Ph^n V: C 6 N G NGHg GEN THgC VAT 345
CAU TRUC vector plasmid MANG GEN KHANG SAU VA iTNG DUNG TRONG TAO CAY TRONG CHUYEN GEN
THONG QUA VI
K H U A NAgrobacterium tumefaciens
Le Tan Dire, Nguyen Hiru Ho, Nguyen Van Uyen Phdng Cdng nghe gen thuc vdt, Vien Sinh hgc Nhiet dai
i,
MdDAU
Hien nay, ben canh phuong phdp tao gidng cdy trdng truyin thdng nhu lai hiiu tinh, gay dot bien..., nganh cdng nghe sinh hgc vdi sy phdt trien ciia cdng nghe gen da vd dang tao ra nhiing budc dot phd do cdng nghe gen cho phep dua cdc gen cd Igi vdo thyc vdt de tao ra nhirng cdy trdng cd tinh trang ma chiing ta mong mudn.
Hdng ndm tren the gidi tdn rdt nhieu chi phi frong viec phdng trir sdu hai. Cac nha khoa hgc da vd dang nghien cim chuyen nap gen Bt (Bacillus thuringiensis) vdo cay frdng de cay frdng ty sdn sinh cdc protein gay chet sdu hai. Cdy trdng mang gen khdng sau Bt Id mgt frong nhimg thdnh tyu ciia cdng nghe sinh hgc frong viec cai tien cdy frdng. Cay mang gen tao ddc td ndy cd khd ndng khdng vdi cdc sdu hai chinh thudc bd Lepidoptera, Coleoptera vd Diptera. Hien nay ngodi viec cdi tien ky thuat chuyen gen Bt cdn phdi cdi tien gen thich hgp sao cho gen Bt, mdt gen ciia vi khudn cd the bilu hien cd hieu qud frong thyc vat. Phuong phap cd hieu qud de tao cdy chuyen gen Id su dung Sling ban gen; tuy nhien, ddi vdi nhimg co sd nghien ciiu chua the trang bi dugc thiet bi nay thi viec chuyen gen bdng vi khuan Agrobacterium tumefaciens Id phuong phap dugc lya chgn; ngodi ra phuong phdp dimg vi khudn Agrobacterium tumefaciens cdn cd cdc im diem rieng ciia nd md cdc phuong phdp khdc khdng cd dugc.
Theo hudng dimg vi khudn de chuyen nap, chiing tdi da nghien ciiu tdi cdu tnic vector plasmid mang ba gen Id gen bar (gen chgn Igc), gen gusA (gen chi thi) vd gen Bt crylA nhdm tao mdt sd vector plasmid mdi - dugc ddt ten Id plasmid pITB (Institute of Tropical Biology).
Ndi dung bdi bdo nay, chiing tdi trinh bdy ket qud nghien ciiu tdi cdu true va sir dung cdc plasmid qua tai cdu tnic trong nghien cim tao cay cay hdng vd cay cdi nggt chuyen gen.
VAT LIEU VA PHLTONG PHAP 1. Cau triic plasmid moi
• Chimg Agrobacterium tumefaciens EHA 105 dugc sir dung de chuyen gen.
• Chudn E. coli DH5: dugc dimg ldm vi khudn cd khd ndng bien nap.
• Plasmid pCAMBIA 3301, plasmid pUbi.cryL\(b) vd plasmid pUbi. crylA(c).
