TAP CHI SINH HOC 2013, 35(4): 504-510
T H I E T K E V E C T O R C O V U N G T I - D N A M A N G G E N CODA V A M O T S O C A U T R U C H O T R O C H O N L O C C A Y C H U Y E N G E N Biii Thi Thu Huong''^', Ho Thj H u o n g ' , Biii Van T h i n g \ Le Van So•n^ Le Tran Binh^
'Trudng Dai bgc Ndng nghiep Hd Ndi, *[email protected] Trudng Dai hgc Lam nghiep
"'vien Cong nghe sinh hgc, Vien Han lam KH & CN Viet Nam
TOM TAT: Nhirng yeu to bat loi tir moi trudng nhu han, man, iing, nhiet do cao thirdng lam mat can bang ap sual iham thau, gay anh huong nghiem trong den sinh Irudng, phat trien va nang suat cua thirc vat noi chung va cay trong noi neng Glycine betaine da duoc chiing minh la cd kha nang giiip cay chong lai dieu kien bat loi tren Vi vay, nham phuc vu cong tac tao gidng cay co kha nang chdng chm nhiing yeu 16 bat loi phi sinh hgc khac nhau, cong nghe gen hien nay dugc cho la mpt phucmg tien giai quyet hieu qua nhiem vu tren, Bai bao nay trinh bay ket qua thiet ke vector tai td hop mang gen ma hoa choline oxidase, tham gia vao sinh long hop glycine betam (codA) Ngoai ra, d viing T-DNA ciia vector duoc thiet kS chiia gen chi thi glucuronidase (Gus) va gen hygromycm phosphotransferase cd the giup su lira chgn cay chuyen gen dc dang hem. Cau tnic vector tai to hop da duac duac chuyen vao vi khuan A. tumefacien thanh cong va budc dau tao dugc cay thuoc la chuyen gen co kha nang chiu man.
Tir khoa. Choline oxydase, gcn codA, gcn chgn loc. gen chi thi. glycine betaine MO DAU
Bien ddi kbi hau dang la mdt Ihach thiic Idn doi vdl nhan loai, ciing niiu doi vdi ndng nghigp. San luong ciing n b u chat lugng ndng san giam do cay Irdng dang d i m nbirng yeu to bat lgi tir mdi trudng, U'ng dung cong nghe gen trong vigc nghign cim nham tang c u d n g kha nang chdng chiu cua cay trdng bang each kicb Ihicb cay trdng tdng bgp cdc chat Ibam t h i u tuong Ihich, giiip cbo le bao cd the vuot qua cdc dieu kien cue doan la mdt yeu cau cap tbiel cbo cac nha khoa hpc. CodA la gen ma hda choline oxidase, mdi enzyme chia khda trong qua trinh Idng bgp va lich luy glycine betaine, duy tri ap suat tham Ihau trong te bdo [6, I I ] Gen codA da duac chuyen vao mdt sd loai cay trdng va chiing dugc lang cudng d u g c kha nang chdng cbiu cac dieu kien mdi irudng bat Igi nhu cay Arabidopsis thaliana tang c u d n g kba nang cbiu lanh, nhiet va bang gid [2, 8], cay cai be, bach dan, ca chua cd khd nang cbiu man va oxy hda [ 1 , 7 , 1 2 ] ,
Nham giup cbo vice ebon Igc cay chuygn gen de dang va thuan lgi, trong bdi bao nay chiing tdi irinh bdy kcl qud lliigl kg mdt sd cku Inic vector chuygn gen chua gen codA, gen chi thi va gen chgn Igc d vimg T-DNA, nhdm tang cudng tinh chdng chiu dieu kien bat loi ctia mdi
trudng, Nhiing ket qua nay se la lien dg Ihiic day nhanb chdng cdng tdc chuyen gen Iao ra cac gidng cay trdng cd kha nang chdng chiu tot vdi cac yen Id phi smh hgc bat lgi phuc vu cho cdng Iac chpn gidng,
\ ' \ T LIEU \ \ PHUONG PHAP NGHIEN CUTJ Sir dung p C A M B I A 1 3 0 1 mang gen Gus va 2 veclor pB1121 {pB\\2\ll,5S-?-TP-codA-cmyc, p B n 2 l / "iSS-codA-cmyc) mang gen codk nhan tao cd bd sung mdt doan 30 nucleotide ma hoa doan peptide c-myc giup cho phat hien miic do bieu hien gen chuyen. Rieng veclor p B I I 2 l / 3 5 S - TP-codA-cmyc da dugc bd sung Ihem mgl doan TP dai 216 nucleotide ma hoa tin hieu dan (signal peptide) giup van cbuygn enzyme vao trong luc lap. Cac vector ndy do Phdng Cdng nghe Ig bao Ihuc vat, Vien Cdng nghe sinb hoc cung cap,
Cac cap mdi dac hicu cho 2 gen TP-codA- cmyc va codA-cmyc d u g c Idng hop tir hang Macrogen (Han Quoc), cd Irinh tu n h u bang I.
