HO' NGH! KHOA HOC C O N G N G H g SINH HOC T O A N Q U O C 2013
, ^GHIEN CO'U XA.Y DUNG MA VACH DNA CHO VIEC PHAN LOAI NHAN DANG CAY SAM NGOC LINH
V i i Huyen T r a n g * , Hoang D a n g H t l u , C h u H o a n g Ha Vi^n Cdng nghi slnh hgc
T O M T A T
Sam Ngpc Linh (Panax vielnanicnsLi) hay cda goi la sam Vi?t Nam, la vi thuoc dac biet quy, voi chSt lupng dung tren ca iiim Trieu Tien, sam T/ung Qu6c va sam My. Tuy nhien, Irong nhimg nam gan day sam Ngpc Linh dang bj khai ih3c qud mirc do gia iri kinh le cao, Viec bao ton va phat trien sam Ngpc Linh dang gap nhifiu kho khan do ihieu mgt phuong phap dang tin i^y de nhan biel chinh xac loai thuc vat n i y Hien nay, ky thugt D N A ma vgch ( D N A barcode) dang dugc su- dung rgng rai tren the gidi de phSn loai va nhan dang mpt each don gian va cbinh xac die loai thuc v | i , Trong nghien ciru nay. chiing loi da sii dicing 5 chi ilij D N A ma vach nem nang la p.sbA-lmH. maiK. ImL, rhcL va ]TS de djnh danh 5 mau sam Nggc Linh thu thap dugc o Quang N a m , Da Lai va Konlum Phan tich kel qua va so sanh v d i cac trinh ly; da dirge cong ho tren ngan hang Genbank, chung l6i da chung minh rang trong 6 chi ihj nghien cim, p.ibA-imH la chi thi c6 tiem nang nhat, cho phep phan biet sam Ngpc Linh vol cac loai :iam khae tren the gidi v(!n dg chinh xac cao
Tirkhoa. DNA barcode, Panax vietnamensis, psbA-tmH, Sam Nggc Linh
Cho t6i nay vi0c djnh danh dc loai sinh vgt thuo'ng chu yeu di/a tren dinh gia cac d^c diem hinh th^i - ca sa chO ylu cho cdng tac phdn lo^i. De phin logi mdt loai sinh v|it si> dgng phucrng phap phin loai hoc truyen thong ydu ciu nhd nghien ctru phai c6 nhO'ng hilu bilt chuyen sau vi loai cdn phdn tlch. Trong trufi'ng hop mlu vgt c l n phan tich thieu cdc d$c dilm hinh thdi can thiet. vi#c phan logi trd nen die biet kho khdn, ngay ca doi vd'i cdc chuyen gia de dinh danh loai m$l cdch chinh xac. Vi?c su dung cac cht thj DNA md dgc bi§t Id chl thj DNA ma vadi da khic phyc dirg'c phkn idn cac nhuoc dilm cua phuang phdp phdn Iogi hgc tnjyln thing. Hi§n nay ky thudt DNA ma vgch dang duo'c si> dung rOng rai vd CO hi^u qud trong mot s l nghidn cuu phyc vg cong tdc djnh danh lodi khdng dua trdn cdc dac dilm hinh thdi (Liu et al., 2012; Zhang ef a/., 2013), Ky thugt DNA ma vgch Id m§t cong cy hO-u ich cho vi§c phan logi, giup ich dang k l cho Hnh vuc phan logi hgc. O ddng vdt gen COI ma hod tilu dan vi cytochrome c oxidase 1 trong ty the dugc BLF dgng lam chi thi DNA md vach co khd ndng phdn bi?t da s l cdc lodi ddng v|it (Hebert et ai, 2004). Doi vai thi/c v§t nhiiu vung gen da duoc nghien CCPU vd dd xuat lam chi thj DNA cho thyc vdt (Taberlet et al., 2007) tuy nhien chua c6 cht thj DNA ndo dugc da phan cac nhd phan logi hgc thi^c vgt hoan todn chip nhdn (Ole & Gitte, 2009). Mdc dii vdy. cdc nha nghidn CU'U cung da di tai mdt quan a i m thing nhit Id se can khong cht mdt md nhilu vOng DNA chi thj d l dinh danh loai doi vdi thyc v^t (Taberlet ef al„ 2007) vd hd gen Igc Igp la nai chiia cdc vung DNA chi thj tilm ndng. trong dd mot so viing DNA luc Igp da dug'c nhieu nha nghien cuu d l xuit Id chi thj DNA (Chase efa/., 2005). Trong