JOURNAL OF SCIENCE OF HNUE DOL 10 1S173/2354-1O59.2O16-00I8 N a m r a l S c i . 2016, Vot. 6 1 , No. 4, pp. 116-121
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XAC DINH, PHAN LOAI VA XAY DUMO BAN DO GEN CUA HQ DEHYDRIN b CAY QUYT (Citrus Clementina)
Cao Phi Bang' va Tran Thi Thanh Huyen^
Khoa Khoa hgc Tu nhiin, Trucmg Dgi hoc Himg Vucmg
^Khoa Sinh hoc. Tnrcmg Dai hoc Suphgm Hd Ndi
Tom tdt. Cac dehydrin (DHN) thupc nhdm II cua ho LEA. Chung ddng vai tro quan trpng trong vi?c bao V? te bao trudc cac tac ddng bat loi ciia moi tradng thong qua kha nang giir nudc va bao ve cac mang sinh hpc cung nhu cac phan tu protein. Trong cdng trinh nay, nhd su dung cac phuong phap nghien cuu in sihco, chiing toi da xac dmh duyc tdng so sau gen mS hda cho cac DHN trong h? gen cua cay quyt (Citrus clemenlina) Sau DHN ciia cay quyt deu cd mang phan manh K dac trung cho cac protein thuoc hp DHN da duoc bao cao. Cac gen trong ho DHN cua cay quyt phan bo d 5 trong tdng sd 9 nhiem sac the. Ket qua phan ti'ch cac motif bao thii cung nhu phan tfch cay pha h^ da chi ra rang cac DHN nay duac xep vao ba nhom SKn, KS va YnSKn Moi nhdm gom cd hai gen. Hai gen CclDHNl va CclDHN6 cd the la san pham ciia mpt sy kien nhan ddi phan manh nhiem sac the dien ra sau qua trinh phan hda loai giiJa quyt va Arabidopsis.
Tir khda: Dehydrin (DHN), ban do gen, cay pha he, in sihco, quyt. xae dmh gen
l.Md-dau
Quyt (Citrus clemenlina) la cay thupc hp Cam, mpt Irong nhiing hp cay an qua quan trgng nhat tren the gidi Theo thdng ke, nam 2009, san lugng ciia cac cay trong hg nay dat 122,3 ttieu tan (FAO 2009). C) Viet Nam, thdng ke cho thay san luang hang nam cua cae cay thupc hp cam, quyt dal khoiing 700 nghin tdn (Thdng ke Viet Nam 2012). Do cd gia in cao, cay quyt la mpt trong cac cay dugc lya chpn tam dai dien frong dy iin giai trinh ty he gen cac cay thudc hp Cam [ I ] .
Dehydrin (DHN) ma hda cac protein thufic nhdm II ciia ho Late Embryogenesis Abundant protein (LEA) [2]. Chiing ddng vai trd quan frgng trong sy fra Idi Ihfeh nghi cua thuc vai vdi cac dieu kien bat Igi Clia mdi tnrdng, dac bi?t lii vat trd bao ve te bao frong qud trinh mat nudc [2, 3]. Cac protein DHN dugc bao cao cd vai trd quan frpng ddi vdi cac ehdi ngii va qua frinh luyen lanh eua cay gd [2].
Cac DHN d u g c dac trumg bdi su cd mat ciia it nhiit mdt frinh tu bao thii gdm cd 15 axit amin giau lysine, EKKGIM(E/D)KIKEKLPG (phan mdnh K). Phan mdnh K thudng phan bfi d ddu carboxyl ciia phan t u protein va cd khd nang hinh thanh cau true xoan luong cyc, cau iruc giii vai tro quan trpng trong sy tuomg tac ciia DHN vdi cac mdng sinh hpc va cac protein [2]. Ngoai ra, DHN cung cd the co cac motif bao thit khdc la phan manh Y va phan manh S. Phdn mdnh Y giau tyrosine, (V/T)D(E/Q)YGNP, phan bd d gdn ddu tan ciing amin va cd mgt bp phan axit amin gidng vdi motif d vi Iri gdn nucleotide ciia cdc protein chaperone d nhieu loai sinh vat [2]. Phiin manh S giiiu serine, gom tir 4 tdi 10 serine). Dua vao edu tnie, cac DHN cd the dugc chia thdnh 5 phan nhdm: Kn, SKn, KnS, YnKn va YnSKn [2]. Gdn day. phan nhdm SKS ldn dau lien da dugc bao cao d fren ddi m a n g cay d u a n g [4].
