TAP CHI SINH HOC, 2013, 35(1)

TAP CHI SINH HOC, 2013, 35(1): 92-9

N G H I E N ClTU K H A N A N G T l T O N G T A C IN VIVO C U A P R O T E I N N L I - I F L I E N Q U A N D 6 N T I N H C H 6 N G C H f U S T R E S S 6 L U A

Nguyin Duy Phuong, Najaren Tutcja, Ph^m Xudn H$i*

Vifn Di truyin ndng nghifp, *xuanhoi.pham@gmail.com

TOM T A T : Trong nghien ciru sir dyng trinh ly DNA dich nlm trong trjl ty Idi cua 2 promoter cam ufng vdi dieu kifn han JRC0528 vd JRC0332, chiing toi da phan ldp dugc mpt gen ma hda protein NLI-IF (Nuclear LIM Interactor-Interacting Factor). Trong cong trinh ndy, chung tdi dua ra cac kit qud nghifin ciru dac diem tuang tdc vdi DNA va protein cua NLI-IF. Bang ky thudt lai phan tir trong te bao nlm men, chiing toi da chimg minh dugc kha nang lien kit d^c hifu vdi trinh ty DNA dich c6 trSt ty Idi CCTCCTCC ciia NLl-lF trong dilu kifn in vivo. Sang Ipc thu vifn cDNA xir Ii han, chung toi da phan lap dugc 8 done cDNA ma hoa cho cac protein tuang tac vdi NLI-IF. Cdc kit qua nghien ciru da cimg cd cho gia thuygt ciia chung toi: protein NLI-IF Id mpt nhan to phicn ma tham gia vdo qua trinh chong chiu stress d thyc v5t.

Tir khoa: Chju hgn, DNA dich, nhan td phien m§, NLI-IF, yeast one hybrid, yeast two hybrid.

MdDAu

Cac ylu td bit lgi cua mdi frudng hifn dang la nhOng thdch thirc Idn cho myc tieu duy tri sy phat trien ben vung trong san sudt luong thyc ciia loai ngudi. Han va mSn la hai trong sd nhung nlian td quan trgng nhat kim ham sy phat triln san suit ndng nghifp, ddc bift la san xult liia gao. Do dd, viec nghien cuu dac tinli cac gen dap img han va man cung nhu vifc danh gid kha nang dp dung cua chiing la mgt yeu clu hit sire cdp thiet. Cdch tiep cdn hudng nghien cim vl sfress thuc vat tren thi gidi hifn nay dang tap trung vdo viec phan lap va nghien cim dac tinli mgt tap hgp day du cac gen lien quan din bit lgi mdi trudng va mdi lien he giua cdc bit lgi mdn, han va nhift do. Hang tram gen dugc cam img trong cac dieu kien bat lgi khac nhau vd san phdm ciia cdc gen cam img vdi dilu kifn bit lgi dugc chia lam hai nhdm: nlidm cac protein chirc nang giup thuc vat chdng lai bit lgi ciia mdi trudng va nlidm cac protein dilu khiln lam nhiem vy dieu hda bilu hien gen vd truyin tin hifu trong qua trinh dap img dilu kifn bit lgi.

Cdc nlian td phien ma thugc nhdm thir hai vd la hg gen Idn [12]. Gdn day, rIt nhilu ngliien cim ve nhan td phien ma dugc thyc hifn fren cay md hinh Arabidopsis va cac loai thyc vat kliac da chirng minh vai frd quan frgng ciia chiing trong qua trinh dilu hda phan img ciia thuc vat trong cdc dilu kifn bit lgi mdi trudng.

Ddc diem ddc tnmg cua cdc protein dilu khien (nhan td phien md) Id cd hai vimg ho^t dgng (domain): vimg ho^t hod cdc protein chirc ndng (activation domain) vd vimg Uen kit (binding domain) vdi cdc trdt tu DNA ddc hifu (cis-acting element) tren viing dilu khiln cua gen (promoter). Dya vao dac tinh bam DNA, ky thuat sdng Igc phep lai don trong t l bdo nlm men dugc hinh thdnh d l phdn lap cdc nhdn tl phien ma. Nhdn td phien md dlu tien (OLF-1) dugc phan lap blng ky thuat sang Igc phep lai don trong te bao nam men [16] vd ngay l^ip tire trd thdnh phuong phdp day tiem nang trong vifc phan lap cac gen ma hda cdc protein cd kha nang bam DNA [19].