nhung nam gin ddy nhieu nhd nghidn cuu Tmng Quoc da thdnh cdng trong vide si> dgng cht thi DNA ma vgch psbA-trnH de xdc djnh cac cdc lodi dug'c li$u thudc chi Polygonaceae vd chi Dendrobium (Ma etal., 2010, Yao etal., 2009). Ndm 2010 Chen vd dong tac gid da chung minh ring cht thj ITS la mdt dii thj tilm nang cd t h i xdc dinh chinh xdc din 92,7% tm-ang hgp trong 6600 mlu thu$c 7 ngdnh (Angiosperms, Gymnospemis. Ferns, Mosses. LivenAiorts. Algae, and Fungi). K l tu do, vung ITS da dugcsudungr^ngraitrongphdnloginhilulodi thuc vgt khae nhau (Liu e(a/., 2012, Pang efa/.. 2011)
Sdm Nggc Linh tdn khoa hgc Id Panax vietnamensis Ha et Grushv la cdy ban dja ddc hifU cua nOi rCfng Nggc Linh, theo cdc tdi ligu nghidn cuu tnrdc ddy, nhirng a i m cd sam Nggc Linh mgc ty nhidn deu a dg cao tu 1.500 - 2.200 m (tgp tmng chu yeu b dg cao 1.800 - 2.000 m) thudc dia bdn cua hai huyen Ddk Td {tinh Kon Turn) vd Trd My (tinh Qudng Nam). Dugc phdt hidn lln diu tien vao nam 1973. ti> do den nay c6 rdt nhilu cdng trinh nghiSn cu-u chi>ng minh Sdm Ngoc Lmh la lodi sdm quy c6 gia tri hang diu, dirng trdn ca Nhdn sdm Trilu Tidn, Nhdn sam Tmng Quoc vd Nhdn sdm My. Sdm Nggc Linh c6 nhilu cdng dgng nhu tang thi lye, phgc hdi sy suy gidm chirc nang; khdng cdc dgc t l gdy hai t l bao, giiip keo ddi sy song cua t l bdo vd tdng cac t l bao mai. Bgc bidt sdm Ngoc Linh c6 nhung tinh nang ma sSm Trilu Tien vd sdm Tmng Quoc khdng c6 Id tinh khdng khuin, chong trim cam, gidm lo du, chdng oxi hda vd hidp lye tot vd'i thuoc khdng sinh. thudc trj bgnh tilu duifng. V6i thanh phdn cdc hyp chit hoa hge da dgng vd Id "thdn duye" ddi vdi sue khde con ngudi sdm Nggc Linh hien nay duyc ban tren thi trudng vdi gia rat cao, cd nh&ng thdi diem 1 kg tu'ai sdm j Nggc Unh-Qudng Named gid trj 1 2 - 1 5 trieu ding, nh&ng liic khan hiem ed thi Idn tdn 25 - 30 bi§u ddng. trong khi dd 1 kg tuai sam Tneu Tidn ehi cd gid tri khodng 3-4 trieu dong. Do lyi ich qua Idn, ndn sam Nggc Linh trd thdnh doi tuyng ,san tim cua con ngudi va din tdi nam 1993 sdm Ngoc Linh duyc xep diu bang trong Sach do thyc vdt Vidt Nam o6 nguy ea tuyet chung. Tuy nhien vigc xde dinh" sam Nggc'Lfnhehu ylu phy thudc vdo eac d|c diem hinh thdi va kinh nghigm cua cd nhan. vi#c xac dinh ndy se gap nhilu kho khan khi m l u vdt thu duyc khdng hodn thidn hodc ton tgi nhu :md_t thdnh phin trorig hon hyp. Vi vdy vide dinh danh chinh xdc sdm Nggc Linh se tgo dieu kidn thu$n lyi eho cong tdc fluan ly m$t each ddng bd va cd hg thdng lodi cdy duge lieu quy hilm ndy nhim, gdp phin bdo ton su da dgng. nang Kao gid trj su dgng phgc vu xult khiu cua sam Vigt Nam. Trong thdi gian gin ddy mdt s6 nghidn eiru eua Hdn QuIc da tien hanh nghidn cu-u xdy dyng md vgch eho mgt s l loai ehinh thudc chi sdm nhu P. bipinnatifidus, P. ginseng. P.
notoginseng. P. stipuleanatus va P. pseudoginseng... tuy nhidn khdng d l c§p din sdm Nggc Linh cua \fldt Nam. Trong [nghien edu ndy chiing tdi dd sir dgng 5 ehi thj DNA ma vgch tilm ndng {psbA-tmH, rbcL, rpoB, rpoCI vd ITS) nhdm , jJjnh danh vd phdn bigt sdm Nggc Linh {Panax vietnamensis) vai cdc loai sdm khde trdn thd ^di eung thudc chi Panax.
