HOI NGHI KHOA HOC CONG NUMt SIMH nv^.
PHAN LAP GEN MA H 6 A CHITINASE VA T H I E T K ^ C A C VECTOR BlEU HIEN THLfC VAT
Hd H ^ Hanh, Ld Thanh Htrimg, Le Thj Thu Hiln
Vi^n Nghidn dhi hi gen, Viin Hdn lam Khoa hgc vd Cong ngh$ Viit Nam
T6M TAT
H9 chitinase diiidng cd mil trong nhieu m6 ciia nhieu loai cay trong khic nhau, trong dd cd c ^ ca cao (pieobroma cacao L). Mft trong nhimg vai trd caa duiinasc trong divrc v|t bac cao la bao vl cay chong lai s^ tSn cdng cua cdc yeu to gay b^nh, d?c bigt linaoL Vdi rate dich sir dyng cac vector Ti-plasmid tai tl hpp de chuyen gen ma boa chitinase vio c5y trflng ndi chung vi cfiy ca cao o6i rieng, chiing tdi d3 tien hanh phan % vi tgo ding gen ma hda chitinase tCr cay ca cao (TcChil - toin bp viln^ mang ma; TcChil-U- toin b$ vung mang mi vi mpt phin vung khong djch ma). Sii dung vector gdc pCB301, Ti-plasmid tii to hpp mang gen TcOiV dirpc tgo ra, trwig do TcChil dupc lai vdi mpt do?n ma hoa pqitid c-Myc KDEL vi dupc dieu khiSn bieu hi?n bdi CaMV3SS promoter. Ngoii la, Tl-plasmkl tii td hpp mang gen TcChil-U dudi syr dilu khiln cua FMV34S promoter cung dirpc diict kl trin co so su dyng h? vector gdc pPIPRA. cic vector bilu hien thvc vat smi khi thilt kl da dupc su dyng dl t?o cic cbilng vi kliiiii Agrobactenum tumefadais tii td hpp, kiem tra bilu h i ^ tam thdi tr&i cay tiiu^ li dong C9-1 vi chuyin vio cay ca cao.
Tw khoa • Agrobaclerium ban^aaais, cay ca cao, chitinase, Ti-plasmid.
MdreAU
Hp chitinase Id cdc enzyme tham gia vSo qui trinh thuy phfin N-acetylglucosamine potymer chitin vd thifd-ng cd ni9 bung nhidu md ttiyc v$t cua nhiiu toai cfly tr6ng khac nhau, hong dd cd ca cao (Theobroma cacao L.) (Snyder et al., 1992). CSc enzyme chibnase c6 thi 6irge bilu ht§n ccr djnh trong cfiy vd'i mi>c dO thip. Tuy nhiSn, mile dO bilu hi^
cOa cdc enzyme ndy c6 thi dif^c tfing ldn d$t biln khi cfiy phdi chiu tfie dpng dia cfic y l u td vd sinh (nhu- ettiylene, salicylic ackl, cdc dung djch mu6t, ozone, tia UV) vd cdc y l u to h(hj sinh (nhu nam, vi khuan, virus). M$t trong nhOng vsi trd dugrc cho Id cua chibnase trong thi/c v$t b?c cao Id bdo v# cfiy ching Igi sg tin cdng cua cfic ylu t6 gfiy b|nh, djc bi$t Id nim, do cdc chibnase du^rc bilu hl$n mgnh lgn ddng k l trong cdy sau khi cay bj nhilm bgnh (Punja & Zhang, 1993). Gen mfi hda chibnase tdp 1 ducrc phdn l$p tur cfiy ca cao gom 3 exon (cd kich thucrc lan lu^t Id 417 bp, 153 bp vd 393 bp) vfi 2 Inbon (cd kich thudc Id 88 bp vfi 104 bp). VOng mang md cda gen cd kfch thudc 963 bp, ma hda phfln ti> protein TcChil g6m 320 ammo add Protein TcChil bilu hi|n dr cdy ca cao chuyin gen cd khfi ndng i>c chl s^ phfl triln cua n ^ Co/tefofrKhum ^oeosporioides, ddng th6i hgn chl sg phdt biln cua cac dim hogi tCr or cdc lfi dfi bj nhiilni nim (Maximova et al., 2003, 2006).