Cac chung vi khuan E coli (DH5a), Agrobacterium tumefaciens EHA 105 va giong thudc Id K326 vdl cay con da phdt trign d u a c 3-4 la thai dugc su dung dg tign hanh cdc Ihi nghiem phuc vu cho chuygn gen do phdng Cong
Bui Thi Thu Huong et al nghe te bao thuc vat, Vien Cdng nghe sinh hgc
cung cap.
Phirong p h a p
Thuc hien cac phan iing cat enzyme gidi han, lai tag vector tai td hgp. Plasmid tai to hcrp duac bien nap vao tg bdo vi khuan tbeo phuang phap sdc nhiet hoac xung dien ciia Cohen et al.
(1972) [4]. DNA plasmid dugc tach chiel va lam sach Iheo p h u a n g pbap ciia Sambrook et al (2001) [9] DNA tai Id hgp duoc kigm tra bang phuang phap PCR vdi cap mdi dac theo chu
trinh nhiet nhu sau 94''C/3 phut, 94''C/30 giay, (57-60''C)/30 giay, 7 2 X / 1 phiit 30 giay; 72°C/7 phut, 4''C/30 phlil, 25 chu ki.
Chuyen vector tdi td hgp vao thudc la qua A. tumefaciens theo phuong pbap cua Topping (1998) [10], Cac ddng cay thudc la cbuyen gen dugc kiem tra bang phucmg phdp nhudm md te bao dg kiem tra bieu hien ciia gen Gus [5] vd danh gia khd nang chiu man Iheo phuong phap cua Wang et al. (2003) [13] va Barunava et al (2010)[3]
Bang I Cac cap mdi dac hieu cho 2 gen TP-codA-cmyc va codA-cmyc Ten cap
moi yiTP-XbaS IKjcmyc- Sacl
¥lcocU- Xbal lUcmyc- Sacl
Trinh tir nucleotide S ' O C T C T A G A A T G G C A C A A A T T A A C A B ' 5 ' C G A G C T C T C A A T T C A G A T C C T C T T C 3 ' 5 ' G C T C T A G A A T G C A C A T C G A T A A T A T T G A 3 ' 5 ' C G A G C T C T C A A T T C A G A T C C T C T T C 3 '
Nhan gen TP-coilA
codA
Kich thadc doan DNA nhiin(bp)
1904
1688
KET QUA V A THAO LU \ N
Ket qua thu n h a n cac d o a n gcn q u a n t a m bang enzyme gidi h a n
Nghien ciiu da xac dinb dugc 2 plasmid pBII2l/35S-7-/'-co(M-c7»j't, p B I I 2 b 3 5 S - t o J . ' l -
cmyc va vector nhan pCAMBIA1301/35S-gi«
cd cimg diem cat gidi ban HindWl va EcoR\, cb the thu dugc doan gen muc lieu vd md vong veclor nhan, chiing Idi lien banh xu ly chiing dong llidi 2 enzyme nay Dien di kigm tra san pham cat enzyme gidi han dugc chi ra d hinh I.
