CHAO THY VA CAC BAN N

VI BUI THUYT TRINH CUA

NHOM 3
Tai:
Thc Phm Bin
i Gene
Cy Trng Chu
Hn
GVHD: Hoang Anh Hoang
Nhm 3
Trn Th Dim Hng
L inh Tm
L Thanh Thin
Vo Ngoc Thnh
 Ni dung chnh
1 i tng nghin cu
2 C ch biu hin
3 Phng phap nghin cu
4 Kt qua
5 Kt lun
1. i tng nghin cu
Basmati la ging la dng ht dai
c trng ph bin n .
Ging la nay la c mi thm,
hng v c trng va c cht lng
tt.

Ging la Pusa Basmati 1 sinh

trng va pht trin tt iu kin
kh hu Vit Nam, c tim nng v
nng sut va cht lng.
Cac chung vi khun va cac vector
Chuyn gene OsNAC6 nh t bao
kha bin cua vi khun A.
Tumefaciens

Vector chuyn gene pBCKH.

Vector nhn dong pSK II, pUC19-
Ubi.
Vi khun nhn dong E.Coli DH5.
Ha cht
Mi trng c s dng trong nghin cu nui cy m t bao va nui
cy vi khun dng cho th nghim chuyn gene bao gm: MS, LB, YEM.
Thit bi
Tu cy v trng.
Tu sinh trng.
My PCR.
My giai trinh t.
My quang ph k.
My o pH.
My ly tm.
B n nhi t.
Cc thit b khc
2. C ch biu hin
Thay i v hinh thi, sinh l, sinh ho va phn t

V hinh thi, sinh l: ng cc kh khng, giam h hp va quang

hp, giam th tch nc trong cc m.

Mc phn t: gia tng mc biu hin va tch lu cua cc

gene/protein chng chu stress.
2. C ch biu hin
Cc protein tham gia vao qu trinh chng hn cua thc vt c chia
lam 2 loi:
Nhm protein chc nng trc tip tham gia chng li vi iu
kin hn (ABA, LEA, proline, mannitol, sorbitol...)

Nhm protein iu khin s biu hin cua cc gene chc nng

lin quan n tnh chu hn (nhn t phin m, kinase...)
2. C ch biu hin
* Nhn t phin m OsNAC6

Ho NAC cha trinh t ng nht (c pht hin t petunia

NAM va t Arabidopsis ATAF1, ATAF2 va CUC)
- C vai tro quan trong trong vic xc nh m phn sinh nh
chi
- Phan ng vi iu kin b tn thng va tc nhn gy hi tn
cng
- Tng cng tnh chu hn
2. C ch biu hin
* Nhn t phin m OsNAC6

Khng tham gia trc tip vao phan ng p ng vi iu kin hn

S biu hin cua chng li c vai tro kch hot s biu hin cua rt
nhiu gene chc nng khc

Bm vao trinh t ADN c hiu trn vng khi ng gene cua cc gene
chc nng => hot ha s biu hin cua cc gene nay => tng cng
tnh chu hn
2. C ch biu hin
* Nhn t phin m OsNAC6
iu kin hn hn

Nhn, truyn tn hiu

Cc gene iu khin
biu hin
Cc nhn t phin m
Cc gene chc
nng biu hin
Cc gene chc nng

Thc vt tng cng tnh chu hn

 S THC HIN
Gene
OsNAC6

Phan ng PCR

San phm PCR gene

OsNAC6 ln 1 E.Coli
DH5
Phan ng PCR
Nui trong LB
San phm PCR gene
OsNAC6 ln 2 Sinh khi E.Coli
DH5
Tinh sch
Vector nhn X l CaCl2
San phm PCR
dong pSK II tinh sch
E.Coli kha np
Ct RE + Tinh sch
Ct RE + Tinh sch T4 Ligase
Ti t hp
Sc nhit
Trai a LB.amp.X-gal
Cc dong E.Coli cha
plasmid

Sang loc bng PCR

1 dong E.Coli cha
plasmid mang gene
OsNAC6
 S THC HIN
1 dong E.Coli cha plasmid
mang gene OsNAC6
T bao kha bin
Thit k vector chuyn Agrobacterium
Ht la
gene mang OsNAC6

