Tqp chi Congnghe Sinh hoc 10(1): 49-58, 2012
P H A N L A P V A X A C D J N H T R I N H TV" H O A N C H I N H c D N A M A H 6 A H E M O C Y A N I N L I £ N Q U A N D E N D A P ITNG M I E N D ( C H (3 T 6 M S C (PENAEUS MONODON)
H o i n g Thj Thu V l n ' , Kim Thj Phutmg 0 a n h ^ Ph?m Anh I u S n \ Nong VSn Hii^
Dqi hgc Thai nguyen
Vifn Cong ngh^ sinh hoc, Vi^n Khoa hgc vd Cong ngh? Vi^t Nam Tong cue Thuy sdn. Bo Nong nghi4p va Pliat trien nong llion
T 6 M T A T
Hemocyanin la mpt lo?i protein ngoai bdo chixa ddng ton lai d trong mdu ciia dpng vat than mem vd chan khdp Dau tien, hemocyanin dupe biet Id protein van chuyen oxy phan tii trong mdu. okn nay, protein ndy dupe chimg minh co nhieu chiJtc ndng, d^c bi^t Id cdc chirc n5ng lien quan d^n mien djch khdng nam, khdng khuan vd virus. Mgt phan cDNA hemocyanin 6 torn sii da dupe c6ng bo tren GenBank ma ,s6 AF431737 c6 kich thudc 1470 bp gom ci ma ket thuc vd mpt do?n ngdn 3'-UTR. Trong nghien ciiru ndy, chiing toi tien hinh phan lap gen hemocyanin hodn chinh dya tren mpt phdn thong tin da biet ndy. B3ng ky thugl 5'RACE (5' Rapid Amplification of cDNA Ends) chiing toi da phan lap vd xdc djnh trinh tu doan gen keo ddi vc phia ddu 5' gom ca ma md ddu vd mpt doan ngdn 5'-UTR Tir kdt qua 5'RACE vd trinh ty AF431737, do?n ORF hoan chinh cua gen hemocyanin dupe phan lap bing ky thuat RT-PCR vd xac d|nh trinh ty. Trinh ly ddy dii ciia gen hemocyanin co tdch thuoc 2052 bp, ma h6a 683 amino acid. Hemocyanin dupe du dodn co 3 domain va 6 vi tri amino acid histidine quyet djnh cau true va chiic ndng ciia phan tir protem. Khi so sanh trinh ty gen hemocyanin mdi xac dinh vdi mpt phan trinh ty da cong bo trudc (AF431737), chiing toi thdy co sai khdc 6 33 vi tri nucleotide. Sy sai khac trinh ty nucleotide nay din din sy sai khdc trinh tu amino acid d 7 vi tri 242H^D, 304N-.D, 360V-»L, 443K-*L, 453Y^F, 472A--P vd 576G—A K6t qud nghien cuu cua ch'iing tfli g6p phan vao viec n ^ i e n cim chiic nang ciia hemocyanin vd nhiing gen/protein Hen quan den kha nang mien djch ciia tflm su.
Tir kk6a: protein vdn chuyen oxy, gen lien quan din mien dich. Penaeus monodon, hemocyanin, tom su
MCiDAU
San xuat tom la hoat ddng kinh te quan trpng ddi vdi cac nude dang phat trien. Viec phdt trien nghe nuoi torn qua nhieu thap ky gan day luon di cung vdi su phat hipn ciia cac benh dich do virus, vi khuan v i mpt sd trudng hpp do vi nam (Destoumieux et ai, 1999, Lightner, Redman 1998, Lighmer 1983). Cac loii vi khuan thupc chi Vibrio l i nhung tic nhan gay bpnh vi khuan chii yeu v i cd the gay td vong cao nhu: benh phat sang tren torn s\i {V harveyi, V.
parahaemolyticus, V. alginolycus), bpnh hoai tu bg phan v i an mon vd kitin (V. parahaemolyticus, V.
alginolycus, V. aguillarum). Ci torn nudi cd khoang 20 loai virus gay benh, trong dd virus gay h6i chiing ddm trdng (White Spots Syndrome Virus - WSSV) la ngudn bpnh gay ra ti le td vong cao trong thdi gian ngdn, d i giy nen su thiet hai Idn nhat ve kinh te cho tom nuoi trong thap ky gan day (Zhang et ai, 2002).
Do d6, hi€u bill ro hon vk d i p utig miln dich cua tom chdng l?i cac vi sinh v^t xam nhiem se giiip dua ra cac giai phdp di^u khi€n b^nh hipu q u i de nham muc dich phit trien dn dinh lau dii ngjie nuoi torn
tren the gidi. Mac du da cd nhieu nghien cuu nhung cho den nay, viec kiem soil benh virus d torn van con la thii thdch Idn ddi vdi ngdnh thuy sdn (Shekhar, Lu, 2009).
