Tgp chi Cong ngheSinh hoc 11(3): 547-554. 2013
T H I E T K E \ E C T O R p B i n G l y B a r l \ L A N G G E N G - 7 M r P H L C \ L C H U Y E N G E N T H U C \ .\T Tham Thj T h u Nga, Le Hoang Due, Le Thj Bich Thiiy, Nguyin Diic T h a n h
Vien Cong nghe sinh hoc. Vien Hdn ldm Khoa hoc va cdng nghe Viet Nam
TOM TAT
Gen ma hoa cho gamma-tocopherol methyl transferase {G-TMT) hen quan den tich luy tocopherol la li^n chat san sinb ra vitamin E cr hat Day la chat chong oxi hoa Ur nhien tan trong d^u. rat cin thi^ cho sire khoe con ngirm Gen G-TMT cb vai tro quan trong trong chuyen hoa gamma-tocopTierol thanh alpha-tocopherol. co boat tinh vitamin E cao hon. Muc dich cua ngbien cuu nay la lao dong gen G-7.U/" phuc vu chuyen gen a thuc vat CO dau, tao cay trong chuyen gen co kba nang sinb vilamin E cao. dong thoi nghien cuu ung dung plasmid pBinGlyBarl trong chuyen gen da gen vao thuc \al ma truuc lien la gen G-TMT Gen G-TMT da duuc phan lap lit Arabidopsis thaliana bing RT-PCR su dung bg kit Reverse Transcriptase va cap moi dac hieu duoc thiet ke dua tren trinh tu gen G-TMT cua Arabidopsis cong b6 Iren ngan hang gen quoc tl voi ma s6 dang ky NM_105171,3-GenC-7"M7'sau khi phan lap dirac gan vao vector pJET I 2 Blum dl \.k dmh trinh tu Kit qua so sanh trinh tu gen G-TMT phan lap duue cho thay muc do tucmg dong so voi trinh tu G-TMT da duuc cong bo la 99%. Gen G-TMT Avtac gan vao \eclor pBinGlyBarl tai vi tri cat cua hai en/yme £roRl va Xho\ irons;
vector nay nham tao vector chuyen gen pBmGlyBarl-C-rMT" Vector pBinGlyBarl-(;-7l/7 se tilp tuc duoc gSn bo sung gen muc tieu khac nham tao vector chuyen da gen vao thirc vat
cliuvcn gen llurc i •(•/( G-TMT, itiiC-l ke v iliincndaiicn. i
M6DAU
Tocopherols la chat chong oxi boa lu nbicn co mat trong hat ciia nhung loai cay c6 dau. Chung gom bon dang dong phan co boat tinh vitamme E la alpha-, beta-, gamma-, va delta- tocopherol Bon dang tocopherol khac nhau boi so luong va vi tri gan ciia nhom methyl trong vong thom 6-chroinanol Phan lon cac nghien ciiru deu cbo rang alpha-tocopherol the hien boat tinh chong oxi hoa cao bon trong dau mo o nong dp thap, con o nong do cao tbi gamma- tocopberol the hien hoat tinh cao hon (Ujnc et ed..
2005). Gamma-tocopherol methyl transferase (G- TVIT) la mpt enzyme dong vai tro quan trong Irong long hiTp tocopherol, xiic lac cho phan irng chuyen hoa delta, sigma-tocopherol thanh alpha, beta- tocopherols, tuong ting, 6 thuc v :it Nguoi la da cbiing mmh s^r dot bien cua gen G-TMT da lam gian doan qua trinh smh long hpp alpha-tocopherol, va do do lam tang lich luy gamnia-tocopherol. Su bieu bien cua gen G-TMT trong cay dau tuong chuyen gen da lam tflng ham lugng alpha-tocopherol len toi tren 90%
tocopherol long so va kel qua la lam tiing hoat tinb cua vilammc I. Ien g^p 5 Isin so voi cay khong chuyen gen (Van Eenennaam et ai, 2003: Tawa et al 2007) Cac bien the (genclic variation) cua gen G-IMT se quycl d|nh ham lugng alpha-tocopherol, va dua tren nhCtng kcl qua nghien ciiu do nguoi ta \:1\ dung nen
cac chuong trinh nham cai thien ham lirong tocopherol Irong hat di\ co dau (Dwiyanti cl ai.
