Nguyen Vii Thanh Thanh vd Dtg Tap chi KHOA HOC & CONG NGHS 107(07): 6 3 - 6 8
TACH DONG GEN ZmNACl TlT MAU NGO DIA PHl/ONG VI XUYEN -HA GIANG, V I £ T NAM
Nguyen Vii T h a n h Thanh , Nguyen Van Tufrng , Pham Thanh T i l n g \ Lg Van Sffn^ Chu Ho^ng Mau^
' Trudng Dgi hoc Khoa hgc- DH Thdi Nguyen; ^Vien Cong nghe sinh hpc Truong Dai hoc Su phgm - DH Thai Nguyen T O M T A T
NhSn t l phien ma NAC (bat nguin tir ba chiJ NAM-no apical meristem, ATAF-Arabidopsis transcription activation factor, CUC-cup-shaped cotyledon) 1^ m^t hp protein c6 chirc nSng r^t da dgng, giU vai trd quan trong trong vifc dieu h6a sy sinh trudng v^ ph^t triln ciia thuc v§t, dilu chinh noi tilt to va phan irng vdi nhOng ^p lyc kh^c nhau, qud trinh ISo h6a, phdt triln hinh thdi, con dudng truyen tin hieu va c6 thi d6ng vai tr6 quan trpng trong con dudng khdng cdc ylu t l sinh hpc vd phi sinh hpc. Trong nghign ciru ndy chung toi dd thilt kl cdp mii NACZm-F/NACZm-R, khuech dai, tdch dong thanh c6ng v^ xdc dinh trinh tu nucleotide ciia gen ZmNACl tir DNA he gen mau ngo dja phuang VX (Hd Giang). Gen ZmNACl co kich thudc Id 1035 bp, viing md hod gim 939 bp ma hod 312 amino acid. Gen ZmNACl c6 2 exon va 1 intron; exon 1 co kfch thudc 471 bp, exon 2 - 468 bp, intron co 96 bp. Gen ZmNACl ciia miu ng6 VX xuSt hifn 6 vi tri nucleotide thilu hut d viing intron (487, 556, 557, 558, 559, 560) vd protein suy diln c6 vj tri amino acid 181 sai khac so vdi protein NACI tren GenBank (md s6 ACF33136). Trinh tu gen ZmNACl thu dupe la ca sd de tiep tuc nghien ciru ddc diem ciia gen ZmNACl va mii lien quan giita su thay doi trong cau tnic gen ZmNACl vdi dac tinh chiu han d cay ngo.
Tukhoa: chiu han. genNACl. ngd dia phuong. nhdn td phiin ma, tdch ddng.
M O D A U
Stress phi sinh hpc n h u hgn han va do man cao anh h u d n g x a u den su sinh t r u d n g , phat trien va n a n g suat ciia t h y c vat. Trong nhirng nam gan day, nhieu tien bd da d u a c t h y c hien theo h u d n g x i e dinh tiem nang cac gen lien quan den kha nang tang s u e chju d u n g cua thyc vat doi vdi cac stress phi sinh hoc tii' mdi trudng. N h a n td phien ma NAC la mdt hp protein cd chirc n a n g rat da dang, giif vai trd quan trgng trong viec dieu hda sy sinh t r u d n g va phat trien c u a t h y c vat, q u a trinh lao hda, phat trien hinh thai, con d u d n g truyen tin hieu, dieu chinh npi tiet td va phan irng vdi nhifng tac ddng khac nhau til' ngoai canh [2], [4], [5]. N h a n td phien ma NAC cd mdt logt cac chuc nang quan trpng khdng chi trong su phat trien t h y c vat ma cdn trong phan u n g vdi cac stress phi sinh hpc. Gen NAC da d u o c chiing minh cd lien quan den tinh chdng chiu stress phi sinh hoc [6]. K i t q u a nghien ciru ciia Liu va dtg (2009) c u n g chi ra rang gen
Tel- 0912664126
