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B Y T

TRNG I HC DC H NI

NGUYN HU TH

NGHIN CU TNH A DNG SINH

HC CA CY D

KHA LUN TT NGHIP DC S

H NI 2013

B Y T

TRNG I HC DC H NI

NGUYN HU TH

NGHIN CU TNH A DNG SINH

HC CA CY D

KHA LUN TT NGHIP DC S

Ngi hng dn:

PGS.TS. Trn Vn n

Ni thc hin:

B mn Thc Vt Trng H

Dc H Ni

H NI - 2013

LI CM N

Trc ht, ti xin by t lng knh trng v bit n su sc ti PGS.TS.

Trn Vn n Ging vin B mn Thc Vt, Trng i Hc Dc H Ni

trc tip hng dn v ch bo tn tnh cho ti trong sut thi gian thc hin ti

kha lun.

Ti xin gi li cm n chn thnh ti cc thy c TS. Hong Qunh Hoa,

ThS. Phm H Thanh Tng, DS. Nghim c Trng, cng ton th cc thy c

gio v cc ch k thut vin B mn Thc vt, nhit tnh ch bo v gip , to

iu kin thun li cho em hon thnh kha lun tt nghip.

Ti xin gi li bit n su sc ti s dy d, du dt ca cc thy c gio

trong nh trng trong sut 5 nm hc va qua. Ti cng xin gi li cm n c

bit ti b m, ngi thn v bn b lun ng vin tinh thn cho ti trong sut

thi gian hc tp v nghin cu.

H Ni, ngy 19 thng 05 nm 2013

Sinh vin

Nguyn Hu Th

MC LC

Trang

T VN 1

CHNG 1 TNG QUAN 3

1.1 Cy d 3

1.1.1 V tr phn loi ca chi Coix L. 3

1.1.2 c im thc vt v phn b ca chi Coix L. 3

1.1.3 Thnh phn ha hc 5

1.1.4 Tc dng sinh hc 8

1.1.5 Cng dng v s dng ca cy d 10

1.2 DNA v phng php xc nh trnh t gen 12

1.2.1 Mt s tnh cht v chc nng ca DNA 12

1.2.2 Phng php xc nh trnh t gen 14

1.3 Phng php sc k lp mng hiu nng cao 17

1.3.1 Sc k lp nng hiu nng cao 17

1.3.2 Du vn tay sc k 17

1.3.3 ng dng HPTLC 18

CHNG 2 PHNG PHP NGHIN CU 19

2.1 Nguyn vt liu, thit b, ho cht 19

2.1.1 Nguyn vt liu 19

2.1.2 Thit b, ha cht 19

2.1.3 Phn mm h tr 21

2.2 Ni dung v phng php nghin cu 21

2.2.1 Nghin cu a dng v c im hnh thi v nng hc 21

2.2.2 Nghin cu a dng v di truyn 24

2.2.3 Nghin cu v ha hc bng phng php HPTLC 25

CHNG 3 KT QU NGHIN CU V BN LUN 27

3.1 Tnh a dng v c im hnh thi v nng hc 27

3.1.1 c im hnh thi ca cc mu d thi im ngy 02/05/2013

27

3.1.2 c im nng hc ca cc mu d 35

3.1.3 Kt qu nghin cu tnh a dng v c im hnh thi v nng hc

36

3.2 S khc bit v thnh phn ha hc trong ht 38

3.3 Tnh a dng v di truyn 40

BN LUN 42

KT LUN

KIN NGH

45

47

DANH MC CC K HIU, CC CH VIT TT

A Adenin

C Cystosin

CTAB Cetyltrimethylammonium (cetrimonium) bromide DNA Acid deoxyribonucleic

dATP Deoxy Adenosine Triphosphate

dCTP Deoxy Cytidine Triphosphate

dGTP Deoxy Guanosine Triphosphate

dTTP Deoxy Thymidine Triphosphate

ddATP Dideoxy Adenosine Triphosphate

ddCTP Dideoxy Cytidine Triphosphate

ddGTP Dideoxy Guanosine Triphosphate

ddTTP Dideoxy Thymidine Triphosphate

ddNTP Dideoxy Nucleoside Triphosphate

dNTP Deoxy Nucleoside Triphosphate

EDTA Ethylen Diamin Tetraacetic Axit

FDA C quan qun l cht lng thuc v thc phm

GAP Good Agricultural Practices - Thc hnh tt trng trt cy thuc

HPTLC Sc k lp mng hiu nng cao

IC50 Inhibitory Concentration 50 - Nng c ch 50% i tng th

ITS Internal Transcribed Spacer

KLT Kanglaite

mARN Messenger Acid Ribonucleic ARN thng tin

MeOH Methanol

Nu Nucleotide

PCR Polymerase Chain Reaction - Phn ng chui trng hp

Rf Retention Factor H s lu tr

STZ Streptozocin

T Thymin

TLC Thin Layer Chromatography- Sc k lp mng

DANH MC CC BNG

TT K hiu Tn bng Trang

1 Bng 1.1 Cc phn ng ha hc c hiu khi thc hin gii trnh

t gen bng phng php Maxam v Gilbert

15

2 Bng 2.1

Danh sch cc mu nghin cu 19

3 Bng 2.2

Cc ch tiu nng sinh hc c theo di 22

4 Bng 2.3

Cc bc thc hin tch DNA t mu l d 24

5 Bng 3.1

Bng tng kt cc ch tiu nng sinh hc v c im

hnh thi so snh 7mu d

36

6 Bng 3.2 Din tch peak ca mu Y1 ti Rf = 0,14; mu Y2 ti Rf

= 0,66 v mu Y5 ti Rf = 0,63

38

7 Bng 3.3 Din tch peak ph HPTLC dch chit methanol cc

mu d ti Rf = 0,69

39

DANH MC CC HNH

TT K

hiu

Tn hnh Trang

1 Hnh 1.1 Cng thc cu to ca Coixol v -monolinolein 5

2 Hnh 1.2 Cng thc cu to ca Coixenolide 6

3 Hnh 1.3 Cng thc cu to ca cc hp cht (4) (8) 7

4 Hnh 1.4 Cng thc cu to ca cc hp cht (9) (13) 8

5 Hnh 1.5 Thuc tim Kanglaite 12

6 Hnh 1.6 Cu trc ca vng ADN ribosom ITS 14

7 Hnh 2.1 Chu nha dng trng cc mu d 22

8 Hnh 3.1 c im hnh thi mu Y1 (Mai Chu Sn La) 27

9 Hnh 3.2 c im hnh thi mu Y2 ( Bc Ha Bnh) 28

10 Hnh 3.3 Thn mang l mu Y3 (o Tr Vnh Phc) 29

11 Hnh 3.4 Thn mang l mu Y4 (Hong Mai H Ni) 29

12 Hnh 3.5 c im hnh thi mu Y5 (Chiang Rai Thi Lan) 30

13 Hnh 3.6 c im hnh thi ca mu Y6 (Thun Chu Sn La) 31

14 Hnh 3.7 c im hnh thi mu Y7 (Mng Thanh in Bin) 32

15 Hnh 3.8 c im hnh thi mu Y8 (Thnh ph Hi Dng) 33

16 Hnh 3.9 c im hnh thi mu Y9 (Vn Quan Lng Sn) 34

17 Hnh

3.10

Thn mang l mu Y10 (Ba V H Ni) 34

18 Hnh

3.11

Cc giai on pht trin ca cy d 35

19 Hnh

3.12

Cy phn loi 7 mu d da trn c im hnh thi v

nng hc

38

20 Hnh

3.13

Sc k HPTLC ca cc mu d 39

21 Hnh

3.14

Cy phn loi 9 mu d da trn trnh t nucleotid 41

DANH MC PH LC

TT K hiu Tn ph lc

1 Ph lc 1 Cc ch tiu nng sinh hc theo di c t thng 12/2012

n thng 05/2013

2 Ph lc 2 Bng m ha cc bin s c im hnh thi v ch tiu nng

sinh hc

3 Ph lc 3 Kt qu chng peak sc k 10 mu d

4 Ph lc 4 7 on trnh t nucleotid dng so snh t ngn hng gen

quc t

5 Ph lc 5 Trnh t nucleotid ca 9 mu d

6 Ph lc 6 tng ng di truyn ca 9 mu d

7 Ph lc 7 Kt qu ging hng trnh t nucleotid ca 9 mu d v 7

on trnh t so snh

1

T VN d (Coix lachryma-jobi L.), h La (Poaceae), l cy thuc c s dng

lu i v ph bin trn Th gii cng nh Vit Nam vi nhiu cng dng. R

d dng cha vim nhim ng niu, si thn, thy thng, phong thp au

xng, tr em a chy, bch i, ri lon kinh nguyt, kinh b, tr giun a, au

bng giun. Ht d c dng cha p-xe phi, rut tha, vim rut a chy, phong

thp sng au, lot d dy, lot c t cung, mn cc, eczema. d l thuc b v

bi dng c th tt, dng b sc cho ngi gi v tr em, dng li sa cho ngi

ph n sinh . Gn y, cc nghin cu trong v ngoi nc pht hin tc dng

h ng huyt [10], [35] v tc dng chng ung th [29] ca cy ny.

Mc d c s dng ph bin vi nhiu cng dng quan trng nh vy,

nhng ngun dc liu d s dng trong nc ch yu c nhp khu t

Trung Quc. min Bc Vit Nam, d ch yu mc hoang v c trng ri rc

vi qui m nh, cha p ng c nhu cu s dng trong nc. V vy, cn phi

m rng qui m trng cy d theo cc quy chun ca Thc hnh tt trng cy

thuc (GAP), nhng min Bc Vit Nam c nhiu th/ging d khc nhau, vi

hnh thi kh ging nhau m cho n nay vn gy ra s nhm ln gia cc

th/ging. Mi th/ging c th cho nng sut v cht lng khc nhau, dn n

hiu qu kinh t v iu tr c th khc nhau.V vy, vic xc nh tnh a dng sinh

hc ca cy d min Bc Vit Nam l cn thit. Nm 2012, Dc s Nguyn

Kim Khanh nghin cu s a dng sinh hc ca 6 mu d min Bc Vit

Nam. Kt qu thu c l 6 mu d t c s a dng v ha hc nhng c s khc

bit v c im hnh thi, nng hc v di truyn [11]. Tuy nhin nghin cu ny

mi ch nghin cu trn 2 th l: Coix lachryma-jobi L. var. lachryma-jobi v Coix

lacryma-jobi L. var. stenocarpa Oliv.

Do ti Nghin cu tnh a dng sinh hc ca cy d c tip tc

thc hin vi 2 mc tiu chnh l:

1. Xc nh tnh a dng v c im hnh thi, nng hc v di truyn ca

mt s ging d.

2

2. Kho st s khc bit v thnh phn ha hc trong ht ca mt s ging

d bng phng php HPTLC.

3

CHNG 1. TNG QUAN 1.1. Cy d

1.1.1. V tr phn loi ca chi Coix L.

Chi Coix L. thuc h La (Poaceae), b La (Poales), phn lp Hnh

(Liliidae), lp Hnh (Liliopsida), ngnh Ngc Lan (Magnoliophyta) [3].

1.1.2. c im thc vt v phn b ca chi Coix L.

Cy tho sng hng nm hay lu nm. Thn mc thng ng cao 1,5 2m,

phn nhnh nhng ngn c hoa. gc thn c nhiu r ph. L mc so le, mt l

rp, gn l song song khng c cung. Hoa n tnh cng gc. Bng hoa c ngn

mu lc nht trng ta mt nhnh ca bng la; hoa ci nm trong mt l bc dy

v cng, t mu xanh chuyn sang mu nu n tm en. Qu hnh trng hi nhn

u (thng gi nhm l nhn) c bao bc bi mt l bc cng (thng gi nhm

l v) [5], [18].

B phn dng: Ht (Semen Coicis), thng gi l d nhn. R, l cng c

dng [5].

Phn b: Mc ph bin vng nhit i, c bit l chu nhit i, mc

hoang ven b nc, ni m nh bi, rung. Cy cng c trng ch yu bng

ht [5].

Ma hoa qu: Thng 5 - 12 [2].

Vit Nam, xc nh d c 4 th sau: [5], [9]

- Coix lachryma-jobi L. var. lachryma-jobi - Cm go: Cy c sng nhiu

nm, thn c ng knh 8-10mm. L phng, cung di 40-50cm, rng 4-5cm, gn

gia to, gn bn rt mnh; b nhn, li nh 1mm. Cm hoa ngn nhnh. Bng

cht c trn, di 6-7cm. Nh vng. Bng cht ci di, c bao hnh bu, di 8-

9mm, ng knh 6mm, khi chn nu en ri trng, rt cng. Th ny mc cc t

hoang n vng nc l.

- Coix lacryma-jobi L. var. mayuen (Rom) Stapf (C.chinensis Todaro ex

Balansa) d, Bo bo: Cy c sng hng nm. Thn phn nhnh, nhn, to, xp,

cao 1-2m. L phng, thun di, hnh tim gc, nhn u, di 10-50cm, gn gia

4

ln ni r, gn bn rt mnh; b l nhn, ko di, thng rt rng. Cm hoa l bng

mc thng ng, c cung. Hoa n tnh cng gc. Bng cht c mc chm 2-3

chic mt ch trn cung chung mnh, c my cng bao bc. Bng cht ci hnh

bu dc; l bc rt dy, cng m trng nht hay xanh xm. Qu hnh cu hay hnh

bu dc, c v dng giy hay ha cng. Phn b n , Vit Nam, Lo, Indonexia,

Philippin. Vit Nam, cy mc hoang v cng c trng, thng gp cc b

nc, ni t m ven rng, ven ng. Ra hoa t thng 7 n thng 12.

