J O U R N A L O F SCIENCE OF HNUE DOI: 10.18173/2354-1059.2015-00018 Natural Sci. 2015, Vol. 60, N o . 4, pp. 127-131
This paper is available online at http://stdb hnue.edu.vn
XAC DINH VA PHAN TICH IN SILICO CAC GEN DREB2 Cf CAY QUYT {Citrus Clementina)
Cao Phi Bang
Khoa Sinh hoc. Tru&ng Dai hpc Himg Vuong. Phd Tho Tom lat Cac gen DREB2 thuoc ve phan nhom A2 ciia ho tac nlian phicn ma DREB (dehydration- responsive element binding), giir vai tro quan irong Uong ira Idi vdi cac slress vo sinh 6 thirc vaL Trong cdng uinh nay, chiing loi su dung each tigp can /;i silico dd xac dinh va phan tich bon gen DREB2 ciia cay quyt (Cilru.s clemenlina) Chiing deu co chiJa viing bao thu AP2 (PF00847) va mouf dac trung KGCMlRKJGKGGPxN. Cac gen DREB2 cd do dai tir 633 tdi 1581 pb Chi gen CclDREB2-2 cd mot intron. ba gen con lai ma hda hen tuc. Cac protein suy dien co do dai tir 210 tdi 339 axit amin Cac protein nay co tinh axit vdi cac gia tri pl dao dong irong khoang tir 4,80 tdi 6,99. Cay di truyen cho thay cac gen nay phan bd iren ba nhanh khac nhau. Trong cac protein suy diln DREB2, khong tim thay molif PEST nhung lai cd cac axil amin la cac vj iri phosphoryl hoa tiem nang cua cac protein kinase. Ben canh do, cac axit amin dac tnmg cho vi ui ublquitin hoa dirge tim thay a cac protem DREB2. ngoai trir CclDREB2-3.
Tirkhoa: Gen DREB2, cay di truyen, dac diem hoa - li, in silico, quyt
1. Mor aau
Cay quyt (Citrus clemenlina) thudc hg Cam, hg cay an qua quan trong tren the gidi. San lugng cua cdc cay trong bp Cam dat 122,3 trieu tdn ndm 2009 (FAO 2009). 6 Viet Nam, sdn lugng hang nam cua cdc cdy thupc bg cam dat khodng 700 nghin tdn (Thdng ke Viet Nam 2012).
Mgt trong nhirng con dudng tra ldi cac stress mat nudc va nhiet dp cao d thuc vdt dugc biet Id con dudng phu thupc axit abxixic (ABA) thdng qua cac tac nhdn phien ma DREB2 [ I ] . Cac DREB2 thudc ve nhdm A2 cua bp tdc nhan phien ma D R E B (dehydration-responsive element binding) [2].
Gidng n h u cdc protein cimg ho khac, cac DREB2 cd mpt viing bdo thu cd kha ndng gan vdi ADN goi la viing AP2 nhung chiing khac vdi cdc protein ciing hp D R E B bdi sy cd mat cua motif dac trung GKGGPxN nam phia trudc vimg AP2. Cac DREB2 gan vdi yeu td cis G C C G A C , dugc goi ten la DRE (Dehydration Responsive Element), trong vung khai dgng cua cdc gen dich va kich boat su bieu hien cua chiing.
Cdc DREB2 co khoi lugng phan Xd bien ddi trong mdt pham vi rdng tiiy loai thuc vat. Phan tich cay di truyen cua cac D R E B 2 nhieu lodi thuc vat khac nhau cho thay chung cd the duac xep vdo ba phdn nhdm kbdc nhau. Cac nghien ciiu ve bieu hien gen cho thdy cac DREB2 thugc phan nhdm I bieu hien dudi tac d d n g cua dieu kien mdt nudc, nhiet dp cao. Cac DREB2 thudc phdn nhdm 2 va 3 hau nhu khdng bi cam iing bdi dieu kien mat nudc. Nghien cuu chuc nang bang each chuyen gen DREB2 lam tang kba nang chdng chiu vdi cac stress vd smh n h u khd ban, nhiet dp cao vd man a cac cay chuyen gen ciia nhieu lodi thuc vat [3]. Su cd mat cua cac vi tri phosphoryl hda va ubiquitin hda gai y
Ngay nhdn bai: 2/3/2015. Ngay nhan dang- i 7/5/2015 Tac gia lien l?c: Cao Phi BSng, dja chi e-mail phibang.c
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Cao Phi Bang
lam tang kha nang chdng chiu vdi cac suess vd sinh nhu kho hsin, nhiet dp cao va man d cac cay chuyen gen cua nhieu loai thuc vdt [3], Su cd mat cua cdc vj tri phosphoryl hda va ubiquitin hda ggi y ve cac ca che dieu hda sau phien ma va sau djch ma [ I ]. Trong nhihig nam gan day, nhieu nghien ciru dang ke da dugc thuc hien de xac dinh cac gen DREB2 trong he gen cua thuc vat ciing vdi toan bd cac gen thupc sieu hp AP2, trong dd cd cac loai thdn gd hay thyc vat khac [2,4].
