CHUYN GEN

CHUYN GEN
V CC PHNG PHP CHUYN GEN
V CC PHNG PHP CHUYN GEN
THC VT
THC VT
K THUT CHUYN GEN V CC PHNG PHP CHUYN GEN THC VT K THUT CHUYN GEN V CC PHNG PHP CHUYN GEN THC VT
KHI NIM CHUNG
MC ICH BIN NP GEN THC VT
CC PHNG PHP BIN NP GEN THC VT
XU HNG P DNG V MT S THNH TU BIN NP GEN THC
VT
CC AC IM U VIT CUA CC PHNG PHP BIN NP GEN SO
VI CC PHNG PHP TO GING TRUYEN THNG
K thut di truyn K thut di truyn
L k thut chuyn gen to
ra ging cy trng, vt nui
hay vi sinh vt nh mun ca
con ng!i
"#t $hn on %&' tr(n
nhi)m s*c th ch+u tr,ch
nhi-m v. m#t /c t0nh
ca sinh vt
Gene
Khi nim chung
Gen l g?
Gen l mt don v| cua vt cht di truyn. Ban thn n
ho}c kt hp vi cc gen khc quy d|nh mt tnh trng
cua co th.
V m}t phn tu, mt gen l mt don ADN (o mt s
virt l ARN) m ho ho}c trc tip ho}c tham gia vo
vic tng hp nn mt phn tu protein, hay trong mt s
trung hp l cc phn tu ARN ribosom (rARN), ho}c
ARN vn chuyn (tARN), ho}c mt s cc phn tu ARN
cu trc khc.
Gen (ADN) -> ARN -> Protein -> Tnh trng
Bin np gen (chuyn gen / k thut di truyn)
o thc vt l khi nim dng m ta qu trnh
chuyn mt ho}c mt s gen ngoi lai vo trong
t bo thc vt nhm to ra mt tnh trng mi
m truc d co th thc vt d khng c.
Khi nim chung
Bin np gen
o thc vt
l g?
Cc gen bin np c th
c ngun gc khc nhau.
N c th duc tch ra t
mt ngun t bo khc c
trong t nhin, ho}c duc
tng hp bng cc k
thut sinh hc phn tu.
Ngun gc cua
gen bin np
Qu trnh bin np gen duc coi l thnh cng khi
gen bin np sau qu trnh chuyn gen kt hp n
d|nh vi ADN cua h gen nhn cua t bo bin np.
T bo bin np ny sau d duc ti sinh thnh cy
hon chinh vi s biu hin cua gen bin np, v duy
tr n d|nh trong cc th h sau nh qu trnh th
tinh bnh thung.
MC ICH BIN NP GEN THC VT MC ICH BIN NP GEN THC VT
1. Nghin cu v lm sng to chc nng cua mt gen duc quan tm hay
tng phn cua gen d.
2. Lm thay di mc d biu hin cua mt gen ni bo.
3. Chuyn cc gen quy d|nh cc tnh trng mong mun vo t bo d thu
nhn duc cc tnh trng mi o t bo v cy chuyn gen.
Trong d, cc gen quy d|nh cc tnh trng mong mun duc quan tm
nghin cu nhiu hon ca, gm:
1 2,c gen kh,ng 3-nh 4virus, n5m, vi khu6n, su 3-nh, giun tr7n89
1 :en ch+u hn, ;nh, v c,c <i.u ki-n 35t ;=i kh,c ca mi tr!ng
1 :en kh,ng thuc <i-t c>
1 :en c?i to c,c /c t0nh v. ch5t ;=ng 4thay @i thnh $hAn aB0t 3Co, tDng c!ng
thnh $hAn aB0t khng no, tDng c!ng thnh $hAn c,c aB0t amin khng thay thE,
gen ch0n chm, vFv89
QU TRINH BIN NP GEN THC VT C THC HIN NH TH NO? QU TRINH BIN NP GEN THC VT C THC HIN NH TH NO?
:iai on GF Giai don chuyn gen (giai don bin np).
Trong giai don ny, gen mong mun thung duc chuyn vo t bo ho}c
m thc vt.
:iai on HF Giai don ti sinh cy.
Trong giai don ny, cc m t bo duc chuyn gen duc chn lc ra v
cho ti sinh d pht trin thnh cy.
Iai giai on 3iEn n$ v t,i sinh cJng cK nghLa Muan trNng v MuyEt +nh
thnh cng ca m#t th0 nghi-m 3iEn n$F 'Eu sO 3iEn n$ B?y ra m khng
cK sO t,i sinh kPm theo, ho/c sO t,i sinh <i)n ra m khng kPm theo sO 3iEn
n$ thQ th0 nghi-m 3iEn n$ cha thnh cngF
CC YU CAU C BN I VI H THNG TI SINH CC YU CAU C BN I VI H THNG TI SINH
- Cc mu t bo, m dng cho qu trnh chuyn gen cn phai c kha nng phn chia
in vitro nhanh.
