UNG DJNG KY THUAT SINH HC PHAN

TU DE XC DJNH MT S LOI SAM

THUC CHI PANAX
Nguyn Thanh Thun*, V Thanh Thao*, Nguyn Vn Thanh*, Trn Cat Dng*
TM TAT
Mo du: Nhn sm duoc dung trong y hoc phuong Dng hang ngan nm. R cua nhn sm co
tac dung tng kha nng thich ung cua co th, tng hiu qua diu tri bnh tiu duong type II, kich
thich ham mun tinh duc, tng cuong suc khoe va diu tri tiu duong cng nhu ri loan tinh duc o
nam gioi. Sm thuong duoc ban duoi dang r kh hay ct lat. Cac loai sm khac nhau thi hoat tinh
cng khac nhau. Do do cn mt phuong phap khoa hoc d xac dinh cac loai sm o muc phn tu.
Mc tiu: Xy dung cac phuong phap thun tin va co d lp lai cao d xac dinh cac loai sm thuc
chi Panax va ngun gc cua chung.
Phuong php: Chung ti dung phuong phap giai trinh tu, PCR-Restriction Fragment Length
Polymorphic (PCR-RFLP) trn vung ITS va 18S, va Random AmpliIied Microsatellite (RAMS).
Kt qua: Phn tich trinh tu vung ITS co th duoc dung d xac dinh cac loai sm. Ngoai ra, k thut
RFLP co th giup phn bit ngun gc cua chung.
Kt lun: Cac phuong phap phn tu d su dung thich hop d xac dinh va phn bit cac mu sm.
T kha: PCR-RFLP, sm, RAMS, di truyn hoc.
ABSTRACT
AUTHENTICATION OF GINSENG SPECIES BY BIOMOLECULAR METHODS
Nguyen Thanh Thuan, Vu Thanh Thao, Nguyen Van Thanh, Tran Cat Dong
* Y Hoc TP. Ho Chi Minh * Vol. 14 - Supplement oI No 1 - 2010: 129-133
Background: Ginseng has been used in Iolk medicine Ior thousands oI years. Its root is taken as
adaptogens, aphrodisiacs, nourishing stimulant and in the treatment oI type II diabetes, as well as
sexual dysIunction in men. The root is most oIten available in dried Iorm, either whole or sliced.
DiIIerent ginsengs have diIIerent active substances and eIIects. ThereIore, a scientiIic method oI
identiIying ginseng cultivars at the molecular level is required.
Objectives: To develop some convenient and reproducible approaches Ior diIIerentiation between
Panax species and their sources.
Methods: Using sequence analysis, PCR-Restriction Fragment Length Polymorphic (PCR-RFLP)
with primers rDNA 18S and ITS, and Random AmpliIied Microsatellite (RAMS).
Results: Sequence analysis oI ITS region can be used Ior identiIication at species level. RFLP
method can diIIerentiate the origin oI samples.
Conclusions: These methods were useIul Ior identiIying and diIIerentiation ginseng samples
Keywords: PCR-RFLP, gingseng, RAMS, genetics
M O DAU
Nhn sm la mt loai thuc b, dong vai tro quan trong trong vic du phong va diu tri nhung
bnh ly man tinh, cn diu tri dai ngay nhu bnh tiu duong, ung thu, bnh xo vua dng mach,
huyt khi, cao lipid mau, cao huyt ap, thiu nng tun hoan no. do tac dung tng cuong th
luc va gia tng suc tu d khang khng dc hiu cua co th, tac dung chng oxi hoa mt cach truc
tip hay gian tip. Y hoc hin dai d chung minh Nhn sm duy tri su hng dinh ni mi cua h
thn kinh trung uong, h ni tit va h min dich. Tuy cac tac dung tri liu khng din hinh,
tuong di yu so voi cac thuc dc tri Ty y nhung vic it gy tac dung phu khi su dung dai
ngay, nht la o nhung di tuong tuong bnh nhn cao tui la uu dim cua Nhn sm(1).
Mt loat k thut sinh hoc phn tu d duoc dung d nghin cuu da dang di truyn cua chi Panax
nhu random ampliIied polymorphism DNA (RAPD)(11), ampliIied Iragment length
polymorphism (AFLP)(4), PCR-restriction Iragment length polymorphism (PCR-RFLP)(3),
arbitrarily primed PCR (AP-PCR) (10), direct ampliIication oI length polymorphism (DALP)(5),
inter-simple sequence repeat (ISSR)(6) va giai trinh tu(2).
Trong b gen thuc vt, cac gen RNA ribosome (rDNA) duoc sp xp trong nhung don vi lp lai
ni tip nhau voi mi don vi gm 18S rDNA-ITS1-5.8S rDNA-ITS2-26S rDNA. Vung m hoa cua
ba gen rDNA duoc bao tn cao hon hai vung ITS.
Mt k thut khac dua trn PCR la random ampliIied microsatellite (RAMS). K thut RAMS la su
kt hop dc tinh phn tich RAPD va vung vi v tinh. Ban cht cua k thut nay la cac du n duoc
phat hin khi khuch dai hai vung vi v tinh ngu nhin cung voi trinh tu xen giua hai vung nay.
Phuong phap nay co d lp lai cao va cho phep phat hin da hinh cua DNA trong cung mt loai
hay giua cac loai(7).
VAT LIJU V PHUONG PHP
Cac mu sm
Cac mu sm tuoi hay kh duoc thu mua tu cac ngun sau: cac mu P. ginseng duoc mua o Vit
Nam (Pg1, Pg2) va Han Quc (Pg3, Pg4); P. quinqueIolius L. (Pq), P. notoginseng (Pn1, Pn2) va P.
vietnamensis (Pv) tu Vit Nam.
Tach chit DNA.
Tach chit DNA tu cac mu Panax theo phuong phap cua Manian duoc bin di (9). Thanh t
bao duoc pha v bng cach lam lanh trong nito long va nghin min trong ci su. Mang t bao
duoc pha v bng cach u trong dm chit o 65
o
C trong 30 phut. Sau do, DNA duoc chit bng
hn hop chloroIorm-isoamyl alcohol (24:1). Tua DNA bng isopropanol va rua bng ethanol
70. DNA duoc hoa tan lai trong 1003l TE.
Thuc hin PCR-RFLP.
Vung ITS duoc khuch dai voi cp mi chung ITSAF (5`- GGA AGG AGA AGT CGT AAC
AAG G -3`) va ITSBR (5`- CTT TTC CTC CGC TTA TTG ATA TG -3`) (8). Phan ung PCR
duoc thuc hin voi tng th tich la 253l gm 13M/mi (IDT); 1U %aq polymerase (BioLabs);
0,4mM dNTP (BioLabs); 1X PCR buIIer (BioLabs); 2mM MgCl
2
(BioLabs). Chu trinh phan ung
la 1 chu ky: 94
o
C 5 phut; 30 chu ky: 94
o
C 30 giy, 45
o
C 1 phut, 72
o
C 1 phut; 1 chu ky: 72
o
C 7
phut.
Vung 18S cng duoc khuch dai voi cp mi chung 18SCOMF (5`- TGC ATG GCC GTT CTT
AGT TGG TGG -3`) va 18SCOMR (5`- CAC CTA CGG AAA CCT TGT TAC GAC -3`) (12).
Phan ung PCR duoc thuc hin voi tng th tich la 253l gm 0,43M/mi (IDT); 1U %aq
polymerase (BioLabs); 0,4mM dNTP (BioLabs); 1X PCR buIIer (BioLabs); 2mM MgCl
2