Vdi vai trd d^c bict quan trpng frong vice bao vc tc bao vd sy cam img m a n h b d i cac dicu kicn bat lgi vd sinh ciing nhu hiru sinh, cac DHN dugc coi la chi thi phiin tii ciia tinh chdng chiu vdi cac dieu kien bdt lgi ciia mdi frudng a Ihuc vat [3]. Trong nghien ctiu nay, chiing tdi hudng tdi xac djnh trinh ty Ngay nhan bai: 24/3/2015 Ngay nhan dang: 26/4/2015
Tac gia lien tac: Cao Phi Sdng. dja chi c-mail: [email protected] 116
Xdc dinh, phan logi vd xdv dung hdn do gen ciia ho Dehydrin er cdy quyt (Citrus clementma) Clia cac DFTN trong he gen eiia cay quyt, xay dyng cay pha he va phan loai cac DFIN. Ddng thdi lap ban dd gen ciia ciic gen trong hp nav,
2. N o i d u n g n g h i e n c i h i
2.1. V a t lieu vii p h u w n g p h i p n g h i e n ciru
* Cff sd da lieu
Trinh ty he gen ciia cay quyt dugc lay tir Wu et al [5]. Cac frinh m DHN cua cay Arabidopsis dugc lay tir Hundertmark va Hincha [6].
* Phuffng phdp
Xdc dinh cdc gen DHN d cdy quyt. Cdc protein DTIN ciia cay Arabidopsis [6] duoc diing liim khudn dd. chuang trinh T B L A S T N dugc sir dung de tim kiem cac gen m a n g dong tren dii li?u he gen Clia cay quyt. Pfam (ca s o dv: lieu cae hp protein [7] dugc sti dung de xac nhan sy hien dien ctia cac
\-ung bdo thli trong trinh ty protein suy dien cita eac iing vien DHN.
Xdc dinh cdc motif bdo thu: Phan mem M E M E dugc su dyng de lach cdc motif tir cac protein DTTN d cay quyt va cay Arabidopsis [8].
Tgo cdy phd he: Cac trinh ty protein DFTN eua cay quyt va cay Arabidopsis dugc sdp day bdng each diing phan mem M A F F T [9]. Cay pha he dugc xay dyng tir cae frinh ur protein DHN da sdp day nhd phan mem M E G A 5 , dir lieu dugc xii li la tat ca cac vj tri va phuang phap Bootstrap vdi 1000 tan lap lai [10].
Lap bdn do gen- Ban dd c u tni ciia gen fren cae nhiem sac the dugc thiel lap nhd phan mem M a p C h a r t [ I l ] .
2.2. Ket q u a v a t h a o l u a n
* Xdc dinh hp gen BHN trong hi gen cua cay quyt
He gen hoan chinh ciia cdy quyt da duge tim kiem vdi cac protein DHN khuon dd ciia cay Arabidopsis nhd sir dung c h u a n g trinh TBLASTN Tdng sd, 6 gen cd the ma hda eho cac DHN da duac xac djnh (Bang I). Ket qua phan tich viing bao thii nhd sti dung Pfam cho thay chi ed 4 DHN la CcU3HN2, CcIDHN3, CclDHN4 va CelDHN5 la cac DHN dien hinh (PF00257). Hai DHN cdn Iai la CclDHN I va CcIDHNd m a n g ddng vdi nhieu DHN thudc nhdm KS da d u a c chi ra d nhieu loai thye vat khac dd khfing tim thay vimg bao thu dac trung PF00257.