Vdi myc dich chuyin gen mfi hda chitinase dudri sg dilu khiln cua cdc logi promoter mgnh nhu CaMV3SS vd FMV34S vfio cdy b ^ g , cdc vector tfii t l hop mang gen ma hda chitinase hgn ca sit pCBSOl vfi pPIPRA558 da duQrc thiif kl.Trong cdng blnh ndy, chung tdi thdng bfio kit qufi phfin idp gen mfi hoa chitinase tO cdy ca cao, thilt k l cdc 71- plasmkl tdi t l hgrp cung nhu tgo chung vi khuin A. tumefaciens mang nhung vector ndy, dong thdi nghlSn cuu k i ^ tra bilu hi$n tgm ttidi cua gen ma hda chibnase trdn cay thulc la Nicotiana tabacum ddng C9-1. Cdc chiing A. tumefaaem t&i t l tigp dang dugc su dyng d l chuyin gen ma hda chitinase vfio cfiy ca cao nhfim tfing cudng khfi ndng khdng nln cua cdy ca cao.
VJBIT L I $ U VA PHlfONG PHAP NGHIEN CIJU V$t lif u
V$t li$u tfi(«: v$t: Ld aja ddng ca cao thu'orng mgi TD3 ducrc thu thdp tai Vudn isam ca cao, Trudng Dgi hpc Nflng Ldm Thfinh phd Hd Chi Minh. Cfiy thulc la ddng C9-1 do phong Cdng nghg T l bdo thyc vat. Vign Cdng nghg sinh Hoc
cung cap. . C$p m6t. Ba cdp ml! TcChi1-W_F/R; TcChi1.U_F/R; TcChil F/R thilt k l di^a trgn trinh tu gen chitinase md 1
U30324.1 vd 6irgc dgt ting hgrp tgi Hang Alpha (Canada). 1 TcCtii1-W_F S-acgtagatetagtcaatggctcgatglgdcca.a' TcChil-W^R: 5'-actgaaatctgaaaatgaaaatcaaaggaacca-3' 3
^ 1 - Bgfll, J TcChi1_U_F: ff-tta^ccacacattgacaaaatcacaTT-S' TcChi1_U_R- S'-gaa^tetatcgatctttgfEtaccaaagtca-a'
^ ' Bgfll T=0,M_F !r-Bt<x=^t«9agc01cgg9ccng-3' TcChn_R:5-scactaaaacggccflcgclacltgaglcca.3'
I
' « " ' Wolf
^ S ^ ! j r > ; ^ „ ' S ' ^ i * * ™ n : Vector tso dSrg pJET1.2ftlunt d w c mua t» Hang Fermentas: Vector pRTRA 7/3, n P I P ^ ™ ^ p S r S ^ T I T ^ ' f ' ' * ' ^ " ' * " "^y " " 9 ' Gatersleben, CHLB OCc cung d p . Vector pPIPRASffl rumeftciensEHA105vSLBA44(M(Ii«re mua tir Hang Clontectiva Life Technologies.
Ptiirang phdp
tir as lao DNA 14, t6 h5p nh„. tich chfSl vS tlnh s,ch DNA plasmid, xC ly DNA plasiiild bing enzyme hgn ch«, gir. n*
,, ,-.11, ,k;, I ' 1^, OUINVJ wvsnc oiiNn n v ^ " - ' " " ' w u v j ^ £u lo
cac dogn DNA vao vector, dign di ben gel agarose... duoc tiln hdnh theo Sambrook vfi Russell (2001). Ti-plasmid tfii to hgp dugrc biln ngp vfio t l bao E. coli theo Draper vd d ^ g tdc gifi (1988), vdo A. tumefaciens theo Holster vd ddng tdc gid {1987). Thi nghigm chuyin gen 7cCh/T vdo thulc lfi thdng qua vi khuan A. tumefaciens dugrc thuc hign theo phucrng phdp mfinh lfi (Walden, 1988).
