CVv157V60S422015121.pdf

JOURNAL OF SCIENCE OF HNUE DOI. 10,18173/2354-1059.2015-00017 NamralSci. 2 0 l 5 , V o l . 6 0 , N o . 4 . p p . 121-126

This paper is available online at blip //stdb.hnue.edu.vn

XAC DINH, XEP LOAI VA NGHIEN ClTU SlT BIEU HIEN CUA CAC GEN DEHYDRIN (3 Ck\ YlTNG {Sesamum indicum L.)

Tran Thi Thanh Huyen', Cao Phi Bang^ va D6 Thi H5ng'

Khoa Sinh hge. Truang Dat hoc Suphgm Hd Not 'Khoa Sinh hpc, Tnr&ng Dgi hoc Hiing Virong

Tom tat. Cac dehydrin (DHN) thuoc nhom II cua hp LEA Chiing dong vai tro quan Irong irong viec bao vc te bao tnrdc cac tac dong bat Igi ciia moi inrdng thong qua kha nang giii nuac va bao ve cac mang sinh hgc cung nhu cac phan tii protein Trong cong trinh nay, chiing loi da xac dinh dime tong so 6 gen ma hoa cho cac DHN trong h? gen ciia cay virng (Sesamum indiaim L ). Ca sau DHN ciia cay vimg deu co mang phan manh K dac Inmg cho cac protein thuoc ho DHN da dugc bao cao Ket qua phan tich cac motif bao thu cung nhu phan tich cay phat sinh chung loai da chi ra rang cac DHN nay dugc xep vao 3 nhom. YnSKn {ba genl. SKn (hai gen) va KS (mgi gen) Ket qua khao sal sir bieu hien gen cho thay cay virng co it nhai ba gen DHN bieu hien trong hai dang phat tnen.

TirAAoo.'Dehydrin (DHN), cay virag, cay phat sinh chung Ioai, bicu hien gen.

1. Mff d a u

Vimg (Sesamum indicum L.) la lodi cay thudc gidng vimg (Sesamum). hp Pedaliaceae, la mpt trong nhung cay trdng cd hat chua ddu cd nhat va dupc trdng a cdc vung nhiet ddi vd can nhiet ddi cua chau A, chdu Phi vd Xam MI [1]. Vimg cd ham lugng dau rdt cao, tir 45 tdi 6 3 % cua hat da tach vd.

Hat vimg gidu protem, cdc vitamin, bao gdm ca niacin, chdt khoang va cac lignan nhu Id sesamolin va sesamin. Hat virag dupc su dung ldm tbuc an ciing nhu trong dupc hoc [ i ]. Nhd cd gia tri ldn ddi vdi ddi sdng con ngudi, cay vijmg duoc trdng rpng rai tren the gidi. Theo thong ke cua td chuc Ndng - Luang the gidi (UN FAO 2010), tdng dien tich udng vimg dat 7,8 trieu ha va tdng san lugng thu duac la 3,84 trieu tdn. He gen ciia cay vimg da dupc giai trinh tir todn bp vao nam 2013 [2]

Dei^drin (DHN) diudc nhdm II cua ho protein cd sd lugng ldn trong pha phdt trien phdi mupn (Late Entfwyogenesis Abundant protein. LEA) [3]. Cdc protein nay ddng vai trd quan Uong trong su ua ldi Uiich n ^ ciia thuc vat vdi cac dieu kien bdt lgi ciia mdi trudng [3]. Cac DHN dugc dac tmng bdi su cd mat cua h nhdt mdt trinh tu 15 axit amin bao thu giau lysine (phdn mdnh K), EKKGIM(E/D)K1KEKLPG, d diu carboxyl cua phdn tu protein va cd kha ndng hinb tbanh cau tnic xoan ludng cue, cau tnic giir vai u d quan trpng trong su tuang tdc ciia DHN vdi cac mdng sinh hpc va cac protein [3]. Ben canh dd, DHN ciing cd the cd phdn manh Y va phdn mdnh S Phan manh Y gidu tyrosine, (V/T)D(E/Q)YGNP, phdn bd d gan ddu tan cdng amin va cd mdt bd phdn axit amin gidng vdi motif o vi tri gan nucleotide cua cac protein chaperone d nhiSu loai sinh vat [3], Phan manh S giau serine, gdm tu 4 tdi 10 serine.

