KHOA HQC CONG NGHE
TACH DdlMG V A THIET KE VECTOR CHUYEIM GEIME CHfU HAIXI AtYUCCAB TLT CAY Arabidapsis thaliana
Ma Thi Thu W, Tnrcmg Kim Oanh\ Ta Phuong Thiiy^' ^ Ng6 Bao Oanh\ Nguyen Tlii Tinh^ NgO Xuto Binh^'^
Biii Tri ThiicS Nguyen Ti^n Diing' T6MTAT
0 thuc vai, auxin dbng vai tro rat quan trpng trong qua trinh sinh tnrong va phat tri^n cua cay. Mac dii rat nhieu con ducmg tdng hop auxin da duoc chiing minh, nhimg gan day gen AtYUCCAO thupc nhom gen YUCCA ma hoa cho protein flavin monooxygenase dupe cho rang co vai tro chinh trong qua trinh sinh truang va piiat tn^n cua cay. Khi gen duoc lang cuong bieu liien da thiic day qua trinh tong hpp auxm lAA giiip cho cay chong chiu tdt vcri stress moi Iruong. dac biet la han. Bang ky thuat PCR va enzyme cat gioi han da ti^n hanh tach dong thanh cong gen AtYUCCA6 \.ii cay Arabidopsis su dung vector pBluescnpt.
Viing gen ma hoa c6 kich thuoc 1,25 kb dupe cat bang enzyme No& va SpA va noi voi promoter UBQ14 cr cimg di^m cdt trong vector tach dong pBS. Ket qua dircrc kiem tra b^ng enzyme cat gioi han va giai trinh lu gen. Cau tnic pUBQ14::AtYUCCA6 sau do dupe cat enzyme AsA va SpA va chuy&n sang vector chuyen gen pER8 b ciing vi tri cat. K^t qua duoc kiem tra bang PCR va enzyme cat gioi ban. Cau true vector chuydn gen pER8-pUBQ14::AtYUCCA6 sau do dirp^ chuyen vao chung vi khuan Agrobactenuni GV3101 d^ phuc vu chuydn gen ti^&n cky.
Tit kh6a: Arabidopsis. AtYUCCA6, chiu ban, tach ddng, vector
I.DATVANDE
Auxin la mot trong nhiing hormone sinh truong CO anh hudng lon d^n qua trinh sinh h-ucmg ciia te bao va ca the thirc vat. Auxin kich thich su phat trien ciia rh, thiic day su hap thu va van chuyen nuox: trong cay, giup cay vugt qua dieu kien han han, bat loi [1].
6 thuc vat, khi hi stress gay ra nhieu phan ung, tir viec thay doi bieu hien gen, qua trinh trao doi chat trong t^ bao cho den nhiiiig thay doi lon hen quan den kha nang sinh truong va nang suat. O miic do phan tu, yeu t6 moi tntong bat lpi lam gia tSng mtrc dp bieu hien va tich luy ciia hang loat cac gen dap ung dieu loen han, bao gom cac gen lien quan den tong hop chat di&u hoa tham thau, cac gen ma hoa cho cac protein, cac phan tii tin hieu va cac y^u td phien ma [7]. Trong do, cac y6u to phien ma dugc nghien cuu nhieu boi hoat dpng cua chting gay tac dong d^n su bieu hien cua hang loat cac dap img vol dieu kien bat lgi cua moi trucmg.
' Truong Dai hpc Nong Liim, Dai hpc Thai Nguyen ' Truang Cao dang Y le Thai Nguyen
•• 86 Kho.i hpc va Cong nghe Email: nguyentiendungi3>luai.edu.vn
[AA la mot hoc mon chinh trong thuc vat dupe tong hop thong qua axit amin tryptophan (Trp) theo 4 con duong gian tiep khac nhau lien quan d^n axit indole-3 pyruvic QPA); indole-3-acetaldoxime, indole- 3-acetamide va tiyptamine (TAM). Tuy nhien vai tro ciia timg con duong van chua dupt lam sang to [15].