KtT QUA VA T H A O LUAN Tfich c h i l t DNA t i n g s l tir lfi cdy ca cao
Ld ca cao tuori duvc su dgng ldm nguygn ligu de tach chilt DNA ting s l do wgc nghiin md Id de ddng hon so vdi cac md khdc. Riiromg phfip tdch chilt DNA ting so sO dgng CTAB 2% va PVP 1%. Kit qud dign di cho thiy sfin pham DNA ting s l dugrc tdch chiet khd sgch va nguygn vgn, khdng lln tgp chat, it b| phan hOy vd dgt ygu cau cho cdc thi nghigm tilp theo.
Nhan gen ma hda chiflnase TcChil-Viftir hd gen cfiy ca cao
Chiing tdi dd bin hanh PCR si> dyng Pfu polymerase vdi khudn Id mau DNA ting so tdrfi tCr Id ca cao ddng TD3, si>
dung cdp mdi la TcChil -i/V_FIR d a l u kign nhigt dg gin moi Id 58'C. Trinh tif cfip mdi dugrc thilt k l nhim nhfin todn bg trinh ty gen mfi hda chitinase 7cC/i/J-lVvdi kfch thudc 1856 bp bao gdm vung 5' khdng djch ma (5' UTR), vung tin hieu TATA, ba exon md hda todn bd protein chitinase, hai intron, vung tin hieu poly(A) vd vCing 3' khdng djch ma (3' UTR) (td vj trf 1 den 1856 cda trinh tg U30324.1),
1 m i
Hlnh 1. Di^n dl san phim PCR nhfln gen m i hda TcChll-W Hlnh 2. Difn dl sin phim PCR nhin gen mi hoa TcCMf va (A), vfi sin phim xtr ly plasmid pJET-t- TcCMMV btng Bgfll TcChif-U (A), v i sin phim xd I^ plasmid pJET+TcC/iff bing (B) trftn gel agarose 0,8%. A: M Thang DNA chuin 1 kb; 1. Bgfll v i NoA (Bl trgn gel agarose 0,8 % A: M. Thang DNA chuin Sin phim PCR nh§n gen mS hda TcChll-W. B- M. Thang DNA 1 kb; 1. San phim PCR nhan gen ma hda TcChi7-U vd TcChil. B:
chuin 1 kb;1,2.3 Plasmid pJET+7cCh;7-W/1.7,13/Sgfll M. Thang DNA chuan 1 kb; 1, 2 ,3. Plasmid pJET+TcC/i/t/1, 2.
4/flgni+Nofl
Kit qud dign di hdn Hinh IA cho thiy sdn phim PCR dfic higu. cd kfch thudc khodng 1,8 kb ddng nhu tfnh todn 1;^
thuylt. Ket qud xu \'if enzyme han chl Bgli\ d ba ddng pJET+TcCftrt-VWI, pJET+7cCh/MV/7 vd pJET+7cC/i/t-lV/13 b-gn hinh IB cho thiy gen ma hda chitinase dfi dugrc tach ddng thdnh cdng bong vector pJET1.2. Cdc plasmid tdi td hgrp trdn dirgrc tfich chilt vdi lugrng Idn vd Sirgc tinh sgch de su dgng trong thf nghigm xdc i^nh trinh tg' gen. Kit qud phan tfch trinh tM' gen cho thiy gen mfi hda chitinase da dugrc phfin ldp va tgo ddng thfinh cdng
Nhan gen ma hda chitinase TcChil vd Tcchll-U tif dbng pJET+TcChil-iflf/l
Sau khi gen ma hda chitinase TcChil-W duvc tgo ddng bong vector pJET 1.2 vd dugrc xac djnh trinh tir hofin chlnh, ChOng tdi da su dyng khudn mlu Id plasmid tdi t l hgrp ddng s6 1 (pJET+7cCh/1-lV/1) vd cgp mdi TcChi1_F/R d l nhdn dogn gen mfi hda TcChU c6 kfch thudc 1159 bp (cht chua viing mang mfi), cap mdi TcChil-i/_F/R d l nhdn doan gen TcChi1-Uc6 kich thudc 1223 bp (chd-a vClng mang ma vfi m$t phin viing khdng djch mfi). San pham PCR nhdn gen ma hda TcChil vd TcChil-U 6irgc trinh bdy trdn Hlnh 2A.