346 [ HQI nghi KHOA HQC VA C O N G NGHg 2007
2. Chuyen gen vao cay I Vat lieu va dong vi khuan: Cay frong dng nghiem dugc diing ldm vat lieu chuyin gen.
Sir dung chung vi khudn Agrobacterium tumefaciens (At) EHA 105 chiia plasmid ITB
mang gen crylA(c), gen bar va gen gusA. 1 Phuong phap chuyin gen vao cay hdng (Paulownia fortunei): Sir dung yi khudn At
dugc lac qua dem dl gay nhiem manh la, sau dd cdc mdnh la dugc nudi cdy tren moi trudng tai sinh tao chdi (mdi trudng MS cd 0,1 mg/l NAA vd lOmg/1 BA) cd bd sung chat acetosyringone ndng do 100 pM frong hai ngdy. Sau dd rira va diet vi khudn bang dung dich khdng sinh 500mg/l cefotaxime frong thdi gian 30 phiit, chuyen cac mdnh la sang mdi trudng tai sinh tao chdi cd chiia 4mg/l PPT vd 500mg/l cefotaxime. Cay truyen 2 tudn/ lan tren cimg loai mdi trudng. Sau 6 tuan, mau la cd chdi tdi sinh dugc cay truyen sang mdi trudng cho ra rl cd chiia 4mg/l PPT. Cac cay ra re tdt dugc dua ra frdng d vudn uom. b vM Phirong phap chuyen gen vao cay cai nggt (Brassica integrifolia L.): La mam vdi cudng la dai l-2mm ciia cay con tir hat ndy mdm 5-6 ngdy tudi dugc sii dyng ldm mau chuyen gen. Sir dung vi khuan da ldc qua dem de gay nhiem mdu, sau do cac la mam dugc nudi cay fren mdi trudng tdi sinh tao chdi (mdi trudng MS cd 2mg/l NAA, 4mg/l BA va 3,3mg/l AgNOs) frong hai ngay (cd bd sung acetosyringone ndng do 100 pM).
Sau dd rira va diet vi khuan bdng dung dich khang sinh 500m^l cefotaxime frong th6i gian 30 phiit, chuyen cdc mdu sang mdi trudng tdi sinh tao chdi cd chiia chdt chgn Ipc PPT va 500mg/l cefotaxime. Cay truyen 2 tuan/ lan tren cimg loai mdi trudng. Sau 4-5 tudn, mdu cd chdi tdi smh dugc cdy truyen sang mdi trudng ra re (mdi trudng MS c6 0,1 mg/l NAA va 6mg/l PPT).
Phuong phap Idem tra cay chuyen gen: •J:
• Kiem tra gen chi thi gusA bang dung dich X-Gluc: bdng cdch ngam cdc mdnh la chdi nhd vdi dung dich X-Gluc khoang 15 gid d 37°C, mdu chuyin gen se cd mdu xanh cham dac trung, mau ddi chimg se khdng chuyin mdu.
• PCR gen crylA(c): DNA thyc vat dugc chay PCR vdi cap mdi chuyen biet, quy trinh tach DNA thyc vat dugc thyc hien theo phuong phdp cua Dellaporta (1983).
• Cac cay chuyin gen dugc kiem tra sy bilu hien ciia gen crylA(c) qua khd nang khang sau bang each tha sdu xanh Heliothis armigera len cac mau la frong dia p^tri.
KET QUA VA THAO LUAN 1. Cau true plasmid moi
Viec gdn gen cryIA(b) vd crylA(c) vko plasmid pCAMBIA 3301 dk tao plasmid mdi sau:
Vecto plasmid GAMBIA 3301 cd dg ddi 12 Kb chira gen gusA va gen bar. C6 I]
enzyme gidi han tai vi fri cloning site frong vimg T-DNA, d do cho phep tach hodc i "
Ph^n V: CdNG NGHg GEN THgC VAT 347
cac doan gen mong mudn. Trong thi nghiem nay chiing tdi da sir dyng enzyme gidi ban Hind ni (A/AGCTT) de cat tao hai ddu Hind JR cua chuoi plasmid pCAMBIA 3301.
• Plasmid pUbi.cryIA(b) va plasmid pUbi.crylA(c) chiia gen crylA cd do ddi 4 kb, d hai '.->; ddu ciia toan bd gen nay (promoter-gen-terminator) cd vi tri cat bdi enzyme gidi han
Hind III. Nen chimg tdi da sir dung enzyme gidi ban Hind III dl cdt d hai vi tri ndy.
• Sau khi cdt bdi enzyme gidi ban Hind III cdc doan plasmid DNA da dugc chay dien di cho ket qua: vdi plasmid pCAMBIA da dugc md ra vdi hai ddu cdt bdi Hind III vd dugc ,. xir ly bdi AP (alkaline phosphat) de chiing khd cd thi ty ndi lai vdi nhau. Cdn plasmid , It pUbi.crylA do cd hai vi tri cdt nen tao ra hai doan: 6 kb DNA tuong iing vdi than cdn lai
r' ciia plasmid vd doan 4 kb tuong iing vdi doan gen crylA.
Sau dd tien hdnh ndi ket 2 doan DNA plasmid pCAMBIA 12 kb vd doan gen crylA 4 kb vdi nhau bdi enzyme ligase d nhiet do 4°C trong thdi gian 16 gid. Ket qud da tao dugc 2 plasmid mdi ddi khodng 16 kb.