I /AT LIEU VA PHlfONG PHAP ' /gt lieu
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HOl NGHI KHOA HOC CONG NGHE SINH HOC TOAN Q U 6 C 2013
Cac mil (primer) duyc dat tip hdng Epoehlifescience (My) tdng hap. '^^M
° Chung Ecoli va vector pBT, vector tach dong do phong Cdng nghe t l bdo, Vidn Cong nghe sinh hoc c i j n g ^ H ^
•• 5 m i u la cdy sdm Nggc Linh duyc thu thap tai Quang Nam. Dd Lat, Kon Turn S P
Phu-ong phap nghien ciru ^ ^ | Tach chiet DNA tong so
DNA ting s6 tir cdc mlu Id duac tiln hanh tach chilt bang phuang phap CTAB theo Dellaporta va dong tac gia (1983}
Phan ling PCR
DNA tong so sau khi tach chiet thanh cong duyc sir dung lam khudn cho phan irng PCR vdi nhu sau: budc 1: 94''C, 5 phut; buac 2: 94 C. 30s; budc 3- 54''C, 50s, budc 4: 72°C. 1 phut; tu budc 2 din budc 4 igp lai 30 chu ki, budc 5:
72 C. 10 phut; budc 6: 4''G bao quan mau.
San pham PCR sau do duyc didn di trdn gel agarose 1% frong ddm IX TAE, nhudm gel frong dung dich ethidium brommide va tinh sach bing kit thdi gel ciia QIAamp,
Chiing tdi da tiln hanh phan dng PCR vdi cac mdi sau:
Bang 1 . Trinh t y cac cap moi s i f dgng trong nghien cii'u STT Ten moi Ky hieu Trinh t y mSi (5'- 3')
psbA-tmH matK ITS rbcL trnL pUC18
PI OF PI OR P9F P9R P12F P12R P2F P11F P11R pUCISF pUCISR
GTTATGCATGAACGTAATGCTC CGCGCATGGTGGATTCACAATCC ATCCATCTGGAAATCTTAGTTC CTTCCTCTGTAAAGAATTC ACGAATTCATGGTCCGGTGAAGTGTTCG TAGAATTCCCCGGTTCGCTCGCCGTTAC ATGTCACCACAAACAGAAAC TCGCATGTACCTOCAGTAGe CGAAATCGGTAGACGCTACG GGGGATAGAGGGACTTGAAC CAGGGTTTTCCCAGTCACGA GGGGATAACAATTTCACACA Tich dong va xac dinh trinh tu- gen
Sdn pham PCR sau tinh sach du-yc gan vdo vector tdeh ddng pBT vd biln ngp vdo te bao kha bien E.coli DH5-a theo phuong phdp sic nhigt. Chgn Ipc khuin Igc xanh/trang fren mdi trudng thach LB eci bo xung 50mg/l Carbenicilin, 0,004% X-gai vd O.ImM IPTG. Tdch plasmid theo hudng dan cCia bd Kit tach plasmid (Bioneer, Han Qucic), Kilm tra sy ed mat cua DNA tdi to hyp bing phuong phdp clony-PCR vdi cap mdi pUC18F vd pUCISR. Trinh tu DNA duac xdc djnh bing mdy phan tich trinh ty ty ddng ABI PRISM 3100 Avant Genetic Analyzer theo nguyen li cOa Sanger vdi bd kit BigDye Terminator v. 3.2 Cycle Sequencing (Macrogen Inc. HanQuoc). de so sanh trinh tu gen va dyng cdy phdt sinh chOng logi tren ca sd du* ligu thu duyc.
Phin tich kit qui
Trinh ty DNA da giai duyc xd ly va phan tich bang phin mdm BioEdit va DNAstar. Do bien thidn giCi-a cac loai trong mpt chi vd khae chi duyc xdc djnh su dung phln mem Speciesldentifier 1.7.8, Cdy phdn loai duyc xay dung theo phuang phap l l i da (Maximum Parasimony (MP) tren ca sd sd ligu thu duyc.