YUCCA thupc nhom enzyme flavin monooxygenases (FMOS) CO vai tro chuyen hoa IPA thanh lAA. C^c nghien cuu truoc day cho rgng hoat dgng ciia protein YUCCA trong con duong tong hop TAM dua tr^n kha nang tai to hpp cua protein YUCCA sir dung TAM nhu mgt co chat [6], [9]. Nhung gkn day cac nghien cuu ve di truyen va hoa sinh cho rang YUCCA cimg vai tryptophan aminotransferase (TAA/TARs) CO cimg vai tro trong con duong tong hgp IPA [10], [12]
Nhom YUCCA gom co 11 gen thanh vien dugc phat hien tren cay Arabidopsis [10]. Cac nghien ciiu cho thay khi cac gen nay hoat dgng qua miic se lam tang ham lugng lAA cdng nhu cac auxin khac trong qua ti-inh sinh tiiiang ciia cay [31, [14]. Su gia tang qua trinh tong hpp cac auxin do gen YUCCA giiip cho cay thich ung dugc voi di6u kien han o lua (51, [13], khoai tay [4], dau tuong [8]. De nang cao kha nang ch6ng chiu han cho cay tr6ng da tien hanh tach dong va thi^t ke vector mang gen YUCCA6 tir cay Arabidopsis de ung dung tao cay tr6ng chuyen gen
KHOA HOC CONG NI
nhu lua, ngo. dau iinmg... Ihich ung voi dieu kien kho han bXi^t Nam.
2. VAT LCU VA PMime PHAP r » « l CUU 2.1. V$tli^l nghien cuu
Ca>- Arabidopsis thahana (Columbia), vector tach dong pBluescnpt va vector chuyen gene pER8 do Giao su Park Soon Ki. Truong f)ai hgc Quoc gia Kyungpook. Han Quoc cung cap trong khuon kho hpi) tac vcri Truang Dai hpc Nong Lam, Dai hpc Thai Nguyen.
2.2. Phuong ph4p nghifen ciiu
22.1. Tach chidt RS.A tdng sd va khuech dai genetucDNA
liNA tong so dugc tach lu nu hoa cay Arabidopsis thaliana bang bp kit RNcasy Plant Mini cua Hang Qiagene (htlp://wv™'.qiagene.com/). Quy trinh tach chi^t dugc thuc hien theo huong dan ctia nha san xuat. Nong dp RNA tong so dugc do bang may quang phci ke Biorad Specphotometer b hum- song OD260/2S0. lug RNA long so dugc dimg lam khuon de tong hgp cDNA Iheo hirdng dan cua bg kit Inipromll Reverse transcription System Hang Promega (ht1ps://woridwide.promega.com). cDNA sau khi long hpp dugc sir dung lam khuon cho phan img PCR khu&ch dai gene AtyZlCCAtivbi cap moi dac hi^u AtYUC6Notl-F va AtYUC6SpeI-R (Bang I).
Phan ung dugc thyc inen trong 25 chu ky a 94"C; 2 phut, SS-C: 30 giay, 72"C: 2 phuL San pham PCR dugc ki6in tra tr^n gel agarose va tinh sach bang bp kit PCR purification ciia Hang SolGenel (http://www.solgenet.com).
2.2.2. Gan vio vector tiich ddng
De tach dong gene At^VCCXd da su dung vector nhan dong pBluescript co chiia promoter UBiJ14 dugc cung cap boi Giao su Park Soon Ki.
TrucVng Dai hpc Quoc gia K>'ungpook. Han Quoc.
Toan bp chieu dai gene .At)TCC.Ati ma hoa dugc khuc-ch dai bang PCR voi cap moi dac hieu co chen vi tn' nhan bi^t ciia 2 enzyme tuang ung la .\oil\i\
Spel. ky hieu la AtYUC6NoU-F va AtYl'C6SpeI-R (Bang 1). San pham PCR nhan gene At)TCCAt>'va vector tach dong pBluescript duoc cat d6ng thai bang hai enz>ine .\(>tl va Sprl cua Hang BioLabs flittps://www.neb.com). San pham cit sau do duoc tinh sach bang kit ciia Hang SolGenet (htti)://www. solgenet.com) va ghep noi bang
enzyme T4 ligase. Phan ung ghep noi duoc tlurc hi^n a nhiet dp phong d^ong 12 gia voi the tich 10 pi bao gom: DNA chen + DNA vector + 1 pi T4 DNA ligase.
San pham ghep noi sau do dupe biein nap vao le bao E. cobDHSa kha bien theo phuong phap sue nhiet a 42" C, 30 .giay. Te' bao bien nap dugc nuoi qua d^m a 37"C tren moi trucmg LB (Luria-Bertani gom 10 g/l NaCl. 10 g/l trytone, 5 g/l yeast extract. 15 g/! agar) b6 sung khang sinh ampiciUin 100 mg/l.