Gin ddy, sir dyng ky thuat sang Igc phep lai don trong tl bdo n l m men vdi frinh tu dich co chieu dai 50 nucleotide chira trdt ty DRE fren viing khdi dgng gen JRC2606 chiing tdi da phan lap dugc gen md hda nhan td phien ma OsRap2.4B tu gidng lua Mdc tuyIn [10].

OsRap2.4B cd klia ndng lien kit ddc hifu vdi trinh ty dich JRC2606 trong cd 2 nghien ciiu in VIVO va in vitro. Cdy md hinh dugc chuyen gen OsRap2.4B bilu hifn khd ndng chdng chju vdi stress cao hon so vdi ddi chimg (sd lifu chua cdng bd). Mgt nghien ciiu sau dd, sir dyng frinh ty DNA JRC0528 vd JRC0332 dh sang Igc thu vifn cDNA xir Ii stress, chiing tdi da phan lap dugc mgt gen ma hda protein NLI-IF 1 (Nuclear LIM Interactor-hiteracting Factor) [9]. Trong nghien cihi nay, chiing tdi tilp tyc sir dyng phep^

1

Nguyen Duy Phuong. Najaren Tuteja, Pham Xuan Hoi lai phdn tii frong t l bao nlm men d l chimg

minh kha ndng lien kit d | c hifu DNA, ddng thdi xdc dinh tuong tac protein-protein cua NLI- IF frong dieu kien in vivo.

VAT Lifu vA PHU^ONG PHAP NGHI£:N ciru V | t lieu

Trinh ty ma hda nhdn t l phien md NLI-IF da dugc tdch ddng gift trong vector pGEMT vd cdc vector bilu hifn frong tl bdo nlm men pAD-GAL4, pGBKT7 do phdng Bfnh hgc Phdn tir, Vifn Di truyen ndng nghifp cung d p .

Chimg nlm men AH109, YM4271 vd thu vifn cDNA xir Ii sfress so d p diing trong kT thu§t sang Igc phan tir (Yeast one hybrid/ Yeast two hybrid) dugc cung d p bdi Phdng thi nghifm Sinh hgc phdn tu thyc v§t thugc Trung tam Cdng nghf sinh hgc vd KJ thuat di truyin Qudc te (International Cenfre of Genetic Engineenng DNA Biotechnology), New Delhi, AnDg.

Phuomg phap

Thiel ke vector bieu hi^n pAD-GAL4/NLl-IF vd pGBKT7/NLI-IF

Vector tai td hgp pGEMT/NLI-IF vd vector bieu hifn trong ndm men (pAD-GAL4 vd pGBKT7) dugc xir ly ddng thdi vdi enzyme cIt gidi ban (EcoRI/XhoI vd Pstl/Ncol). Trinh ty ma hda NLI-IF dugc ghep ndi vao vector bieu hifn nhd enzyme T4 Ligase.

Bien nap plasmid tdi to hop vdo te bdo ndm men Vector tai td hgp mang trinh ty dich vd gen dich dugc ddng biln nap (co-transformation) vdo tl bdo nlm men YM4271, su dyng LiAc [19] va dugc chgn Igc fren mdi trudng SD khuylt duong axit amin SD-His/-Leu/-Ura (ddi vdi ky thudt sdng Igc phep lai don-YlH) hay SD-His/-Leu/-Trip (ddi vdi ky thudt sang Igc phep lai kep-Y2H).