£^^SE3
Hmh 1 Sdn phdm ci\ vector boi Hindlll'EcoRI (A) va san pham unh sach doan gcn (B) M Thang marker DNA 1 kb; 1.2.3 (A) San phSm c51 pBil21/35S-TP-codA-cmyc,pBn2l/35S-codA-cmyc va pCAMBIA1301/35S-Gus; 1,2.3 (B)- San phdm tinh sach doan gen 35S-TP-codA-cmyc, 35S-eodA-cmyc
vapCAMBIAI301/35S-Gus Cac mau cdt b^ng enzyme gidi han dugc dien di iren gel agargse cho cac bang vach kha
ro rang, kich thudc phu hgp Iinh todn Iheo li Ihuyet (hinh IA). Cac doan gen quan Iam gdm
TAP CHI SINH HOC 2013, 35(4)-504-510 35S-TP-corfA-cmyc, 35S-co(:/.-l-cmyc va
pCAMBIA1301/35S-Gus co kich thudc tuong ling kboang 3036, 2820 va 11783 bp duoc thu nhan va Iinh sach. Sir dung 5 pi sdn pham tinh sach dien di kiem tra (hinh IB). Kgt qua Ihu dugc cho thay, san pham tinh sach cd ham luong cao, khong bl diil gay, phii h c ^ vdi kich thudc nhu tinh todn ly thuyet. Mdi gieng cd mdt vach bang Urong ung vdi kich thudc nhu mong mudn, chimg td qua Irinh tinb sach tbanb cdng, sdn pham dil dieu kien de Ibuc hien phdn iing ghep ndi bang T4 ligase dg tao veclor tai td bgp.
K e t q u a tao v e c t o r c h u y e n gen
Cac doan gen sau khi linh sach dugc tien hdnh pban irng ghep noi nhd T4 ligase San pham ghep ndi dugc bien nap vao Ig bao kba bien E coli D H - 5 K bang phuong phdp sdc nhiet va duac chgn loc tren nidi Imdng chda khang sinh
kanamycin. Cdc plasmid diu dugc lir cac khuan lac dugc kigm tra bang phdn iing cat bdi H i n d n i / E c o R I , ket qud d u g c trinh bay d hinh 2.
Ket qua dien di cho cdc vach bang sang, rd, khdng dtii gay va cd kich tbudc nhu ly tbuygt da tinh toan, digu nay chiing td qud trinh thigt kg vector tdi td hop tbanb cdng n h u hinh 3.
T a o cay t h u o c ' a c h u y e n gen t h d n g qua A. tumefaciens
Tao chung A. tumefaciens mang vector tdi to hcrp Vector tdi td h g p d u a c bien nap vao A tumefaciens va d u a c chpn Ipc trgn moi Irudng cbiia k b a n g s m h . Chgn mdt sd khuan lac mgc Iren dTa thach de lien hanh pban irng colony-PCR b a n g cap mdi dac bieu. San pham phdn u n g d u o c dien di kiem tra trgn gel agarose, ket qud the hien d hinb 4.
i
^
Hmh 2 San phdm cat plasmid tai td hgp bSng Hindlll/EcoRI^ 1
(A). pCA.MBlAl?lH 35S-TP-rad4-cmyc/35S-Gus. iB) pCAMBiA1301/35S-ra,M-cmyc/35S-Gus
V I ), l i i . i u M I ! (IC
I
Hinh 3. S a dd T- DNA tai td hgp
II true gen 35S-TP-codA-cmyc, 1*2), mang cdu tnic 2en 3 ^S-cod A-cmyc M. marker DNA 1 kb, gicny DC. doi chimg ducmg; gieng 1,2: cac dong khuan lac bien nap vector pCAMBIA1301/35S -TP-codA-cmyc/35S-Gus (A), gieng 1,2,3,4,5,6,7: cac dong khuan lac bien nap vector pCAMBIA 1301 /35S-codA- cmyc/35S-Gus (B) Hinh -I. San pham PCR- colony cac khuan Iac A. lumefacicns
Bui Thi Thu Huong el al.
Hinh 4A cho thay, d 2 khudn lac phan tich cd xuat hign bang D N A kich thudc tugng duong vdl ddi chiing khoang 1904 bp. T u a n g tu d hinh 4B, 6-/7 ddng khuan lac cd bang kich thudc khoang 1688 bp dung vdi kich thudc ly thuyet tinh toan. Ket qua Iren chijng Id, da tao thanh cong cdc chung A. tumefaciens mang plasmid tai td hop.
