Phần V. Công nghệ Gen Thực vật

PHN V

CNG NGH GEN THC VT

Phn V: CNG NGH GEN THC VT

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CU TRC vector plasmid MANG GEN KHNG SU V NG DNG TRONG TO CY TRNG CHUYN GEN THNG QUA VI KHUN Agrobacterium tumefaciens

L Tn c, Nguyn Hu H, Nguyn Vn Uyn Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i M U Hin nay, bn cnh phng php to ging cy trng truyn thng nh lai hu tnh, gy t bin, ngnh cng ngh sinh hc vi s pht trin ca cng ngh gen v ang to ra nhng bc t ph do cng ngh gen cho php a cc gen c li vo thc vt to ra nhng cy trng c tnh trng m chng ta mong mun. Hng nm trn th gii tn rt nhiu chi ph trong vic phng tr su hi. Cc nh khoa hc v ang nghin cu chuyn np gen Bt (Bacillus thuringiensis) vo cy trng cy trng t sn sinh cc protein gy cht su hi. Cy trng mang gen khng su Bt l mt trong nhng thnh tu ca cng ngh sinh hc trong vic ci tin cy trng. Cy mang gen to c t ny c kh nng khng vi cc su hi chnh thuc b Lepidoptera, Coleoptera v Diptera. Hin nay ngoi vic ci tin k thut chuyn gen Bt cn phi ci tin gen thch hp sao cho gen Bt, mt gen ca vi khun c th biu hin c hiu qu trong thc vt. Phng php c hiu qu to cy chuyn gen l s dng sng bn gen; tuy nhin, i vi nhng c s nghin cu cha th trang b c thit b ny th vic chuyn gen bng vi khun Agrobacterium tumefaciens l phng php c la chn; ngoi ra phng php dng vi khu n Agrobacterium tumefaciens cn c cc u im ring ca n m cc phng php khc khng c c. Theo hng dng vi khun chuyn np, chng ti nghin cu ti cu trc vector plasmid mang ba gen l gen bar (gen chn lc), gen gusA (gen ch th) v gen Bt cryIA nhm to mt s vector plasmid mi - c t tn l plasmid pITB (Institute of Tropical Biology). Ni dung bi bo ny, chng ti trnh by k t qu nghin cu ti cu trc v s dng cc plasmid qua ti cu trc trong nghi n cu to cy cy hng v cy ci ngt chuyn gen. VT LIU V PHNG PHP 1. Cu trc plasmid mi Chng Agrobacterium tumefaciens EHA 105 c s dng chuyn gen. Chun E. coli DH5: c dng lm vi khun c kh nng bin np.

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Hi ngh KHOA HC V CNG NGH 2007

Plasmid pCAMBIA 3301, plasmid pUbi.cryIA(b) v plasmid pUbi. cryIA(c). 2. Chuyn gen vo cy Vt liu v dng vi khun: Cy trong ng nghim c dng lm vt liu chuyn gen. S dng chng vi khun Agrobacterium tumefaciens (At) EHA 105 cha plasmid ITB mang gen cryIA(c), gen bar v gen gusA. Phng php chuyn gen vo cy hng (Paulownia fortunei): S dng vi khun At c lc qua m gy nhim mnh l, sau cc mnh l c nui cy trn mi trng ti sinh to chi (mi trng MS c 0,1mg/l NAA v 10mg/l BA) c b sung cht acetosyringone nng 100 M trong hai ngy. Sau ra v dit vi khun bng dung dch khng sinh 500mg/l cefotaxime trong thi gian 30 pht, chuyn cc mnh l sang mi trng ti sinh to chi c cha 4mg/l PPT v 500mg/l cefotaxime. Cy truyn 2 tun/ ln trn cng loi mi trng. Sau 6 tun, mu l c chi ti sinh c cy truyn sang mi trng cho ra r c cha 4mg/l PPT. Cc cy ra r tt c a ra trng vn m. Phng php chuyn gen vo cy ci ngt (Brassica integrifolia L.): L mm vi cung l di 1-2mm ca cy con t ht ny mm 5-6 ngy tui c s dng lm mu chuyn gen. S dng vi khun lc qua m gy nhim mu, sau cc l mm c nui cy trn mi trng ti sinh to chi (mi trng MS c 2mg/l NAA, 4mg/l BA v 3,3mg/l AgNO 3) trong hai ngy (c b sung acetosyringone nng 100 M). Sau ra v dit vi khun bng dung dch khng sinh 500 mg/l cefotaxime trong thi gian 30 pht, chuyn cc mu sang mi trng ti sinh to chi c cha cht chn lc PPT v 500mg/l cefotaxime. Cy truyn 2 tun/ ln trn cng loi mi trng. Sau 4-5 tun, mu c chi ti sinh c cy truyn sang mi trng ra r (mi trng MS c 0,1mg/l NAA v 6mg/l PPT). Phng php kim tra cy chuyn gen: Kim tra gen ch th gusA bng dung dch X-Gluc: bng cch ngm cc mnh l chi nh vi dung dch X-Gluc khong 15 gi 37 C, mu chuyn gen s c mu xanh chm c trng, mu i chng s khng chuyn m u. PCR gen cryIA(c): DNA thc vt c chy PCR vi cp mi chuy n bit, quy trnh tch DNA thc vt c thc hin theo phng php ca Dellaporta (1983). Cc cy chuyn gen c kim tra s biu hin ca gen cryIA(c) qua kh nng khng su bng cch th su xanh Heliothis armigera ln cc mu l trong a ptri. KT QU V THO LUN 1. Cu trc plasmid mi Vic gn gen cryIA(b) v cryIA(c) vo plasmid pCAMBIA 3301 to plasmid mi nh sau:


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Vect plasmid CAMBIA 3301 c di 12 Kb cha gen gusA v gen bar. C 14 enzyme gii hn ti v tr cloning site trong vng T-DNA, cho php tch hoc ghp cc on gen mong mun. Trong th nghim ny chng ti s dng enzyme gii hn Hind III (A/AGCTT) ct to hai u Hind III ca chui plasmid pCAMBIA 3301. Plasmid pUbi.cryIA(b) v plasmid pUbi.cryIA(c) ch a gen cryIA c di 4 kb, hai u ca ton b gen ny (promoter-gen-terminator) c v tr ct bi enzyme gii hn Hind III. Nn chng ti s dng enzyme gii hn Hind III ct hai v tr ny. Sau khi ct bi enzyme gii hn Hind III cc on plasmid DNA c chy in di cho kt qu: vi plasmid pCAMBIA c m ra vi hai u ct bi Hind III v c x l bi AP (alkaline phosphat) chng kh c th t ni li vi nhau. C n plasmid pUbi.cryIA do c hai v tr ct nn to ra hai on: 6 kb DNA tng ng vi thn cn li ca plasmid v on 4 kb tng ng vi on gen cryIA. Sau tin hnh ni kt 2 on DNA plasmid pCAMBIA 12 kb v on gen cryIA 4 kb vi nhau bi enzyme ligase nhit 4 oC trong thi gian 16 gi. Kt qu to c 2 plasmid mi di khong 16 kb. kim tra kt qa ny chng ti chuyn 2 plasmid mi vo vi khun bin np E.coli, nhn bn E. coli v tch chit ADN. S dng li enzyme gii hn Hind III ct cho kt qu mi plasmid cho c hai bng DNA vi 1 on 12 kb v 1 on 4 kb (xem nh 2 in di) hon ton ph hp vi kch thc plasmid mi c chng ti t tn l plasmid pITB-CRY (Institute of Tropical Biology) ch a 3 gen v c chuyn vo dng vi khun Agrobacterium tumefaciens EHA 105.Chuyn gen vo cy hng:

Sau khi chuyn gen bng vi khun At, mu l c t trn mi trng c PPT nng 4mg/l, a s cc mnh l bt u vng sau 2 tun nui cy, mt s mnh l vn duy tr mu xanh nhng khng hnh thnh m so, ch mt s t khong 10% h nh thnh m so v mt s trong bt u ti sinh sau 5 tun nui cy. S au giai on chn lc ny cc chi con c chuyn sang mi trng MS ra r vi 4mg/l PPT. V ch c vi dng cy tip tc pht trin v ra r, c chng ti gi nh l cy chuyn gen. Kt qu qu trnh chuyn gen c th hin nh sau:Bng 1: T l ti sinh chi v cy chuyn gen ca cc mnh l trn mi trng c 4mg/l PPT sau khi nhim vi Agrobacterium tumefaciens v mu l i chngTh nghim Chuyn gen i chng S mu l 580 30 S mu l to m so 52 0 S mu l ti sinh chi 21 0 S lng dng cy chuyn gen 5 0 Tn s chuyn gen (%) 0,8 0

khng nh y l nhng cy hng chuyn gen, chng ti cy cy hng gi nh chuyn gen v cy i chng vo mi trng ra r vi cht chn lc PPT nng 4mg/l th kh nng chng chu, sau 2 tun ton b cy i chng cht hon ton, trong khi cc cy chuyn gen vn tip pht trin v ra r, gip chng ti khng nh y l nhng cy hng c chuyn gen.

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S biu hin gen gusA ca cy chuyn gen: Cc chi nh, mnh l ca cy chuyn gen c x l vi dung dch X-Gluc, kt qu cho thy chi v mnh l ny c mu xanh chm c trng khng nh s biu hin ca gen gusA trong cy chuyn gen. S biu hin ca gen bar trong cy chuyn gen: gen bar to tnh trng khng thuc tr c c khng nh trong cy in vitro, tuy nhi n chng ti mun tip tc kho st s tn ti n nh v kh nng biu hin ca gen ny trn cy sau khi c trng ra mi trng t nhin ngoi vn m. Chng ti s dng nng 300mg/l PPT phun l n cc cy i chng v cy chuyn gen 1 thng tui ti vn m. Sau 2 tun nhn thy cc cy hng chuyn gen tip tc sinh trng v pht trin bnh thng, ch c mt dng cy hi b vng l pha di nhng vn sng v pht trin cho php chng ti khng nh mt ln n a y l nhng cy hng nhn c gen chuyn. S hin din ca gen cryIA(c) v kh nng khng su ca cy chuyn gen: Phn tch PCR vi cp mi chuyn bit cho gen cryIA(c) cho thy cc mu DNA ca cy chuyn gen sau khi chy in di c xut hin cc bng c kch thc 0,65 kb ging nh mu i chng dng tnh plasmid, chng l kt qu ca s khuch i gen cryIA(c) trong khi mu cy i chng hon ton khng c bng. Qua chng ti kh ng nh s hin din ca gen cryIA(c) trong nhim sc th ca cy chuyn gen. Sau cc cy hng chuyn gen c chuyn ra trng trong vn m. th nghim tnh khng su cc l nh c t trong a ptri cng su xanh Heliothis armigera. Sau 3 ngy, vi cc l i chng din tch l b hi ln, kch th c su tng r rt, trong khi l cy chuyn gen din tch l b su n nh, su tng tr ng rt km v sau 3 ngy su hon ton khng cn n v cht nhiu vo ngy th 5 hoc 6. Chuyn gen vo cy ci ngt: Vi phng php s dng l mm ca cy con t ht n y mm 5-6 ngy tui, cy l mm vo mi trng ti sinh 3-4 ngy trc khi nhng cung l mm v o dch vi khun ly nhim, thi gian vi vi khun l hai ngy trn mi trng c b sung 100 M Acetosyringone v sau khi r a c cy trn mi trng ti sinh c Cefotaxime 500mg/l v 4mg/l PPT th sau 3-4 tun cc chi ti sinh hnh thnh v chng c cy truyn trn mi trng MS c 6mg/l PPT kim tra kh nng khng PPT ca cy. Chng ti thc hin th nghim chuyn gen nhiu ln vi s mu trun g bnh t 80120 mu/ln. Kt qu c trnh by bng 2.Bng 2. T l ti sinh ch i v cy, trn mi trng c 4mg/l PPT, ca cc l mm c nhim vi Agrobacterium tumefaciens v ca mu l mm i chngTh nghim Chuyn gen i chng S l mm th nghim 1380 30 S l mm to m so 52 (3,7%) 0 S l mm ti sinh chi 21 ( 1,5%) 0 S lng dng cy gi nh chuyn gen 2 0 Tn s chuyn gen (%) 0,14 0

Qua th nghim, chng ti nhn thy s lng l mm chng chu vi cht chn lc 4mg/l PPT hnh thnh m so khong 3,7% nhng s lng c th ti sinh chi ch mc 1,5% v gn nh a s cc chi u khng pht trin v cht cc ln cy truyn sau, theo chng ti y l hin tng c coi l escape kh ph bin trong qu trnh chuyn


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gen hoc chi th khm v ch cn 2 chi tip tc pht trin trn mi trng MS vi 6mg/l PPT - iu ny cho thy tn s chuyn gen thp (0,14%). Chng ti cho rng khi chuyn gen trn cc i tng cy trng khc nh cy thuc l v cy hng th mu l c kh nng ti sinh chi cao t xung quanh ra l nn vi khun Agrobacterium tumefaciens c th xm nhp vo nhiu v tr c kh nng ti sinh n y, trong khi cy ci ngt l mm ch c th ti sinh ti cung l nn vi khun Agrobacterium tumefaciens ch c mt v tr duy nht c th xm nhim to cy chuyn gen. Cc cy ci gi nh chuyn gen c phn tch PCR vi cp mi chuyn bit ca gen cryIA(c) - cho thy cc mu DNA ca cy gi nh chuyn gen sau khi chy in di c xut hin cc bng c kc h thc 0,65 kb ging nh mu i chng dng tnh plasmid, chng l kt qu ca s khuch i gen cryIA(c) trong khi mu cy i chng hon ton khng c bng. KT LUN Vi vic s dng vi khun Agrobacterium tumefaciens EHA 105 mang plasmid ITB chuyn gen, chng ti nhn c mt s dng cy hng v cy ci ngt chuyn gen mang gen cryIA(c) khng su xanh Heliothis armigera. B ng cc k thut sinh hc phn t v sinh hc chng ti xc nh c ca gen ny hin din trong cy hng v cy ci ngt chuyn gen v gen chuyn c biu hin tnh trng kh r rt.