- Cc m, t bo ny phai c kha nng tip nhn gen mi.
- Quy trnh ti sinh cy phai c hiu qua cao, khng ho}c t ph thuc vo kiu gen.
- Cy ti sinh phai c t l sng (khi dua
ra ngoi dt trng) cao, tn s bin d|
thp (ti thiu), v kha nng hu th cao
d c th su dng lm ngun nguyn liu
ban du d tip tc tin hnh chuyn gen
trong diu kin in-vivo sau ny.
- Cc phuong php chuyn gen (bin np gen) du chuyn gen vo cc t bo, hay m
ni cch khc t bo v m l don v| tip nhn gen mi.
h thng ti sinh l diu kin
tin quyt d thc hin thnh
cng bin np gen
Cc loi m su dng lm h thng ti sinh:
Phai c kha nng ti sinh cao v thun li cho vic chuyn
gen.
M so c ngun gc
khc nhau.
M l, thn mm
Phi non,
phi truong thnh
VI D VE PHNG PHP THU NHN CC CY BIN NP T CC T BO KHC NHAU
Dng t bo / m dch PHNG PHP THU NHN CY BIN NP
T bo ho}c m nui cy in vitro
(cultured cells / tissues)
Thng qua qu trnh pht sinh co quan (organogenesis),
ho}c pht sinh phi (embryogenesis).
Phi non (immature embryos), ho}c
cc t bo dang pht sinh co quan
(organogenic cells)
Ti sinh cy in vitro t cc dng t bo bin np
Cc t bo o phi non, v m phn
sinh hoa
S pht trin tip tc cua phi, chi, hay hoa s cho mt cy
kham.
Ht phn c ngun gc t cc dng t
bo bin np duc su dng cho vic
to ra cc ht cy duc bin np
thng qua qu trnh th tinh bnh
thung
To cc cy bin np trc tip qua con dung th tinh vi
ht phn chn (mature pollens) dang pht trin m ADN cua
n d duc u l bin np
Ht phn To cc cy bin np trc tip qua con dung th tinh vi
ht phn chn (mature pollens) dang pht trin m ADN cua
n d duc u l bin np
GIAI ON CHUYN GEN GIAI ON CHUYN GEN
Cc vcto chuyn gen
Cc vcto chuyn gen l cc phn tu ADN mang don gen cn
bin np.
Ngoi ra chng cn c cc don ADN c cu trc d}c th nhm
tng hiu qua cc cc qu trnh bin np gip qu trnh bin
np c th thc hin duc.
Cu trc cua
vcto chuyn gen
1. C mt don ADN khoi dng (promoter). y l mt don ADN c lin
quan v cn thit cho s khoi du phin m. N thung c mt v| tr bm
cho enym ARN polymeraa, mt dim khoi du phin m, v mt s v| tr
bm khc cua cc protein diu khin / diu ho qu trnh phin m (CaMV-
3S-promoter, nos-promoter )
Vng gn enyme
gii hn (MCS)
Gen khng
khng sinh
Vi khun
im ti ban
Vi khun
Prokaryote
Promoter
Gen c bi n n p
Eukaryote
Promoter
Vng 3 cha tn hiu
polyadenine ho
Gen chi th|
chn lc
Eukaryote
Promoter
3- PolyA
S CHUNG CHO VCT S CHUNG CHO VCT
CHUYN GEN THC VT CHUYN GEN THC VT
ATG TAG
Cu trc cua vcto
chuyn gen
Vng gn enyme
gii hn (MCS)
Gen khng
khng sinh
Vi khun
im ti ban
Vi khun
Prokaryote
Promoter
Gen c bi n n p
Eukaryote
Promoter
Vng 3 cha tn hiu
polyadenine ho
Gen chi th|
chn lc
Eukaryote
Promoter
3- PolyA
S CHUNG CHO VCT S CHUNG CHO VCT
CHUYN GEN THC VT CHUYN GEN THC VT
ATG TAG
2. C mt don ADN kt thc (terminator). y l don ADN cn thit cho
s kt thc cua qu trnh phin m v giai phng phn tu mARN khoi si
khun ADN. on kt ny thung c mt trt t tn hiu polyadenin, nhu
AATTAA ho}c AACCAA, gip cho phn tu mARN bn vng hon.
. Mt don ADN cha cc trt t nucleotid d}c trung cho s nhn bit
cua cc enyme gii hn, thung k hiu l MCS (multi-cloning sites).
Nh don ADN ngui ta c th dng enym gii hn d}c hiu d ct v ti
t hp phn tu ADN cua vcto vi don ADN mang gen cn bin np.
3. Mt don ADN ch|u
trch nhim cho qu trnh
ti ban (replication im
ti ban) thung c ngun
gc vi khun, nhu ColE1.
