정은앞에서서술한cDNA
의합성 방법과동일하다.
Quantitative real-time PCR (qPCR)
참돔
Hsp70 mRNA
의발현을확인하기위하여,
분리된RNA
를사용하여
qPCR
을이용하여분석하였다. qPCR
의수행을위 한primer
는밝혀진참돔Hsp70 full-length cDNA sequence
를 통해설계하였다(Table 1). qPCR
은BIO-RAD iCycleriQ Mul- ticolor Real-Time PCR Detection System (Bio-Rad, USA)
과iQ
TMSYBR green Supermix (Bio-Rad, USA)
를이용하여95℃
에서5
분동안denaturation
과정을거친후에, 95℃
에서20
초동안denaturation, 55℃
에서20
초동안annealing
을총40
회실시하였다.
대조구의유전자로는House-keepieng gene
으로알려진참돔β-actin
을사용하였고, qPCR
을통해calcu- lated threshold cycle (ΔCt)
값을결정하여β-actin
에대한발현 량을2
-ΔΔCtMethod, [ΔΔCt= 2
^-(ΔCt
sample– ΔCt
internalcontrol)]
Hsp70 family signature 1 ATG/GTP binding site motif
Hsp70 family signature 2 Bipartite nuclear localization signal sequence
EEVD consensus sequence
Red seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Black porgy 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMK 88 Humphead snapper 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Goldlined seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Mandarin fish 1:MAPAKGVAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPSNTVFDAKRLIGRKMDDQVVQADMK 90
..**.*.***************************************.*************.**.************..**.***.***
Red seabream 89:HWPFNVINDNTRPKVQVEYKGESKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Black porgy 89:HWPFNVINDNTRPKAQVEYKGESKSFYPEEVSSTVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Humphead snapper 89:HWPFNVINDNTRPKVQVEYKGESKTFYPEEISSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Goldlined seabream 89:HWPFNVINDNTRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Mandarin fish 91:HWPFKVVGDGGKPKIQVEYKGEDKTFYPEEISSMVLVKMNEIAEAYLGQQVSNAVITVPAYFNDSQRQATKDAGVIAGLNVLKIINEPTA 180
****.*..*...**.*******.*.*****.**.**.**.********..*.**********************.*.*****.*******
Red seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Black porgy 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Humphead snapper 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIGEFKRKYKKDISDNKRAVRRLRTACE 268 Goldlined seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Mandarin fish 181:AAIAYGLDKNKTGERNVLIFDLGGGTFDVSILTIEDGIFEVKATAGDTHLGGEKFDNRMVNHFVEEFKRKHKKDISHNKRPLRRLRTACE 270
*********....*****************************.**********.*********..*****.*****.***..********
Red seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGLIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Black porgy 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Humphead snapper 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Goldlined seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Mandarin fish 271:RAKRTLSSSTQASIEIDSLFEGIDFYTSITRARFEELNSELFRGTLDPVEKALQDAKLDKSKIHEVVLVGGSTRIPKIQKLLQDFFNGRE 360
*******************.**.***************..***********.*.***.**..**..**********************.*
Red seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Black porgy 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Humphead snapper 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Goldlined seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Mandarin fish 361:LNKSINPDEAVAYGAAVQAAILMGDTSENVQDLLLLDVAPLSLGIETAGGVMTPLIKRNTTIPSKQTQIFSTYSDNQPGVLIQVYEGERA 450
**********************.**.************.**************.*********.****.*.*******************
Red seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Black porgy 449:MTRDNSLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKIQITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Humphead snapper 449:MTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Goldlined seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Mandarin fish 451:MTKDNNLLGKFELTGIPPAPRGVPQVEVTFDIDANGVLNVSAVDKSTGKENKITITNDKGRLSKEEIERMVQDSEKYKAEDDMQREKITA 540
**.**.*******************.**********..***************.***********.******..********.**.*..*
Red seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVIGWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Black porgy 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDGYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Humphead snapper 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAEKDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMGGG 628 Goldlined seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMPGG 628 Mandarin fish 541:KNSLESYAYNMKSSVEDENMKGKISEEDKKMVIDKCNQTISWLENNQLAGKEEYQHGQKELEKVCQPIISGLYQ--GGAPAGSCGEQAGS 628
**.*****.****.****...****..**....****..*.**..**.*....*.*.********.***..***..**.*.*...*.
Red seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Black porgy 629:FPGAGGAAPGGGSSGPTIEEVD 650
Humphead snapper 629:FPGAGGAAPGGGSSGPTIEEVD 650
Goldlined seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Mandarin fish 629:GSQGPTMEEVD--- 639
...
