Tgp chi Cong nghe Sinh hgc 12(1): 113-123, 2014
XAC DINH VA P H A N TICH INSILICO HO GEN KNOX 0 CAY QUYT DlTOfNG {CITRUS CLEMENTINA)
Cao Phi Bang', Christophe D u n a n d ^ ' Truang Dgi hoc Hiing Vuong
^ Laboratoire de Recherche en Sciences Vegetales, Universite de Toulouse, UPS, UMR 5546, BP 42617, F- 31326, Castanet-Tolosan, France
^ CNRS UMR 5546. BP 42617, F-3I326, Castanet-Tolosan, France N g i y nhan bai: 21.01.2014
N g a y n h a n d a n g : 15.3.2014 T 6 M TAT
Cac gen KNOXmS. hoa cac tac nhan di€u hoa phiSn ma homeodoniain, cac tac nhan nay dong vai tro quan frong trong viec duy tri hoat tinh cua mo phan smh dinh 6 thuc vat Dong thdi cac protein nay cung co vai tro trong su phat sinh hinh thai va sir phat tn£n thii cip a nhieu loai ciy xanh. Trong cong trinh nay, chung toi sir dung cac phucmg phap nghien cihi in silico de xac dinh va phan tich cac gen KNOX a trong he gen ciia cay quyt dudng (Citrus Clementina), loai cay co gia trj ldn trong ho Cam. Tong so 10 gen KNOXda dugc xac dinh cr cay quyt duong nhd cac phan tich tren toan bo he gen Tat ca cac gen nay deu co chira hai viing bao thu KNOXl (PF03790) va KN0X2 (PF03791), nhung chi 9 trong t6ng s6 10 gen co chila viing bao diii ELK (PF03789) vi Homeodomain (Homeobox^KN, PF05920) Cac gen KNOX ma hoa khong lien mc, chua tir hai tcri nam infron. Cac gen CclKNOXphan bo tren 5 trong tong so 9 nhiem sac the ciia cay quyl duong Chi c6 hai gen CclKNOXI-I va CciKNOXl-2 dugc hinh thanh nho su kien lap doan cua nhiem sac the. Cac gen CclKNOX CO the dugc phan chia thanh ba phan nhom la phan nhom KNOX I, ph^n nhom KNOX II va phan nhom KNOXM voi lan lugt 6, 3 va 1 thanh vign trong cAc phan nhom nay. K§t qua phan tich in silico viing promoter cua cac gen cho thay chiing co the it chiu su dieu hoa cua cac tac nhan vo sinh cCia mfii tnrong nhimg c6 the chiu sir dilu hoa cua mpt so phytohormon nhir auxm, gibberellin va dac biet la cac hpp chat brassinosteroit. Sau trong tong so 10 gen CclKNOX b\eu hi^n frong cac co quan co chua mo phan sinh dinh, trong do cac gen KNOXphan nhom II bieu hien manh nhat,
Tu khda: bieu hien gen, cdu trdc exonhntron, cay di truyen, he gen. in silico, KNOX, mo phan .•unh dinh.
promoter, quyt du&ng
MCJ DAU ciia cay, Trong qua trinh sinh truong scr cap, cac lap vo va the ciia mo phan sinh dinh choi san sinh ra cac Cay quyt ducmg (Citrus clementina) la mot thu tign biSu bi va tign tucmg tkng, khi cac lop l6 bao nay frong nh6m quyt, thuoc ho Cam, mot frong nhimg hp keo dai va biet hoa se sinh ra vo va cac mo dSn (Evert, cay 5n qua quan frpng nhai Iren the giai. Theo thong 2006).
ke ciia Fao, nam 2004, san luong ciia cac cay trong ^ ^ ^ (KNotted-like homeobOX) la ten goi tat hp nay dat 108,535 ti tan, frong do 22% thuoc ve cac , . , , , gi6ng qu;^ (Khan, 2007). Cl Viet Nam, th6ng ke cho ^^^ ^^^ P™'^'" ^'^ ^.^"^ ™"g ^ao thu homeodomam thdy san lupng hang nam ciia cac cay thupc ho cam, co kha nang hen ket vai ADN, cac protem nay co quyt dat fren duai 700 nghin t^n (Th6ng ke Viet ck\x friic giSng vai protein dupc ma hoa boi gen Nam 2012). Vai gia tri cao, cay quyt ducmg la mot knoitedl ditac phal hien lan dau each day khoang hai trong cac cay dugc lira chpn lam dai dien trong du an muai nam a cay ngo (Vollbrecht et al, 1991), Cac giai frinh tu he gen cac cay Ihupc hp Cam (Aleza el ^^^ ^ g ^^^ ^^^ j ^ ^ , ^^^ p^jgn ^na nay dong vai fro '• quan trong frong sir phat trien choi cay (Groover et al, 2006; Du el al, 2009). Su phat hien cac gen KNOX cung cap nhung hieu biet o mirc phan frr ve Mo phdn sinh dinh ch6i la mot tap hpp cac Ie bao
nam 6 chop sinh tnrong ciia cac canh cay, dam bao
qui trinh phat sinh phoi lien tuc frong su6t cupc doi viec duy Iri chuc nang ciia mo phan sinh dinh choi.