Kit qud dign di cho thiy. sdn phim PCR rat dgc hieu. cd kich thudc khoang 1,2 kb (TcChil-U) vd 1,15 kb (TcChil) dung nhu tfnh todn ly thuylt. Cdc sfin phim PCR ndy sau dd dugrc tao ddng trong cfic vector tgo ddng pPlPRA522 vfi pJETI.2 (Hlnh 28), xdc djnh trinh ty va luu giO phuc vg cac thi nghigm t i p theo.
Thiet ki vector biiu hiin thuv v#f pCB301 mang gen ma hoa TcChil
Oe cd t h i bilu hign trong thyc v$t. gen ma hda TcChil can duvc dat dudi sy dieu khiln cua promoter vd temiinator thich hgp Trong nghien ci>u ndy, gen m§ hda 7cCh»7 dug'C dua vdo vector tmng glan d l tao cau trdc hoan chlnh (bao gdm promoter, gen dich vd terminator). Sau dd c l u tn^c hodn chfnh cda gen dugc chuyin vdo vector bilu hign thyc vgt gdc pCB301. Vector trung gian dugrc lya chgn Id pRTRA. Vector pRTRA cd mang cfic vCing trinh ty cin thilt nhu CaH4V35S promoter, signal peptide hudng protein vdo ludi ndi chit, viing ma hda cho chuli peptide cMyc KDEL liin kg vdi vimg terminator tgo thu^n Igri cho vigc kilm tra sy bilu hign gen.
Thiet k l vector trung gian pRJRA+TcChi1: Vector pRTRA (chira CalVIV35S+Sp+IV11+cMyc KDEL) kfch thudc 4247 bp Hirgc xir iy bing enzyme BamHI vd Not\ d l logi bd gen dfch (Ml cd kich thudc 600 bp). Ding thdi, vector pJET+rcCh/7/1 cung dugrc xd \'j! bfing fig/11 + Wo(l d l thu dugrc dogn gen ma hda 7cCft/t dgng dogn Bgl\i - Wofl. Do enzyme BamHI (G/GATCC) vfi BglU (A/GATCT) la hai enzyme tuong dang ndn doan gen ma hda TcChil cd the dugrc gin vfio vector pRTRA (thay t h i gen Ml). Sdn phim gen ma hda TcChil vfi vector pRTRA sau khi xir {•j enzyme da
MUI f>iiin! i\MUA n y u uumw ,> j M m B ^ o c t j f S S B f l n n T i * . dTOC fai v « nhau. bi«n nsp vio E coli ching DHIOb va tl4n hSnh chon lgc hSng enzyitie hsn ch4 SamHl. VI ticng g«
m§ hoa TcChil ctii o i mOl nSm cat da BamHI vS aiSm cSt cua SamHl trSn vector pRTRA khong cin t6n t?l do lai w Sglll trin gen TcChi) n«n sin phim x» 1) enzyme c4c plasmid lai to hop pRTF!A*Tca»» Uieo tinh toin ly Ihuyll l i mi bing c6 kich thifiic 4,6 kb. Ket qua ai#n d, sin phim »> ly pRTRA*rcC/r/HSamHI xuit hl«n mpt bang 4,6 kb ddng nhi tlnh loin ly thuyet
Hlnh 3. S« d i thi^ k i vector pCB3«1+rcC/iit(A} vd kit qua difn df ftinh 4 IQim tra pPIPRA522 tif t i h^p bing SarnHf. 1 Bin phim xfr fy pCB301«rcC/i/f bing H/fKlfff trin gef agarose 0.8% pPIPRA522: 2 pPfPRA522+rcChif-Li; M. Thang DN»
(B). B: M. Thang DNA chuin 1 klj; 1. pCB301+7ca?iJ/1/Hindfff; 2. chuSn 1 kb: 3-8. pPIPRA522+rca,iM;/BBmHl pCB30f4rcCAiI/3/Hindfff
