Le Van San vo Dig Tap chi KHOA HOC & CONG NGHE 90(02): 59 - 64
THIET KE VECTOR IVIANG GEN OPHC2 PHI C \ L TAO C.\\ CHIA EN GEN PH.AMHY T i l l ()C TRLT SAU
Le Van Son',.Nguyen \ u Thanh Thanh" , Nguyen Manh C u o n g ' ' I 'leii ('dug nghe smh hoc - Tnr&ng Dai hoc Khoa hoe-Dai h(.)C Thai \gtiyeii
T O M T A T
(Organophosphorus (OP) la mgt chat cd nhieu trong cac loai thuoc trir siiu hien dang dugc dimg rgng rai tren thj truang va no giiy d nhiem mdi trudng nghiem trgng. Enzyme organophosphorus hydrolase (OPHC2) ciia mgt sd loai vi khuan cd kha nang phan hut chat OP. Mot sd cdng trinh nghien ciru dii sir dung gen ma hda cn/yme OPHC2 dc bien nap. bieu hien trong cay thudc lii va lao ra ciit chuyen gen khang dugc thudc trir siiu methyl parathion (Mep)-mgt loai OP. Trong bai biio niiy. chiing tdi trinh bat kel qua toi uu trinh tu gen OPHC2 bieu hien trong thuc tat tii thiet ke vector chuven gen mang cau iriic 35S-OPHC2opl-cmyc-KDEL-NOS (pBlN121 0PHC2). Vector niiy da chuven vao chung vi khuan ,1. lumejaciens CV58 va kiem tra bang PCR t d i cap mdi dac hieu. Diiv lii ca sa de tao ciiy chuyen gen mang gen OPI1C2 de phan hut thuoc trir sau Mep tdn du trong mdi trudng.
Tir khoa: Agrohuclerium. ()PliC2, Organophosphorus hydrolase, rector chitren gen.
M O D A U
\'icc sir dung tran lan tkudc bao ve tkuc vat trong Siin xuiit ndng ngkiep dang k'l mdt trong nkCmg nkiin td giiy d nkiem mdi trudng diit a nkieu kku vuc san xuat ndng ngkitip anh hirong nghieni trpng den sue kkpc eiia con ngirtVi va ciiy Irdng (Wauckopc. 1978;
Kawahigashi. 2009). Nhicu ngkien ciru dii cko tkiiy t iec su dung eay trdng de loai bo cac ckiit d nkiem tir mdi trudng diit vi'i nutVc mang liil nil nkieu uu diem so t d i ciic pkirong pkap xu Iv truven tkong. Ciie ckat d nkiem tkudng dupe kiip tku t i i o city tlidng qua re vii la t i i dutic chuyen kda tkiink ciic chiit kkdng cd kai Vii tich ICiy trong md eua ciiy (Salt et ak. 1998) koac Ciiv Irdng tiet ra ciic loai c n / y m e giup pkiin kuy cliiil d nhiem Irong mdi trudng diit, Organopliosphoi'us (OP) ki mdt ckat cd trong thutie trir siiu hien dang dupe dung rdng rai tren ihi trudng ndng ngkiep (Singk et ak. 2006;
Karpou/as cl ak. 2006) vii dang kim d nhiem mdi tnrdng ngkiem trpng (Kawahara el ak.
2005; Kpnslanlinou et al.. 2006; Pagliuea et ak.
2006; Bondarenko et al,. 2004; Qiao et ak.
2006) I n/vme oiganopkospkoriis kvdrola.sc ( O P I l ) eua mdt sd loiii vi khuan co kha niing phiin huy cliiil OP ( W t i el ak. 2001. Amaya-
lel 0912 (i(>4 126. Email, ihanhthanhdhkhln a gimiil com
Ckavez et ak. 2006). Dii cd mdt sd ngkien cuu sir diing gen ma kda en/ymc OPTI de bien niip va bieu kien trpng cay thut3c ki. Ket qua pkiin tick cko tkiiy. ciiy tkudc lii chuyen gen da san xual ra cn/yme O P I l irong mdi irudng nudi cav. Ciiv chuyen gen khiing dupc thutie trir siiu methyl partuhion (Mep)-indt Ipai (3P (Wang et ak. 2008).
Trong biii bao nay. ckimg tdi trink biiy ket qua tdng lip'p gen ma kda cn/yme OPIIC2 (OPHC2ppt) dirtTC tdi uu bieu kien trpng thuc Viit Va thiet ke t ector ckuyen gen mang eau tnic gen OPHC2opt. D;"iy ki cti sd eko tao ciiy ckuven gen pkan huy thudc trir sau Mep tdn du trong mdi trudng hien nay.