- Coix lacryma-jobi L. var. puellarum Balansa (C.puellarum Balansa) Bo

bo di, Cm go: Cy c sng nhiu nm, cao trn 50cm, phn nhnh nhiu, rut

xp, nhn. L mm, phng, mp nhn nheo, u nhn, gc hnh tim, gn mnh ni

r c 2 mt; b l nhn, thun di. Cm hoa nch l hay ngn thn. Bng cht c

trn nh, hp vi cung chung xp st nhau; my bng, cng. Bng cht ci c l

bc cng bao bc, mu trng tm, bng. Qu c l bc pht trin m ly ton b

phn bn trong nh b, hnh cu,bng, cng rn. Cy mc hoang ven i thp m,

ven b nc hoc trn cc ry. Ra hoa qu gn nh quanh nm.

- Coix lacryma-jobi L. var. stenocarpa Oliv: Cy c sng nhiu nm, sng

trn cn hay di nc. Thn ni hay nm, c r mt di n 30m. L c phin,

di 30 90cm, rng 2 4cm, mt trn c lng; cung khng lng. Hoa mc k l,

vi bng cht c thng, mu xanh. Bng cht c mc chm 3 chic mt ch trn

cung chung mnh. Qu cng, mu vng hay nu.

Nm 2012, Nguyn Kim Khanh tin hnh nghin cu tnh a dng sinh hc

ca 6 mu d, ch yu l cc ging hoang di c ngun gc t 6 tnh min Bc

Vit Nam (H Ni, Qung Ninh, in Bin, Bc Giang, Ha Bnh, Lng Sn). Cc

mu d c gieo trng trong cng mt iu kin v trong cng mt ma v ti

Vn thc vt B mn Thc vt Trng i hc Dc H Ni, v c nghin

cu xc nh tnh a dng v c im hnh thi v nng hc, tnh a dng v di

truyn v tnh a dng v ha hc. Kt qu l 6 mu d c s khc bit v c

chia thnh 3 nhm; nhm 1 gm cc mu Ba V H Ni, Sn ng Bc Giang

v Vn Quan Lng Sn; nhm 2 gm cc mu Ba Ch - Qung Ninh v Mng

5

Thanh - in Bin v nhm 3 gm mu Bc - Ho Bnh. Tuy nhin, s lng

mu nghin cu cn t (6 mu); s khc bit da trn c im hnh thi v nng

hc khng tng ng vi s khc bit v di truyn; da trn thnh phn ho hc,

cc mu d nghin cu khng khc bit r rng v thnh phn, nhng sai khc r

rng v hm lng [11].

1.1.3. Thnh phn ha hc

Qu d cha 50 79% tinh bt, 16 19% protein, 2 - 7% du bo, lipid

(5,67% glycolipid, 1,83% phospholipid, sterol), thiamin, acid amin, adenosine,

cht v c, ; coixenolid (khong 0,25%), coixol, -monolinolein (Hnh 1.1) [2].

R d cha 17,6% protein, 7,2% cht bo, 52% tinh bt, benzoxazolon (cht

c tc dng chng vim r) [16], [2].

V qu cha alcohol coniferyl, acid syringic, acid ferulic, syringaresinol, 4-

ketopinoresinol v mayuenolide [28].

6

5

4

8

9

7

O1

NH

3

2O

MeO

1. Coixol 2. -monolinolein

Hnh 1.1. Cng thc cu to ca Coixol, -monolinolein

1.1.3.1. Coixenolide [36]

Nm 1960, Tyunosin Ukita v cng s tch chit c coixenolide t dch

chit aceton ca ht d (Coix lacryma-jobi L. var. ma-yuen Stapf). Bt nhn ht

d c chit vi aceton nhit phng. Sau tin hnh phn lp v tinh ch

bng sc k ct silicagel thu c hp cht c cng thc C38H70O4, n20D=1.4705,

[]20D=0o. Hp cht ny c tc dng c ch s pht trin ca khi u c gi l

coixenolide (Hnh 1.2).

6

3. Coixenolid

Hnh 1.2. Cng thc cu to ca Coixenolid

1.1.3.2. Cc coixan [35]

Nm 1986, Takahashi v cng s phn lp c coixan A, B, C t dch

chit ht cy d (Coix lacryma-jobi var. ma-yuen). Bt nhn ht d c chit

vi nc nhit phng. Sau tin hnh sc k ct tch ring cc hp cht

coixan A, B, C. Xc nh cu trc coixan A, B, C bng cc phng php phn tch: 1H-NMR (ph cng hng t ht nhn proton), 13C-NMR (ph cng hng t ht

nhn 13C).

1.1.3.3. Cc hp cht lactam [29]

Nm 2008, Ming-Yi Lee v cng s tm ra 5 hp cht c ch t bo ung

th t dch chit methanol thn d (Coix lacryma-jobi L. var. ma-yuen Stapf) l:

Coixspirolactam A (4), coixspirolactam B (5), coixspirolactam C (6), coixlactam (7)

v methyl dioxindol 3 acetat (8). Bt thn d c chit bng methanol, cn

to thnh c lc vi cc dung mi: n-hexan, ethyl acetat v n-butanol. Nm hp

cht trn phn lp t phn on ethyl acetat bng sc k ct silicagel v c tinh

ch bng HPLC (sc k lng hiu nng cao) trn ct Si 60 Lichrosorb. Da vo

cc phng php phn tch quang ph gm IR (ph hng ngoi), 1H-NMR (ph

cng hng t ht nhn proton), 13C-NMR (ph cng hng t ht nhn 13C), 2D-

NMR (ph cng hng t ht nhn hai chiu) v MS (ph khi) ngi ta xc nh

cu trc ca 5 hp cht (Hnh 1.3).

7

6

5

4

3a

7a

7

NH1

3

2O

10

8

O9

O

6

5

4

3a

7a

7

NH1

3

2O

108

O9

O

H11

CH3

6

5

4

3a

7a

7

NH1

3

2O

108

O9

O

H3C11H

4. Coixspirolactam A 5. Coixspirolactam B 6. Coixspirolactam C

6

7

8

8a

4a

5

NH1

2

3

4

O

9

O

OCH3

5

6

7

7a

3a

4

3

NH1

2

HO8

O

9

OCH3

O

7. Coixlactam 8. methyl dioxindol 3 acetat

Hnh 1.3. Cng thc cu to ca cc hp cht (4) (8)

1.1.3.4. Cc hp cht c tnh acid [34]

Nm 1994, Numata M v cng s t dch chit aceton ca nhn ht d

tm thy cc thnh phn c tc dng c ch s tng trng khi u trn chut. Cc

thnh phn ny c tch ring bng sc k ct silicagel v c xc nh cu trc

bng cc phng php phn tch: Quang ph hng ngoi v sc k lng - kh. Cc

thnh phn ny c xc nh l 4 acid bo: Acid palmitic, acid stearic, acid oleic

v acid linoleic.

1.1.3.5. Coixol v cc hp cht benzoxazinones [33]

Nm 1985, Nagao v cng s phn lp c 5 hp cht benzoxazinones (9)

(13) v coixol (1) t phn on chloroform ca dch chit methanol r cy d

(Coix lacryma-jobi L. var. ma-yuen Stapf). Hp cht (9) l 2-hydroxy-7-methoxy-

1,4(2H)-benzoxazin-3-one. Hp cht (10) l 2-O--D-glucopyranoside. Hp cht

(11) l 2-O--glucopyranosyl-4-hydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one.

8

Hp cht (12) l 2-O--glucopyranosyl-4,7-dimethoxy-1,4(2H)-benzoxazin-3-one.

Hp cht (13) l 2-O--glucopyranosyl-7-hydroxy-1,4(2H)-benzoxazin-3 (Hnh

1.4).

7

6

5

9

10

8

N4

3

2O1

R5

R412

R3

OR2

O

Hnh 1.4. Cng thc cu to ca cc hp cht (9) (13)

1.1.4. Tc dng sinh hc

1.1.4.1. Tc dng chng ung th

Nm 2008, Ming-Yi Lee v cs nghin cu tc dng c ch t bo khi u

t thn d v 5 hp cht lactam c tch t dch chit MeOH thn d (Coix

lacryma-jobi L. var. ma-yuen). Cc ng tin hnh th nghim trn cc dng t

bo l A549 ung th phi ngi, HT 29 (Ung th t bo biu m trc trng

ngi) v COLO 205 ung th t bo biu m rut bng phng php th MTT (3

4,5 dimethylthiazol 2 yl 2,5 diphenyl tetrazolium bromid). Kt qu cho

thy dch chit thn d cc phn on v 5 hp cht phn lp c u c tc

dng c ch t bo ung th. Tuy nhin, phn on ethyl acetat c tc dng c ch

mnh hn cc phn on cn li (IC50 trong phn on ethyl acetat t 82% n

153%) [29].

Dch chit cn bt d cng c bo co l lm gim c ngha s lng

cc tiu nang bt thng tin ung th [30].

1.1.4.2. Tc dng h ng huyt

R2 R3 R4 R5

9 H H OMe H

10 Glc H OMe H

11 Glc OH OMe H

12 Glc OMe OMe H

13 Glc H OH H

9

Nm 1986, Michiko Takahashi v cs phn lp c 3 cht l coixan A, B

v C t ht ca d (Coix lacryma-jobi L. var. ma-yuen) v ch ra rng dch

chit ht d v 3 cht ny c tc dng h ng huyt trn chut [35].

Nm 2009, ThS. Phng Thanh Hng v cs nghin cu tc dng h ng

huyt ca dch chit thn d (Coix lacryma-jobi L. var. ma-yuen Stapf) trn chut

bnh thng, chut tim STZ (Streptozocin) v chut tim adrenalin (Liu 20g dc

liu kh/kg). Kt qu thu c nh sau:

- Trn chut bnh thng: Sau khi cho ung dch chit thn cy d v tin

hnh xc nh nng glucose huyt ca l chng v l th thi im 0h, 1h, 2h,

3h, 4h v 5h thy glucose huyt khng thay i theo thi gian.

- Trn chut tim STZ: Sau khi tim STZ 72h, nh lng glucose huyt lc

i ca chut, sau cho chut ung dch chit d v sau 10 ngy nh lng li

nng glucose huyt ca l chng v l th. Tin hnh song song vi l chng

ung nc ct. Kt qu cho thy nng glucose l th gim khong 36%. Nh

vy dch chit thn d c tc dng h glucose huyt trn chut tim STZ.

- Trn chut tim adrenalin: Chut nhn i 12h c cho ung dch chit.

Sau 3h tim adrenalin vi liu 0.6mg/kg, nh lng glucose huyt thi im

trc khi tim v sau khi tim 60ph. Tin hnh song song vi l chng ung nc

ct. Kt qu cho thy sau khi tim adrenalin liu 0.6mg/kg, glucose huyt ca chut

c 2 l u tng, nhng l ung dch chit, mc tng glucose huyt (~72%) thp

hn l chng (~125%). Nh vy, dch chit thn d c tc dng hn ch tng

glucose huyt bi tc nhn adrenalin [10].

1.14.3. Tc dng chng vim

Nm 2009, Din Wen Huang v cs iu tra nhng nh hng ca dch

chit v ht d (Coix lacryma-jobi L. var. ma-yuen Stapf) trn lipopolysaccharide

gy ra p ng vim ti i thc bo RAW 264.7 v cho thy dch chit v ht d

c tc dng chng vim do chng chng li s gia tng sn sinh t bo ca nitric

oxide v prostaglandin E2 gy ra bi lipopolysaccharide bng cch lm gim s sn

xut ra cc enzym nitric dioxide synthase v cyclooxygenase [25].

10

1.1.4.4. Cc tc dng khc :

Tc dng h m mu [21], [24], [27].

Tc dng chng d ng [20], [23].

Tc dng co rt t cung trong thai k: Dch chit nc ht d c kh nng

lm nhim c thai nhi v co rt t cung trong thi gian mang thai [37].

Tc dng trn h ni tit : Dch chit methanol lm gim bi tit

progesterone, dch chit methanol thn d lm gim tit testosterol [22].

1.1.5. Cng dng v s dng ca cy d

1.1.5.1. Tnh v, quy kinh, cng nng, ch tr [14]

- Theo Y hc c truyn, d c v ngt, nht, tnh hi hn; quy vo 5 kinh

T, V, Ph, Can, i trng.

- Cng nng ch tr:

Li thy: Dng tr cc bnh ph thng, tiu tin kh khn, i but.

Dng ht hoc cy, l, r, sc ung.

Kin t ha thp: Dng tr bnh t h, tiu ha km, tit t. d sao

vng cng vi mt s v thuc khc trong bi Ph nhi cam tch, dng tt

cho tr em.

Tr phong thp, au nhc, phi hp vi Ma hong, Phng k, Mc thng.

Thanh nhit c, tr m: Dng iu tr chng ph qun ha m (p xe

phi). R d kt hp vi L cn, o nhn, Dip c.

Th cn gii kinh: Dng khi chn tay b co qup.

Gii c tiu vim: Dng d trong bnh vim rut tha. Phi hp vi

Kim ngn hoa trong bnh ni mn mt, trng c. Ht d nu cho ngy

10g, hoc phi vi thuc thanh nhit gii c.

1.1.5.2. S dng

B phn dng lm thuc ch yu l ht, cn gi l D m, D nhn, d nhn,

ht Bo bo, Bo bo... Ngoi ra, ngi ta cn dng thn, r lm thuc [12].