K£T Q U A V A T H A O LUAN Kit qua tach chiet DNA tong so
ITS, psbA-tmH, matK, YcfS, rbcL, trnL da duoc nhdn ban thanh cdng si> dung mdt cgp mdi cho moi locus td 5 miu sdm Nggc Linh thu duyc. Ty le nhdn ban thdnh cdng dli vdi cae locus ITS, psbA-trnH, matK, rbcL. tmL la 100% (Bang
1).Ty Id eac mlu dgc trinh tu thdnh cdng Id 100% Oe ddm bao tinh chinh xac Hinh1. Kgt qua tach chiet DNA t6ng chiing tdi da lap Igi thi nghiem 3 lan dli vdi mdi mlu sdm Ngpc Linh thu thap
dugc.
Hinh 1 la ket qud DNA ting s l duyc tdch chill tii- 5 mlu Sam Ngpc Linh nghidn ciru, dya tren kit qud thu duye frdn hlnh cdc bdng vgch sang, rd rdng, cd thi thdy DNA thu duyc co chit luyng tdt va do tinh sgch eao, du chit luyng cho cic budc nghidn cuu tilp theo.
110'
HOI NGH! KHOA HOC C O N G N G H E SINH HOC TOAN QU6C 2013
Bang 2, r-lc so lieu v * cac chl th) DN*. ma vach sfr dung frong nghie ITS
S i fuvng m l u 5 T^l# nhdn ban PCR Ihanh cdng (%) 15,'15 (100) Klch Ihudc sdn p h i m PCR ttw Quoc 800 (bp)
T^ l§ aoc trinh t u thanh cong (%) 15/15 (100) Kich thudc floan gen sau khi so sanh 633 (bp)
Od biln thien trung blnh trong nOi bo d i i Dp bi^n thien trung binh gii>a cac chi
15/15(100) 15/15(100)
0.0040 (0 - 0.0054) (0.0005-0.0153) ( 0 - 0 0342) (0-0.0024)
Ket q u a n h a n d o n g , t a c h d o n g v d x a c d j n h t r i n h t y ir c a c d o a n g e n n g h i e n c u o i
DNA sau khi tdch c h i l t se d u y c dung idm khudn de t h y c hien phan d n g PCR v d i eac m d i nghidn c i K i Kdt qua didn di sdn p h i m P C R frdn gel agarose d u g e t h i hien fren H i n h 1A C a c b a n g DNA iiep tuc d u o c tdch d o n g v d k i e m fra b i n g phdn d n g PCR-clony (Hlnh I B ) ,
Hinh 2. Di^n di san p h i m PCR (A) va kiem fra cac khuan lac bang phan u n g PCR-clony (B)
Hinh I A Cho t h i y cdp m l i matK da d u y c s d dgng thdnh cdng trong vigc nhdn b a n , k h u e c h dgi dogn g e n m o n g m u l n (khodng 950 bp) t u D N A cua 5 m l u Sdm Nggc Linh, cdc dogn gel n d y d u y c tinh s g c h v d b i l n ngp vao t l b d o E. Ct^i DH5a. T u a n g t y . cdc m l i c d n Igi cOng d u y c nhan ban thanh cdng ( k i t qua khdng cdng b l )
Hlnh I B Id k i t qua kiem fra cdc ddng khuan lac da b i l n nap vao E. C o / / b d n g phan i m g PCR-clony s u dgng m l i pUC18 (kich t h u d c sdn p h i m PCR thu d u o c khodng 1150 bp), cae d d n g khuan Ige 1. 9. 10 id nhOng ddng k h u i n Igc a m tinh Ididng mang gen ^ i t l h y p , n h d n g d d n g khuan Igc cdn lai Id n h u n g d d n g d d b i l n ngp t h a n h cdng ed c h i r a d o ? n g e n tai to h y p SB d u y e tiln hdnh nudi frong m d i t n / d n g LB Idng c d b l x u n g 50mg/l Carbenicilin nudi qua d d m s a u d d t i e n hanh tdch plasmid. vd g u i dgc trinh t y . T u a n g t y . cdc doan gen edn lai {psbA-tmH, matK, rtxL. tmL) eung d u y c b i l n ngp v a tach ddng thdnh cdng.