2011. Garcia-Moreno et cd. 2012) Tocopherol ciing
\(Vi cac chat chong o\i hoa khac con co \ai trd quan Irong trong vice tang cuong tinh chong chiu voi dieu kien bat lai cua moi trm'mg (Cela el ul. 201 I) Mol bien phap hiru hieu \a nhanh chong tmng cai thicn chat luong cay trong dugc cac nha nghien ciiu lua chon hang dau la chuyen gen. Ky thuat chuyen gen ngay cang duoc hoan thicn va chung to duoc tinh hCtu hieu trong nbieu muc dich cai thicn chat luong cay trong ciing nbu muc dich nghien ciiu. Cac vector chu\en gen cung ngav cang duoc cai ticn. hoan thicn va dugc ihitang mai boa lam cho nghien ciiu chuyen gen ngay cang hieu qua Phan lon cac vector duoc thicl ke cho phep chuven tung gen muc tieu neng lc
\ai mot promoter dac hicu Tu\ nhicn. theo hinVng phat tnen cua cong nghe chuven gen thuc vai, vice chuyen da gen la huiVng iighien ctiu ngay cang duac chli trong \'\ \ a \ . nhicu nghien cuu ihici ke vector chuven da gen da \uat hien lie thong moi lmh lioat hien na\ cho phep ebon lua cac promoter sa terminator CO kha nang bicu hien da gen tu mot vcLtiir bicn nap dtm (Chunga et al. 20051 V'oi muc dich tren. chiing loi nen hanh nghien ciru tliict kc vector chuven da gen dua tren plasmid pBinGlyBarl T rong nghien ciiu nay. chung toi dil tien hanh tach, tao dong gen G-TMT tu cay Aiabidopsis iluihmia va gSn v>io
Tham Thi Thu Nga et ai vector chuyen gen nhu la mpt to hgp mang gen thir
nhal sau nay se dugc gan them gen dich tiep theo tao vector chuyen da gen,
VAT LIEU VA PHU'ONG PHAP Vat lieu
Vat lieu sii dung de tach dong gen G-TMT la cay Arabidopsis ihaliana in vitro do phong Di truyen Ie bao thuc vat — Vien Cong nghe sinh boc cung cap
Vector pJET 1.2/Blunt cua bang Fermentas, dimg de tach dong gen G-TMT.
Vector pBinGlyBarl do Vien Cong nghe sinh hgc cung cap (Hinb 1), diing de thiet ke vector chuyen da gen
P h u a n g p h a p
Thiet ke cac cap moi dac hteu cbo tach dong gen
G-TMT dugc dua vao cac trinh ty ciia cac gen da cong bo sit dung phan mem Primer Design Program,
Tach chiet RNA tong so sii dyng hoa chat Trizol, Iheo hucmg dan ciia nha san xuit (Invitrogen, My)
Tach dong gen G-TMT bang cac phuong phap dugc mo ta bai Sambrook va Russell (2001). Bien nap cac doan phan lap dugc vao vector nhan dong, tach DNA plasmid, tinh sach va xac djnh Irinh ty de kiem tra theo huong dan ciia nha san xuat.
Trinh Iy nucleotide cua gen G-TMT duac xac dinb bang may giai trinh ly ABI PRISM® 3100 Avant Genetic Analyzer, sir dung bg kit BigDye^
Terminator v3.1 Cycle Sequencing
Phan tich trinh ty gen bang phan mem DNAMAN (version 3.0, Lynnon BioSoft), BLAST.
Xmal ( i l N o s - p r o
^''^,SinuJ(ii4!6) BamHI (i)
NOS-term Asel (9943) T-DNA LB
. . ^ ^
Gly-pro EcoRl (703)
'W-^~;
<>.