Dua vdo cdu tnic, cac DHN cd th^ dugc chia thdnh 6 phdn nhdm: Kn, SKn, KnS, YnKn vd YnSKn va SKS[4]. . . ,

Cac DHN dugc coi la chi thi phan tu cua tinh chdng chiu vdi cac dieu kien bat Igi ciia mdi trudng d thuc vdt [3]. Trong nghien ciiu nay, chung tdi hudng tdi xac dinh uinh tu cua cdc DHN trong he gen cua cay vimg, xay dung cay phdt sinh chung loai va phdn loai cac DHN. Ddng thdi chung tdi khao sat Ngay nh|n bai- 5/3/2015. Ngay nhan dang: 27/4/2015

Tac gia lien lac: Tran Thi Thanh Huyen, dia chi e-mail Iranthanhhuyenijijhnue.edu vn

Tran Thanh Huyen, Cao Phi Bang va Do Thi lUng

sv bieu hi^n cua cae gen DHN a loai cay nay. K6t qua nghien cuu thu dugc gdp phdn bd sung dan lieu ve ho DHN d loai cdy trong quan trpng nay, lam tien de cho eac nghien cuu va iing dung kT thuat di tmyen cd lien quan den hp gen nay d cdy virng cdng nhu cdc lodi cdy trdng khac.

2. N o i d u n g n g h i e n c i i u 2.1. Phirong p h a p n g h i e n ciru

* Cff s& dGr lieu

Trinh Ur he gen cua cdy virng dupc Idy tu Zhang *•( al. (20I3]''(2] Cac trinh tu DHN cua cay Arabidopsis dugc lay tir Hundertmark vd Hmcha (2008) [5],

* Xdc dinh ede gen DHN & cdy vimg

Cac protein DHN cua cdy Arabidopsis [5] duac dimg lam khudn dd, chuang trinh TBLASTN dugc su dung de tim kiem cac gen tuang ddng tren dir lieu he gen cua cay vung. Pfam (ca sd dir lieu cac ho-protein, [6]) duac su dung de xac nhan su hien dien cua cac vung bao thii trong trinh tu protein suy dien cua cac ung vien DHN.

* Xdy dung cay phdt sinh chung logi

Cdc Irinh tu protein DHN ciia cay vimg va cay Arabidopsis dupc sap day bang each dung phdn mem MAFFT [7]. Cay phdt sinh chung loai duoc xdy dung tu cac Uinh tu protein DHN da sap day nhd phan mem MEGA5 [8]

* Khao sdt sg bieu hien gen

Su bieu hien cua cac gen dupc khao sal bang each dem sd lupng trinli tu bieu hien dugc danh dau (EST) trong cac tap hgp EST cua cay vimg (taxid:85681) tdn tai tren trang web NCBI nhd chuong trinh TBLASTN.

2.2. Ket qua n g h i e n ciin va thao luan 2.2.1. Ho gen DHN trong he gen ciia cay virng

Chiing tdi su dung cac protein DHN cua cdy Arabidopsis lam kliudn dd de tim kiem cac gen tuong ddng trong he gen cua cay virng nhd su dung chuang trinh TBLASTN. Budc ddu, chung tdi xdc dinh dugc sau gen co the ma hoa cho cac DHN da dugc xac dinh (Bang 1). Ket qua phan tich vung bao thu nhd su dung Pfam cho thay chi cd 5 DHN (SiDHNl, SiDHN3, SiDHN4, SiDHN5 vd SiDHN6) la cac DHN dien hinh (PF00257). DHN cdn lai (SiDHN2) tuong d6ng vdi nhi^u DHN thupc nhom KS da duac chi ra d nhieu loai thuc vat khac du khdng tim thdy vung bao thu dac trung PF00257. So vdi d mdt sd loai thuc vat khac, sd lucmg gen DHN a cay vimg bang vdi a cdy quyl dudng [9], it ban d cdy Arabidopsis [5], cdy duang [4] nhung nhieu bon dcaynlio [10].