Plasmid ITB (lane 2 v 3 mang gen cryIA(b) v cryIA(c)

Phn tch PCR gen cryIA(c) cy hng chuyn gen (bng DNA 0,65 kb)

Phn tch PCR gen cryIA(c) cy ci ngt chuyn gen (bng DNA 0,65 kb)

Cy ci ngt gi nh chuyn gen pht trin trn mi trng chn lc PPT

Cy hng gi nh chuyn gen pht trin trn mi trng chn lc PPT

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LI CM N Cc tc gi xin chn thnh cm n Chng trnh KC-04-13, Chng trnh nghin cu c bn ti tr thit thc cho nghin cu ny. TI LIU THAM KHO 1. L Tn c, Nguyn Hu H, Nguyn Vn Uyn, 2003. To cy hng (Paulownia fortunei) chuyn gen khng su thng qua vi khun Agrobacterium tumefaciens . Tuyn tp Bo co khoa hc Hi ngh Cng ngh Sinh hc to n quc, H Ni, 1617/12/2003. NXB Khoa hc v K thut, H Ni, tr. 1088-1090. 2. L Tn c, Nguyn Hu H, Phm Th Hnh, Nguyn Vn Uyn, 2005. nh hng ca tc nhn chn lc n m cy ci ngt ( Brassica integrifolia) v nghin cu to cy ci ngt chuyn gen. Tuyn tp Bo co Hi ngh khoa hc t on quc Nhng vn nghin cu c bn trong Khoa hc S sng , i hc Y H Ni, H Ni, 3/11/2005, tr. 1194-1197. 3. Phm Th Hnh, L Tn c, Nguyn Hu H, Nguyn Vn Uyn, 2005. Kho st kh nng ti sinh cy in vitro cy ci ngt (Brassica integrifolia) t l mm v tr mm phc v cho nghin cu chuyn gen. Tuyn tp Bo co Hi ngh khoa hc ton quc Nhng vn nghin cu c bn trong Khoa hc S sng , i hc Y H Ni, H Ni, 3/11/2005, tr. 498-500. 4. Nguyn Vn Uyn, L Tn c, Nguyn Hu H , 2003. Nghin cu h thng ti sinh cy hng (Paulownia fortunei) v nh hng ca tc nhn chn lc to cy chuyn gen. Tuyn tp Bo co khoa hc Hi ngh Cng ngh Sinh hc to n quc, H Ni, 16-17/12/2003. NXB Khoa hc v K thut, H Ni, tr. 866-869. 5. Murashige, T., Skoog F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15: 473-497. 6. Pua, E. C., Barfield D. G., 1991. Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation. Plant Cell Reports 10: 308-314.

SUMMARY

Construction of plasmid vector and utilization for production of transgenic plants resistant to insect via Agrobacterium tumefaciensLe Tan Duc, Nguyen Huu Ho, Nguyen Van Uyen Institute of Tropical Biology New plasmid vector pITB-CRY (Institute of Tropical Biology) consisting bar, cryIA(c) and gusA genes was constructed and applied to transfer into Agrobacterium tumefaciens EHA 105 for Paulownia and Brassica plants genetic transformation. Many transgenic lines of both plant cultivars were obtained. PCR analyses and indigogenic GUS assay were performed to confirm the presence and the expression of bar, cryIA(c) and gusA genes. Insect bioassays with larvae showed an improvement of resistance against Heliothis armigera.


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TO CY THUC L KHNG THUC TR C V CN TRNG

Phm Th Hnh, Phan Tng Lc, L Tn c, Nguyn Hu H Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i

T VN Mt trong nhng yu t lm gim nng sut cy trng trn ng rung hin nay l nhng tc hi do su bnh gy ra v vic phng tr dch hi ny thng gy s nhim mi trng ngy cng nghim trng. Vn ny ch c th c gii quyt thng qua s kt hp gia khoa hc hin i v thc gi gn sinh thi mi trng. Mt trong nhng ng dng ca cng ngh sinh hc gii quyt vn tr n l to ra nhng cy trng t bn thn c kh nng chng chu vi cc loi su bnh. Trong cc loi cn trng gy hi th ry, rp chim vai tr quan trng, vic chch ht nha ca chng lm cy khng pht trin ng thi chng l ngun ly lan mt s bnh virus quan trng nh virus gy bnh khm cy thuc l... Hin nay, phng php chuyn gen vo cy trng bng vi khun Agrobacterium tumefaciens c xem l mt phng php c hiu qu, l phng php qua k t qu chuyn gen thng biu hin kiu tch hp gen (DNA integration) t ng i n gin, ph hp vi mun ca cc nh cng ngh gen. Ni dung bo co ny, chng ti nghin cu ti cu trc plasmid mi mang gen bar (gen khng thuc tr c), gen gna (gen khng cn trng chch ht) v gen gusA; chuyn plasmid mi cu trc vo vi khun Agrobacterium tumefaciens v dng dng vi khun bin np trong nghin cu to cy thuc l chuyn gen khng thuc tr c v cn trng. VT LIU V PHNG PHP 1. Vt liu Vi khun E. coli cha plasmid pBluescript SK +-GNA mang cassette gen nguyn vn [Bao gm Promoter Ubiquitin (Ubi) + Trnh t m ho protein GNA (gen gna) + Terminator Nopaline synthase (nos)], kch th c khong gn 4 kb. Chng vi khun E. coli cha pCAMBIA 3301 mang gen bar (gen khng thuc tr c) v gusA (gen ch th). Gen ch th gusA: c phn lp t vi khun E. coli (Jefferson & ctv., 1987), m ha enzym -glucuronidase (khng mu). Enzyme ny xc tc phn ng phn gii c cht glucuronide (khng m u) to thnh

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sn phm c mu xanh chm c trng d nhn bit. Glucuronide th ng dng gi l X-gluc, cng thc ha hc: 5-bromo-4-chloro-3-indolyl- -D-glucuronide. Gen bar khng cht phosphinothricin (PPT) c phn lp t vi khun S. hygroscopicus. Gen ny m ha cho enzym phosphinothricin acetyl transferase (PAT), gip bin i PPT t dng c ch sinh tng hp glutamine gy cht cy trng sang dng b acetyl ha khng c n gy c cho cy. V vy, ngoi tc dng chn lc trong qu trnh chuyn gen thc vt, gen bar cn c vai tr khng thuc tr c Basta trong sn xut nng nghip. Chng vi khun E. coli DH5 c dng trong bin np nhm khuch i s l ng bn sao cc plasmid. Chng vi khun Agrobacterium tumefaciens LBA 4404 dng bin np plasmid mi. Mi trng nui cy: Mi trng Luria-Bertani (LB) cha khng sinh c s dng trong nui cy v bin np. Enzym gii hn SacI, KpnI v enzym ni DNA ligase T4. 2. Phng php a. Cu trc cc plasmid mi Phng php x l enzym ct DNA plasmid theo qui trnh ca Sambrook v ctv. (1989). Cc phn ng ni kt c thc hin nhit 14 0C trong thi gian 3-4g. Bin np E. coli v Agrobacterium tumefaciens theo phng php sc nhit, 42 0C trong 2 pht. Mi trng LB c b sung cc khng sinh thch hp chn lc c th bin np. Trong th nghim chng ti s dng cc khng sinh nh kanamycin (50mg/l) v streptomycin (25mg/l). Cc hp cht ha sinh nh IPTG (Isopropyl-D-thiogalactoside) 50mg/l v X-gal (5-Bromo 4-chloro 3-indolyl -D galactopyranoside) 40mg/l c b sung vo mi trng nui cy cc th bin np hot ho enzym -galactosidase, cho php chn lc cc khun lc xanh/trng. Ph ng php tch DNA plasmid cc khun lc kim tra cloning theo phng php ca b kit cng ty Promega. b. To cy thuc l khng thuc tr c v khng cn trng S dng ging thuc l si v ng Kutsaga 326. L cy thu c l trong ng nghim c ct kch thc khong 1x1cm c dng lm nguyn liu chuyn gen. S dng vi khun lc qua m gy nhim mnh l, sau cc mnh l c nui cy trn mi trng ti sinh to chi (mi tr ng MS c 0,1mg/l NAA v 1mg/l BA) trong hai ngy. Sau r a v dit vi khun bng dung dch Cefotaxim e 500mg/l trong thi gian 30 pht, chuyn cc mnh l sang mi tr ng ti sinh to chi c 10mg/l PPT v 500mg/l Cefotaxim e. Cy truyn 2 tun / ln trn cng loi mi trng. Sau 6 tun, mu l c chi ti sinh c cy truyn sang mi tr ng ra r c cha 20 mg/l PPT. Cc cy ra r tt c a ra trng vn m. Phng php kim tra cy chuyn gen Kho st kh nng pht trin v ra r ca cy chuyn gen v cy i chng in vitro trn mi trng MS c cha cht chn lc nng 20mg/l PPT.


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Kim tra gen ch th gusA bng dung dch X-Gluc: bng cch ngm cc mnh l v chi nh vi dung dch X-Gluc khong 15 gi 370C, mu chuyn gen s c mu xanh chm c trng, cn mu i chng s khng chuyn m u. S hin din ca gen gna trong cy c kim tra qua phn ng PCR vi cp mi chuyn bit nh sau: Mi 1: 5CGG ATC CAT GGC TAA GGC AAG TCT CCT C-3 v mi 2: 5CGG TAC CTC ATT ACT TTG AAG TCA CAA G -3. Kch thc on DNA ca gen gna khuch i ca phn ng PCR c mong i l 0,47 kb. S hin din ca gen bar c kim tra qua phn tch PCR vi cc cp mi nh sau: Mi 1: 5 ATG AGC CCA GAA CGA CG 3 v mi 2: 5 TCA GAT CTC GGT GAC GG 3. Gen bar cy chuyn gen qua phn tch PCR s c on DNA c khuch i l 0,5 kb. Cc cy chuyn gen v i chng c phun dung dch Basta (PPT nng 4g/l) c b sung cht bm dnh Tween 80 kim tra tnh khng thuc tr c.

KT QU V THO LUN 1. Cu trc plasmid mi Trong th nghim ny chng ti x l cassette gen nguyn vn [Bao gm Promoter Ubiquitin (Ubi) + Trnh t m ho protein GNA (gen gna) + Terminator Nopaline synthase (nos)], kch th c khong gn 4 kb nm tr n plasmid Bluescript SK+ c ct bi hai enzym gii hn Kpn I v Sac I. Cassette ny s c chn vo v tr Lac Z alpha (trong vng T-DNA, cng c ct bi Kpn I v Sac I) plasmid pCAMBIA 3301. Tip theo chy in di trn gel agarose 0,8% kt qu in di sau khi ct bi 2 enzym cho thy plasmid pBluescript SK+ -GNA c ct lm hai on, on khong gn 4 kb l nguyn cassette cha gen gna cn plasmid pCAMBIA ch c 1 on khong 12 kb. Tch v lm sch on cassette v plasmid backbone trc khi a vo ni kt (ligation). Phn ng ni kt gia gen gna v vector pCAMBIA bng enzym ligase T4 14 oC trong 3 gi. Sau cc sn phm tr n c bin np vo E. coli DH5 kh bin trong mi trng LB c 50 mg/l khng sinh kanamycin, nui 37 oC qua m. Kt qu cho thy trn cc a i chng (bip np khng c plasmid), cc t b o E. coli DH5 do khng c b sung plasmid khi thc hin bin np n n khng c kh nng khng Kanamycin, do vy chng khng sinh trng c trn mi trng LB c b sung kanamycin. Ngc li, cc th c bin np mang plasmid mi pht trin c trn mi trng chn lc ny chng t chng chnh l cc th mong mun. Bn cnh , mi trng chn lc c b sung X-gal, da trn nguyn tc sng lc, cc khun lc trng tr n a bin np s c s chn nhn bn plasmid mi. xc nh kt qu, chng ti ly

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mt s khun lc mu trng nui trong mi trng LB lng c 50mg/l khng sinh kanamycin, nui lc qua m 370C. Sau khi tch DNA plasmid, ti n hnh x l 2 enzym SacI v KpnI cho plasmid m i v pCAMBIA. Kt qu x l enzym, chy in di trn gel 0,8% cho thy plasmid mi cha 2 on DNA: mt on 12 kb (t ng ng pCAMBIA 3301 backbone) v m t on kch thc khong gn 4 kb (tng ng cassette cha gen gna); cho thy plasmid c hnh thnh c kch thc khong gn 16 kb. Nh vy c th khng nh gen mc ti u c lp ghp thnh cng v plasmid mi c nhn bn trong E. coli, plasmid ny c k hiu l p3301-GNA. Sau chng ti to chng Agrobacterium tumefaciens mang plasmid n y. 2. To cy thuc l khng thuc tr c v khng cn trng Trn mi trng c PPT nng 10mg/l, cc mnh l i chng b mt m u mu xanh sau 2-3 tun. Ngc li, mt s mnh l x l vi khun chuyn gen vn c n mu xanh v hnh thnh dn cc cm m so v chi nh. Sau 6 tun chn lc, cc chi ny c chuyn sang mi trng ra r c 20mg/l PPT. Chi thuc l qua x l chuyn gen vn tip tc pht trin v ra r. Trong khi th nghim i chng, chi t m l khng x l chuyn gen trn mi trng ra r cha 20mg/l PPT dn dn vng v cht trong 2-3 tun. Do chng ti cho rng nhng chi v cy pht trin trn mi trng 20 mg/l PPT l nhng c th gi nh chuyn gen. Qua th nghim chuyn gen, cy sinh trng tt trn mi trng c 20mg/l PPT c s biu hin ca gen gusA, l ca cc cy ny c mu xanh c trng.Bng 1: T l ti sinh chi ca c c mnh l trn mi trng c 20mg/l PPT sau khi nhim vi Agrobacterium v mu l i chng