11

Do c lng protid, cht bo v tinh bt kh cao nn qu d c coi l

ngun lng thc c gi tr, ng thi l mt v thuc qu. d thng c dng

cha ri lon tiu ha, ph thng, b i, trng ung, t l, au bng, phong thp

lu ngy khng khi, gn co qup kh vn ng. Nhn dn Vit Nam thng dng

d lm thuc bi dng c th, nht l i vi tr em. Dc liu thng c mt

trong nhiu n thuc v bit dc, nh 54.5% trong bt b t tr giun, 40% trong

bt cam tr em v 10% trong vin Ph nhi lin hon hoc ko b t. d hm vi

ht Sen v tht nc l mn n v thuc cho nhng ngi c th gy yu, suy dinh

dng [2].

Liu dng 8 30g. Thng dng di dng thuc sc. Dng ring hay kt hp

vi cc thuc khc.

1.1.5.3. Mt s bi thuc trong Y hc c truyn

Cha trng ung bng trng y tiu tin kh: d, Qua lu nhn, mi v

6 9g; Mu n b, o nhn, mi v 6g. Sc nc ung [2].

Cha thy thng: d, Xch tiu u, ng qua b, mi v 30g; Hong k,

Phc linh, mi v 15g. Sc nc ung [2].

Cha tiu tin ra mu: d 20g, nc 600ml, sc n khi cn 20ml.

Ung lin tc cho n khi thy tiu tin bnh thng l c [12].

Cha bnh phi, nn ra mu: d 40g, nc 400ml (2 bt) sc cn

1 bt (200ml) thm t ru vo ung lm 2 ln trong ngy. Ung lun 10

ngy [12].

B, cha lao lc: d 5g, Mch mn ng 3g, Tang bch b 3g, Bch b

3g, Thin mn ng 3g, nc 600ml. Sc cn 200ml chia lm 3 ln ung

trong ngy [12].

Cha t thp: d nhn 40g, Th phc linh 20g, nc 600ml sc cn

200ml. Chia lm 2 ln ung trong ngy. Ung lin 10 ngy nu thy tiu

tin nhiu l bnh gim [12].

1.1.5.4. Mt s sn phm t d

12

Thuc tim Kanglaite (KLT) (Hnh 1.5) [38] l thuc chng ung th mi c

chit xut t ht d. FDA (C quan qun l cht lng thuc v thc phm) ca

Hoa K ph duyt giai on II th nghim ca KLT kim tra hiu qu ca n

trong iu tr ung th phi khng phi t bo nh. Mt s nghin cu cho thy n

c th c ch mt s gen chng qu trnh t dit t bo v kch hot mt s gen tin

t dit t bo.

Hnh 1.5. Thuc tim Kanglaite

Thuc tim ny c th c ch ng k nhiu loi t bo ung th khc nhau, do

, n tr thnh phn ct li ca nghin cu l lm th no tip tc khm ph

thuc tim KLT thc y s t hy dit t bo khi u bng cch tc ng ln cc

gen c lin quan [31].

Thuc tim KLT c ch s tng trng t bo HepG2 bng cch gy cht t

bo thng qua s kch hot con ng Fas/FasL [32].

Tm li, cc nghin cu trong nc v ngoi nc nghin cu nhiu tc

dng v cng dng ca cy d, ch yu trn th Coix lachryma jobi L. var.

mayuen v nghin cu trn thn v ht. V a dng sinh hc ca cy d, nc

ngoi nghin cu a dng cc mu d da vo du vn tay DNA, hay du vn

tay TLC [26]. Vit Nam c ti nghin cu a dng sinh hc cy d vi

cc ch tiu du vn tay DNA, du vn tay TLC v c im hnh thi, nng hc

[11].

1.2. DNA v phng php xc nh trnh t DNA [6], [8], [15]

1.2.1. Mt s tnh cht v chc nng ca DNA

Cu to ca DNA m bo cho n thc hin chc nng "gi" c thng tin

di truyn.

13

DNA l 1 chui xon kp. Trn mi mch n ca DNA cc Nu lin kt vi

nhau bng lin kt phophodieste bn vng. Trn 2 mch ca DNA, cc Nu lin kt

vi nhau bng lin kt cng ha tr theo nguyn tc b sung. A lin kt vi T bng 2

lin kt H, G lin kt vi C bng 3 lin kt H (tuy lin kt H khng bn, d dng b

ct t bi 1 loi enzym trong qu trnh tho xon nhng vi s lng Nu ln

lm cho cu trc khng gian n nh). T 4 loi Nu nhng do s sp xp khc nhau,

s lng v thnh phn khc nhau, to ra s a dng ca cc phn t DNA cc

loi sinh vt khc nhau.

Tnh cht ca DNA m bo n " truyn t" c thng tin di truyn: DNA

c kh nng t nhn i, nh vy NST s nhn i m bo thng tin di truyn

c truyn t qua cc th h sau. DNA cha cc gen cu trc, cc gen ny c kh

nng phin m to mARN ri t hnh thnh protein, quy nh tnh trng cho

sinh vt.

DNA c kh nng t bin, hnh thnh nhng thng tin di truyn mi, cc

thng tin di truyn ny t nhn i nh c ch ti bn ca DNA.

DNA c s bin tnh thun nghch: Khi dung dch DNA b un nng trn

nhit ring th cu trc t nhin b ph hy, hai chui b sung tch khi nhau

v to ra cu trc n xon ngu nhin. Nu lm lnh nhanh v duy tr nhit

khong 25C di Tm trong mt khong thi gian nht nh th hai si n DNA

bin tnh s bt cp tr li theo nguyn tc b sung.

Trong c chui ADN nhn ca t bo thc vt, khu vc c nghin cu

nhiu nht l vng ADN ribosom 18S-26S v vng phin m ni (Internal

Transcribed Spacer ITS) vi nhiu on lp v trnh t nucleotid . Vng phin m

ni ITS ca ADN ribosom (ITS-rADN) c s dng ph bin trong cc nghin

cu phn loi loi ca thc vt. Vng ITS c 3 phn: ITS1, 5.8S v ITS2 (Hnh 1.6)

vi kch thc thay i t 500 n 700bp.

14

Vng ITS

Hnh 1.6. Cu trc ca vng ADN ribosom ITS

Trnh t nucleotid vng ITS c nghin cu nhiu v s lng cc trnh

t nucleotid ca vng ny c cng b trong ngn hng gen quc t kh phong

ph, thun li cho phn tch so snh. Vng ITS c u im l: Vng ITS c lp

li cao trong ADN nhn ca thc vt, sn phm PCR ca ADN nhn c th s dng

cho thng tin nghin cu pht sinh loi ca nhiu loi, v vng ITS c kch thc

nh v tnh bo th cao nn d dng khuch i.

1.2.2. Phng php xc nh trnh t gen

1.2.2.1. Khi nim

Phng php gii trnh t gen (DNA sequencing) l phng php xc nh v

tr sp xp cc nucleotid trong phn t DNA.

1.2.2.2. Nguyn tc: Da trn 1 trong 2 nguyn tc sau

- Nguyn tc ha hc: Da vo cc phn ng ha hc thy gii c hiu phn

t DNA, to thnh mt tp hp nhiu phn on c kch thc khc nhau.

- Nguyn tc enzym hc: Da vo s tng hp mch b sung cho trnh t cn

xc nh nh ADN polymerase. Vi vic s dng thm cc dideoxynucleotid cng

vi cc deoxynucleotid thng thng, kt qu tng hp cng l s hnh thnh mt

tp hp nhiu on DNA c kch thc khc nhau.

1.2.2.3. Cc phng php gii trnh t gen

a. Phng php Maxam v Gilbert

- Phng php ny c thc hin da theo nguyn tc ha hc.

- Cc bc thc hin:

26S ADNr nhn

18S ADNr nhn

15

Bc 1: nh du phng x 32P u 5 ca mch khun, bin tnh v

in di tch ly mt mch DNA lm mch khun, cho cc x l tip theo.

Bc 2: Thc hin cc phn ng ha hc c hiu (Bng 1.1)

C th s dng 4 5 ng nghim thc hin cc phn ng ha hc c hiu

khc nhau.

Bng 1.1. Cc phn ng ha hc c hiu khi thc hin gii trnh t gen bng

phng php Maxam v Gilbert

ng X l c hiu V tr ct

1 Dimethyl sunfat Ct ti G

2 Acid PH = 2 Ct ti A v G

3 Hydrazin Ct ti C v T

4 Hydrazin + NaCl Ct ti C

5 NaOH Ct ti A hoc C (A>C)

Trong mi ng nghim c hm lng nht nh cc mch n DNA khun

nh du phng x, c x l ha hc c hiu khc nhau, to cc on DNA di

ngn khc nhau.

Bc 3: Ly sn phm x l trong mi ng nghim em chy in di, hin

hnh phng x v xc nh kt qu.

Bc 4: Tp hp kt qu thu c tt c cc ng nghim, thu c cc

on polynucleotid di ngn hn km nhau 1 nucleotid, t xc nh c trnh t

ca mch DNA khun.

Phng php xc nh trnh t nucleotid bng cc phn ng ha hc c hiu

ca Maxam v Gilbert c chun xc km, cn phi thc hin nhiu ln v loi b

cc sai st chn mt kt qu gn ng nht. Do , phng php Maxam v

Gilbert t c ng dng trong thc tin.

b. Phng php enzym hc thng qua vic s dng cc dideoxynucleotid ca

Sanger v cng s

- Phng php ny c thc hin theo nguyn tc enzym hc.

- Cc bc thc hin:

16

Bc 1: Bin tnh DNA khun, in di trn gel polyacrylamid (c b sung

ure) thu cc mch n DNA.

Bc 2: Chun b phn ng tng hp DNA

Ly bn ng Eppendorf, cho cc thnh phn cn thit vo mi ng (Mch

khun, mi c nh du phng x, enzym ADN polymerase v dNTP (dATP,

dTTP, dGTP, dCTP) v dung dch m thch hp). Thm vo mi ng tng ng

mt lng nh (khong 1%) mt loi ddNTP nht nh: ng 1 b sung 1% ddATP,

ng 2 thm 1% ddTTP, ng 3 thm 1% ddGTP v ng 4 thm 1% ddCTP.

Bc 3: Thc hin phn ng tng hp DNA vi cc thit b tun hon

nhit.

Bc 4: in di kt qu, so snh cc kt qu cc chui mch n DNA

c tng hp xc nh trnh t ca mch khun.

Tng hp kt qu 4 ng, thu c cc on mch n hn km nhau mt

nucleotid, thu c trnh t on mch n mi c tng hp v xc nh c

trnh t ca mch DNA khun.

Phng php gii trnh t gen do F.Sanger v cng s pht minh c chnh

xc tng i cao, l c s ca cc my gii trnh t gen t ng.

c. Gii trnh t DNA bng my t ng (Sequencer)

- Nguyn tc: My gii trnh t gen bn t ng hoc t ng hon ton c

thit k trn nguyn tc s dng ddNTP do F.Sanger v cng s pht minh.

Trong qu trnh tng hp DNA c s dng cc mi v dNTP nh du hunh

quang thay cho nh du phng x, mi loi dNTP c nh du hunh quang

khc nhau, biu th cc mu sc khc nhau.

My thc hin tng hp cc mch n DNA mi trn c 2 mch khun DNA

ng thi s dng cc phn mm tnh ton trn my tnh x l kt qu gip kim

sot c cc sai st, m bo chnh xc cao.

- Cc bc thc hin:

Bc 1: Chun b DNA khun, DNA khun phi m bo tinh sch cao,

c kch thc thch hp (thng s dng cc mu DNA c tch dng

17

c chnh xc cao).

Bc 2: Chun b cc iu kin cn thit cho phn ng tng hp DNA (theo

protocol ca cc hng cung cp ha cht hoc thit b...) v thc hin nhn

DNA bng thit b PCR.

Bc 3: Np sn phm PCR vo my gii trnh t gen v chy my gii

Trnh t gen. Trong qu trnh my hot ng cc nucleotid c kim sot nh

detecter bo v b phn x l ca my tnh, phn mm x l kt qu v v th

ca cc loi nucleotid i qua detecter.

Bc 4: Phn tch kt qu t cc d liu do my tnh cung cp, so snh kt

qu xc nh trnh t c 2 mch n mi, ng thi cn c vo th sa cc

sai st v chn kt qu ng nht.

1.3. Phng php sc k lp mng hiu nng cao

1.3.1. Sc k lp mng hiu nng cao

Sc k lp mng hiu nng cao (HPTLC) l dng nng cao v t ng ha ca

sc k lp mng (TLC) cho php chm mu ln bn mng chnh xc hn v nh

gi nh lng cc vt c tch. S ci tin v k thut ny lm tng hiu lc tch

v gim thi gian phn tch. S khc nhau gia TLC v HPTLC ch kch thc

v mc ng u ht ca cht hp ph. TLC thng c phm vi kch thc ht

t 10-50 m, trung bnh khong 20 m nhng vi HPTLC phm vi kch thc ht

nh hn v kch thc ht trung bnh khong 5 m. Phng php a mu ln bn

mng cng c ci tin vi thit k dng c chm mu chnh xc lm gim ng

knh vt chm ban u. Nhng ci tin ny lm gim ng k thi gian v tng hiu

lc tch ca peak [1], [17].