\ A T U E U \ ' , \ PHU'ONG PHAP NGHIEN C U L
\ ' a t lieu
Gen 0 P H C 2 t i i mdi (primer) dat tir kang Epocklifescienee (My ) tdng kap.
Chung E.coli D H 5 u . .Igrohacleriuin C V 5 8 va vcctoi' p B I I 2 1 do pkdng Cdng ngke tc bao- N'ieii Cdng ngke sink kpc cung cap
Phu'ong p h i i p nghien c i i u
l i i c k DN \ . plasmid va tink sack dupe thuc hien dua tkeo plunuig pkiip eua Sambrook (1989) koiie kit tiick pkismid cua (,)1 \GT.N Thu doan D N A mong mudn t u gel agarose vdi kit thdi gel OTAquick ( ( J I A G l . N )
59
Le \'an Son vJ D:; Tap chi KHOA HOC cC C(4NG NGHE 90(02): 59 - 64 Cac phir.sui; phap trong sinh hoc phan t u nhu Trinh t u D N A diroc xac dinh tren may doc cat hang e n / \ me .at gioi kan. ghep ndi gen trink t u t u Jong M i l P R I M S K .'100 \ t a n t dutitc ili'jc hicn dija tkeo phuong phap cua Genetic .Analyzer ;\"iiig c.icli su dung bd hoa samhroc^k i 2 U i ) l ) . c h i t sinh chuiin HigDvcK Terminor \ 3 1 Bidn nap plasmid vao trong E.coli dua theo '-~>-'l'-' "is-qucn.-iig - \ ien Cdng nghe Smh hpc, phuang phap bien nap bang sde nhiet (Cohen. K E T QL'.A \ A TH.AO L l .\N
19-2). vao lj;''/N;./c;•;;;;;; bang phuong phiip y g j „.„ ^., ,5no hop gen O P H C 2 bicu hien
^""gdien. trong thu^c Mit:
- 0 ; o 3 0 - i - FC eA' " 0 SO - ! E i - ' I t 0 P H C 2 A T & G C C G C A C C e G C A O . - . C \ G . \ A G A C C C , . t ; t . T A C C & G f r C r - C l ^ C C G T sTGGC.AC I C G G T G A C T T C G A A G T C A C C 6 C T C T O P H C ? o p t . . . A . . T . . A A . A . A . C A . . T . T A . A . . T T . G 1 . . . . G . . r - A . . A . .
0 P H C 2 G T A T G A C C G C T A C G I C & \ C C I G C < 1 G C C A G C C I GC r C A A G G G C A T C G A T G A C A ^ l - i . ^ ( f-',-.. U f i C 1 f-v i 0 P H C 2 o p t T . C . . T . . T T . TT A T C A T . T G . . . A . T . . . . T A . I . T . G A G r . . l T .
O P H C ? I G T T C G I G G C G I C M . I.. - - ( ; r . < r . l G C A G A C T C C C G T C A A C C ; C C I A C C I G , . I C A A C A C I G G C G A C A A C C T C G T C C TGA TC O P H C ? o p t A T . A . C . A . A . . T . . A T . . T . . . - . . A . T T . . T . . A . . T . . T
O P H C ? C A T A C C G G C G C C G C C C A C I G O T T C G G C C C G A C T C T C G G C G I G G T G C A G A C C A A C C I C . A C C C A T C C G G C I A C C A C C C G G A O P H C ? o p t C A T . T . . T . I , C - T - . A . X . I T I I G A A A .
3 " o ; 4 0 -5.3 :ۥ:< 3 7 r . - 9 0 : y o 4 0 0 i 1 , - . - - I . . . . I . - , I . . . I . . . I . . . 1 . . . I O P H C ? GCAGGT T G T A C C G T C C T C C T C A C C C A C C T G C J ^ C C C A G A C C A T C C C T C C G C C C T G G . C C G C C G / i C G G C A G C C C G C C C T O P H C ? o p t A A . T T G . A . T T . T . . T . . C . . A . . T . . T . T . f. . . T . T . . T T C A . . T . . . .
O P H C ? A C C C C A A C G C G A C C G T G C ; . G G T G C C G C A C C C C C A C G f T G A A T T C T G C C T C C - . C G A G G C G A C C A T C G C C / J i C C C C C C C G A A O P H C ? o p t T . C - A . . T . T . A . . C . A T . . . T . G C . T . . . . A . A . . A . . A .