2.2.3. Chon loc dong tdbio tai td hop Te bao tai to hgp dupe sang lgc bang PCR voi cap m6i dac hi^u tir cac khuan lac (colony) mpc ti-^n dia moi truong LB chiia khang sinh ampicilhn 100 mg/l. Ti^p den te bao tai to hpp dugc nuoi tang sinh, tach plasmid de kiem ti'a cac doan chen bang enzvTiie cat han ch^ va giai trinh tu gene. Trinh tu gene sau do dugc ki^m tra bang each so sanh vai trinh tu gene goc trfen ngan hang gene NCBI bang cong cu BLAST. Trinh tu tach dong hoan hao duoc gan vao vector chuydn gene.
2.2.4. Gin vao vector chuyen gene
\ecior bi^u hic^n pER8 dupe su dung de chuyen gene .Ai)VCCA6vao cay trong. Cau tiiic gene pBS- pUBQ14::AlYUCCA6 va vector pERB dugc cat ddng thoi bang enzyme Asclva Spel sau do ghep noi bai enzyme T4 DNA ligase. Phan ung ghep noi tao cau Uiic pFR8::pUBQ14-AtYUCCA6 duac tien hanh tuong tu nhu ti-inh bay a phan tach dong gene. San pham ghep noi dupe chuyen vao te bao vi khuan E.
cob DHSa va nuoi cay tren moi tiniong LB dac bo sung 50 mg/l kanamycin. Te bao tai to hgp dugc kiem tra bang PCR va enzyme cdt gioi han. D l chuydn vao cay ti-6ng. plasmid tai t6 hgp duoc chuyen vao te bao vi khuan GV3101.
3. KET QUA NGHeV CUU VA THAU LUAN
3.1. Phan tich gene AtYUC6trtn ca sa du li^u database
AtYUCCA6 ma hoa cho protein nhom flavin monuoxyenases co vai tro quan trgng trong qua tiinh long hpp auxm. Gene AtYUCCA6 nSm tren nhi^m sac the s6 5, kich thuoc 3719 bp. co 5 exon va 4 inti-o (TAIR. https://seqviewer.arabidopsis.org). Gene bieu hien o tat ca cac bg phan cua cay. Trong do manh nhat b ca quan sinh san nhu cum hoa, hat phan (Hinh 1).
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 10/2019
KHOA HOC CONC NGHE
i n i U i i
'iii
Hinh 1. Bilu hi6n cua gene AtYUCCA6a cay Arabidopsis thahana 3.2. Tdch d6ng gene AtYUCCAd
Dua tren ket qua phan tich da ti^n hanh tach dong gene AtYUCCAd de ling dung cho chuyen gene. Bang phan ung triing hgp chuoi (PCR) voi cap moi dac hieu AtYUC6Noa-F va AtYUC6SepI-R toan bo chieu dai 1,25 kb trinh tir gene ma hoa (coding sequences-CDS) dupe khuech dai tren khuon cDNA.
San pham PCR dupe hnh sach bang bg kit cua Hang Solgenet (http://vvTvw.solgenet.com) de su dung cho tach dong gene. Su dung vector tach dong pBluescript (pBS) mang promoter UBQ14 da duoc chen san vao vi tri giija Asci va Notl. Gene .^/?Z7([rC4(ydupc dua vao vi ti'i giira hai enzyme No&
va Spel sau khi dupe cat va noi bang enzyme T4 DNA ligase (Hinh 2A). Ket qua tach dong gene dugc kiim tra a cac khuan lac mpc tren moi ti'uong LB dac CO khang sinh chpn lpc Ampicilin 100 mg/l. Sau khoang 16 gia nuoi cay a nhiet dp 37"C, tiln hanh kiem tra ngau nhien 16 khuan lac bang ky thuat PCR tai cho vai cap m6i AtYUC6NotI-F va AtYUC6SpeI-R.
Kel qua dien di san pham PCR tren gel agarose 0.8%
cho thay tat ca diu cho kit qua duong tinh voi bang Bang 1. Trinh tu moi sir
vach CO kich thuoc khoang 2,25 kb tuong ung vol kich tiiuoc cua doan gene AtVUCCAd (Hinh 3A). De kiem tra chinh xac, da nuoi tang sinh khuan lac so 8 va so 13, tach DNA plasmid va cat bang enzyme gioi ban Notlva SpeL Kit qua dien di cho thay tr&n ban gel CO hai doan tuang ung vai kich thuoc khoang 4 kb va 1,25 kb day la kich thuoc cua vector tach dong pBS mang promoter UBQ14 va gene AfVUCCAdlihi duoc cat boi Notlva Spel (Hinh 3B). Kit qua giai tidnh tiJ gene voi cap moi M13 (-40), AtYUC6- R cho thay gene AtYUCCAff co do tuang d6ng 100% voi trinh tu goc va trinh tu amino axit dupe bao toan.