1.3.2. Du vn tay sc k [7],[13], [19]

lp du vn tay TLC ca dc liu, cn chn iu kin chit, iu kin

chy sc k, pha ng, hin vt. Tin hnh chy nhiu ln xem xt lp, c

hiu ca cc vt hin v tch r trn sc k . Tin hnh chn la vt c trng, lp

18

bng Rf v mu sc cc vt cng sc k l du vn tay TLC ca dc liu theo

iu kin sc k chn.

xy dng mt du vn tay ca dc liu bng phng php HPTLC, ta

chn cc iu kin chit mu, iu kin sc k nh ct, pha ng, loi detector pht

hin thch hp vi cc thnh phn ha hc ca dc liu . Tin hnh sc k nhiu

ln vi nhiu mu th v mu chun i chiu xem xt lp li, tinh khit

v n nh ca cc peak c ra gii trn sc k . Sau tin hnh chn la

cc peak c trng cho dc liu, cc peak ny m bo cc yu cu sau:

- Peak phi xut hin n nh.

- lp li tng i ca thi gian lu phi nh hn 1%.

- Peak c tinh khit cao (>95%)

- Din tch peak phi tng i ln ( >1% tng din tch cc peak chnh).

- phn gii vi cc peak cnh khng nh hn 1,0.

1.3.3. ng dng HPTLC

- Phng php HPTLC dng nhn dng dc liu bng cch xy dng du

vn tay ha hc ca dc liu. V mi dc liu c thnh phn ha hc vi t l

nht nh cc cht v cha cc hot cht c trng cho dc liu .

- Phng php HPTLC cn c dng kim nghim dc liu, xc nh s

ging nhau v khc nhau gia thnh phn ha hc ca dc liu cn nhn dng so

vi dc diu chun thng qua vic xy dng du vn tay ha hc ca dc liu.

Qua tng quan nghin cu v cy d c bit l nghin cu ca Nguyn

Kim Khanh thc hin nm 2012, chng ti tip tc m rng nghin cu ti ny

xc nh chnh xc s a dng sinh hc ca cy d; s lng mu l 10, trong

gi nguyn 4 mu c nghin cu nm 2012 (Bng 2.1), v nghin cu

thm 6 mu c ngun gc t o Tr Vnh Phc (Y3), Hong Mai H Ni (Y4),

Thnh ph Hi Dng (Y8), Mai Sn Sn La (Y1), Thun Chu Sn La (Y6) v

Chiang Rai Thi Lan (Y5).

19

CHNG 2. PHNG PHP NGHIN CU 2.1. Nguyn vt liu, thit b, ho cht

2.1.1. Nguyn vt liu

Tng cng c 6 mu nghin cu, c thu thp 3 vng sinh thi Ty Bc,

ng Bc, ng bng Sng Hng v 1 mu c ngun gc t Thi Lan (Bng 2.1).

Nhm trnh tc ng ca ngoi cnh tt c cc mu c gieo trng ti Vn thc

vt B mn Thc vt Trng i hc Dc H Ni, v c theo di hng tun

trong giai on t thng 12/2012 n thng 5/2013. Ngoi ra so snh vi nghin

cu thc hin nm trc, c 4 mu c nghin cu lp li, gm cc mu Y2,

Y7, Y9 v Y10 (Bng 2.1).

Bng 2.1. Danh sch cc mu nghin cu

TT M s

mu

a im thu mu c nghin

cu nm 2012

Ghi ch

1 Y1 Mai Sn Sn La (Sn La 1) Cy trng

thng mi

2 Y2 Bc Ha Bnh X

3 Y3 o Tr Vnh Phc

4 Y4 Hong Mai H Ni

5 Y5 Chiang Rai Thi Lan Cy trng

thng mi

6 Y6 Thun Chu Sn La (Sn La 2) Cy trng

thng mi

7 Y7 Mng Thanh in Bin X

8 Y8 Thnh ph Hi Dng

9 Y9 Vn Quan Lng Sn X

10 Y10 Yn Sn Ba V X

2.1.2. Thit b, ha cht

2.1.2.1. Nghin cu c im thc vt

20

- Knh lp soi ni Leica EZ4, knh hin vi Leica CME.

- My nh k thut s Canon, thc k, a petri, giy v th, kim mi

mc,

2.1.2.2. Nghin cu ha hc

a. Dng c

- My HPTLC (Camag), bn mng silicagel (CF254).

- B dng c ct hi lu, cn phn tch (Shimadzu AY220).

- Thit b chit siu m, bnh nn dung tch 25ml.

- Cc c m dung tch 100ml,ng ong dung tch 25ml.

- ng nghim, pipet cc loi: 1ml, 2ml, 5ml, 10ml, t sy.

- Cc chy sc k lp mng, giy lc,

b. Ha cht

- Methanol, ethanol 96, ethanol tuyt i, dichloromethan, ethylacetat, acid

sulfuric c, vanilin.

2.1.2.3. Nghin cu sinh hc phn t

a. Dng c

- Ci chy, nit lng, t m, my li tm (Eppendorf 5415R), t sy dng c.

- ng eppendoff (2ml; 1,5ml; 0,2ml), micropipette (1000, 200, 100, 20,

10l).

- L vi sng (Saiko), my in di (Consort EV222), b soi chp gel (UVP

Doc-It) (UV 254).

- My gii trnh t gen t ng ABI 3100 (Applied Biosystems).

b. Ha cht

- CTAB buffer (Gm NaCl 1.4 M, CTAB 2%, PVP 1%, Na2EDTA

20mM,Tris Base 0.1 M) (Sigma).

- Dung dch 24:1 (Gm Chloroform : isoarylalcohol = 24:1).

- Nit lng, SDS 10%.

- Isopropranol (Merk), cn 70 (Merk).

- 10X PCR buffer, MgCl2 25mg, dNTPs 10mM mi loi.

21

- Tag DNA polymerase (1 U/l).

- TAE:1 (Pha dung dch gc 1Lit TAE:10 gm Tris base 48,4g; Acid acetic

glacial 11,4ml; Na2EDTA 3,7g; pH (8-8.3).

- Agarose.

- Ethidiumbromid 10mg/ ml.

- 6X loading dye (10mM Tris HCl (pH 7,6), 0,03% Bromophenol Blue,

0,03%.

- Xylene cyanol/ FF, 60% glycerol, 60mM EDTA).

- Nc ct pha tim.

- Cp mi ITS1 v ITS4.

2.1.3. Phn mm h tr

- Phn mm phn loi: Phn mm PCORD vesion 4, phn mm PAUP.

- Phn mm x l HPTLC: WinCat version 1.7.4.

- Phn mm ging hng trnh t nucleotid Bioedit.

2.2. Ni dung v phng php nghin cu

2.2.1. Nghin cu a dng v c im hnh thi v nng hc

2.2.1.1. Mc ch

Xc nh s a dng v c im hnh thi v nng hc ca 10 mu d

(Bng 2.1) c gieo trng ti Vn thc vt, B mn Thc vt Trng i hc

Dc H Ni.

2.2.1.2. Cch lm

- Thu mu: Tin hnh thu trc tip ht trn cy. Chn ht chc, hong n

kh iu kin thng, dng lm ging.

- Gieo ht: Ht ca 10 mu d (Bng 2.1) c ngm trong nc m 4-6

ting (3 si/2 lnh) v c gieo trong cc chu nha c chun b sn t

(Hnh 2.1). Mi mu gieo 15 ht.

22

Hnh 2.1. Chu nha dng trng cc mu d

- Chm sc cc mu d v theo di cc ch tiu nng sinh hc v c im

hnh thi t khi gieo n ngy 02/05/2013 (mi mu theo di 3 cy ly kt qu trung

bnh). Tng cng c 20 ch tiu c theo di (Bng 2.2)

Bng 2.2. Cc ch tiu nng sinh hc c theo di

TT Ch tiu n v o Cch o lng

1. S chm qu/cy Chm m s chm qu/cy thi im

thu hoch

2. S qu/chm Qu m s qu/chm thi im thu

hoch

3. S qu/cy Qu m s qu/cy vo thi im thu

hoch

4. Khi lng 100 qu Gam Cn 100 qu

5. Khi lng 1000 ht Gam Cn 1000 ht

6. T l v tru/qu % Cn qu, bc/xt, cn ht tnh t l

7. T l ny mm ca ht % Gieo ht, tnh t l ny mm

8. Thi gian c l u tin Ngy Theo di hng ngy

23

9. Chiu cao theo cc thng Cm o chiu cao sau mi thng

10. S l theo cc thng L m s l sau mi thng

11. Thi gian nhnh Tun Mi tun theo di 1 ln

12. Chiu cao thi im

nhnh

Cm o chiu cao thi im nhnh

13. S l/thn thi im

nhnh

L m s l/thn thi im

nhnh

14. Thi gian ra hoa Tun Mi tun theo di 1 ln

15. Chiu cao thi im ra

hoa

Cm o chiu cao thi im ra hoa

16. S l/thn thi im ra

hoa

L m s l/thn thi im ra hoa

17. S nhnh/thn thi

im ra hoa

Nhnh m s nhnh/thn thi im ra

hoa

18. Chiu di ging cy

trng thnh

Cm o chiu di ging thi im ra

hoa

19. S ging cy trng

thnh

Ging m s ging/thn thi im ra

hoa

20. Tng s l/cy L Tnh t khi mc n khi t s l

cui cng, nh du cc l m

- c im thc vt: M t theo phng php m t chn on, ch tp trung

vo cc c im khc bit quan st c trn tt c cc mu nghin cu [3], [4].

24

- Tn khoa hc ca 10 mu d do PGS.TS. Trn Vn n (B mn Thc vt

Trng i hc Dc H Ni) thm nh.

- Da vo cc ch tiu theo di c, cc c im khc nhau c m ha

to thnh ma trn d liu v c phn tch bng php phn tch chm (Cluster

Analysis), s dng phn mm PCORD.

2.2.2. Nghin cu a dng v di truyn

2.2.2.1. Mc ch

- Xc nh s a dng v di truyn da vo trnh t DNA ca cc mu d

bng cch so snh trnh t gen ca cc mu nghin cu vi nhau hoc so snh trnh

t gen ca cc mu nghin cu vi trnh t gen cng b trong ngn hng gen

quc t ca chi Coix L.

2.2.2.2. Cch lm

- c tin hnh qua 2 bc sau:

+ Bc 1: Tch DNA. Cc bc tch DNA c tch theo qui trnh 12 bc

(Bng 2.3).

Bng 2.3. Cc bc thc hin tch DNA t mu l d

TT Quy trnh thc hin Yu cu Ghi ch

1 Chun b mu l L bnh t (0,04g)

2 Nghin S dng Nit lng Ly 1/2 ng

3 B sung: 800l CTAB

buffer v 56l SDS Lc u

4 my cch thy 65C/ 60 pht Kim tra, lc

u

5 ngui nhit

phng

6 B sung hn hp 24:1 800 l Lp li 1 ln

t b6-b8 7 Ly tm 11000 vng /pht30pht/6C

8 Thu dch ni S dng micropipette

9 Ta DNA S dng isopropranol

25

Bo qun t lnh su 3h gi

10 Ly tm 12,000 vng/pht30 pht/6C

11 Ly ta, ra ta Ra ta bng cn 70

12 Bo qun DNA

Ha tan trong nc ct tit trng

hoc TE

Bo qun t lnh su.

+ Bc 2: Gii trnh t DNA ca 10 mu d v x l kt qu

DNA ca 10 mu c gi n phng th nghim Macrogen, Hn Quc

gii trnh t DNA. Qu trnh gii trnh t DNA c thc hin trn my gii trnh

gen t ng ABI 3100 (Applied Biosystems). Trnh t nucleotid ca cc mu d

c so snh vi 7 on trnh t c cng b trong ngn hng gen quc t ca

chi Coix L. bao gm cc on trnh t 300669377, 68164101, 451173147,

451173146, 451173145, 451173144 , 1314374 (Ph lc 4). Sau khi x l, on

trnh t nucleotid cui cng a vo so snh l ITS1 5.8S ITS2. Tt c cc

trnh t nucleotid c ging hng trong phn mm Bioedit xc nh s khc

nhau gia cc on nucleotid nghin cu, v s dng phn mm PAUP xy dng

cy phn loi.

2.2.3. Nghin cu v ha hc bng phng php HPTLC

2.2.3.1. Mc ch

- Da vo s tng ng peak sc k ca cc mu d sau khi tin hnh

HPTLC chia thnh cc nhm khc nhau.

- S b nh gi s khc bit v hm lng ca cc hot cht c trong ht ca

cc mu d da trn vic so snh din tch ca cc vt c cng Rf xut hin trn

sc k sau khi tin hnh HPTLC.

2.2.3.2. Cch lm

a. Chit hot cht trong nhn ht d bng phng php chit siu m

- Nhn ht d c nghin thnh bt mn bng chy ci s.

- Cn chnh xc 0,5gam bt d vo bnh nn 25,0ml ri thm chnh xc

26

5,0ml dung mi methanol.

- bnh nn vo b chit siu m, thi gian chit l 30 pht.

- Lc dch chit methanol qua giy lc, c n cn ri thm 1,0ml

dung mi methanol ha tan cn lm dch chm sc k.

b. Chy sc k HPTLC

- Chun b: Bn mng silicagel hot ha 105C/30 pht, h dung mi

chy sc k: Dicholoromethan: ethanol: ethylacetat (95:5:5), thuc th hin mu:

Ha tan 4,0g vanilin trong 200ml ethanol 96; thm t t 4,0ml acid sulfuric c.

- Chy sc k:

Lng dch chm: Mi mu c chm lp li, ln 1 chm 1,5l v ln 2

chm 3,0l.

Lng dung mi cn thit l 35ml, thi gian bo ha dung mi l 5 pht

Thi gian treo bn mng: 5 pht.

Thi gian trin khai sc k: 7 pht.