NACI cd vai trd quan trgng trong phan ung vdi cac stress tii' ngogi canh, tang c u d n g kha nang chong chju cac yeu td bat lai sinh hpc va phi sinh hgc [4]. Nghien ciiu ciia Lu va dtg (2012) cho ket qua gen ZmNAC d u p e cam irng manh me bdi nhiet do thap, dp man cao, khd han, va axit abscisic ( A B A ) [5]. Cac nghien cuu vai trd ciia gen NAC cdn xem xet d khia canh nhan va truyen tin hieu cac yeu to mdi tru'dng de t h u c hien chuc nang boat hda qua trinh phien m a [1]. N h u n g ket qua nghien ciru ciia cac tac gia cung da chi ra rang protein N A C I cd chiJrc nang hogt hoa qua trinh phien ma ciia nhdm gen chiu han trong mdi t r u d n g thieu n u d e cue doan va cd the dng dyng nham cai thifn tinh chiu han ciia thyc vat va cay ngd b a n g ky thuat chuyen gen [5], [6]. Cac thao lugn ve vai tro va muc tieu sii dung cac protein N A C trong chien l u a c cai tien gidng cay trong thdng qua su can thiep cua cdng nghe sinh hpc dang la van de mang tinh thdi sy [8], [10]. C o hai xu h u d n g tim kiem gen lien quan den tfnh chdng chju cua cay trdng ddi vdi cac yeu td ngoai canh bat lai
63
Nguyen Vu Thanh Thanh vd Dtg
Tgp chi KHOA HQC & CONG NGH$
107(07): 6 3 - 6 8phi sinh hpc ( I ) gen lien quan tryc tiep den tinh chju han, chju nhift, chju m(ln va (2) gen tone hpp san pham protein cd vai trd dieu khien qua trinh phien ma. Gen NAC la hp gen trong nhdm gen dieu ho^ hogt dgng phien mS cua nhdm gen chong chju. Trong nghien ciiu n^y chimg tdi trinh b^y kcl qu^ t^ch ddng vk phan tich trinh t y gen ZmNACl phan Igp tir mau ngd dja phuang V j Xuyen - Ha Giang, Viet Nam.
V A T L I E U V A P H U O N G P H A P N G H I E N C U U
Vat lieu
Mau ngd dia phuong V j Xuyen, Ha Giang ( V X ) do nhdm nghien ciru suu tap tgi V j Xuyen - Ha Giang, cd hgt trSng nga, hgt d?t va dai, khoi lupng 100 hat la 29,56 ± 0,26 (g), ham lupng protein 10.05 ± 0,045 (%>), ham lupng lipid la 4,39 ± 0,062 (%).
Phuo'ng phap
Tach chict D N A tdng sd tir la ngd trong d f m 2% C T A B , lOOmM Tris H C l , 1,4M NaCl, 20mM E D T A , 2 % PVP, 0 , 0 1 % 2- mercaptoethanol. Gen ZmNACl dugc khuech dai bang PCR vdi cap mdi NACZm- F/NACZm-R dugc thiet ke dua tren trinh t y gen duac cdng bd tren GenBank cd ma sd EU8I0024 [3]. Cap moi nhan gen ZmNACl cd trinh tir nucleotide la:
NACZm-F:
5'GGGATCCATGGGTCTGCCGATGAGGA3'
- llOObp -lOOObp
- SOObp
NACZm-R:
5 ' G A G C T C T C A G A A C T G T C C C A A C C C G 3 ' Thanh phan phan iirng PCR nhan gen ZmNAC! trong the tich 25^il, gom: 12,5^1 GoTaq® Green Master M i x 2 X ; l.Ofil NACZm-F ( l O p M / n O , 1,0 NACZm-R ( l O p M / n l ) ; 1.0^l D N A tong so; 9,5^1 H2O.
Tach ddng gen duprc thyc h i f n theo phuang phap ciia Sambrook va Russell (2001) [9].
Trinh t y gen ZmNACl dupe xac djnh tren thiet bj giai trinh t y nucleotide t y dpng ABI P R I S M ( ^ 3 1 0 0 Genetic Analyzer (Apphed Biosystem) tgi V i f n cdng n g h f sinh hpc. Ket qua dpc trinh t y dupe x u ly bang phan mIm BioEdit.
K E T Q U A V A T H A O L U A N
T a c h d d n g gen ZmNACl tir D N A he gen cay ngo
La ngd non dupe su d y n g de tach chiet DNA tong so. K i e m tra chat lupmg tach chiet DNA bang phucmg phap dipn di tren gel agarose 0,8% va xac djnh ham lupng D N A tren may quang phd d budc sdng 260nm. Ket qua cho thay D N A tdng sd tach chiet dugfc cd ham lugng va dp tinh sach dam bao va cd the sir dung cho cac thi nghiem phan tich D N A tiep theo. Sir dung D N A tdng sd de nhan gen ZmNACl bang phan irng PCR v d i cap moi dac hieu NACZm-FX.4CZm-R. k i t qua thu dupe dogn D N A cd kich thudc khoang l l O O b p ( H i n h I A ) .