Hinh 4 the hien mpt phdn kit qua tach dong gene AtYUCCA6 dugc kiem tra bang cong cu Blast tren ngan hang gene (NCBI) voi m6i ngugc M13 (-40) cung cap boi Cong ty COSMOGENETECH. Tnnh ty gene AtYUCOhit dau tir vi tri 381. xen giira vi tri tii 375 den 380 la trinh tu enzyme Spel (ACTAGT) noi giira gene AtYUCCAGva vector pBS (Hinh 4). Tir cac kit qua thu dugc da khang dmh gene AtYUCCAd duoe tach dong thanh cong, ky hieu la pBS::pUBQl4- AtYUCCA6.
dung trong nghien ciiu
Ten moi Trinh tir (5'-3')
AtYUC6-NF TTGGCGGCCGCATGGATITCTGTTGGAAGAG
AtYUC6-SR TCAACTAGTTCAATTCCCACCACAATCACTCTC
PER8-R ACG TTG TCG AAA CCG ATG ATA CGG
KHOA HOC CONG N:
nhu lua, ngo, dau tmmg,.. thich ling voi diiu ki?n kho han a V u t Nam.
2. VAT UEU VA PHDONG PHAP I K H f i l o n 2.1. V4tli§u nghien cuu
Ca\' Arabidopsis thahana (Columbia), vector tach dong pBluescnpt va vector chuyen gene pER8 do Giao su Park Soon Ki, Truang Dai hpc Quoc gia Kyungpook. Han Quoc cung cap trong khuon kho hgp lac voi Truimg Dai hpc Nong Lam, Dai hpc Thai Nguy&n.
2.2. Phuong p h ^ nghifin ciiu
2.21. Tich chidt RNA tdng sd ii khuech dai gene ttic DNA
RNA long so dugc tach tir nu hoa cay Arabidopsis thaliana bang b6 kit RNcasy Plant Nhni ciia Hang Qiagene (http://vmw.qiagene.com/). Quy ttrinh tich chiet dugc Ihirc hi&n theo huong dan ciia
\ nhd san xuat. Nong dg RNA tong so dugc do bang may quang phri k l Biorad Specphotometer a buoc IsAng OD260/280. lug RNA tong so dugc dimg lam khu6n d l long hgp cDNA flieo huong dan cua bg kit Inipromll Reverse transcnption System Hang
|;Eromega (https://worldwide.promega.com). cDNA sau khi long hgp dupe sii dung lam khudn cho phan ling PCR khulch dai gene AtWCC.Ati voi cap moi d$c hi|u AtYUC6NoU-F va AtYliC6SpeI-R (Bang 1).
Phan ling dupe thyc hi?n trong 25 chu ky a 94"C: 2 phiit, 55"C: 30 giay, 72'C: 2 phiiL San pham PCR dupe kiim tra tr&n gel agarose va tinh sach bang bp kit PCR purification ciia Hang SolGenet Oiltp://www.solgenet.com).
2.2.2. Gin vao vector tach ddng
De tach dong gene AtWCCAd da sir dung vector nhan dong pBluescript co chiia promoter UBQ14 dugc cung cap boi Giao su Park Soon Ki, Truang Dai hgc Qudc gia Kyungpook, Han Quoc.
T o ^ bO chiiu dai gene At'iJ'CC.Ad ma hoa duoc khulch dai bang PCR voi cap moi dac hieu co chen vi tri nhan biit cua 2 enzyme tuong ung la .\oil\a Spel k\- hieu la AtYUC6NotJ-F va At^TJCBSpel-R (Bang 1). San pham PCR nhan gene AtYUCCAS va vector tach dong pBluescript dugc cat dong thoi bang hai enzyme .\oil va Spel cua Hang BioLabs (https://www.neb.com). San pham cat sau do dupe tinh sach bang kit ciia Hang SolGenet (http://\\'\\'\v. solgenet.com) va ghep n6i bang
enz>-me T4 ligase. Phan ling ghep noi du«?c thuc hi^n a nhi&t dp phong ti'ong 12 giu voi thi tich 10 pi bao gom: DNA chen + DNA vector + 1 pi T4 DN \ ligase.
S ^ pham ghep noi sau do diroi- biln nap vao ie bao
£ cvb DH5a kha biln theo phuong phap soc nhi|t a 42 C, 30 giay. Te bao biln nap duoc nuoi qua d&m a 37"C tren moi tiirong LB (Luna-Bertani g6m lH g/l NaCl. 10 g/l trytone. 5 g/l yeast extinct. 15 g/l agai") bo sung khang sinh ampicillin 100 mg/i.
