Ddo Xuan Tan vd Dtg Tgp chi KHOA HQC & CONG NGHE 138(08): 67-72
PHAN T i C H T R I N H TlT N U C L E O T I D E CUA GEN GmDREBl PHAN L A P TU*
M O T SO G I O N G DAU TU*OfNG VIET NAM
Ddo Xudn T3n', Hh Manh Ttrdng^, Vfl Thj Thu Thuy^ LS Vdn Son^ Chu Hoang M^u^' S& Gido due va Ddo tgo Thdi Nguyen: ^ Vien Cong nghf sinh hgc - VAST
^Truang Dgi hgc Suphgm - Dgi hoc Thai Nguyen TOM T A T
DREB la hp protein dugc tim thdy nhilu trong tl bdo khi thuc vdt gdp cdc dilu kien bdt lpi tir ngoai canh. Cdc cdng trinh nghifin ciiu diu nhgn djnh DREB la nhan td phien md co chiic ndng dilu khiln su bilu hifin ciia mpt sd gen cam (mg vdi hgn, mdn vd lanh. Gen GmDREB2 d cdy ddu tuong cd chiic ndng kich hogt sy phien ma ciia nhdm gen chju han vd sdn phim bilu hien gen GmDREB2 dugc tim thay khi cdy ddu tuong gdp han, lanh vd man. Trong bai bao nay, chiing toi trinh bay ddc dilm phdn tu cua gen GmDREB2 phan lap tir mRNA cua 6 gidng ddu tucmg Viat Nam (DT26, DT51, DVN5, CB, CBD, DT2008). Gen GmDREB2 (cDNA) cd kich thudc la 480bp, xdc djnh chuoi polypeptid ddi 159 amino acid. Miln AP2 cua DREB2 gdm 59 amino acid vd co 11 vj tri amino acid lifin kit vdi DNA. He sd tuong ddng vl trinh tu nucleotide la 94,6% - 99,6%, vl trinh ty amino acid suy diln la 92,5% - 99,4%. Cd 17 vj ui sai khde vl amino acid trong protein DREB2 cua cdc gidng dau tuang nghien ciiu da dugc xdc djnh. Gen GmDREB2 (cDNA) ciia gidng ddu tuong DT2008 chiu ban tdt dugc su dung de thilt kl vector chuyen gen phuc vu tao ddng cay d^lu tuong chuyen gen nhdm cai thifin ddc tinh chiu han ciia cac gidng ddu tuong dja phuong.
Tir khoa. chiu hgn. ddu tuang, gen GmDREB2, mien AP2. tdch ddng cDNA MCJDAU
Dgu tuong (Glycine max (L.) Merrill), mgt loai eay hp ddu, khdng ehi cd gia trj kinh te vd dinh dudng cao ma cdn giu vai trd quan trpng trong viec eai thien do phi va sir dung ben vihig tai nguyen ddt canh tdc. Trong nhiing ndm gdn ddy, sy biln ddi khi hdu va sy ndng len cua trai ddt da anh hudng den sinh trudng, phat trien va nang sudt cay ddu tuong. Ddu tuong thudc nhdm cdy trdng cd kha ndng chju han kem. Vi vdy, ebon tgo gidng dau tuong cd khd ndng ehiu hgn cao va tim kiem bien phap cai thipn kha nang chju han cua cay dau tuong la chii de thii vj ciia nhieu nhd khoa hpc fren the gidi va d Viet Nam. Mdt sd gen ciia ddu tuong da dugc md ta ed phan ung tmdc tdc ddng eiia hgn d muc phien md [ 1 ].