Gen
CclDHNl CclDHN2 CclDHN3 CclDHN4 CclDHN5 CclDHN6
Bdng I. H
Ten locusClcleN 10024637 m.g Ciclevl0002349m.g Ciclevl0026675m.B CiclevI0029952m.g Ciclevl0029285 m.g
-
o DHN d cdv iluvt Nhilm siic the
3 5 7 8 8 9
Vi tri 41697369..41697776 42490019..42490852 1699891. 1699246 550850..551472 5894816..5896074 7493081..7493375
Cac protein suy dien dugc dimg Iam khudn de tim cac protein mong ddng d cac loai khdc (ortholog) cd trong c a sd dii lieu cua NCBI Phep kiem tra nay khang dinh cdc trinh tu lim dugc deu thufic ho DTTN. Thuc vay, eac protein da d u a c bao cdo gidng nhat vdi cac CclDHN deu la cac DHN (Bang 2).So lugng gen DHN a cdy quyt it hon d eay Arabidopsis [6], cay d u a n g [4] n h u n g nhieu han d cay nho [12].
Cac gen ma hda DHN cua eay quyt dugc phan bd tren 5 trong tdng sd 9 nhiem sac the Trotig do, chi duy nhit n h i l m sdc t h i so 8 cd mang hai gen la CclDHN4 va CclDHN5. Cdn iai, cac nhiem sac t h i 3; 5; 7 vd 9 chi mang mdt gen thufic hp nay. Dua iren do tuang ddng cao, cd the hai gen CclDHNl va CclDHN6 dugc hinh thanh do mdt sy kien nhan ddi phan manh nhiem sac the.
Cao Phi Bdng va Trdn Thi Thanh Huyen
Protein CclDHNl CclDHN2 CclDHN3 CclDHN4
CclDHN5
CclDHN6
Bdng 2. Cdc gen tirffng dong gan nhat vdi cdc DHN cua cdy quyt cd trong cff sd dii lieu cua NCBI
Mieu tii tren NCBI C O R l 5 [Citrus Clementina X Citrus reliculala]
dchydrm [Cifni.'i x parodist]
dehydrin \Jalropha curcas^
dehydrin 3 [Vitis veshanensis]
>gb]AFI38957.l'| dehydrin 3 [ VitL\- yeshanensi.^]
dehydrin 4 [Vitis yeshanensis] >
gblAF138958.l| dehydrin 4 [ Vitis yeshanensis]
cold stress protem [Citrus trifoliata]
Chi .so circ
237 432 120 81.3
139
108 Chi
so tong 237 432 120 81.3
139
108 Mijc (1$ so sanh 100%
100%
100%
49%
98%
69%
Gia tri E- value 7 E - 7 9 S E - 153
2 E - 3 2 2 E - 1 7
1 E - 3 8
1 E - 2 9 Mirc
a?
tinmg ddng 95%
99%
56%
57%
50%
86%
Ki hieu Genbank AAQ92310.!
AAN7M1251 ADT65201.I AEQ199051
AHQ199061
AAA99963,I
Hmh I. Bdn dd gen cua cdc dehydrin trong he gen cua cdy quyt
* Xdc dinh cdc motif bdo thu, phdn tich cdy phd he vd phdn loai cdc dehydrin
Kgt qua phan tich motif bao thu bang phSn mgm MEME cho chay. phan manh K (trinh tit
KKG[IFL1M[ED]KIKEKLPG[HG1H. hinh 2A) co mat a tit ci cac DHN cua cay quyt voi so i k Ijp
lai khac nhau. Trong do, CclDHNl va CclDHN6 chi co 1 phan manh K, CclDHN2 va CclDHN4 cdng
CO 2 phan manh K Irong khi 3 phan manh K duoc lim thay o CclDHN3 va CclDHN5 Chi mol phan
manh S ([LG]H[RD]S[GN]SSSSSSS[DE][ED][DE], hinh 2C) duoc tim thay a ca sau DHN ciia cay
quyt, nhung vi tri cua phan manh nay lai rat khac nhau, njm cr dau tan cimg carboxyl ciia FsDHNl va
FsDHN6 trong khi nim o phia dau tan ctmg amin ctia bdn DHN con lai Rigng CclDHN4 co mang
phan manh S khong hoan chinh, chi vol hai axit amin Serine. Phan manh Y (DEfYFHlGNP, hinh 2B)
chi duoc tim thiy 6 CclDHN3 (2 ban sao) va CclDHNS (3 ban sao). Nhu vay. cac DHN ciia cay quyt
CO mang cac phan manh bao thti. dac trung giSng nhu a cac DHN ciia loai khac da duoc mieu ta [3].