Gen

SI DHN 1 SiDHN2 SiDHN3 SiDHN4 SiDHNS SiDHN6

Bdng 1. Cdc DEN a cdy vimg vddac diem Ten

locus

SIN 100291)2 SIN 1003537 SIN 1007393 SIN_1009023 SIN 1014753 SIN 1022224

Phan nhom

YnSKn KS SKn SKn YnSKn YnSKn

Chieu dai gen/CDS

(bp) 654/807 327/327 708/1008 639/735 483/615 453/539

Chilu dai protein

(aa) 217 108 235 211 160 150

Khoi luong protein (kD) 22,05 12.09 26,76 23,66 17.06 15,76

cua c

Pl

7,87 6,51 5,07 5,22 7,99 6.79

hung

GRAVY

-0,99 -1,92 -1,53 -1,57 -1.29 -1.29

Chl so beo 33,46 30,74 43,23 38.91 42.75 31.40

So intron

1 0 1 I 1 1 Cac DHN cua cdy vimg cd cd dd dai tu 327 tdi 1008 nucleotit Trong sd 6 gen DHN cua cay vimg, nam gen ma hda khdng lien tuc, cd mdt intron (Hinh 1). Protein suy dien cua chung cd chi6u dai tu 108 tdi 235 axit amin, tucmg ung vdi khdi lugng phdn tu tu 12,09 kD tdi 26,76 kD, trong do, SiDHN2 cd kich thudc nhd nhdt, SiDHN3 cd kich thudc Idn nhat. Cac SiDHN cd di£m ddng dien (pl) 122

Xdc dinli. .xep Ioai vd nghien ciru .sir hieu hieu cua cdc gen deltydiin a cdy virng (Sesamum iiuiwum L )

dao ddng tir 5,07 tdi 7,99 (Bang 1). Trong dd, bdn DHN cd tinh axil yau (SiDHN 1, SiDHN3, SiDHN4 vd SiDHN6), SiDHN2 vd SiDHN5 cd tinh kiem y6u. Ca sau SiDHN deu c6 tinh ua nudc cao vdi cac gia trj G R A V Y n i m trong pham vi tir -0,99 tdi -1,92, trong do SiDHN2 c6 do ua nudc cao nhdt Cac chl sd beo cua cac DHN d cdy vung kha cao, trong khoang tii' 30,74 tdi 43,23. Chi sd beo dai dien cho the tich t u o n g ddi bi chiSm bdi cac axit amin cd chudi ben beo nhu alanine, valine, leucine va isoleucine. Chi sd nay thudng cao d cdc protein ben nhiet ban so vdi cdc protein thudng. Cac dac didm cua cac DHN cua cdy virng phii hpp vdi cdc DHN cua cdy d u a n g da dupc bao cao [3, 4, 9].

—SiDHH? •

— SiDHH? •

—SiDHNJ*

Hinh I. Cau true exon/intron cda cdc gen dehydrin &cdy vimg

2.2.3. Xac dinh cac motif b a o thu, phan tich cay phat sinh chung loai va phdn loai cac DHN SiDHN3 MA^•EVKr;'!K---^HFs^:7DF;^F:A.^?^KTF,F./;\AVF,s3n?.----[,r•[•^M ' S1DHN4 MftDEAQ-fKH HDENKSGEPCDSSS P/^VEATLiRG-~Ll= DfG S1D.HN6 MAQ^GDQVG BaTIffiirGIWlMT-~IMBrca5P','KHBPGG

SiDHNl 1-lA aVG RKTDEYGNPIRiT lJEyGMPIliRT3AGGGTI,GDYGPITGAr'HTP??>

SiDHN5 y.1DLROBSeBPICLADQirGJ9F,/i<LT---DBlri3HP'.-"IT3 JA SID:-IH2 HS

SlDUNT SiDflN4

S.!.D;HN6 TCC.-bTTCAYGI TrC::-TIGTT'5AY'., SlD-lNl TAG?Y.».NQYAATGAyATDFhGi?TGA'J'GTjPYGATGTTGAYGTD;>rJATGTTGflY3TDr';