2.2.3. Chon loc ddng tdbio tai td hop T I bao tai to hgp duoc sang loc bang PCR vai cap moi dac hi&u tir cac khuan lac (colony) mpc ti-&n dia moi trucmg LB chiia khang sinh ampicillm 100 mg/l. Tilp din t l bao tai to hgp dupe nuoi tang sinh.
tach plasmid dl kiim tra cac doan chen bang enzyme cat hgn chi va giai ti-inh hr gene. Trinh tu gene sau do dugc kiim tra bang each so sanh vai trinh tu gene gdc ti-&n ngan hang gene NCBI bang cong cu BLAST. Trinh tu tach dong hoan hao dugc gan vao vector chuyin gene.
221. Gin vao vector chuyen gene
Wclor bieu hien pER8 dugc sii dung de chuyen gene AtYUCCAdvao cay ti'dng. Cau tnic gene pBS- pUB(J14::AtYUCCA6 va vector pER8 dugc cat ddng thoi bang enzyme AscI va Spel sau dd ghep ndi bai enzyme T4 DNA ligase. Phan ling ghep ndi tao cSu tnic pER8::pUBQ14-AtYUCCA6 dupe tien hanh tucmg tu nhu fa-inh bay a phin tach dong gene. San pham ghep ndi dugc chuyen vao t l bao vi khuan K cob DH5a va nuoi cay tren moi tiuong LB dac bd sung 50 mg/l kanamycin. T l bao tai td hgp dugc kiem tra bang PCR va enzyme cat gioi ban. Be chuyen vao cay trdng, plasmid tai td hgp dugc chuyen vao te bao vi khuan GV3101.
3. KFT QUA NGHHI CUU VA THAO LUAN
3.1. Phan tich gene AiYUC6tr€n ca sa dii li^u database
AtYUCCAd ma hoa cho protein nhom flavin monooxyenases co vai tro quan ti-png trong qua trinh tdng hgp auxin. Gene AtYUCCAS nam tren nhiim sac thi so 5. kich thuoc 3719 bp, c6 5 exon va 4 mti-o (JAIR. htti)s://seqviewer.arabidopsis.org). Gene bilu hien a t^l ca cac bg phan cua cay. Trong do manh nhat b ca quan sinh san nhu cum hoa. hat phain (Hinh 1).
NONG NGHIEP VA PHATTRIEN NONG THON - KY 1 - THANG 10/2019
KHOA HOC CONG NGHE
Hinh 1. Bilu h i | n ciia gene MYUCCA6b cSy Arabidopsis thahana 3.2. T^ch d6ng gene AtYUCCA6
Dua tr&n ket qua phan ti'ch da tiln hanh tach dong gene AtYUCCAS de ung dung cho chuyin gene. Bang phan ling trimg hgp chu6i (PCR) vol cap moi dac hieu AtYUC6NotI-F va AtYUC6SepI-R toan bg chieu dai 1,25 kb trinh tu gene ma hda (coding sequences-CDS) dup-c khulch dai ti-en khuon cDNA.
San pham PCR dugc tinh sach bang bg kit ciia Hang Solgenet (http://wTvw.solgenet.com) de su dung cho tach dong gene. Sir dung vector tach dong pBluescript (pBS) mang promoter UBQ14 da dugc chen san vao vi tri giira Asd va No&. Gene At)fUCCA6 Auac dua vao vi tri giiia hai enzyme No&
va Spel sau khi dupe cat va ndi bang enzyme T4 DNA ligase (Hmh 2A). Kit qua tach dong gene dupe kiem tra d- cac khuan lac moc tren moi trudng LB dac cd khang sinh chgn lpc Ampicihn 100 mg/l. Sau khoang 16 gid- nuoi cay o- nhiet do 37''C, tiln hanh kiem tra ngdu nhien 16 khuan lac bang ky thuat PCR tai chd voi cap mdi AtYUC6NotI-F va AtYUC6SpeI-R Kit qua dien di san pham PCR tren gel agarose 0,8%
cho thay tat ca deu cho kit qua duong tinh vai bang
Bang 1. Trinh tu mdi su dung trong nghi&n cuu
vach cd kich thudc khoang 2,25 kb tuang ling vdi kich thudc cua doan gene AtYUCCAS (Hinh 3A). De kiim tra chinh xac, da nuoi tang sinh khuan lac sd 8 va sd 13, tach DNA plasmid va cat bang enzyme giai ban Notlva SpeL Kit qua di&n di cho thay tr&n ban gel cd hai doan tuang img vdi kich thude khoang 4 kb va 1,25 kb day la kich thudc cua vector tach ddng pBS mang promoter UBQ14 va gene AtYUCCAS]<hi dupe cat boi Notl va Spel (Hinh 3B). Kit qua giai trinh hr gene vdi cap mdi M13 (-40), AtYUC6- R cho thay gene AtYUCCAS cd do tuong dong 100% vdi trinh tu gdc va trinh tu amino axit duoc bao toan.