Phep lai dom trong te bdo ndm men (Yeast one hybrid assay)

Kha ndng tuong tac DNA ciia NLI-IF dugc danh gid dya fren mire dg bilu hifn cua gen thdng bao HIS khi nudi cly thi biln nap fren mdi trudng SD chgn Igc khuylt dudng axit amin cd bd sung chit ire chl canh franh 3-AT

30 mM (SD -His/-Leu/-Ura + 3-AT 30 mM) vd mire dg bilu hifn ciia gen thdng bdo lacZ tren mdi trudng cd bd sung co chit X-Gal 20 mg/ml nhu dd md td trong nghien cim trudc [9].

Cdc vector pHis-Si/pLacZ mang 2 trinh ty DNA ldp 191 cua trinh ty Idi JRC0528 vd JRC0332 (WT) trong viing promoter dd sir dyng dl phdn l^p gen NLI-IF [9] dugc tilp tyc sir dyng trong nghien cim ndy. Ngodi ra, hai trinh ty DNA dich dOt biln dd thay ddi ylu td cis- acting bdi do§in trinh ty AAAAAAAA (MU) dugc sir dyng dl chirng minh khd ndng lien kit DNA d^c hifu cua protein NLI-IF. Vector pAD- GAL4 (khdng mang gen) dugc sir dyng lam ddi chimg dm trong nghien cim ndy.

Phep lai kep trong le bdo ndm men (Yeast two hybrid assay)

10'-10^ te bao ndm men tdi td hgp mang vector pGBKT7/NLI-IF dugc sang Igc theo quy trinh ciia nha san xuat [20], sir dyng thu vifn cDNA thir d p (-10* pfii/ml) dugc tao tir thu vifn cDNA so d p xir ly sfress ciia liia PBl. 33 thi ddng biln nap (mang vector pGBKT7/NLI- IF vd pAD-GAL4-cDNA) sinh trudng manh nhdt dugc chgn Igc tu 537 khudn lac duong tinh xudt hifn fren mdi trudng SD-His/-Leu/-Trip dugc sdng Igc lan hai fren mdi trudng SD-His/- Leu/-Trip/-Ade. Su dyng phuang phap ddnh gia boat tinh ciia P-Galactosidase de sang Igc 27 the biln nap sinh trudng tren mdi frudng sdng Igc thir d p , 19 the bien nap cd bieu hifn ciia gen thdng bdo lacZ. Sir dung ky thudt PCR de sang Igc kich thudc 19 ddng cDNA thu dugc, 8 ddng cDNA cd kich thudc >500 bp dugc chgn Igc, nlidn ddng vdo vector nhdn ddng pGEMT va gidi trinh ty nhu da md td trong nghien cim trudc day [9].

Ktl QUA VA T H A O LUAN

Thilt ke vector bieu hifn trong nam men mang trinh t y md hda NIL-IF

Khi phdn l^p frinh tu ma hda NLI-IF cd kich thudc 1320 bp tu thu vifn cDNA, chiing tdi da sir dyng cdp mdi dugc thiet ke mang 2 vj tri nhdn bilt cua EcoRl va Xhol [9]. Do dd, d l dua trinh ty gen NLI-IF vdo vector bilu hifn pAD- GAL4 vd pBGKT7, chung tdi da xir ly ding thdi cdc vector pGEMT/NLI-IF vd pAD-GAL4

TAP CHf SINH HQC. 2013,35(1): 92-98 vdi EcoRVXhol (hinh lA), pGEMT/NLI-IF vd ciia vector bilu hifn blng e n ^ m e T4 ligase vk pGBKT7 vdi Pstl/NcoI (hinli 2A). Sau khi tinh biln n?p vdo t l bdo khd biln E. coli chung sach sdn phim cdt gidi h ^ tfr gel agarose blng DH5a. Cdc khuIn l^c duong titvh dugc chimg bO kit Gel Exfraction Kit (Fermentas), frinh ty tdi sdng Igc blng PCR vdi c$p mdi d$c hifu ciia md hda NLI-IF dugc ch6n vdo vj fri da dilm cdt NLI-IF.

m m t '