T bao m so Bin np

Xm nhim

Chon loc

Ti sinh

Kim tra cy
chuyn gene

Trng ngoai
thc a
 NHN GENE OsNAC6 T TH VIN BNG K THUT PCR
H2O : 34.5 l
Gene m 10X : 5 l
OsNAC6 Mi F (10 pmol) : 2 l
Mi R (10 pmol) : 2 l
Phan ng PCR
dNTPs 10 mM : 5 l
San phm PCR : 0,5 l
Taq DNA polymerase
gene OsNAC6
Th vin cDNA : 1 l
ln 1
Phan ng PCR Tng th tch : 50 l
San phm PCR Chu trnh Nhit Thi gian Chu k
gene OsNAC6
( oC ) ( pht )
ln 2
Tinh sch Khi u 94 5 1

San phm
Bin tnh 94 45
PCR tinh sch Gn mi 55 45 30
Ko dai 72 60
n inh 72 7 1
 Tinh sch sn phm PCR t gel agarose bng
ct GeneluteTM Minus Etbr (Sigma Cat # G-2166)

Bng DNA sau khi in di xc nh ng kch thc s c

ct va chy qua kim tim 1ml lam nh trc khi chuyn

qua ct.
Ly tm ct cha agarose va DNA 12.000 rpm, 10 pht.

San phm thu c chnh la DNA c tinh sch c th s

dng cho cc phan ng tip theo.

 KT QU

1 2 M 4 5

900 - 1000 bp 926 bp

Hnh : Kt qu in di sn phm PCR nhn gene OsNAC6 t th vin.

M- Thang ADN chun 1kb (invitrogene);
Ging 1, 2 - Sn phm ADN khuch i ln 1;
Ging 4, 5 - Sn phm PCR ln 2.
GN SN PHM PCR VO VECTOR NHN DNG pSK II

Vector nhn San phm PCR

dong pSK II tinh sch
Ct RE + Tinh sch

Ti t hp T4 Ligase
Ct RE + Tinh sch
m cua enzym (10X) : 5l
San phm PCR/vector pSK II (1 g/l) : 20 l
BamH I (10 unit/l) : 2 l

dH2O : 23 l

Tng th tch : 50 l

Phan ng ct c tin hanh 37oC trong 1 gi

TINH SCH BNG PHNG PHP PHENOL LNH
B1: Ct ly mu gel cha on DNA c kch thc cn kim tra cho vao ng
tim 1 ml, dng p lc y gel agarose cha DNA qua ng tim 1 ml, san
phm c thu trong ng eppendorf 1,5 ml.

B2: Thm 800 l phenol (pH = 8,0), trn u bng vortex, u -80 oC,1h

B3: Lam tan hn nhit phong, ly tm 12.000 rpm, 5 pht.

B4: Ht dch ni chuyn sang ng eppendorf mi va b sung mt lng tng

ng dung dch chloroform : isoamyl alcohol vi t l 24 : 1. Lc u va tip
tc ht dch ni chuyn sang ng eppendorf mi.

B5: Thm 1/10 th tch CH3COOK, pH = 5,2 va 2,5 ln th tch cn tuyt i,

gi lnh. U hn hp -20oC, 1 gi.

B6: Ly tm 12000 rpm trong khoang 30 pht, thu kt tua va lam sch DNA
bng cn 70%. Ly tm 12000 rpm trong 5 pht, thu kt tua, lam kh va hoa tan
DNA trong th tch nc hp l.
GN SN PHM PCR VO VECTOR NHN DNG pSK II

Vector nhn San phm PCR

dong pSK II tinh sch
Ct RE + Tinh sch

Ti t hp T4 Ligase
Ct RE + Tinh sch
m cua enzym (10X) : 1 l
San phm PCR (50 ng/l) : 1 l
Vector pSK II II (200 ng/l) : 1 l
T4 ligase (10 unit/l) : 1 l

dH2O : 6 l

Tng th tch : 10 l
Phan ng gn c tin hanh 25 C trong thi gian 1
0
BIN NP GENE OsNAC6 VO T BO E. coli
E.Coli
DH5
Nui trong LB
Sinh khi
E.Coli DH5
X l CaCl2
E.Coli kha
np
Ti t hp
Sc nhit
Trai a LB.amp.X-gal
Cc dong E.Coli
cha plasmid
Sang loc bng PCR
1 dong E.Coli cha plasmid
mang gene OsNAC6
 CHUN B T BO KH BIN E. coli DH5