Th nghim Chuyn gen i chng

S mu l th nghim 120 20

S lng mu l ti sinh chi 11 0

Tn s chuyn gen( %) 9,16 0

Kim tra s hin din ca gen mc ti u gna trong cy thuc l chuyn gen: Phn ng PCR c thc hin vi cp mi c hiu cho gen gna. i chng dng l plasmid pUbi-GNA v i chng m l DNA b gen tch chit t nhim sc th ca cy khng c chuyn gen. Kt qu cho thy cc dng cy gi nh chuyn gen c s hin din ca vch DNA mong i c kch th c bng vi kch thc ca vch i chng dng tnh l 0,47 kb (so vi thang chun). Khng thy vch ny trng hp cy thuc l i chng. Kt qu n y chng minh gen mc tiu gna t plasmid ca vi khun Agrobacterium c chuyn v sp nhp vo b gen ca cy. S hin din ca gen bar v kh nng khng thuc tr c Basta: Kt qu phn tch PCR cho thy cc mu DNA ca cy chuyn gen xut hin bng DNA vi kch thc khong 0,50 kb l kt qu ca vic khuch i gen bar. Do , chng ti c th khng nh bc u gen bar c chuyn vo nhim sc th ca cy. Sau khi trng ti


355

vn m khong 30 ngy, cc cy thuc l i chng c kim tra kh nng chng chu vi thuc tr c Basta bng cch phun thuc vi cc n ng 4 g/l PPT. Sau 14 ngy, tt c cy thuc l i chng u b cht nng ni tr n nn chng ti chn nng 4g/l PPT x l s cy thuc l chuyn gen. Sau 14 ng y, cy thuc l chuyn gen vn tip tc sinh tr ng bnh thng. KT LUN Nh vy bng cc k thut sinh hc phn t chng ti to c chng Agrobacterium tumefaciens mang gen bar, gen gna v gen gusA. Sau s dng vi khun ny to cy thuc l chuyn gen mang gen bar khng thuc tr c v gen gna khng cn trng chch ht. LI CM N Cc tc gi xin chn thnh cm n S Khoa hc v Cng ngh Tp H Ch Minh v Trung tm pht trin KHCN Tr - Thnh on Tp H Ch Minh ti tr cho nghin cu ny. TI LIU THAM KHO 1. Gleave A. P., 1992. A versatile binary vector syste m with a T-DNA organisational structure conductive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology 20: 1203 -1207. 2. Hilder V. A., Gatehouse M. R., Gatehouse J. A., Van Damme E., Boulter D., 1995. Expression of snowdrop lectin (GNA) in transgenic tobacco plants results in added protection against aphids. Transgenic Research 4: 18 -25. 3. Kumar K. K., Sudhakar D., Raija J. A., Balasubramanian P., 1999. Engineering resistance in elite indica rices to major diseases through Agr obacterium -mediated transformation.General Meeting of The International Program on Rice Biotechnology, September, 1999, Phuket, Thailand. 4. Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473-497. 5. Rao K. V., Christou P., Gatehouse M. R., Gatehouse J. A, 1998. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper. Plant Journal 15(4): 469 -477. 6. Sambrook J., Fritsch E. F., Maniatis T., 1989. Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory Press.

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SUMMARY

Production of transgenic tobacco plants ( Nicotiana tabacum L.) resistant to insect and Basta R herbicide via Agrobacterium tumefaciensPham Thi Hanh, Phan Tuong Loc, Le Ta n Duc, Nguyen Huu Ho Institute of Tropical Biology

Intact gna gene cassette was collected from the plasmid pBluescript SK + by restriction enzymes KpnI v SacI and inserted in Lac Z alpha region on T-DNA of the plasmid pCAMBIA 3301. Consequently, newly for med plasmid was introduced into competent E. coli and Agrobacterium tumefaciens LBA 4404 (for transformation). After co-cultivation, tobacco leaf disks were cultured on selection medium containing 10 mg/l PPT (Phosphinothricin). Many transgenic tobacco plants with aphid resistance gene gna, herbicide resistance gene bar, and reporter gene gusA were obtained via Agrobacterium tumefaciens LBA 4404 carrying the plasmid pCAMBIA 3301-GNA. PCR analyses and indigogenic GUS assay were pe rformed to confirm the presen ce and the expression of gna, bar and gusA genes.


357

.

Hnh 1. nh in di ca plasmid pCAMBIA 3301 GNA (1) v pCAMBIA 3301 (2)

Hnh 2, 3: Kt qu phn tch PCR gen bar (h. 2) v gna (h. 3) cy thuc l chuyn gen. L: thang DNA 1 kb, PC: i chng dng tnh, NC: i chng m tnh, 1,2,3,4: cy chuyn gen

Hnh 4: Cy thuc l chuyn gen v i chng trn mi trng cha 20 mg/l PPT

Hnh 5: Chi cy thuc l chuyn gen biu hin gen gusA qua ngm trong dung dch X-gluc

Hnh 6: Cy thuc l chuyn gen v i chng sau khi phun dung dch Basta

358


NGHIN CU TI SINH in vitro V BC U PHN TCH CY CC Dendranthema grandiflorum L. MANG GEN ipt to cytokininNguyn Hu H, L Tn c, Phan Tng Lc, Phm c Tr, Nguyn Th Thanh Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i

M U Trong ngnh trng hoa thng mi, mt trong cc vn c quan tm nhiu nht l nghin cu gi cho cy hoa ni chung v hoa ct cnh ni ring sao cho lu tn. Cc nh sn xut v kinh doanh hoa cnh s dng mt s tc nhn l m cho hoa c ti lu nh dung dch cht iu ho sinh trng (HST) cytokinin (dihydrozeatin hoc benzyladenin), dung dch ng, dung dch st khun,,, x l hoa ct cnh hoc x l i vi c cy hoa. Cy hoa cc l i tng cy hoa c nhu cu tiu th rt cao v c hoa p, a dng, nhiu mu sc nhng thi gian bn ti ca hoa ct cnh khng di nht l khi a vo tiu th ging cc n i hoc trng by, bo qun hoa trong iu kin khng thch hp nn nghin cu lm tng bn ti ca cy hoa ny l vn cn c quan tm. Nh chng ta bit, cc cht iu ho sinh trng nh auxin, gibberellin, ethylen, acid abscisic, cytokinin c ngha rt ln trong vic iu ho s lo ho. Trong cc nhm cht nu trn, cytokinin chim vai tr quan trng [5,9,10,11]. Trong cng ngh nui cy m t bo in vitro, cytokinin c vai tr rt r rt trong vic gi cho m l tch ri chm thoi ho dip lc t - c xanh ti lu. Trong thc tin cng tc ging cy trng, vic x l cytokinin, li n quan mt thit n s ti lu ca rau va thu hoch v ko di tui th ca hoa ct cnh, mang ngha thng mi rt cao. Trn th gii, cc nh khoa hc nghin cu chuyn gen np ipt (m ho enzym isopentenyl transferase c lin quan n sinh tng hp cytokinin isopentenyl adenin, zeatin v dihydrozeatin) vo cy tr ng mc ch lm m t bo ca chng t sn xut cytokinin. Thc t nghin cu trn th gii chng minh rng, mt s tr ng hp nh cy thuc l [25], Petunia [5,10] cy bng ci [11], cy ci x lch [13,14] k c cy cc [8,9,10], tc dng ca cytokinin ni sinh (sn phm ca gen c chuyn np) i vi tui th ca l v hoa ca cy chuyn np gen l rt c ngha. trnh s to d tha (overproduction) cytokinin gy thay i h nh dng cy, cc nh khoa hc s dng mt s loi promoter khc nhau to s biu hin gen khi cy iu kin c


359

th [1,5,9,10,13] trong c promote r SAG12 (to s sinh tng hp cytokinin khi m i vo trng thi lo ho) [5,13] m chng ti s dng trong nghin cu ny. Theo xu hng trn, nghin cu ny, qua kt hp cng ngh nui cy m v cng ngh gen, chng ti nghin cu ti sinh cy in vitro v chuyn gen ipt vo cy hoa cc Dendranthema morifolium L. Vi hy vng s hin din v biu hin ca gen ipt ni trn s c tc dng gp phn lm cho cy hoa lu ho tn. VT LIU V PHNG PHP Ging lan: S dng ging cc i o Lt c hoa mu vng. Phng php Mi trng nui cy m v th tnh chng chu i vi khng sinh hygromycin 1. 2. 3. 4. Mi trng nui cy: MS (Murashige -Skoog, 1962) [15 ] khng ch t iu ho sinh trng (HST). Mi trng ti sinh chi: MS c cc cht HST NAA , BA vi cc nng khc nhau. Mi trng vn chi to cy v ra r: nh mi trng s 1. Mi trng th tnh chng chu i vi khng sinh hygromycin: nh mi trng 1 nhng c hygromycin t 5 - 10mg/l. iu kin nui cy: 9 gi chiu sng/ng y, nhit 25-28 oC. Dng vi khun v mi trng nui cy S dng dng vi khun Agrobacterium tumefaciens LBA 4404 cha plasmid pVDH396 (kch thc 15,9 kb) (do TS. Nguyn Th Thanh thit k) mang gen ipt (c ngun gc t vi khun Agrobacterium tumefaciens ) to enzyme isopentenyl t ransferase [c promoter SAG12 (senescence associated gene 12, t Arabidopsis thaliana), gen gusA (c intron, promoter CaMV35S) v gen khng hygromycin hph (promoter CaMV35S). Mi trng gi ging vi khun l mi trng LB c 50mg/l kanamycin. nhn ging cho nghin cu chuyn gen, vi khun c nui cy lc qua m trong mi trng khong AB (Chilton v cs., 1974) cng c nng kanamycin nh trn. Nhit nui cy: 25 - 280C. Quy trnh chuyn gen Cc mnh l vi kch thc 0,6 x 0,6cm, t cy in vitro trn mi trng MS khng cht iu ho sinh trng, c s dng lm vt liu chuyn gen. Tm tt quy trnh chuyn gen 1. Nui cy mnh l trn mi trng c 0,5mg/l NAA v 1mg/l BA trong thi gian 2 ngy.

360


2. 3. 4. 5.

Gy nhim cc mnh l vi vi khun trong thi gia n 10 pht. Vt mu ra, thm bt dch vi khun tha bng giy lc. Nui chung mnh l vi vi khun trong 2 ng y. S dng mi trng nh trn nhng c acetosyringone 100 M. Ra tht sch vi khun bng n c ct v bng dung dch cefotaxime 500 mg/l (kt hp lc nh). Vt mu ra, thm ro nc v cy cc mnh l trn mi trng chn lc: nh mi trng bc 1 ca quy trnh nhng c b sung 10 mg/l hygromycin, 500 mg/l cefotaxime. Thi gian nui 15 ngy / chu k chn lc v thc hin 2 chu k. Tip tc thc hin s chn lc thm 3 chu k (15 ngy/chu k) trn mi trng chn lc nh trn nhng c 5mg/l hygromycin n khi c chi ti sinh. Cy truyn cc chi / cm chi ti sinh (cao khong 1 cm) sang mi trng MS khng cht sinh trng cng cha 5mg/l hygromy cin n khi vn chi, thi gian nui khong 1 thng. Tch cc chi vn cao (khong 2cm) v cy truyn cng sang mi tr ng MS khng cht sinh trng c 5mg/l hygromycin: theo di s vn chi v ra r trong thi gian khong 20 ngy. Nhum GUS (Jefferson v ctv, 1987) [7]: m l trong thuc th GUS qua m 370C (t m). PCR: S hin din ca gen hph, gen gusA v gen ipt c kim tra dng cc cp mi c hiu.

6. 7.

Kim tra cy chuyn gen 1.

2. 3.

Gen hph: HPH1: CGTCGTTCGAGAAGTTTC, HPH2: 0 TACTTCTACACAGCCATC; chng trnh nhit: 94 C: 5 pht; 35 chu k (940C: 1 pht, 62 0C: 1 pht, 72 0C: 1 pht); 72 0C: 5 pht; khuch i on DNA 800 bp. Gen gusA: GUS1: CCTGTAGAAACCCCAACCCGTG, GUS2: 0 CCCGGCAATAACATACGGCGTG; chng tr nh nhit: 94 C: 3 pht; 30 chu k (940C: 30 giy, 560C: 45 giy, 72 0C: 1 pht); 720C: 10 pht; khuch i on DNA 365 bp . Gen ipt: IPT1: TCAACCGGAAGCGGACGACC, IPT2: 0 0 GCCATGTTGTTTGCTAGCCAG; chng tr nh nhit: 90 C:10 pht; 40 chu k (94 C: 15 giy, 450C: 45 giy, 720C: 50 giy); 720C: 7 pht; khuch i on DNA 355 bp. KT QU V THO LUN a. Tnh chng chu t nhin ca m l i vi hygromycin Theo mt s ti liu cng b v chuyn np gen qua s dng tc nhn chn lc l hygromycin th cy cc l i tng rt mn cm i vi tc nhn chn lc n y. Qua trin khai th nghim tnh chng chu i vi hygromycin d ng vt liu l mnh l v cy con in vitro, kt qu cho thy cc mnh l (kch thc 0,6 x 0,6cm) v cy con (cao