Hinh 1 Ket qua gh^p ndi trinh ty ma hda NLI-IF vdo pAD-GAL4 A. Kit qud difn di sdn phim sdn phim clt gidi h ^ pGEMT/NLI-IF (gilng 4 va 5) vd pAD-GAL4 (gilng 2 vd 3) bang EcoRVXlwl, gilng 2 vd 4: vector nguyen ban, gieng 3 vd 5: sdn phim clt gidi h?ui; B. Kit qud difn di san phdm PCR kilm tra vector pAD-GAL4/NLI-IF, sir dyng c§p mdi ddc hifu ciia NLI-IF (giing 2 ya 4) vd cap mli AD-Fw/AD-Rv (gilng 3 va 5), gilng 2 va 3: khudn Id pAD-GAL4/NLI-IF, gilng 4 va 5: dii chimg dm (khdng cd DNA khudn); C. Kit qud cat gidi han kilm tra pAD-GAL4/NLI-IF blng EcoRVXhol.

Ket qud kiem tra plasmid tinh sach tu cdc thi biln nap duong tinh blng PCR vdi cdp mdi ddc hifu vector (AD-Fw/AD-Rv ddi vdi vector pAD-GAL4 vd BD-Fw/BD-Rv ddi vdi vector pGBKT7) va c | p mdi ddc hifu gen NLI- Fw/NLI-Rv chiing tdi da thu dugc sdn pham cd kich thudc tuong img lln lugt la 1,5 kb (hinh IB-gieng 3 vd hinh 2B-gieng 2) vd 1,3 kb (hinh IB-gieng 2 va hinh 2B-gieng 5) diing vdi kich thudc tinh toan Ii thuylt. Kiem fra plasmid tai td hgp pAD-GAL4/NLI-IF blng phan &ng clt gidi

han vdi EcoRVXhol, chimg tdi thu dugc 2 bdng DNA cd kich thudc 1,3 kb (tuong ling vdi trinh tu md hda NLI-IF) vd 7,6 kb (tuong dng vdi khung vector pAD-GAL4) (hinh IC, gilng 1).

Kit qua tuong ty thu dugc khi kilm fra plasmid tdi td hgp pGBKT7/NLI-IF blng phan ting clt gidi h?m vdi Ncol (hinh 2C-gilng 2) vd Pstl/NcoI (hinh 2C-gilng 3). Cdc kit qua ndy chiing td chiing tdi da gdn thanh cdng trinh ty md hda nhdn t6 phien md NLI-IF vdo vector bilu hifn pAD-GAL4 vd pGBKT?.

I 2 J 4

Hinh 2 Ket qud ghep ndi trinh ty ma hda NLl-lF vdo pGBKT7

A. Kit qud difn di sdn phim sdn phim clt gidi h?n pGEMT/NLI-IF (gilng I vd 2) vd pGBKT7 (gilng 4 vd 5) bang Pstl/NcoI, gilng 1 vd 5: vector nguyen bdn, gilng 2 va 4: sdn phim clt gidi h^n; B. Kit qud difn di sdn phim PCR kilm tra vector pGBKT7/NLI-IF, sir dyng cap mli ddc hifu ciia gen (gilng 2 vd 3) va cap mli vector (gilng 4 vd 5), gilng 2 va 4: khuon la pGBKT7/NLI-IF, gilng 3 vd 4: dii chimg dm (khdng co DNA khu6n); C. Kit qud clt gidi h?ji kilm tra pGBKT7/NLI-IF blng Pstl/Ncol (gilng 4) vd Ncol (gilng 3).