113
Cao Phi B ^ g & Christophe Dunand Smith va dong tac gia dS chi ra rang protein KNl a
frong choi cua cay ngo bi giii lai trong cac te bao phao sinh va bi loai trir khoi cac te bao phat sinh la (Smith et al, 1992). Hoat ttnh cua KNl cin thiat di ngan chan cac te bao goc thuc frong cac mo phan sinh dinh choi ngo khoi sir biet hoa dinh san ciia cac te bao, tucmg tu nhu protein SHOOT MERISTEMLESS (STM) a cay Arabidopsis, cac dot bien mat chuc nang ciia cac gen nay, stm va knl, khong the thiet lap va duy tn mo phan sinh dinh choi (Long et al, 1996; Vollbrecht el al, 2000).
Dua tren cau tnic cua gen, mo hinh bieu hien va cac phan tich cay di truyen, cac KNOX duoc xep vao hai phan nhom (Mukherjee et al, 2009), Phan nhom 1 gom cac gen co homeodomain gan giong vai gen KN I (>71%) va duac bieu hien frong cac vimg giao nhau ciia cac mo phan sinh dinh choi nhung bj giam bieu hien a la a ca thuc vat mot la mam va hai la mam. Phan nhom 11 gom cac gen co chiia mpt infron trong viing ELK, khac biet vai phan nhom 1 va c6 cac mo hinh bieu hien da dang (Hake el al, 2004; Zhong et al, 2008). G k day, mot nhom nho cac gen KNOX moi dugc xac dinh ma hoa cho cdc protein thieu homeodomain va bieu hien 6 cac vimg gan-ben cua cac tien co quan va a lap bao ciia cac ca quan thanh thuc (Magnani, Hake, 2008). Vai Iro ciia cac gen KNOX phan nhom 1 dupc biet kha ro, chiing giu vai tro quan trpng trong viec duy tri chiic nang ciia mo phan sinh dinh choi (duy tri cac te bao goc) va trong qua trinh phat sinh ca quan (Abraham-Juarez el al, 2010;
Chatterjee et al, 20! 1). Cf Arabidopsis, phan nhom I g6m CO cac gen KNATl (BREVIPEDICELLUS), KNAT2. KNAT6 va STM (SHOOTMERISTEM- LESS). Trong cac gen nay, STM giu vai tro chu yfiu trong viec hinh thanh va duy tri mo phan sinh dinh (Hay, Tsiantis, 2010). Cac gen KNATI va KNAT6 tham gia vao viec duy tri chuc nang cua mo phan sinh dinh choi dong thoi tham gia diSu khign sir phat frien cua mam hoa (Venglat el al., 2002; Mele et al, 2003; Ragni et al, 2008), Trong nhihig nam gan day, vai tro ciia cac gen KNOX phan nhom 1 cilng dupc nghien cuu tren nhieu dfii tugng thuc vat khac nhu cay cp dau (Jouarmic el al, 2007), cay tequila (Abraham-Juarez el al, 2010), cay dau lay (Chatterjee et al, 2011), cay thuoc la va cay man chau Au (Srinivasan et al, 2011), cay dao (Testone et al, 2012). Trong khi do, thong tin thu dupc ve vai fro cua cac gen KNOX phan nhom II hien nay chua nhieu. O Arabidopsis, phan nhom nay gom 114
cac gen KNAT3, KNAT4. KNAT5 va KNAT7 (Hake et al, 2004). Cac gen KNAT3. KNAT4 ya KNATS dupc biet CO vai tro frong sir phat frien ciia re (Truemit, Haseloff, 2007). Gen KNAT7 co thi Iham gia diiu hoa qua trinh sinh tong hgp lignin va thanh th bao (Zhong et al, 2008; Li et al, 2012). M$t s6 it nghien cim ve vai fro cua gen KNOX phan nhom II cflng dupc tien hanh fren cac Ioai cSy khac nhu cay dao (Li et al, 2012). Tac dgng ciia cac gen KNOX gian tiep thong qua viec dieu hoa ho?it Iinh cac phytohormon gibberellin va cjlokinin (Hay, Tsiantis, 2010). Cac protem KNOX hoat dpng thong qua tucmg tac vai BELL, mpt nhom cac protein co homeodomain khac thupc ve hg TALE, nha qua trinh di-me hoa dac hieu hoac to hpp (Rutjens el al, 2009; Hay, Tsiantis, 2010).