_1300bp
Hinh 1. Hinh dnh diin di sdn phdm PCR nhdn gen ZmNACl (A) vd sdn phdm colony~PCR chgn d khudntdi to hop
M- Thang DNA Ikb; VX: Dogn DNA nhdn bdn tir DNA he gen cda mdu ngd VX,
!. 2. 3, 4. 5, 6. Cdc ddng khudn lac chiia vector tdi id hop
64
Nguyin Vii Thanh Thanh va Dtg Tap chi KHOA HQC & C 6 N G N G H E 107(07): 63 - 68
E D a i O 0 2 4 a i G G G T C T G C C e a i G S G G S G G G B G A G G G a C G C G G S G G C G G A G C T G A f . C d S C C G C C G G G G T T C C G G I T C C
E D B 1 O 0 2 4 A C C C C J l C C G A C G A C G A G C I G G T G G a G C A C T A C C I S T G C C G C A A G G C G G C G G G G C A G C G a r r C C C C G I G C C
E U S 1 0 0 2 4 C A T C a T C G C C G A G G T G G A C C T G T A C A G G T T C G A C C C C T G G G A C C T G C C G G A G C G C G C G C T C l I C G G G G C C
E n 8 1 D 0 3 4 C G C G A G T G G T f t c m C T T C R C G C C C A G G G A C C G C A A G T f i C C C C A A C G G C T C C C G C C C T A A C C G C S C C G C C G
E U 8 1 0 0 Z 4 G C A A C G G e T A C T G G A A G G C C A C C G G C e C C G A C A A G C C C G T C G C G C C G C G C G e C C G C A C e d r T d s G A i a ^ A
B U 8 1 0 0 Z * G A S G G C G C I T G T C T T C T A C G C C G G C A J i G G C G C C G C G C G G G G T C A A G ; , C 3 E ; . : : T a 3 J
E O S 1 0 a 2 4 A G G C T C G C C G R C G C T S G C C G C G C C G C G GCCGCCAAG A ; , G G G G T C G C T T n G G G T AAGC (
. 0 0 2 4 f J ^ . T C G T C G I G C r t G T I G G . ^ T G £ C T G S G T G C T G T S C C G G C T G T A C a i . c a A G A A G A A C G . ? . G T G G G A G A A G A T G
B D B 1 0 0 2 4 CAGCTGGGG A A E A t t C G C C G T C G C C G G C G I CGGCGCCACCJiAGGAGGAGGCGATGG;iC A T G G C C A C C I CGC
E n s i 0 a 2 4 A C A C G C A C T C C C A C I C C C A A I C A C A C I C G C A C X C G T S S G G C G A G A C G C G C A C G C C A G A G T C G G A G A T C G T
E U e i 0 0 2 4 G G . ^ C ^ J . C G A C C C G T T C C C G G A G C I G G A C I C G I I C C C G G C G n C C A G G A C C C G G C G A I G A T G A T G A C G G T G
,GGAGCA> GG A C G G C l GC AGCGCCA^' GAGCGGC A A C C T G T T C G I G G A C C I C A G C T ACGACG
B D B 1 0 0 2 4 A C A I C C f ^ G G C A T G T A C A G C G G C C i a i f t C S T G C T G C C G C C G C C C G G G G A G G a C I T C T A C T C C T C G C T C T T
E D 8 1 Q 0 2 4 C G C G T C T C C C f t G G G T C A A G G G G A A C C A G C C C G C C G G A G C C G C C G G G I T G G G A C A G T I C I G A
Hinh 2. So sdnh trinh lugen ZmNACl
(EU810024. Trinh tu gen ZmNACl tren GenBank mdsSEU810024. VX Trinh tu gen ZmNAC 1 phdn Igp tu DNA he gen mdu ngd dia phuang Vi Xuyin-Hd Giang)
Nguyin VQ Thanh Thanh vd Dtg Tgp ch( KHOA HpC & CONG NGHE 107(07): 63 - 68
JiC F-3 3 1 . 3 6 -
A c r 3 3 1 3 6 -
iKi^KKtKrl. j k x S v
. c r 3 3 a . 3 6 - MUP.