Hsp70 family signature 3
Austrofundulus limnaeus XP_013888455 Nothobranchius furzeri XP_015816161 Fundulus heteroclitus XP_012712369 Poecilia formosa XP_007541686 Poecilia reticulata XP_008400612 Maylandia zebra XP_004574972
Pagrusmajor AKS36893
Ctenopharyngodon idella ACJ03596 Hypophthalmichthys molitrix ACJ03595 Rattus norvegicus CAA54422
Capra hircus AEX55800 AEX55800 Ovis aries AEX55801 AEX55801
Pelophylax lessonae ACY69994 Xenopus laevis NP_001121147 Genlisea aurea EPS60056
Medicago sativa AAV98051 Camellia sinensis ACD93209
Arabidopsis thaliana CAA05547 Paeonia lactiflora AEK70336 Paeonia suffruticosa AFA51946 100
92 46 51 82 100 99 90 100
99 96 100 99 63 97 95
0.2
Fish
Mammalia Reptilia
Plant
0 20 40 60 80 100
Muscle Intestine Pituitary Spleen Gill Trunk-kidney Liver Head-kidney
Relative expression
Tissues
a ab
b
ab ab
c
d
e
0 5 10 15 20
Head-kidney Liver Intestine
Relative expression
Tissues
Control Infection
a a a
b
b
b
Fig. 2. Neighbour-joining tree of Hsp70 members constructed with MEGA4. The bootstrap confidence values presented at the nodes of the tree are based on 2,000 bootstrap replications. GenBank acession numbers of selected genes are indicated within brackets. Hsp70, heat shock protein 70; MEGA4, multiple-sequence software.
Table 1. Primers used for qPCR amplification
Genes (Accession no.) Primer DNA sequences
Hsp70 (KT334554) Forward 5’-AGC TCA ATG CTG ACC TCT TC-3’
Reverse 5’-GGA TCA GCC CTT TAT CCA TCT T-3’
β-actin (JN226150) Forward 5’-GAT CTG GCA TCA CAC CTT CTA C-3’
Reverse 5’-TCT TCT CCC TGT TGG CTT TG-3’
구두충 감염에 따른 참돔의 Hsp70 mRNA 발현
167
(Livak and Schmittgen, 2001)
를이용하여정량화하였다. 통계분석
각 실험결과로부터 얻어진 자료값 사이의 유의차 유무는
SPSS
통계처리프로그램(version 10.0; SPSS Inc, USA)
에의 한One-way ANOVA
및Duncan’s multiple range test
를실시 하여참돔의정상개체와감염개체간의수치를평균간의유의 성(P<0.05)
을검정하였다.
결과 및 고찰
본연구에서는참돔으로부터
Hsp70 full-length cDNA
를건 강한 어체의간으로부터RACE
법을 통해 밝혔으며,
구두충 이참돔의스트레스에미치는영향을확인하기위하여대조구인미감염개체와구두충에감염된실험어를비교하여
Hsp70
mRNA
발현분석을수행하였다.
우선,
예상크기의단일PCR
산물(Hsp70: 408 base pairs, bp)
은RT-PCR
에의해분리되었 고,
분리된단일PCR
산물을이용하여(3’
및5’ RACE) Hsp70 full-length cDNA
를분리하였다. Open reading frame (ORF)
이포함된전장의Hsp70 cDNA
는650
개의아미노산으로이 루어진단백질로이루어져있었으며, NCBI
의Blast algorithm
(Blastp)
을이용하여타어종과의아미노산배열을비교한결과
,
참돔의Hsp70
아미노산배열은다른어종과유사함을확인 할수있었다(Fig. 1).
참돔Hsp70 full-length cDNA
의계통발 생학적분석은Mega 4 software package
와neighbor joining (NJ)
법을사용하여분석하였다.
다른어종들과의계통수분석 결과다양한경골어류들과cluster
를형성했고,
이들중zebra
umbuna Maylandia zebra
와는98.6%, mummichog Fundulus heteroclitus 97.8%
의높은상동성을보였다(Fig. 2).