O P H C ? C G C A I G C , ^ \ G G C A I G I T C A A C A T G G C G C A A C A G G C A G T C G C A C C C T A T G C C A A G A l G A A C A A G C T C v \ A G C C C T A C A / i G A C n i ' H C ? o p t . . I T A . . . . A . G . . . . r . . A T . . . . A . T . 1 G . . . I .
t U ' H C ? A G . - ^ i , G C C G A A T I G T r G C C T C C C G r C A G C C T G G l / GC G A G C C C G G a ' \ C A C A C G C C C C G . CA f . C C I C 7 I AC C I G F I C A A A I O P H C ? o p t . . A . CC TC I . - G . - T I C T . T G I T C I . . I . . . . I . T . . T . . C - T - . A . I . . GA
O P H C ? C G G G T G G A C A G A G C C K . C T G G I A T G G G G C G A C A T 1 C 1 GC T T A / i C C A C C C C G T G C A G T T C C C C A A G C t i U . G I f . l . K I T C O P H C ? o p t C I . . . A T C i r I . . . T A . I I . A A A . . M . C . « . , T . . .
O P H C ? ( . A t I T C C A T G I C G A C GC G f . C C A G G C C A G G C A A T C C C G C C A / C G C A I C C TGGCC G . , G C G G C C A C A G A C A A G C I G T C C G T O P H C ? o p t A . T . . G . I C - . I . . , T . . . . A A . G . G A . A A I . T G . . A . . . . C . T . T . . . .
O P H C ? f G C I C G l G C G C A C C T G C C C T T C C C C G G C C T G G G C C A C G t A C G C / V . G G A A G C C C / - . , G C C ! A C G C C T G G G T A C C C G T C G A G T O P H C ? o p t i C . . 1 . . T T A . A . T T . . T . T . . . G T . . T C . . A
O P H C ? I C A G C C C G A T C C G T A G C G A C C G C T G A O P H C ? o p t I I C T . T T A . A I , . T A . A A
Hinh I. V ^Jsnh tnnh lie •c^n OPHC: cua P.ps.hJt.jl.jhzenes iOPHC:i vu ()rHC2 JJ sira Jd n)P}K':t>p!,
M l
I.e van Strn vi:'/ Dig Tap chi KHOA HOC & CONG NGHE 90(02): 5 9 - 6 4
\ l \ M'Al^tJKKJVPC'i'i KMAl.GDl I V I M.AIKj'i \ DIP V^l.l.kdlDDKDl (,)Sl,l..\R\ir\ \sl K(i\t,)l \ \ \ \ M l \ l ( i l ) \ l \1.1
DTGA \(;( I (,Pll,(iV\'(/l\l.KAS(iYgi'h(;Vl)lVl.l.lHI.HPDH-\CGL\'N \ D ( i S l ' , \ M ' \ ATVI \"PQ \l \l F\\ I 11 1 \ I \ I \ K A I ' I
(iMUfiVll K V l v g y W A P Y VKXIKKI.KPYKTEGELl.PGVSI V VSPGHTPGITI SM I KSddgsl I.X'W (iDlI.EMlA Vt;tAKPT,VVE
I 1 lIVDSDDARl^SRtJRII \ l , \ , \ I I ) K I W VAC IAI II PFPCil Cil IV RKI. \(;(A" WW IA I I M'lRsllR, Hinh 2. Trinh tu amintj add cua prolein ()PI-IC2
Mdi amino acid dupe mii hda boi cac bd ba nucleptide kkac nhau. Mi're dp bieu hien eua mdi bd ba trong t u n g ddi t u p n g , tirng loai rat khac nhau. C i m g mdt amino acid, ed nhung bd ba bicu hien trong vi kkuiin miic dp eao nkung trpng tkuc vat lai bieu kien d mirc dp thiip Vii ngup'c lai (Kavvabe va Miyaskita.
2003: Strcatneld, 2007). Do viiy. dd bieu hien protein eao trong cay trdng can pkai ldi uu ma bd ba pkii ktip. M i i c bicu hicn protein ciio la tiin dc dupe nhicu cdng trink nghien cuu. ung dung t i l eko ket qiiii Itit (Tennoy vii cs. 1993;
I agiiia-Bceker vii cs. 2010; .labeen va cs.
2010). Dua t i i o trink ttr cua gen OPHC2 (kich tlui'dc 906 bp) pkiin kip tir cluing Pseudoinotias pseiidoalcaligaiies li'cii N C B I VtVi niii so A.I605330. cluing tdi dii tkiet ke moi dc nkiin gen ()PIIC'2. Irinh ttr gen ()PIIC2 dii'tic sua ddi tie bicu hien tdi uu trong tlure t i i t (kinh 1).