361

khong 1,5 - 2cm) u b cht nu trn mi trng MS khng cht sinh trng c 10mg/l hygromycin. Trn mi trng c nng 5mg/l hygromycin, mnh l c s hnh thnh m so nhng m so en dn v khng c kh nng ti sinh chi, v nu c s ti sinh chi xy ra th chi rt yu t, xanh nht; i vi th nghim tr n cy con th cy tuy cng c s pht trin nht nh nh ng chm, r ra ngn v c mu nu en, cc l gn gc trng thi ho nu, r. Kt hp th nghim ring trn ging cc th nghim v theo ti liu cng b, chng ti chn nng hygromycin 10mg/l chn lc t bo chuyn gen giai on u (1 - 2 chu k) qua s dng phin l (khng mang phn cung) l m vt liu x l chuyn gen. Tuy vy, chng ti cng quan tm cn i vic gia gim nng tc nhn chn lc ny m bo va c s ti sinh mc t ng i chp nhn c va khng b lng tng do vic ti sinh nhiu cy khng phi l cy chuyn gen. Cng qua thc t trin khai th nghim ny trn ging cc i o vng ng Thp (ging chu nhit) th ging ny li t ra t mn cm hn ging cc ni trn (s liu c nhn). b. Nghin cu nui cy m ti sinh cy phc v nghi n cu chuyn gen Ni chung, trong nghin cu nui cy m v chuyn gen th vic quan tm trng thi sinh l ca l l yu cu quan trng. Tuy ging cc n y pht trin rt tt trong bnh nui cy, l xanh m v to to thun li cho vic ly mu nh ng theo kinh nghim nghin cu th vic ly cc l khng v tr ngn cng nh gn gc l iu cn thit nht l i vi nghin cu chuyn gen. iu ny rt r i vi nghin cu nui cy m v chuyn gen i vi cy hoa ct t ng (ti liu c nhn). Chng ti b tr mt s mi trng th nghim ti sinh tr n c s khong MS vi cc nng NAA 0,1; 0,5 v 1mg/l kt hp vi BA 1 v 2mg/l. Kt qu cho thy, ni chung, khi NAA cao (0,5; 1mg/l) kt hp vi BA 1 hoc 2mg/l th s ti sinh chi xy ra theo kiu pht sinh c quan (organogenesis) [22] thng qua giai o n trung gian l m so. Ngc li, khi nng NAA thp h n (0,1mg/l) kt hp vi BA 1 hoc 2mg/l, nht l khi dng 2mg/l, th s ti sinh chi xy ra t qua giai on m so, mt s chi c v hnh thnh trc tip t mnh l [20,22,23]. c bit, s hnh thnh trc tip ny l thng theo kiu sinh phi s-ma [19], xy ra nhiu khi ch dng BA t 1 - 2mg/l m khng b sung NAA (s liu c nhn). Theo mt s tc gi nc ngoi nghin cu chuyn gen trn cy cc, s ti sinh theo dng trc tip ny khng c li cho nghin cu. Kt qu nghin cu ca chng ti ph hp kin nu trn. V vn ny, thc t nghin cu cho thy kh nng nhn c cy chuyn gen s cao h n khi iu chnh s ti sinh thng qua m so v theo chng ti t bo chuyn gen, trc ht, cn c nhn ln v nh vy s to c hi cao cho s ti sinh cy mang gen chuyn. Ni chung, hin nay tr n th gii, nghin cu nui cy m phc v nhn ging [2], s pht sinh h nh thi [4,8, 20,22,26] v ph c v chuyn np gen [17,18] v ang c cc nh khoa hc quan tm rt nhiu i tng cy trng ny. c. Quy trnh chuyn gen Nh ni trn do tnh rt mn cm vi hygromycin nn chng ti, nh trnh by trong phn quy trnh chuyn gen, s chn lc c thc hin giai on u vi

362


10mg/l hygromycin, sau gim xung cn 5mg/l nhm to c hi cho s ti sinh. Cch lm ny ca chng ti ph hp vi cch thc hin ca mt s tc gi nc ngoi v theo cc tc gi y, trn ging cc c th cng b, th s ti sinh khng th xy ra d c s chuyn np gen (gen hph). Cng do qu mn cm i vi hygromycin n n qu trnh chn lc t bo chuyn gen t sau x l vi khun n khi nhn c cy th kh di. iu ny l do trn mi trng c hygromycin t bo tng sinh chm v cy khi c y thn l pht trin cng rt chm. V thc t cng cho thy khi l cy chuyn gen nhng chng cng pht trin kh chm trn mi trng c nng hygromycin d khng phi mc qu cao (5mg/l). Theo chng ti, y l i m p ng c th ca cc ging cc khi s dng tc nhn chn lc l hygromycin. Qua qu trnh chuy n gen, chng ti nhn c 3 dng chi c kh nng vn cao thnh cy. Chng c kim tra v kh nng sinh trng, ra r trn mi trng c hygromycin s c trnh by di dy. Hin nay, trn th gii, cc nh khoa hc dng nhiu phng php khc nhau [3, 6, 12, 16, 17, 21, 23, 24, 27] chuyn mt s gen khc nhau nhm to ra cc c tnh mi cho cy cc nh cy chm lo ho [5, 9, 10, 13], to hoa c mu sc mi l, khng virus [27]. d. Kim tra kh nng ra r trn mi trng c hygromycin Cc cy vn cao t 2cm tr ln c cy sang mi trng MS khng cht sinh trng c 5mg/l hygromycin theo di s sinh trng tip theo. So snh vi cy i chng, s sinh trng tip tc vi biu hin k m theo l c s hnh thnh r l hai hin tng xy ra ng thi i vi cy thc s l cy chuyn gen; ngc li, cy i chng mt hon ton kh nng sinh trng dn n s cht sau tr n cng loi mi trng. e. Th GUS im gy s ch y l cc dng chuyn gen u cho kt qu m tnh i vi th nghim GUS. im c bit na l khng ch ring ging cc ny trn cc dng chuyn gen khc nhau m i vi ging cc khc (i o v ng ng Thp) to ra cng bng phng php dng vi khun Agrobacterium v bng phng php chuyn gen khc (l bn gen) th tt c cc dng chuyn gen cng u cho kt qu m tnh (ti liu ca phng). Theo mt ti liu nc ngoi, hin tng m tnh i vi th GUS ny vn cha gii thch c khi dng dng vi khun LBA 4404 chuyn np v khi dng promoter CAMV35S cho gen gusA. Tuy nhin, theo chng ti, y ch l gen ch th nn d c biu hin hay khng l iu khng quan trng, ch c iu n gy cho ng i nghin cu t nhiu kh khn khi mun kim tra nhanh cy chuyn gen khi ch a cn lm PCR. f. Phn tich PCR cc gen hph, gusA v ipt Cc dng cy c kim tra bng PCR ch khi chng qua giai on to r v c sinh trng bnh thng trn mi trng c 5mg/l hygromycin. Kt qu phn tch PCR cc gen hph, gusA, ipt v i cc cp mi c hiu cho thy c s hin din ca cc bng DNA c khuch i tng ng l 800 bp, 365 bp v 355 bp.


363

hph

gusA

ipt

Hnh hng trn (t tri sang phi): Phi s -ma, chi hnh thnh trc tip v gin tip t mnh l Hnh hng di (t tri sang phi): Cm chi khng hygromycin; phn tch PCR gen hph (800 bp), gen gusA (365 bp) v gen ipt (355 bp)

KT LUN Qua nghin cu nui cy ti sinh cy v chuyn np gen trn cy cc i o vng Lt nh vi khun Agrobacterium tumefaciens, chng ti c mt s kt lun sau: Trong s cc mi trng kho st, mi trng thch hp ng thi cho vic ti sinh cy v cho nghin cu chuyn np gen ( kha cnh cn s ti sinh cy thng qua m so) l mi trng MS c 0,5mg/l NAA v 1mg/l BA. Ging cc ni trn rt mn cm i vi hygromycin n n vic chn lc p lc cao (10mg/l hygromycin) ch nn c thc hin trong 1 - 2 chu k u, sau cn gim xung (5 - 6mg/l hygromycin) giai on ti sinh c th nhn c chi ti sinh. nhn c 3 dng cy cc mang gen ipt, gen hph v gen gusA. S hin din ca chng c kim tra bng phn tch PCR. LI CM N Cc tc gi xin chn thnh cm n S Khoa hc v Cng ngh Thnh ph H Ch Minh ti tr cho nghin cu ny. TI LIU THAM KHO 1. Aida, R., S. Nagaya, K. Yoshida, S. Kishimoto, M. Shibata , A. Ohmiya, 2005. Efficient transgene expression in chrysanthemum, Chrysanthemum morifolium Ramat., with the promoter of a gene for tobacco elongation factor 1 protein. JARQ 39(4): 269-274. Bhattacharya, P., S. Dey, N. Das, B. C. Bhattacharya, 1990. Rap id mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants. Plant Cell Rep. 9: 439-442.

2.

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Boase, M. R., J. M. Bradley, N. K. Borst, 1998. Genetic transformation mediated by Agrobacterium tumefaciens of Florists chrysanthemum (Dendranthema grandiflorum) cultivar Peach Margaret. In vitro Cell. Dev. Biol.- Plant 34: 46-51. Bush, R., E. D. Earle, R. W. Langhans, 1976. Plantlets from petal segments, petal epidermis and shoot tips of the periclinal chimera, Chrysanthemum morifolium Indianapolis. Am. J. Bot. 63: 729-737. Chang, H., M. L. Jones, G. M. Banowetz, D. G. Clark, 2003. Overproduction of cytokinins in Petunia flowers transformed with P SAG12-IPT delays corolla senescence and decreases sensitivity to ethylene. Plant Physiology 132: 2174-2183. De Jong, J., W. Rademaker, M. F. Van Wordragen, 1993. Restoring adventitious shoot formation on chrysanthemum leaf explants following co -cultivation with Agrobacterium tumefaciens . Plant Cell Tiss. Org. Cult. 32: 263-270. Jefferson, R. A., T. A. Kavanagh, B. W. Bevan, 1987. GUS fusion: glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6: 3901-3907. Kaul, V., R. M. Miller, J. F. Hutchinson, D. Richards, 1990. Shoot regeneration from stem and leaf explants of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.). Plant Cell Tiss. Org. Cult. 21: 21-30. Khodakovskaya, M. V., R. McAvoy, H. Liu, Y. Li, 1998. Ethylene -regulated expression of the ipt gene increases flower number in trans genic Dendranthema grandiflorum. Abstracts, Society for In Vitro Biology.

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8.

9.

10. Khodakovskaya, M. V., Y. Li, J. Li, R. Vankova, J. Malbeck, R. McAvoy, 2005. Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petunia hybrida and Dendranthema grandiflorum. Plant Growth Regulation 00: 00-00. [email protected] . 11. Kim Hong, N. T., E. J. Kane, P. J. Dix, 1998. Hormonal regulation of senescence in cauliflower (Brassica oleracea var. Botrytis) (Abstract). In: Altman A., Ziv M., Izhar S. (eds), Plant Biotechnology and In Vitro Biology in the 21 st Century, IX International Congress on Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, The Netherlands, p. 164. 12. Ledger, S. E., S. C. Deroles, N. K. Given, 1991. Regeneration and Agrobacteriummediated transformation of chrysanthemum. Plant Cell Rep. 10: 195-199. 13. McCabe, M. S. et al., 2001. Effects of P SAG12-ipt gene expression on development and senescence in transgenic lettuce. Plant Physiol., 127: 505-516. 14. McCabe, M. S., U. Mohapatra, F. Sch epers, K. Van Dun, J. B. Power, M. Davey, 1998. Delayed senescence in transgenic lettuce using an autoregulated ipt gene. J. Exp. Bot. Suppl. 49: 49. 15. Murashige, T., F. Skoog, 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant .15: 473-497.


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16. Renou, J. P., P. Brochard, R. Jalouzot, 1993. Recovery of transgenic chrysanthemum (Dendranthema grandiflorum Tzvelev) after hygromycin resistance selection. Plant Science 89: 185-197.i1 17. Sherman, J. M., J. W. Moyer, M. E. Dau b, 1998. A regeneration and Agrobacterium-mediated transformation system for genetically diverse chrysanthemum cultivars. J. Amer. Soc. Hort. Sci. 123: 189-194. 18. Shinoyama, H., T. Kazuma, M. Komano, Y. Nomura, T. Tsuchiya, 2002. An efficient transformation system in Chrysanthemum [ Dendranthema x grandiflorum (Ramat.) Kitamura] for stable and non -chimeric expression of foreign genes. Plant Biotechnology 19(5): 335-343. 19. Tanaka, K., Y. Kanno, S. Kudo, M. Suzuki, 2000. Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Plant Cell Rep. 19:945-953. 20. Teixeira da Silva, J. A., 2003. Thin cell layer technology for induced response and control of rhizogenesis in chrysanthemum. Plant Growth Regulation 39: 67-76. 21. Teixeira Da Silva, J. A., S. Fukai, 2002. Increasing transient and subsequent stable transgene expression in chrysanthemum following optimization of particle bombardment and Agroinfection parameters. Plant Biotechnology 19(4): 229 -240. 22. Teixeira da Silva, J. A., S. Fukai, 2003. Chrysanthemum organogenesis through thin cell layer technology and plant growth regulator control. Asian Journal of Plant Sciences 2(6): 505-514. 23. Teixeira da Silva, J. A., S. Fukai, 2003. Four gene introduction methods affect the shoot regeneration and localization of transgene expression in greenhouse stem explants and in vitro-grown chrysanthemum stem thin cell layers. African Journal of Biotechnology 2(5): 114-123. 24. Urban, L. A., J. M. Sherman, J. W. Moyer, M. E. Daub, 1994. High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora). Plant Sci. 98: 69-79. 25. Wang, J., D. S. Letham, E. Cornish, K. R. Stevenson, 1997. Studies on cytokinins action and metabolism using tobacco p lants expressing either the ipt or the gus gene controlled by a chalcone synthase promoter: 1. Developmental features of the transgenic plants. Aus. J. Plant Physiol. 24: 661-672. 26. Xiaohan, Y., J. Bo, Z. Yan, M. Ding, T. Xuemei, 2002. Enhancement of direct shoot regeneration from internode segments of chrysanthemum by silver nitrate. Propagation of Ornamental Plants 2(1): 28-37. 27. Yepes, L. C., V. Mittak, S -Z. Pang, C. Gonsalves, J. L. Slightom, D. Gonsalves, 1995. Biolistic transformation of chrysanthemum wit h the nucleocapsid gene of tomato spotted wilt virus. Plant Cell Rep. 14: 694-698.