Thi gian sy kh bn mng: 5 pht.

Nhng bn mng trong thuc th hin mu, ri sy 105C/5 pht.

- X l s liu bng phn mm Win Cat version 1.7.4.

27

CHNG 3: KT QU NGHIN CU V BN LUN 3.1. Tnh a dng v c im hnh thi v nng hc.

3.1.1. c im hnh thi ca cc mu d thi im ngy 02/05/2013

3.1.1.1. Mu Y1 (Mai Chu Sn La)

Thn hnh tr, cao 115cm, mu xanh nht, phn nhnh, chia thnh nhiu t,

ng knh thn 0,8cm. L mu xanh m, hnh mc, hi hp, thun di, u nhn,

di 50-55cm, rng 3-4cm. B l khng lng, di 15cm; gn song song, gn gia ni

to v gn 2 bn mnh.

Hoa n tnh cng gc, cung hoa di 5-6mm. Trn bng cht c c nhiu

hoa c mc thnh chm gm 2-3 hoa c, trong c mt hoa c c cung

mnh. Nh 3 ri u mu vng. Bng cht ci hnh cu c bao bc bi 1 l bc rt

dy,cng, mu xanh xm hay mu tm nht, di 8-9 mm, ng knh 5-6mm. Bu

nhy hnh bu dc, mu trng xanh, di 3-4mm, ng knh 1-2mm; vi nhy mu

tm, c lng bao ph; 2 nh lp mu trng xanh.

Hnh 3.1. c im hnh thi mu Y1 (Mai Chu Sn La)

1: Mt bng nh; 2: Bng hoa c; 3: Mt hoa c nguyn vn; 4: My ln hoa

c; 5: Mt bao phn; 6: Hoa ci; 7: Hoa ci (phi) v my chung ca cm hoa

(tri); 8: B nhy.

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix lacryma-jobi

L. var. mayuen (Rom) Stapf (C.chinensis Todaro ex Balansa).

3.1.1.2. Mu Y2 ( Bc Ha Bnh)

28


ng knh thn 0,7cm. L mu xanh nht, hnh mc, hi hp, thun di, u nhn,

di 40-50cm, rng 3-4cm. B l khng lng, di 16cm; gn song song, gn gia ni

to v gn 2 bn mnh.

Hoa n tnh cng gc. Trn bng cht c c nhiu hoa c mc thnh chm

gm 2-3 hoa c, trong c mt hoa c c cung mnh. Nh 3 ri u mu vng.

Bng cht ci hnh bu dc thun di, c bao bc bi 1 l bc rt dy, cng, mu

xanh, di 10-11mm, ng knh 4-5mm. Bu nhy hnh bu dc, mu trng xanh,

di 1-2mm, ng knh 1-1,5mm; vi nhy mu trng, c lng bao ph; 2 nh lp

mu trng xanh.

Hnh 3.2. c im hnh thi mu Y2 ( Bc Ha Bnh)



(tri); 8: B nhy.

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix lacryma-jobi

L. var. stenocarpa Oliv.

3.1.1.3. Mu Y3 (o Tr Vnh Phc)

Thn hnh tr, cao 30cm, mu xanh nht, chia thnh nhiu t, ng knh

thn 1cm. L mu xanh nht, hnh mc, hi hp, thun di, u nhn, di 45-50cm,

29

rng 3- 3,5cm. B l khng lng, di 12cm; gn song song, gn gia ni to v gn 2

bn mnh.

Hnh 3.3. Thn mang l mu Y3 (o Tr Vnh Phc)

Hoa cha thy (Do cy cha n hoa ti thi im bo co).

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix lachryma-jobi

L. var. lachryma-jobi.

3.1.1.4. Mu Y4 (Hong Mai H Ni)


thn 1cm. L mu xanh nht, hnh mc thun di, rng, u nhn, di 45-50cm,

rng 4-5cm. B l khng lng, di 14cm; gn song song gn gia ni to gn 2 bn

mnh.

Hnh 3.4. Thn mang l mu Y4 (Hong Mai H Ni)




3.1.1.5. Mu Y5 (Chiang Rai Thi Lan)


thn 0,9cm. L mu xanh nht, hnh mc thun di, rng, u nhn, di 50-55cm,

30

rng 3-3,5cm. B l khng lng, di 16cm; gn song song, gn gia ni to v gn 2

bn mnh.



Bng cht ci hnh cu hi dt, c bao bc bi 1 l bc rt dy, cng, mu xanh,

di 15-16mm, ng knh 11-12mm. Bu nhy hnh cu hi thun, mu trng xanh,

di 3-4mm, ng knh 1-2mm; vi nhy mu trng, c lng bao ph; 2 nh lp

mu trng xanh.

Hnh 3.5. c im hnh thi mu Y5 (Chiang Rai Thi Lan)



(tri); 8: B nhy.

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix sp.

3.1.1.6. Mu Y6 (Thun Chu Sn La)


ng knh thn 0,5cm. L mu xanh m, hnh mc, hp, thun di, u nhn, di

25-30cm, rng 2-2,5cm. B l khng lng, di 14cm; gn song song gn, gia ni to

v gn 2 bn mnh.



31

Bng cht ci hnh cu hi thun, c bao bc bi 1 l bc rt dy, cng, mu

xanh, di 12-13mm, ng knh 5-6mm. Bu nhy hnh cu hi thun, mu trng

xanh, di 2-3mm, ng knh 1-2mm; vi nhy mu trng, c lng bao ph; 2 nh

lp mu trng xanh.

Hnh 3.6. c im hnh thi ca mu Y6 (Thun Chu Sn La)



(tri); 8: B nhy.

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix lacryma-jobi L.

var. mayuen (Rom) Stapf (C.chinensis Todaro ex Balansa).

3.1.1.7. Mu Y7 (Mng Thanh in Bin)


thn 0,7cm. L mu xanh nht, hnh mc thun di, hi hp, u nhn, di 50-

55cm, rng 3-3,5cm. B l khng lng, di 13cm; gn song song, gn gia ni to v

gn 2 bn mnh.


gm 2 - 3 hoa c, trong c mt hoa c c cung mnh. Nh 3 ri u mu

vng. Bng cht ci hnh cu, c bao bc bi 1 l bc rt dy, cng, mu xanh,

di 7-8mm, ng knh 5-6mm. Bu nhy hnh bu dc, mu trng xanh, di 2-

32

3mm, ng knh 1-2mm; vi nhy mu tm, c lng bao ph; 2 nh lp mu trng

xanh.

Hnh 3.7. c im hnh thi mu Y7 (Mng Thanh in Bin)



(tri); 8: B nhy.

- Tn khoa hc: Mu c thm nh c tn khoa hc l Coix lacryma-jobi L.

var lacryma-jobi.

3.1.1.8. Mu Y8 (Thnh ph Hi Dng)


thn 1cm. L mu xanh nht, hnh mc thun di, rng, u nhn, di 60-65cm,

rng 4-5cm. B l khng lng, di 15cm; gn song song, gn gia ni to v gn 2

bn mnh.



Bng cht ci hnh cu, c bao bc bi 1 l bc rt dy, cng, mu xanh, di 8-

9mm, ng knh 5-6mm. Bu nhy hnh bu dc, mu trng xanh, di 1-2mm,

ng knh 1mm; vi nhy mu tm, c lng bao ph; 2 nh lp mu trng xanh.

33

Hnh 3.8. c im hnh thi mu Y8 (Thnh ph Hi Dng)



(tri); 8: B nhy.



3.1.1.9. Mu Y9 (Vn Quan Lng Sn)


thn 0,8cm. L mu xanh nht, hnh mc thun di, hi hp, u nhn, di 45-

50cm, rng 3-4cm. B l khng lng, di 15cm; gn song song, gn gia ni to v

gn 2 bn mnh.


gm 2 - 3 hoa c, trong c mt hoa c c cung mnh. Nh 3 ri u mu

vng. Bng cht ci hnh cu, c bao bc bi 1 l bc rt dy, cng, mu xanh,

di 7-8mm, ng knh 4-5mm. Bu nhy hnh cu hi thun, mu trng xanh, di

1-2mm, ng knh 1- 1,5mm; vi nhy mu tm, c lng bao ph; 2 nh lp mu

trng xanh.

34

Hnh 3.9. c im hnh thi mu Y9 (Vn Quan Lng Sn)



(tri); 8: B nhy.



3.1.1.10. Mu Y10 (Ba V H Ni)


thn 1cm. L mu xanh nht, hnh mc thun di, hi hp, u nhn, di 40-45cm,

rng 2,5-3cm. B l khng lng, di 14cm; gn song song, gn gia ni to v gn 2

bn mnh.

Hnh 3.10. Thn mang l mu Y10 (Ba V H Ni)




35

3.1.2. c im nng hc ca cc mu d

Cc giai on pht trin ca cy d (Hnh 3.11): Nhn chung cc mu d

u pht trin tt khi c gieo trng ti Vn thc vt B mn Thc vt Trng

i hc Dc H Ni. Thi gian ny mm t 6 15 ty theo tng mu (Ph lc 1);

trong tun u tin sau khi ny mm cc ging d bt u ra l th nht (Ph lc

1), trong mu Y3 v Y5 c thi gian ra l u tin nhanh nht (7 ngy sau khi

gieo). Tc pht trin ca cc mu l khng ng u, trong mu Y6 c tc

pht trin nhanh nht; sau 8 19 tun th cc mu bt u nhnh v ra hoa. Ti

thi im ngy 02/05/2013 c 7 mu ra hoa l Y1, Y2, Y5, Y6, Y7, Y8 v Y9.

A. Ny mm B. Ra l u tin. C. Ra l th 2, th 3

D. Ra nhiu l E. Khi ra hoa F. Khi nhnh

Hnh 3.11. Cc giai on pht trin ca cy d

S bin i chiu cao ca cc mu d theo thng (tnh t thi im ny

mm): Trong vng 1 n 3 thng u tin sau khi ny mm, chiu cao ca cc mu

d tng chm, nhng t thng th 4 tr i chiu cao ca cc mu bt u tng

nhanh. Tuy nhin, s pht trin chiu cao ca cc mu d l khng ng u,

36

trong mu Y5 c tc pht trin chiu cao nhanh nht v mu Y7 c tc

pht trin chiu cao chm nht.

S bin i s l ca cc mu d theo thng (tnh t thi im ny mm):

Trong vng 3 thng u tin sau khi ny mm, s l ca cc mu d tng chm

(mi thng mc thm 1 3 l ty theo mu), t thng th 4 tr i s l ca cc mu

tng nhanh (mi thng mc thm 3 5 l ty theo mu). Tuy nhin tc mc l

ca cc mu d l khng ng u, trong mu Y6 c tc mc l nhanh nht

v mu Y7 c tc mc l chm nht.

- Cc ch tiu nng sinh hc theo di c t thng 12/2012 n thng

05/2013: Trong giai on t thng 12/2012 n thng 05/2013, ch mi theo di

c cc mu d t khi ny mm n khi c hoa (Ph lc 1), trong mi ch c

7 mu ra hoa l Y1, Y2, Y5, Y6, Y7, Y8 v Y9.

3.1.3. Kt qu nghin cu tnh a dng v c im hnh thi v nng hc

- Ti thi im bo co (thng 05/2013) mi c 7 mu d ra hoa bao gm

mu Y1, Y2, Y5, Y6, Y7, Y8 v Y9; nn nghin cu ny ch dng li vic xc

nh tnh a dng v c im hnh thi v nng hc ca 7 mu ny.

- Sau khi quan st c im hnh thi v theo di cc ch tiu nng sinh hc

ca 7 mu d ra hoa trong giai on t thng 12/2012 n thng 05/2013, a

ra bng tng kt cc c im hnh thi v ch tiu nng sinh hc so snh 7

mu d vi nhau (Bng 3.1). Cc c im khc nhau c m ha thnh ma trn

d liu (Ph lc 2).

Bng 3.1. Bng tng kt cc ch tiu nng sinh hc v c im hnh thi

so snh 7 mu d

c im Mu d

Y1 Y2 Y5 Y6 Y7 Y8 Y9

Thi gian ny mm

(ngy)

9 15 6 9 15 16 15

Thi gian ra l u

tin (ngy)

12 23 7 12 21 23 21

37

Chiu cao sau thng

th nht (cm)

6 3 12 10 5 4 4

S l sau thng th

nht (l)

4 2 4 6 2 2 2

S l/thn thi im

nhnh (l)

12 10 12 12 10 12 12

S l/thn thi im

ra hoa (l)

14 15 12 14 12 14 13

S nhnh/thn thi

im ra hoa (nhnh)

4 6 2 4 4 4 3

Chiu di b ca cm

hoa u tin (cm)

2 3 2 2 2 2,5 2,5

Hnh dng l

Hnh

di,

hi

hp,

Hnh

di,

hi

hp

Hnh

di,

rng

Hnh

di,

hp

Hnh

di,

hi

hp

Hnh,

di,

hi

hp

Hnh

di,

rng

Mu sc l Xanh

m

Xanh

nht

Xanh

nht

Xanh

m

Xanh

nht

Xanh

nht

Xanh

nht

Hnh dng qu

Hnh

cu

Hnh

bu

dc

Hnh

cu

Hnh

cu

Hnh

cu

Hnh

cu

Hnh

cu

c im b mt qu X x Trn

X x Trn Trn Trn Trn

c im v qu Mng Dy Mng Mng Dy Dy Dy

Mu sc vi nhy Mu

tm

Mu

trng

Mu

trng

Mu

trng

Mu

tm

Mu

tm

Mu

tm

- S dng phn mm PCORD phn tch kt qu. Da vo c im hnh

thi v nng hc, tng ng xp x 5% 7 mu d c chia thnh 2 nhm:

38

Nhm 1 gm 4 mu Y2, Y7, Y8 v Y9, trong mu Y2 c phn thnh 1 nhm

ring tng ng xp x 80%; 3 mu Y7, Y8 v Y9 c xp thnh 1 nhm

ring vi tng ng xp x 98%, trong 2 mu Y7 v Y8 trng khp nhau.