•• D I kilm fra kit qua ndy chimg tdi da chuyen 2 plasmid mdi vdo vi khudn biln nap E.coli, nhdn bdn E. coli vd tdch chiet ADN. Sir dyng lai enzyme gidi ban Hind III dk cdt cho kit qud mdi plasmid da cho dugc hai bang DNA vdi 1 doan 12 kb vd 1 doan 4 kb (xem dnh 2 dien di) hoan todn phii hgp vdi kich thudc plasmid mdi dugc chung tdi ddt ten la plasmid pITB-CRY (Institute of Tropical Biology) chiia 3 gen vd dugc chuyin vdo ddng vi liiman Agrobacterium tumefaciens EHA 105.
Chuyin gen v^o cay hong:
Sau khi chuyin gen bdng vi khudn At, mdu Id dugc ddt fren mdi trudng cd PPT ndng dp 4mg/l, da so cac mdnh la bdt ddu vdng sau 2 tuan nudi cdy, mdt sd mdnh Id van duy tri mdu xanh nhung khdng hinh thdnh md seo, chi mdt sd it khoang 10% hinh thanh md seo va mdt sd frong dd bdt ddu tai smh sau 5 tuan nudi cdy. Sau giai doan chgn loc ndy cdc chdi con dugc chuyin sang mdi frudng MS de ra re vdi 4mg/l PPT. Vd chi cd vdi ddng cdy tilp tiic phdt triln vd ra rl, dugc chiing tdi gid dinh Id cdy chuyen gen. Ket qud qua trinh chuyin gen dugc the hien nhu sau:
Bang 1: Ty I? tai sinh chdi vd cay chuyen gen cua cac mdnh la tren moi tru'ang co 4mg/l PPT sau khi nhilm vd'i Agrobacterium tumefaciens vd mdu la ddi chu-ng
Thi nghiem Chuyin
gen Ooi chCrnq
Sd mdu la 580
30
Sd mlu la t^o md
SQO
52 0
Sd mlu la tai sinh
chdi 21
0
Sd lu'gng dong cay chuyen gen
5 0
Tin sd chuyen gen (%)
0,8 0
Dl khdng dinh day Id nhimg cdy hdng chuyin gen, chung tdi cay cay hdng gid dinh i chuyin gen vd cay ddi chiing vdo mdi trudng ra re vdi chdt chgn Igc PPT ndng do 4mg/l de
thiir khd ndng chdng chiu, sau 2 hian todn bd cay ddi chimg chit hodn todn, frong khi cdc cay chuyin gen van tilp phdt tiiln vd ra re, giup chimg tdi khdng dinh day Id nhimg cay hdng da dugc chuyin gen.
348 HCl nghi KHOA HOC VA C O N G NGHg 2007
Su bilu hien gen gusA cua cay chuyin gen: Cdc chdi tihd, manh Id cua cay chuyen gen da dugc xir ly vdi dung dich X-Gluc, kit qua cho tiidy chdi vd manh la nay cd mdu xanh cham dac frung khdng dinh sy bilu hien ciia gen gusA frong cay chuyen gen.
Sy bilu hien cua gen bar frong cay chuyin gen: gen bar tao tinh ti^g khang thudc frir cd da dugc khdng dinh frong cay in vifro, tuy nhien chiing tdi mudn tiep tiac khdo sat sy tdn tai dn dinh va khd nang bilu hien ciia gen nay fren cay sau khi dugc frdng ra mdi tiircmg ty nhien ngoai vudn uom. Chung tdi sir dung ndng do 300mg/l PPT dk phun len cac cay ddi chung va cay chuyin gen 1 tiiang tudi tai vudn uom. Sau 2 frian nhdn tiidy cac cdy hdng chuyen gen tilp tuc sinh tmdng va phat triln bmh tiiudng, chi cd mdt ddng cay hoi bi vang la phia dudi nhung van sdng va phat triln cho phep chung tdi khdng dinh mdt Idn nua day la nhimg cay hdng da nhan dugc gen chuyen.