Tgo cay thuoc Id chuyen gen thong qua A. tumefaciens
Manh thudc Id sau kbi d u o c bign nap vdi chiing A tumefaciens mang vector tai to hop dugc theo ddi mdt sd chi lieu tbu d u g c n h u bang 2
Manh cay thudc la dugc nudi cay tren mdi tmdng dinh dudng se lai sinh cbdi Trong mdi Imdng tai sinb cd bd sung Hygromycin, chi cd tg
bao nao dugc le bao vi khuan A. tumefaciens xam nhap mdi cd kha nang phdt trien chdi. Cac manh Id dugc chuyen hai cau tnic vector nay cd ty le lao chdi kha cao (> 97%) cdn vdi cay W T l , ty le manh Id phat sinh chdi trong mdi trudng cd khang sinh Id 0% (bang I, hinh 5C). Tuy nhien, su chuyen cau tnic gen vao he gen te bao thdnh cdng hay khdng the hien su sdng sdt ctia cdc chdi va su tao re trong mdi trudng ra re cd khang sinh chgn Igc. 0 day, ty le cac cbdi sdng sdt va ra re trong mdi trudng nay doi vdi cay dugc chuyen cau tnic *l la 52,46% va cau tnic *2 la 46,15%, cdn WT2 (ty le choi ra rg Irong mdi Irudng khong cd khang sinh la 93,6%). Ben canh nhiing choi dugc binh thanh Irong mdi trudng lai sinh choi nhung khdng phat trign (heo, ua hay khdng ra re-binh 5D,E va F) la nhirng chdi dugc chuygn gen thanh cong
^ 2. Su phat sinb binb thai ctia cdc man thudc la chuyen gen in vitro
Doi
Wong
TNI TN2 WTl WT2Kha niing hinh thanh choi So manh la
thi nghiem (manh)
137 96 62 60
Ty le manh la hinh thanh choi
(%)
98,73 97,77 0 97,2
Kha nang hinh thanh re Tong so choi nuoi ciiy (choi)
182 158 129
Ty le so choi ra re
(%)
52,46 46,15 93,6
Ty le so dong duong tinh X-
glux
(%)
19,78 18,99
-
TNI Manh la duge chuyen eau tnic *l tren moi trudng co Hygioniycin; TN2 Mjnh l;i duoc ehuyen t
*2 ticn moi trudng co Hygromyein; WTl(DC) Manh la khong chuyen gen tren moi trucrng co Hygrc WT2(DC) Manh ia khong ehuyfin gen tren moi trucmg khong c6 Hygromycin
D
' ^
^
F Hinh 5 Mau cay tbuoc Id irong mdi Irudng lai sinb chdi (A. B va C)
va mdi trudng ra re (D, E vd F) cd khdng sinh Hygromycin
A, B va C Cac manh la cay thu6c la da chuygn ciu tnie •|(35S-TP-codA-cmyc). *2(35S-codA-cmyc) ya manh thuoc la khdng chuyan gen (DC) trong moi truong tai sinh ehoi sau 2,5 tuan, D, E. Cae choi cay thuoe la phat In^n tir manh thudc la da chuydn c5u tnic •l(35S-TP-cod.'\-cmye), *2(35S-codA-cmyc) trong moi tnrcmg ra re sau 2.5 tu4n; F: Cac ch6i cay lhu6c la phat tnen tir manh thuoc la da chuyen cau tnic *1 hoac *2 (cay I. 2, 3) va ch6i thuoc la phat tnSn tir manh thuoc la khong chuyen gen (DC) Irong moi trudng ra re sau 3,5 tuan.
TAP CHI SINH HOC 2013, 35(4) 504-510 Tuy nhien, cac ddng thudc Id chuyen gen cd
kha nang sdng sdt tren mdi trudng cd Hygromycin d u g c khang dinb da chuyen cdu tnic Tl hay khdng, dugc tien tign hdnh nhudm hoa mo Ie bdo vdi X-glux, ket qud thu d u a c ty Ie sd ddng duong tinh (co mdu xanh dac tnmg) khoang 18,89% vdi cay d u o c chuyen cau tnic
*2 va 19,78% vdi mau cay d u g c chuyen cau tnic *1 (bang 2 va binh 6).