Ct miic dp he gen hoan chinh, lan lugt 9, 10 va 15 gen thupc hp KNOX d\iqc xac dinh frong he gen cila cac cay hai la mam, Arabidopsis, cay dao va cay duang (Hake el al, 2004; Hay, Tsiantis, 2010;
Testone et al, 2012), so lupng cac gen tuang iing la 13 va 12 a cay ngo va cay liia, cac dai dien cho lop mpt la mam (Hay, Tsiantis, 2010).
Vai nhirng vai tro dupc chi ra, theo Du va Groover, cac gen KNOX co the tac dpng len san lupng ciia cay Irong rimg va cay an qua than go, frong do CO cay quyt duong, thong qua cac chi lieu nhu su phat tnen sinh duong, chieu dai canh, ham lupng lignin va sire khoe cita than cay (Du, Groover, 2010). Hien nay, trong so cac cay an qua than go, cac gen KNOX mai buac dau dupc nghien cim a cay tao va cay dao (Watillon el al, 1997; Testone el al, 2008) va moi chi dugc khao sat toan di$n tren quy mo he gen a cay dao (Testone el al, 2012).
Trong nghien ciiu nay, chiing toi da xac djnh dupc 10 gen KNOX Irong toan bo he gen cua cay quyt duang. Ban do gen, kdt qua phan lich cay di truyen ciia cac gen trong hp, cac dac trung Ii hoa.
Cling nhu cau tnic promoter ciia cac gen se dugc gioi thieu frong cong Irinh nay.
NGUYEN LIEU VA PHLTONG PHAP NGHIEN CLTU
Cff sd dfr lifu ve cic trinh tw gen d cSy qu^
dutrng, cay dao v^ Arabidopsis
Trinh tu h? gen ciia cay quyt duong ton t^i fren
frang web phytozome
(http .//www.ph ytozo me. net^c lemenline. phpl. phiSn
ban 1.0 ICGC (Interaafronal Citrus Genome
Tgp chi Cong ngh$ Sinh hgc 12(1): 113-123, 2014
Consortium), dfl lieu nay dugc ma ngay 15 thang 1 nam 2011. Tap hpp cac EST (Expressed Sequence Tags) cua Ioai cSy nay duac tai vk tir trang web NCBI
(ftp7/ftp.ncbi.nlm.nih.eov/repositorv/UniGene/Citru X&c dinh cac gen KNOX d cay quyt du-ong (CclKNOX)
Be nhan dugc ho gen KNOX d i y du tu he gen cua cay quyt duang, lan lugt 10 trinh tu protein KNOPEl lay tir Testone va dong tac gia (2012) dupc dung lam khu6n do, chucrag trinh TBLASTN dupc sir dung de tim kiem cac gen titong dOng fren dii lieu nucleotit ciia toan he gen (httpV/phvtQzomenet/5earch.php?show^blast&raeth od^Ore Cclementinal.
Lap bSn do gen
Ban d6 cu tru ciia gen fren cac nhiem sac the dugc thiet lap nha phan mem MapChart (Voomps, 2002).
Tao cay di truydn
C^c trinh tu protein cua cac gen AW^OX nghien ciru dupc sap day bang each diing phan mem MAFFT, phien ban true tuyen vai cac cai dat mac dinh (Katoh, Standley, 2013). Cay di truyin dupc xay dung tiir cac trinh tir protein KNOX ciia cac to^i Arabidopsis, dao va quyt duong da sap day
s Clementina/), dii lieu dugc ma tir ngay 22 thang 8 nam 2011, Cac frinh tu KNOX ciia cay Arabidopsis va cay dao dugc lay tir Hake va d6ng tac gia (2004), Magnani va Hake (2008) va Testone va dong tac gia (2012).