SiDfJNb TIGQTA AVE?QSPPPP?OQ000EOL SiDHNZ

SiD'lN3 GLI.EKIHRSWSSSSSS5J5EEEVEEGGFKKKK-KKG SiD.HN4 S-L0KLS'RSSSSS5SSSnEEEVEEGCLKKKK-KKG S-iKHNS GGAPPAAIJyVPAAH¥5S--E:)DGSGi;RR-KKG SiDilNl A P G T T G A Y G T : 1 C C S G G G : A A G T T D V G - L C G V M B R P 5 G S S S 5 S S S S - - E 3 D G L G G R R - K K G

SitiHN5 PKi;GSSSSSSS5"S--E?ljGCGGR»KKKG

SiDKfJ2 G

SiD:HN3 LKEKIKDKLSCE:<KEE---EKAi;EVKirEt)TAVP lEKCJDArTH IHEFEBKK SiDHN4 LKEK1KEKVSGEKKEE---D KKCDETiTTH OSPEEKK SiDHN6 LKEKIKEKUGGGHRD---DQAHa-GAATTTPrjf-GGYGYi:HGG EH KKK SiDHHl LKBKIKBKLPGG ITST(;GGGYG:-irYGGAEB?HH EKK SiD.HNS lKKKlKBTFGGG:-(HKliKGEEA/iHS-TPATlTAASiKi;3TrVPG--0A!?EH EKK SiDHNS IIHKIEEKLGMGGHKE-GEKHGEDHK.»AJHSEHK?EHGV---GE-)K?EHGfGEh-<Pf BKE S.I.D:4N3 GFLDKIKBKI.PCGi<K7EF.VA.i..-P.HHAAKCATTPEAEGKBKItGPLEiaKEKIPGYri3K.';rjF SiD>IN4 GLIJ)KIK15KLP'iGKKEEEVEAPPPTFAAPi::YTTJAEGKEKKGEl«KIKEKIPGY;:GKaDE S I D : 4 N 6 Q I M D K I K E K L P G G H K H

S.LD:IHI OdMEKIKEKLPGriOliK SlD.-lNS GVMERIKEKLPt.HGHKH^AKEF

SIDHN2 GLlBKIKDKlH..EGGEHGSKij Ei<KK:<KFKK'<HEKGHEHGK5SSS0S

S i D H H i EKEKEKEGAYH s.lD:^^J'l EKEEAAEACK- yiDHNfr SiDHNl S.1.DHH5 SiDHN2 050

Wnh 2. Cac dehydrin cda cay virng (Ddu sao chi cdc axit amin bdo thd, phdn mdnh K chir in dam, phdn mdnh YchU in dam nghieng, phdn mdnh S chir in nghieng)

Tran Thanii Huyen, Cao Phi BSng va Dd Thi HSng

Ket qua phdn tich motif bao ihu cho Ihay, phan manh K co mat d tat ca cac DHN ciia cay vimg vdi sd ldn lap lai khac nhau. Trong dd, SiDHN3 vd SiDHN4 cd ba ldn lap lai. SiDHN 1, SiDHNS vd SiDHNd cd hai ldn lap lai, rieng SiDHN2 chi cd 1 lan l^p lai. Tuang tu, phdn manh S dugc tim thdy d ca sau DHN cua cdy vimg, nhung vj Ui cua phdn mdnh nay lai khde nhau. ndm d dau t ^ ciing carboxyl cua SiDHN2 nhung ndm d trudc phdn manh K cua cac gen cdn lai Rieng SiDHNd cd mang phdn manh S khong hodn chiiJi. chi vdi hai axit amin Serine. Phan mdnh Y chi duac lim thdy d SiDHNI, SiDHN6 (2 ban sao) va SiDHNS (3 ban sao) (Hinh 2). Nhu vdy, cdc DHN cua cdy vung cd mang cac phdn manh bao thu, d^c trung gidng nhu d cac DHN cua loai khac da dugc mieu td [3].