Trin ca s* d u tnjc CaMV35S+rcChil+cMyc KDEL da thiit k i v i veclor biiu hiin thvc v i t gic pCB301, chung Iii aa bin hinh thlit k i pfasmid t i , t i hgp pCB301 mang gen ma h()a TcCn/l du^l s\f diiu kiin cija CaMVSSS promoter. Vung MCS cija pRTRA c i vf fif cit ctia enzyme Hrndlli, do viy. toin b$ ciu true gen CaMV35S+rcCh/f+cMyc KDEL kldt lhif6c - 2 kb tir vector pRTnA+rcC/i/f di/gc chuyin vio veclor pCB301 difdrl ding doin HIndWi tio vector tii t i hpp pCB301+7cC/)/l, kich thi^^c - 8 kb. So- d i thiit k i di/grc trlnh biy IrSn hinh 3A. Sau khi so bO sing Ipc cic ding mang plasmid kich Ihiffic l*n han pCB301, chijng tii da xu- 1^ enzyme hgn chi H/ndlll d dong pCB301+7"cCWW1 vi pCB301+TcCWI/3. Theo b'nh loin 1^ thuyit sin phim w> ly enzyme niy s i la hai bang: mit bang c i kich IhiFdc - 2 kb tifong ong wW kich t h i n ^ oia ciu tnic CaMV35S+rcC/jil+cMyc KDEL v i mot bing co kich thifirc ~ 6 kb tirong i>ng vii kich thuic cua vector pCB301. K i l qui trin hlnh 3 (gling 2) cho thiy s\f phii hap v i i tlnh loin ly thuyit ching l i Chung tOi d i chuyin diroc ciu tnic CaMV35S+rcCWI+cMyc KDEL vio vector pCB301 v i tgo du-oc plasmid Iii l6 hpp pCB301+rcCh/r.
r w i r k i irector biiu M^ thiKv$t pPIPRA mang gen ma hda TcChll-U
H i vector biiu hiin thoc vit pPfPRA do phia dii lic Hoa Ky chuyin giao co mang FMV34S promoter va Eg lenninalw diiu khiin biiu hi«n gsn dich. Diy l i cic dogn khii dpng v i kit thic c i gia trj. V i i muc dich lao cic ciu tnjc gen ma hia chitinase khic nhau. ngoii pCB301+rcCW», chijng til da tiin hinh thilt k i va tgo vector biiu hi«ii pPIPRA5S8+7cC/i/l-U, tipng d i TcChl1-U nim difii si/ diiu khiin Ciia FflW34S pramoler v i E9 temiinalor.
Thiit k i veclor Injng gian pPIPfiA522*rcC/i/Hy: Veclor pPIPRA522 dirpc m i ving bing enzyme SamHl v i ghip n i v i i TcChi1-U (dang doan Ball). K i l qui x i i> cio plasmid lai l i hop pPIPRA522troC/iiH/ bing enzyme BsmHI (enzyme c i m i l vi tri nhin biit trin gen TcChlt-U) l i mpl bing c i kich thiric 5,2 kb (Hinh 4).
P? ' ^ ' ^ ^ ^ FMV34STCCW-U+E9 vip vector biiu hiin thirc vit pPIPRA558, ching t i i da Hin hinh logi b i ci«
FMV^StGUS.Eg ra khi, vector pPIPRA55e bSng enzyme hgn chi Pad. Theo tinh toin iy thuyit san phim xi) l(
" S T / 1 ? , ^ ' ' ^ " J . ' " ^ ' "^"8 c i kich Ihiric - 3,4 kb luong i n g v i i kich Ihoic ciia c i u M c FMV34S+l3UStE9 vJ mot bing c i kfch tfioftc - 9 i kb lirong irng v i i phin cin Igi cua veclor pPIPF!A558. Oogn veclor c i kich thi/ic 9 J kb.
diroc Ihu hii v i tnh sgch. Join b i d u tnic FMV34S+7cC/irt-(/4-E9 trong veclor tgo ding pPfPRA522+TcC/l/(-UdiK»!