Dch t bao DH5 gi -800C c cy trai trn mi trng LB c,

nui qua m 370C, sau chon mt khun lc n nui lc 200
rpm qua m 37oC trong 5 ml mi trng LB lng.
500 ml mi trng LB lng c b sung vao 5 ml dung dch nui
qua m va tip tc nui lc 200 rpm 370C khoang 2 gi (OD600=0,4
- 0,6).
Dch khun c em ly tm 3.000 rpm trong thi gian 10 pht, thu
cn t bao va hoa tan trong 20 ml CaCl2 0,1M lnh.
T bao c u trong dung dch CaCl2 0,1M; gi hn hp trn trong
khoang 2 gi, tip tc ly tm tc 3.000 rpm trong 10 pht 4oC.
Cn t bao c hoa tan trong 20 ml dung dch CaCl 2 0,1M va 10%
glycerol. T bao kha bin DH5 va chun b c chia thanh tng
lng nh 80 - 100 l vao cc ng eppendorf va bao quan -80oC.
 Bin np gene OsNAC6 vo t bo E. coli bng
phng php sc nhit
10 l vector nhn dong pSK II mang gene OsNAC6
chun b trn (mc 2.2.1.3) c b sung vao t bao
kha bin DH5.
Hn hp c trn nh va u trong 20 pht to iu
kin cho cc vector bm ln thanh t bao.
Sau , c thc hin phan ng sc nhit bng vic
chuyn hn hp phan ng trn vao b n nhit 42 0C
trong 60 giy, ngay lp tc u hn hp vao trong 5 pht.
Hn hp phan ng sau c trai trn a mi trng
LB c c b sung 100 g/ml ampicilin, nui trong tu m
370C qua m.
 Kim tra gene OsNAC6 trong khun lc
bng phn ng PCR.
San phm PCR va vector pSK II ch c gn vi nhau bi mt v tr
enzym gii hn BamH I nn san phm PCR c th gn vao vector theo
chiu xui (forward) hoc ngc (reverse).
Vi vy, phan ng PCR c hiu vi mi xui la mi cua promoter T3
(T3-F) va mi ngc la mi c hiu cua gene OsNAC6 (OsNAC6-R)
c tin hanh xc nh s c mt xui chiu cua gene OsDREB2A
ti t hp vi vector pUC19-Ubi trong khun lc.
Cc khun lc cho san phm PCR la cc bng DNA c kch thc
tng ng vi kch thc cua gene OsNAC6 c tip tc nui tch
plasmid.
 1 dong E.Coli cha
plasmid mang gene
OsNAC6

Tch DNA plasmid t

E.Coli DH5

Giai trinh t gene

OsNAC6

Thit k vector chuyn

gene mang OsNAC6
 TCH DNA PLASMID T VI KHUN E. coli DH5
B1 : Nui cy lc 1 khun lc n trong 2ml mi trng LB lng, lc
200 rpm qua m 370C.

B2 : Chuyn dung dch nui qua m vao ng eppendorf 1,5 ml, ly tm

tc 6.000 rpm trong 5 pht.

B3 : Thu cn t bao, hoa tan trong 150 l dung dch I lnh (50 mM
glucose, 25 mM Tris-HCl (pH = 8,0) va 10 mM EDTA).

B4 : B sung 300 l dung dch II (0,2 N NaOH, 1% SDS), ao nh.

B5 : Tip tc b sung 200 l dung dch III lnh (60 ml CH3COOK

5M; 11,5 ml axit acetic m c; 28,5 ml nc), ao nhe.

B6 : Dung dch t bao c u trong khoang 10 pht va ly tm 12.000

rpm nhit phong.
 TCH DNA PLASMID T VI KHUN E. coli DH5
B7 : Thu dch trn chuyn sang ng eppendorf 1,5 ml sch va b sung 2 l enzym
RNase (10 g/ml).

B8: B sung mt lng tng ng dung dch phenol : chloroform : isoamyl

alcohol theo t l 25 : 24 : 1, ly tm 12.000 rpm trong khoang 5 pht nhit
phong.

B9 : Chuyn pha trn cng sang ng eppendorf 1,5 ml mi ri b sung dung dch
chloroform : isoamyl alcohol theo t l 24 : 1 vi th tch tng ng. Ly tm
12.000 rpm trong khoang 5 pht nhit phong.

B10 : Thu pha trn cng va chuyn sang mt ng eppendorf 1,5 ml mi va b sung
CH3COOK 3M (pH = 5,2) vi t l 1/10 th tch. B sung 2,5 ln th tch cn tuyt
i gi lnh, kt tua DNA -80oC trong 1 gi. Ly tm 12.000 rpm khoang 30 pht
4oC.

B11 : Ra DNA bng cn 70%, sy kh, hoa tan DNA trong 50 l nc / TE.
 KT QU

Hnh : S tach dng gene OsNAC6 trong vector nhn

dng pBluescript II ( PSK II)
 KT QU GII TRNH T GENE OsNAC6

Mc tng ng Nucleotide cua on DNA la 100% vi

gene m ha OsNAC6 la Japonica, 93% vi NAC cy ng, 91%
vi NAC bc ha ng.