Cu trc cua vcto
chuyn gen
Vng gn enyme
gii hn (MCS)
Gen khng
khng sinh
Vi khun
im ti ban
Vi khun
Prokaryote
Promoter
Gen c bi n n p
Eukaryote
Promoter
Vng 3 cha tn hiu
polyadenine ho
Gen chi th|
chn lc
Eukaryote
Promoter
3- PolyA
S CHUNG CHO VCT S CHUNG CHO VCT
CHUYN GEN THC VT CHUYN GEN THC VT
ATG TAG
. Cc gen chn lc (selectable marker genes) v gen chi th| (reporter genes).
Cc gen ny thung tng hp nn cc phn tu protein gip phn bit cc t
bo d duc bin np khoi cc t bo khng duc bin np (gen chn lc),
ho}c ghi nhn s hot dng cua gen bin np (gen chi th|).
Cu trc cua vcto
chuyn gen
Vng gn enyme
gii hn (MCS)
Gen khng
khng sinh
Vi khun
im ti ban
Vi khun
Prokaryote
Promoter
Gen c bi n n p
Eukaryote
Promoter
Vng 3 cha tn hiu
polyadenine ho
Gen chi th|
chn lc
Eukaryote
Promoter
3- PolyA
S CHUNG CHO VCT S CHUNG CHO VCT
CHUYN GEN THC VT CHUYN GEN THC VT
ATG TAG
Cc gen chn lc thung c tnh tri, thng thung c ngun gc t vi
khun nhung hot dng cua chng duc kim sot boi cc promoter kiu
Eukaryote.
Cc gen chn lc ph bin gm:
Cc gen khng khng sinh nhu kanamycin (neomycin
$hos$hotransReraseSn$tTT), hygromycin (hygromycin $hos$hostransReraseS
h$t), streptomycin (stre$tomycin $hos$hotransReraUa),...
Cc gen khng cht dit co, nhu glyphosate ho}c bialaphos
(phosphinothricin, BASTA): gen 3ar c ngun gc vi khun m ho cho
enyme lm bt hot BASTA l phosphinothricin acetyltranseraa (PAT).
Cc gen khc (gen $mi phosphate manose isomerase- chuyn ho
manose thnh glucose. Nh vy, cy c th sng trn mi trung khng c
glucose (manose l nhn t chn lc)
GII THIU VE GEN CHN LC (selectable marker gene)
BIN NP GEN
BIN NP GEN
THC VT
THC VT
Cc nhn t chn lc Cc nhn t chn lc
-
Kanamycin Kanamycin
-
Hygromycin Hygromycin
-
Basta Basta
-
Manose Manose
GEN CH TH (reporter gene)
Cc gen gen chi th| l cc gen c trch nhim thng bo l gen cn
bin np d gn vo h gen thc vt v bt du hot dng hay chua.
Cc gen chi th| thung duc dng bao gm:
- galactosidase
- glucuronidase (GUS)
Neomycin-phosphatase (NPT)
Chloramphenicol-acetyl transerase (CAT)
Alkaline phosphatase
Lucierase
Protein pht hunh quang anh lc GP
(green luorescence protein) v cc dn ut cua n.
Protein chuyn trng thi nng lung cng huong pht hunh
quang RET (luorescent resonance energy transer)
Lactose Galactose + Glucose
- galactosidase
-D-glucuronide + H2O Axit glucuronic + Glucose
- glucuronidase
X-Gal
-Brom--chlor-3-idolyl--D-
galactoside

-

g
a
l
a
c
t
o
s
i
d
a
s
e
-Brom--chlor-indigo
X-Gluc
-Brom--chlor-3-idolyl--D-
glucuronid

-

g
l
u
c
u
r
o
n
i
d
a
s
e
-Brom--chlor-indigo
Khng mu
Mu anh sm

- galactosidase & - glucuronidase

Su dng gen chi th| gus
Vector chuyn gen
NGUYN TAC
S biu hin
ca gen gus
m hoa! m l
"# phi ng
non $%&c
chuyn gen
Su dng gen chi th| Lucierase
Nguyn tc
'uci(e)a*e l# m+t lo,i p)otein
c- t).ng l%&ng /0 12a pht
)a nh *ng b%3c *-ng 4/0
nm 5m#u "#ng-6anh l7c89
'# gen $%&c pht hin "#
tch )a t: lo#i cn t)ng
Photynis pyralis.
Su dng gen chi th| Lucierase
Su dng gen chi th| GP (green luorescene protein)
;<= l# m+t lo,i p)otein c-
t).ng l%&ng 30 12a pht )a
nh *ng b%3c *-ng 4>? nm
5m#u 6anh l7c *ng89
'# gen $%&c pht hin "#
tch )a t: lo#i *a Aequorea
victoria9
Su dng gen chi th| GP (green luorescene protein)
thc vt
CHUT THI NGHIM
CC VCT MANG GEN BIN NP C CHUYN VO T BO
THC VT NH TH NO?