bang phan mem MEGA5 nho su dung phuang phap Maximum Likelihood va tuan theo cac tham bien: mau Jones-Taylor-Thomton (JTT), dii lieu d u a c xir li la tat ca cac vi tri va phuang phap Bootstrap vdi 1000 l i n lap lai (Tamura et al, 2011),
PhSn tich in silico ve ho gen KNOX
Cac dac diem vat li, hoa hpc ciia cac gen/protem nghien cfru dugc khao sat nha cac phan mem ciia ExPASy (Expert Protein Analysis System;
(Gasteigere/a;,2005)), PhSn tich promoter
Trinh tu 1000 nucleotit nam tru6c ATG cua gen dupc su dung de tim cac yeu to cis nha six diing phan m i m PLACE (Higo el al, 1999)
Khao sat in silico sy bieu hien gen
Su bieu hien cua cac gen duoc khao sat bang each dem so lugng EST dua fren du lieu ton tai Iren Irang web NCBI (taxid:85681) nhii chucmg Irinh TBLASTN
Bdng 1. Dac dilm cCia hp KNOX&cSy qu^t dyang
Chieu d^i Khoi liro'ng protein (aa) protein (kD) ^ CclKN0X2-1 11
CclKNOX2-2 11 CclKNOXI-1 1 CclKNOXM M CclKNOXI-2 1
CclKN0C1-3 1
CclKNOX2-3 II
C c l K N O X H 1
CclKNOXI-5 1 CclKNOXI-6 1
Cidev10008269m,g Ciclevt 0008821m,g Ciclevi 0015818m g Ciclev10017185m-g Ciclevi 0015581 m.g Ciclevi 0024581 m,g
& c l e v 1 0 0 2 2 7 8 1 m g ) Ciclevi 0021319m g Ciclevi 0033752m g (+
Ciclevi 0033524m, 9) Ciclev10001779mg Ciclevi 0001508m g
450 345 345 124 384
282
303
329
334 379
50,6 39,3 38,4 13,7 43,0
31,8
34,0 37,3
38,0 43,3
5,78 5,74 5,71 4,44 5,96
5,09 6,16
5,12
4,6 6,04
1 1 2 2 2 3
3
4
5 5
1461909 1465475 28782248, 28785369 22765392. ,22769634 26900230..26901255 35642594 35646764 3273325 3278876
6655487 6659893
22456126 22463589
34037645 34040678 34911260,34916032
5 5 3 2 3
4 4
4
4 4
Cao Phi Bang & Christophe Dunand KET QUA VA THAO LUAN
Xdc dinh ho gen KNOX fr cSy quyt dirtrag He gen hoan chinh cua cay quyt duong da dugc tim kiim vai cac protein KNOX khuon do cua cay dao nha su dung chuong trinh TBLASTN. Tong so, 10 gen CO the ma hoa cho cac tac nhan dieu hoa phien m% KNOX da dugc xac dinh (Bang 1), Ket qua phan tich vung bao thii nha su dung Pfam chi ra ca 10 gen nay deu c6 chua hai vimg bao thu KNOXl (PF03790) va KN0X2 (PF03791), 9 ttong t6ng s6
10 gen nay co chua vimg bao thii ELK (PF03789) va Homeodomam (Homeobox^KN, PF05920). So s&ih vdi Homeodomain cua gen knl a cay ngo (Vollbrecht et al, 1991), cac gen nay co miic do ttrong d6ng tir 58 toi 88% (Bang 2). Cac KNOX ciia cay quyt ducmg co miic dp giong vai cac gen tuang d6ng a cay Arabidopsis tu 42 toi 81% (Bang 3), Nhu vay, so lupng gen KNOX duoc tim thSy 6 cay quyt dirong bang voi cr cSy dao (Testone el al, 2012), nhiiu hem a cay Arabidopsis nhimg lai it hon 6 cay ducmg va cac cay mpt la mam nhu Iiia va ngo (Hay, Tsiantis, 2010),
B a n g 2. So sanh viing Homeodomain cua cac CclKNOX & cay quyt d u o n g vai KN1 d cSy ngfl.
C h i s o t o n g P h a n t r a m t r i n h tip du'O'c s o s a n h
Gia trj m o n g do-i
Mii'e d o tu'O'ng d d n g CclKNOXI-1
CclKN0X1-2 CclKNOCI-3 CclKN0X1-4 CclKN0X1-5 CclKN0X1-6 CclKN0X2-1 CclKNOX2-2 CCIKNOX2-3
92.4 94.7 90.9 90,5 80,5 99 79,3 77,4 80,9
92,4 94,7 90,9 90,5 80,5 99 79,3 77,4 80,9
8 3 % 8 3 % 8 3 % 8 3 % 8 3 % 8 3 % 9 6 % 9 6 % 9 6 %
3,00E-31 3,00E-32 1,00E-30 1,00E-30 1,00E-26 8.00E-34 4,00E-26 2,00E-25 8,00E-27
83%
87%
79%
79%
63%
88%
60%
58%
62%
Bang 3. So sanh cac CclKNOX & cay quyt d u a n g v a i cac gen tu-ang dong a cay Arabidopsis.