Nguyen Duy Phuong. Najaren Tuteja. Pham Xuan Hoi Nghien cihi khd n9ng lien k i t DNA in vivo

trong t l b&o n l m men ciia NLI-IF Trong mdt nghiSn cihi trudc, chung tdi dd phdn l§p dugc gen md hda nhdn t l phien md NLI-IF bdng ky thudt sang Igc phep lai don frong t l bdo nlm men, sir dyng 2 frinh ty dich 50 nucleotide Id trinh ty Idi ciia 2 promoter JRC0332 vd JRC0528, cd cung mOt ygu t6 cis- acting ndm frong viing boat hda (hijp TATA) cd frinh ty CCTCCTCC [9]. Trong nghien ciru ndy, chung tdi tilp tyc su dyng ky thuf t lai phdn tir

in vivo trong tl bdo nlm men d l xdc djnh khd ndng lien kit d$c hifu in vivo cua NLI-IF vdi trinh ty dich. Bk thyc hifn myc dich ndy, chiing tdi dd biln ngp plasmid dung hgp pAD-GAL4/NLI-IF vdo chung tl bdo nlm men tdi td hgp cd hai gen thdng bdo (HIS vd lacZ) da dugc dung hgp vdi m0t promoter chira 2 trinh ty l$p l^i cua trinh ty DNA dich (JRC0332 ho^c JRC 0528) dd bj thay thi ylu td cw-acting CCTCCTCC blng AAAAAAAA (hinh 3A).

A JRC0332 - WT. CGAAAACGGAACOCtCCCCCCCTCCTCCCCTCTCCACGTCACTGCGCGGT JRC0332 - MU- CGAAAACGG.^ACGCCCCCCCAAAAAAAACCTCTCCACGTC.^CTGCGCGGT JRC0328 - WT: GTGGCCTCGCCTCCTCCTCTTCCTCCACTCCACCACCCACCCGCCCGG JRC0332 - \ t U : GTGGCCTCGAAAAAAAATCTTCCTCCACTCCACCACCCACCCGCCCGG

Hinh 3 • Ket qud ddnh gid bieu hien cua gen thdng bao trong te bao ndm men tai td hgp A. Trinh ty DNA dich nguyen bdn (WT) va trinh ty DNA dich dpt biln (MU) da thay trinh ty I6i CCTCCTCC blng poly-A; B Nudi cay tren mdi trudng YPD; C. Nuoi cly tren moi trudng SD-His/-Leu/- Ura; D. Nudi cly tren mdi trudng SD-His/-Leu/-Ura co bl sung 3-AT 10 mM; E. Danh gia hoat tinh P- galactosidase; 1. Ddi chimg am (YM427I + pHis/pLac/pAD-GAL4); 2. JRC0332-MU; 3. JRC0528-MU; 4.

JRC0528-WT; 5. JRC0332-WT; 6. Dli chiing duang.

Cdc te bdo nam men tdi td hgp cd the sinh frudng binh thudng fren mdi frudng thilu histidine (hmh 3B), tuy nhien, khi cd bd sung chdt ire chl canh tranh 3-AT ndng dg 10 mM vdo mdi trudng chgn Igc, chi cd cdc t l bao mang gen thdng bao dugc dung hgp vdi trinh ty dich nguyen bdn (WT) mdi cd khd ndng sinh trudng buih thudng (hinh 3D). Ngodi ra, hoat tmh P-galactosidase cung chi bilu hifn d nhijmg tl bao nlm men mang trinh ty dich nguyen bdn (hinh 3E). Kit qud nghifin cim in vivo ndy chimg td NLI-IF lien kit ddc hieu vdi ylu td c/s-acting CCTCCTCC n l m frong trinh ty dich ciia 2 promoter JRC0332 va JRC0528, khdng lien kit vdi frmh ty poly-A hay dlu 5' vd 3 ' cua trinh ty dich.

Sing Igc protein tuong tdc vdi NLI-IF ti^ t h u vifn cDNA xir Ii stress

Chung tdi sir dyng phuong phdp sang Igc thu vifn cDNA blng phep lai phdn tu trong t l bdo nlm men (yeast two hybrid) dl xdc djnh protein tuong tdc vdi NLI-IF. Chiing tdi dd bien nap plasmid tdi td hgp pGBKT7 mang gen NLI- IF vdo te bdo nam men AH 109 vd sir dyng chiing nam men tdi td hgp ndy de sang Igc thu vifn cDNA. Sau khi sdng Igc thu vifn cDNA, chiing tdi dd thu dugc 8 ddng te bdo nlm men tdi td hgp mang cdc doan cDNA cd kich thudc

> 500 bp, cd khd nang sinh trudng binh thudng fren mdi trudng chgn Igc khuyet dudng axit amin (SD -His/-Trip/-Leu/-Ade) vd cd bieu hifn boat tinh P-galactosidase (hinh 4). Cdc ddng cDNA ndy dugc chiing tdi nhdn ddng vdo vector pGEMT de gidi trinh ty vd so sdnh vdi cdc trinh ty dd cdng bd tren GeneBank (bang 1).