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SUMMARY

In vitro plant regeneration and transformation of Dendranthema grandiflorum L. with the ipt gene for cytokinin productionNguyen Huu Ho, Le Tan Duc, Phan Tuong L oc, Pham Duc Tri, Nguyen Thi Thanh Institute of Tropical Biology

The leaf disks of Dendranthema grandiflorum L. (0.6 x 0.6 cm) were co -cultivated with Agrobacterium tumefaciens strain LBA 4404. This Agro-strain contains the plasmid pVDH396 harbouring the ipt gene (with SAG12 promoter) for the isopentenyl transferase enzyme, the nptII gene (for kanamycin resistance) and the reporter gene gusA. After 2 day co-cultivation, The leaf disks were transferred to the medium containing hygromycin of 10 mg/l for sel ection. Through 4 - 5 rounds of selection, 3 independent transformed plants were obtained. The presence of the hph, gusA and ipt genes was checked by the PCR analyses and / or the GUS assay [In the case of gusA gene: positive in the PCR analysis; negative for the expression of gene in the different organs of three in vitro transformed plant lines, in the GUS assay]. These transgenics are being grown in the greenhouse for further studies.


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MT S NGHIN CU HNG N KH NNG TO vc-xin VIN GAN B T CY TRNGNguyn Hu H, Trng Thin Tr, L Tn c, Phm Th Hnh, Nguyn Hu Tm Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i

M U Thi gian gn y, cng ngh sinh hc c bit l cng ngh gen c nhng bc tin vt bc, qua cc nh khoa hc nhiu nc (M, Cuba, c, Hn Quc, Trung Quc, Ty Ban Nha) c th sn xut vc -xin ti t hp t cy trng [9,10,11,15,16,17,19,20]. H ng nghin cu sn xut vc-xin ny c xem l hng c tim nng v cng ln v theo tnh ton gi thnh sn phm s thp hn rt nhiu so vi gi thnh cc sn phm hin dng do c th sn xut quy m ln, li kh n gin trong khu t chc sn xut, vn chuyn v bo qun ngun vc-xin, sn phm khng cha cc virus gy bnh cho ngi - to iu kin thun li phc v tim chng vcxin m rng cho ngi dn cc nc ngho v cc nc ang pht trin. Nu ti nghin cu ny, chng ti hy vng kt qu nghin cu ca mnh, cng vi kt qu nghin cu theo hng ny ca cc nh khoa hc khc trong nc, s gp phn nht nh vo s pht trin ca lnh vc nghi n cu, sn xut vc-xin t cc ngun vt liu truyn thng ni chung v vc-xin c sn xut t cy trng ni ri ng. Nh chng ta bit, nc ta l vng lu hnh cao ca bnh vim gan do siu vi B (HBV). T l ngi mang mm bnh vo khong 10-15 % dn s (trn di 10 triu ngi). Tnh trng nhim ny gy ra nhiu hu qu nghim trng. Bnh ny ly lan theo hng a dng v phc tp, trng hp bnh mn tnh c th dn n x gan v ung th gan, thi gian cha tr ko di hn na chi ph iu tr cn rt cao so vi thu nhp. Cch tt nht l phng nga v mt trong cc bin php ph ng nga quan trng l chng nga. Tuy nhin, chi ph cho chng nga bnh cng cn kh cao nu tun th phc iu tr tiu chun (tim nhiu mi cch qung thi gian). Protein HBsAg ca HBV l thnh phn rt quan trng gip cho HBV bm dnh v o mng t bo v sau i vo t bo v huyt tng ngi bnh v huyt tng c cha HBsAg l ngun vt liu quan trng sn xut thuc chng nga c ngun gc huyt tng (ngoi huyt tng, vc-xin ny cn c sn xut t nm men, t b o ng vt hu nh). c mt s cng b khoa hc li n quan n nghin cu chuyn np gen HBsAg (m ho protein v - HBsAg), dng cng ngh chuyn gen vo nhn v lc lp t bo, vo mt s cy trng, sn xut sinh khi m t b o cy chuyn gen, chit tch protein tinh khit v nghin cu kh nng p ng min dch c th ng vt qua tim

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chch protein tinh khit hoc/v n trc tip sn phm cy chuyn gen. Qua cc nghi n cu nu trn, nhn thy protein khng nguyn HBsAg s dng qua ng tiu ho c kh nng to p ng min dch tt - m ra trin vng nghin cu chuyn np gen ny vo cc cy trng c b phn n ti c nh qu, l, thn, c Cng qua mt s cng b nu trn, kt qu nghin cu cn c mt hn ch l lng protein (sn phm ca gen chuyn) trong cy chuyn gen c n cha cao nn trong nghin c u ny, chng ti t vn a h thng sao chp T7 ca thc khu n th vo nghin cu i tng thc vt (vn gy c s quan tm c bit ch trong v i nm gn y) kt hp vi promoter PDS to s biu hin chuyn bit qu nhm hng lm tng hm lng protein khng nguyn trong lo i m ny. Tuy nhin, nghin cu ny, gen HBsAg vi promoter PDS c nghin cu s tch hp v biu hin trc ht cy thuc l tuy rng protein biu hin chuyn bit qu cy thuc l khng c ngha s dng nh vc-xin n c v kt qu nghin cu chuyn gen ny trn cy c chua qu bi l kt qu khoa hc mang tnh mi m. VT LIU V PHNG PHP Ging thuc l, c chua: S dng ging thuc l si v ng K.326 v c chua bi qu di (mua ngoi ch). Ht c kh trng bng nc Javel 50 % (v/v) trong 5 pht, ra sch bng nc ct v c cy trn mi trng khng cht sinh tr ng. Cc mnh l cy in vitro vi kch thc khong 0,7 x 0,7cm (i vi thuc l) v l mm ca cy mm 7-10 ngy tui (i vi c chua) c s dng lm nguyn liu chuyn gen. Mi trng nui cy m v iu kin nui cy: S dng mi tr ng c bn MS (Murashige v Skoog, 1962) [13]. Gieo ht sau kh trng v nui cy giai on to r: MS khng cht sinh tr ng. 1/ Ti sinh chi, cy t mnh l: MS + 0,1mg/l NAA + 1 mg/l BA (i vi thuc l), MS + 0,5mg/l NAA + 2mg/l BA (i vi c chua). iu kin nui: Nhit 26-28C, cng nh sng khong 3000 lux. Dng vi khun Agrobacterium tumefaciens: 2/ LBA 4404 cha plasmid pITB-HBsAg ( 16,78 kb) (do TS. Nguyn Hu Tm thit k) mang gen HBsAg (promot er T7, terminator T7; 0,7 kb), gen gusA (promoter T7, terminator T7) v gen nptII khng kanamycin (promoter CaMV35S). Gen HBsAg m ho protein nh S v gen gusA c iu khin bi h thng [promoter PDS (phytoene desaturase, 2 kb) + T7-polymerase (2,7 kb)] to s biu hin chuyn bit qu. Vi khun c nui trong mi trng LB c 50mg/l kanamycin trc khi gy nhim m. Quy trnh chuyn gen: Cy cc mnh l (i vi thuc l) v l mm (i vi c chua) trn mi trng 2; nui 2 ngy ngoi sng.


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Gy nhim m vi vi khun trong 10 pht v nui chung m v vi khu n trong 2 ngy trn mi trng 2; ngoi sng (trng hp c chua th mi trng c b sung acetosyringone 100 M). Ra vi khun bm vo m bng nc ct + 500mg/l cefotaxime, thm kh nh m. Cy m l / l mm trn cng mi trng 2 c 500mg/l cefotaxime v tc nhn chn lc kanamycin 100 mg/l (i vi thuc l) hoc tng dn t 20 -50mg/l (i vi c chua); ngoi sng. Cy truyn m hnh thnh trn mi trng c kanamycin sang mi tr ng chn lc nh trn (thc hin 3-4 chu k, 15 ngy / chu k) n khi hnh thnh chi v cy con. Kim tra s hin din v biu hin ca gen chuyn : Nhum GUS (Jefferson v ctv, 1987) [7]: m l, chi trong thuc th GUS 37C (t m) trong 24 gi. i vi c chua, mnh l ca cy gi nh chuyn gen c cy trn mi trng s 2 c 50mg/l kanamycin theo di kh nng to m so v ti sinh. PCR: S hin din ca gen nptII, gen gusA v gen HBsAg c kim tra dng cc cp mi c hiu. Gen nptII: K1: ACACGCTGAAATCACCAGTCTC, K2: CTCGTCCTGCAGTTCATTC; khu ch i on 600 bp; chng trnh nhit: 94 C: 5 pht; 35 chu k (94 C: 1 pht, 55 C: 1 pht, 72 C: 2 pht); 72 C: 7 pht. Gen gusA: GUS1: CCTGTAGAAACCCCAACCCGTG, GUS2: CCCGGCAATAACATACGGCGTG; chng tr nh nhit: 94 C: 3 pht; 30 chu k (94 C: 30 giy, 56 C: 45 giy, 72 C: 1 pht); 72 C: 10 pht; khuch i on DNA 365 bp . Gen HBsAg: S dng cp mi: HBV1: CTACACTCGTGGTGGACTTCTC, HBV2: AACGCCGCAGACACATCC; khu ch i on DNA 680 bp; ch ng trnh nhit: 94 C:10 pht; 40 chu k (94 C: 50 giy, 45 C: 45 giy, 72 C: 50 giy); 72 C: 7 pht; hoc cp mi HBsAg1: ATGGAGAACACAACATC, HBsAg2: GGATCCTTTTGCGGAAGCCCA; khu ch i on DNA 480 bp; ch ng trnh nhit: nh trn. Sn phm PCR gen HBsAg (680 bp) c n c kim tra bng my phn tch t ng Agilent 2100 Bioanalyzer - Automated Analysis System (cng ty Agilent Technologies, M) vi b kit DNA 12000 LabChip (cng ty Caliper Life Science, M). S hin din ca protein HBsAg c kim tra bng my phn tch Agilent 2100 Bioanalyzer vi b kit Protein 200 Plus LabChip ca cc cng ty ni tr n. Kim tra nhanh s hin din ca protein HBsAg trong cy chuyn gen bng que th c hiu ca cng ty Clinotech Diagnostics (M). KT QU V THO LUN Chuyn gen vo cy thuc l: Sau khong 15 ngy nui cy chn lc trn mi trng c hygromycin, nhn chung cc phin l b mt dn mu dip lc, ng thi c s hnh thnh cc cm m so nh mp l v

370


sau l cc chi nh c ti sinh. Trn mi trng c nng tc nhn chn lc kanamycin ng ngng (100mg/l), mu sc tng phn ca chi ti sinh (mu xanh lc m) v phin l (mt mu xanh) l hin tng rt d nhn bit; iu ny cho thy cc chi mi hnh thnh rt c th l nhng chi mang gen chn lc. Kh nhiu trng hp c nhiu hn mt chi / cy hnh thnh trn 4 ng ct (chu vi) ca mt mnh l. Sau , cc cy con c cy truyn sang mi trng nui (mi trng 1) cng c nng kanamycin nh trn. bo co ny, cy gi nh chuyn gen (GCG) c nh ngha l nhng cy ti sinh trn mi trng ti sinh c nng chn lc ngng, c kh nng pht trin tt r thn l trn mi trng nui cy (MS khng cht sinh tr ng) vi cng nng chn lc ngng. Thc ra, cc dng cy ti sinh trn mi trng chn lc thng cng c kh nng pht tin tt r thn l trn mi trng nui cy chn lc. Tt c cc d ng cy ti sinh qua x l chuyn gen u c kim tra theo cch ny trc khi c em phn tch tip theo. Do thuc l l mt i tng c p ng kh cao i vi chuyn gen n n sau mt s chu k chn lc chng ti nhn c kh nhiu dng cy GCG. Theo d kin ca chng ti th gen gusA (cng vi gen HBsAg) s khng biu hin m sinh d ng nh l, thn, r do chng c iu khin bi promoter PDS ch to s biu hin chuyn bit qu. Kt qu th nghim GUS i vi cc loi m ni tr n u cho kt qu m tnh vi thuc th X -gluc, hon ton ph hp vi d kin. Ngc li, phn tch PCR gen gusA cho kt qu d ng tnh vi bng DNA 365 bp. PCR gen chn lc nptII v gen mc tiu HBsAg cng c thc hin vi cc trnh t DNA c khuch i theo th t l 600 bp v 680 bp (i vi cp mi HBV1 v HBV2) hoc 480 bp (i vi cp mi HBsAg1 v HBsAg2). S hin din cng nh kch thc on khuch i gen HBsAg (680 bp) qua kim tra bng h thng phn tch t ng cho kt qu khp vi kt qu chy in di trn gel agarose. Cng bng h thng phn tch t ng n u trn, mt peak protein c trng lng phn t khong 23 kDa c ghi nhn [trng lng phn t ca protein thuc loi nh (S) trong s ba loi protein cu thnh protein khng nguyn b mt ca virus (S, preS1, preS2)]; ngc li, trng hp i chng th khng c. Nh chng ta bit, protein khng nguyn b mt S l protein quan trng trong to p ng min dch c th ngi v ng vt. Trong nghin cu ny, do mun kim tra s chnh xc ca cu trc plasmid, gen v vai tr ca promoter PDS nn chng ti, trc ht, nghin cu chuyn gen ny vo cy thuc l. Hin chng ti ang cu trc gen HBsAg vi promoter cu trc (constitutive) CaMV35S trong h thng T7 nhm to biu hin cao tt c cc b phn ca cy vi hy vng trong tng lai c th nghin cu chit tch protein khng nguyn tinh khit t l cy thuc l - mt i tng cy trng cho sinh khi l rt cao t ng t nh mt s nghin cu trn th gii [9,11,15,20]. Vic s dng promoter to biu hin chuyn bit mt b phn ca cy trong lnh vc nghin cu to vc-xin t cy trng cng c cng b [4]. Ngoi nghin cu sn xut vc-xin vim gan siu vi B, hin nay cc nh khoa hc cn ang nghin cu to vc-xin phng bnh vim gan C t cy trng [14]. Cc cy chuyn gen ang c trng v theo di vn m n khi ra hoa, kt qu. Chng ti s thc hin mt s nghin cu lin quan n s biu hin ca gen HBsAg v gen gusA giai on qu.