KNOX A r a b i d o p s i s
a C h i s o CLFC C h i s o t o n g
Gia trj m o n g M I F C d 9 t i r c n g do'i d o n g CclKN0X1-1
CclKNOXI-2 CclKNOCI-3 CclKN0X1-4 CclKNOXI-5 CclKN0X1-6 CclKNOXM CclKN0X2~1 CclKNOX2-2 CclKNOX2-3
AtSTM AtSTM AtKNAie AtKNATG AtKNATG AtKNATI AtKNATM AtKNATS AtKNAT4 AtKNAU
355,00 398,00 308,00 382,00 239,00 450,00 60,50 583,00 453,00 451,00
355,00 398,00 308,00 382,00 254,00 450,00 60,50 583,00 467,00 451,00
6 8 % 9 8 % 7 6 % 100%
8 1 % 100%
6 9 % 9 8 % 6 9 % 9 0 %
2,00E-124 1,OOE-140 4,00E-108 3,00E-136 3,00E-80 5,00E-161 3,00E-17 0,00E+00 8,00E-163 3,00E-164
68%
63%
64%
64%
54%
62%
42%
72%
8 1 % 79%
Tao cly di truyen
Cac protein CclKNOX dupc sap day vai phan mem MAFFT phign ban 7 (Hinh 1). Kit qua cho thay cac 10 gen CclKNOX duoc phan chia lam ba phan nhom, phan nhom I voi sau gen, phan nhom II vai ba gen va mot gen thupc phan nhom M. Cac protein tuong iing co cac axit amin chung frong cac vimg bao thii KNOXl, KNOX2, ELK va Homeodomain (dau sao), Cac axit amin dac trung cho phan nhom I va phan nhom II dugc to mau xam nhat va xam dam Cay di truyen dugc xay dung nen
tu cac protein KNOX cua Arabidopsis, cay dao va
cay quyt duong (Hinh 2} cho thay cac protein nay
dupc xep vao ba nhanh khac nhau. Tren moi nhanh,
ton tai cac gen tucmg dong ciia ca ba Ioai. 0 cay cjuyt
duong, so lupng thanh vien cua m6i nhom cua hp
KNOX bang vai 6 cay dao. Khi so vdi cay
Arabidopsis, cay quyt ducmg co nhieu hon hai gen
thufic phan nhom 1 nhung lai co it hon mpt gen Ihupc
phan nhom 11, Ket qua nay co the giai thich bJng
hien tugng nhan doi hoac bien mat cua cac gen frong
qua trinh tien hoa.
Tgp chi Cong nghe Sinh hgc 12(]): 113-123,2014
dSngv Hai gen CclKNOXl-I va CclKNOXl-2 tirang thcri dilm phan hoa Ioai giira cay quyt duong va cay
%vin mQX gen AtSTM ciizzky Arabidopsis, &&y\d, dao, Truong hgp ngupc lai o phan nhom 2, khi s\t kit qua cua su kien nhan doi gen diln ra sau thai kien nhan doi gen dien ra a cay Arabidopsis nhung diem phan hoa Ioai giiia hai Ioai nay, nhung trudc khong dien ra o cay quyt duong cflng nhu a cay dao.
K N O X l C c l K H O X l - S
C c l K H O X l - S C o l K t l O X l - 4 C c l K N O C l - 3
C c l R N O X l - l
C c l K N O X l C e l K N O X : C c i K N o x : C c l K K O C : C o i K H o x : C c l K N O X M
C c l K N O X 1 - C e l K N O X 1 - C c l K N O X 1 - C c l K N O C l - C c l K N O X l - C c l K H O X l - C o l i a K » H C a l K N 0 X 2 -
caixsaxz-
C c l i a K I X 2 -
C e l K N O X l - C o l K N O X l - C c l K H O X l - C c l K N O X l - C c l K N O X M C a l K N O S 2 -
C c l K N O X l - C c l K H O X l - C a l K H O X l -
C a l X H O X l - C B I K N O X I - C o l K M O X M CC1KII0X2- C C U C N O Z 2 -
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V M M D A A Y P Q Y Y H E N G V - G N P F F M D C T T L A P P M V M D A T Q P Q Y Y I D S T V M G N P E
Hinh 1. Kdt quS sdp ddy cac protein trong hp KNOX 6 cdy quyt dirdng
Cao Phi Bang & Christophe Dunand
AtKHATS AIKHAT3 AKNATH ' PpKNOPEM KHOX phan nhom M
KKOX p h i n nhfim II
Hinh 2. Cay di truyen dup'c thiet lap tiJ" cac gen KNOX a ba loai Arabidopsis thaliana. Citrus Clementina vd Prunus persica
C i c dac diem cua cac KNOX if cay quyt d i r ^ g Cac gen CclKNOX co chiia tu hai toi nam infron (Hinh 3), frong do nam gen mang 4 infron, hai gen mang 3 infron, hai gen mang 5 infron, duy nhat mpt gen mang 2 infron la gen thupc phan nhom M, Cac
gen CclKNOX ma hoa cho cac protein c6 dp dai hi 124 tai 450 amino acid, tucmg ung vai khoi lupng phan tli tit 13,7 kDa toi 50,6 kDa. Cac protein KNOX CO diem dang dien (pi) dao dpng tir 4,44 tdi 6,16 (Bang 1).