Mc tng ng acid amine la 94% vi nhn t phin m

NAC la m, 94% vi nhn t phin m NAC bc ha ng va
87% vi nhn t phin m NAC cy ng.

Da theo cc kt qua thu c, c th khng nh phn lp

thanh cng on DNA m ha nhn t phin m OsNAC6 iu
khin tnh chu hn, mn la.
Thit k vector chuyn gene mang gene OsNAC6 bng k thut Gatewaya
To vector trung gian

Gene OsNAC6 trong vector tch dong pSK II-OsNAC6 c thu nhn li bng
phan ng PCR s dng cp mi c hiu OsNac6-F/OsNac6-R nhn trinh t
nucleotide m ha gene OsNAC6. Phan ng PCR c tin hanh nh m ta phn
phn lp gene OsNAC6, ch khc la thanh phn phan ng s dng si khun la 50
ng plasmid pSK II-OsNAC6.

San phm PCR c in di trn gel Agarose 1%, tinh sch bng ct
GeneluteTM Minus EtBr, x l enzym gii hn Bam HI, gn vao vector pUC19-Ubi
x l Bam HI va loi gc phosphate trc to vector trung gian pUC19-
Ubi-OsNAC6. Chiu gn cua gene OsNAC6 trong vector pUC19-Ubi c kim tra
bng cp mi Ubi-F/OsNac6-R. Vector pUC19-Ubi-OsNAC6 c chiu gn xui
c bin np vao t bao kha bin E. coli DH5, nui lng thu sinh khi, tch
plasmid va giai trinh t trc khi gn vao vector chuyn gene.
Thit k vector chuyn gene mang gene OsNAC6 bng k thut Gatewaya

To vector chuyn gene

Vic gn cu trc gene gm promoter Ubiquitin, OsNAC6 va trinh
t kt thc phin m trong vector pUC19-Ubi-OsNAC6 vao vector nhn
pBCKH to ra vector chuyn gene pBCKH-Ubi-OsNAC6 c thc
hin theo quy trinh Gateway kit (Invitrogene).

Phan ng gateway c tin hanh trong hn hp phan ng 5 l

vi cc thanh phn phan ng :
LR clonase buffer : 1 l
Vecor cho (pUC19-Ubi) 100 ng/l : 0,5 l
Vector nhn (pBCKH) 20 ng/l : 0,5 l
TE : 2,5 l
LR clonase : 0.5l

Tng th tch : 5 l
Thit k vector chuyn gene mang gene OsNAC6 bng k thut Gatewaya

Hn hp phan ng c u 250C trong thi gian 1gi, sau b sung

0,5 l proteinase K u 370C trong 10 pht loi b protein.

1,5 l hn hp phan ng c dng bin np vao t bao kha bin

E.coli DH5 nh trinh bay mc 2.2.1.4. Cc khun lc moc trn mi
trng LB cha 50 g/ml kanamycine c kim tra s c mt cua gene
OsNAC6 trong vector chuyn gene pBCKH bng phan ng PCR vi cc
cp mi: OsNac6-F/Out-R, Ubi-F/OsNac6-R, Ubi-F/Nos-RT50, OsNac6-
F/OsNac6-R (bang 1).

Cc khun lc cha vector chuyn gene pBCKH mang gene OsNAC6

c sang loc trong mi trng LB lng cha 50 g/ml kanamycin.
To t bao Agrobacterium ti t hp c mang gen
OsNAC6

A. CHUN B T BAO KH BIN

B. BIN NAP BNG XUNG IN
Chun b t bao kha bin
Bc 1: Nui cy 5ml A.Tumefaciens trong 500ml YEM, lc
200v/p, 280C, 8h, OD=0.5 0.8 ->Tng sinh vi khun
Bc 2: Ly tm lnh 6000v/p, 5 pht, 40C -> Thu sinh khi t
bao(cn)
Bc 3: Hoa tan bng 5 - 10ml nc lnh, trn u, them tip
n 500ml nc lnh, ly tm 6000v/p, 5 pht, 40C -> Thu cn t
bao
Bc 4: Thm 250 ml nc , ly tm 6000v/p, 5 pht, 4 0C ->
Thu cn t bao
Bc 5: B sung 5ml glycerol 10% -> bao v t bao
Bc 6: Bao quan trong Nit lng -800C
Bin np gene OsNAC6 vao t bao kha bin
Bc 1: Trn 100ng plasmid ti t hp vao cc t bao kha bin Agrobacterium
EHA105, trn u, hn hp trong 30 pht