Cdy phdt sinh chung loai dugc thiet lap tir cac protein DHN cua cdy virng (Si) vd cay Arahidopis (At) dugc the hien d Hinh 3. Cac SiDHN nay chi phdn bd tren bdn nhdnh chinh cua cdy phat sinh chung loai. Can cu vao vi tri phan bd tren cay phat sinh chung loai ciing vdi phdn tich cac motif bao thii, cdc DHN cua cay virng dugc xep vao ba nhdm. Theo sir phdn loai cua cac lac gid khac [4, 5, 9].

sau DHN ciia cdy virng dugc xep vao ba nhdm KS, SKn va YnSKn Trong dd, ba protein SiDHNI, SiDHNS vd SiDHNd nam cung nhdnh vdi cac DHN thudc phdn nhdm YnSKn cua cdy Arabidopsis.

nhdm ndy gom cdc DHN cd ca ba phan manh bao thu cua DHN, Hai gen SiDHN3 va SiDHN4 nam cung nhanh vdi cac DHN thudc phan nhdm SKn, nhdm chi cd cac phdn manh bao thu thudc S va K, trong dd phdn mdnh K nam sau phdn manh S (Hinh 2). Cudi ciing, SiDHN2 duoc xep vao phan nhdm KS, nhdm nay khac vdi nhdm SKn d vi Ui tuong ddi cua hai phan manh bdo thu K va S (nhdm KS cd phan mdnh S d phia sau ciia phan mdnh K) Phan tich cay phat sinh chung loai ciing cho thdy co hai hien hrgng nhan ddi gen da xay ra gan day trong hg DHN ciia cay virng, tao thdnh cap gen SiDHNI va SiDHNd, SiDHN3 vd SiDHN4. Hien tugng nban ddi gen DHN a cay virng ciing gidng nhu d nhieu loai khac da dugc nghien cuu [4, 5, 9].

AT3G50980 - AT5G66400 SIDHM - SiDHNS

78 I

YnSKn

SiDHNS AT2G21490

AT4G39130|YK AT3G50970 |Kn -AT1G54410

991 SiDHN2 AT4G38410

-AT1G20450 AT1G20440

AT1G76180 -SiDHN3 - S I D H N 4

KS

SKn

Hinh 3. Cdy phdt sinh chung loat dui;c xdy dirng tir cac DHIW cua cdy Vitng (Si) vd cay A. thaliana (At),

(vdi cdc tham bien: thugt loan Maximum Likelihood, mo hinh Jones-Taylor-Tharnton (JTT), phuangphdp Bootstrap vdi 1000 lan Idfj lai. gid trf bootstrap duac the hien

tren moi nhdnh, li le xich la so ammo acid thay the tren mat vi trii

Xdc djnh, xep logi vd nghii «• bidu hien cua cdc gen dehydrin cr cdy virng (Sesamum indicum I..) 2.2.4. S y bieu hien c u a cac D H N ciia cay v i m g

T a p hcrp cac EST d u g c xay dung bdi nhieu nhd khoa hoc tir cac loai md khac nhau cua cdy vimg, trong dd cd hat dang phat trien. Day la cac hat dirge thu thap trong khoang thdi gian tir 5 ngay tdi 30 ngay sau thdi di6m nd hoa. N h d su dung chuang trinh TBLASTN. chung tdi tim thay cac EST ciia it nhdt 3 trong tdng .so 6 gen DHN cua cay virng, la cac gen SiDHNl, SiDHNl va SiDHNS (Hinh 4), trong do sd iugng E S T cua gen SiDHN2 (12 EST) nhieu gap ddi .so vdi hai gen con lai (ciing 6 EST).

Cdc EST nay thudc vd cac tap hgp EST thu duac tir mo hat dang phat trien cua cay^ virng Khdng cd EST nao d u g c lim tbay tucmg ung vdi ba gen SiDHNi, SiDHN4 va SiDHNd. Cac kel qua nghien cuu ve sir bieu hien cdc gen DHN cua cay vimg tuong ddng vdi sy bieu hien cua nbieu gen DHN ciia cac loai thuc vat khac dd d u a c bdo cao n h u d cay dai mach, cay thudc la va cdy ca chua hay cay quyt dudng [3, 9]. Dang chu y, cac gen khdng cd EST gdm mdt gen phan nhdm YnSKn [SiDHNS) vd ca hai gen phan nhdm SKn (SiDHN3 va SiDHN4). Su bieu bien khdng gidng nhau trong hat dang phdt trien cua cdc DHN ggi ^ vai trd kbdc nhau cua chung trong qud trinh phat trien hat cua cdy vimg. ddy cung la tbdi ki cd su bien ddng ve bdm lugng nuoc trong md hat.