Xiuiiio acid eua O P I I C 2 opt cd kick thud'c va trinh til kkdng tkay tldi t d i c n / y m e t3PIIC2 cua P.pseitdoalcaligcnes (kink 2),
Trinh t u gen bi Ikiiy ddi nkiim tiing kka niing bieu kien trong Ciiy trdng. song trink l u protein O P I I C 2 t i i n g i u nguyen n k u ban dau.
do tiiv koat link protein kkdng tkiiy d d i , tink chiit phiin huy cluil OP viin dupe g i d nguyen.
Kill O P I I C 2 o p l bieu kien trong ke tkong tc hao tkuc t i i t . dt; giup eko protein dupe luian ihieii c i u irtic, koiil t m h . cluing ldi bd sung them mtit peplid tin kieu K D I T , v.i mtit doan nkiin biet c-mye (hink 4).
Dc dam bao sir ckinh xiie trinh tir gen tdi uu.
OPIIC2opt dii dutic tdng hap nluin tao boi Cdng IV TpockliTescienee ( M v ) C\iu true luiy dli duoc gkep trong vector lack ddng pBluescript II SK(-)
Sau k k i nkan dupe pBluescnpt 11 SK(-) ( i P I IC2ppt. cluing tdi dii tien kank bien nap va nhan trong E.coli, sau do kiC'in tra ciii thu vector bang en/y m e Bain\U Sctcl. Ket qua iku dupc ed 2 doan D N , \ cd kick tkutie kkoang I kb va 3 kb. tuong ting t d i kick tkut'ie cua gen OPHC2opt Vii t c c t o r pBlueseripl II SK(-) (kink 3).
Hinh 3. //;;;/;;;;;/; i;/;c';7 (/; san phdm cdt pBluescript ll SKt-) OPIK ':opl himg BcnnHI SucI
\l: llicing DX.I clnidii I kh: 1-2 san pham cdi rector pBliiescriplIISK(-) ()PllC2opl
Gen OPHC'2opt ciing dii dupc kiem tra lai trink t u . ket qtia tku dirtie gidng t d i cau trtic dii tkiet ke. trink tu gen dii dupe tdi uu mii bd ba tkeo veu ciiu ban diiu Diiy la co so eko thiet ke vector chuyen gen.Thiet ke vector chuyen gen m a n g gen ma h d a c h o O P H C 2 o p t
Ds} tkiet ke vector cluiyeii gen. plasmid pBluescriptll SK(-) O P I I C 2 o p t dupe c i t ddng thd'i biing . enzyme /i(;;;;lll .S'(;cl. Cau tnic O P I I C 2 o p t - e i n y c - K D F , L tku dupc cd kich tkudc kkoang 1.0 kb (hink I). Ddng thoi vector ckuyen gen p B I I 2 l cd ckua promoter 35S cung duoc ciit boi 2 e n / y m c tuong irng 61
Le \ ."in Son va Dig
Tap chi KHOA HOC cV: CONG NGHl90(02): 59-64 Banii\\,Sac\. Ciiu tnic 0PHC2apt-emye-
KDEL va vector p B l l 2 l ma vdng dupe thu lai va tinh sach khoi agarose. Sau dd 2 san pham dupe ghep ndi vao nkau bang enzvme T4 huasc
35S ; P H C : C-riKc KDELNos
Hinh 4 Cdu Irtic gen chuyen Irong reclor pBll2l San pkam lai nay dugc bien nap vao E.coli D115 Vii dupc ckpn loc dong bien iiiip bang kkang sink Kanamieinc
( ac ddng bien nap dirtie kiem tra biing Ciicli lack ckiet plasmid. sau dd PCR toi ciip nidi diic kit;u OPHC2-FOPIIC2-R va ciit boi enzyme kan die BanilW Sctc\ (kinh 3).
Hinh 5 Hinh anh dien di sun phdm PCR I ll va cdi rcclor nil Id hoppBII2l OPHC2opt (Bl .\l lining D.\ I chudn 1 kh:
1-2 III sciii phdm PCR nhdn gen OPIK. '2opl rot cap nidi ddc hieu
1-2 Ipl san phdm cat cdc ddng vector ldi id htrp pBll21 ()PHC2opl himg enzyme gtiti hgn
Hink 5 cko tkay san pkam PCR cd kick thirdc khoang 1.0 kb tirong dirong vdi kich tkudc can ti'uc gen OPH( 2cipt-cmyc-KDE.L. Tren kink ank dit:ii di cit vector lai td kop bang en/viue gioi han ckung tdi tku dutie cd 2 biing kick ikiitK' kkoang 1.0 kb va 12 kb luong tmg vol cau true 0PIIC2apl-cmyc-
KDEL va vector p B l l 2 l Nku tax. ciu ti'iic OPIiC2opt-emyc-KDEL dii dutie tkiet kc thiink cdng v ao v ector ckuy cn gen pBI I 21.