1. 1. Phuong php chuyn gen gin tip Phuong php chuyn gen gin tip
- Thng qua vi khun - Thng qua vi khun %gro3acterium tumeRaciens V %F rhiUogens %gro3acterium tumeRaciens V %F rhiUogens
2. 2. Cc phuong php chuyn gen trc tip Cc phuong php chuyn gen trc tip
- Phuong php dng sng bn gen (particle inlow gun / - Phuong php dng sng bn gen (particle inlow gun /
bombardment) bombardment)
- Phuong php chuyn gen nh ung din (electroporation) - Phuong php chuyn gen nh ung din (electroporation)
- Phuong php chuyn gen nh PEG (polyethylene glycol) - Phuong php chuyn gen nh PEG (polyethylene glycol)
- Phuong php vi tim (microinjection) - Phuong php vi tim (microinjection)
Cc buc thc hin phuong php chuyn gen nh Cc buc thc hin phuong php chuyn gen nh %gro3acterium %gro3acterium
1. Thit k vcto mang gen bin np 1. Thit k vcto mang gen bin np
2. Nhn (tch dng cloning) vcto nh vi khun 2. Nhn (tch dng cloning) vcto nh vi khun WF co;i WF co;i
3. Chuyn vcto mang gen bin np t vi khun 3. Chuyn vcto mang gen bin np t vi khun WF co;i WF co;i sang sang %gro3acterium %gro3acterium
. Ly nhim . Ly nhim %gro3acterium %gro3acterium mang vcto cha gen bin np vi t bo / m mang vcto cha gen bin np vi t bo / m
thc vt d tin hnh qu trnh chuyn gen bin np sang m / t bo dch thc vt d tin hnh qu trnh chuyn gen bin np sang m / t bo dch
. Chn lc cc t bo / m d duc bin np thnh cng . Chn lc cc t bo / m d duc bin np thnh cng
6. Ti sinh m / t bo d duc bin np thnh cng thnh cy bin np 6. Ti sinh m / t bo d duc bin np thnh cng thnh cy bin np
hon chinh (v dnh gi s biu hin cua gen bin np) hon chinh (v dnh gi s biu hin cua gen bin np)
Phuong php chuyn gen nh Phuong php chuyn gen nh %gro3acterium %gro3acterium
- Vi khun %F tumeRaciens khng san ut duc mt s acid
amin nhu octopin, nopaline.
tn ti, n chuyn gen cua n vo thc vt, thc vt san
ut cc cht cn thit, vi khun phn hu cc cht ny d
ly nng lung.
Trong qu trinh cng sinh ny, vi khun cng chuyn cc
gen tng hp cc cht kch thch sinh truong lm cc t bo
nhim vi khun phn chia v t chc, gy pht sinh khi u
o thc vt.
Vi khun %F tumeRaciens v hin tung khi u o thc vt
Ban cht phn tu cua qu trnh pht sinh khi u do vi khun %gro3acterium tumerRaciens gy
ra o thc vt: trong qu trnh nhim khun o vng t bo b| thuong cua cy cc t bo b|
thuong tit ra hp cht thu ht cc t bo vi khun %gro3acterium tumerRaciens. Cc t bo vi
khun khng thm nhp vo t bo thc vt m chi chuyn np vo chng mt loi plasmid
d}c hiu gy nn tnh trng khi u - l plasmid Ti (tumor inducing).
Ti plasmid mang cc gen tng hp IAA, cc amino acide nhu octopine hay nopalin d gip cho vi
khun pht trin.
Con ngui li dng tnh cht ny cua %grgo3acterium d chuyn gen vo t bo thc vt.
Phuong php chuyn gen nh Phuong php chuyn gen nh %gro3acterium %gro3acterium
Gii thiu v vi khun %gro3acterium tumeRaciens
%F tumeRaciens
Ti
plasmid
T-ADN
H gen
nhn VK
S ly
nhim t bo
thc vt v
truyn T-
ADN
ADN nhn
TBTV
T-ADN
C 3 nhn t lin quan dn s to khi u cua Ti-plasmid
T-DNA duc chuyn vo t bo chu o dng mt nhn t di dng
Cc gen vng vir (virulence) l noi c cc gen san ut cc protein cn cho
s bin dng t bo thc vt d Ti-plasmid c th thm nhp.