Zhang et al. (2011)
의보고와마찬가지로,
참돔의Hsp70
아 미노산배열에서도3
가지Hsp70 family signature (1, IDLGT- TYS; 2, IFDLGGGTFDVSIL; 3, IVLVGGSTRIPKIQ)
와ATP-GTP binding site
로추정되는AEAYLGKT (aa 129-136), consensus sequence (EEVD)
가포함되어있었다.
본연구에서 밝혀진 참돔의Hsp70
도Hsp70 family signature, ATP-GTP binding site, consensus sequence, N-terminal
및C-terminal
이 존재함은물론,
타어종과도높은상동성을보이는점으로보아, Hsp70 family
와일치하는것으로판단된다.
참돔
full-length Hsp70 sequence
를 바탕으로,
본 연구에서 는 참돔의조직별Hsp70 mRNA
발현을분석하였다. Hsp70
mRNA
발현은건강한참돔의두신과간조직에서주요하게높은값을나타내었고
(Fig. 3),
구두충에감염된실험어의두신,
간 및장조직에서Hsp70 mRNA
발현을조사한결과,
모든조직 에서대조구인미감염개체보다유의적으로높은발현을확인 할수있었다(Fig. 4).
두신은 어류의 장기에 있어 주요 조혈기관이며
(Zapata, 1979),
다량의식세포(
호중구및단핵세포)
가두신에자리하 고있어주변의백혈구들은물리적인자극이나미생물의감염 시침입된항원의인식을통해제거를위한염증반응을유도 한다(Grinde, 1989; Ellis, 1990; Alexander and Ingram, 1992;
Dalmo et al., 1997). Black bullhead Ameiurus melas
와blu- gill sunfish Lepomis macrochirus
를이용한연구에서Hsp70 mRNA
는계절적인변화에따라두신에서의발현이지속적으 Fig. 3. The Hsp70 mRNA expression in various tissues of healthy red seabream Pagrus major, gill, head kidney, intestine, muscle, pituitary, spleen and trunk kidney. Hsp70 transcript levels were quantified and expressed relative to the β-actin transcript level. Values with different characters are significantly different between control and infection (P<0.05). All values are means±SD (n=10). Hsp70, heat shock protein70.Hsp70 family signature 1 ATG/GTP binding site motif
Hsp70 family signature 2 Bipartite nuclear localization signal sequence
EEVD consensus sequence
Red seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Black porgy 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDAVVQSDMK 88 Humphead snapper 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Goldlined seabream 1:--MSKGPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDSERLIGDAAKNQVAMNPTNTVFDAKRLIGRRFDDTVVQSDMK 88 Mandarin fish 1:MAPAKGVAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPSNTVFDAKRLIGRKMDDQVVQADMK 90
..**.*.***************************************.*************.**.************..**.***.***
Red seabream 89:HWPFNVINDNTRPKVQVEYKGESKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Black porgy 89:HWPFNVINDNTRPKAQVEYKGESKSFYPEEVSSTVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Humphead snapper 89:HWPFNVINDNTRPKVQVEYKGESKTFYPEEISSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Goldlined seabream 89:HWPFNVINDNTRPKVQVEYKGETKSFYPEEVSSMVLTKMKEIAEAYLGKTVNNAVITVPAYFNDSQRQATKDAGTISGLNVLRIINEPTA 178 Mandarin fish 91:HWPFKVVGDGGKPKIQVEYKGEDKTFYPEEISSMVLVKMNEIAEAYLGQQVSNAVITVPAYFNDSQRQATKDAGVIAGLNVLKIINEPTA 180
****.*..*...**.*******.*.*****.**.**.**.********..*.**********************.*.*****.*******
Red seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Black porgy 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Humphead snapper 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIGEFKRKYKKDISDNKRAVRRLRTACE 268 Goldlined seabream 179:AAIAYGLDKKVGSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFIAEFKRKYKKDISDNKRAVRRLRTACE 268 Mandarin fish 181:AAIAYGLDKNKTGERNVLIFDLGGGTFDVSILTIEDGIFEVKATAGDTHLGGEKFDNRMVNHFVEEFKRKHKKDISHNKRPLRRLRTACE 270
*********....*****************************.**********.*********..*****.*****.***..********
Red seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGLIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Black porgy 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Humphead snapper 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Goldlined seabream 269:RAKRTLSSSTQASIEIDSLYEGVDFYTSITRARFEELNADLFRGTLDPVEKSLRDAKMDKGQIHDIVLVGGSTRIPKIQKLLQDFFNGKE 358 Mandarin fish 271:RAKRTLSSSTQASIEIDSLFEGIDFYTSITRARFEELNSELFRGTLDPVEKALQDAKLDKSKIHEVVLVGGSTRIPKIQKLLQDFFNGRE 360