Trinh ty cis va nhdn td trans giu' vai trd quan trgng trong sy bieu hien gen dap iing vdi tae ddng ciia ban [2], Thdng bao eiia Yamaguchi va Shinozaki (1994) cho thay mpt trinh ty cis - ylu td dap ling sy mdt nudc (DRE) dugc dilu khiln bdi promoter COR78/RD29A lien
* Teh 0913 383289: chuhoangmau@tnu edu vi
quan den phdn iing cdc stress hgn, nhiet dd thdp va ndng dp mudi cao. Nhan td trans - protein DREB (Protein DREB - Nhan td cd tac dpng trans) cd the lien ket vdi trinh ty cis de kich bogt bieu hien gen d thue vat khi cd tin hieu sfress td ngoai canh [3]. Protein DREB la mdt phdn hp ciia nhdn td phien ma AP2/ERF cd chdc nang dieu khien sy bieu hien ciia mdt sd gen cam iing vdi hgn, nhiem man va nhiet dp thip tao ra tinh chdng chju cdc stress ciia ngoai ednh d nhieu loai thuc vat [4]. Thudc tinh chu yeu ciia gen DREB Id viing bao thu AP2 lien ket vdi eae nhan to phdn img vdi cac stress. Nhieu gen DREB dd dugc phan lap tu cac Ioai thyc vgt kbac nhau, nhu cay Arabidosis, Iua {Oryza sativa L.), ngd (Zea mays L.), Iiia mi {Triticum aestivum), ddu tuang va nhieu cay frdng khac [2], [5], [6]. It nhat cd 10 thanh vien cua phdn hg gen DREB dugc xdc djnh cd trong he gen cay ddu tuong (GmDREBa, GmDREBb, GmDREBc, GmDREBl, GmDREB2.
GmDREB3, GmDREBS, GmDREBd, GmDREBT) [7]. Cae gen ndy ddng vai frd quan frgng trong sy khang hgn cua eay ddu 67
Dao Xuan Tan vd Dtg Tap chi KHOA HQC & CONG NGHE 138(08): 67-72 tuang. Chen vd cs (2007), da phdn lap gen
GmDREBl tir ddu tuong va dya tren sy gidng nhau ve miln AP2, gen GmDREB2 xep vao phdn hg A5 trong nhdm gen DREB. Gen GmDREBl dugc bieu hien manh frong mdi trudng cd hgn, ndng dp mudi cao, nhiet dp thdp va ABA [8]. Su bilu hien eua GmDREB2 a cdy thudc la chuyin gen the hien d sy tich liiy ham lugng proline cao [9].
Trong Ngdn hang gen qudc tl, trinh ty gen GmDREB2 dd dugc cdng bd bdi Wang va cs (2006) cd kich thudc 480 bp ma hda 159 amino aeid, viing AP2 cd 59 amino acid, tu vi tri amino acid 34 din 92 [10]. Gen GmDREB2 a cay dau tuang cd chuc nang kich hoat sy phien ma cua nhdm gen myc tieu lien quan den kha nang chju hgn. San phdm bieu hien ciia gen GmDREB2 dugc tim thiy khi cay dau tuong gap han, Ignh, man va gen GmDREBl da dugc chung minh la tich eye tham gia vao viec chdng han d cay ddu tuong [U) [8], [9]. Trong nghien euu nay, chiing tdi frinh bay mdt sd dac diem ca ban cila trinh ty gen GmDREBl phan lap tu mpt sd giong ddu luong Viet Nam, tao nguyen lieu va eung cdp thdng tin phyc vy thiet kl vector chuyen gen trong chien luge nghien ciiu cdi thipn kha ndng chju han ciia cay ddu tuong.
VAT LIEU vA PHUONG P H A P Su dung 6 gidng dau tuong trdng phd biln d mien Bdc Viet Nam lam vat Ijeu nghien ciru.
Cac gidng dau tuong nhu Vang Cao Bdng (CB), Cao Bdng den (CBD), DT5I, DT26 do Trung tdm nghien cdu va phat trien ddu dd, Vien Khoa hpe Ndng nghiep Viet Nam cung cap; gidng ddu tuang DVN5 dugc cung cap bdi Vien Nghien ciru Ngd vd giong DT2008 do Vien Di truyen Ndng nghipp cung cap.
Tdch chiet RNA tdng sd tu la cdy ddu tucmg bdng bp kit GeneJET Plant RNA Purification Mini (Hang Thermo Scientific). Tao cDNA tu RNA tdng sd bdng bd kit RevertAid First Strand cDNA Synthesis (Thermo Scientific).