Cc gen khc c trong nhim sc th cua Agro3acterrium ch|u trch nhim
lin kt t bo vi khun vi m thc vt
Phuong php chuyn gen nh Phuong php chuyn gen nh %gro3acterium %gro3acterium
Cu trc cua Ti-plasmid v T-ADN
%F tumeRaciens
Ti
plasmid
ADN
nhn
VK
Vng gy khi u (Onc) Vng gy khi u (Onc)
T-ADN
Vng phn
giai nopaline
Vng ti ban Vng ti ban
Vng tip hp plasmid Vng tip hp plasmid
Vng Vng
gy dc gy dc
vir vir
A
B
C
D
pTiC8
(Nopaline)
Ti plasmid l mt phn tu ADN vng tuong di ln, gm 10.000-200.000 c}p nucleotit. Tuy
nhin chi c mt don gm 20.000-30.000 c}p nucleotit c kha nng thm nhp vo b genom cua
t bo chu, d l don T-ADN. T-ADN c cha cc gen gy ra s sinh truong v diu khin cua
cc t bo thc vt. d l cc gen m ho auin v cytokinin gy ra s sinh san v t chc cua t
bo b| nhim.
Nhng enym ch|u trch nhim chuyn np don T-ADN t plasmid vo trong genom cua t bo
chu duc m ho boi cc don vir (virulence)
Phuong php chuyn gen nh Phuong php chuyn gen nh %gro3acterium %gro3acterium
Cu trc cua Ti-plasmid v T-ADN
Cu trc
T- ADN
TGGCGGATATATATGTGGTGTAAAC TGACAGGATATATGGCGGGTAAAC
Tms 1 Tmr Tms 2
nos
LB RB
Vng gy khi u (Onc) Vng gy khi u (Onc)
Cc Ti - plasmid cai bin duc ct bo cc don gen gy pht trin khi u, chi d li
don vir v phn T-ADN ti thiu mang dim ghp gen. Loi vector ny dang duc
su dng rt hiu qua cho vic dua ADN l vo t bo thc vt o ca cc loi cy mt
v hai l mm.
Ngui ta dua gen cn chuyn vo Ti plasmid, sau d dua vo %gro3acterium
Dng agrobacterium ly nhim m thc vt. Theo d}c tnh cua mnh, vi khun
chuyn cc gen vo t bo thc vt.
Nhu vy vic chuyn gen d duc hon thnh
Nhim v cua ngui thc nghim l ti sinh t bo hay m thnh cy, sau d kim
tra s c m}t cua gen chuyn o cy ti sinh.
u tin, l s hnh thnh mt vt dt (nick) ti v| tr vng bin phai RB cua don T-
ADN. on gen ny sau d duc tch ra khoi plasmid nh s hnh thnh mt don
dt th hai. on dt ny thung khng c d|nh di vi cc plasmid khc nhau.
Trong khi cc at nucleic trng to ra trn plasmid duc lp dy theo nguyn tc b
tr cua cc at nucleic th don T-ADN duc tch ra duc bm boi mt protein bm
si don (SSBP - single strand binding protein). Nh phc hp vi SSBP ny m don
gen T-ADN duc chuyn vo nhim sc th cy chu theo mt co ch dn nay chua
bit r.
Ti
plasmid
Vng Vir Vng Vir
T-ADN T-ADN
Vt dt 1
Vt dt 2
SSPB SSPB
Chuyn vo t
bo thc vt
S kt hp cua gen bin
np vi gen nhn thc vt
Phuong php chuyn gen nh Phuong php chuyn gen nh %gro3acterium %gro3acterium
Qu trnh chuyn T-ADN vo t bo thc
vt
Thit k vcto Ti plasmid mang gen bin np
Phuong php bin np nh vi khun Phuong php bin np nh vi khun %gro3acterium %gro3acterium
ADN nhn VK
Enym
gii hn A
Enym
gii hn A
XE 3o cho
gen 3iEn n$ H ADN
gen nhn
mang gen
3iEn n$
:en 3iEn n$
Y;asmi< trng
4Z =c ;oi
3> c,c gen gy
khi u S [nc9
Ti plasmid mang gen bin np
Qu trnh chuyn gen vo t bo thc vt v ti sinh cy
Phuong php bin np nh vi khun Phuong php bin np nh vi khun %gro3acterium %gro3acterium
Ti plasmid mang gen bin np
T bo thc vt
ADN
nhn
:en
chuyn
n$
QU TRINH
BIN NP
T bo mang gen chuyn np phn chia
2y chuyn gen 2y chuyn gen
1. S ban sao cua gen bin np duc
chuyn vo t bo thc vt thp. Do vy,
giam ti thiu s khng biu hin cua gen
duc chuyn, tng kha nng chuyn gen
bn vng, hiu qua chuyn gen cao.
2. Trnh duc s hnh thnh cua cc cy
chuyn gen kham.
3. K thut don gian, d thc hin
. Khng di hoi thit b| dt tin
Nhuc dim u dim
Phuong php ny duc su
dng thnh cng o nhiu cy
hai l mm. Nhung hiu qua
chuyn gen o cc cy mt l
mm cn thp.