*******************.**.***************..***********.*.***.**..**..**********************.*
Red seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Black porgy 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Humphead snapper 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Goldlined seabream 359:LNKSINPDEAVAYGAAVQAAILSGDKSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTTIPTKQTQTFTTYSDNQPGVLIQVYEGERA 448 Mandarin fish 361:LNKSINPDEAVAYGAAVQAAILMGDTSENVQDLLLLDVAPLSLGIETAGGVMTPLIKRNTTIPSKQTQIFSTYSDNQPGVLIQVYEGERA 450
**********************.**.************.**************.*********.****.*.*******************
Red seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Black porgy 449:MTRDNSLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKIQITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Humphead snapper 449:MTKDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Goldlined seabream 449:MTRDNNLLGKFELTGIPPAPRGVPQIEVTFDIDANGIMNVSAVDKSTGKENKITITNDKGRLSKEDIERMVQEAEKYKAEDDVQRDKVSA 538 Mandarin fish 451:MTKDNNLLGKFELTGIPPAPRGVPQVEVTFDIDANGVLNVSAVDKSTGKENKITITNDKGRLSKEEIERMVQDSEKYKAEDDMQREKITA 540
**.**.*******************.**********..***************.***********.******..********.**.*..*
Red seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVIGWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Black porgy 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDGYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMAGS 628 Humphead snapper 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAEKDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMGGG 628 Goldlined seabream 539:KNGLESYAFNMKSTVEDEKLAGKISDDDKQKILDKCNEVISWLDKNQTAERDEYEHQQKELEKVCNPIITKLYQSAGGMPGGMPEGMPGG 628 Mandarin fish 541:KNSLESYAYNMKSSVEDENMKGKISEEDKKMVIDKCNQTISWLENNQLAGKEEYQHGQKELEKVCQPIISGLYQ--GGAPAGSCGEQAGS 628
**.*****.****.****...****..**....****..*.**..**.*....*.*.********.***..***..**.*.*...*.
Red seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Black porgy 629:FPGAGGAAPGGGSSGPTIEEVD 650
Humphead snapper 629:FPGAGGAAPGGGSSGPTIEEVD 650
Goldlined seabream 629:FPGAGGAAPGGGSSGPTIEEVD 650
Mandarin fish 629:GSQGPTMEEVD--- 639
...
Hsp70 family signature 3
Austrofundulus limnaeus XP_013888455 Nothobranchius furzeri XP_015816161 Fundulus heteroclitus XP_012712369 Poecilia formosa XP_007541686 Poecilia reticulata XP_008400612 Maylandia zebra XP_004574972
Pagrusmajor AKS36893
Ctenopharyngodon idella ACJ03596 Hypophthalmichthys molitrix ACJ03595 Rattus norvegicus CAA54422
Capra hircus AEX55800 AEX55800 Ovis aries AEX55801 AEX55801
Pelophylax lessonae ACY69994 Xenopus laevis NP_001121147 Genlisea aurea EPS60056
Medicago sativa AAV98051 Camellia sinensis ACD93209
Arabidopsis thaliana CAA05547 Paeonia lactiflora AEK70336 Paeonia suffruticosa AFA51946 100
92 46 51 82 100 99 90 100
99 96 100 99 63 97 95
0.2
Fish
Mammalia Reptilia
Plant
0 20 40 60 80 100
Muscle Intestine Pituitary Spleen Gill Trunk-kidney Liver Head-kidney
Relative expression
Tissues
a ab
b
ab ab
c
d
e
0 5 10 15 20
Head-kidney Liver Intestine
Relative expression
Tissues
Control Infection
a a a
b
b
b
박형준
ㆍ
민병화168
로증가하였고
(Yoo and Janz, 2003), silver carp Hypophthal- michthys molitix, grass carp Ctenopharyngodon idellus
및Nile tilapia Oreochromis nilotica
에서는독성물질인마이크로 시스틴(microcystin)
의노출에의해간에서높은발현을보였다(He et al., 2010).
Roubal (1993)
의보고에의하면,
구두충에감염된어류의장 에서lymphocytes
와macrophage-like cells
를동반한세포성 염증반응이관찰되어,
장내기생충감염시숙주는mast cell, rodlet cell, eosinophilic granule cells
등다양한염증반응세포 를형성하며(Reite, 1997),
구두충에감염된어류는숙주의특이 적인granulocyte, macrophage
등의생성과구두충의proboscis
의
hook
구조와외피에의해서면역반응이나타난다고보고된바있다
(Mehlhorn, 2011).