Hinh 3. C^u tnic exon/intron cua cac CclKNOX. Cac exon duoc the hi#n bai hgp m^u trlng, cdc inlron dugc thd hi$n bin duang ke mau den.
Tgp chi Cong nghg Sinh hoc 12(1): 113-123, 2014
Sy phSn bo cua c&e gen trong ho KNOX v c l y q u ^ 6vtmg
Cac gen CclKNOX chi dugc phan b6 fren 5 frong tdng s6 9 nhilm sac the (Hinh 4). Nhilm sdc th^ s6 1 c6 hai gen (CclKN0X2-I va CclKNOX2-2), nhilm sSc tha s6 2 CO ba gen (CclKNOXl-I, CclKNOXMva
CclKNOXl-2), nhiem sac the so 3 co hai gen (CclKNOXI-3 va CclKNOX2-3), nhilm sic thl so 4 chi CO mpt gen (CclKNOXl-4) va cuoi cung la hai gen CclKNOXl-5 va CclKNOXl-6 nSm fren nhilm sac the so 5. Hinh 4 ciing chi ra rang hai gen CclKNOXI-l va CclKNOXl-2 la k i t qua ciia su kien nhan doi manh cua nhiem sac the so 3.
Hinh 4. Vj tri cCia cSc gen KNOXU&n cac nhilm sac the cua cay quyt ducrng
XAc djnh c i c yeu to cis trong promoter cua cac gen KNOX
Ket qua frong bang 4 ggi y rang cac gen CclKNOX it chju su dieu hoa boi cac stress vo sinh cua moi truong. Khong mpt promoter ciia gen KNOX n^o mang yen to cis HSE (vj tri gan ciia tac nhan dieu hoa phien ma HSF, cam img bai nhiet dp cao) frong viing promoter ciia chiing cung nhu cac yeu to cis MYBR (vi tri gdn cua lac nhan dieu hoa phien ma MYB), NACR (vi tri gin cua tac nhan dieu hoa phien ma NAC) va ABRE (vi Iri gan cua tac nhan dieu hoa phien ma ABF), Irong do cac tac nhan dieu h6a phien ma MYB, NAC va ABF duac cam img boi dieu kien thieu nuoc ciia moi truang Mot so it gen CclKNOX c6 thl bi dilu hoa bai nhiet dp thdp v6\ Stf c6 mat cua cac ylu 16 as trong promoter cua chiing, nhu gen CclKNOXl-3 co chua mpt yeu to CRT (vj Iri g5n ciia cac lac nhan dieu hoa CBF), cac gen CclKN0X2-l, CclKNOXM, CclKNOXl-3 ya CclKNOXl-5 CO mang mpl ylu l6 MYCR (vi tri gSn ciia tic nhan dieu hoa phien ma ICE) Irong khi do
khong promoter ciia gen nao co chua yeu to CM2 (vi tri gan cua tac nhan dieu hoa phien ma CAMTA).
Cac gen CclKN0X2-l, CclKNOXI-I, CclKNOXl-3 va CclKNOXI-5 c6 the bi dieu hoa bai anh sang v6i su CO mat ciia ylu t l SORLIP (vi tri gan ciia cac tac nhan bi dilu hoa boi cac phytochrome A) Irong promoter ciia chiing.
Trong moi lien he voi cac phytohormone, su co mat cua cac yeu to cis trong vimg promoter gpi y rdng mot vai gen CclKNOX c6 the bi dieu hoa boi mpt so phytohormone khac nhau, Yeu to cis AuxRE (vi Iri gan ciia tac nhan dieu hoa phien mg ARF, tra lai vai auxin) chl Ion tai tren promoter cua ba gen CclKNOXI-1, CclKNOXMvh CclKNOX2-3.