Tom giong nhu cdc ddng v^it khong xuong khdc khong cd hp d i p ling miln dich thich ling thuc sy (Hoffinann et al, 1999). Tuy nhifin, do tom sdng Irong moi trudng nude giiu c i c vi sinh vat nen da phdt trien h^ thong bdo ve co the hi^u qui. Budc ddu tien va can thiet ciia q u i trinh niy l i sy nhan biet cac vi sinh vat xam nhiem (Vargas-Albores, Yepiz- Plascencia, 2(X)0) tiep theo do l i nhiing phdn ling nham loai bd chiing ra khoi c o the, hp thdng nay dupe gpi l i mien dich tu nhien. Hp miSn dich tu nhien chia ra l i m hai he thong bao v? chinh: miln djch te b i o (cellular barriers) va miln dich thg dich (Humoral barriers). Miln djch ik b i o bao g6m he thong thyc b i o (phagocytocis), apoptosis, tao khoi u bao bpc v^it the la xam nh|tp (encapsulation) vd sau dd dupe pha huy thong qua h^ thdng ho^t hoa Pro- phenoloxidase (proPO activating s ^ t e m ) . Miln dich the djch bao gom cac he thdng: bd thfi (complement 49
HoingThiThuYenefa/.
system), dong mdu (hcniolymph coagulation system), ydu lo dinh k^l le bio (agglutinin-lectin system) vd qua trinh tdng hpp cdc lo^ii peptide khdng khuin, khing nam vi khdng virus (Iwanaga. Lcc, 2005; Jiravanichpaisal el al, 2006; Liu ci al. 2009;
Tassanakajon et al, 2010).
Hemocyanin li mpl loyi protein Idn chda dong, cd d mdu ciia dpng vat chan khdp vi thin mem (Voil. Feldmaicr-Fuchs. 1990). Hemocyanin dupe xac djnh d trong mdu ciia l^l ca taxa chan khdp (Hagner-Holler et ul. 2004; KUSLIIC, Burmcslcr 2001; Kusche et al, 2002; Pick vt ai. 2009). Trong Idp gidp xdc (Crustacea) hemocyanin d mpt so phan Idp chi cd vai tr6 v(in chuyen oxy, cdc hemocyanin ciia phdn Idp Malacostraca dupe nghiSn cuu nhieu vc cau triic vi chiic ning (Manguin, 1985; MarkI, Decker. 1992) Hemocyanin cua phan Idp Malacostraca ludn hinh thanh cau iriic d^ng hexamcr ho^c dodecamcr, m^c dau cau triic 4x6 da dupe cong b6 (Markl. Decker, 1992; Markl et al. 1986. Miller et ai, 1977). Trong phan Idp nay cdc hemocyanin dupe phan lo^i thinh 3 loai khic biet bang phuong phdp mien d)ch li alpha, beta vi gama (Markl et ai, 1986). Phan tu hemocyanin gdm 630-660 amino acid, vdi khoi lupng phan tii dao dong khoang 75 kDa. Moi phan tu thudng co chua 3 domain (Gaykema et al, 1984, Markl, Decker 1992;
Volbeda, Hoi, 1989). Domain thu nhit ddu N (-150- 180 amino acid) can thiet cho sy hinh thinh soan a.
Domain thii 2 (-220 amino acid) bao gom cdc vi tri bam ciia ddng (Cu) li CuA va CuB. Su van chuyen 1 phan tu oxy dupe trp giiip bdi 2 ion Cu, moi phan tu Cu lien ket vdi 3 amino acid hislidin tuong ling trong cac vi tri d domain 2. Domain thu 3 cd chiia nep gap beta (~260 ammo acid) quy d|nh sy hinh thinh nen cau tnic bac 2.
Dau tien hemocyanin dupe biet den Id mpt protein v$n chuyen oxy. Sau d6, no dupe chimg minh li mpt loai protein da chiic nang lien quan den cac qua trinh sinh ly nhu dy tru protein, dieu khien tham thau, chu ky Ipt xac vi sy hinh thdnh bp xuong ngoii (Adachi et ai, 2005; Jaenicke el al, 1999;
Paul, Pirow, 1998). Den nay, phan tu hemocyanin vi cac peptide bat nguon tir trinh ty amino acid dau C cua hemocyanin da dupe chiing minh co chiic ndng khdng khuan vi khang nam (Decker, Jaenicke 2004;
Decker, Rimke, 1998; Decker el al. 2001;
Destoumieux-Garzon el ai. 2001; Jiang et al, 2007;
Lee et ai, 2003; Nagai, Kawabata, 2000; Nagai el al, 2001). Nhieu gen ma hda cac tieu phan ciia hemocyanin da dupe tao ddng vi xdc dinh trinh ty d
Idp giip xdc Crustacea, 0 t6m cing xanh (Mar.supenaeus japonicus) cd 2 tieu phau hemocyanin (Lei et al. 2008), torn th6 chan trdng (Pi'iiai'u.\ vannamei) cd 2 tieu phan (Sellos et al, 1997), lom Trung Quoc (Fenneropenaeus chinensis) c6 1 ti^u phlin (FJ594414), tom sii Penaeus monodon c6 I lieu phan (AF431737), torn Odontodactylus scyllarus cd 4 lieu phan (Schcrbaum et ai. 2010).