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Kt qu th nhanh bng que th ELISA cho thy c s h nh thnh vch dng tnh (test line) ngoi vch i chng (control line) cy chuyn gen. Theo cng ty Clinotech Diagnostics, que th c chnh xc rt cao (99,8%), tnh c hiu tuyt i (100%) i vi tt c cc subtype virus c cc quyt nh khng nguy n (determinants) ph bin hin nay. Chuyn gen vo cy c chua bi: Sau khi c ct ra t cy mm, cc l mm c nui 2 ngy trn mi trng s 2 nhm lm tng kho (pre-conditioning) trc khi ly nhim vi vi khun. Cc khu x l, nui chung vi vi khun, chn lc c thc hin tng t trng hp cy thuc l v c trnh by phn nguyn liu v phng php. Ni chung, cy c chua bi c p ng khng cao i vi s chuyn np gen mc d kh nng ti sinh in vitro kh cao (s liu cha cng b). Ti thi gian u nghi n cu, sau khi nui chung, m c cy chn lc ngay trn mi trng (MS + 1mg/l NAA + 2 mg/l BA) c nng kanamycin cao - 50 mg/l. Kt qu cho thy, tt c cc l mm u cht. Sau , chng ti p dng ch chn lc vi nng kanamycin t ng dn t thp n cao - t 20mg/l n 50mg/l qua 4 chu k chn lc v bc u nhn c 2 dng cy ti sinh. Mnh l ca hai dng cy ny, c kch thc khong 0,6 x 0,6cm, c cy trn mi trng cha cc cht iu ho sinh trng NAA v BA v c 50mg/l kanamycin theo di kh nng hnh thnh m so v ti sinh cy. Kt qu cho thy, khong 7 - 10 ngy sau nui cy c s hnh thnh m so v c s ti sinh chi sau khong 20 -30 ngy nui cy; ngc li, l cy i chng, ho n ton khng c s hnh thnh m so. Phn tch PCR cc gen ang c thc hin khng nh chng l cy chuyn gen. Nh chng ta bit, vic to mt s vc-xin n c nhm phng bnh vim gan B v mt s bnh khc [11,16,18,19, 21] c thc hin trn mt s i tng cy trng c sn phm n c trc tip (i vi ng vt) nh khoai ty [1,2,3,8] th cy c chua [6,12] l i tng cy trng c quan tm v c th n trc tip qu i vi nghin cu to p ng min dch cho ng i. Nghin cu ca chng ti nhm tip cn xu hng ny.

Hnh 1: PCR gen nptII v i bng. DNA 600 bp

Hnh 2: PCR gen HBsAg v i bng DNA 450 bp

KT LUN Qua s dng phng php chuyn gen bng vi khun Agrobacterium tumefaciens , chng ti nhn c mt s dng cy thuc l v cy c chua bi mang gen m ho protein khng nguyn b mt virus vim gan B. Cc dng ni trn c bn qua kim

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tra s hin din v biu hin ca gen chuyn bng mt s phng php nh tnh v sinh hc phn t. Nghin cu ny nhm hng n vic to vc-xin n c. TI LIU THAM KHO 1. 2. Arakawa, T., 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Research 6: 403-413a3 Bose, B., N. Khanna, S. K. Acharya, S. Sinha, 2006. Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen. J. Gastroenterology and Hepatology 21: 1439-1447. Diminsky, D., R. Schirmbech, J. Reiman n, Y. Barenholz, 1997. Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells ( Hansenula polymorpha): composition, structure and immunogenecity. Vaccine 15(6-7): 637-647. Domansky, N., P. Ehsani, A. H. Salmanian, T. Medvedeva, 1995. Organ -specific expession of the hepatitis B surface antigen in potato. Biotechnol. Lett. 17: 863-866. Ehsani, P., A. Khabiri, N. Domansky, 1997. Polypeptide of hepatitis B surface antigen in transgenic potato. Gene 190: 107-111. Gao, Y., Y. Ma, M. Li, T. Cheng, S -W. Li, J. Zhang, N-S. Xia, 2003. Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg. World journal of Gastroenterology 9(5): 996-1002. Jefferson, R. A., T. A. Kavanagh, B. W. Bevan, 1987. GUS fusion: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6: 3901-3907. Joung, Y. H., et al., 2004. Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum. Plant Cell Rep. 22: 925-930. Kapusta, J. et al., 1999. Biotechnological approaches to making vaccine in plants. Plant Biotechnology and In vitro Biology in the 21 st Century, A. Altman et al. (eds.), Kluwer Academic Publishers, 571 -574. Kong, Q., L. J. Richter, Y. F. Yang, C . J. Arntzen, H. S. Mason, Y. Thanavala, 2001. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc. Natl. Acad. Sci. USA 98(20): 11539-11544. Mason, H. S., C. J. Arntzen, 1995. Transgenic plants as vaccine production systems. Trends in Biotechnology 13: 388-392. McGarvey, P. B., J. Hammond, M. M. Dienelt, D. C. Hooper, Z. F. Fu, B. Dietzschold, H. Koprowski, F. H. Michaels, 1995. Expression of the rabies virus glycoprotein in transgenic tomatoes. Bio/Technology 13: 1484-1487. Murashige T., F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. Nemchinov, L. G., T. J. Liang, M. M. Rifaat, H. M. Mazyad, A. Hadidi, J. M. Keith, 2000. Development of a plant -derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145: 2557-2573.

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4. 5. 6.

7. 8. 9.

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13. 14.


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15. Ramrez, N., et al., 2003. Expression and characterization of an anti -(hepatitis B surface antigen) glycosylated mouse antibody in transgenic tobacco ( Nicotiana tabacum) plants and its use in the immunopurification of its target antigen. Biotechnol. Appl. Biochem . 38: 223-230. 16. Richter, L. J., Y. Thanavala, C. J. Arntzen, H. S. Mason, 2000. Production of hepatitis B surface antigen in transgenic plants for oral immuniza tion. Nature Biotechnology 18: 1167-1171. 17. Sakakibara, K. Y., K. Saito, 2006. Genetically modified plants for the promotion of human health. Biotechnol. Lett. 28: 1983-1991. 18. Smith, M. L., L. Richter, C. J. Arntzen, M. L. Shuler, H. S. Mason, 2003. Structura l characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies. Vaccine 21: 4011-4021. 19. Tripurani, S. K., N. S. Reddy, K. R. S. Sambasiva Rao, 2003. Green revolution vaccines, edible vaccines. African Journal of Biotechnology 2(12): 679-683. 20. Valds, R., et al., 2003. Hepatiris B surface antigen immunopurification using a plant-derived specific antibody produced in large scale. Biochemical and Biophysical Research Communication 310: 742-747. 21. Webster, D. E. et al., 2002. Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine. Virology 76: 7910-7912.

SUMMARY

Towards evolving production of Hepatitis B vaccine from plantNguyen Huu Ho, Truong Thien Tri, Le Tan Duc, Pham Thi Hanh, Nguyen Huu Tam Institute of Tropical Biology Towards production of vaccine from the plant cultivars, the leaf disks of tobacco Nicotiana tabacum L. and the cotyledons of tomato Lycopersicon esculentum were cocultivated with Agrobacterium tumefaciens strain LBA 4404 containing the HBsAg gene (with T7 system and PDS promoter) along with the nptII gene (kanamycin resistance gene) and the reporter gene gusA. After 2 - 3 day co-cultivation, tissues were transferred to the medium containing kanamyc in (100 mg/l for tobacco, 20-50mg/l for tomato) for selection. Through 3 -4 cycles of selection, several transgenic plants were obtained. The presence of the HBsAg, nptII and gusA genes was checked by PCR analyses and / or GUS assay [GUS assay: positive for the expre ssion in the fruit but negative in the leaf, stem and root]. The presence of HBsAg protein was carried out by the Agilent 2100 Bioanalyzer Automated Analysis System and by the quick ELISA test using the dip stick of the Clinotech Diagnostics Inc. The trans genics are being grown in the greenhouse for further studies.

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CHUYN GEN PHT SNG gfp VO CY HOA PHONG LAN Dendrobium cv. Burana White NH VI KHUN Agrobacterium tumefaciensV Phan Mi Sa, L Tn c, Nguyn Th Thanh, Nguyn Hu H, Nguyn Vn Uyn Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i

M U Phong lan l loi hoa p do a dng v m u sc, kiu dng; hoa c tiu th ph bin trn th gii v trong nc, kinh doanh hoa lan trong n c c kh nng t doanh thu rt cao (1 t ng/ha/nm). Ri ng ti Thnh ph H Ch Minh, Ban Ch o v pht trin hoa lan c thnh lp nhm xy dng chng trnh ging v xc nh vng trng hoa lan vi quy m h ng ho a cy hoa lan tr thnh cy nng nghip th ch lc ca thnh ph. Trong tin trnh chuyn i c cu cy trng, Thnh ph H Ch Minh s phn u m rng din tch trng lan l n 200 ha vo nm 2010. Trn th gii cng nh nc ta, ngoi vic nhn ging phong lan phc v sn xut bng ph ng php nui cy m, vic to ging (dng) mi nh ging khng bnh (nm, vi khun, virus), ging c dng cy nh (mini) v p ng cc tiu chun i hi ngy cng cao ca ngi tiu dng nh hoa c mu s c v c tnh mi l l nhng vn ang v s c quan tm nghin cu. Hin nay, bn cnh phng php to ging cy trng truyn thng nh lai hu tnh, gy t bin..., phng php cng ngh sinh hc (chuyn np gen) v ang chng t l cng c h tr rt c lc cho nghi n cu to ging v song hnh vi cc phng php to ging truyn thng. n nay c kh nhiu ging cy trng bin i gen (gen nhn) c trng trn din rng nhm mc ch th ng mi. Nghin cu to ging bng cng ngh gen v ang c thc hin i vi cy lan mt phn v vic to ging lan bng phng php lai hu tnh tiu tn nhiu thi gian v ngun gen ch mong mun dng lai to khng phong ph [12]. Trn th gii, c kh nhiu thng bo cp chuyn mt s gen v o cy hoa lan (vo nhn) nh gen bar, gen gusA, gen nptII, gen hpt...[11, 14, 16, 17, 18, 24, 25], gen m ho protein v virus (papaya ringspot virus ) [12], trnh t ACC antisense [1]; cng ghi nhn c cng trnh nghin cu chuyn gen pht sng gfp [16], luc [5,6]. Cy phong lan Dendrobium lai (hybrid) mang gen luc pht sng sinh hc (bioluminescent orchid) c rao bn u gi trn th trng th gii vi gi khi im 200.000 -la Singapore [4] [ lnh vc chuyn gen v o ng vt, c cnh Zebra - Zebra fish


375

Brachydanio rerio mang gen gfp [1] pht sng c iu kin (chiu tia UV) cng c thng mi ho vi gi 5 -la/con ti Singapore, M]. Nh chng ta bit, gen gfp (green fluorescent protein gene) t o protein GFP c kh nng pht sng - c phn lp (t sa bin Aequorea victoria), c xc nh trnh t v dng ho vo u nhng nm 90 [2, 13]. T , gen n y c bin np v biu hin nhiu loi c th sinh hc khc nhau nh vi khun, nm, ng vt c / khng c xng sng v thc vt [3, 8, 13]. Ngoi gen gfp, gen luc (m ho luciferase, phn l p t om m) [4, 5, 6, 20] cng to s pht sng - theo c ch khc nhng t c nghin cu hn do k thut to pht sng m t b o cy chuyn gen phc tp hn. lnh vc chuyn np gen vo cy trng, gen gfp c xem l gen ch th sng (vital) [21, 22, 23], mi [22] v c tim nng ng dng ln trong nghi n cu [7, 8, 13, 19]. c kh nhiu cng trnh nghin cu chuyn gen ny bng phng php bn gen [23], bng vi khun Agrobacterium tumefaciens [10, 13] vo mt s cy trng v c th nghin cu s biu hin ca gen ny mc ty th t bo. Do vy, theo chng ti, gfp l i tng gen cn c xem xt s dng trong cc nghi n cu v bin np di truyn. n nay, nc ta, cha ghi nhn c cng trnh cng b chuyn cc gen ni chung, gen gfp ni ring vo cy hoa lan. V v y, theo chng ti, nghi n cu chuyn gen gfp cy phong lan (Dendrobium Burana White) l vn cn c quan tm thc hin theo xu hng chuyn gen ny vo mt s i tng cy hoa trn th gii hin nay. VT LIU V PHNG PHP Ging lan: S dng ging lan Dendrobium Burana White c hoa mu tr ng. Phng php: Mi trng nui cy m 1. 2. 3. Mi trng nui, nhn protocorm -like body (PLB): 1/2 x MS (Murashige -Skoog, 1962) [15] c1 mg/l BA. Mi trng to s vn chi: 1/2 x MS c 2mg/l BA. Mi trng nui cy: 1/2 x MS khng c cht sinh tr ng. iu kin nui cy: 9 gi chiu sng / ngy, nhit 25-28oC. Dng vi khun v mi trng nui cy S dng dng vi khun Agrobacterium tumefaciens LBA 4404 cha plasmid pCAMBIA 1303 (kch th c 12,36 kb) (CAMBIA) mang gen gfp5 to protein pht sng GFP (green fluorescent protein) , gen gusA v gen khng hygromycin hph. Mi trng gi ging vi khun l mi trng LB c 100mg/l kanamycin. nhn ging cho nghin cu chuyn gen, vi khun c nui cy lc qua m trong mi trng khong AB (Chilton v cs., 1974) cng c nng kanamycin nh trn. Nhit nui cy: 25 - 28oC.