Do cd vai trd dac biet quan trong trong tra Idi vdi cac stress vo sinh ciia cac DREB2, trong nghien cuu nay, ehung tdi hudng tdi xac djnh trinh tu ciia bdn DREB2 cua cdy quyt. Cac dac irung, cdc motif bdo thu cung nhu c4u tnic cua chiing ciing dugc xac dinh nhd cdc phuang phap in silico. Ben CEinh do, ket qud phan tich cdy pha he cung se duoc gidi thieu trong cdng trinh nay
2. Noi dung nghien cuu 2.1. Phu'ong phap nghien cmi
* Ca s& die lieu: Trinh tu he gen cua cdy quyt dugc lay tir Wu va nnk (2014) [5]. Cac trinh tir DREB2 cua cay Arabidopsis dugc lay tir Sakuma va nnk (2002) [2].
* Xdc dinh cdc gen DREB2 & cdy quyt: Cac protein DREB2 ciia cay Arabidopsis [2] dupc dimg lam khudn dd, chuang trinh TBLASTN duoc su dung de tim kiem cdc gen tuang ddng tren du lieu he gen cua cdy quyt vdi gia tri e-value a mdc c'^.
* Xdc dinh cdc motif bdo ihu: Cdc motij tir cdc DREB2 & cdy quyt vd mpt sd thuc vdt khde dugc tim bang phdn mem MEME [6]
* Tao cdy phd he. Cdc trinh tu protein DREB2 dugc sap day bang each diing phan mem MAFFT [7].
Cdy pha he dugc xay dung tu cac DREB2 da sdp ddy bdng phan mem MEGA5 nhd su dung phucmg phap Maximum Likelihood, dir lieu dugc xu li la tdt ca cdc vi tri vd phuang phap Bootstrap vdi 1000 lan lap lai [8].
* Cdc dac diem hda - li vd cdu true khdng gian- Cac dac diem vat li, hda hoc cua cac gen/protein duoc khdo sat nhd cac phdn mem cua ExPASy [9]. Cau tnic khdng gian ciia viing AP2 dugc xdy dung nhd phdn mem Phyre2 [ 10], Cdc vi tri phosphoryl hda trong phdn tir protein suy dien dugc xac dinh bdng cac phan mem Musite [ I I ] . Cdc vj tri ubiquitin hda duac xdc dinh bang chuang trinh UbPred[12], 2.2. K e t q u a n g h i e n c u u v a thao l u a n
2.2.1. Xac dinh bon gen DREB2 a cay quyt
Budc dau chiing ldi xac dinh duoc bdn gen cd the ma hda cho cac DREB thudc phan nhdm A2 (Bang I). Nhu vay, so lirong gen DREB2 duoc tim thdy o cay quyt it ban d cay Arabidopsis. cdy duang, cay nho hay cay tao [2,4].
Ten CclDREB2-l CclDREB2-2 CclDREB2-3 CC1DREB2.4
Bdng I. Cdc DREB2 d cdy quyt vd ddc diem cua chdng Lncus
Cidevl0022244m.g Ciclevl0032029m,g Cidev10002280m g Ciclevl0005549mg
Kich thiTirc Gen(bp)
633 1581 747 882
Kich thuo-c protein (aa)
210 339 248 293
Khoi luong phan tir (l<Da)
23,25 38,20 27.12 32.64
Pl 6,09 4,80 6,99 6,27
So I n ^ g Intron
0 0 0 1 2.2.2. Dac diem cua cdc DREB2 ciia cdy quyt
Cac gen DREB2 cua cay quyt cd dd dai tir 633 tdi 1581 bp, quy dinh cdc protein cd kich thudc tiir 210 tdi 339 axit amm, tucmg ung vdi khoi lugng phan tir tu 23,25 kD tdi 38,20 kD, trong dd, DREB2- 2 cd kich thudc ldn nhit. Cac CclDREB2 co tinh axit y^u vdi cac gia tri diem dang dien (pi) dao ddng
Xdc dinh vd phdn tich in silico cdc gen DREB2 n cdy quyt (Citrus clemenlina)