Khuech dai gen DREB2 (cDNA) bdng phan img PCR vdi cap mdi dugc thiet ke dya trSn frinh tu nucleotide cua gen DREBl cd md so 68
DQ054363 fren Ngdn hang gen qudc te [10], Chu ky nhipt eua PCR: 95°C/4 phut, (95^/30 gidy - 57°C/30 giay - 72°C/45 giay)
^°, 12°Cn phut. San phim PCR duge dien di tren gel agarose \% frong dem IX TAE. Gel dugc nhudm trong dung djch ethidium bromide vdi ndng dg 0,1 |ag/ml.
Tach ddng gen dupc tien hanh theo Sambraok (2001) [11]. San phdm PCR dugc tach khoi gel, tinh sach theo kit DNA extraction ciia hdng Fermentas va gdn tryc tiep vao vector tach ddng pBT, sau dd dugc bien nap vao tl bdo E. Coli DH5a. Chpn lpc eac khuin lac duong tinh tren mdi trudng LB cd bd sung 50 mg/I ampicillin, 30 mg/l X-gal va 0,1 mM IPTG. Su ed mat eiia DNA tai td hgp duoc kilm tra bdng phan ung cdlony-PCR vdi cap mdi ddc hieu. Taeh chilt plasmid dugc thirc hien theo bd kit Plasmid Miniprep ciia hang Qiagen. Trinh ty ciia gen dugc xdc djnh tren may dgc trinh tu nucleotide ty dpng ABl PRISM@ 3100 Advant Genetic Analyzer (Applied Biosystem), Phan tich trinh ty nucleotide, trinh ty amino acid bang phan mem Blast frong NCBl va BioEdit.
KET QUA vA T H A O L U A N
Nhan ban va giai trinh tur gen GmDREB2 (cDNA) phan lap tir cac giong d^u tuong Dya tren trinh ty nucleotide ciia gen DREB2 ed ma sd DQ054363 tren Ngan hang gen quoc te [2] ehiing tdi da thiet ke cap mdi DREB2- F/DREB2-R cd trinh ty Id:
GmDrebl-Ncol-F:
5'CATGCCATGGATGGAAGAAGCGGGTFrAG3' GmDrebl-Notl-R:
5TrcCGGCCGCCTAATCTTCAGGTrTGGGA3'
Ket qua nhdn bdn dogn eDNA bang phan iing
RT-PCR da nhan dugc 6 doan DNA ciia 8
gidng dgu tuang nghien ciru deu cd kich
thudc khoang 0,48kb, diing nhu tinh toan ly
thuyet va tucmg ung vdi kfch thude doan ma
hod thudc gen GmDREBl cua giong dju
tuong mang ma sd DQ054363 cdng bo tren
Ngdn hang gen qudc tl (Hinh 1 A).
Ddo Xudn Tan vd Dtg Tap chi KHOA HQC & CONG NGHE 138(08): 67-72
<o/aUi
Hinh \.A: Kel qud diin di kiem tra sdn phdm PCR khuech dgi cDNA cua gen GmDREB2 tir 6 gidng ddu tuang; B: Kit qud dien di hem tra sdn phdm colony-PCR lii 8 ddng khudn lgc (M. thang DNA chudn I kb,
I- DT2008: 2'CB: 3-CBD: 4-DT26; 5-DT51: 6- DVN5
Bang I. Cdc vi tri sai khde vi trinh tu amino acid suy dien lir trinh tu gen GmDREB2 giua 6 gidng ddu tuang vd gidng dgu tuang cd md sd DQ054363
TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
P r - P r - P r - P r - P r - P r - P r -
VI tri 3 14 21 32 68 79 90 106 114 119 136 139 140 146 147 153 158
D Q 0 5 4 3 6 3
DQ0S4363 DT2008 E
T E K T F P K K V T I S 1 E E E
R I D R E B 3 - D T 2 0 0 e DREB2-CB DR2B2-CBD DBSB2-DT26 DRJEB2-DVN5 D B B B 2 - 0 T 5 1
D S D K A F P N K V S A N S D E E . 0 SD y-IR R . K -u E I R K
CB V T E K A F A N K V S A N S D E E
KR R[
Hinh 2. Trinh tir ami CBD
V T E K A F A N K V A
s
N S D E E
60 70
••L '--5 - TPVT
10 acid cua mier DT26
E T E K A F P N K V S A N S D E E
so R DT V HL R
. 1 . . AP2
DVN5 E R E N A F P N Q V
s
A N S D G Eso . - Ri . P
DT51 E T E K A L P N K 1 S A N S D E G
SL
Kit qua chpn ddng khuan Igc chii'a vector tai to hgp dya tren kilu hinh va bdng phan img colony-PCR vdi cgp mdi dac hieu cho thdy xuit hien bang DNA cd kich thudc khoang 0,48kb tu 6 ddng khudn lgc (Hinh IB) va plasmid tdi td hgp da dugc tdch va dem gidi trinh ty nucleotide. Kit qud xae djnh trinh tu nucleotide va bdng Blast trong NCBl da khdng djnh dogn gen phdn lap tir mRNA cua 6 giong dgu tuong vdi kfch thudc 480bp, ma hda cho 159 amino acid Id GmDREBl. Cac
frinh ty gen da dugc dang ky tren Ngan hang gen qudc te vol ma sd la: LK936507, LK936508, LK936509, HG965097, HG965098, HG965099.