Ket q u a p h d n t i c h t r l n h t y
K i t qua xdc dmh 90 frinh t u thu d u y c t u 5 m l u sdm Ngpc Linh v d i 5 cap m l i {psbA-tmH, matK. rbcL, tmL, ITS) v d phan tich frinh t y s i r dung phan m d m DNAstar ehiing toi nhgn t h i y : v d i tat ea 5 egp m l i d a SLT d g n g . trinh t y cua c d c mau sdm Ngpc Linh thu ttigp d u y e tgi cae dja d i l m khae nhau (Quang N a m , D d LgL K o n h j m ) d e u c d h g s l t u a n g d d n g di tmyen cao ( l u 99.8-100%). Sau khi phdn tfch frinh t y ehiing tdi da s o sdnh v d i cdc frinh t u t u a n g d d n g d u a c cdng bo trdn ngan hang Genbank.
Theo Moritz v d Cicera. 2004, mdt ehi thi DNA m d vach d u y c coi Id ed kha ndng phdn b i i t lodi tdt n l u c d the p h a n bigt r o rang, khdng c h l n g cheo cdc loai b o n g mgt chi (intergenus) va khde chi (intragenus). C h i i n g tdi da phan Kch s y p h d n b d cdc bien thidn giu'a cdc lodi trong m p t ehi v d khae chi d l i vdi 5 chi thi D N A m a vgch s u d y n g p h l n m i m Speciesldentifier j i .7-8. (Hlnh 3), d a v d i eht thj tmL d o d d bien thidn giira cae loai khae ehi bang 0 ndn c h i i n g tdi khdng t h i hidn k i t qua trdn Hinh 3. K i t qua phdn tich cho t h i y s y b i l n thidn cua ede loai frong m g t chi va khde chi s u dung 4 ehJ thi ITS, psbA- tmH. matK vd rtxL cho t h i y d l i v d i cdc c h i thj ITS, matK, va rbcL c d s u chdng eheo dang k l miipc d d bien thidn g i i i a eac_
I c d i frong ciing mpt c h j ^ d khae c h i . frong khi do c h i thi psbA-tmH su c h o n g cheo I h l p h o n dang ke so v d i 4 ehf thj c 6 n
^Ig^ (Hinh 3 ) r N f r u vdy. so v d i cae ch? ttij edn lai. chi thj psbA-tmH c d u u t h i phdn bigt cac lodi eiing m d t chi v d k h d e c h i .
^3ay cd t h i coi Id chi thi t i l m nang de phdn biet s d m Ngpc Linh v d i c a c loai khae tnang chi s d m (Pana^r).
HOI NGH! KHOA HOC C O N G NGHE SINH HOC TOAN QUOC 2013
matK
rhcL
HllBJ.i!!, a g il g; g g g g § g i g g g g g g s g g g g
i ? ^ S ^ ^ ^ ^ ^ ^ s 5 S s 5 s s s ? S s j e
Hinh 3. Phan b l s y bien thidn giii'a cac loai trong mot chi va khae chl d6i v d l 4 chi thj DNA m i vach Old trj frung binh chl so biln thidn nucleotide giu-a cdc loai trong mgt ehi va giua cac chi duyc the hien trong Bang 2, chiing toi nhgn thiy chl s l bien thien cua cdc lodi trong mdt chi ciia chi thi psbA-trnH la cao nhit (0.0382) tilp theo lin luat la ITS (0.0186), riJcL (0.0205), frnL (0.0040) vd mafK (0.0024) (Bang 2). Tuong ty, gia frj trung binh chi so biln thidn nucleotide giii'a cdc lodi khde chi, chi so biln thidn cuaxhi thj psbA-trnH Id cao nhlt (0 0154) tiep theo lin luyt Id /TS (0.0057), rbcL (0.0034), mafK (0.0013) va fm/. (0 0001) (Bang 2). Nhu vdy, so vdi cdc chi thj cdn lai, chi thj psM- tmH cd gid tri trung binh chl s l bien thien cCia cdc loai trong mpt chi va khae chi Id cao nhlt.