tu 4,80 tdi 6,99 (Bang 1). Nhiing ket qua nghien cuu ndy kha phd hgp vdi cac nghien cuu vi cdc DREB2 ciia mpt sd loai khac [ 2 , 4 ] ,
2.2.3. Xac dinh cac motif bao thu vd cau triic khong gian ciia cac DREB2 cua cdy quyt
Hinh L Biiu tirgng trinh tu axit amin cda vitng bdo tbu AP2 (A) vd motif dgc trung (B) cua cdcDREB2
C C 1 D R B B 2 - 1 M EK\.-SEAASLSQKGTKKPNMG C C 1 D R E B 2 - 3 M SKALMGGIGEKKQLKKPAQfi C c l D R E B 2 - 2 MAIQSKDSSPSLMMPLSSDRKRKBRDGVNVAETLERWRRYNESLESGNGECKPMRBVPAK
C C 1 D R E B 2 - 4 M ESYKKSPLK
CC1DBEB2- CC1DREB2- CC1DREB2- CC1DREB2-
•1 RSF^GCMKOTGGPENjjLCDTrKGVRQRTWGKWVAEIElEPNRGRRLWLGSraTSLDAALR'rD
•3 SSE^GCMRGKGGPENflLC riKGVRQRTWGKWVAEIREPNRGARLWLGTFDTSHEAAMAYD
•2 GSKJtGCMKGKGGPENtRC ^YRGVRQRTWGfWVAEIREPNRGNRLWLGTFPSAVEAALAYD
•4 PBatGPTRGKGGPLKksCDY RGVRQRTWGKWVAEIREPKKRTRLHLGS FATAEEAAMAYD
KtliM
CC1DREB2- CclDREB2' CC1DREB2-
C C 1 D R E B 2 - 4 EAflRRLYGPDRYLHLPhLHTDNNKPSSMN- - 1 ELAARKLYGDYAKLNLP JVQ- - 3 AAARKLYGPDAKLNLP IL—
HAARAMYGPCRRLNLP JVS-
•QHNRPSSNN INNNNHITAS QV PSA QSHHLPAS SV
•RLNESSKDSDSTTSSNQSEIEDRKVKNDARERES K I KSHNKFEWVPSKNFISMFPSCGLLNV
C C 1 D R B B 2 - 1 FTTDHIVPIQQQQQl ESSNE S C c l D R E B 2 - 3 NCTOHVAQRRNQPQMIHNSGI—TCSSNSPLPRSSNNDAIT2VYNFNYNNSAM3FLDNGS
C C 1 D R E B 2 - 2 lAQPEAELLSSPVKP KAKDE C c l D R E B 2 ~ 4 NAQPSVHVIHQRLEEIJKNGVFNQTSSSSSSSGESKVEAPTTCIGDKTLLERVSVREKEV C c i D B E B 2 - l VGNSSRF GESSVPGHFTS
C c l D R E B 2 - 3 VGSUGKL—AENEVKFSLNEQETDGFWEHT.NV I.^-=VFDF.ST
C C 1 D R S B 2 - 2 AEDNEKYYWGEQ LDC K r S A H D E P I L G S l C D V K f i T G D E C C 1 D R E B 2 - 4 EISSGNMLGWREEKKQIDLME ELQQLGILKEESQAEVTEGTEG
C c X D R E B 2 - l WISGEDNLYWP—ESLEANDFLDVND KJVSLS—DLMMGGNDL C C 1 D R E B 2 - 3 WA—EAAMSVDYPVMGE QGL-LN—RNFVDASCC C C 1 D R E B 2 - 2 EKDflTGIDWLEGYD WSKDEMFDVNDI.LDLLDNNPLCGSEQQSE-LSYYNLQAGSLDQ C C 1 D R E B 2 - 4 SSGRAESESSMKD Y G E - L — R A F A D T S - F
C C 1 D R E B 2 - 1 DWDGLQAP RSL C C 1 D R E B 2 - 3 GWDMLPTP KCV C C 1 D R E B 2 - 2 SQFGYEKPLDPFFQLRNPEWELPVGVNYAEHGASDYGLQFLKEE ESDFt.f
C C 1 D R E B 2 - 4 NWDAL lEMHGNGIAGHHQLGGFQLYDVQEEVTLPTSIWNF Hinh 2. Kit qua s&p day cdc DREB2 cda cay quyt
Vung bao tbii AP2 ndm Uong d cd dudng ke liSn. Motif d | c trung nam uong d cd dudng ke ddt. Dau sao: cac axit amin bao thii, ddu sao uong d udn' cac vi tr'i gan AND, ki Or in dam, nghieng: vi Ui phosphoryl hda. Id tu in ddm, gach chdn: vi tri ubiquitin hda
Viing bao thu A P I (PF00847) va motif dac trung cho cac DREB thudc phan nhom A2 duac tim thdy d ca bdn DREB2 ciia cay quyt. Trinh tu cua vung bao thii cung nhu motif dac tnmg duoc gidi thieu Uong Hinh I va Hinh 2. Ket qua sap day cdc CclDREB2 nhd su dung phan mem MAFFT mmh hoa ro b a n ve cac motif v a v i tri ciia chung tiong phan tu protein. Cac axit amin cd kha nang gan vdi ADN Cling d u g c xdc dmh ndm trong viing bao thii AP2 (Hinh 2).