ApaU(SiS2) j T , Rep Origin 2 ^ bom site from pBR322
ApaUiytSA) ^
etal {67J7) Rep Origin •
3inGlyBarl
11418 bp
U %
Xhol (2363) Giy-term Clal (2690)
^' H i n d l i l (2821) M u i (2860) Pstl (2870}
STA region from pVSI piasmid Hinh 1 S o 66 plasmid pBinGlyBarl
KET QUA VA THAO LUAN
K i t qua thi^t k i cap mfli n h a n gen G-TMT De tien hanh nhan gen muc Ueu G-TMT bang ky thuat PCR, truoc lien phai Ihi^l ka cac cap m6i
dac hieu cho gen muc tieu nay dua vao Irinh Vf nucleotide gen G-TMT ciia loai Arabidopsis Ihaliana cong bo tren ngan hang gen quoc te vai m3 so dang ky N M _ I 0 5 I 7 I . 3 . Ket qua thidt k^ cac c^p moi dac hteu nhan gen G-TMT dugc th^ hien a
Tap chi Cong ngh? Stnh hgcU{i): 547-554, 2013
Bang 1. Trinh t i / c$p moi d^c hi$u nhan gen G-TMTXir A. Thaliana Ki hieu
G-TMTF G-TMTR
T r i n h t i r nucleotide cua mol EcoRl Start
5'-GCGAATTCATGAAAGCAACTCTAGCAGC-3' X/iol Stop
5*- GCCTCGAGTTAGAGTGGCTTCTGGCAA-3'
Tm 60,3
60.9
Moi xuoi dimg de nhan gen G-TMT {G-TMT?) dai 28 bp, nhiet do nong chay Tm = eO^C, ty le GC - 45%, CO thiet ke Irinh tu cat ciia enzyme gioi ban EcoRl (GAATTC) 6 dku 5' M6i ngugc diing d6 nhan gen G-TMT (G-TMTR) dai 27 bp, nhiet do nong chay 60,9''C, ty lc GC = 47,4%, co thigt kg trinh ty cat ciia enzyme 2C/ioI (CTCGAO) a dau 5' Kef qua tach RJNA tong so tii Arabidopsis va nhan g e n C - T A / r t i i c D N A
RNA long so dugc tach chiet tir Arabidopsis ihaliana bang phuang pbap su dung hoa chat Trizol Reagent va lam theo huong dan ciia nha san xuat (Invitrogen, My). RNA long so sau kbi dugc kiem tra tren gel agarose, dugc sir dung cho phan irng RT- PCR long hgp cDNA sir dyng bg kit RevertAid H Minus Reverse Transcriptase (Fermentas). Tiep theo, cDNA duac diing lam khuon de nhan gen G-TMT bang cap m6i dac hicu, Ket qua nhan gen G-TMT dugc ki^m tra bSng di?n di tren gel agarose 1%, the hien 6 hinh 2.
Hinh 2. K6t qua nhSn gen G-TMT ti> cDNA cua A. thaliana.
M- DNA marker 1 kb (BioLabs); 1' sSn ph^m PCR nhSn gen G - ™ r t i > c O N A
Kcl qua tren anh dien di cho Ihay san pham PCR chi CO mgt v^ch duy nh5l co ki'ch thuoc khoang 1 kb, luong ducrng vat kich thuoc ciia gen G-TMT theo ty thuyet. Nhu vay, buac dau co the k^l lu|in: Da long hgp dugc cDNA lir RNA tdng s6 tach tir Arabidopsis
thaliana va da nhan dugc gen muc lieu G-TMT tit nguon cDNA voi hieu qua cao. cap moi thiet ke de nhan gen G-TMT co linh dac hieu cao.
Ket qua gan gen muc tieu G-TMT vao vector pJET1.2/Blunt
San pham nhan gen G-TMrduoc gan vao vector tach dong pJET 1,2/Blunt ciia hang Fermentas, vector tai to hgp pJET1.2/Blunt mang gen G-TMT duoc bicn nap vao vi khuan E. coli chung DH5a nuoi trong moi truang chpn lpc co bo sung 50pg/ml khang sinb Ampicilin. Trong mot truang chpn lpc nay theo ly thuyet tbi cbi nhiing te bao E coh nao chira vector lai to hgp mang gen kbang Ampicilin tbi mai song sot. Dong that, Irong vector pJET ciing chita gen gay chet nen nhirng te bao mang vector ty dong vong cung se dugc loai bo
Hinh 3. Ket qua colony PCR. M: DNA marlter 1 kb (BioLabs); 1: san phSm PCR nhan gen G-TMT, 2-5: tuong ung v6i cac dong plasmid G l , G2, G3. G4.