(BioLabs). Chu trinh phan ung la 94
o
C 5 phut / 35 chu ky 94
o
C 30 giy, 52
o
C 30 giy, 72
o
C 1 phut
/ 72
o
C 7 phut.
San phm PCR duoc tinh ch qua ct Promega Wizard SV Gel and PCR Clean-Up System va ct
bng hn hop enzyme HaeIII va HindIII (BioLabs) trong 1 gio o 37
o
C. San phm ct duoc din
di trn gel polyacrylamide 15.
Giai trinh tu
Cac san phm PCR duoc giai trinh tu boi cng ty Macrogen (Han Quc). Kt qua duoc BLAST
trn NCBI d tim cac trinh tu tuong dng
Thuc hin RAMS
Cac mi cho phan ung RAMS: RAMS1 (ACA)
5
, RAMS2 (CCA)
5
, RAMS3 (CGA)
5
, RAMS4
(GT)
5
G (7). Phan ung PCR duoc thuc hin voi tng th tich la 253l gm 5ng DNA khun;
0,53M/mi (IDT); 1U Taq polymerase (BioLabs); 0,2mM dNTP (BioLabs); 1X PCR buIIer
(BioLabs); 2mM MgCl
2
(BioLabs). Chu trinh phan ung la 95
o
C 5 phut / 35 chu ky gm 95
o
C 30
giy; 38,5
o
C (RAMS1), 53
o
C (RAMS2), 53
o
C (RAMS3), 33,5
o
C (RAMS4) 45 giy; 72
o
C 2 phut
/ 72
o
C 7 phut. San phm PCR duoc din di trn gel agarose 1.
Phn tich kt qua
San phm din di duoc chup hinh bng may Dolphin (Wealtec Corp.). Phn tich kt qua bng phn
mm BioNumerics 3.0 d tao cy phat sinh loai lam cn cu phn bit.
K ET QUA V BN LUAN
PCR voi cp mi ITS cho san phm co kich thuoc phu hop voi du doan trong khoang 750bp.
Ring cac mu Pg1 va Pg2 khng cho san phm. Con cp mi 18S cho san phm khoang 500bp
nhung mu Pn2 khng cho san phm.
Kt qua giai trinh tu va BLAST trn NCBI cho thy cac mu du thuc chi Panax. Tuy nhin, kt
qua trn vung 18S khng th phn bit o muc loai. Diu nay co th la do su bao tn cao cua vung
nay giua cac loai co quan h gn. Dua vao kt qua trn vung ITS, chung ti co th xac dinh loai
co quan h gn nht voi cac mu (bang 1) nhung khng th phn bit ngun gc cua cac mu.
Bang 1. Kt qua BLAST trinh tu vung ITS trn NCBI
Mu Loi Query
coverage
()
Max
ident
()
Pq Panax
quinqueIolius
98 98
Pg3 Panax ginseng 98 99
Pg4 Panax ginseng 96 99
Pn1 Panax
notoginseng
97 98
Pn2 Panax
notoginseng
98 97
Pv
Panax
quinqueIolius
Panax
vietnamensis
96 98
Mc du mu Pn1 va Pn2 la P. notoginseng, kt qua phn tich RFLP trn vung ITS cho thy
chung co muc tuong dng khng dang k (duoi 50). Nguyn nhn co th la do ngun gc, diu
kin sinh truong, th nhung khac nhau. Cac mu Pq, Pg3 va Pg4 co muc tuong dng cao, cho
thy chung co quan h gn voi nhau (hinh 1, 2).