Cdc ddng thudc la cbuyen gen chiu dugc hygromycin cd bieu hien d u a n g tinh hay am tinh vdl X-glux d u g c thu nghiem kha nang chiu muoi (NaCI), ket qua thu d u g c chi ra d bang 3.
o o
Hinh 6. Mau thudc Id in vitro cbuygn cau true *1 (A) va *2 (B) khi dugc x u ly X-glux Bdng 3 KJia nang chju mudi cua cdc ddng thudc la chuyen gen
Dot tircfng TNl.l TNI.2 TN2.1 TN2.2 WTl(DCl) WT2(DC2)
So dong thi nghiem
36 30 30 30 1 1
Ty le manh thuoc ia song tren MT muai NaCI 50mM sau 1 tuan nudi cay (%)
77,78 10,00 50,00 3,33 0,00 0,00
Ty le choi thuoc la song tren NaCI 250mM sau 1 tuan nuoi cay (%)
83,33 16,67 53,33 6,67 0,00 0,00
TNI 1 Manh la duoc chuyen cau tnic *1 tren moi trudng cd Hygromycin duong tinh X-glux; TNI.2. Manh la duoc chuyen cau tnic *i tren moi trucmg co Hygromycin am tinh X-glux; TN2,1, Manh la duoc chuyen cau tnie *2 tren moi trudng co Hygromycin duong tinh X-glux; TN2.1. Manh la duoc chuyen cau tnic *2 tren moi trudng c6 Hygromycin am tinh X-glux; WTl(DCl). Manh la khong chuyen gen tren moi trucmg co Hygiomycin. WT2(DC2) Manh la khong chuyen gen tren moi trudng khong co Hygromycin.
Nhirng ddng Ihudc la chuyen gen cd bigu hien duong tinh vdi X-glux cd ty le ve kha nang chiu muoi cao (tren 50%). Digu nay cbiing td cd the su dung su bieu bien ciia gen Gus Id mot dau hieu nhan biet cay chuyen gen. Ben canh dd, mdt sd ddng thudc la am tinh vdi X-glux van cd khd nang cbiu mudi (vdi ty lc ihap chi dat 3%-10%). Ddng ihudc Id cd man la khdng Ihay bieu hicn ciia gen Gus nhung van cd kba nang chm mudi cd the dugc giai thich bdi cdu tnic Ti-DNA cd the da duoc chuygn vao mdt vj tri nao dd Irgn gennome, nliung gen Gus chua cd dieu kien bieu hicn gen (ihe bien d mdt bp pban khac ngoai mau Id dem xii ly X-glux hay d mdt giai doan phdt trien nao dd cua co thg) nhung gen codA da boat ddng giup cay cd kha
nang chju d u g c mudi. Nhirng cay khdng chuyen gen (WT) khdng cd kha nang chiu d u a c mudi.
Thiet ke thanh cdng 2 vector pCAMBIA1301 CO viing T-DNA mang cau tnic gen 35S-(TP)-codA-cmyc va mdt sd cau tnic ho irg chuygn gen vao thirc vat.
Chuygn thanh cdng veclor tdi td hgp vao cbung VI khuan A. lumefaciene, s5n sang cho viec chuyen vdo thuc vat.
Vector tai td hgp chiia gen CodA budc dau d u g c chuyen vao thudc la va xac dinh duoc vai trd ciia mdt sd cau tnic bd tra viing T-DNA trong chgn Ipc cay chuyen gen n b u gen khdng
Bui Thi Thu Huong et al.
Hygromycin, a-glucuronidase (GUS). Cac dong Ihudc la chuygn gen b u d e dau d u o c chiing minh cd kha nang chdng chiu dugc digu kien man (ndng do mudi NaCI cao) cua mdi trudng nudi cay.
TAI LIEU THAM KHAO
1 Ahmad R., Kim M. D., Back K. H , Kim H.
S , Lee H. S , Kwon S Y . Murata N., Chung W. 1., Kwak S. S., 2008. Stress- induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, alt and drought stresses. Plant Ceil Rep., 27: 687-698.