TiSp tuc lien hanh kiem tra bang phan itng colony PCR (PCR tryc li^p lir khuan lac) d6i vdt 4 dong khuan lac mpc tren moi truong co bo sung khang sinh Ampicilin (50 pg/ml) Kfit qua_di?n di san phdm colony PCR tren binh 3 cho thay, ca 4 dong VI khudn E. coh I. 2. 3, 4 deu cho bang vach CO ki'ch thuoc khoang 1 kb, tuang duong voi kich 549
Tham Thi Thu Nga et a, thudc ciia gen G-TMT, nhu vay budc dau chting toi
CO Xhk kSt luan 4 dong vi khuan tren deu mang gen G-TMT.
De khang dinb chac chan ket qua tren, cac dong E. coli G l , G2, G3 va G4 dem nuoi trong moi trudng LB long cd bd sung khang sinh Ampicilin 50 ^ig/mL sau do tien hanh tach chiet plasmid.
Hinh 4 Ket qua cat plasmid bSng cgp enzym £coRI/Xhol M DNA marker 1 kb (BioLabs); 1 - 4 c^c dong plasmid G l
Ti^p tyc cat plasmid bang cap enzyme gidi han EcoRi va Xhol ki6m Ira sy cd mat ciia G-TMT. Kk qua dien di san pham cat plasmid bang EcoRl va -V7ioI tren hinh 4 (A, B) cho Ihay, san pham cat gom cd 2 bang, trong do 1 bang cd kich thudc khoang 3 kb luang ung vdi vector pJET 1.2/Blunt, bang con lai CO kich thudc khoang 1 kb tuang ung vdi kich thudc cua gen G-TMT.
Ket q u a xac dinh t r i n h tir ciia gen G-TMT Sau khi kiem tra plasmid bang phuang phap cat enzyme giai han va sir dung ky thuat clony-PCR, budc dau CO the ket luan cac ddng G l , G2, G3 vaG4 mang gen muc lieu G-TMT. De khang dinh ket luan tren la chinh xac, lien hanh xac dinh trinh tir nucleotide ciia doan cben vao d cac mau plasmid 02.
Plasmid duac tinh sach bang kit (AccuPrep Gel Purification Kit) ciia hang Bioneer, sau dd dugc giti di xac djnh trinh ty d Vien Cong nghe sinh bpc.
Trinh lu nucleotide ciia doan gen phan lap dugc so sanh vdi trinh ty nucleotide ciia gen G-TMT d cay Arabidopsis thaliana cdng bd tren ngan hang gen quoc te vdl ma sd dang ki NM_105171,3 bang phan m^m BLAST.
ref IHt-l 105171.31 Arabidopsis t h a l i a n a G-TMT (GAMMA-TOCOPHEROL METHYLTRANSFERASE) ; 0 - m e t h y l t r a n s f e r a s e (G-TMT) mRNA, complste e d s ; Length=1415
GEHE I D : 8 J 2 B 0 5 G-TMT I G-TMT (GAt*lA-TOCOPHEROL METHYLTRANSFERASE); t o c o p h e r o l thaliana] (10 o
lethylt PubMed linl
Score = 1901 bits (1029), Expect = 0.0 Identities = 1045/1053 (99%), Gaps = 0/1053 Strand=Plus/Plus
Query 1 TAAATGAAAGCAACTCTAGCAGCACCCTCTTCTCTCACAAGCCTCCCTTATCGAACCAAC 60 I I I I I I I I I i I I I I I I I I 1 1 I I I I M I I I I I I I I I I i I I I I I I I M M I I I I I I I I I I I i Sbjct 114 TAAATGAAAGCAACTCTAGCAGCACCCTCTTCTCTCACAAGCCTCCCTTATCGAACCAAC 17 3 Query 61 TCGTCTTTCGGCTCAAAGTCATCGCTTCTCTTTCGGTCTCCATCCTCCTCCTCCTCAGTC 120