Trong khi nhiu cy mt l
mm l nhng cy luong
thc quan trng nhu la,
ng, la m ...
Phuong php bin np nh vi khun Phuong php bin np nh vi khun %gro3acterium %gro3acterium
Phuong php bin np gen bng sng bn gen Phuong php bin np gen bng sng bn gen
LUNG KHI LUNG KHI
HELI HELI
LUNG KHI LUNG KHI
HELI HELI
Mng n
Mng mang
Ht mang ADN
Mng dng
Bung chn khng
M th c v t
Mi trung nui cy
m TV
Phuong php bin np gen bng sng bn gen Phuong php bin np gen bng sng bn gen
=)e**u)e gauge
2i*1 @ith 2AB-
coated pa)ticle*
Stop plate
Sample goe* he)e
Vacuum line
;a*
line
Cuptu)e di*1
Vacuum chambe)
Nhuc dim u dim
D9 E- th p d7ng "3i hFu hGt cc lo,i m! tG b#o9
Hu t)Inh chuyn gen nhanh! $Jn giKn "L mMt 1N
thuOt9
09 E- th 6P lQ m+t l%&ng mRu l3n t)ong thSi gian
ngTn9
39 Ec "UctJ mang gen ti tV h&p c- cWu t,o $Jn
giKn9 Xhng $Yi hZi cWu t)[c gen 1iu \-B2A nh%
t)%Sng h&p chuyn gen b]ng Agrobacterium.
^9 Eh_ cFn m+t l%&ng nhZ pla*mid B2A9
49 `iu hin t,m thSi ca gen biGn n,p c- th auan
*t thWy t)ong "Yng "#i ng#y *au biGn n,p9
D9 AhiLu bKn *ao ca gen biGn n,p
c- th $%&c chuyn "#o tG b#o
cng l[c! gby 1h- 1hcn cho "ic
phbn tch biu hin ca gen! dRn
$Gn * biu hin ca cc gen
1hng bLn "dng9
09 eiu auK chuyn gen thWp9
39 fYi hZi thiGt bg $Tt tiLn 51hng
phKi =\A n#o chng c- th $Fu t%89
Ehi ph "Ot t% $Tt 5h,t "#ng!
"ol()am! 9998
Phuong php bin np gen bng sng bn gen Phuong php bin np gen bng sng bn gen
Phuong php bin np gen bng ung din Phuong php bin np gen bng ung din
Phuong php chuyn gen nh ung din thung duc su
dng cho vic chuyn gen vo cc t bo trn
(protoplast).
T bo trn l t bo d duc loi bo thnh t bo bng
vic u l vi cc enym (cellulase, pectinase) d thu
duc cu trc t bo chi cn cht nguyn sinh v mng
nguyn sinh cht.
Ngui ta thung su dng dng din c hiu din th cao
pht ung trong thi gian cc ngn (khoang - 6/1000
giy) d to ra trn b m}t t bo trn tm thi cc l c
dung knh khoang 30 m, m qua d cc ADN bin
np c th thm nhp vo t bo.
T bo thc vt c vo enlulo gi cho t bo c hnh
dng nht d|nh. Cc hp cht pectin nm trong vo lm
nhim v kt gn cc t bo vi nhau

T bo TV T bo trn
pectinase
cellulase
My xung i n
Phuong php bin np gen bng ung din Phuong php bin np gen bng ung din
Hiu qua chuyn gen cao,
n d|nh, d}c bit di vi cc
gen don.
Nhuc dim u dim
H thng ti sinh cua cc t bo
trn cua nhiu loi cn g}p kh
khn.
Cho dn nay, nhn chung cn
chua duc p dng nhiu. Mi
duc su dng trong cc nghin
cu tm thi cua gen.
Phuong php bin np gen bng ung din Phuong php bin np gen bng ung din
Phuong php bin np gen nh PEG (polyethylene glycol) Phuong php bin np gen nh PEG (polyethylene glycol)
Cng ging nhu phuong php chuyn gen
bng ung din, phuong php chuyn gen nh
PEG thung duc su dng d chuyn gen vo
cc t bo trn.
nng d cao, PEG lm ADN cn bin np
khng cn o trng thi ho tan na m kt dnh
li trn mng sinh cht. Sau d, bng cch loi
bo PEG v u l nng d cao cua Ca2 ho}c o
d pH cao, ADN bin np s duc chuyn np
vo trong t bo protoplast.
ADN plasmid
PEG
Hiu qua chuyn
gen cao, n d|nh nu
qu trnh bin np
thnh cng.
Khng di hoi thit
b| dt tin
Nhuc dim u dim
i
Qu trnh bin np kh diu khin.