^kT££'„Z^p'^^T ^ f ' . ? " J ' " '^" "^"^ '=' "^ * ' " '"=• " " " " 9 plasmid pPIPIW65e.rcCm-U«upc rt 9 2^b ( Z ^ ^ S I S ^ " " ! p ^ ^ r ^ f i ^ ' ^ / " " " " " ^ i>ng v i l kich thiric ciia c i u tnic FMV34S+7-CCM-WE9) k i M n h c S ^ PPIPRA558 khuyit FIW34StGUS.E9) cho thiy pPIPRA558+rcC/«-U da dooc I h *
lnPW^r^]^,l''.S!"l!^ vector biiu hiin pCB301*7cC/,/), gen TcCh,1-U Irong vector biiu hiS»
E h S ^ S d t ^ ™ S , n T ? ° ^ " ' ? r * " " * '^'^ '^^ " " ^ S c i so Ihay dii trinh to nucleotide trong qui rt-da i?c dmh M n h ^ S ^ Z , ? ?"? "^ ! ° ' ^ " ' ' " ' " " ^^ ^ "^ " " = ^'"^'"^- ToChll-Uwa gen ma hia Tp«)- IV da xic dmh trtnh V b o i c d i cho Ihay hoin loin khong c i so sai khic v i trinh to gen.
Tgo Chung VI khuin A tumetaciens mang cic vector t i l l i hop da thiit k i
b W O T v i e n S e l a n ^ i * n * f ' ' ^ ' ' ' ^ ' ; « " ' ' ' " * ° '"• " " " " ^ " ^ ' ^ chijng EHA105 v i LBA4404. Kit qui kiim W c i J i i a T i - i t e S S S l ^ c J c l I-- ; ^ f ' "^ ".•"" '"^f ' ^ """S " • '"riiefaclehs ching EHA105 v i LSA4404 S S ^ L S o nT Hing ^ " " ^ " « " "^V " " W SIT dgng d i chuyin gen m i hia chilinase iSo ciy IrSng nii chi»9 Chuyin gen TcChIt v i o ciy thuoc l i
C CONG NGHE SINH HOC TOAN QUOC 2013
Chdng A. tumefydens EHA 105 mang vector pCB301+rcC/Mt da dugrc chuyin vfio cfiy thuoc Id ddng C9-1 theo phuvng phap manh la nhfim budc diu nghien ciru sg bilu hi#n cua cau true gen chuyin trong cSy (Hinh 5). DNA ting so cda cfic cfiy "thudc lfi chuyin gen dugrc tfich chiet vfi su dung lam khudn mSu cho PCR vdi c^p mdi nhfin gen TcChi1_F/R nhfim xfic djnh sv c6 m^t cua gen TcChil trong cfic cfiy thudc la. Chdng toi da phan tich bang PCR 18 cay thuoc Id chuyin gen vfi nh$n dugc 16 cay duong tinh vdi PCR chimg td hi#u qufi chuyen gen tot (Hinh 6). Cfic cfiy thuoc la chuyin gen dang dugrc nghien ci>u muc dd bieu hidn cua gen chuyin.
Hlnh 5: Kit qufi chuyin gen vfio ciy thulc td. A: MSnh id dat trfin mfii tnremg cfim Ung, B: Cgm ch6i xuat hi#n tr*n mfli tn/dng chpn Ipc; C: TSch chol chuyin sang mfli truirng ra r l ; D: CSy trin m5i trucrng ra rfi
Hlnh 6: Dl$n dl sin pham PCR nhin gen ma hda TcChil tit thuoc ii. M. Thang DNA chudn 1 kb; 1 San phim PCR nhdn gen ma hfla TcChil tir ca cao dflng TDS; 2. Sdn phdm PCR nhdn gen mi hda TcOilt tCr cSy thudc 1^ dflng C9-1 d<^i chdng fim; 3-20. Sfin phim PCR nh3n gen md hfla TcChil tir thudc lfi dflng C9-1 chuyin gen
K £ T LUAN
Gen mfi hda chitinase dfi dugrc nhdn tir h§ gen cCia cfiy ca cao. Gen ma hda chitinase vdi kich thudc khfic nhau (vimg mang ma cd kdm ho^c khdng kfim vOng khdng dich mfi) dfi dugrc t^o ddng thfinh cdng trong vector pJET1.2 vfi pPIPRA522 cDng nhu dugc xfic (finh trlnh tg hofin chlnh. Hal cSu tnJc vector bieu hidn th<^c v^t da dugrc thilt ki thfinh cflng: (1) Vector pCB301 chii-a gen TcChU dugrc lai vdl mdt dogn mfi hda peptid c-Myc KDEL, dilu khiln bilu hi#n bdi CaMV35S promoter va NOS temiinator; (2) Vector pPIPRA558 chira gen TcChil-U dug'C dllu khiln bilu hidn l>di FMV34S promoter vfi E9 terminator. Vector tfii td hg'p pCB301+7cCh/1 dfi dug'C so' b$ kilm tra trdn cfiy thulc \d ddng C9-1. Cfic vector dang dugrc sir di^ng d l tang cudng bieu hi$n gen ma hda chitinase hen cay ca cao.