376


Quy trnh chuyn gen Cc cm nh PLB (c ct t cm to, nhm to vt th ng) vi kch thc 0,3 x 0,3cm c s dng lm vt liu chuyn gen. Tm tt quy trnh chuyn gen 1. 2. 3. 4. 5. Nui cc cm PLB trn mi trng s 1 trong thi gian 2 ngy. Gy nhim cc cm PLB vi vi khun trong thi gian 10 pht. Vt mu ra, thm bt dch vi khun tha bng giy lc. Nui chung cc cm PLB vi vi khun trong 7 ng y. S dng mi trng s 1 c b sung acetosyringone 100 M. Ra tht sch vi khun bng nc ct v bng dung dch cefotaxime 500 mg/l (kt hp lc nh). Vt mu ra, thm ro nc v cy cc cm PLB trn mi trng chn lc: mi trng s 1 c b sung 20mg/l hygromycin, 500 mg/l cefotaxime. Thc hin 2 chu k chn lc, thi gian chn lc 20 ngy / chu k. Tip tc thc hin s chn lc th m 3 chu k (20 ngy / chu k) trn mi trng chn lc nh trn nhng c 30mg/l hygromycin. Cy truyn cc cm PLB khng hygromycin sa ng mi trng s 2 cng cha 30mg/l hygromycin n khi vn chi, thi gian nui khong 2 thng. Tch cc chi vn cao (khong 1-1,5cm) v cy truyn sang mi trng s 3 c 30mg/l hygromycin: theo di s vn chi v ra r. Nhum GUS (Jefferson v ctv, 1987) [ 9]: m PLB, chi trong thuc th GUS 37C (t m) trong 24 gi. PCR: S hin din ca gen hph, gen gfp c kim tra dng cc cp mi c hiu. S pht sng ca PLB, chi chuyn gen ( c ghi nhn trong iu kin ti) bng cch chiu tia cc tm sng di (365nm) t n cc tm Blak-Ray (cng ty UVP, San Gabriel, CA, M) hoc s dng knh hin vi hunh quang Nikon. Chp nh qua knh lp bng my nh k thut s Nikon 8700 8 Mpixels (khi d ng n cc tm cm tay) hoc dng camera (khi dng h thng knh hin vi hunh qua ng).

6. 7.

Kim tra cy chuyn gen 1. 2. 3. 4.

Gen hpt: HPH1: CGTCGTTCGAGAAGTTTC, HPH2: TACTTCTACACAGCCATC; chng tr nh nhit: 94 C: 5 pht; 35 chu k (94 C: 1 pht, 62 C: 1 pht, 72 C: 1 pht); 72 C: 5 pht; khuch i on DNA 800 bp. Gen gusA: GUS1: CCTGTAGAAACCCCAACCCGTG, GUS2: CCCGGCAATAACATACGGCGTG; chng trnh nhit: 94 C: 3 pht; 30 chu k (94 C: 30 giy, 56 C: 45 giy, 72 C: 1 pht); 72 C: 10 pht; khuch i on DNA 365 bp .


377

Gen gfp: GFP1: TGGAGAGGGTGAAGGTGATG, GFP2: TGTGTGGACAGGTAATGGTTG; chng tr nh nhit: 94 C: 5 pht; 30 chu k (94 C: 1 pht, 55 C: 1 pht, 72 C: 1 pht); 72 C: 10 pht; khuch i on DNA 500 bp. KT QU V THO LUN Thc tin nghin cu trn th gii v s biu hin (pht sng c iu kin) ca protein GFP c th cy trng cho thy s pht sng xanh lc ca protein GFP b che khut bi s pht sng ca dip lc t khi cy c chiu nh sng cc tm sng d i hoc nh sng lam. V vy, i vi cy trng ly hoa trang tr (hng, ct t ng), cc nh khoa hc thng chn i tng c hoa mu trng (khng c dip lc t). Trong nghin cu ny, chng ti cng s dng ging phong lan Dendrobium Burana White c hoa mu trng. Bc u nghin cu cho thy, ging phong lan n y tuy nui cy m khng l vn kha cnh nhn ging v to chi / cy t PLB nhng tng i kh thc hin s dung np gen gfp hn so vi mt s ging khc nh Dendrobium Candy Stripe x Madam Cherry (s liu cha cng b) trong cng iu kin plasmid s dng, c ng iu kin chuyn np v phng php chuyn np. a. Tnh chng chu t nhin ca m PLB i vi khng sinh hygromycin Tuy c thng tin v nng khng sinh hygromycin d ng chn lc m PLB i vi mt s ging lan l 30mg/l nhng yu t nhy cm i vi khng sinh n y, theo chng ti, c khc nhau tu ging. Thc t nghin cu ca chng ti cho thy ging Burana White c tnh nhy cm cao hn so vi ging khc l Candy Stripe x Madame Cherry. iu ny to thun li v nh hng cho qu trnh chn lc l cn s dng nng hygromycin t thp n cao (tng dn t 15 mg/l n 30mg/l) - khc vi cc nghin cu trn th gii l s dng nng cao 30mg/l hygromycin ngay chu k chn lc u tin; v thc t cng cho thy rng nu dng ngay nng 30mg/l hygromycin th th nghim khng t kt qu. Thm ch c mt s l th nghi m, ton b PLB cht ht khi nng hygromycin ch 15mg/l c s dng sau khi x l chuyn gen bng vi khun. b. Giai on nui chung Theo nhiu cng b v chuyn np gen cy trng, giai on nui chung m vi vi khun thng ch ko di t 2 - 3 ngy. Nhng i tng phong lan chng ti nghi n cu, trn mi trng nui chung m l mi trng 1/2 MS c 1mg/l BA v 100 M acetosyringone th sau 2 ngy chng ti nh n thy vi khun mc rt yu, th ng khng nhn bit c bng mt thng. V vy, chng ti ko di thi gian nui chung ln 7 ngy v i khi lu hn. Thi gian nui chung ko d i nghin cu ca chng ti ph hp vi mt s nghin cu trn th gii (c l th nghim ko d i c thng); theo chng ti c th l do m PLB kh cng, tng i trn lng to bm t, c th trong cy lan c hp cht khng thch hp cho s pht trin ca vi khun; h n na mi trng nui chung li kh n gin (khong 1/2 MS). S x l ko d i thi gian nui chung thng t c s dng tr mt vi i tng cy trng v tu loi m nh trng hp

378


nui chung vi khun vi l cy hoa ct tng - c th ln n 5 ngy. Ni tm li, nng 30mg/l hygromycin c th c dng chn lc ging lan ny. c. Quy trnh chuyn gen Nh ni trn do tnh mn cm vi hygromycin, tng t nh trng hp chn lc t bo la bng hygromycin dng nng tun t t thp n cao, tr c ht, chng ti dng nng tng i thp 15mg/l chn lc. Trn mi trng ny, sau khong 1 thng nui chn, c s hnh thnh mt s cm PLB mi tng sinh nhng a s PLB cn sng nhiu. S chn lc d ng 15mg/l hygromycin c thc hin 2 ln; tip theo l 3 ln chn lc trn mi trng c hygromycin 30mg/l. Sau thi gian chn lc ( 3 thng), mt s cm PLB khng hygromycin tng sinh. The o chng ti, cn p dng ch chn lc theo kiu tng dn nng hygromycin v bn cht t bo cu thnh PLB khng phi l t bo m so nn theo chng ti tnh c lp ca chng trong h thng khng cao so vi t bo m so nn chng cn c iu kin v thi gian tng sinh. Nhn thy cc m c kh nng tng sinh mnh tr n mi trng 30mg/l hygromycin (do c chuyn gen) th chng vn c kh nng tng sinh tr n mi trng c nng hygromycin cao n 50mg/l. d. Kim tra kh nng ra r tr n mi trng c hygromycin Cc cy nhn c trn mi trng chn lc ng ngng (30mg/l) c cy truyn sang mi trng s 3. Kt qu cho thy cc cy chuyn gen ra r v c sinh trng bnh thng trn mi trng c 30 mg/l hygromycin; ng c li cy i chng b cht sau khong 15-20 ngy nui. e. Th GUS Hu ht cc m PLB / m chi khng hygromycin u c kt qu d ng tnh vi thuc th GUS (hnh 1, 2). Cng ghi nhn c mt s tr ng hp m tnh vi thuc th nhng c kt qu dng tnh vi PCR gen gusA, tuy nhin do gen ny khng l gen mc tiu nn chng ti khng quan tm l y s liu thng k l bao nhiu phn trm. y l trng hp thng nhn thy cc nghi n cu chuyn np gen c bit i vi gen gusA. f. Phn tch PCR cc gen hph, gusA v gfp Cc dng cy c kim tra bng PCR ch khi chng qua giai on to r v c sinh trng bnh thng trn mi trng c 30mg/l hygromycin. Kt qu phn tch PCR cc gen hph, gusA, gfp v i cc cp mi c hiu cho thy c s hin din ca cc bng DNA c khuch i tng ng l 800 bp, 365 bp v 500 bp (hnh 5). g. S pht sng ca m chuyn gen Cc cm PLB v chi nh c trong ti trong khong thi gian 1 thng ( gim hoc hn ch s hnh thnh dip lc t) c chiu tia cc tm sng d i 365nm. Kt qu cho thy c th ghi nhn c s pht sng xanh lc bng mt th ng v s pht sng ny c chp qua knh lp bng my nh k thut s hoc my nh thuc h thng knh hin vi hunh quang Nikon (hnh 3, 4).


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Hnh 1, 2: Th GUS dng tnh PLB v chi. 3, 4: PLB v chi pht sng xanh lc qua x l tia UV sng di 365 nm. 5: Kt qu PCR dng tnh gen gfp v i bng DNA c khuch i 500 bp

KT LUN Qua s dng chng vi khun Agrobacterium tumefaciens LBA 4404 cha plasmid pCAMBIA 1303 mang gen gfp5, gen gusA v gen hph chuyn gen vo PLB cy hoa phong lan Dendrobium Burana White, chng ti nhn c mt s dng cy chuyn gen. Cc dng cy ny c sinh trng in vitro bnh thng trn mi trng c hygromycin 30mg/l, nhum xanh vi thuc th GUS v c s pht sng mu xanh lc khi c x l bng tia UV sng d i (ca n cc tm) hoc sng lam ca h thng knh hin vi hunh quang. LI CM N Cc tc gi xin chn thnh cm n Vi n Khoa hc v Cng ngh Vit Nam ti tr cho nghin cu ny. TI LIU THAM KHO 1. BioThailand 2005: Biotechnology - Challenges in the 21 st Century (The 3rd International Biotechnology Trade Exhibition and Conference, Bangkok, T hailand, Nov. 2-5/2005; Conference on Biosafety of Genetically Modified Organisms Plant Genetic Engineering Unit at Kasetsart University, Kamphaengsoen Campus). Baulcombe D. C., Chapman S., Santa Cruz S. 1995. Jellyfish green fluorescent protein as a reporter for virus infection. The Plant Journal. 7: 1045-1053. Chalfie M., Tu Y., Euskirchen G., Ward W., Prasher D. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802-805. Chia T. F. 2000. Bioluminescent orchid, Natural Science Academic Group, NTU, Singapore. E-mail: [email protected]; http://www.interauct.com. Chia T. F., Chan Y. S., Chua N. H. 1990. In: Bonham D. G., Kemohan J. (eds). Proceedings of the 13 th World Orchid Conference. 13 WOC Proceedings Trust, Auckland, p. 284.

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SUMMARY

Genetic transformation of Dendrobium Burana White with the gfp gene through Agrobacterium tumefaciensVo Phan Mi Sa, Le Tan Duc, Nguyen Thi Thanh, Nguyen Huu Ho, Nguyen Van Uyen Institute of Tropical Biology

Protocorm - like bodies (PLBs), about 3 mm in size, of the Dendrobium Burana White (White flower) were cut and co -cultivated with the Agrobacterium tumefaciens strain LBA 4404 containing the gfp5 gene (green fluorescent protein gen e), gusA gene and hph gene (hygromycin resistance gene) (from CAMBIA). Following co -cultivation (for one week), PLBs were cultured on the 1/2 MS (plus 1 mg/l BA) se lection medium containing 20-30mg/l of hygromycin. After 5 rounds of selection, several tran sfomed PLBs were obtained. The transformed PLBs / in vitro small plants were checked for green fluorescence using the long - wave UV lamp (365nm) and/or the Nikon fluorescence microscope. They also showed the positive results on the GUS assay and the PCR analyses of the hph and gfp genes.

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BC U NGHIN CU CHUYN GEN ipt TO cytokinin NHM KH NNG LM CHM S LO HO CY BP CI (Brassica oleracea var. capitata)Bi nh Thch, Nguyn Hu H, Nguyn Th Thanh, L Tn c, Phan Tng Lc, Nguyn c Minh Hng Phng Cng ngh gen thc vt, Vin Sinh hc Nhit i

M U Nh chng ta bit, gen ipt (phn lp t vi khun Agrobacterium) l gen to ra enzyme isopentenyl transferase v c ng l enzyme quan trng ca con ng sinh tng hp cytokinin tro ng thc vt [4,5,7,20,22]. i vi s l o ho thc vt, cytokinin c vai tr iu ho theo hng tch cc l lm chm qu trnh ny [6,7,9,10,13,16,20]. Nghin cu chuyn gen ipt v o thc vt c thc hin thnh cng trn mt s i tng cy trng nh Arabidopsis, thu c l, Petunia, bng ci, ci x lch, cc,...[6,7,9,10,12,13,15] v k t qu cho thy cy chuyn gen c l xanh t i hn, cc l gi do dip lc t chm b phn hu n n l chm chuyn sang m u vng. iu ny m ra trin vng khai thc tim nng ca gen n y trong nghin cu chuyn gen to ging cy trng chm l o ho, tng sinh trng v nng sut [10,17]. Ngoi ra, gen ipt cn c kh nng lm tng hm lng hp cht th cp cy d c liu chuyn gen [19] v c th c s dng nh gen c kh nng thay th cho gen chn lc [8]. Trc y, gen chuyn ipt to tnh trng d tha cytokinin (overproduction) dn n s thay i kiu hnh ca cy [9] nhng gn y tnh trng ny c khc phc do s dng cc promoter nh cor15a, SAG12... ch to biu hin trong nhng iu kin nht nh [12,13,15]. cy bp ci, trn ng rung, cc l gi gn gc thng chuyn vng sm (mt dip lc t do lo ho) to iu kin cho nm bnh xm nhp; ngo i ra, vic gi sn phm sao cho c xanh ti lu trong qu trnh thu hoch, vn chuyn v bo qun l yu cu rt quan trng i vi cc nh trng trt v kinh doanh cy trng ny. Ni dung bi ny, chng ti trnh by k t qu bc u vic nghin cu chuyn gen ipt vo cy bp ci nh vi khun Agrobacterium tumefaciens nhm kh nng lm gim cc nh hng khng tt do hin tng lo ho gy ra nh ni trn v y l cng trnh u tin v nghin cu chuyn gen ny vo cy bp ci.