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Cao Phi B5ng
Cac vi tri phosphoryl hda vd ubiquitin hda dugc tim thdy trong ca bdn protein DREB2 cua cay quyt. Kel qua nay phii hgp vdi nhieu bao cao trudc dd v6 cac gen DREB2 [3]. Ddng thdi, ggi y rang cac geri DREB2 ciia cay quyt cd th6 dugc digu h6a bdng cdc co cha di§u hoa sau phien ma,
Cdu tnic khdng gian viing bao thii AP2 cua cdc DREB2 dugc xdy dung nhd chuang trinh Phyre2 (Hinh 3). Ket qua chi ra cd bdn DREB2 deu ed 3 chuoi p vd I xodn a. Cdu tnic nay tucmg ddng vdi cdc DREB thupc nhdm Al, cac DREB cung cd khd nang gdn len yiu to cis GCCGAC gi^ng nhu cac DREB2ll,2],
Hinh 3. Mo hinh 3D cdu true bgc bai cda cdc DREB2 cua cay quyt (A) DREB2-1, (B): DREB2-2, (C): DREB2-3, (D): DREB2-4
1.1.4. Phdn tich cay phd he va phan loai cac DR£B2
Hinh 4. Cdy pha hi dugc xdy dung tvr cdc DREB2 cua cay quyt (Cci), cay tdo (Md), cay dmmg (Pt), cdy nho (Vv) vd eay Arabidopsis (At)
Cay phd be duoc thiet lap tir cac protein DREB2 cua cau quyt, cdy tao, cay duang va cdy Arabidopsis duoc the hien d Hinh 4. Ket qua cho thay cac DREB2 cua cJy quyt ndm tren ba nhanh khac nhau cua cay di truyen. Dieu nay ggi y rang cdc DREB2 cua cay quyt cd the dugc xip vao ba phdn nhdm khac nhau. Ket qud nay phu hgp vdi su phdn loai cac DREB2 o nhieu loai thuc vdt khac [1]. Cdy phd he cUng cho thdy d cdy quyt khdng cd su kien nhdn gen nao xdy ra sau thdi di6m biet hda lodi nhu d cdc cay Arabidopsis, cdy duong vd dac biet la cay tao. Day cd the la nguyen nhan gidi thich tai sao so lugng gen DREB2 a cdy quyt it ban so vdi d cac loai cay tren.
3. Ket luan
Trong cdng uinh nay chiing tdi da xac dinh dugc bdn gen DREB2 cua cay quyt. Dac di^m li-hda
ciia cdc gen nay da duac khdo sat, Cac motif bao thu va cdu tnic cua chung cung duoc bao cao Uong
bai bao ndy. Hon nua, kit qua phdn tich cay pha he cho phep kel luan cac DREB2 ciia cdy quyt dugc
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Xdc dinh vd phan lich in silico cdc gen DREB2 & cdy quyt (Citrus eiemenlina) TAI L I E U T H A M K H A O
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A B S T R A C T
Identiflcation and in silico analysis of DREB2 genes in C l e m e n t i n e o r a n g e s {Citrus clemenlina)
DREB2 genes belong to the A2 subgroup of D R E B (dehydration-responsive element binding) transcription factor family which plays an important role in the response to abiotic stress in plants. In this work, w e used the in silico approach lo identify and analyze four DREB2 genes in Clementine oranges (Citrus clemenlina). These genes contain an AP2 conserved domain (PF00847) and typical KGCM[RK]GKGGPXN motif. The full-length genomic sequence of Clementine DREB2 genes includes those from 633 to 1581 pb. Clementine DREB2 genes are a continously coding gene except for CclDREB2-2 which has an intron region. In silico analysis results showed that the full-length predicted protein sequences contain 387 to 392 amino acids. All of the DREB2 proteins are acidic or neuUal with a p i value ranging from 4.80 to 6 99 A phylogenetic Uee analysis showed four CclDREB2 genes disuibuted on three different branches We did not find any PEST motif in the CclDREB2 proteins, however, the potentially targeted sites of protein kinases were present on all four proteins. Tn addition, characterized residues for the ubiquitination sites were found in the DREB2 proteins, except for CclDREB2-3.
Keywords: DREB2 genes, phylogenetic tree, physico-chemical properties, tn silico, Clementine.