Ket qua so sanh trinb tu gen GmDREB2 va trinh tir amino acid suy dien
Ket qua so sanh trinh ty gen GmDREBl cua 6 gidng nghien cuu vdi nhau va vdi trinh ty gen cd md sd DO054363 tren Ngdn hang gen qudc te cho thay, he sd tuong ddng ve trinh ty nucleotide dao ddng td 94,6%) din 99,6%., cdn
69
Dao Xuan Tdn vd Dig Tap chi KHOA HOC & CONG N G H $ 138(08): 6 7 - 7 2 he sd sai khac t u 0,4%o d i n 6 , 3 % vdi 26 vj tri
sai khac ve nucleotide. Gidng D T 2 0 0 8 xudt hien n h i l u vj tri nucleotid sai khac nhat so vdi trinh t y nucleotide gen DREBl cua gidng mang ma sd D Q 0 5 4 3 6 3 vd cac gidng cdn Igi.
He sd t u o n g d d n g ve trinh t y amino aeid suy dien giifa 6 gidng ddu tucmg nghien cdu va trinh ty amino acid suy dien t u trinh t y gen DREBl mang ma sd D O 0 5 4 3 6 3 Id 9 2 , 5 % d i n 9 9 , 4 % , he sd sai khac t u 0,6% d i n 7,9% va x u i t hien 17vj tri sai khac (Bang 1).
Thudc tinh chu yeu eua D R E B la m i l n bao thu A P 2 lien k i t vdi cac nhan td phan iing vdi cac stress Phan tich trinh t y amino acid mien AP2 ciia D R E B 2 d 6 gidng dgu t u o n g Viet Nam vd gidng cd ma sd D Q 0 5 4 3 6 3 cho t h i y mien AP2 gdm 59 a m i n o acid, t u vi trf t h u 34 d i n 92 vdi dd t u o n g d d n g rat cao, chi sai khac d 3 vj trf amino acid (Hinh 2). D i l u nay da khdng djnh, m i l n AP2 cOa D R E B 2 cd tinh bao thil cao vd nhiing thay ddi v l thanh p h i n amino acid tai mien ndy rdt cd t h i Iam thay ddi dac tinh ciia protein.
Ket qua d hinh 2 cdn cho thdy, cac d i l m lien k i t cua DREB2 vdi D N A (DNA binding site) d 11 amino acid, d o la cdc vi trf 35, 36, 3 8 , 40, 42, 44, 4 8 , 50, 57, 59, 6 2 . Tuy nhien, c h u a tim thay sy khae biet ndo trong trinh t u amino acid cua mien AP2 gitra cdc gidng dau tuong.