Nhm 2 gm 3 mu Y1, Y5 v Y6; trong 2 mu Y1 v Y6 c tng ng xp

x 96%, 2 mu ny gn ging vi mu Y5 vi tng ng xp x 85% (Hnh

3.12).

Hnh 3.12. Cy phn loi 7 mu d da trn c im hnh thi v nng hc

3.2. S khc bit v thnh phn ha hc trong ht

Sc k sau khi tin hnh HPTLC ca cc mu d (Hnh 3.13).

Kt qu chng peak sc k cc mu d cho thy cc peak sc k c hnh

dng tng i ging nhau; khc nhau v chiu cao v din tch peak (Ph lc 3).

Trong , Rf = 0,63 mu Y5 (Chiang Rai, Thi Lan) cha 1 cht c din tch ln

hn so vi cc mu cn li; ti Rf = 0,66 mu Y2 cha 1 cht c din tch ln hn

so vi cc mu cn li; Rf = 0,14 mu Y1 cha 1 cht c din tch ln hn so vi

cc mu cn li.

Bng 3.2. Din tch peak ca mu Y1 ti Rf = 0,14; mu Y2 ti Rf = 0,66 v

mu Y5 ti Rf = 0,63

TT Mu Rf Din tch peak

1 Y1 0,14 3766,5

2 Y2 0,66 18582,7

3 Y5 0,63 128672,4

39

Hnh 3.13. Sc k HPTLC ca cc mu d

Mi mu chm 2 vt, vt pha bn tri chm 1,5l v vt pha bn phi chm 3,0l.

Cc mu ti Rf = 0,69 u c 1 vt m hn cc vt khc, do c chn

lm i din so snh cc mu. Din tch peak chim t l ln th lng cht tng

ng vi peak trong mu cng ln. Trong , mu Y4 c din tch peak ln nht

v mu Y10 c din tch peak nh nht (Bng 3.2).

Bng 3.3. Din tch peak ph HPTLC dch chit methanol cc mu d ti

Rf = 0,69

TT Mu Din tch peak

1 Y1 39297,5

2 Y2 14495,9

3 Y3 37710,4

4 Y4 46567,2

5 Y5 19505,5

6 Y6 41646,1

7 Y7 20252,7

8 Y8 30519,0

9 Y9 16967,2

10 Y10 7760,3

40

3.3. Tnh a dng v di truyn

Trong 10 mu d c gi n phng th nghim Macrogen, Hn Quc

gii trnh t gen th mu Y8 qu trnh gii trnh t gen khng thnh cng, nn ta ch

xc nh tnh a dng di truyn ca 9 mu cn li gm Y1, Y2, Y3, Y4, Y5, Y6,

Y7, Y9 v Y10 (Bng 2.1).

- Sau khi tin hnh gii trnh t gen thu c trnh t nucleotid ca 9 mu

d (Ph lc 5) .

- Da vo trnh t nucleotid, xc nh c tng ng di truyn ca 9

mu d (Ph lc 6). Trong on trnh t kho st l ITS1 5.8S ITS2 th trnh

t nucleotid ca 9 mu d t c s khc bit vi tng ng di truyn gia cc

mu dao ng t 97% n 99%.

- Trnh t nucleotid ca 9 mu d v trnh t nucleotid ca 7 on so snh

c ging hng trong phn mm Bioedit, trong cc nucleotid bi mu vng l

cc nucleotid khc bit ca cc on trnh t nghin cu (Ph lc 7). S dng phn

mm PAUP xy dng cy phn loi, kt qu 9 mu d c chia thnh 4 nhm

(Hnh 3.14): Nhm 1 gm mu Y1 (Mai Sn Sn La). Nhm 2 gm cc mu Y2

( Bc Ha Bnh) v Y3 (o Tr Vnh Phc). Nhm 3 gm cc mu Y4

(Hong Mai H Ni), Y9 (Vn Quan Lng Sn) v Y10 (Yn Sn Ba V);

trong mu Y10 c xp thnh 1 nhm ring v 2 mu Y4 v Y9 c xp

thnh 1 nhm ring. Nhm 4 gm cc mu Y5 (Chiang Rai Thi Lan), Y6 (Thun

Chu Sn La) v Y7 (Mng Thanh in Bin); trong mu Y6 c xp

thnh 1 nhm ring v 2 mu Y5 v Y7 c xp thnh 1 nhm ring. Da co cy

phn loi ta thy mu Y5 v Y7 c trnh t ging vi cc on trnh t c

cng b nht.

41

Hnh 3.14. Cy phn loi 9 mu d da trn trnh t nucleotid

42

BN LUN V c im hnh thi v nng hc: Cc kt qu theo di ch tiu nng sinh hc cho thy cc mu d u pht

trin tt trong iu kin tin hnh thc nghim. Trong cng mt iu kin tin hnh

th nghim cc mu d c tc pht trin khc nhau, ti thi im bo co

(thng 05/2013) mi c 7 mu ra hoa l mu Y1, Y2, Y5, Y6, Y7, Y8 v Y9. Mt

khc, cc mu ny do c trng khng ng thi v nn cy b p ra hoa khi mi

trng trong thi gian ngn. Ni chung ti ny mi ch dng li qui m nh (s

lng mu l 10), do cn m rng nghin cu thm cc mu min Bc Vit

Nam. Tuy nhin, ti ny c c im ni bt l cc mu c trng ti cng mt

a im, cng mt ma v nn hn ch c s tc ng ca mt s yu t ngoi

cnh nh: t ai, a hnh, kh hu, cao,v c s nghin cu lp li 4 mu

c tin hnh nm 2012 (Bng 2.1). Nhn chung, v c im hnh thi ca cc c quan sinh dng (l, thn), cc mu d t c s khc bit; ch n mu Y6 hnh

dng l hp hn hn so vi cc mu cn li, mu sc l ca mu Y1 v Y6 c phn

m hn so vi cc mu khc. V c im hnh thi ca cc c quan sinh sn (hoa,

qu , ht), 7 mu d ra hoa ti thi im bo co (thng 05/2013) c s khc

bit v cc c im: Hnh dng qu (hnh bu dc hay hnh cu), mu sc vi nhy

(mu tm hay mu trng), b mt v qu (trn nhn hay x x), chiu di b l ca

cm hoa u tin, c im v qu (dy hay mng); trong mu Y2 ( Bc

Ha Bnh) qu c hnh bu dc khc vi cc mu cn li qu c hnh cu. Nghin

cu ny mi ch dng li vic xc nh tnh a dng v hnh thi v nng hc ca

7 mu d ra hoa ti thi im bo co (thng 05/2013). V vy, cn phi tip

tc theo di s sinh trng v pht trin ca 10 mu d n khi thu hoch, tin

hnh xc nh li s a dng ca 10 mu d v c im hnh thi v nng hc. Da v c im hnh thi v nng hc 7 mu d ra hoa gm Y1, Y2, Y5,

Y6, Y7, Y8 v Y9 c chia thnh 2 nhm nh trn. Trong 3 mu c nghin cu

lp li ra hoa gm Y2 ( Bc Ha Bnh), Y7 (Mng Thanh in Bin) v

Y9 (Vn Quan Lng Sn) th mu Y2 c s khc bit so vi 2 mu cn li, kt

43

qu ny cng ging vi kt qu ca nghin cu c thc hin nm 2012 [11]; tuy

nhin s khc bit gia mu Y2 so vi 2 mu Y7 v Y9 l khng nhiu vi

tng ng xp x 88%, trong khi theo nghin cu c thc hin nm 2012 th

s khc bit ny l rt nhiu [11]. iu ny c th c gii thch l do nghin cu

nm 2012 tp trung so snh cc c im hnh thi v thn, l, hoa, qu, ht [11],

m t quan tm so snh n cc ch tiu nng sinh hc nh: Thi gian ra l u

tin, thi gian ny mm, chiu cao v s l ca cc mu d sau mi thng, s l

thi im ra hoa, s l thi im nhnh, nn kt qu gia 2 nghin cu c s

khc nhau.

Nhn chung cc mu c nghin cu lp li nm 2012 (Bng 2.1) u pht

trin bnh thng khi c gieo trng ti Vn thc vt B mn Thc vt Trng

i hc Dc H Ni trong giai on t thng 12/2012 n thng 05/2013; v 2

nghin cu ny c thc hin trong 2 nm lin tc thi gian khng cch xa nhau,

nn c im hnh thi ca cc mu d t c s thay i, ch khc nhau 1 s ch

tiu nng sinh hc nh: Thi gian ny mm, thi gian ra l u tin, thi gian

nhnh,thi gian ra hoa, s bin i chiu cao theo cc thng v s bin i s l

theo cc thng; c s khc bit ny l do thi im gieo trng khc nhau (Nghin

cu nm 2012 cc mu c gieo vo thng 08/2011 [11], cn nghin cu nm nay

cc mu c gieo vo thng 12/2012), dn n iu kin ngoi cnh khc nhau,

m cc ch tiu ny ph thuc nhiu vo cc iu kin ngoi cnh nh: nhit ,

m, lng ma,

Da vo kt qu nghin cu tnh a dng v c im hnh thi, nng hc v

kt qu nghin cu tnh a dng v di truyn, ta nhn thy s khc bit v c im

hnh thi v nng hc khng tng ng vi s khc bit v di truyn. Kt qu ny

cng ging vi kt qu ca nghin cu c thc hin nm 2012 [11]. Tuy nhin,

xt trong on trnh t kho st l ITS1 5.8S ITS2 th s khc bit v trnh t

nucleotid gia cc mu l khng ng k (h s tng ng di truyn gia cc mu

dao ng t 97% n 99%), nn khng c c s s dng on trnh t ITS1

5.8S ITS2 phn chia cc mu cp di loi.

44

V thnh phn ha hc: Da vo kt qu phn tch bng HPTLC vi dch chit methanol, cc mu

d c hnh dng peak sc k tng i ging nhau, ch sai khc v chiu cao v

din tch peak. iu ny cho thy cc mu d t c s a dng v ha hc, nhng

c s sai khc v hm lng cc hot cht c trong mu. Kt qu ny cng ging

vi nghin cu ca Dc s Nguyn Kim Khanh tin hnh nm 2012 trn 6 mu

d vi dch chit methanol v dch chit aceton [11] v nghin cu ca K. Lakkham

c tin hnh nm 2009 trn 4 mu d vi dch chit methanol v dch chit

aceton [26]. Tuy nhin, cn lu n mu Y5 (Chiang Rai Thi Lan), mu Y2

( Bc Ha Bnh) v mu Y1 (Mai Chu Sn La); bi v ti Rf = 0,63 mu Y5

cha 1 cht c hm lng cao hn nhiu so vi cc mu cn li, ti Rf = 0,66 mu

Y2 cha 1 cht c hm lng cao hn nhiu so vi cc mu cn l, ti Rf = 0,14

mu Y1 cha 1 cht c hm lng cao hn so vi cc mu cn li. Mt khc, so vi

nghin cu c tin hnh nm 2012 th s lng vt xut hin trn sc k nhiu

hn; iu ny c th c gii thch l do nghin cu ny tin hnh chit hot cht

trong cc mu d bng phng php chit siu m, nn trong dch chit cha

nhiu cht hn v c th cha c tp. Cc mu d t c s a dng v ha hc,

iu ny gi c th s dng cc mu d thay th cho nhau. Tuy nhin, trong

Thc hnh tt trng cy thuc (GAP) th s sai khc v hm lng li rt quan

trng, do n nh hng n nng sut hot cht thu c trn mt n v din tch

trng (trong trng hp chit xut hot cht), cng nh hiu lc iu tr ca thuc

(trong trng hp dng dch chit tng). iu ny cho thy vic chn ging vn cn

thit, thm ch cn lai to ging mi to ra ging ti u hn. Nghin cu ny ch

mi dng li xc dnh du vn tay sc k, do khng c cht chun nn khng xc

nh c chnh xc hot cht ang quan tm (Coixan A, B, C; coixenolide v

coixol) nm Rf bao nhiu. V vy cn tin hnh phn tch bng HPTLC cc dch

chit d ng thi vi cc cht chun (Coixan A, B, C; coixenolide v coixol)

nh gi chnh xc hm lng ca cc hot cht c trong cc mu d da trn vic

so snh din tch cc vt xut hin trn sc k gia mu th v mu chun

45

KT LUN 1. Tnh a dng v c im hnh thi v nng hc

- m t c im hnh thi v nng hc ca 10 mu d v xc nh chng

thuc cc th sau: Coix lacryma-jobi L. var. lacryma-jobi, Coix lacryma-jobi L.

var. mayuen (Rom) Stapf (C.chinensis Todaro ex Balansa), Coix sp. v Coix

lacryma-jobi L. var. stenocarpa Oliv, trong nh chnh li tn khoa hc ca

cc mu nghin cu Trng i hc Dc H Ni t nm 2008 n nay. y l

nghin cu u tin Trng i hc Dc H Ni xc nh c th Coix

lacryma-jobi L. var. mayuen (Rom) Stapf (C.chinensis Todaro ex Balansa).