I I I I 1 I I I I I I I I I I 1 1 I I I I I I I I I I I I M I I I I I I I I I I I I I I I I I I I i I 1 I I I I I-I Sb^ict 174 TCTTCTTTCGGCTCAAAGTCATCGCTTCTCTTTCGGTCTCCATCCTCCTCCTCCTCAGTC 233 Query 121 TCTATGACGACAACGCGTGGAAATGTGGCTGTGGCGGCTGCTGCTACATCCACTGAGGCG 180 Sbjct 234 TCTATGACGACAACGCGTGG.AAACGTGGCTGTGGCGGCTGCTGCTACATCCACTGAGGCG 293 Query 181 CTAAGAAAAGGAATAGCGGAGTTCTACAATGAAACTTCGGGTTTGTGGGAAGAGATTTGG 240 Sb:)Ct 294 CTAAGAAAAGGAATAGCGGAGTTCTACAATGAAACTTCGGGTTTGTGGGAAGAGATTTGG 353 Query 241 GGAGATCATATGCATCATGGCTTTTATGACCCTGATTCTTCTGTTCAACTTTCTGATTCT 300 3b] ct J54 GGAGATCATATGCATCATGGCTTTTATGACCCTGATTCTTCTGTTCAACTTTCTGATTCT 413 Que•y 301 GGTCACAAGGAAGCTCAGATCCGTATGATTGAGGAGTCTCTCCGTTTTGCCGGTGTTACT 360
I I I I I I I I I I I I I I I 1 I I I I I I I 1 I I I M I I I I I i I I I I I I I I M I I I I M I I I I I I 1 1 :L -•?- Ill CGICACAAGGAAGCTCAGATCCGTATGATTGAAGAGTCTCTCCGTTTTGCCGGTGTTACT 473
Tgp chi Cong nghe Sinh Inn H i 3 ) 547-554,2013
Q u e r y 3 6 1 S b j c t 4 7 4 Q u e r y 4 2 1 S b j c t 5 3 4 Q u e r y 4 8 1 S b j c t 5 9 4 Q u e r y 5 4 1 S b j c t 6 5 4 Q u e r y 6 0 1 S b j c t 7 1 4 Q u e r y 6 6 1 S b ^ c t 7 7 4 Q u e r y 7 2 1 S b j c t 8 3 4
Q u e r y 7 8 1 S b j c t 894 Query 8 4 1 S b j c t 9 5 4 Q u e r y 9 0 1 S b j c t 1 0 1 4 Q u e r y 9 6 1 S b ] c t 1 0 7 4
GATGAAtSAGG^ "-G - ~ -jy^AAGATAAAGAAAGTAGTGGATGTTGGGTGTGGGATTGGAGGA 4 2 0 i l l i l l l l l l l l . M l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l t l l l l l l l l l GATGARGAGGAGGAGRAAAAGATAAAGAAftGTAGTGGATGTTGGGTGTGGGATTGGAGGA 5 3 3 AGCTCAAGATATCTTGCCTCTAAATTTGGRGCTGAATGCATTtMCfiTAACTCTCAGCCCT 4 8 0
l l l l l i l i l l l l j j l l l l l l i l l i l l i i l l i i i i i i i i i i i i i i i ! I M I I M I I I I I AGCTCAAGATATCTTGCCTCTAAATTTGGAGCTGAATGCATTGGCATTACTCTCAGCCCT 593 GTTCAGGCCAAGAGAGCCAATGATCTCGCGGCTGCTCAATCACTCGCTCATAAGGCTTCC 540
I I I I I I I I M I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I M M I I I I I I I GTTCAGGCCAAGAGAGCCAATGATCTCGCGGCTGCTCAATCACTCGCTCATAAGGCTTCC 653 TTCCAAGTTGCGGATGCGTTGGATCAGCCATTCGAAGATGGAAAATTCGATCTAGTCTGG 600
I I M I I I I i l l l l l l l l l l l l M I I I I I I I I I M M I I I I M I I I I I I I I I I I I I M l TTCCAAGTTGCGGATGCGTTGGATCAGCCATTCGAAGATGGAAAATTCGATCTAGTGTGG 713 TCGATGGAGAGTGGTGAGCATATGCCTGACAAGGCCAAGTTTGTAAAAGAGTTGGTACGT 660
I I I I M I I I I I I I I I I I I I I M I I I I I I I I I I I I I M I I I I I I I I I I I I I I M I I I I I I I TCGATGGAGAGTGGTGAGCATATGCCTGACAAGGCCAAGTTTGTAAAAGAGTTGGTACGT 773 GTGGCGGCTCCAGGAGGTAGGATAATAATAGTGACATGGTGCCATAGAAATCTATCTGCG 72 0