Tao chiing Agrohaclerium mang vector pBI121/OPIIC2opt
Vector pBll21 (iPHC2opt da dut.ie ckuven Viio ckung .Agrohiicteriiini C \ 5S biing pkii'ong phiip xung dien va diroc ckpn lpc tren mdi trudng chua kkang sink Rit'aniieiiie va Kanamieinc.
U b I'.S kb
•
C
•
m
-.
^ ,
'
>
1
; **^„
^:
i 1
I
r%
Hinh 6. Hinh unh dien di sun phdm PCR lir chung Igrohcn teriiim dd bien nup pBII2l OPIIC2iipl
\l: thang / A . I chudn 1 kh: ddt chimg chnnig (plasmid pBI121 ()l'IIC2opli. -: doi chimg dm (iiirircl: 1-4: cdc ddng
Igrobacleriitm bien ngp
( ac kkuan lac mang t cclor chuy en gen dirtic kiem tra biing PCR biing cap mdi tkic hieu ()PIIC2-F OP1IC2-R. Dien di san pkiim PCR cho thiiy. vi kkuan .'Igrohacleriuin da mang t cclor tai td kgp (kink 6). Ket qua nay cho tkiiy. ckiing tdi da bien nap tkiink edng vector pBI I 2 1 OPl l( 2opt V ao Agrohaclerium.
KETI.L,\N
Dua tren trink tu gen ma hda eii/vme OPIIC2 cua P.pseiidoalcaligencs. eliiing tdi dii tdi uu ma bieu kien trong tkuc vat vit tdng hgp dugc gen OPHC2opt. Cau tnic gen OPIIC2o|it- cmye-KDEL da dupe tkiet kt: \ao vector ckuyen gen p B I I 2 l . Diiy la ngudn vat lieu de su diiiig ckuyen gen viio thue vai. nham niing cao kha nang phiin kuy thudc Irir siiu Mep gay d iihit;ni mdi trudiii;.
o:
Le Van San ea Dig Tap chi KHOA HOC & CONG NGHI 90(02): 5 9 - 6 4
L d i c a m o'n; Cdng Irinh dirge ihirc hien trong khudn khd de tdi cdp Vien Cimg nghe sinh hoc " Xgltti'it cim phinrng phdp Igo cdy .xoan la (Melia azeduruch Lin; chuyen gen C)PHC2 nhdm inig dung Irong xir ly <i nhiem ihui'ic hao ve thirc vdl" Cdc thi nghiem dirge lien hdnh cd sir dung trang ihii'l hi cua Phdng Thi nghieni trgng diem vi' cdng nghe gen, I'ien ('dng nghe sinh hgc.
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\'an San vd Dig Tap chi KHOA HOC cV C(iNG NGHE 90(02): 59 - 64
SUM\1AR>'
CONSTRUCTION OF BINARY VECTOR CARRYING OPHC2 GENU FOR PESTICIDES RESISTANCE TR-VNSCiEMC
Le \ an Son', Nguyen \ u Thanh T h a n h ' " , Nguven Manh Cuong"
Insniute of Biotechnology 'College o- s,,,' , ..v - Thai \giiyen I 'ntrersit\
Organophosphorus (OP) compounds are ttidelv used as pesticides in agriculture but cause broad- area environmental pollution. Bacterial organophosphorus hydrolase (OPHC2) could degrade OP compounds in environment. OPHC2 gene is isolated and transferred inio lobacco. \n assay of enzyme actititx showed that transgenic plants could secrete OPH into the grottth medium. The transgenic plants ttere resistant to methyl pariuhion (Mep) as an OP pcsiicide In this paper, tte present the results on 0PHC2 and binary tcctor construction. In this paper. OPHC2 gene has successfully optimi/ed codon in plants and consirucled binary vector caring 35S-OPHC2opt- cmtc-KDET-\C)S ( p B l \ i 2 1 (iPHC2opt). This tcctor ttas transferred into A. tunicfaciens C\'58 and confirmed by PCR ttith specific primers "This results leads to Turther study for OPHC2opl gene oterexpression and Mep degradation in transgenic plants.
Ke.\ words: Igrnhucterium.hinuiy reclor. OPHC2, Orgunophosphorus hvdrolase
' e,/,_^ ; 26 I " all thanhlhanhdhkhln dgmail com (.4