Hinh 4 thi hien mpt phin kit qua tach ddng gene AtYUCCAS dugc kiim tra bang cong cu Blast tr&n ngan hang gene (NCBI) voi mdi ngupe M13 (-40) cung cap bdi Cong ty COSMOGENETECH. Trinh tu gene AtYUCtihit dau tir vi tri 381, xen giira vi tri tir 375 den 380 la trinh tu enzyme Spel (ACTAGT) noi giiia gene AtYUCCASva vector pBS (Hinh 4). Tir cac ket qua thu duge da khang dinh gene AtYUCCAO dugc tach ddng thanh cong, ky hi&u la pBS::pUBQ14- AtYUCCA6.
Ten ni6i AtYUC6-NF AtTOCU-SR PER8-R
Trinh tu (S'-Sl TTGGCGGCCGCATGGATTTCTGTTGGAAGAG TCAACTAGTTCAATTCCCACCACAATCACTCTC ACG TTG TCG AAA CCG ATG ATA CGG
KHO* HOC C 6 N C li«Vi<
(A| /-
pBluescnpt il SKC*)]
I 1 1 1 I M 1 (5) I 1 t
1
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.1 li
Hinh 2. Hinh ve c^u trtic vector nhdn ddng v i vector chuy&i gene AtYUCCAB (A)-vector nhan dong pBluescipt duoc cit mo vong vi chen do^ gene pUBQ14-AtYl!CCA6 t^i vi tri enzyme cat gioi h^ Ascl va Spel trong viing MCS
(B)-vector chuySn gene pERS duoc chen cau tnic pUBQ14-AtYUCCA6 cd gene chi Ihi chon loc kanamycin trong viing RB va LB.
Hinh 3. Kft qua i n t o tra khuSn l?c mang gene AtYUCCAShiiDg PCR v4 enzyme gi6i h ^ (Al-PCR khuan lac sii dung cfp moi M13 vii AtYUCCAS.
MOl ten chi doan khuich dai kich ±uac khoang 1,25 kb tuong ting vol kich thuoc mong mudn khi di^n di tren gel agarose 1%.
(B)- Khuan lac so 8 va 13 duoc kiem tra bing enzyme cit gioi han Notl va Spel cho kich thuoc luong ung voi gene .-Uii'CCAfi VI! vector tach dongpBS.
, \(im(^M , :.\jy'f-y;A,J[.iv{Miff)k^/]i
I I I I I I I I I M I I I I I i i l l l i 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 l l t l l l l l l l l l l l l l l l l l l l l l l
#yfn-"v
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 l l I I I L U l l J L I I I I I I I I I 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
• spel
Hinh 4. Kit qua giii trinh ti;r gene t ^ h dong AtYUCCAS vbi c ^ mdi ngugc M13 (40) Trinh tir nucleotid tivng hop mau do la vi tri enzyme cdt Spel ndi giOa gene AtYUCCAS vi vector pBluescript tuong ting.
3.3. G ^ vio vector chuyin gene
Sau khi tach ddng tiianh cong. gene AiWCCAO dugc dua sang vector chuyin gene pER8 kich thuoc 11,9 kb cd cac vi tri enzyme cat gidi han Asd va SpA.
cat ddng thdi pBS::pUBQ 14-AtYUCCA6 va pER8 vector voi enzyme .Asd va Spei va ghep ndi bang enzyme T4 DNA ligase theo s a dd md ta d hinh 2A va 2B. Phan ung ghep ndi duoc biln nap vao t l bao kha biln E. cob DH5a va nuoi cay trai tren dia thach cd
khang sinh chpn lpc kanamycin 50 mg/l. Kit qua kiim tra ngau nhien 8 khuan lac bang PCR su dung cap mdi AtYUC6-F va pER8-R cho thay: ca 8 khuan lac d&u cho kit qua duong tinh vdi kich thudc gene tirong ling khoang 1,25 kb (Hinh 5A). Khuan lac s6 1, 3 va 6 dugc nuoi t ^ g sinh va kiem tra bang enzyme cat Ascl va SpeL Kit qua di&n di san pham cat plasmid tir 3 khuan lac tr&n d&u thu dugc 2 bang co kich thuoc khoang 11,9 kb va 2,25 kb tuang ung voi kich thudc oia vector pERB va cau tnic \^ lVj]4- NONG NGHIEP VA PHATTRIEN NONG THON - KY 1 -THANG 10/2019
KHOA HOC CONG NGHE
AtYUCCA6 (Hinh 5B) chiing td gene AtYUCCAS dupe chen thanh cong vao vector chuyen gene pERB.