- Da vo c im hnh thi v nng hc, 7 mu d c hoa c chia

thnh 2 nhm: Nhm 1 gm 4 mu Y2 ( Bc Ha Bnh), Y7 (Mng Thanh

in Bin), Y8 (Thnh ph Hi Dng) v Y9 (Vn Quan Lng Sn). Nhm 2

gm 3 mu Y1 (Mai Sn Sn La), Y5 (Chiang Rai Thi Lan) v Y6 (Thun

Chu Sn La).

2. S khc bit v thnh phn ha hc trong ht

- xc nh c s khc bit v thnh phn ha hc ca cc mu d bng

phng php HPTLC. Nhn chung, cc mu d nghin cu t c s khc bit v

thnh phn ha hc, nhng sai khc r rng v hm lng. Tuy nhin cn quan tm

n mu Y5 (Chiang Rai, Thi Lan), mu Y2 ( Bc, Ha Bnh) v mu Y1 (Mai

Chu, Sn La); bi v ti Rf = 0,63 mu Y5 cha 1 cht c hm lng cao hn

nhiu so vi cc mu cn li, ti Rf = 0,66 mu Y2 cha 1 cht c hm lng cao

hn nhiu so vi cc mu cn li, ti Rf = 0,14 mu Y1 cha 1 cht c hm lng

cao hn so vi cc mu cn li.

3. Tnh a dng v di truyn

- tin hnh gii trnh t DNA ca 9 mu d, v da vo trnh t

nucleotid th 9 mu d c chia thnh 4 nhm: Nhm 1 gm mu Y1. Nhm 2

gm cc mu Y2 v Y3. Nhm 3 gm cc mu Y4, Y9 v Y10. Nhm 4 gm cc

46

mu Y5, Y6 v Y7. Tuy nhin, xt trong on trnh t kho st l ITS1 5.8S

ITS2 th trnh t nucleotid ca 9 mu d t c s khc bit.

47

KIN NGH

- Tip tc theo di s pht trin v sinh trng ca cc mu d n khi thu

hoch, xc nh li tnh a dng v c im hnh thi v nng hc ca 10 mu

d.

- Tip tc theo di cc v sau, nh gi s bin i theo thi gian ca cc

mu, m rng s lng mu nghin cu.

- Xc nh thm mt s ch s tinh bt, in hnh l o nhit h ha, da

trn my qut nhit vi sai DSC - Differential Scanning Calorimetry, nhm xc nh

tnh cht tinh bt ca tng mu.

- Tip tc nghin cu su hn v ha hc, xc nh du vn tay ha hc, du

vn tay DNA , d dng kim nghim Dc liu, so snh cc thnh phn c tc

dng nh coixan, coixenolid,trong cc mu.

Ti liu tham kho Ting vit 1. Trn T An, Thi Nguyn Hng Thu (2006), Ha phn tch, NXB Y hc, tr. 125

146, 173 210.

2. Huy Bch, ng Quang Chung, Bi Xun Chng,(2006), Cy thuc v

ng vt lm thuc Vit Nam, tp II, NXB Khoa hc k thut, tr. 1156 1157.

3. DS. L nh Bch, TS. Trn Vn n (2007), Thc Vt hc, NXB Y hc.

4. DS. L nh Bch, TS. Trn Vn n (2004), Thc tp thc vt v nhn bit cy

thuc,Trung tm Thng tin Th vin i hc Dc H Ni.

5. V Vn Chi (2003), T in cy thuc Vit Nam, NXB Khoa hc v k thut H

Ni, tr. 741 742.

6. H Hunh Thy Dng (2005), Sinh hc phn t, NXB Gio dc, tr. 200 205.

7. Nguyn Th Hng Hnh (2011), Nghin cu nhn bit sm Ngc Linh bng sc

k vn tay ( HPTLC), Kha lun tt nghp Dc s.

8. Nguyn Nh Hin (2011), Cng ngh sinh hc, NXB Gio dc, tp 1.

9. Phm Hong H (2000), Cy c Vit Nam, NXB Tr, tr. 734 735.

10. Phng Thanh Hng v cs (2009), Tc dng h glucose huyt ca cy d

(Coix lachryma-jobi L.var. mayuen Stapf) trn chut nht trng, Hi ngh khoa

hc Tui tr ln th XV, nm 2009.

11. Nguyn Th Khanh (2012), Nghin cu a dng sinh hc v tc dng ca cy

d min bc Vit Nam, Kha lun tt nghip Dc s, nm 2012.

12. Tt Li (1999), Nhng cy thuc v v thuc Vit Nam, NXB Y hc tr. 844

846.

13. Nguyn Th Minh Phng (2011), Nghin cu phng php vn tay sc k

(TLC, HPTLC) gp phn nh gi v thuc an sm, Kha lun tt nghip

Dc s, nm 2011.

14. Phm Xun Sinh, Phng Ha Bnh, Dc hc c truyn, NXB Y hc, tr. 269.

15. Khut Hu Thanh (2006), K thut gen nguyn l v ng dng, NXB Khoa hc

v k thut H Ni, tr. 173 179.

16. Ng Vn Thu (2004), Bi ging dc liu tp 1, Trung tm Thng tin Th

vin i hc Dc H Ni, tr. 42 43.

17. Thi Nguyn Hng Thu (2010), Sc k lp mng, Ti liu dng sau i hc,

Trung tm Thng tin Th vin i hc Dc H Ni.

18. T in Bch Khoa Dc hc (1999), NXB T in Bch khoa H Ni, tr.

718.

19. Nguyn Vn Tu (2009), Nghin cu pht trin b d liu tiu chun ca

mt s dc liu thng dng phc v cng tc kim tra kim sot cht lng

dc liu v thuc ng dc, ( ti nghin cu cp B), pp.10 11, 37.

Ting anh 20. Chen HJ, Shih CK, Hsu HY, Chiang W. (2010), Mast cell-dependent allergic

responses are inhibited by ethanolic extract of adlay (Coix lachryma-jobi L. var.

ma-yuen Stapf) testa, Journal of Agriculture and Food Chemistry, 58(4): 2596-

601.

21. Ha do T, Nam Trung T, Bich Thu N, Van On T, Hai Nam N, Van Men C, Thi

Phuong T, Bae K. (2010), Adlay seed extract (Coix lachryma-jobi L.)

decreased adipocyte differentiation and increased glucose uptake in 3T3-L1

cells., Journal of Medicinal Food, 13(6):1331-9.

22. Hsia SM, Tseng YW, Wang SW, Kuo YH, Huang DW, Wang PS, Chiang W.

(2009), Effect of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull

extracts on testosterone release from rat Leydig cells., Phytotherapy Research,

23(5):687-95.

23. Hsu HY, Lin BF, Lin JY, Kuo CC, Chiang W. (2003), Suppression of allergic

reactions by dehulled adlay in association with the balance of TH1/TH2 cell

responses., Journal of Agriculture and Food Chemistry, 51(13):3763-9.

24. Huang BW, Chiang MT, Yao HT, Chiang W. (2005), The effect of adlay oil

on plasma lipids, insulin and leptin in rat., Phytomedicine, 12(6-7):433-9.

25. Huang Din-Wen, Cheng-Pei Chung, Yueh-Hsiung Kuo, Yun-Lian Lin,

Wenchang Chiang (2009), Identification of compounds in Adlay (Coix

lachryma-jobi L. var. ma-yuen Stapf) Seed Hull Extracts That Inhibit

Lipopolysaccharide-induced inflammation in RAW 264.7 macrophages.

26. K. Lakkham et al(2009), "Identification and quality of four varieties of adlay",

Songklanakarin Journal of Science and Technology, 31 (4), 425-431.

27. Kim SO, Yun SJ, Jung B, Lee EH, Hahm DH, Shim I, Lee HJ. (2004),

Hypolipidemic effects of crude extract of adlay seed (Coix lachrymajobi var.

mayuen) in obesity rat fed high fat diet: relations of TNF-alpha and leptin

mRNA expressions and serum lipid levels., Life Science, 75(11):1391-404.

28. Kuo CC, Chiang W, Liu GP, Chien YL, Chang JY, Lee CK, Lo JM, Huang SL,

Shih MC, Kuo YH. (2002), 2,2'-Diphenyl-1-picrylhydrazyl radical-scavenging

active components from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf)

hulls., Journal of Agriculture and Food Chemistry, 50 (21): 5850-5.

29. Lee MY, Lin HY, Cheng F, Chiang W, Kuo YH. (2008), Isolation and

characterization of new lactam compounds that inhibit lung and colon cancer

cells from adlay (Coix lacryma-jobi L. var. ma-yuen Stapf) bran, Food and

Chemical Toxicology, 46 (6): 1933-9.

30. Li SC, Chen CM, Lin SH, Chiang W, Shih CK. (2011), Effects of adlay bran

and its ethanolic extract and residue on preneoplastic lesions of the colon in

rats., Journal of the Science of Food and Agriculture, 91(3):547-52.

31. Lu Y, Li CS, Dong Q. (2008), Chinese herb related molecules of cancer-cell-

apoptosis: a minireview of progress between Kanglaite injection and related

genes., Journal of Experimental and Clinical Cancer Research, 27:31.

32. Lu Y, Wu LQ, Dong Q, Li CS. (2009), Experimental study on the effect of

Kang-Lai-Te induced apoptosis of human hepatoma carcinoma cell HepG2.,

Hepatobiliary and Pancreatic Diseases International, 8(3):267-72.

33. Nagao (1985), Benzoxazinones from coix lachryma-jobi var.ma-yuen,

Elsevier.

34. Numata M ( 1994 ), Antitumor components isolated from the Chinese herbal

medicine Coix lachryma-jobi (palmitic,oleic,stearic,linoleic) , Planta media.

35. Takahashi Michiko, Chohachi Konno v Hirosh hikino (1986), Isolation and

Hypoglycemic Activity of Coixans A, B, C, glycans of Coix lacryma-jobi var.

ma-yuen seeds, Planta Medica, (1):64-5.

36. Tyunosin Ukita (1960), Studies on the anti-tumor component in the seeds of

Coix lachryma-jobi L.var.ma-yuen(Roman.) stapf. I. Isolation and Anti-tumor

activity of coixenolide ,Faculty of pharmaceutical science, Universiry of

Tokyo.

37. Tzeng HP, Chiang W, Ueng TH, Liu SH. (2005), The abortifacient effects from

the seeds of Coix lachryma-jobi L. var. ma-yuen Stapf., Journal of Toxicology

and Environmental Health, A, 68(17-18):1557-65.

Ti liu t internet 38.http://www.google.com.vn/search?q=thu%E1%BB%91c+ti%C3%AAm+kanglai

te&source=lnms&tbm=isch&sa=X&ei=1wivUfrgGa31iQeQqYHwAg&ved=0CAc

Q_AUoAQ&biw=1280&bih=641#facrc=_&imgrc=4N4gi3imif4wDM%3A%3B9z

QSLmHewFdFkM%3Bhttp%253A%252F%252Fimages-

en.busytrade.com%252F1792720010000100%252FKanglaite-Injection-klt-

%3Bhttp%253A%252F%252Fvi.busytrade.com%252Fproducts-search%252FAnti-

cancer.html%3B100%3B100.

PH LC

Ph lc 1. Cc ch tiu nng - sinh hc theo di c t thng 12/2012 n

thng 05/2013

TT c im Mu

Y1 Y2 Y3 Y4 Y5 Y6 Y7 Y8 Y9 Y10

1 Thi gian ny mm

( ngy)

9 15 6 15 6 9 15 16 15 14

2 T l ny mm (%) 56 20 86 20 80 70 70 80 100 60

3 Thi gian c l u tin

(ngy)

12 23 7 23 7 12 21 23 21 20

4 Chiu cao sau thng th 1

(cm)

6 3 8 3 12 10 5 4 4 5

5 Chiu cao sau thng th 2

(cm)

12 4 14 5 26 43 7 6 6 14

6 S l sau thng th 1 (l) 4 2 3 2 4 6 2 2 2 2

7 S l sau thng th 2 (l) 8 3 8 3 8 10 3 3 3 7

8 Thi gian nhnh (tun) 19 16 - - 15 8 15 18 16 -

9 Chiu cao thi im

nhnh (cm)

72 33 - - 99 33 60 100 90 -

10 S l/thn thi im

nhnh (l)

12 10 - - 12 12 10 12 12 -

11 Thi gian ra hoa (tun) 20 19 - - 15 9 16 19 17 -

12 S l/thn thi im ra 14 15 - - 12 14 12 14 13 -

hoa (l)

13 S nhnh/thn thi im

ra hoa (nhnh)

4 6 - - 2 4 4 4 3 -

14 Chiu cao thi im ra

hoa (cm)

72 75 - - 99 33 60 100 90 -

15 S ging/thn thi im

ra hoa (ging)

12 15 - - 12 11 11 12 11 -

16 Chiu di ging thi

im ra hoa (cm)