Hnh 1. Kt qua RFLP vung ITS. M: thang 100bp

Hnh 2. Kt qua phn tich su da hinh san phm RFLP doan ITS

Phn tich RFLP trn vung 18S cho thy cac mu Pg3, Pg4 (thu mua tu Han Quc) co muc tuong
dng cao hon cac mu Pg1, Pg2 (thu mua tai Vit Nam) khoang 20 (hinh 3, 4). Nhin chung
phn tich RFLP trn vung 18S cho kt qua tuong dng cao hon vung ITS do vung nay duoc bao
tn cao hon.

Hnh 3. Kt qua RFLP vung 18S. M: thang 100bp

Hnh 4. Kt qua phn tich su da hinh san phm RFLP doan 18S
Thu nghim cac mi RAMS thi chi co mi RAMS1 cho san phm khi phn tich trn gel. Do do,
chung ti chon mi nay cho cac thu nghim tip theo. Khao sat nng d MgCl
2
cho thy nng d
MgCl
2
o 2mM cho kt qua PCR r nht.
Trn co so cac thng s nhu trn, chung ti tin hanh RAMS trn cac mu. Kt qua phn tich trn
BioNumerics cho thy cac mu co muc da dang thp (duoi 15). Diu nay la do kich c mu
thp va chi co mt mi duoc thu nghim (hinh 5, 6).

Hnh 5. Kt qua PCR mi RAMS1. M: thang 1000bp

Hnh 6. Kt qua phn tich da hinh san phm khuch dai RAMS1.
Tu cac kt qua trn, chung ti nhn thy d co th kt lun chinh xac v loai va ngun gc cua
cac mu cn kt hop nhiu phuong phap. Phn tich trinh tu vung ITS co th giup xac dinh loai
cua cac mu. Ngoai ra, su kt hop giua phn tich RFLP va giai trinh tu co th phn bit ngun
gc cua cac mu nay.
K ET LUAN
Trong nghin cuu nay, cac mu Panax sp. duoc xac dinh loai va phn bit ngun gc nho cac k
thut sinh hoc phn tu. Nhin chung, cac mu sm thu mua tu Han Quc co muc tuong dng cao hon
cac mu thu mua tai Vit Nam.