Hai tieu phan hemocyanin (AF43173) d tdm cang xanh (MjallcL vi MjaHcY) cd sy bieu hipn khac nhau d miic dp phien ma khi tdm nhiem WSSV, lieu phan MjaHcL tang ddng ke d nhdm torn nhiem WSSV (Lei ('/ ai, 2008). Nim 2006, Zhang vi dong tic gia chi ra hemocyanin ctia tdm th^ co the hiong tac vdi X loii vi khuan gay bpnh d tom (Zhang el al, 2006). Cling nhdm nghien cim ndy da chung minh hemocyanin cd hoat tinh phan giii h ^ g cau (Zhang el ai, 2009). Hemocyanin dupe tich chiet d torn su vd cho thay khi nang khang nhieu I091 virus bao gom cd virus RNA vd DNA, Tuy nhien co ch^ khang van chua dupe sing td (Zhang et ai. 2004).
Ndm 2003, mpt doan gen hemocyanin khong hoan chinh da dupe phin l^p d tdm sii cd chieu dai 1470 bp (ma so GenBank AF431737), ma hda 499 amino acid dau C vi mpt ma ket thuc TAG. Do do, de gop phan nghien ciiu vai tro cac gen lien quan den khi nang mien dich ciia tom su, trong nghien cuu niy, chiing tdi lien hanh 190 dong va xac djnh trinh ty gen hemocyanin hoan chinh tren ddi tupng tom sii Viet Nam dya tren thdng tin mpt doan gen da cong bo tren GenBank nham phyc vy cho nghien ciru bieu hi^n va chiic nang.
NGUYEN LI$U vA PHLTONG PHAP Nguyen li^u
Chiing tdi da lien hanh thu th§p mau torn bj b?nh ddm trang tgi Tram Nghien ciiu thuy sdn nude Iff, km 10 cao loc Hdi Phdng - Dd Son vd dam nuoi tu nhan vung Ian c^n. Ngay sau khi nhan torn nguyen con, md gan da dupe tach rieng vi bdo quin trong dimg djch nita long.
Phutmg phip
Tach chiet RNA tdng so, tinh sach mRNA vd tong hpp cDNA
Phuong phap tach chiet RNA tdng so, tinh s^ch mRNA va tdng hop cDNA dupe thyc theo md ti cua Hoang Th] Thu Yen va ddng tic gia (Hoing Thj Thu YSn era/., 2010).
Tap chi Congnghe Sinh hgc 10(1): 49-58, 2012
Nh&ngen bang ky thuat 5'RACE va RT-PCR Dua tren thSng tin mpt doan gen hemocyanin da cong b6 trSn GenBank (ma s6 AF431737), chimg toi thi6t vk m6i dac hieu GSPl (Hinh 1, Bing 1). M6i
dac hieu niy ket hpp vdi m6i UPM (Universal Primer A Mix), cung cap bdi kit "SMARTer™
RACE cDNA Amplification Kit" (Invitrogen) d6 nhan do^n gen hemocyanin k^o ddi vk phia dau 5' (gpi tat la: do^n 5'-hemocyanin).
~ 4 0 0 b p
AF432737 U P M
G S P H e m o c y n F l
1470 b p 1
3 ' U T R
1
Hemocyn R l
Hinh 1 . S o dfi thiet ke moi.
Bang 1. Danh sach va trinh tif cdc moi du-o'c SLP dung trong nghien CCPU.
Trinh ty nucleotide (5'-3') Kich thipo^ ircpc tinh
GSP1 CCATGTGGATCACTCTGTCGAC Hemocyn F l ATGGGGACCGTCAACATGAAGGTCTTG Hemocyn R1 CTAATCATGTTGGATATATTCGCCATG
5'-hemocyanin > 400 bp Hemocyanin ORF day dii ~ 2,0 kb
Phin img 5'RACE dupe thuc hien bang enzyme Dream Taq DNA Polymerase (Fermentas) vdi chu trinh nhiet nhu sau: 94''C -3 phiit; (94°C -1 phiit;
55''C -1 phiit; 72''C -I phut) x 30 chu ky; 72°, 10 phiit; ket thuc vi giii d 4''C.
Sau khi tach dong va dpc trmh tu cua doan 5'- hemocyanin, frinh ty diy dii cua gen hemocyanin se dupe xac dinh bing each ghep vdi thdng tin da biet AF431737. Dua tren ket qua phan tich ly thuyet nay, chiing tdi thiet ke cap mdi dac hi6u Hemocyn Fl vi Hemocyn Rl At phan lap gen hemocyanin hoin chinh tu miu tom sii Viet Nam (Hmh 1, Bing 1).
Phin ling RT-PCR dupe thirc hien bang enzyme Dream Taq DNA Polymerase (Fennentas) vdi chu trinh nhiet nhu sau: 95''C/3 phiit; (95''C/1 phiit;
55''C/45 giay; 72''C/1 phut 30 giay) x 30 chu ky;
72''/10 phut; ket thiic va gid d 4''C.
Tdch dong gen
Phuong phap tach ddng cac doan 5'~hemocyanin vi gen hemocyanin ddy du duoc thyc theo mo ti ciia Hoing Thi Thu Yen vi dong tic gia (Hoing Thj Thu Ykn et al, 2010).
Xdc dinh trinh ttfgen
Trinh tu nucleotide cua cac gen dupe xac dinh tren miy ABI PRISM® 3100 Avant Genetic Anlalyzer (Applied Biosystems). Gen hemocyanin dupe phan l|.p va xac dinh trinh ty tir mau mo gan cua 1 ci the tom sii bi nhilm benh dom trang. Trinh ty dupe dpc d 3 dong plasmid tai to hop, d m6i dong dpc lap lai 2 Ian xuoi ngupc vi 2 m6i nam trong gen.