Ngodi ra. dya vdo cdc frinh ty da xdc dinh duyc va cdc trinh ty tuang dong tren ngan hdng Genbank chiing tdi da tien hanh phdn tich djnh danh lodi dya trdn phuang phap xdy dyng cdy phdn loai. Vdi cdc edy phdn logi da xdy dung dugc.
chiing tdi da kit ludn ring 5 mau sam Ngpc Linh thu dugc d Viet Nam phdn bigt vdi cdc lodi khde thudc chi sdm trdn the gidi (kit qud khdng cdng bd). So sdnh 5 cdy phdn logi sir dgng 5 chi thj DNA ma vgch psbA-tmH, matK. rbcL, tmL, ITS -chting tdi nhdn thdy su dgng cay phdn loat dya vao kit qua xac djnh frinh tu nucleotide vdi ehi thj psbA-tmH cho kit qua
cd d$ lin cdy cao nhlt, cho phep phdn bidt sdm Nggc Linh vdi cae loai sdm khde trdn thi gidi,
Quan sat cdy phdn log! xdy dung frdn ea sd so sdnh trinh ty cua dogn gen psbA-tmH (Hlnh 4), chiing tdi nhan thiy t i t ed cdc mlu sdm Ngpc Unh thu thgp duyc nghien cii'u ed t h i thude cOng mgt nhdm phdn logi hay cd the xem thudc ciing mgt lodi vd phdn bidt vdi lodi P.blpinnatifidus (vdi dp tin cgy cao, chi sd bootstrap 96,2%). Ngodi ra chi thj psbA-tmH cdn cd thi phdn bi^t cac loai khde thugc chi sdm (Panax) vdi chi s l tin cay khd cao Trdn cay phdn logi ta cd the phan bidt duoc sdm Ngoc Linh vdi cac loai P.japonicus vd lodi P.bipinnaUfidus, P.quinquefolius, P.ginseng, P.stlpuleanatus, P.notoginseng (vdi dO tin cdy cao, chi s6 bootstrap trong khoang td 70,3 - 96,2%) vd sii dung chi thj nay cung cho phdp phdn bidt giua cdc lodi P.quinquefolius, P.ginseng, P.stlpuleanatus, P.notoginseng, P.trifolius v&i chi sd bootstrap frdn 70% Dieu nay khlng djnh, vdi chi thj psbA-trnH cac mlu sam nghien cuu cd sy bien thien giua cac lodi trong ciing mgt ehi khd eao va sy biln thidn trong cOng lodi khd thap. Day la chi thj tot de phdn biet cac mau sam Nggc Linh nghien cuu vdi cac ioai sam khae tren thi gidi
Hinh 4. Cay phan loai cdc loai thuoc chi sam Panajr s u dgng chi thj psbA-tmH
HOI NGH! KHOA HOC C O I ^ NGHE SINH HQC TOAN QUOC 2013
KET LUAN
Ky thuat DNA ma vach eho phep dinh danh sinh vgt tai bit ky thdi dilm ndo frong qua binh sinh trudng vd phdt friln eung nhu trong tinh trgng khdng hoan chinh. ddy Id mdt uu diem Idn so vdi cdc phuang phdp dinh danh truydn thing dya trdn d|c d i ^ hinh thdi. Ky thugt ndy da khie phye ede nhuae diem va lam tang ddng k l tinh ehinh xdc cho cac phuang phap phan logi hpc truyln thing. Ngodi ra, ky thuat DNA ma vgch cdn eho phdp phdn tich mgt cdch dan gian, chinh xic. nhanh ehdng vd so sanh true tilp dij lieu thu duye vdi ca sd dtp ligu toan c l u (Genbank). Nghidn ciru nay ciia chiing tdi cung khing djnh duyc eac uu thi nay cua chi thj DNA md vgch d l xac djnh sam Ngpc Linh, phdn biet vdi cac loai sdm khae frdn thd gidi.
So sdnh 5 chi thi DNA ma vgch chung tdi dd xac djnh duyc chi thi psbA-tmH la chi th] tdt nhlt, cho phdp nhgn dgng sam Nggc Linh. Chi thj nay cho phep nhdn ban doan gen cd kich friudc frung binh, khoang 600bp, friudn tidn cho cdc bude phdn tich doi vdi cac lodi thude ehi sdm nhu tach chilt DNA, nhdn ban bing PCR, va dpc frinh tu, ^eu nay r i t ed y nghTa eho vide xdy dyng mdt phuang phap chuin d l xac dinh ehinh xae, nhanh chdng vd ty ddng hoa, nhinn djnh danh cac lodi thuc vat noi chung vd ede loai sdm frdn thd gidi ndi rieng. Ngodi ra ehi thj psbA-tmH con cho phep phdn biet phdn biSt rd rang, khdng chdng cheo cdc lodi frong mpt chi (intergenus) vd khde ehi (infragenus). Gid trj tmng binh chi s l biln thidn nucleotide giua eac lodi trong mdt chi vd giOa cdc ehi eiia eh! thj psbA-frnH Id cao nhat frong 5 ehi thj chiing tdi sir dgng d l nghien cuu. Lya chgn st> dyng chi thj psbA-tmH la chi thj tiem nang cho phep phdn logi cde lodi thyc vat frong nghien cuu ciJa diiing tdi cung phu hap vdi lua chgn cua nhilu nghidn cdu khde trdn thd gidi d l dinh danh cac loai duyclidu quy (Ma etal., 2010, Yao efa/., 2009. Liu etal., 2012, Pang etal.. 2012).