So lirgng EST 1 4

I

6

I

S i D H N l S i D H N 2 S i D H N B S i D H N 4 S i D H N 5 S 1 D H N 6 Hinh 4. Kit qud khdo sdt subieu hiin gen cua cdc dehydrin cda cay virng

3. Ket luan

Trong cdng trinh nay chiing tdi da xac dinb dugc long sd 6 gen DHN trong toan bd he gen ciia cay vijng. Kdt qua phdn tich cdc motif bdo tbii trong cac protein suy dien cho thay cdc DHN ciia cdy vimg cd mang cdc motif bao thu dac trung nhu phdn manh K, phan mdnh Y va phan manh S tiiy gen. Dua tren su cd mat ciia cac motif bao thu, k6t hgp vdi phan tich cay phat sinh chung loai da chi ra rdrig cac gen nay thuoc vd ba nhdm KS vd YnSKn va SKn Cac gen DHN ciia cay vimg bieu hien khdng gidng nhau Uong qua trinh phat uidn ciia hat. Day la nhdng k^l qua budc ddu cd y nghTa quan uong, md dudng cho viec nghien cuu chiic nang cua cdc DHN d loai cay nay.

T A I L I E U T H A M K H A O

fl] Anilakumar K. R,, Pa! A., Khanum F. and Bawa A, S., 2010. Nutritional, medicinal and industrial uses of sesame (Sesamum indicum L) seeds-an overmew Agriculturae Conspectus Scientificus (ACS), 75(4), pp. 159-168.

f21 Zhang H., Miao H., Wang L , et al., 2013, Genome sequencing oJ the important oiLseed crop Sesamum indicum L. Genome Biol, 14(1), p. 4 0 1 .

125

Tran Thanh Huy^n. Cao Phi Biing va D6 Thi H5ng

[3] Hanin M., Brini F„ Ebel C, Toda Y.. Takeda S. and Masmoudi K,, 2011. Plant dehydrins and stress tolerance: versatile proteins for complex mechanisms. Plant Signaling &

Behavior, 6(10), pp, 1503-1509.

[4] Liu C, C , Li C. M,, Liu B. G., et al., 2012. Genome-wide identification and characterization of a Dehydrin genejamily in Poplar (Populus trichocarpa). Plant Moi Biol Rep, 30(4), pp.

848-859,

[5] Hundertmark M and Hincha D. K., 2008 LEA (late embiyogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC genomics, 9(1), p. I !8.

[6] Bateman A., Coin L., Durbin R. and Finn R. D.. 2004. The PJam protein families database.

Nucleic Acids Res, 32. pp. D138-D141,

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improvements in performance and usability," Moi Biol Evol. vol. 30, pp. 772-80, Apr 2013.

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[9] Cao Phi Bdng, 2014. Ddc trimg hoa vd khao sal su bieu hien cua hp gen dehydrin & edy quyt duffng (Citrus clemenlina). Tap chi Khoa hgc va Phat trien, 12(7), pp. 1134-1139, [10] Yang Y., He M,, Zhu Z,, et ai, 2012 Identification of the dehydrin gene family from

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A B S T R A C T

Identification, classification and expression of dehydrin genes in sesame {Sesamum indicum L.)

Dehydrins (DHN) belong to Group II of Late Embryogenesis Abundant protein (LEA).

These proteins play a crucial role in protecting cells against the adverse effects of environmental stress by increasing water retention capacity. They are also important in membrane and molecule protection. In this study, we identified six DHN genes in the Sesame genome (Sesamum indicum L.). All of these DHN proteins contain the K-segment which was characterized in all previously reported DHN proteins. Based on the results of conserved motif analysis as well as phylogenetic tree analysis, six sesame DHN genes were classified into three subgroups of YnSKn (two genes), SKn (two genes) and KS (one gene). The result of the expression study shows that sesame contains at least three DHN expressed genes in its developing seed.

Keywords: Dehydrin (DHN), sesame, phylogenetic tree, gene expression.