Ket qua trinh ty gen dupe phan tich, so sinh bang 51
Hodng Thi Thu Yen el al p h ^ mem sinh hpc chuyen dyng (Sequence Scanner,
BLAST, Bioedit, GenDoc).
KET QUA vA THAO LUAN Phan l^p doan S''-hemocyanin
Mau mRNA tdch chicl vd tinh sgch td mo gan ciia torn su bj bpnh d6in trdng dupe diing lam khudn tdng hi;^ spi cDNA thu nhat bing cdch su dyng moi 5'-RACE CDS A, SMARTer II A Oligonucleotide vi enzyme Reverse Transcriptase Ihco kit
"SMARTerTM RACE cDNA Amplification Kil"
(invitrogen). Dya tren thdng tin mpt dogn gen hemocyanin di cong b6 trfin GenBank (md s6 AF431737), chiing tdi thiet kc moi d$c hi?u GSPl (Hinh I, Bang 1) Mdi d^c hipu ndy kcl hpp vdi mdi UPM (Universal Primer A Mix), cung cap bdi kit
"SMARTer™ RACE cDNA Amplification Kit"
(Invitrogen) de nhan dogn gen hemocyanin keo ddi ve phia dau 5' (gpi tdt ii: do^n S'-hemocyanin). Phdn ling 5'RACE dupe tiin hanh vdi c$p mdi GSPl/UPM. Sin phim 5'RACE dupe dipn di kiam
tra tren gel agarose 0,8% (Hinh 2A).
Ket qui d hinh 2A cho th^y sin phlm 5'RACE khuyech d^i do^n 5'-hemocyanin cd kich thu6c khoing 1200 bp phii hpp vdi tinh todn ly thuy^
(Bing 1). Nhu vyy, td vj tri m6i GSPl chiing toi dd keo dii them vdo trinh ty d3 biet khoing 700 bp vk phia dau 5'. Sin phdm 5'RACE niy dupe tinh s^ich tir agarose gel vi tich d6ng phin tii trong vector pCR2.1 (Invitrogen). De kiem tra cdc ddng plasmid tii td hpp mang gen hemocyanin chung tdi sit dyng enzyme gidi h^n HindUl vi Xhol, hai enzyme niy sS cat do^n 5''-hemocyanin (neu cd) ra khdi vector tii t6 hpp.
Sau khi phin tich cdc d6ng plasmid tai id hpp bing enzyme Hindlll vi ATtcI, ket qui dipn di cho thay moi plasmid bj cdt thanh hai doan: mpt doan I6n cd kich thudc tuong ling vdi vector pCR2.1 (-4 kb) vi mpt d o ^ nho hon cd kich thudc khoing 1,3 kb luong ling vdi sin pham 5RACE vd mpl phan ciia vector pCR2.1 (Hinh 28). Nhu vay, budc diu chung toi khdng djnh cdc ddng plasmid ndy di cd gdn do^n S'-hemocyanin.
b M 1
Hinh 2. K4t qud tdch d6ng doan 5'-hemocyanin. A. S i n p h i m nhdn doan 5'-hemocyanin b i n g k j ttiu^t 5'RACE (M: Marker
I
1 kb, 1 Sdn phdm 5'RACE nhdn doan 5'-bemocyanin). B KiAm Ira s y c6 m^t ciia sdn p h i m 5'RACE trong vector Idi t6 hpp (M, Marker 1 kb; 1-5 Plasmid pCR2.1 mang doan 5'-hBmocyamn).
Xdc djnh trinh ty do^n 5''-hemocyanin Cdc ddng plasmid mang do^n S'-hemocyanin dupe tinh sach vi lim khudn cho chuoi phin ung dpc trinh ty. Ket qui xic djnh trinh ty dupe Irinh biy d hinh 3.
Ket qua dpc trinh ty cho thiy d o ^ 5'- hemocyanin chung toi phan l^p dupe vdi c$p mdi UPM/GSPl c6 chigu dai 1212 bp, trong do doan tu
v} tri 755-1212 trimg khdp vdi d o ^ dau ciia trinh tu da h\ki (AF431737). Chung tdi di xic dinh dupe them 709 bp ve phia dau 5' bao g6m 7 bp thupc do?n 5'-UTR vi 702 bp thupc ORF cua gen hemocyanin.
Nhu vay. k^t hpp vdi 1350 bp dS biet (AF431737) ta cd the suy ra trinh ty gen hemocyanin diy du la 2052 bp. Trinh ty nucleotide d o ^ 5'-/iemocycrn(n baogfim ca doan 5'-UTR ma chiing tdi da phin lap dupe la mdi va chua dupe cong bd fren GenBank.