L o i cam am
Nghien cm dufrc thuc hien vdi sir ho trfr kinh phi cua cac de tai "Nghien cuu xdy dung ma vgch DNA (DNA barcode) phuc yu cho viec dinh danh sdm Ngpc Lmh vd cdy Bd b?nk " va "Nghien cuu xdy difng quy trinh tgo sinh khoi thong qua h4 thong nuoi cdy re la (hairy roots) ciia mpt so lodi cdy dufrc fipw quy Idm nguyen lieu sdn xudl che pham phuc vu sdc khoe cong dong " thupc chuang trinh nghien cuu khoa hpc vd phdt trien cdng nghf ciia i/ien Khoa hoc vd Cdng ngh? Viet Nam ". Cac thi nghiem dugc tiin hdnh sir dung trang thiel bf ciia Phong thi nghiem trong diem Cdng nghe gen. Vien Cong ngh4 smh hpc
T A I LIEU T H A M K H A O
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Chen SL, Yao H, Han JP, Uu C. Song JY, Shi LC, a i u YJ, Ma XY, Gao T, Pang XH, Luo K, Li Y, U XW. Jia XG. Un YL, Leon C (2010).
Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant Species PLoSONE. 5;e8613.
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Liu Y, Zhang L. Liu Z, Luo K, Ctten S. Chen K (2012). Species klenliflcation of Rhododendron (Ericaceae] using the chloroplast deoxynbonucleic acid PsbA-tmH genetic marker. Pharmacogn Mag. 29: 29-36
Ma XY. Xie CX. Uu C, Song JY. Yao H. Luo K, Zhu YJ. Gao T. Pang XH. C«an J , Chen SL (2010). Species identification of medicinal pteridophytes by a DNA barcode marker, the chloraplast psbA-tmH intergenic region. BidPharm Bull. 33(11): 1919-24.
Morilz C and Cicero C (2004). DNA barcoding; promise and pitfalls- PloS Biology 2'1529-1531
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Pang X, Song J, Zhu Y. Xu H. Huang L. Chen S (2011). Applying plant DNA barcodes for Rosaceae species identification. Cladistics 27(2): 165-170
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Yao H,Song J.Liu C,Luo K.Han J.U Y.Pang X.Xu H.Zhu Y.Xiao P.Chen S.. (2010). Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS O N E . 2010.5(10): e13102
, APPLICATION OF DNA BARCODES IN IDENTIFICATION OF PANAX t VIETNAMENSIS
I; Vu Huyen Trang, Hoang Dang Hieu, Chu Hoang Ha 13 Institute Of Biotechnology
I Panax vietnamensis or Vietnamese ginseng (also known as "Sam Ngoc Linh" in Vietnamese) is a very precious herb with its quality
£ even better than thai of North Korean, Chinese and American ginseng. However, in recent years, P. vietnamensis has been over- t exploited due to its highly economic importance. Preservation and development of this plant have faced many di^culties because of ' lack of a reliable idenlificauon method of P. vietnamensis. Currently, DNA barcode technique has been widely used in simply
classifying and idennfying a variety of plant species around the world. In the current study, we used six potential DNA markers which are psbA-tmH, matK. yep, trnL. rbcL and TTS to identify five sampless collected in Quang Hinh. Da Lat and KonUjm provinces. By analysing the results and aligning wilh published sequences in Genbank, we identified that all five samples studied belonged to Panax vietnamensis. In addition, our results showed diat of investigated six markers, psbA-lmH was the best candidate that could differentiate P.vietnamensis with other Paiia.x species in the world with high precision.
"Author for corresspondence: vuhuyentrang@ibt ac vn
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