2. Alia H. H., Sakamoto A., Murata N., 1998.
Enhancement of Ihe tolerance of Arabidopsis to high temperatures by genetic engineering of Ihe synthesis of glycinebetaine Plant J., 16 : 155-161.
3 Barunava P., Sudipla R , Andreas R . Anm L M . 2010 Enhanced salt tolerance of transgenic tobacco plantsby co-expression o f P c I N O l and McIMTl is accompaniedby increased level of myo-inosilol and methylated inositol, Proloplasma, Spnnger- Veriag DOI I0.1007/s00709-010-0163-3.
H Up ://www. academia.edu/302 60 6/
4, Cohen S., Chang N., A. C. Y.. Hsu L., 1972.
Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA Proceedings of the National Academy of Sciences of the United Slates of Amenca, 69:2110-2114.
5. Jefferson R. A , Kavanagh T. A , Bevan M.
W., 1987. GUS fusion- a-glucuromdase as a sensitive and versatile gene fusion marker in
higher plants. E M B O J., 6: 3901-3907.
Hasegava P. M., Bressan R. A., 2000. Plant cellular and molecular responses to high salinity Annual Review of Plant Physiology and Plant Molecular Biology, 51. 463-499 Tran Thi Phuong Lign, 2010 Protein va tinh chdng chiu d thuc vat. Nxb. Khoa hgc tu nhign va Cdng nghe, Ha Noi.
Prasad K V. S. K,, Sbannila P,, Kumar P.
A , Pardha Saradhi P., 2000. Transformation of Brassica juncea ( L } Czem with a bacterial codA gene enhances ils tolerance lo salt stress. Mol. Breed, 6: 489-499.
Sakamoto A., Murata N., 2002 The role of glycine betaine in the protection of plants form stress clues from transgenic planis Planl, Cell and Environment. 25: 163-171.
t. Sambrook J., Fritsch E. F., Manialis T., 2002 Molecular Cloning: A Laboratory Manual New York' Cold Spring Harbor Laboratory Press.
.Topping J S . 1998 Tobacco transformation Methods of molecular biology, Plant virology protocols, 81. 365- 372.
; Yu X,. Kikuchi A , Matsunaga E., Monshita Y . Nanio K., 2009, Eslablisbmeni of the evaluation system of sail tolerance on transgenic woody plants in the special netted house. Plant Biotechnol, 26, 135- 141,
1. Wang Xian Yan, Shan Xiao Yi, Yang Ai Fang, Zhang Ju Zen, 2003. Construction of planl expression vector and analysis herbicide resislance and salt tolerance of transgenic tobacco China Joumal of Agricultural Biotechnology, I (2): 85-91.
TAP CHi SINH HOC 2013, 35(4): 504-510
CONSTRUCTING VECTOR H A V m G TI- DNA CONTAINED CODA GENE AND SOME TRANSPLANTS SELECTION ASSITANCE STRUCTURES
Bui Thi Thu H u o n g ' \ Ho Thi H u o n g ' , Bui Van T h a n g ^ Le V a n S o n \ L e Tran Binh^'Hanoi University of Agriculture
"Vietnam Forestry University
•'institute ofBiotechnology, V A S T SUMM.ARY
Adverse factors from the environment, such as, drought and salinity, water logging and high temperatures often cause serious effects on the growth and development of planl or crop yield. CodA gene found in Aiihrobactei globiformis encode for choline oxidase involved in the biosynthesis of glycine betaine have been shown to be transferable to some plants which help them resist such adverse conditions Veclor recombinant pCAMBIA1301 has been successfully designed wilh the codA gene. Additionally, Ihe Ti-DNA region contains the remarkable gene Gus and a hygromycme resistance selected gene that also can help researcher select transgenic plants quickly and easily The recombinant vector stmcture is also successfiilly transferred into A. tumefacien that have tested by PCR The vector was ready transferred in to tobacco in order to produce valuable tolerance vaneties to salt
Keywords CodA gene, choline oxidase, glycine belaine, transgenic veclor, selection gene, remarkable gene.
Ngdy nhdn bdi- 16-3-2013