S ban copy cua gen trong t bo bin
np c th ln.
i
Tn s bin np thnh cng bin
dng gia cc th nghim.
i
D dn dn hin tung dung hp cua
cc t bo trn, gy kh khn cho
vic phn tch biu hin cua gen.
i
i hoi mt h thng ti sinh t bo
trn, vn cn g}p kh khn o nhiu
loi cy trng.
Phuong php bin np gen nh PEG (polyethylene glycol) Phuong php bin np gen nh PEG (polyethylene glycol)
Phuong php chuyn gen bng vi tim (microinjection) Phuong php chuyn gen bng vi tim (microinjection)
Phuong php chuyn gen nh vi tim l phuong php su dng cc thit b|
hin vi v my vi nhu dng d chuyn gen trc tip vo t bo. n nay,
cc t bo d duc su dng phuong php ny d chuyn gen gm cc t
bo protoplast, cc t bo tin phi cua hp tu hay ht phn
+ Lung ADN duc bin np l
tu v c d|nh.
+ ADN duc dua vo dng v| tr
mong mun, thm ch vo nhn
t bo.
+ C th p dng di vi cc t
bo c kch thuc nho b, nhu
ht phn, phi non m cc k
thut khc khng th thc hin
duc.
Nhuc dim u dim
+ Mi ln bin np chi dua duc ADN
vo mt t bo duy nht, do vy dy l
mt phuong php tn nhiu thi gian
v cng sc.
+ Chi c th thc hin boi cc k thut
vin c k nng cao.
+ i hoi thit b| tuong di dt tin.
Phuong php chuyn gen bng vi tim (microinjection) Phuong php chuyn gen bng vi tim (microinjection)
Cc phuong php chuyn gen khc Cc phuong php chuyn gen khc
+ Ngoi cc phuong php trn dy, ngui ta cn su dng
phuong php siu m (ultrasonication). Nhung ni chung,
dn nay phuong php ny duc su dng hn ch v mi dng
o mc phng th nghim.
+ Trong mt s trung hp ngui ta c th kt hp cc
phuong php vi nhau. Chng hn trong bin np gen o ng
ngui ta su dng kt hp phuong php sng bn gen d gy
tn thuong t bo bin np truc khi chuyn np gen bng
%gro3acterium.
L la v m thc vt
duc chuyn gen chi th|
GUS
Cy chuyn gen trn
mi trung chn lc
theo tnh khng
hygromycin
Cy cc chuyn gen trn mi trung chn lc
XU HNG P DNG CC PHNG PHP BIN NP GEN
Phuong php i tung p dng
Chuyn gen thng
qua vi khun
Agrobacterium
p dng di vi cc loi cy hai l mm, c h thng ti sinh
tt v p dng cho cc phn m d phn ho
Chuyn gen bng
sng bn gen
Khi p dng cho cc loi cy 1 l mm cn cc chung
Agrobacterium c kha nng bin np cao, ho}c kt hp mt s
k thut lm tng tn s bin np (phi hp sng bn gen,
v.v)
Phuong php ung
din v PEG
p dng di vi cc loi cy c h thng ti sinh tt. C th
chuyn gen vo cc loi m, t bo khc nhau (phi truong
thnh, phi non, t bo m so, )
Phuong php vi tim p dng di vi cc loi cy m m vic nui cy co th c th
to ra qun th cc t bo trn c tnh ton nng cao.
Phuong php siu
m
p dng cho trung hp cc t bo c kch thuc hin vi, ho}c
khi cn dua ADN vo v| tr chnh c trong t bo.
~S kin chuyn gen
(Transormation Events)
Events
i
j+t tG b#o $Jn *k phbn chia "#
phbn h-a th#nh m+t cby ho#n ch_nh9
Xhi nim le"entl th%Sng $%&c
dng $ phbn bit cc gimng cby
t)nng t,o )a b]ng cng ngh gen9
j+t oe"entp l# * chqn "#o ca m+t
gen cFn chuyn c7 th "#o m+t "g t)
$Mc t)%ng t)rn m+t AS\9 j+t 1hi
m+t oe"entp $%&c t,o )a t)ong m+t tG
b#o $Jn! n- $%&c duy t)I t: tG b#o
n#y *ang tG b#o 1hc 1hi nhism *Tc
th t nhbn lrn "# cc tG b#o phbn
chia ti *inh th#nh cby ho#n ch_nh
A single cell will divide
and differentiate into an
entire plant.
i
Mt s kin ~event sau d duc truyn t b m
sang con chu qua cc t bo giao tu mang mt ban
copy thng tin di truyn cua b m. Cui cng, cc s
kin ny kt thc o trn cnh dng cua nng dn v
chng duc truyn qua qu trnh lai to ho}c san
ut ht.