Tqp chi Cong nghe Sinh hgc W{\):49-5S, 2012
10 20 30 50
CTAATACGACTCflCTATAGGGCAAGCAGTGGTATCAACGCAGAGTGkTCCftCl
90 100 110 120 130
M G T V N M K
L L F A L V A A A A A W P N F G F Q 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0
Q K Q H D V N F L L H K I Y G D I R D S N L K G K A D 2 5 0 2 6 0 270^ 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 S F D P E A N L S K Y S D G G K A
3 3 0 3 4 0 3 5 0 3 6 0 3 7 0
Q K L M R D L K 3B0 3 9 0 4 0 0 Q Q R
4 2 0
CTGCGdT?3i
D T F V S N 510 520
P G K F K 710 F T G T
730 760 770 780 790 800
[GACAAATACAGCCATCATCTGGATCGCAAAGGAGAGAATTTCTTCT K Y S H H L D R K G E N F F 810 320 S30 340 850 860 870 880 GGGTACATCATCAACTTACCGTTCGTTTTGATGCTGAACGTCTCTCCAATTATTTGGATCCCGTCGACGAACTTCACTGG W V H H Q L T V R F D A E R L S N Y L D P V D E L H W
890 900 910 920 930 940 950 960 GAGAAGCCAATCGTACAAGGTTTTGCTCCCCACACCACTTACAAGTATGGAGGTCAGTTCCCCTCTCGTCCAGATAATGT
E K P I V Q G F A P H T T Y K Y G G Q F P S R P D N V 970 980 990 1000 1010 1020 1030 1040 AGACTTCGAGGATGTGGATGGTGTTGCTCGTATTCGAGACTTGCTTATTGTAGAGAGCCGAATCCGCGATGCTATTGCAC
D F E D V D G V A R I R D L L I V E S R I R D A I A 1050 1060 1070 1080 1090 1100 1110 1120 ATGGTTATATCGTCGACAGGGCTGGTAATCATATTGATATCATGAATGAGCGTGGAATTGACGTTCTTGGAGATGTTATT H G Y I V D R A G N H I D I M N E B G I D V L G D V I
1130 1140 1150 1160 1170 1180 1190 1200 GAATCATCTTTGTACAGCCCTAATGTGCAGTACTATGGAGCCTTGCACAATACTGCTCACATTGTACTTGGTCGACAGAG
E S S L Y S P N V Q Y Y G f l L H N T A H I V L G R Q S 1210
TGATCCACATGG D P H
Hlnh 3. Trinh t y nucleotide vd amino acid suy d i i n ciia doan ^'-hemocyanin. PhSn gach chan la trinh t y cua hai moi UPM v G S P L Phan in d^m la trinh t y 5'-UTR. P h i n c6 mau n&n xdm la trinh t y m& hda hemocyanin mdi du-pc xdc d|nh t h i m .
Hoing Thj Thu Yen e/o/.
Phan l$p gen hemocyanin hoin chinh Dya tren co sd trinh ty do?m gen hemocyanin da cdng bd tren GenBank (AF43I737) vd dotin 5'- hcmocyantn di ph;iii Ipp dupe, chiing loi thi^t k^ m^i dc nhan gen liemocyanin hoin chinh (Hlnh I; Bing 1). MiVi mRNA tich chiel vi tinh s^ch lu mo gan ciia lom sii bj bpnh dom tring dupe dimg lim khudn tong hpp spi cDNA thii nhat bing cich su dyng mdi Oligo(dT). Phin ung RT-PCR sau dd dupe lien hinh
\di cDNA thu dupe. Sdn phim RT-PCR dupe di^n di ki6m tra trfin gel agarose 0.8% (Hlnh 4).
Ket qui d Hinh 4A cho thiy sdn phim RT- PCR nhan gen hemocyanin c6 kich thudc khodng
2,0 kb diing vdi tinh todn ly thuyet. San phim ndy dupe tinh s^ch tir gel agarose vi tich ddng vdo vector pJETI.2 (Fermentas). De kiem tra cdc plasmid tii to hpp chung tdi sir dyng hai enzyme gidi h^n Xbalvi Xhol, cho ph^p cdt gen hemocyanin (n^u c6) ra khdi vector. Sau khi phan tich cic ddng plasmid tdi t6 hpp bing enzyme Xhal vi Xho\, kkt qud dipn di cho thay mSi plasmid bj cdt thinh hai do^n: m$t do^n Idn c6 k(ch thudc tuong img vdi vector pJET L2 (~3kb) vi mpl do^n nhd hon cd kich thudc 2,0 kb tuong ling vdi sin phim RT-PCR gen hemocyanin (Hinh 4B). Nhu v^y, budc diu chiing tdi khftng djnh cic ddng plasmid ndy di cd gin gen hemocyanin.
b M 1
I
Hinh 4. K^ qud tSch ddng gen hemocyanin. A. Sin phim nhan gen hemocyanin bing k? thuit RT-PCR (M' Marker 1 kb, 1:
Sdn phdm RT-PCR nhdn gen hemocyanin), B Kilm tra sy c6 m$t ciia sdn phim RT-PCR trong vedor Idi l4 hpp (M. Marker 1 kb; 1-4. Plasmid pJET1.2 mang gen hemocyanin)
Xic djnh trinh tu vi phdn tich gen hemocyanin Chiing toi xic dinh trinh ty cua gen hemocyanin diydu gin trong vector pJET 1.2 vi tich dong. Phan tich trinh ty, da khing dinh dupe chdc chdn ring do^n cDNA phan lap dupe li trinh tu ORF hodn chinh ma hda hemocyanin. Trinh ty gen hemocyanin day dii cd kich thudc 2052 bp, mi hda 683 amino acid va mpt md ket thiic la TAG. Doan gen dS cong bd tren GenBank mi sd AF431737 tuong ling vdi vj tri 703 - 2052 cua gen tach dong.