Hinh 3. Trinh tir protein NACI {ACF33I36- Trinh lu amino acid ciia protein NACI tren GenBank mdsd ACF33136. NAC I-IX Trinh tu amino acid cua protein NAC I suy diin tir gen ZmNACl
phdn Idp mdu ngd dia phirang Vj Xuyen-Hd Giang) San pham PCR dupe lam sgch bang ky thuat
thoi gel (theo KIT GeneJET Gel Extraction ciia hang Thermo) va dupe gan vao vector tach ddng pBT tao vector tai td hpp. Vector tai td hpfp mang gen ZmNACl dupe bien ngp vao vi khuan E.coli DH5a va de khang dinh chac chan dogn gen ZmNACl cd mat trong cac ddng khuan lac mau trang thu dugc, chimg tdi tien hanh phan irng PCR tryc tiep tir khuan Igc (colony-PCR) vdi cgp moi Ml3.
San pham colony-PCR true tiep tir khuan lac dugc dien di tren gel agarose 0,8%). ket qua cho thay ca 6 ddng khuan lac deu cho kk qua duang tinh vdi phan ung PCR va kich thirdc nhan len theo diing tinh toan ly thu\cl doan gen dai khoang i300bp (Hinh IB). Cae ddng khuan lac sau khi dupe chgn Igc bang phuong phap PCR tryc tiep tir khuan Igc, dugc nudi trong^ mdi tirong LB Idng de tach chiet plasmid phuc vy giai trinh tu gen.
Xac dinh trinh ttr nucleotide cua gen ZmNACl
Sau khi xac djnh trinh ty nucleotide cua gen ZmNACl, dit Meu gen dupe xii li b^ng phin
mem BioEdit va so sanh vdi trinh ty gen NACI cdng bd tren GenBank cd ma so EU810024, k^t qua dvajc trinh bay d Hinh 2.
Ket qua giai trinh ty gen ZmNACl va so sanh vdi trinh tu gen ZmNACl cd ma sd tren GenBank EU810024 d Hinh 2 cho diay gen ZniN.iCl phan Igp tir miu ngd VX co kich thudc I035bp. Gen ZmNACl phan lap tir mau ngd VX cd 2 exon va I intron, exon 1 dai 471 bp \a exon 2 dai 468 bp; intron dai 96 bp (tir vi tri 472 den 567). So vdi trinh ty gen NACI cd ma sd tren GenBank EU810024 tir vi tri m§ md dau den ma ket thiic, thi gen ZmNACl ciia miu VX thieu hyl 6 bp d vi tri 487 vattr 556 -^ 560. Tuy nhien su thieu hut 6 nucleotide nay deu thupc vung intron nen khdng anh hudng den trinh tu amino acid.
Cac vi tri nucleotide cd su thay ddi la: T -^ C (vi tri 342), C -> T (vj tri 479 ), A ->G (vj tri 480), C -> A (vi tri 502), G -> C (vj tri 505), A ^ G (vj tri 552), C ^ T (vi tri 606), A ->
G (vj tri 643). Vimg ma hda ciia gen cd kich
thudc 939 bp ma hoa cho 312 amino acid va
Nguyin Vu Thanh Thanh vd Dtg Tap chi KHOA HOC & CONG NGHE 107(07): 6 3 - 6 8
ket qua so sanh a m i n o acid suy dien ciia gen ZmNACl d u g c thd hifn d Hinh 3 . Hinh 3 cho thay trinh t u a m i n o acid cua protein N A C I suy d i l n tir gen ZmNACl phan Igp m i u ngd dja p h u a n g Vj X u y e n - H a G i a n g cd dp tucmg d d n g c a o s o vdi protein N A C 1 cd mS sd A C F 3 3 1 3 6 tren G e n B a n k (chi khac mpt a m i n o acid d vj tri 181) vdi dp t u o n g ddng cua 2 mau so sanh la 9 9 , 6 8 % . K E T LUAN
DS thiet ke cap moi NACZm-F/NACZm-R, khuech dgi, tach d d n g thanh c d n g va x a c djnh trinh t y nucleotide cCia gen ZmNACl tir D N A he gen mau ngd dja p h u o n g V X ( H a Giang).
Gen ZmNACl phan Igp d u g c cd kich t h u d c la 1035bp, viing ma hoa cd kich t h u d c 9 3 9 b p ma hoa cho 312 a m i n o acid. Gen ZmNACl phan Igp tir mau ngd VX cd 2 exon va I intron, exon I dai 471 b p va exon 2 dai 468 bp; intron dai 96 bp. Gen ZmNACl ciia mau VX cd 6 vj tri nucleotide thieu hut d vimg intron (487, 556, 557, 558, 559, 560) va protein suy dien cd su sai khac d mpt vi tri amino acid (vi tri 181) so vdi trinh t y protein NACI cd mS sd A C F 3 3 1 3 6 tren G e n B a n k . Trinh t y gen ZmNACl la c o sd d l t i l p tyc nghien cuu dgc diem ciia gen ZmNACl va mdi lien quan giu'a s y thay ddi trong cau true gen ZmNACl vdi dgc tinh chju han d cay ngd.