또한, Hatai et al. (1987)
은구두충 에감염된참돔의장에세포적침투및염증반응이나타났으 며,
이는세포의necrosis
와밀접한연관성이있음을의미한다(Chaicharn and Bullock, 1967).
이러한연구보고에따르면기 생충감염에따른염증반응이생성되어이에대한면역반응이 일어나게되는데이때, Hsp70
이강한매개체와같은면역반응 을자극시키는것으로알려져있다(Basu et al., 2001).
이와같이
Hsp70
은감염질병에대한면역반응을일으키는데주요한역할을수행함에따라
(Suzue and Young, 1996),
본연구에서 도구두충에감염됐을때이에대한면역반응을유도하기위하 여Hsp70 mRNA
발현이유의적으로증가한것으로사료된다.
따라서,
본연구결과를요약하면구두충에감염된참돔은감 염으로인한염증반응으로체내유의적인스트레스를유도하는 것으로보이면서스트레스로인한Hsp70 mRNA
의높은발현이확인됨에따라
,
구두충감염에따른참돔의생체유지및방 어기작의조절을수행하는것으로여겨진다.
본연구는이러한 결과를바탕으로기생충감염에의한어류의분자생물학적메 커니즘을밝히는데기초적인연구보고가될것으로사료된다.
사 사
이논문은국립수산과학원수산과학연구사업동해특성품종 양식기술개발
(R2018012)
의지원으로수행된연구입니다.
References
Alava JJ and Aguirre WE. 2005. Scanning electron microscopy of Neoechinorhynchus sp. (Acanthocephala: Neoechino- rhynchidae), a possible new species of intestinal parasite of the tallfin croaker Micropogoni asaltipinnis (Günther, 1864).
Parasitol Latinoam 60, 48-53. http://dx.doi.org/10.4067/
S0717-77122005000100007.
Alexander JB and Ingram GA. 1992. Noncellular nonspecific defense mechanisms of fish. In: Faisal M, Hetrick FM (Eds), Ann Rev Fish Dis 2, 249-279. https://doi.org/10.1016/0959- 8030(92)90066-7.
Basu S, Binder RJ, Ramalingam T and Srivastava PK. 2001.
CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14, 303-313.
https://doi.org/10.1016/S1074-7613(01)00111-X.
Basu N, Todgham, AE, Ackerman PA, Bibeau MR, Nakano K, Schulte PM and Iwama K. 2002. Heat shock protein genes and their functional significance in fish. Gene 295, 173-183.
https://doi.org/10.1016/S0378-1119(02)00687-X.
Beckmann RP, Mizzen LE and Welch WJ. 1990. Interaction of HSP70 with newly synthesized proteins: implications for protein folding and assembly. Science 248, 850-854.
Chaicharn A and Bullock WL. 1967. The histopathology of acan- thocephalan infections in suckers with observations on the intestinal histology of two species of catostomid fishes. Acta Zool 48, 19-42. https://doi.org/10.1111/j.1463-6395.1967.
tb00130.x.
Dalmo RA, Ingebrigtsen K and Bogwald J. 1997. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis 20, 241-273.
https://doi.org/10.1046/j.1365-2761.1997.00302.
Ellis AE. 1990. Lysozyme assays. Tech Fish Immunol 1, Fl101- Forsyth RB, Candido EPM, Babich SL and Iwama GK. 1997. 103.
Stress protein expression in coho salmon with bacterial kid- ney disease. J Aquat Anim Health 9, 8-25.
Frank SN, Godehardt S, Nachev M, Trubiroha A, Kolas W and Sures B. 2013. Influence of the cestode Ligula intestinalis and the acanthocephalan Polymorphus minutus on levels of heat shock proteins (HSP70) and metallothioneins in their fish and crustacean intermediate hosts. Environ Poll 180, Fig. 4. Hsp70 mRNA expression in head-kidney, liver and intestine
tissues from red seabream Pagrus major infected by Longicollum pagrosomi. Hsp70 transcript levels were quantified and expressed relative to the β-actin transcript level. Values with different char- acters are significantly different between control and infection (P<0.05). All values are means±SD (n=10). Hsp70, heat shock protein 70.