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VT LIU V PHNG PHP Ging bp ci: S dng ging F1 TN5 (ca Cng ty ht ging Trang Nng).Phng php: Kh trng ht

Trc ht, ht c ra vi nc ct v trng 2 ln, kh trng bng nc Javel nng 50% (v/v) trong 7 pht. Sau , ht c ra sch bng nc v trng 3 ln v c gieo trn mi trng.Mi trng nui cy m v th tnh chng chu i vi khng sinh hygromycin

1. 2. 3. 4. 5. 6.

Mi trng gieo ht v nui cy: khong MS (Murashige - Skoog, 1962), vitamin Morel (Morel, 1948), khng c ch t iu ho sinh trng. Mi trng ti sinh chi t l mm: khong MS, vitam in B 5 (Gamborg v cs., 1968) c 0,1mg/l IBA , 3 mg/l BA. Mi trng vn chi v to r: nh mi trng s 1. Mi trng th tnh chng chu i vi khng sinh hygromycin: nh mi trng 1 nhng c hygromycin t 5 - 10mg/l. Cc loi mi trng trn c ng 30g/l, thch 9g/l, pH 5,8. iu kin nui cy: 12 gi chiu sng/ng y, nhit 23 - 25oC.

Dng vi khun v mi trng nui cy

S dng dng vi khun Agrobacterium tumefaciens LBA 4404 cha plasmid pVDH396 (kch thc 15,9 kb) (do TS. Nguyn Th Thanh thit k) mang gen ipt (c ngun gc t vi khun Agrobacterium tumefaciens ) to enzyme isopentenyl transferase [c promoter SAG12 (senescence associated gene 12, t Arabidopsis thaliana), gen gusA (c intron, promoter CaMV35S) v gen khng hygromycin hph (promoter CaMV35S). Mi trng gi ging vi khun l mi trng LB c 50mg/l kanamycin. nhn ging cho nghin cu chuyn gen, vi khun c nui cy lc qua m trong mi trng khong AB (Chilton v cs., 1974) cng c nng kanamycin nh trn. Nhit nui cy: 25 - 28oC.Quy trnh chuyn gen

Cc l mm, t cy mm 7 - 10 ngy tui trn mi trng MS khng cht iu ho sinh trng, c s dng lm vt liu chuyn gen.Tm tt quy trnh chuyn gen

1.

Nui cy l mm trn mi trng s 2 (0,1mg/l IBA, 3mg/l BA) trong thi gian 2 ngy.

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2. 3. 4. 5.

Gy nhim cc l mm vi dch vi khun (OD= 0,6 -1) trong thi gian 15 pht. Vt mu ra, thm bt dch vi khun tha bng giy lc. Nui chung l mm vi vi khun trong 2 ng y. S dng mi trng nh trn nhng c acetosyringone 50 M. Ra tht sch vi khun bng n c ct v bng dung dch cefotaxime 500 mg/l (kt hp lc nh, 15 pht). Vt mu ra, thm ro nc v cy cc mnh l trn mi trng s 2 b sung 5-10 mg/l hygromycin, 250mg/l cefotaxime. Thi gian nui 15 ngy/ chu k chn lc v thc hin 3 chu k chn lc dng tun t 5-10-5mg/l hygromycin. Cy truyn cc chi/ cm chi ti sinh (cao t 1 -1,5cm) sang mi trng MS khng cht sinh trng cng cha 5mg/l hygromycin n khi vn chi, thi gian nui khong 1 thng.

6.

Kim tra cy chuyn gen

1.

Tch cc chi vn cao (khong 2cm) v cy truyn sang mi trng MS khng cht sinh trng c 5mg/l hygromycin: theo di s vn chi v ra r trong thi gian khong 20 ngy. Mnh l cy gi nh chuyn gen c nui cy trn mi trng s 2 c 5mg/l hygromycin v theo di kh nng hnh thnh m so v ti sinh sau khong 20-30 ngy (so vi i chng). Nhum GUS (Jefferson v cs., 1987) [11]: m l trong thuc th GUS qua m 37C (t m). PCR: S hin din ca gen hph, gen gusA v gen ipt c kim tra dng cc cp mi c hiu.

2.

3. 4.

Gen hph: HPH1: CGTCGTTCGAGAAGTTTC, HPH2: TACTTCTACACAGCCATC; chng tr nh nhit: 94 C: 5 pht; 35 chu k (94 C: 1 pht, 62 C: 1 pht, 72 C: 1 pht); 72 C: 5 pht; khuch i on DNA 800 bp. Gen gusA: GUS1: CCTGTAGAAACCCCAACCCGTG, GUS2: CCCGGCAATAACATACGGCGTG; chng tr nh nhit: 94 C: 3 pht; 30 chu k (94 C: 30 giy, 56 C: 45 giy, 72 C: 1 pht); 72 C: 10 pht; khuch i on DNA 365 bp. Gen ipt: IPT1: TCAACCGGAAGCGGACGACC, IPT2: GCCATGTTGTTTGCTAGCCAG; chng trnh nhit: 90 C: 10 pht; 40 chu k (94 C: 15 giy, 45 C: 45 giy, 72 C: 50 giy); 72 C: 7 pht; khuch i on DNA 355 bp. KT QU V THO LUN Do khng c ging bp ci thun c bn trn th trng nn chng ti s dng ging lai F1 TN5 nghin cu. Do vy, vic nhn ging cy chuyn gen d ng cho cc nghin cu tip theo s c thc hin bng phng php nhn ging v tnh in vitro /


385

ex vitro. Sau 7-10 ngy nui cy ht trn mi trng gieo ht, cc l mm c s dng lm vt liu chuyn gen. Qua tham kho mt s ti liu cng b v nui cy m cy bp c i trong v ngoi nc, chng ti b tr mt s cng thc mi tr ng vi nng cht iu ho sinh trng (HST) IBA, BA khc nhau. Kt qu cho thy, i vi ging F 1 TN5, t hp HST IBA (0,1mg/l + BA 3mg/l) c xem l t hp cho t l ti sinh chi rt cao ( 60%). Theo mt s nghin cu trong v ngoi nc [1,3,18,21], nng BA cao (2-4mg/l) c xem l thch hp, to s ti sinh vi tn s cao; nng 3mg/l BA trong nghin cu ca chng ti ph hp vi khong dao ng nng BA s dng ca cc tc gi n u trn. Qua th nghim tnh chng chu t nhi n ca m i vi khng sinh hygromycin, kt qu cho thy m so cng nh chi/ cy con rt mn cm i vi khng sinh n y (nng dng chn lc ch t 5-10mg/l) tng t nh trng hp cy bng ci [2]; ngc li, i vi ging bp ci B. oleracea cv. Alboglabra [3] v i ging ci Brassica rapa [13] th nng hygromycin s dng cao hn, theo th t l 30mg/l v 25mg/l. L mm, sau khi x l v nui chung vi vi khun, c cy trn mi trng ti sinh c b sung 5mg/l hygromycin. Nhn thy sau khong 7-10 ngy nui cy, mt s l mm c phn gc cung ph nh to v khong mt thng sau c s ti sinh chi (h nh 1); ngc li, ton b l mm i chng u cht. Cc chi/ cm chi ti sinh trn mi trng c 5mg/l hygromycin c cy truyn tip sang mi trng c nng hygromycin cao h n (10mg/l) s chn lc c trit hn. Kt qu nghin cu cho thy c mt s chi cht giai on chn lc tip theo ny. Cc chi/ cm chi tuy ti sinh tr n mi trng c hygromycin nh ng kh nng pht trin ca chng khng nhanh tr n mi trng c 10mg/l hygromycin. Do vy, chu k chn lc th ba, chng t i ch dng nng hygromycin 5mg/l (nh i vi chu k 1) tip tc kim tra tnh khng ca chi/cy. n nay, chng ti nhn c mt s dng chi/ cy c kh nng sinh tr ng bnh thng trn mi trng c 5mg/l hygromycin (dng cy gi nh chuyn gen). Nh ni trn, do tnh mn cm cao i vi hygromycin n n sau khi qua 3 chu k chn lc, chng ti cy truyn chi/cy sang mi tr ng nui cy khng c hygromycin chi/cy pht trin nhanh. Cc dng gi nh chuyn gen c kim tra mt ln na qua s dng cc mnh l cy trn mi trng ti sinh c 5mg/l hygromycin nhm theo di kh nng to m so v ti sinh. Kt qu cho thy, cc mnh l cy gi nh chuyn gen c kh nng h nh thnh m so v ti sinh chi; ngc li, mnh l cy i chng ho n ton khng c kh nng to m mi v cht sau . M l ca cc chi c kh nng pht trin li n tc trn mi trng c hygromycin c nhum vi thuc th X -Gluc kim tra s biu hin ca gen gusA. Kt qu cho thy chng c mu xanh c trng vi thuc th (hnh 2); ngc li, cc mu l cy i chng u m tnh kt qu th GUS n y. Cng ghi nhn mt s m l m tnh vi thuc th d chi c s pht trin bnh thng trn mi trng chn lc v theo chng

386


ti c th y l do hin tng im lng ca gen gusA thng gp trong nghin cu chuyn np gen ny. Chng ti ang tin hnh phn tch PCR nhm kim tra s hin din ca cc gen hph, gusA, ipt vi cc mi c hiu nh ghi trong phn nguyn liu v phng php.

Hnh 1: Chi chuyn gen ti sinh tr n mi trng chn lc c 5 mg/l hygromycin

Hnh 2: Mu l/ chi cy chuyn gen dng tnh vi thuc th GUS

LI CM N Cc tc gi xin chn thnh cm n Trung tm Pht tri n Khoa hc - Cng ngh Tr Thnh on Thnh ph H Ch Minh (Chng trnh Vn m sng to) ti tr cho nghin cu ny. TI LIU THAM KHO 1. Nguyn Th Lin Chi, Nguyn Hu H, Nguyn Th Than h, Lao Th Nga, Nguyn Quc Bnh, Nguyn Vn Uyn, 1994. Chn lc cc mi tr ng nui cy nng cao hiu sut ti sinh cy bp ci Brassica oleracea var. capitata dng cho h thng chuyn gen ca Bacillus thuringiensis khng su t. Tp ch Khoa hc v Cng ngh 22(1): 1-7. Kim Hong, N. T., E. J. Kane, P. J. Dix, 1998. Hormonal regulation of senescence in cauliflower (Brassica oleracea var. Botrytis) (Abstract). In: Altman A., Ziv M., Izhar S. (eds), Plant Biotechnology and In Vitro Biology in the 21 st Century, IX International Congress on Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, The Netherlands, p. 164. Nguyn Th Thanh, Nguyn Vn Uyn, Maria Dolores Sacristan, 1997. To hai dng ci Brassica oleracea L. cv. Alboglabra mang gen khng hygromycin bi Agrobacterium tumefaciens. Tp ch Sinh hc 19(4): 13-16.

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3.


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Akiyoshi, D. E., H. Klee, R. M. Amasino, E. W. Nester, M. P. Gordon, 1984. T -DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81: 5994-5998. Barry, G. F., S. G. Rogers, R. T. Fraley RT, L. Brand, 1984. Identification of a cloned cytokinin biosynthetic gene . Proc. Natl. Acad. Sci. USA 81: 4776-4780. Chang, H., M. L. Jones, G. M. Banowetz, D. G. Clark, 2003. Overproduction of cytokinins in Petunia flowers transformed with P SAG12-IPT delays corolla senescence and decreases sensitivity to ethylene. Plant Physiology 132: 2174-2183. Chang, H., M. L. Jones, G. M. Banowetz, D. G. Clark, 2003. The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis. Plant Physiol. 131(1): 167-176. Ebinuma H., K. Sugita, E. Matsunaga, M. Yamakado, 1997. Selection of marker free transgenic plants using the isopentenyl transferase gene. Proc. Natl. Acad. Sci. USA 94: 2117-2121. Eklof, S., C. Astot, F. Sitbon, T. Moritz, O. Olsson, G. Sandberg, 2000. Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin -and cytokinin-overproducing phenotypes. The Plant Journal 23(2): 279-284.

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10. He, S. S., A. Hoelscher, J. Liu, D. ONeill, J. Layton, R. McCarroll, S. Dotson, 2005. Cell cycle specific isopentenyl transferase expression led to coordinated enhancement of cell division, cell growth and development in transgenic Arabidopsis. Plant Biotechnology 22: 261-270. 11. Jefferson, R. A., T. A. Kavanagh, B. W. Bevan, 1987. GUS fusion: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6: 3901-3907. 12. Khodakovskaya, M. V., R. McAvoy, H. Liu, Y. Li, 1998. Ethylene -regulated expression of the ipt gene increases flower number in transgenic Dendranthema grandiflorum. Abstracts, Society for In Vitro Biolo

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