Lin cim ona
Cdng Irinh dugc thifc ki$ii trong khudn khd nhifm vy hvp Idc quoc le: "Nghiin ciiu lai sinh in vitro vdt^c^ cacao (Theobroma cacao L> chuyen gen" thupc "Chucrng trinh trpng diem phdi Irienvd ing di/ng cang nghe sinh hpc trong linh v^ nong nghi^vd phdt trien nong thon den nam 2020".
TAI LI^U THAM KHAO
Draper J, Scott R, Armitage P (1988). Plant genetic transfomiation and gene expression A Laboratory Manual, Blackwell Scientific Publications. 3-63
Holster M, De Waele D, Deplcker A, Messens E, van Montagu M, Schell J (1987). Transfection and transfomiation of A. tumefaciens.
Mol Gen Genel 163:161-187.
Maximova S. Miller C, Antunez de Mayolo G, Pishak S, Young A, Guiltinan MJ (2003). Stable transfomiation of Theobmma cacao Land influence of matrix attachment regions on GFP expression. P^anfCe//Rep 21:872-883
Maximova SN, Marelli JP. Young A. Pishak S, Venca JA, Guiltanan MJ (2006). Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen Colletotrichum gloeospadoides. Ptanta 224(4)' 740-749.
Michiels A Vanden EW, Tucker M, Van RL, Van LA (2003). Extraction of high quality genomic DNA fram latex containing plants Anal Biochem 315:85-89
Punja ZK. Zhang Y (1993) Plant chitinases and their roles in resistance to fungal diseases J Nemalol 25(4): 526-540.
Sambrook J, Russell DW (2001) Molecular cloning. A Laboratory Manuel Cold Spnng Harbor Laboratory, Cold Spring Hariior, NY.
Walden R (1988) Genelic transformaton in plants. Open University Press, Milton Keynes.
HOI NGH! KHOA HQC C O N G N G H ^ SINH HOC TC'Z^ t"
ISOLATION.OF CHITINASE-ENCODING GENE AND CONSTRUCTION OF
PLANT TRANSFORMATION VECTORS *
Ha Hong H a n h , Le Thanh Huong, Le Thi T h u Hien' Instttule of Genome Reseach. VAST
S U M M A R Y
Chilinase-cncodiiig gene family has been found in various plant tissues, including Theobroma cacao L. The role of chitinase m vascular plants is to protect plant against pathogen attack, especially fiingi. For the puipose of introducmg chitinase gsne in lo plants using Ti-plasmid-derived system, the chitinase gene from cacao was isolated and cloned (TcChil- coding region, TcChil-U- coding region and parts of untranslated regions). The recombinant Ti-plasmid containmg TcChil gene was constmcted based on pCB30l vector. In this recombinant Ti-plasmid, the rcC/iiV gene was placed under the control of CaMV35S promoter and ligated with c-Myc KDEL cassette. Using the pPlPRA vector system, the recombinant Ti-ptasmid containing TcChil-U gene under the control of FMV34S promoter was also contructed. Hie resulting recombinant vectors were success&Uy trassfered into Agrobacterium tumefaciens strains, and checked the icn^rary expression in tobacco plants (Nicotiana tabacum, genome C9-1). Cacao transformation experiments using ihese A. tumefaciens strains obtained are in progress.
Key words: Agrobacterium lumrfaciens. cacao, chitinase, Ti-plasmid
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