I I I M I I I M I I I I I I I M I I I I I I I I I I I I I I I I I I I I I I I I M I M I I I I I I I M I I GTGGCGGCTCCAGGAGGTAGGATAATAATAGTGACATGGTGCCATAGAAATCTATCTGCG S 3 3 GGGGAGGAAGCTTTGCAACCGTGGGAGCAAAACATCTTGGACAAAATCTGTAAGACCTTC 78 0
I I M I I M I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I M I I I I I I 111 GGGGAGGAAGCTTTGCAGCCGTGGGAGCAAAACATCTTGGACAAAATCTGTAAGACGTTC 8 93 TATCTCCCGGCTTGGTGCTCCACCGATGATTATGTCAACTTGCTTCAATCCCATTCTCTC
. . J l i i l l l l l l l l i i i i i J i i i i i i i i i i i l l l l l l l l l l M I I I I I I M I I I I I I M I
TATCTCCCGGCTTGGTGCTCCACCGATGATTATGTCAACTTGCTTCAATCCCATTCTCTC CAGGATATTAAGTGTGCGGATTGGTCAGAGAACGTAGCTCCTTTCTGGCCTGCGGTTATA . I M I I I I I I I I I I I M I I I I I I I I I I I I I I I I l l l l l l l i i i l l l l l l i i i l l l l M i l
CAGGATATTAAGTGTGCGGATTGGTCAGAGAACGTAGCTCCTTTCTGGCCTGCGGTTATA CGGACTGCATTAACATGGAAGGGCCTAGTGTCTCTGCTTCGTAGTGGTATGAAAAGTATT l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l i l l l l l l l M I I I I I l l l l l CGGACTGCATTAACATGGAAGGGCCTTGTGTCTCTGCTTCGTAGTGGTATGAAAAGTATT AAAGGAGCATTGACAATGCCATTGATGATTGAAGGTTACAAGAAAGGTGTCATTAAGTTT I I I I I M I I I I I I I I I I I I I I I I I I I I I I I I I I M I I I i l l l l l M l l l l l l l l l l l l l l AAAGGAGCATTGACAATGCCATTGATGATTGAAGGTTACAAGAAAGGTGTCATTAAGTTT
1073 1020 1133 Query 1021 GGTATCATCACTTGCCAGAAGCCACTCTAAGTC 1053
" " M I I M I I I I I I I I I I I i l l l l l l l l l l l 1134 GGTATCATCACTTGCCAGAAGCCACTCTAAGTC 1166
Hinh 5, K^t qua so sanh trinh t u nucleotide gen G-TMT (dong G2) vai cac trinh t u nucleotide ciia gen G-TMT a cay Arabidopsis thaliana dS dugc cong b6 tren Ngan hang gen bang chuong trinh Blast
Query Trinh ty nucleotide cua gen G-TMTlach dong t u Arabidopsis Sbjct Trinh ty nucleotide gen G-TMT ciia Arabidopsis, ma so NM_105171 3
K^l qua so sanh trinh tu nhu hinh 5 cho thay trinh ty nucleotide ciia gen G-TMT tach dong tir A.
thaliana tuong ddng 9 9 % vdi trinh lu nucleotide ciia gen G-TMT ciia A. thaliema cong bd tren ngan hang gen qudc te (ma so dang ky NM_I05171 3). Nhu v^y. chiing Idi khang dmh da tach dong thanh cdng gen G-TMT tir A. thaliana Phan tich gen G-TMT
cho k§t qua khung dpc ldn nhat (max ORF) nam trong doan nucleotide 4 d^n 1050 ma hoa cho 348 amino acid co khdi luong phan ty 38,014kD Tnnh tu amino acid suy dien ciia gen G-TMT phan lap dugc cd dp tuong ddng 100% so vdi doan trinb ty amino acid tuong irng ciia gen G-IMl NM 105171.3 cd ORF lu vi Iri nucleotide 117 den
551
Tham Thi Thu > a etal 1163 Gen G-TMTsau khi xac djnh trinh ty de khang
dinh da lach thanh cong, se dugc gan vao vector bieu hien pBinGlyBarl.