Y I U to GARE (vi tri gin ciia tac nhan dieu hoa phien ma GAMyb, tra loi vai gibberellin) Ion Iai tren promoter cua bon gen CclKN0X2-l, CclKNOX2-3, CclKNOXI-5 va dac biet la ciia CclKNOXM (3 trinh Iu lap lai), Chi duy nhdt mpt ylu to CRE (vi Iri gan ciia tac nhan dieu hoa phien 119
Cao Phi Bdng & Christophe Dunand ma ARR type B, tra lai voi cytokinin) va mot yeu
to JARE (vi tri gan cua tac nhan dieu hoa phien ma BBDl, tra lai vai jasmonat) dugc lan lugt tim thay trong promoter cua gen CclKNOXM-va CclKN0X2- 3. Co thl cac gen CclKNOX bi dilu hoa nhieu nhat boi cac hgp chat Brassinosteroid voi sir co mat ciia yeu to BRRE (vi tri gan cua tac idian dieu hoa phien ma BZRl) tren promoter ciia ba gen va cua E-box (vi Iri gan cua tac nhan dieu hoa phien ma BZR2} tren promoter cua bay gen khac nhau, cac tac nhan dieu hoa phien ma nay dupc cam ung bai cac hpp chat Brassinosteroit.
Cac kit qua phan tich in silico viing promoter cua cac gen CclKNOX ggi y cac gen nay it chju anh huang ciia hiu het cac tac nhan stress vo sinh ciing nhu cac phytohormon. Gia thuyet nay can dupc kiem chung bang thuc nghiem, nhung it nhat cho tdi hieu nay, su cam ung bieu hien gen KNOX duai anh huong cua cac tac nhan vo sinh hoac phytohormon van chua dugc bao cao. Trong khi do, cac KNOX dugc biet den nhieu han trong vai fro la gen Idem soat sir sinh tong hop mot so phytohormon, KNOX dieu tiet su can bang giira gibberellin va cytokinin (Hay, Tsiantis, 2010)
Bang 4. Cac yeu to cis tfin tai trong promoter cua cac gen CclKNOX.
Gene HS E
CR T
MYC R
SORLI C M 2 P
MYB R
NAC R
ABR E
ERE/
GCC AuxR
•box E CAR E
GARE s
CR E
JAR E
G- bo X
BRR E
E- ho X
SA E CclKN0X2-1
CclKNOX2-2 CclKNOXl-1 CclKNOXM CclKNOXl-2 CclKN0C1-3 CCIKN0X2-3 CclKNOXI-4 CclKNOXI-5 CclKNOXl-6
2 1 1 1 1
2 4 3 1 PhSn tich su- bieu hien gen
Chiing toi nhan thay co 6 trong t6ng s6 10 gen CclKNOX bieu hien trong cac co quan co chiia mo phan sinh dinh a cay quyt duang, voi so EST dupc tim thay tir 2 cho tai 22 tiiy thugc vao tirng gen va nhom gen (Bang 5), Chi co ba trong t6ng s6 6 gen KNOX phan nhom I bieu hien, voi s6 EST duoc tim thdy la 2; 2 va 8 ciia ldn lugt cac gen CCIKN6X1-2;
CclKNOXI-4 va CclKNOXI-6. Khong co EST nao ciia gen CclKNOXM dupc tim thdy frong khi ca ba gen KNOX phan nhom 11 dlu bilu hien voi s6 EST lan lupt la 8; 22 va 15 tuong ung vai cac gen CclKN0X2-l; CclKNOX2-2 va CclKNOX2-3. Nhu vay, cac gen KNOX Ihupc nhom II cua cay quyt duong bieu hien manh a cac co quan co chua mo
phan sinh dinh. Cac ket qua nay kha phii hpp voi nhiing ket qua thuc nghiem thu dupc ve sir bieu hien cua cac gen KNOX a cac loai cay khac, Irong do cac gen phan nhom 1 chi bieu hien a mo phan sinh dinh va hau nhu khong bieu hien a la, cac gen phan nhom II bieu hien a nhilu mo khac nhau vii phan nhom M chu yeu co vai Iro Irong su phat Inen ciia la kep (Hay, Tsiantis, 2010; Testone el al, 2012). Ket qua nay cho phep dat ra gia thuyel rang cac gen thupc phan nhom II giii mpt vai fr6 quan trpng trong su phat Inen ciia cay quyt ducmg.
Trong khi chuc nang ctia cac gen thupc phan
nhom I va phan nhom M dugc nghien ciiu kha
nhieu o nhieu loai cay khac nhau, chiic nang ciia
cac gen thupc phan nhom II con it dupc bilt den.
Tgp chi Cong nghe Sinh hgc 12(\): 113-123,2014
B i n g S. K & qua k h i o sdt s i / biSu hi#n ciia cdc gen CclKNOX trong cdc m6 c6 chira mo phan sinh dinh.