Ddc tinh chiu han eua cdy dau t u o n g la tinh trang da gen, bao gdm cac gen ma san pham ciia chimg tac dpng tryc t i l p tdi qua trinh chiu hgn hoac kich hoat qua trinh phien ma ciia cac gen chju hgn. D R E B 2 tuy khdng tryc t i l p tham gia vdo qua trinh chju ban, nhung nd Id nhan td kich hogt sy phien ma ddng thdi len cac gen lien quan d i n tinh chju han [10], [12], Ket qua phdn tfch da chi ra su sai khac d nhieu vj tri trong trinh t y nucleotide va trinh ty amino acid cua gen GmDREBl. Tuy nhien, chua tim t h i y tinh quy luat ciia thay ddi vd s y thay ddi dd lien quan the nao vdi hoai dpng phien ma cua cac gen m u c tieu quy dinh tinh chiu hgn cua cay dau t u o n g , day la v i n d l cdn tiep tuc nghien cuu. Tuy nhien, cdng bd ciia Vinh va cs (2010), Hd va cs (2013), Sulieman
70
vd es ( 2 0 1 5 ) da k h a n g djnh, g i d n g dau tucmg D T 2 0 0 8 la g i d n g chju han tdt [13], [14], [15]
cho nen trinh t y g e n GmDREBl ciia gidng D T 2 0 0 8 da d u g c s d d u n g t h i l t k l vector phuc vy chuyen gen n h d m eai thipn kha nang chiu han eiia cdy dau t u o n g .
K E T L U A N
Gen GmDREBl phan lap t u m R N A eiia 6 gidng dgu t u o n g ( D T 2 0 0 8 , C B , C B D , DT26, D T 5 I , D V N 5 ) cd kich t h u d e ia 480bp, xac dinh chudi polypeptid dai 159 amino acid.
Protein D R E B 2 ed c h u a mien A P 2 gdm 59 a m i n o acid vdi 11 vi fri a m i n o acid lien ket vdi D N A . H e sd t u o n g d d n g ve trinh tir nucleotide Id 9 4 , 6 % 9 9 , 6 % , v l trinh tir a m i n o acid suy dien la 92,5%o - 99,4%, Protein D R E B 2 ciia cae g i d n g ddu tuotig nghien c u u cd 17 vj tri sai khac v l amino acid. Dogn gen GmDREBl phdn lap t u gidng ddu tuong chiu hgn tdt DT2008 dupc su dung thiet ke vector ehuyen gen phuc vu lao ddng cay chuyin gen trong m u c dich cai thien ddc tfnh chiu han cita cac gidng dau t u o n g dja phuang.
T A I L I E U T H A M K H A O 1. Mizoi J, Ohori T, Moriwaki T, Kidokoro S, Todaka D, Maruyama K, Kusakabe K, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K. (2013), GmDREB2A,2, a canonical DEHYDRATION- RESPONSIVE ELEMENT-BINDING PR0TEIN2-type transcription factor in soybean, is posttranslationally regulated and mediates dehydration-responsive element-dependent gene expression. Plant Physiol., 161(1); 346-361.
2. Li X.P. , A.G. Tian, G Z. Luo, Z.Z. Gong, J.S.
Zhang and S.Y. Chen (2005), Soybean DRE-binding transcription factors that are responsive to abiotic stresses. Theor Appl Genet., 110(8): 1355-1362.
3. Yamaguchi-Shinozaki K. and K. Shinozaki (2994), A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt suess.
Plant Cell, 6: 25\-264.
4. Shinozaki K., K. Yamaguchi-Shinozaki, (1996), Molecular responses to drought and cold stress.
Current Opinion In Biotechnology, 7(2): 161-167.
5. Andeani J.K., S. Mohsenzadeh and H!
Mohabalkar, (2009), Isolation and Characterization of Partial DREB gene from Four
Dao Xudn Tan vd Dtg Tap chi KHOA HQC & CONG NGHE 138(08): 6 7 - 7 2 Iranian Triticum aestivumCultivars. World
Journal of Agricultural Sciences, 5(5). 56\-566.
6. Chen M., Q. Wang, X.G. Cheng, Z. Xu, L. Li, X.G. Ye, L.Q. Xia, Y.Z. Ma (2007), GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-salt tolerance in fransgenic plants. Biochem Biophys Res Commun., 353(2): 299-305.
7. Riechmann J.L., J. Heard, G. Martin, L. Reuber, C. Jiang, J. Keddie (2000), Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science, 290: 2 1 0 5 - 2110.