TAP CHI SINH HOC, 2013, 35(1): 92-9 Bdng I Cac ddng cDNA ma hda protein tuong tac vdi NLI-IF dugc phdn lap blng ky thuat Y2H

Ddng cDNA C201 C239 C246 C265 C284 C314 C370 C410

Protein dugc ma hda Polyubiquilin

Protein kep chiia domain pho.sphatase ddc hifu cho protein Piolcin hen kll kem, thugc nhdm Alcohol dehydrogenase Protein chua dugc cong bd

Prolem lien kit chlorophyll a/b nhdm 1, tien chit cua chloroplast Polyubiquilin

Cyclophilm 2

U5 small nuclear ribonucleoprotein 200 kDa helicase

Hinh 4 Ket qua sang Igc protein tuong tac vdi NLI-IF tu thu vien cDNA A Sang Ipc tren moi trudng SD-His/-Leu/-Trip co bl sung 3-AT 10 mM; B. Sang Ipc Uen moi trudng SD- His/-Leu/-Trip/-Ade co bl sung 3-AT 10 mM; C. Danh gia ho?it tinh p-galactosidase; D. Sang Ipc blng PCR vdi cap mli AD-Fw/AD-Rv.

Ddc bift, ket qua thu dugc cho thdy, NLI-IF cd tuong tdc vdi protein ubiquitin, mgt protein cd vai frd tham gia vdo qud trinh phien ma, sira

chOa DNA vd dap img stress. Kit qua nay cimg TAI Ll$U THAMKHAO cd them cho gia thuylt ciia chirng tdi vl khd

ndng NLI-IF la mgt nhdn td phien ma cd tham gia vao dap img stress d thyc vat.

protein NLI-IF Id mdt nhan td phien md tham gia vao qud trinh chdng chju sfress cua thyc vat

KET LUAN

Bdng ky thuat lai phan tir trong tl bdo nlm men, chiing tdi da chimg minh kha ndng lien kit ddc hifu vdi trinh ty DNA dich co chira ylu td c/5-acting CCTCCTCC ciia NLI-IF. Chung tdi ciing da phan lap dugc 8 ddng cDNA tir thu vien cDNA xir ly han ma hda cho cac protein cd tuong tac vdi NLI-IF. Cdc kit qua thu dugc cung cd them cho gia thuylt ciia chiing tdi vl

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TAP CHi SINH HQC, 2013, 35(1): 92-98

E X P R E S S I O N A N D F U N C T I O N A L A N A L Y S I S O F N O V E L S T R E S S - I N V O L V E D T R A N S C R I P T I O N F A C T O R N L I - I F IN R I C E

Nguyen Duy Phuong, Najaren Tuteja, Pham Xuan Hoi Agricultural Genetic Institute SUMMARY

We reported previously that one transcription factor NLI-IF (Nuclear LIM inlcraclor-interacting factor)- encodcd cDNA clone was identified by yeast one hybrid assay, using 2 target sequences JRC0528 and JRC0332 In this paper we reported some results of in vivo analysis of NLI-IF. The core sequence-specific DNA binding ability of NLI-IF was identified, using yeast one hybrid assay. To identify of protein-protein interaction, the rice sUess-lreated cDNA library was screened using yeast two hybrid assay and 8 cDNA clones were isolated. These results support to our expectation that transcription factor NLI-IF is involved in stress responses in plant.

Keywords: DNA target sequence, Drought tolerance, NLI-IF, transcription factor, yeast one hybrid, yeast two hybrid.

Ngdy nhgn bdi: 9-5-2012