Ket qua g i n gen G-TMT vao vector chuyen gen pBinGlyBarl
M 1
Ket qua dien di tren hinh 5 cbo thay, vector pBinGlyBarl sau khi xir ly bang cap enzyme Eco^/Xhol da bj cat thanh 2 doan DNA Mpl doan cd ki'ch thudc khoang 1,6 kb luong duong vdi kich thudc gen LUC va mgl doan co kich Ihudc khoang 10 kb tugng duong vdi kich thirdc vector pBinGlyBarl da loai bo gen LUC.
Dong plasmid G2 de thu gen G-TMT cung dugc cat vdl cap enzyme gidi han su dung de cat vector pBinGlyBarl, dd la EcoRUXhol. K6l qua Xhk hi?n Iren hinh 7.
Hinh 6, Ket qua cat vector pBinGlyBarl bSng cap enzyme gioi hgn EcoRUXhol M DNA marker (BioLabs), V vector pBinGlyBarl sau khi xu ly bang EcoR\IXho\
Vector pBinGlyBarl cd kich thudc 11,4 kb cd 2 dicm cat ciia £'coRI va Xliol nam d 2 dau cita gen LUC Do dd, sau khi duac xu ly bang cap enzyine gidi ban noi tren, vector pBinGlyBarl (bi loai bo gen LUC cd kich thudc khoang 1,6 kb) co kich thudc khoang 10 kb, Kcl qua dien di thu dugc nhu hinh 6.
Hinh 7. Ket qua c l t plasmid dong G2 b^ng c$p enzytne giai hgn EcoR\IXho\. M: DNA marker (BioLabs); 1' san pham PCR nhan gen G-TMT, 2" piasmid dong G2 sau khi xir ly bSng cSp enzyme EcoR\IXho\
Hinh 8. Ket qua di$n di san pham cSt piasmid pBinGlyBar-G- TMTbang EcoRi va Xho\ M- DNA marker Ikb (BioLabs); 1 - 4 cac dong plasmid Bl - B4.
Hinh 9. Di§n di plasmid pBinGlyBarl-G-TAfTvd sin pham PCR tren khuon plasmid. 1, 2; pBinGlyBarl-G- TMT. M, Marker 1 kb (BioLabs); 3: san pham PCR G-TMT tren khuon plasmid.
Tgp chi Cong ngheSinh hgc 11(3| 547-554. 2013 Vector pBinGlyBarl va plasmid G2 mang gen G-TMT sau khi xir ly bang cung cap enzyme gidi b^n EcoRlIXIiol. san pham duoc tinh sach bSng thdi gel su dung bg Kit (AccuPrep Gel Purification Kil) ctia hang Bioneer. Khi xu ly vector va gen G-TMT bang eiing mot cap enzyme gidi ban se tao ra dSu ndi tuong dong, trgn Ian vector pBinGlyBarl va gen G- TMT trong mot Irudng cd mat enzyme T4 DNA ligase se lao vector bieu hien tai to bgp mang gen muc lieu G-TMT: pBinGlyBarl-G-7"Wr.
Vector pBinGlyBarl mang gen G-TMT dugc bien nap vao E coh DH5a Sau kbi bien nap, dich te bao dugc cay Irai tren mdi Irudng LB agar c6 bd sung 50 pg/ml khang sinh Kanamycin Vi khuan sau khi cay trai tren mdi trudng LB agar, nudi 37''C qua dem, chpn ipc khuan iac mgc dugc Iren mdi trudng Kanamycin de nuoi cay tach plasmid. Sang lgc so bp plasmid cd mang gen G-TMT bang each cat vdi cap enzyme EcoRi/Xhol. Ket qua the hien tren hinh 8.
Kiem Ira su cd mat ctia G-TMT trong plasmid bang PCR. Kel qua the hien tren hinh 9
Kel qua nhan gen Iren khuon pBinGlyBarl-G- rMTcho thay xuat hien bang cd kich thudc khoang 1 kb, tuong duong vdi kich thudc ciia gene G-TMT.
Nhu vay, gen G-TMT da dugc gan vao vector bieu hien pBinGlyBarl.
KET LUAN
Da tach ddng thanh cong gen G-TMT tir cay Arabidopsis Ihaliana, trinh tu nucleotide cua gen G- TMT Ihu dugc tuong ddng 99% so vdi trinb ty nucleotide ciia gen G-TMT cua Arabidopsis cdng bd tren ngan hang gen qudc Ie (ma sd dang ky NM_105171.3).