Ky hi|u lan lugt la pER8::pUBQ14-AtYUCCA6-l, 3 va 6. Dl danh gia kha nang bilu hien gene tren cay mo hinh Arabidopsis, da tiln hanh dua vector chuyen pER8::pUBQ14-AtYUCCA6-l vao chung vi khuan GV3101. Kit qua kilm tra ngau nhien 4 khuan lac tren moi tnrong khang sinh rifampicin, spectinomycin va genetamycin cho thay hau hit cac khuan lac diu duong tinh voi cap mdi pER8-R cho kich thudc gene khoang 1.25 kb tuong ling vcri gene AtYUCCAS (Hinh 5C). Tir cac kit qua fa-en chung to gene AtYUCCAS da dugc gdn vao vector chuyen gene va bien nap vao vi khuan san sang cho cac thi nghiem chuyen gene.
Mpt sd nghien cuu trudc day da tiln hanh danh gia kha nang chJu han cua cay chuyin gene .AtUICCAS cho kit qua rat kha quan. Kim et aL (2013) tao cay khoai tay chuyen gene sieu bieu hi&n AtYUCCAS Auoi su diiu khien cua promoter PT cho
kha nang chiu ban cao ban cay ddi chiing khoang 12 ngay [4]. Tuong tu Ke etal. (2015) [2] da sii dung vector PCAMBIA2300 mang promoter cam ung SWPA2 de tao cay hudng duong chuyen gen AtYUCCAti. K&t qua cho thay cay chuyin gene sinh trudng nhanh hon va chdng chiu vdi diiu kien ban hon cay ddi chiing khoang 6 ngay. Park et al. (2019) [15] tao cay dau tucmg chuyen gen AtYUCCAS su dung cau true vector pPZP-p35S::AtYUCCA6 cho thay cay chuyen gen cd kha nang chiu ban sau 14 ngay thieu nubc. Tir cac kit qua nghien ciiu tr&n cho thay gene AtYUCCASco kha nang ling dung cao di tao cay chuyin gene chiu han. Trong nghien ctm nay da thiet ke vector su dung promoter ca dinh Ubiqutin 14 diiu khiln gene AtYUCCA6 nham ling dung tao cay chuyen gene chiu hgn d- 'Viet Nam.
Promoter Ubiquitin cd vai trd tuong tu nhu promoter 35S hien dupe nhilu nha khoa hgc sir dung trong cac nghien ciru chuyen gen.
M 1 2 3 4
Hinh 5. Kit qua giai trinh kilm tra k i t qua thilt ke vector chuyin gen AtYUCCAS
(A)- Ket qua dien di san phdm PCR vcn cap mdlAtYUCS-F vi pERS-R trong E. coh. (B)- Ke't qua didn di sin pham cit bing enzyme gidi han Ascl va SpeL (CJ- Kdt qua didn di sin pham PCR vdi cap mdi AtYUCS-F va pER8-R tiong Agrobactenuni GV3101.
4 . KET LUAN
Gene AtYUCCASva duoc tach ddng thanh cong iii cay Arabidopsis tiiah'ana {CoXumh'xd).
Vector chuyen gene pER8::pUBQ14-AtYUCCA6 da dugc tiiiet k l thanh cong.
Dl phuc vu cho cong tac chuyen gene tren cay trdng chiing toi kiln nghi: can tnen khai tiep cac thi nghiem chuyen gen pUBQ14-AtYUCCA6 tr&n cay mo hinh va danh gia kha nang bilu hien cua gen duoe chuyen AtYUCCAS dudi su dilu khiln cua promoter pUBQ14 trong dilu kien ban.
TAI UEU THAM KHAO
1. Kepinski S va Leyser 0 (2005). The Arabidopsis F-box protein TIRl is an auxin receptor.
Natiire 435: 446-451.
2. Ke Q. Wang Z, Ji CY. Jeong JC, Lee HS, Li H, Xu B, Deng X, Kwak SS (2015). Transgenic poplar expressing Arabidopsis FZ7CC4i?exhibits auxin- overproduction phenotypes and increased tolerance to abiotic stress. Plant Physiol Biochem 94:19-27.