Bc 2: Chuyn hn hp vao cuvet in, u 5 - 10 pht, tin hanh bin np bng

my xung in: in dung 25F, in th 2.4 KV, in tr 200 , Thi gian 5s

Bc 3: Chuyn qua ng falcol, u 4h, 280C

Bc 4: Ly tm, b dch nhng gi li 50 - 100 l, cy trang ln mi trng LB c

c b sung 50mg/l Kanamycin, u trong 2 ngay, 280C

Bc 5: Nui cc khun lc n trong mi trng YEB lng c b sung 50mg/l

Kanamycin, lc 200v/p qua m

Bc 6: Tch chit plasmid, in di kim tra

Bc 7: Bao quan cc khun lc c kt qua dng tnh trong mi trng YEB, c

b sung glycerol 15% trc khi lam lnh n -700C
 Chuyn gene OsNAC6 vao ging la Pusa
Basmati thng qua vi khun Agrobacterium

A. To callus
B. Nui cy Agrobacterium Tumefaciens
C. Ly nhim
D. Chon loc va ti sinh
To callus

Ht la bc v, ra bng nc ct 4-5 ln, ngm

va lc nh trong cn 70%, 1 pht.

Ra li bng nc ct kh trng, ngm trong

Javel 50% c b sung tween 20 trong 20 pht.

Ra li bng nc ct 4-5 ln. Thm kh..

t ht vao mi trng MS c b sung 2,4-D,

BAP, casein, L-proline , gi trong ti 280C, 15

ngay.
Nhim callus vi Agrobacterium EHA105
Bc 1: T bao Agrobacterium EHA105 cha gene OsNAC6 c
nui qua m va o OD600, sau c em ly tm 3000 vong/pht
trong 15 pht, thu tua va hoa tan vao mi trng MS lng, sau
pha long ti nng OD600 = 0,5, b sung Acetosyringone nng
30 g/ml.
Bc 2: Cc callus c cho vao ng fancol cha dung dch MS
c hoa Agrobacterium EHA105 c chun b, lc trong my lc
30 pht
Bc 3: Callus c ra tm li kh trng va c lam kh
bng cch t ln giy thm kh trng loi b Agrobacterium con
li, sau callus c chuyn ln mi trng ng nui cy c ngn
bi giy loc. Cc callus sau khi c nhim Agrobacterium EHA105
cha gene OsNAC6 c nui trong ti 280C trong 3 ngay
Chon loc va ti sinh
Sau qu trinh ly nhim, Callus c ra nhiu ln bng nc
ng kh trng c b sung 500 mg/l cefotaxim, lam kh bng
giy thm loi b Agrobacterium EHA105

Chuyn Callus sang mi trng chon loc 50 mg/l hygromycin,

nui cy 2 tun, 280C, ht tun 1 chuyn sang a mi.

Chuyn Callus sang mi trng chon ti sinh loc, 25 0C, m t

50 - 70%, quang chu k 16 gi/ngay, 3.000 Lux.
Cac giai on to chung la Cha gen OsNAC6
4. KT QU
T 100 ht la c em vao nui cy mi trng MS2, thu

c 95 callus va s dng s lng callus nay tin hanh ly

nhim vi vi khun Agrobacterium.
Sau 15 ngay nui cy trn mi trng chon loc, c 20 callus sng

st va c chuyn sang mi trng ti sinh.

Sau 4 tun ti sinh, kt qua thu c 12 callus c chi sinh trng

binh thng.
Tng s thu c 12 dong la sinh trng va pht trin binh

thng.
 Kim tra cy chuyn gene
A. Tch chit DNA tng s
Thc hin theo phng php tch chit plasmid
B. Thc hin p PCR va in di kim tra
Kt qua:
C 11 dong la c chuyen gene OsNAC6 thanh cng
C 1 dong la khng cho kt qua PCR

Hnh. in di sn
phm nhn gene m
hoa nhn t phin m
OsNAC6 cac dng la
Pusa Basmati chuyn
gene
 5. KT LUN
Vic nghin cu thc hin chuyn gene trn ging la Basmati

thanh cng to ra mt c s, ngun cung cp d liu cho vic

thc hin to ra cc ging la mi, c kha nng chu hn nc
ta.
Gp phn vao khc phc vi tinh trng bin i kh hu anh

hng n thi tit, mi trng sng cua cy trng hin nay.

To ra hng nghin cu tch cc
CM N THY VA CAC BAN
A CHU Y LNG NGHE