Sy hien dien ciia gen crylA(c) va kha nang khang sdu ciia cay chuyin gen: Phdn tich PCR vdi cap mdi chuyen biet cho gen ctylA(c) cho tiidy cac mau DNA cua_cay chuyen gen sau khi chay dien di cd xudt hien cac bang cd kich tiiudc 0,65 kb gidng nhu mau ddi chiing duong tinh plasmid, chiing la kit qua ciia sy khulch dai gen crylA(c) frong khi mau cay ddi chiing ho_an toan khdng cd bang. Qua dd chung tdi khdng dinh sy hien dien cua gen crylA(c) trong nhiem sdc till ciia cay chuyin gen. Sau dd cdc cay hdng chuyen gen dugc chuyen ra frdng trong vudn uom. D I tiiir nghiem tinh khdng sdu cdc la nhd dugc ddt frong dia petii cimg sdu xanh Heliothis armigera. Sau 3 ngay, vdi cac la ddi chiing dien tich la bi hai Idn, kich tiiudc sau tang ro ret, frong khi dd d la cay chuyin gen dien tich la bi sau an nhd, sdu tang frurdng rat kem vd sau 3 ngay sau hoan toan khdng cdn an vd chet nhieu vao ngay tiiir 5 hodc 6.
Chuyen gen vao cay cai ngot:
Vdi phuong phdp sir dung la mam ciia cdy con tir hat nay mam 5-6 ngdy tudi, cay la mdm vdo mdi trudng tai sinh 3-4 ngdy trudc khi nhung cudng Id mam vdo dich vi khuan dk lay nhiem, thdi gian ii vdi vi khudn Id hai ngay tren mdi trudng cd bd sung 100 pM Acetosyringone va sau khi rira dugc cay tren mdi trudng tdi sinh cd Cefotaxiine 500mg/l va 4mg/l PPT thi sau 3-4 tudn cac chdi tdi sinh hinh thdnh va chiing dugc cay truyin tren mdi frudng MS cd 6mg/l PPT de kiem fra khd ndng khang PPT ciia cay.
Chung tdi da thyc hien thi nghiem chuyen gen nhieu lan vdi sd mau trung binh tir 80- 120 mau/ldn. Kit qua dugc trinh bay d bang 2.
Bang 2. Ty le tdi sinh chdi va cay, tren moi truo-ng co 4mg/l PPT, cua cdc la mdm du^gc nhilm vdi Agrobacterium tumefaciens vd cua m l u Id mdm ddi chu'ng
Thi nghifm
Chuyen gen Do! chirng
Sd la mim thi nghi$m
1380 30
Sd la mim tgo md s^o
52 (3,7%)
0
Sd la mim tai sinh chdi
21 ( 1,5%)
0
Sd lipgng ddng cay gia djnh chuyin gen
2 0
Tin sd \ chuyin gen (%)_
0,14 1 0 _
Qua till nghiem, chiing tdi nhan thdy sd lugng la mam chdng chiu vdi chdt chgn loc 4mg/l PPT de hinh thanh md seo khoang 3,7% nhung sd lugng cd till tdi sinh chdi chi ^ muc 1,5% va gan nhu da sd cdc chdi deu khdng phat trien va chit d cdc Idn cdy truyin sau»
theo chimg tdi day la hien tiigng dugc coi la escape kha phd biln frong qua frmh chuy^
gen hoac chdi d tiie kham va chi cdn 2 chdi tiep tuc phat triln fren mdi trudno MS v6i
p^ian V: CdNG NGHg GEN THgC VAT 349 6mg/l PPT - dieu nay cho thdy tdn so chuyen gen tiiap (0,14%). Chiing tdi cho rdng khi chuyin gen fren cac ddi tugng cdy frdng khac nhu cay thudc la vd cdy hdng thi mdu Id cd kha nang tai sinh chdi cao tir ximg quanh ria la nen vi khuan Agrobacterium tumefaciens cd thi xam nhap vao nhieu vi tri cd khd nang tai sinh nay, frong khi d cay cdi ngot la mdm chi CO the tai sinh tai cudng la nen vi khuan Agrobacterium tumefaciens chi cd mot vi tri duy nhdt de cd the xam nhiem tao cdy chuyen gen. Cdc cdy cdi gid dinh chuyen gen dugc phdn tich PCR vdi cap mdi chuyen biet cua gen crylA(c) - cho thdy cdc mdu DNA ciia cay gia dinh chuyen gen sau khi chay dien di cd xudt hien cdc bdng cd kich thudc 0,65 kb gidng nhu mau ddi chiing duong tinh plasmid, chiing la ket qud ciia sy khuech dai gen crylA(c) trong khi mdu cdy ddi chiing hodn toan khdng cd bdng.