Khi so sinh trinh Uf gen hemocyanin diy du phan lap tu tdm sii Vipt Nam vdi trmh ty d o ^ gen hemocyanin (genbank, AF43I737) phan l^p tu torn sii An Dp, chung toi thay cd stf sai khic 33 vj tri nucleotide. Su sai khic trinh ty nucleotide nay dan den sy sai khdc trinh ty amino acid d 7 vj tri 242H-»D, 304N-»D, 360V-»L, 443K-»L, 453Y-»F, 472A-tP vi 576G-»A. Diam ding chu y li hemocyanin d tom su Vipt Nam c6 them 5 amino acid d trinh ty dau N. Ngoii ra, d vj tri 242 (sd thii ty theo trinh tu hemocyanin phan lap tu tom sii Vipt Nam ciia nghien ciru nay), da phan cic loii tdm (tom 54
sii Vipt Nam, tdm sii An Dp, tdm the chin tiing, torn Trung Quoc) deu la D, trong khi dd d tdm su An Dp la N; d vi tri 360 ciia hiu iik cic protein nghien ciiu deu li L tlii d tdm An Dp li V; d vj tri 547, 679 vi 681 tuong img hemocyanin tdm sii li F, Y vd Q thi cdc protein cdn l^i la I va H; ccm d vi tri 576 hemocyanin tom An Dp va cic hemocyanin c6n l^i li G riSng 6 tdm sii Vipt Nam Id A. K^ qua nghien ciiu niy dit ra mpt van de mdi cin nghien cuu sau hon ve cic SNPs trong gen hemocyanin cua tdm SIL M$t khic khi phm tich 6 amino acid histidin th^rc hipn chirc nSng li&i k€t vdi Cu - Vj tri bim cho phin tu oxy, chiing tdi thay ring cic histidin niy mang tinh bio thu rit cao o protein hemocyanin cua cac loai tom (Hinh 5). Theo cic nghien oiu trudc diy, mdi tieu phan hemocyanm deu cd 3 domain churc ning (Ga)4cema el al.. 1984;
Markl, Decker, 1992; Volbeda, Hoi, 1989), khi so sanh 3 domain chiic ning d trinh ty nghifin ciiu chung tdi thiy ring domain I vi 3 cd sy sai khac trinh ty amino acid nhi^ hon domain 2. Trong cic vi tri amino acid sai khac gifta trinh ty hemocyanin Vi^t Nam vdi trinh ty da cong bo mdi domain cd mpt vi tri sai khac amino acid.
Tqp chi Congngh^ Sinh hgc 10(1): 49-58, 2012
Hinh 5. So sanfi (alignment) Irlnh b/ amino acid cOa hemocyanin giOa cdc lodi khac nhau. Trong d6 PmHc (hemocyanin tdm sO An Dp m§ s6 AF431737), PmHc VN (hemocyanin tdm sO Vi§t Nam dupe xic dinh qua nghiSn ci>u nSy), PvaHc va PvaHd (Hemocyanin t6m lh6 mS s6 X82502 vd AJ250830). FchHC (tflm Tung Qu6c ma so FJ594414), MjaHcL vd MjaHcY (tflm cing xanh ma s6 EF375711 vd EF375712 ). Phin trinh ty c6 n^n mau den la phan amino acid gi6ng nhau hodn loan giOa cic loii. Phin trinh ty c6 n^n mdu xim l i phin da so cic loii (60-80%) co amino acid gi6ng nhau. Mui ten chi vj tri khdc nhau giOa Irlnh ty hemocyanin mdi dupe xdc djnh vdi trinh ty da bill AF431737 va vi tri sai khac cua hemocyanin torn vdi cic lodi tflm khic Ddu (*) cht gdc amino acid histidine bdo thu.
55
H o i n g Thj Thu Yen e(a/;
KET LUAN
Chiing loi da lien hinh phiin l§p vd xdc djnh Irinh ty doiin S'-hemocyanin vi trinh tu hoin chinh ciia gen hemocyanin tir mau mo gan ciia lom sii nhiem b§nh dom tring. Gen niy cd kich tliudc 2052 bp mS hda cho 683 amino acid. Trinh ly dogn S'-hemocyanin vi gen hemocyanin hoin chinh d i dupe d i n g k^ tren Genbank vdi ma so luong img li: JF342238 v i JF357966.
Ldi c i m im: Cnni; trinh dugc thijcc hi^n vdi Icinh phi ciia de tdi "Nghien cthi gidi trinh tir mgl phan hg gen vd xdy dipig ca sd dif li^-u genome tdm su (P.
monodon) " thugc Clnrang trinh Cong nghp sinh hgc tiiiiy .sdn 2008-2010. Bg Nong nglii^p vd Phat trien nong than; vd de tdi cap Bg Gido difc vd Dao Iqo Nghien cuu dqc diem cdu true gen .syntenin thugc h^
mien djch torn SH (Penaeus monodon) lien quan den b^nh dom trdng".