1 Xac dinh. phdn Inai vd xei-v dimg han dn gen cua hn Dehydrin a cdv ijuvt (Ciirus clemenlina)
Hmh 2. Bieu tuffng trinh tu axh amin cua phan mdnh K (A), phdn mdnh Y (B) vd phan mdnh S (C)
Cae motif bao_thu va vi tn cua chung trong phan nr protein duac the hien ro hon qua Hinh 3, hinh Iv^^^ bieu diln kel qua sap day cac CclDHN nha su dung phan mem MAFFT
lEKKSHMEK 1 ^EKI.P$(,
:S
Hinh 3, Sir sdp ddy cdc dehydrin cua cdy quyt
(Ddu sao chi cdc axil amm bdo tliii. Cdc pfidn mdnh hdo thu ndm trong d. nen xdm, phdn manh K chir in dam, phdn mdnh Y chit in ddm nghieng. phdn mdnh S chit in nghieng)
Cay pha he dugc thilt lap tir cac protein DHN cua cay quyt (Cci) vd cay Arabidopis (At) dugc thi hien d hinh 4. Cac CclDHN nay chi phan bd tren ba nhanh chfnh cua cay pha he dieu nay cho phep xep loai cdc CclDHN vao ba nhdm. Can cu tren each phdn loai cua cdc tdc gia tdiac [4, 6], sau DHN cua cay quyt duge xep vao ba nhdm KnS. SKn va YnSTCn, Trong dd, hai protein CclDHNl va CcIDHNd nam ciing nhanh vdi DITN thupc phan nhdm KnS cua cay Arabuhpsis. nhdm nay gfim cae DHN khdng cd phan manh Y. Hai CclDITN2 va CclDHN4 nam eiing nhanh vdi cac DHN thufic phiin nhdm SKn, nhdm chi cd cac phan manh bao Ihu Ihudc S vd K, trong dd phiin miinh K nam sau phdn manh S (hinh 3). Cudi citng, hai protein CclDHN3 va CclDfIN5 dugc xip vao phan nhdm YnSKn nhd su cd mat cua phan manh Y d phia dau amin cua cac protein suy dien. Phan tfch cay pha he ciing cho Ihdy cd it nhat mdt hien tuong nhiin dfii gen da xay ra trong hp DHN cua cay quyt, tao thdnh cap gen CclDHNl vil CclDHN6. Ket qua nay phii hgp vdi eac hicn hrgng nhdn dfii gen DHN da duac chi ra d cac Ioai khdc [4, 6]. Can cii vdo vj iri cua gen trong city pha he, cd the ket luan sy kien nhan ddi gen CclDHNl \a CclDHNO xiiy ra gan day, sau thai ki biet hda loai.
119
C a o Phi B d n g v a T r d n T h j T h a n h H u y e n
CdDKNd AT3G5D9S0
AT9GES400
AT2G2H90
AT4G39130 | Y K AT3G60970 | Kn I AT1G54410 I
•I J. CclDHNl I K sTLcclDHNe I
Hmh 4. Cdy pbd he duffc xdy dung tir cdc DHN cua cdy quyt vd cdy Arabidopsis
3. Ket luan
Trong cdng trinh nay chimg Idi da xac dmh dugc tfing sfi 6 gen DHN trong loan bd he gen ciia cay quyt. Cac gen nay hiang ddng cao vdi cac DFTN ciia nhieu Ioai thye vat khac. Ket qua phan tfch cac motif bao thu trong cac protein suy dien cho thay cac DHN cua cay quyt cd mang cac motif bao thii dac trung nhu phan manh K, phiin manh Y va phan mdnh S tiiy gen. Dua tren sy cd mat ciia eac motif bao thu, ket hgp vdi phan tich cay pha he da chi ra rang cac gen nay thufic ve ba nhdm KS va YnSKn va SKn. Ban do gen cua hp DHN d cay quyt da dugc chung tfii xay dimg, trong dd ciic DFTN phan bd tren 5 trong tdng sd 9 nhiem sac the. Day la nhung ket qua budc dau cd y nghia quan trpng, md dudng cho vi?c nghien ciiu chiic nang ciia eiie DHN d loai eay nay.
Loi cdm ffn. Cfing trinh nay duoc hoan thiinh vdi sy hfi trg kinh phi tii chuong trinh nghien ciiu khoa hpc ca ban cua Trudng Dai hgc Himg Vuang.