T A I LIEU T H A M K H A O [1]. Golldack D, LUking I, Yang O (2011). Plant tolerance to drought and salinity: stress regulating transcription factors and their functional significance in the cellular transcriptional network.
Plant Cell Rep . 30(8): 1383-91.
[2]. Hao YJ. Song QX, Chen HW, Zou HF, Wei W, Kang XS, Ma B, Zhang WK, Zhang JS, Chen SY
(2010). Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation. Planta.. 232(5):1033-1043.
[3]. Kumar.M., Dalal.M.. Zaidi.P.H . Chinnusamy,V. and Bansal,K.C. (2008). Cloning and functional validation of abiotic stress inducible NAM. ATAF. and CUC (NAC) transcription factor from maize inbred lines differing in drought resistance.
http://www,ncbi.n!m.nih.gov/nuccore/EU810024.
[4]. Liu ZJ, Shao FX, Tang GY. Shan L, Bi YP (2009). Cloning and characterization of a transcription factor ZmNACl in maize (Zea mays)]. Ki CAua/j., 31(2): 199-205.
[5]. Lu M, Ying S, Zhang DF, Shi YS, Song YC, Wang TY, Li Y (2012) A maize stress-responsive NAC transcription factor, ZmSNACl, confers enhanced tolerance to dehydration in transgenic Arabidopsis. Plant Cell Rep 31 (9): 1701 -1711.
[6]. Nakashima K, Takasaki H, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012). NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta. I819(2):97-103.
[7]. Olsen AN, Ernst HA, Leggio LL, Skriver K (2005). NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci., 10(2):79-87
[8]. Puranik S, Sahu PP, Srivastava PS, Prasad M (2012). NAC proteins: regulation and role in stress tolerance. Trends Plant Set., I7(6):369-81.
[9]. Sambrook J. va Russell D.W., (2001).
Molecular Cloning. A Laboratory Manual 3 ed.
Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
[10]. Tran LS, Nishiyama i\, Yamaguchi- Shinozaki K. Shinozaki i^ (2010). Potential utilization of NAC transcription factors to enhance abiotic stress tolerance in plants by biotechnological approach. GMCrops., l(l):32-9
Nguyin VO Thanh Thanh vd Dtg Tap chi KHOA HQC & C O N G N G H S 107(07): 63 - 68
S U M M A R Y
C L O N I N G OV ZmNACl I S O L A T E D L O C A L C O R N C U L T I V A R IN VI X U Y E N , H A G I A N G , V I E T N A M
Nguyen Vu Thanh Thanh'", Nguyen Van Tuong', Pham Thanh Tung', Le Van W , C h u Hoang Mau ' College of Sciences • TNU. -iiislitiue of Biotechnology ' College ofEduealiim - TNU
NAC transcription factors are a family of functionally diverse proteins. They are unique to plants and play an important role in regulation of plant growth and development, hormone regulation and responses to various stresses, the aging process, morphological development, signal transduction pathways and may play important roles in biotic and abiotic resistance pathways. In this study we have amplified, cloned successfully and identified nucleotide sequencing of ZmNACi gene from DNA genome of local VX corn cultivar (Ha Oiang). Sequence analysis showed that ZmN/ICI gene is l,035bp long, coding region of ZmNACl gene is 939bp size, encoding for 312 amino acids.
ZmNACl gene have 2 exons and 1 intron; exon 1 is 471 bp size, exon 2 - 468 bp and intron IS 96bp size. Comparison with NACI gene on GenBanl< (code number: EU810024), ZmNACl gene of cultivar VX appeared 6 different nucleotide positions at intron (487, 556, 557, 558, 559, 560) and position 181 of deductive amino acid sequence. ZmNACi sequencing is the basis for continue to study on characteristics of ZmNACl gene and relation between changes in the structure of ZmNACl gene with drought tolerance of maize.
Key words: drought tolerance. NACi gene, local corn, transcription/actors, cloning
Ngdynhdnbdi. 20/6/2013. Ngdy phdn bi^n.16/8/2013: Ngdy duyet ddng' 10/9/2013 Phdn bien iihoa iioc: TS. Hodng Thi Thu Yin - Trudng Dgt hoc Khoa hoc - DH Thai Nguyen
Tet 0912664126