15 18 - - 18 14 13 13 12 -

Ph lc 2. Bng m ha cc bin c im hnh thi v ch tiu nng sinh

hc

Q Q Q Q Q Q Q Q C C C C C C

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14

Y1 9 12 6 4 12 14 4 2 2 1 1 1 1 1

Y2 15 23 3 2 10 15 6 3 2 2 2 2 2 2

Y5 6 7 12 4 12 12 2 2 1 2 1 1 1 2

Y6 9 12 10 6 12 14 4 2 3 1 1 2 1 2

Y7 15 21 5 2 10 12 4 2 2 2 1 2 2 1

Y8 16 23 4 2 12 14 4 2,5 2 2 1 2 2 1

Y9 15 21 4 2 12 13 3 2,5 1 2 1 2 2 1

Ph lc 3. Kt qu chng peak sc k 10 mu d

Ph lc 4. 7 on trnh t nucleotid dng so snh t ngn hng gen quc t

- Gi|300669377|dbj|AB571313.1| Coix lacryma-jobi var. ma-yuen genes for 18S

rRNA, ITS1, 5.8S rRNA, ITS2, 26S rRNA, partial and complete sequence

AGGAAGGAGAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCA

TTGTCGTGACCCTTAAACAAAGCAGACCGCGAACCCGTCTCTCGTGCCATCGG

GCTTCGGCCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGGCCGC

AACAGAACCCACGGCGCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGCTCG

GCGGAGCGGTCGGCCTGCCTTCCGCTCCCCGCGCAGCGATGATATCTTAATAC

ACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGC

AAAATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTG

AACGCAAGTTGCGCCCGAGGCCTTCTGGCCAAGGGCACGTCTGCCTGGGCGTC

ACGCCAAAAGACACTCCCAACCCACCCCCGGGGAGGGACGTGGTGTCTGGCCC

CCCGCGCCGCAAGGCGCGGTGGGCCGAAGTTGGGGCTGCCGGCGAATCGTGTC

GGGCACAGCACGTGGTGGGCGACACCTAGTTGTTCTCGGTGCAGCGCCCCGGC

ACGCAGCCAGCACATCGGCCCTAAGGATCCATCGGGCACCGCAGCGCACCGTC

GCTCGGACCGCGACCCCAGGTCAGTCGGGACT

- Gi|68164101|gb|DQ005034.1| Coix lacryma-jobi voucher Skendzic 5074 RSA

internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete

sequence; and internal transcribed spacer 2, partial sequence

CAAAGCAGACCGCGAACCCGTCTCTCGTGCCATCGGGCTTCGGCCCGGCCGAA

GGCCCCCGAGCTCCGTCCCGGGGCGGAGGGGCCGCAACAGAACCCACGGCGC

CTTAGGCGTCAAGGAACACTAATGCTGCCTTGCTCGGCGGAGCGGTCGGCCTG

CCTTCCGCTCCCCGCGCAGCGATGATATCTTAATACACACGACTCTCGGCAAC

GGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAAATGCGATACCTGGT

GTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTTGCGCCCG

AGGCCTTCTGGCCAAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACACTCC

CAACCCACCCCCGGGGAGGGACGTGGTGTCTGGCCCCCCGCGCCGCAAGGCGC

GGTGGGCCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGTGGTG

GGCGACACCTAGTTGTTCTCGGTGCAGCGCCCCGGCACGCAGCCAGCACATCG

GCCCTAAGGATCCATCGGGCACCGCAGCGCACCGTCGCTCGGACC

- Gi|451173147|gb|KC181919.1| Coix lacryma-jobi isolate bead-1 18S ribosomal

RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA

gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal

RNA gene, partial sequence

CGATTGAATGGTCCGGTGAAGTGTTCGGATCGCGGCGACGGAGGCGGTTCGCC

GCCCCCGACGTCGCGAGAAGTCCATTGAACCTTATCATTTAGAGGAAGGAGAA

GTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTGTCGTGACCC

TTAAACAAAGCAGACCGCGAACCCGTCTCTCGTGCCATCGGGCTTCGGCCCGG

CCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGGCCGCAACAGAACCCAC

GGCGCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGCTCGGCGGAGCGGTCG

GCCTGCCTTCCGCTCCCCGCGCAGCGATGATATCTTAACACACACGACTCTCGG

CAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCAAAATGCGATACC

TGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAACGCAAGTTGCG

CCCGAGGCCTTCTGGCCAAGGGCACGTCTGCCTGGGCGTCACGCCAAAAGACA

CTCCCAACCCACCCCCGGGGAGGGACGTGGTGTCTGGCCCCCCGCGCCGCAAG

GCGCGGTGGGCCGAAGTTGGGGCTGCCGGCGAATCGTGTCGGGCACAGCACGT

GGTGGGCGACACCTAGTTGTTCTCGGTGCAGCGCCCCGGCACGCAGCCAGCAC

ATCGGCCCTAAGGATCCATCGGGCACCGCAGCGCACCGTCGCTCGGACCGCGA

CCCCAGGTCAGTCGGGACTACCCGCTGAGTTTAAGCATATAAATAAGCGGAGG

AGAAGAAAC

- Gi|451173146|gb|KC181918.1| Coix lacryma-jobi isolate ABY 18S ribosomal










GCCTGCCTTCCGCTCCCCGCGCAGCGATGATATCTTAATACACACGACTCTCGG









AGAAGAAAC

- Gi|451173145|gb|KC181917.1| Coix lacryma-jobi isolate TC3 18S ribosomal



















AGAAGAAAC

- Gi|451173144|gb|KC181916.1| Coix lacryma-jobi isolate TCS4 18S ribosomal



















AGAAGAAAC

- Gi|1314374|gb|U46660.1|CU46660 Coix ITS1, 5.8S ribosomal RNA, ITS2

TCGTGACCCTTAAACAGAGCAGACCGCGAACCCGTCTCTCGTGCCATCGGGCT

TCGGCCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGGCCGCAAC

AGAACCCACGGCGCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGCTCGACG

GAGCGGTCGGCCTGCCTTCCGCTCCCCGCGCAGCGATGATATCTTAATACACA

CGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGGAGAACGTAGCAAA

ATGCGATACCTGGTGTGAATTGCAGAATCCCGCGAACCATCGAGTTTTTGAAC

GCAAGTTGCGCCCGAGGCCTTCTGGCCAAGGGCACGTCTGCCTGGGCGTCACG

CCAAAAGACACTCCCAACCCACCCCCGGGGAGGGACGTGGTATCTGGCCCCCC

GCGCCGCAAGGCGCGGTGGGCCGAAGTTGGGGCTGCCGTCGAATCGTGTCGGG

CACAGCACGTGGTGGGCGACACCTAGTTGTTCTCGGTGCAGCGCCCCAGCACG

CAGCCAGCACATCGGCCCTAAGGATCCATCGGGCACCGCAGCGCACCGTCGCT

CGGACCGCGA

Ph lc 5. Trnh t nucleotid ca 9 mu d

- Y1

TCGNGTGACCCTTAAACAAAGCAGACCGCGAACCCGTCTCTCGT-GCCGTCGG

GCTTCGG CCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGG CCG

CAACAGAACCCACG GCGCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGCTC

GGCGGAGCGGTCGGCCTGCC TTCCGCTCCCCGCGCAGCGATGATATCTTAACA

CACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGT AG

CAAAATGCGATACCTGGTGTGAATTGCAGAATCC CGCGAA CCATCGAGTT TT

TGAACGCAAGTTGCGCCCGAGGCCTTCTGGCCAAGGGCACGT CTGCCTGG G

CGTCACGCCAAAAGACACTCCCAACCCACCCCCGGGGAGGGACGTGGTGTC

TGGCCCCCCGCGCCGCA AGGCGCGGTGGGCCGAAGTTGGGGCTGC CGGCGAA

TCG TGTCGGGCACAGCAC GTGGTGGGCGACACC TAGTTGTTCTCGGTGCA GC

GCCCCGGCACG CAG CCAGCACATCGGCCCTAGGATCCATCGG---GCACCGC A

GCGCACCG---TCGCTC GGA CCG CGA

- Y2

TCTAGTGACCCTTAAACAAAGCAGACCGCGAACCCGTCTCACGTAGCCGTCGG

GCTTCGGCCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGG GGCCGC

AACAGAACCCACG GCGCCTTAGGCGTCAAGGAACACTAAT GCT GCCTTGCTC

GGCGGAGCGGTCGGCCTGCC TTCCGCTCCCCGCGCAGCGATGATATC TTAATA

CACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCG ATGAA GAACGTA

GCAA AATGCGATACCTGGTGTGAATTGCAGA ATC CCGC GAACCATCGAGT T

TTTGAAC GCAAGTTG CGCCCGAGGCCTTCTGGCCAAG GCACG TCTGCCTGG

GCGTCACGCCAA AAGAC A CTCCCAACCCACCCCCGGGGAGGGACGTG GTG

TCTGGCCCCCCGCGCCGCAAGG C GCGGTGGGCCGAAGTTGGGGCTGCCGGC

GAATCG TGTCGGGCACAGCA CGTGGTGGGCGACACCTAGTTGTTCTCGGTGC

AGCGCCCCG GCAC GCAGCCAGCACATCGGCCCTAAGGATCCATCGG---GCAC

CGCAGCGCACCG---TCGCTCG GACCGCGA

- Y3

TCGNGTGACCCTTAAACAAAGCAGACCGCGAACCCGTATCACGTAGCCGTCGG

GCTTCGGCCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGG GGCCGC

A ACAGAACCCACG GCGCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGCT C

GGCGGAGCGGTCGGCCTGCC T TCCGCTCCCCGCGCAGCGATGATAT CTTAA T

ACACACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCG ATGAAGA ACG

TAGCAAAATGCGATACCTGGTGTGAATTGCAGAATC CC GCGAACCA TCGAG

TTTTTGAACGCAAGTTGCGCCCGAGGCCTTCTGGCCAAGGGCACGTC TGCCTG

GGCGTCACGCCAAAAGACACTCCCAACCCACCCCCGG GGAGGGACGTGG TGT

CTGGCCCCCCGCGCCGCAAG GCGCGGTGGGCCGAAGTTGGGGCTG CCG GCA

ATCGTGTCG GGCACAGCACGTG GTGGGCGACACCTAGTTGTTCTCGGTGCAG

CGCCCCGG CACGCAGC CAGCACATCGGCCCTAAGGATCCATCGG---G CACCG

CAGCGCACCG---TCGCTCGG ACCG CGA

- Y4

TC--GTGACCCTTAAACAAAGCAGACCGCGAACCCGTATCACGT-ACCGTCGG

GCTTCGGC CCGGCCGGAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGGCCG

CAACAGAACCCACGGC GCCTTAGGCGTCAAGGAACACTAATGCTGCCTTGC

TCGGCGGAGCGGTCGGCCTGCCTTC CGCTCCCCGCGCAGCGATG ATATC TT

AATACACACGACTCTCGGCAACGGATATCTCGGC TCTCGCATCGA TGAAGA

ACG TAGCAAAATGCGATACCTGGTGTGAATTGCAGAATCC CG CGAACCAT

CGAGTTTT TGAACGCAAGTTGCGCCCGAGGCCTTCTGGCCAAG GGCACGTCT

GCCTGGGCGTCACGCCAAAAGACACTCCCAACCCACCCCCG GGGAGGGAC

GTGG TGTCT GGCCCCCCGCGCCGCAAGGCGCGGTGGGCCG AAGTTGGGGC

TGCCGGCGAATCGTGCG GGCACAGCACGTGGTGGGCGAC ACCTAGTTGTAC

TCGGGGCAGCGCCCCGG CACGCAGC CAGCACAGCGGCCC TAATGAACCA

GCGG---GCACCGCCACGCACCG---ACG CGCGGACC GCGA

- Y5

TCCCGTGACCCTTAAACAAAGCAGACCGCGAACCCGTCTCTCGT-GCCATC GG

GCTTCGG CCCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGGG CCG

CA ACAGAACCC ACG GCGCCTTAGGCGTCAAGGAACACTAATGCTG CCTTGC

TCGGCGGAGCGGTCG GCCTGCC TTCCGCTCCCCGCGCAGCGATGATA TCTTA

ATACACACGACTCTCGGCAACGGATATCTC GGCTCTCGCATCGATG AAGAA

CGTAGCAAAATGCGATACCTGGTGTGAATTGCAGAATC CCGCGAA CCATCG

AGTTTTTGAACG CAA GTTGCGCCCGAGGCCTTCTGGCCAAGGGA CG TCTGC

CTGGGCGTCACGCCAAAAG ACACTCCCAACCCACCCCCGGGGAGGGACGTG

GTG TCTGGCCCCCCGCGCC GCA AGGCGCGGTGGGCCGAAGTTGGGGCTGCC

GGCGAATCGTG TCGGGCACAG CACGTGGTGGGCGACACCTAGTTGT TCTC GG

TGCAGCGCCC CGGCACGC AGCCAGCAC ATCGGCCCTAAGG ATCCATCGG---

GCACCGCAGCGCACCG---TCGCTCGGA CCGCG A

- Y6

TCTAGTGACCCTTAAACAAAGCAGACCGCGAACCCGTCTCTCGT-GCCAT GGG

CTTCGGC CCGGCCGAAGGCCCCCGAGCTCCGTCCCGGGGCGGAGGG GCCGCA

ACAGAACC CACGGC GCCTTAGGCGTCAAGGAACACTAATGCTG CCTTGCTCG

GCGG AGCGGT C GGCCTG CCTT CCGCTCCCCGCGCAGC GATG ATATCTTAAT

ACACACGACTCTCGGCAACGGATATCTCGG CTCTCGCATCG ATGAAGAAC GT

AGCAAAATGCGATACCTGGTGTGAATTGCAGAA TCCC GCGAACCATCGA GTT

TTTGAACGC AAGTTGCGCCCGAGGCCT TCTGGCCAAGGGCA CGT CTGCCTG

GGCGTCACGCCAAAAGACACTCCCAACCCACCCCCG GGGAGGGACG TGGTGT

CTGGCCCCCCGCGCCGCAA GGC GCGGGGGGC CG AA GTTGG GGCTGCCG GC

GAAT CG TGT CGGGCACAGCACGTGGT GGGCGACACCTAGTT GTTCTCG GTG

CAGCGC CCCGG CA CGCA GCCAGC AC

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