8. Chen M., Wang Q. Y, Cheng X. G, Xu Z. S, Li L. C, Ye X. 0 , Xia L. Q, Ma Y. Z. (2007), GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-salt tolerance in transgenic plants. Biochem Biophys Res Commun., 353(2): 299-305.
9. Kobayashi F, Ishibashi M, Takumi S. (2008), Transcriptional activation of Cor/Lea genes and increase in abiotic stress tolerance through expression of a wheat DREB2 homolog in transgenic tobacco. Laboratory of Plant Genetics, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe, 657- 8501,Japan.
10. WangN., Liu,M., Cheng,X and Ma,Y (2006), Glycine max DREB protein (DREB2) gene,
complete cds. GenBank: DQ054363. i.
http://www.ncbi.nlm.nih.gov/nuccore/DQ054363.
1!. Sambrook J., Fritsch E. F., Man^atis T. (2001), Molecular Cloning: A Laboratory Manual. New York, Cold Spring Harbor Laboratory Press.
12. Chu Hodng Mau, Nguyin Thj T'luj' Hudng, Nguyen Vu Thanh Thanh, Chu Hoang Hd (2011), Gen vd dgc tinh chiu han cua cdy dgu tuang, Nxb Dgi hpc Qudc gia Ha Ndi.
13. Ha CV, Le DT, Nishiyama R, Watanabe Y, Tran UT, Dong NV, Tran LS. (2012), Characterization of the newly developed soybean cultivar DT2008 in relation to the model variety W82 reveals a new genetic resource for comparative and functional genomics for improved drought tolerance. Biomed Res Int., 759657. doi: 10.1155/2013/759657.
14. Vinh MQ. , P. T. B. Chung, N. V. Manh, and L. T. A. Hong (2010), Results of research, creation, drought-tolerant soybean variety, DT2008. Journal of Vietnamese Agricultural Science and Tech-nology, 6: 46-50,
15. Sulieman S., Ha CV., Esfahani MN., Watanabe Y., Nishiyama R., Pham CTB., Nguyen DV, and Tran LSP (2015), DT2008: A Promising New Genetic Resource for Improved Drought Tolerance in Soybean When Solely Dependent on Symbiotic N2 Fixation. Biomed Res Int; 687213.
Ddo Xuan Tan vd Dig Tap chi KHOA HQC & CONG NGH$ 138(08): 67 - 72 ^
S U M M A R Y
S E Q U E N C E A N A L Y S I C O F GmDREBl G E N E I S O L A T E D F R O M S O M E V I E T N A M E S E S O Y B E A N C U L T I V A R S
Dao Xuan Tan', Ho Manh Tuong^, Vu Thi Thu Thuy^
Le Van Son^ Chu Hoang Mau^*
'Thai Nguyen Department of Education and Training.
-College of Education - TNU. ^Institute of Biotechnology. VAST DREB is protein family found in living cells when plants to meet the adverse abiotic conditions from extemai environment. The studies have identified that DREB is a transcription factor functioning as controlling expression of a number of genes sensitive to drought, salinity and cold.
Expression products of gene GmDREB2 in soybeans activates transcription of drought-resistant gene group and are found when the soybean plant encounters drought, cold and sah conditions. In this paper, we present the results of amplification, cloning and determination of the nucleotide sequence of GmDR£B2 gene from six Vietnam soybean cultivars (DT2008, CB, CBD, DT26, DT51, DVN5). The coding region of GmDREB2 gene isolated from mRNA of six soybean cultivars is 480 nucleotides in length, encoding 159 amino acids. Domain AP2 of DREB2 protein is 59 amino acids in length and contains DNA binding sites including 11 amino acids. The similarity coefficient of nucleotide sequences from 94.6% to 99.6% and df aminoacid sequences from 92.5% to 99.4% Seventeen positions difference between the deductive amino acid sequences have been determined. GmDREB2 gene (cDNA) of DT2008 soybean cultivar (drought tolerance in high level) used to transgenic vector design aims at improving drought tolerance of soybean cultivars in Vietnam.
Keywords: AP2 domain, drought tolerance. cDNA cloning, GmDREB2 gene, soybean
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