Da gan dugc gen G-TMT vao vector bieu hien pBinGlyBarl
Loi cam tm: Cong trinh duac hodn thdnh v&i sir tdt tro kinh phi ciia Vien Cong nghe sinh boc. Cdc ldc gid xin cdm an Ban lanh dao Vien dd cung cdp bg plasmid vd ho trg kinb phi cho ngbien cini.
TAI LIEU THAM KHAO
Cela J, Chang C, Munne-Boseh S (2011) Accumulation of g- Rather than "-Tocopherol Alters Ethylene Signaling Gene Expression m the vte4 Mutant of Arabidopiis thaliana Plant Cell Physiol 52(8): 1389-1400.
Dwiyanti MS, Tetsuya Y, Masako S, Jun A. and Keisuke K (2011) Genetic variation of y-tocopheroi methyltransferase gene contributes to ele\ated a- tocopherol content in soybean seeds. B.MC Plant Biol.
II 152-168,
Garcia-Moreno MJ, Fernandez-Martinez JM. Velasco L, Perez-Vich B (2012) Genetic basis of unstable expressmn of high gamma-tocopherol content in sunOower seeds.
BMCPIamBiol 12:71-84
Sambrook J, Russell DW (2001) Molecular Cloning A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spnng Harbor, NY
Chunga S\l, Frankmanb EL. and Tzfira T (2005) A versatile vector system for multiple gene expression in plants Trends in Planl Siienie. 10(8), 357-361 Tawa VS. Kim YH. Kagan IA. Dinkins RD. Kim KH.
Collins GB (2007) Increased alpha-tocopherol content in soybean seed overexpressing the Pcnlia frutescens gamma-tocopherol methyltransferase gene Plani Cell Rep 26(l}.61-70.
Ujiie A. Tetsuya Y . Kenshiro F. Yasiishi E. Keisuke K (2005) Identification of soybean varieties with high a- tocopherol content. Breed i i i 55 12.1-125
Van Eenennaam AL. Lincoln K. Durreti VP. Valentin HE, Shewmakcr CK. Thome GM, Jiang J. Has/is SR, Levering CK. Aasen ED, Hao M. Stem JC, Norris SR, Last RL (2003) Engineenng vitamin E content: trom Arabidopsis mutant to soy oil. Pkmi Cell 15:3007-3019
Tham Thi Thu Nga e^fl/.
CONSTRUCTION OF THE pBinGlyBarl VECTOR CARRYING G-TMT GENE FOR PLANT TRANSFORMATION
Tham Thi Thu Nga, Le Hoang Duc, Le Thi Bich Thuy', Nguyen Duc Thanh In.stitule of Biolechnology. Vietnam Academy of Science and Technology
SUMMARY
Tocopherols are amphipathic, Iipid-soiuble antioxidants that are produced only by photosynthetic organisms They play an important roles in human and animal nuUition. Gamma-tocopberol methyltransferase (G-TMT), one ofthe key enzymes in tocopherol biosyntheUc pathway in plants, catalyzes the methylation of delta- and gamma-tocopherol, converts them into beta-, alpha-tocopherols, which is a committed step to elevate vitamin E activity and nutrition value Introduction of G-TMT gene mto plant could be a promising approach to improve a-tocopherol content m the seed Here, we present the results on the cloning of G-TMT gene from Arabidopsis thaliana and constructing the expression vector pBinGlyBarl carrying cloned G-TMT gene Total RNA was extracted from Arabidopsis ihaliana leaves using TRIZOL reagent and treated with DNase I according to the manufacturer's instructions. The first-strand cDNA was synthesized by using RevertAid H Minus Reverse Transcriptase according to the manufacturer's instructions. Then, PCR product using G-TMT pnmers was inserted into pJET1.2/Blunt vector and the cloned G-TMT fragments were sequenced. The companssion ofthe cloned gene with the published G-TMT gene (accession # NM_105171.3) showed 99% similarity The cloned G-TMT gene was inserted into expression pBinGlyBarl vector and the plant transformanon vector pBinGlyBarl-G-TA/T was constructed. The structure ofthe vector was verified by colony PCR and restriction digesbon using EcoRl and Xhol restnction enzymes.
Keywords: G-TMT, expression vector, multiple transgenes, transgenic plant, vector design, vilamin E
-espondence: E-maiP [email protected]