TAI LIEU THAM KHAO
Adachi K, Wakamatsu K, Ito S, Miyamoto N, Kokubo T, Nishioka T (2005) An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis. Pigment Cell fles 18. 214-219.
Decker H, Jaemcke E (2004) Recent findings on phenol oxidase achvity and antimicrobial activity of hemocyanins. Dev Comp Immunol 28(7-8): 673-687.
Decker H, Rimke T (1998) Taranlula hemocyanin shows phenoloxidase acUvity. J Biol Chem 273(40): 25889- 25892.
Decker H, Ryan M, Jaenicke E, Terwilliger N (2001) SDS- induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma califomicum, and Cancer magister J Biol Chem 276(2\): 17796-17799.
Destoumieux-Garzon D, Saulnier D, Gamier J, Jouffrey C, Bulet P, Bachere E (2001) Crustacean immunity.
Antifungal peptides are generated from the C terminus of shnmp hemocyanin in response to microbial challenge J Biol Chem 276(50): 47070-47077.
Destoumieux D, Bulet P, Slnib JM, Van Dorsselaer A, Bachere E (1999) Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp. Eur J Biochem 266(2): 335-346.
Ga>4cema W. Hoi W, Vereijken J, Soeter N, Bak H, Beintema J (1984) A " stmcture of the copper-containing, oxygen-canying protein Panulims interruptus haemocyanin. Nature 309:23-29.
Hagner-Holler S, Schoen « , Erker W, Marden JH,
Rupprechl R, Decker H, Burmester T (2004) A respuatory hemocyanin from an insect Proc Natl Acad Sci USA 101(3): 871-874,
Hoing Th] Thu Y^n, Kim Thi Phuong Oanh, Tran Tning Thdnh. Pham Anh Tuan, N6ng Vdn Hdi (2010) Phan % vd xdc d|nh trinh ty hodn chinh cDNA md h6a syntenin liCn quan d^n dip img miSn djch d6i vdi b^nh dom trdngd lom SLi (Penaeus monodon). Tgp chi Cong nghe Sink htfc 8(2)' 155-163.
Hoffmann JA, Kafatos FC, Jancway CA, Ezckowilz RA (1999) Phylogcnctic perspectives in innate immunity Science 2'&4(^4\%): 1313-1318.
Iwanaga S, Lee BL (2005) Recent advances in the innate immumly of invertebrate animals. J Biochem Mol Biol 38(2). 128-150.
Jaenicke E, Foil R, Decker H (1999) Spider hemocyanin binds ccdysone and 20-OH-ecdysone. / Biol Chem 274(48): 34267-34271
Jiang N, Tan NS, Ho B, Ding JL (2007) Respiratoiy protein-generated reactive oxygen species as an antimicrobial strategy. Nat Immunol 8(10): 1114-1122.
Jiravanichpaisal P, Lee BL, Soderhall K (2006) Cell- mediated immunity in arthropods: hemalopoiesis, coagulation, melanizalion and opsonizaboo.
Immunobiology 2\ 1(4): 213-236.
Kusche K, Burmester T (2001) Ehplcqwd hemocyanin sequence and the phylogenetic position of the Myriapoda.
Mol Biol Evol 18(8)' 1566-1573.
Kusche K, Ruhberg H, Burmester T (2002) A hemocyanin from the Onychophora and the emergence of respiiatoy proteins. Proc Natl Acad Sci USA 99(16): 10545-10548.
Lee SY, Lcc BL, Soderhall K (2003) Processing of an antibactenal peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. J Biol Chem 278(10)' 7927-7933.
U i K. Li F, Zhang M, Yang H, Luo T, Xu X (2O08) Difference between hemocyanin subunils from shnn^
Penaeus japtmicus in anti-WSSV defense. Dev Comp /mmMno/32(7): 808-813.
Lightner D, Redman R (1998) Shrimp diseases and cunail diagnostic methods. Aquaculture 164: 201-220.
Lightner DV (1983) Diseases of cultured Penaeid shnmp.
In Handbook of Mariculture. Vol I. Crustacean of aquaculture (McVey JP ed): 289-320.
Liu H, Soderhall K, Jiravanichpaisal P (2009) Antiviral immunity in cmstaceans. Fish Shellfish Immunol 27(2):
79-88.
Mangum CP(1985) Oxygen transport in invertebrates.-4"
J Physiol 248(5 Pt 2): R505-514.
Markl J (1986) Evolution and fiinction of structurally
Tgp chi Congnghe Sinh hgc 10(1): 49-58, 2012 diverse subumts in the respiratory protein hemocyanin from arthropods. B/O/BM// 171:90-115.
Markl J, Decker H (1992) Molecular sUuchire of the arthropod hemocyanins. Adv Comp Environ Physiol 13:
325-376.
Markl J, Stocker W, Runzler R, Precht E (1986) Immunological correspondences between the hemocyanin subunils of 86 arthropods: evolution of a multigene protein family In: Linzen B (ed) Invertebrate oxygen carriers,
Spnnger, Heidelberg. 281-292.
Miller K, Elderd N, Arisaka F, van Holde K (1977) Structure and function of hemocyanin from thalassinid shiimp. J Comp Physiol B 115: 171-184.