Gen 6 liryng EST EST
CclKNOXI-1
CclKNOXI-2 3634216 CclKN0C1-3
CclKN0X1-4 CclKNOXI-5
CclKNOXI-6 2866059 CclKNOXM
CCIKN0X2-1 2862702
DY297476FC915799 0X288796,1 FC915316.1
DY272850 DY275868 DY277574 DY292298 FC879697 FC887015 FC895362 FC916864
CX293598 FC889112 DY264276 DY287060 FG880690 FC901590 DY263271 DY304873 FC902136
CX296794 DY272900 DY272968 DY292673 FC879661 FC893951
DY264484 DY267528 DY281097 DY282117 DY287042 DY294266 DY296327 DY302371 DY259266 FC876475 FC884128 FC885327 FC888391 FC896357 FC896823 FC902154
DY272799 FC876239 FC902554
4DY267528DY281097 6DY296327DY302371 8FC885327FC888391 4 FC906935 FC909997 9DY287055DY290610 9 FC882822 FC894168 I 4 FC904889
KET LUAN
Trong cong frinh nay, chung toi da xac djnh va phin tich in silico chi tie! hp gen KNOX a cay quyt duimg, bao gom vi^c xac dinh long so gen Irong he gen, cau tnic gen v i cic dac diem ciia cic protein tucmg img, xep loai cac gen, vi tri ciia cic gen tren cic nhiem sdc the. Dong thai chiing toi ciing phan tich viing promoter v i khao sit sir bieu hien gen ciia cac gen niy, Ket q u i nay se mo duong cho vi?c tach dong gen v i phan tich chuc nang cua cac gen trong hp KNOX a cay quyt duang cilng nhu cac loii ciy khic Irong hp Cam.
LM cam on: Cong trinh ndy dugc hodn thdnh vai su ho trg kinh phi lir chuong trinh nghien ciru khoa hoc eg bdn cua Trudng Dgi hgc Hiing Vuong Chan thanh cdm on Giao su Chantal TEULIERES va Pho gido su Chrisliane MARQUE Dai hgc Paul Sabatier (Toulouse III), Cgng hoa Phdp ve cac trao doi vd gop y quy bdu trong sudt qud trinh thuc hi?n cong trinh nay.
TAI LI$U THAM KHAO
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Tgp chi Cong ngh$ Sinh hgc 12(1)113-123, 2014
GENOME-WTOE IDENTIFICATION AND IN SILICO ANALYSIS OF KNOX GENE FAMILY IN CLEMENTINE (CITRUS CLEMENTINA)
Cao Phi Bang*', Christophe Dunand^"^
'Hung Vuong University
'Laboratoire de Recherche en Sciences Vegetales, Universite de Toulouse, UPS. UMR 5546, BP 42617, F- 31326. Castanet-Tolosan, France
'CNRS UMR 5546, BP 42617, F-3I326. Castanet-Tolosan. France
SUMMARY
KNOX genes encode the homeodomain transcripUon factors that play an important role in maintainmg the activity of shoot apical meristem in plants as well as in morphogenesis and secondary growth in many green plant species. In this work, we used the in silico methods to identify and analyze the KNOX genes in the whole genome of Clementine (Citrus clementina) that has high values. Totally, 10 KNOX genes were genome-wide identified in the Clementine. All often KNOX genes contain two conserved domains, KNOXl (PF03790) and KNOX2 (PF0379]). However, two other conserved domains, ELK (PF03789) and Homeodomam (HomeoboxKN, PF05920), only present in nine out often The Clementine KNOX genes are diseoniinuous coding genes which include from two to five introns depending on the gene. Each gene encodes a protein that has from 124 to 450 amino acids m length. In addition, the KNOX proteins are apparently acidic, their pi are in ihe range of 4,44 - 6,16 These CclKNOX genes are distributed on 5 out of 9 chromosomes Two genes, CclKNOXl-} and CclKNOXl-2, were formed in a chromosomal segment duplication event. The CclKNOX genes are classified into three sub-groups, KNOX 1, KNOX II and KNOXM that has 6, 3 and I genes, respectively. The result of promoter in .silico analysis suggested that the CclKNOX genes were weakly regulated by abiotic stresses of environment but strongly regulated by phytohomiones, auxin, gibberellins and brassinosteroides, in particular. Six out of 10 CclKNOX genes express in the tissues containing the shoot apical menstem, among which Ihe subgroup 11 KNOX genes express strongest
Keywords: Gene expression, exon/inlron structure, phylogenetic tree, genome, in silico, KNOX, shoot apical menstem, promoter, Clementine
'Authorfor correspondence E-mail: phihana caofdhxu edu i