T . \ l LIEU THAM KHAO
[1] Ateza P, Juarez J, Hernandez M, et al., 2009. Recoveiy and characterization of a Citrus clemenlina Hort. ex Tan Ciemenule.s' haploidplant .selected to establish the reference whole O/rw.yge'/iome'.Te^uefice. BMC Plant Biology, 9(1), 110. DOI: 10.11 S6/1471-2229-9-110.
[2] Rorat T, 2006. Plant dehydrins - Tissue location, structure and function. Cellular, Molecular Biology Letters, 11(4), pp. 536-556.
[3] Hanin M, Brini F, Ebcl C, Toda Y, Takeda S, Masmoudi K, 2011 Plain dehydrins and stress tolerance: versatile proteins for complex mechanisms. Plant Signaling & Behavior, 6(10), pp. 1503-1509.
[4] Liu CC, Li CM, Liu BG, ef ai, 2012. Genome-wide Identification and Characterization of a Deliydrin Gene Family in Poplar (Populus inchocarpa). Plant Molecular Biology Reporter, 30(4), pp. 848-859.
[5] Wu GA, Prochnik S. Jenkins J, et at., 2014. Sequencing of diverse mandarin, pummeto and orange gemmies reveals complex histoiy of admixture during civus domestication Nature Biotechnology, 32(7). pp. 656-662.
[6] Hundertmark M, Hincha DK, 200S. LEA (lute embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics, 9( 1), 118.
[7] Finn R D , Balcman A, Clements J, et al, 2014. Pfam: the protein famdies database. Nucleic Acids Res, 42, pp. D222-D230.
Xdc dinb, phan loai vd xdy dung bdn dn gen cua ho Dehydrin d cdy quyt (Cilru.s clemenlina) [8] Bailey T L , Williams N, Misleh C. Li W W , 2006. MEME- discovering and analyzing DNA
and protein sequence motifs. Nucleic acids research, 34(suppl 2). W 3 6 9 - W 3 7 3 .
[9] Kaloh K, Standley D M , 2013. MAFFT multiple sequence alignment .software version 7.
improvements in performance and usahilily. Mol. Biol, Evot , 30(4), pp. 112-7^0 [10] Tamura K, Peterson D, Peterson N, Stecher G. Nei M, Kumar S. 201 L II4EGA5. molecular
evolutionary genetics analysis using maximum likelihood, evolulionary distance, and maximum parsimony methods. Mol Biol Evot 28, pp. 2731 -2739.
[11] Voorrips RE, 2002. MapCharf Sojhi'are for the Graphical Presentation of Linkage Maps and QTLs. Joumal of Heredity 93, pp. 77-78.
[12] Yang Y, He M, Zhu 2 , et al. 2012 Identification of ihe dehydrin gene family from grapevine species and analysis of their responsiveness lo various forms of abiotic and hiolic .j/rgis. BMC Plant Biology.'12. 140. DOL 10 1186/1471-2229-12-140
ABSTRACT
Identification, classification and chromosome mapping o f t h e dehydrin gene family in Clementine oranges (Citrus Clementina)
Dehydrins (DHN) are classified as a group 11 Late Embryogenesis Abundant protein (LEA), These proteins play a crucial role in cellular activity making it possible for cells to survive the adverse effects of environmental stress by means of increased water relenbon, as •well as affording protection to membranes and molecules. Tn this work, w e identified six DHN genes in the whole genome of Clementine oranges (Citrus clemenlina) using the in silica method. Alt six DHN genes contain the K- segment which is characteristic of all reported DFTN proteins. The chromosome map constructed by MapChart show Ihat in Clementine oranges, DHN genes are present in 5 out of 9 chromosomes. Based on the results of conserved motif as well as a phylogenetic tree analysis, six Clementine DHN genes were identified as belonging to three subgroups, SKn, KS and YnSKn. Each subgroup contains two DHN genes. The two closest genes, CclDHN6 and CclDHNl. might be products of a segment duplication event which occurred after speeiation between citrus and Arabidopsis.
Keywords: Dehydrin (DHN), chromosome mapping, phylogenetic tree, in sihco, Clementine, gene identification.