3. Kim, J. I., Sharkhuu, A , Jin, J. B., Li, P., Jeong, J. C , Baek, D., Lee, S. Y., Blakeslee, J. J., Murphy, A.
S., Bohner, H. J., etal (2007). YuccaS, a dominant mutation m Arabidopsis. affects auxin accumulation and auxin-related phenotypes. Plant Physiol 145, 722-735.
4. Kim. J. L Baek, D, Park, H. C, Chun, H. J, Oh, D. H, Lee, M. K, Cha, J. Y, Kim, W. Y, Kim, M.
C, Chung, W. S, Bohnert, H. J, Lee, S. Y. Bressan, R.
A, Lee, S. W.Yun. D. J. (2013). Overexpression of
KHOA HOC C 6 N C I
Increased endogenous .Arabidopsis thaliana causes I Arabidopsis }7't'(.46'in potato results in high-auxin
developmental phenotypes and enhanced resistance to water deficii Moi Plant.6(2):337-349.
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Sugawaraa. S.. Ftawaideb, H.. \':Hsunieb. M..
Hanadaa. A.. Yaenoa. T., Shirasua. K.. Yaod. H.. etal.
(2011). The main auxin biosynthesis pathway in Arabidopsis. Proc. Natl Acad. Sci. U S A. His. 18512- 18.517.
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evoluUoiiaiy and functional perspectives. Trends Plant Sci 10:170-177.
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6,437-439.
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Sandbcrg, G., and Hooykaas, P. J. J. (2003).
CLONING AND VECTOR CONSTRUCTION OF AtYUCCAS GENE FROM Arabidopsis thahana Ma Thi Thu Le, Truong Kim Oanh, Ta Phuong Thuy, Ngo Bao Oanh,
Nguyen Thi Tmh, Ngo Xuan Binh, Bui Tri Thuc, Nguyen Tien Dung Summary
In plant, auxin plays cntical roles in many aspects of plant growth anti cieveiopment. Aitliougii a number of auxin biosynthetic pathways have been identified. Recently AlYUCCAfJ gene belonging to the YUCCA family, encoding putative flavm monoox>'genase enzymes has been reported playing cnUca! roles of plant gtowtlt and development. Overexpressing .il] I CC.Uigene mcreases endogenous lAA levels response to environment stresses .is drought-tolerance. By PCR. we amplified coding sequence (1.25 kb) oi AiyUCCA6 gene from .Arabidopsis thahana and digested with Soft and SpA. AIYUCCA6 gene was then inserted in between .\\>!\ and SpA sites of pBluescript vector that cam'ing LTBQH promoter. Results of cloning were confirmed by enzyme di.gcsuon and sequencing. pliBQlt ..^iMJCCAG construct was cut by Asd and S/jd enz\'iiies and transferred to expression vector pERS at the same posiUon of those enzymes and confirmed by colony PCR and enzyme digesuon. Consequently. pER8-pUBQ14:AtYUCCA6 was transformed into .^grobaderium ti\'3101 sU-am for plant U-ansformation.
V^ynoTA^: Arabidopsis. .AlU iC.-\6. drought-tolerance, cloning, vector Ngtidi phan bifn: PGS.TS. NguySn Van D6ng
NgSynhJn bai: 9/8/2019 Ngiy thing qua phan bifn: 9/9/2019 Ng4y duyet dang: 16/9/2019
auxin production '"
both earlier descnbed aiidnovci auxin-related phenoDTKS. J. Plant tirowlh Regul. 22. 240-252.
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J and Zhao. Y (2011). Conversion of tryptophan to indole^-acetic acid by Tryptophan aminotransferases of .Arabidopsis and Yl'CC.As in Arabidopsis. Proc.
Natl .Acad. Sc-i. U S A 108,18518-18523.
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Conslihitively wilted 1, a member of the nce YUCCA gene family, is required for maintaining water homeostasis and an appropriate root to shoot ratio.
Plant Moi Biol. 65, 125-136.
13. Zliao. Y. (2012). Auxin biosynthesis: a simple two-step pathway converts tryptophan to indol^3- acetic acid in plants. Moi. Plant. 5,334-338.
14. Zhao, Y, Christensen. S. K.. Fankhauser. C . Cashman, J. R., Cohen, J. D., Weigel. D., and Choiy, J. (2001). A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science. 291,306-309.
15. Park, J. S. Kim, H. J, Cho, H. S. Jung, H. W, Cha, J. Y Yun, D. J, Oh, S. W, Chung. Y S. (2019) Overexpression of AtYUCCA6 in soybean crop results in reduced ROS production and increased drought tolerance. Plant Biotech Rep (13). 161-168.
14 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 -THANG 10/2019