KET LUAN
Vdi viec sir dung vi khuan Agrobacterium tumefaciens EHA 105 mang plasmid ITB dl chuyin gen, chung tdi da nhan dugc mdt sd ddng cdy hdng vd cdy cdi nggt chuyen gen mang gen crylA(c) khdng sau xanh Heliothis armigera. Bang cdc ky thuat sinh hgc phdn tir va sinh hgc chiing tdi da xdc dinh dugc ciia gen ndy hien dien frong cdy hdng vd cdy cdi ngpt chuyin gen vd gen chuyen da cd bieu hien tinh frang khd rd r?t.
Plasmid ITB (lane 2 v^ 3 mang
* gen crylA(b) v i crylA(c)
Phan tich PCR gen crylA(c) 6*
c§y hdng chuyen gen (bang DNA 0,65 kb)
Phan tich PCR gen crylA(c) o cay cdi ngpt chuyin gen (bang
DNA 0,65 kb)
cay cai ngpt gia djnh chuyin gen phiit triln tr§n mdi tru'd-ng chpn Ipc PPT
Cay hdng gia djnh chuyin gen p h i t triln tren mdi trud-ng chpn 19c PPT
350 HQI nghi KHOA HOC VA C O N G NGHg 2007
LOI CAM ON
Cac tdc gia xin chan thanh cam on Chuong trinh KC-04-13, Chuong trinh nghien ciiu CO ban da tdi fro thiet tiiuc cho nghien cihi ndy.
TAI LIEU THAM KHAO
1. Le Tdn Dire, Nguyen Hiru Hd, Nguyen Van UyIn, 2003. Tao cay hdng (Paulownia fortunei) chuyin gen khang sau thdng qua vi khuan Agrobacterium tumefaciens.
TuyIn tap Bdo cao khoa hgc Hdi nghi Cdng nghe Sinh hgc toan qudc, Hd Ndi, 16- 17/12/2003. N'XB Khoa hgc va Ky tiiuat. Ha Ndi, fr. 1088-1090.
2. Le Tan Dire, Nguyin Him Hd, Pham Thi Hanh, Nguyen Van UyIn, 2005. Anh hudng ciia tac nhan chgn loc din md cay cai nggt (Brassica integrifolia) vd nghien ciiu tao cay cdi nggt chuyen gen. Tuyen tap Bao cao Hdi nghi khoa hgc todn quoc
"Nhiing van dk nghien ciiu co ban frong Khoa hgc Sy sdng", Dai hgc Y Ha Ndi, Ha Ndi, 3/11/2005, fr. 1194-1197.
3. Pham Thi Hanh, Le Tdn Dire, Nguyen Him Hd, Nguyen Van UyIn, 2005. Khdo sat khd nang tdi sinh cay in vifro cay cai nggt (Brassica integrifolia) tir la mam va try mam phuc vu cho nghien cim chuyen gen. Tuyen tap Bdo cao Hdi nghi khoa hpc todn qudc "Nhiing van de nghien ciiu co ban trong Khoa hgc Sy sdng", Dai hgc Y Hd Ndi, Hd Ndi, 3/11/2005, fr. 498-500.
4. Nguyen Van Uyen, Le Tan Dire, Nguyen Hiru Hd, 2003. Nghien ciiu he thdng tai sinh cdy hdng (Paulownia fortunei) va dnh hudng ciia tac nhan chgn Igc de tao cay chuyen gen. Tuyen tap Bao cao khoa hgc Hdi nghi Cdng nghe Sinh hgc todn quoc, Ha Ndi, 16-17/12/2003. NXB Khoa hgc vaKy thuat. Ha Ndi, fr. 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 fransfer in plants of Brassica juncea using Agrobacterium mmefaciens-mediated transformation. Plant Cell Reports 10: 308-314.
SUMMARY i
Construction of plasmid vector and utilization for production of transgenic plants resistant to insect \ia Agrobacterium
tumefaciens
Le Tan Due, Nguyen Huu Ho, Nguyen Van Uyen Institute of Tropical Biology
New plasmid vector pITB-CRY (Institute of Tropical Biology) consisting bar,^
crylA(c) and gusA genes was constructed and applied to fransfer into Agrobacterium mmefaciens EHA 105 for Paulownia and Brassica plants genetic fransformation. Many fransgenic lines of both plant cultivars were obtained. PCR analyses and indigogenJ^
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 resistafl^
against Heliothis armigera.