Nagai T, Kawabata S (2000) A link between blood coagulation and prophenol oxidase activation in arthropod host defense, J Biol Chem 275(38): 29264-29267.
N a p i T, Osaki T, Kaw^ata S (2001) Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial pepndes. J Biol Chem 276(29): 27166- 27170.
Paul R Pirow R (1998) The physiological significance of respiratory proteins in invertebrates. Zoology 100:298-306 Pick C, Schneuer M, Burmester T (2009) The occurrence of hemocyanin m Hexapoda. FEBS J276{7): 1930-1941.
Scherbaum S, Ertas B, Gebauer W, Burmester T (2010) Characterizadon of hemocyanin from the peacock mantis shrimp Odontodactylus scyllarus (Malacostraca, Hoplocand^). J Comp Physiol B180($): 1235-1245 SeUos D, Lemoine S, Van Wormhoudt A (1997) Molecular cloning of hemocyanin cDNA from Penaeus vannamei (Crustacea, Decapoda): stmcture, evolution and physiological aspects. F£B5Z,erf 407(2): 153-158.
Shekhar MS, Lu Y (2009) Application of nucleic-acid- based therapeutics for viral infections in shrimp aquacultuie. Mar Biotechnol (NY) 11(1): 1-9.
Tassanakajon A, Amparyup P, Somboonwiwat K, Supungul P (2010) Cationic anli microbial peptides in penaeid shrimp. Mar Biolechnol (NY) 12(5)' 487-505.
Vargas-Albores F, Yepiz-Plascencia G (2000) Beta glucan binding protein and Us role in shrimp immune response.
Aquaculture \92: 13-21.
Voit R, Feldmaier-Fuchs G (1990) Arthropod hemocyanins. Molecular cloning and sequencing of cDNAs encoding the taranlula hemocyanin subunils a and e. J Biol Chem 265(32): 19447-19452.
Volbeda A, Hoi WG (1989) Crystal stmcture of hexameric haemocyanin from Panulirus interruptus refined at 3.2 A resolution. J Mol Biol 209(2): 249-279.
Zhang X, Huang C. Qin Q (2004) Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon.
^nfmra/ftes 61(2). 93-99
Zhang X, Huang C, Xu X, Hew CL (2002) Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein. J Gen Virol 83(Pt 5): 1069-1074.
Zhang Y, Wang S, Xu A, Chen J, Lin B, Peng X (2006) Affinity proteomic approach for identification of an IgA- like protein in Litopenaeus vannamei and study on its agglutinatton characterization. J Proteome Res 5(4); 815- 821.
Zhang Y, Yan F, Hu Z, Zhao X, Min S, Du Z, Zhao S, Ye X, Li Y (2009) Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity. Fish Shellfish /m/nM/io/27(2): 330-335.
C L O N I N G A N D S E Q U E N C I N G O F T H E C O M P L E T E c D N A E N C O D I N G H E M O C Y A N I N A S S O C I A T E D W I T H I M M U N E R E S P O N S E O F S H R I M P T O W H I T E S P O T S Y N D R O M E V I R U S {PENAEUS MONODON)
Hoang Thi Thu Yen', Kim Thi Phuong Oanh^ Pham Anh T u a n \ Nong Van Hai^' Thai Nguyen University
Institute of Biotechnology, Vietnam Academy of Science and Technology Directorate of Fisheries, Vietnam Ministry of Agriculture and Rural Development
SUMMARY
Hemocyanin is a large, copper-containing protein present in the hemolymph of both mollusks and arthropods. The traditional role of hemocyanin is the transport and storage of molecular oxygen. To day, it is known that hemocyanin is a multifunctional protein, especial fiinction related to antivirus, anflmicrobial and antifringal immune responses. A partial coding sequence of cDNA encoding hemocyanin consisted of 1470
'Authorfor correspondence- Tel/Fax: 84-4-38363222; E-mail vhnongi
Hodng Thj Thu Yen et al nucleotides including slop codon and 3'-L]TR is nvailahk Irom GenBank with accession number AF431737 In ihis study, we cloned and sequenced of Ihc full lenglh cDNA encoding synlenm from WSSS infected black liger shrimp. Using 5'RACE technique, we dclcrmined 5'-cnd sequence including initial codon and 5'-UTR Based on the 5'-cnd sequence iind Ihc GenBank sequence (AF431737), the complete hemocyanin cDNA was ' • cloned and sequenced. The coding region of hemocyanin cDNA wa.s 2052 bp in length, and was predicted to encode II 683 amino acid prolem The puliitivc protein contnins 3 domains and 6 conversed histidines to set sii-uciiiic iuid function. Thcresuli ofnuclcolide sequence alignment between the newly determined hemocyanin sequeiiLL- and thai of WSSV inrcctcd Penaeus monodon isolated in India showed that there are 33 different iiiiclciiiidc-s in ihc Iwo SL-quciiccs. The nucleotide differences were found to result in 7 amino acid substitutions including 2421t-.D. 304N -1), 360V—L. 443K--.L, 453Y-.F, 472A-.P and 576G-*A. Our results would be useful lor further sludy ufihc funciion of hemocyanin and rclulcd proteins in ihc shrimp immune response.
WSSV Keywords: Black liger \hiimp. iinmune-relaledgene, Penaeus monodon, Hemocyanin,