3 yu t d phn bit cc ~event l:
GF :en no Z =c chPn vo \
HF :en =c chPn vo v+ tr0 c] th no tr(n '^X 4;ocus9 \
_F `ao nhi(u 3?n sao ca gen Z =c chPn vo ;ocus K\
i
Hnh anh dui dy m ta mt t bo duc chuyn gen khi gen m nhp vo
nhn v chn vo NST. V| tr chn vo trn NST l khng th don truc. S
lung ban chn vo khi su dng %gro3acterium duc d don l khoangt 1-2
trong khi su dng sng bn gen l nhiu hon.
Hon na t bo duc chuyn nh Agrobacterium thung cao hon sng bn
gen. V th phuong php chuyn gen thng qua Agrobacterium thung duc
la chn d su dng.

Cc Events mong mun thung l him
i
Cc s kin mong mun l cc s kin c mang gen duc chuyn
duc chn vo mt phn cua NST cho php s th hin cua gen d m
khng anh huong g dn cc gen khc cua cy trng (gen c sn).
i
Tuy nhin hu ht cc s kin du b| loi bo ho}c do thai trong qu
trnh pht trin cua cc ging cy trng ho}c cy lai. Mt t l ln cc
s kin duc to ra bng cng ngh gen thm ch chi dng li o mc
d phng th nghim hay nh knh m thi. Cc s kin khc duc
loi bo sm trong qu trnh lai to hay cc giai don dnh gi trn
dng rung.

i
i khi, trong qu trnh chuyn
gen, gen duc chuyn dy mt gen
khc ra v n chim v| tr cua gen
ny. S thc l khi mt gen duc
chn vo ADN cua NST theo cch
tuong t nhu chng ta chen ngang
khi p hng o siu th|.ADN cua
NST b| ct dt v gen cn chuyn
chn vo d. Mt hay nhiu ban
copy bao gm cc gen chi th| s
chn vo v| tr ny.
i
M}c d vy, mt vn d c th ay
ra l nu gen mi t n chn vo
gia gen c sn. Trong trung hp
ny, m ha ADN cua gen c sn
s b| thay di v gen ny s khng
th m ha cho ARN thng tin
chnh c.
A new transgene inserts
into the chromosome
without replacing any of
the resident DNA. This
transgene is inserting into
the middle of a resident
photosynthetic enzyme
gene. This will disrupt the
resident gene preventing
its expression and
possibly causing a
reduction in yield potential
or even death to the plant.
i
C th so snh cc s kin nhu khi chng ta thm mt ch vo gia
mt t v t ny khng cn nghia na. Nu gen ny l cn thit cho s
sinh truong v pht trin, cy s b| cht. Nu gen d quan trng di vi
mt tnh trng, v d nhu s pht trin cua r trong diu kin dt c
pH thp, dt bin s khng th duc nhn bit cho dn khi cy duc
d}t trong cc diu kin ny. Chnh v th cn phai loi bo cc dt bin
c hi trong qu trnh chn lc.
i
Mt kha nng khc l cy duc chuyn gen nhung mc th hin cua
gen ny li rt thp. NST c cha mt s don gi l 'junk DNA'. Cc
don ny c th m ha cho cc gen khng su dng thung uyn ho}c
don ADN khng r mc dch. Khi gen cn chuyn chn vo cc v| tr
ny, s th hin cua chng thung l rt thp ho}c thm ch khng tn
ti. V vy,cc cy chuyn gen cng cn phai duc chn lc d th hin
duc dng mc d. S biu hin thp cua gen cng c th ay ra khi
nu m di truyn cua n b| thay di ho}c b| can thip trong cc buc
chuyn gen.
Tm tt cc s kin c kha nng ay ra trong qu trnh chuyn gen
Dng Event Kt qua S loi bo cc Event
1) Gen cn chuyn duc chn vo
trong 1 phn cua NST m c cha 1
gen cn thit cho s sng v pht trin
cua t bo vn nm sn trn d
("resident gene~)
"Knocks out" gen sn c trn
NST v t bo b| cht ho}c khng
phn chia
Qua qu trnh nui cy m sau chuyn
gen cc t bo ny s t cht.
2) Gen cn chuyn chn vo mt phn
cua NST v khng cho php bt k s
biu hin no cua gen
Cy ti sinh t s kin ny c th
cha gen nhung khng bao gi
biu hin
Qua qu trnh chn lc c th tch cc
cy khng biu hin gen cn chuyn m}c
d qua phn tch ADN vn tn ti gen d.
3) Gen cn chuyn chn vo v| tr mt
gen c sn vn cn thit cho s sinh
san (reproduction).
Cy chuyn gen c th tro thnh
bt th
Boi cc nh di truyn trong giai don
sm cua qu trnh t th ho}c lai li
) Gen cn chuyn chn vo NST,
duc th hin v truyn qua di sau
nhung b| cm ("silenced" ) o th h
sau d
Th h sau cua cy duc chuyn
gen khng biu hin duc gen d
chuyn
Boi cc nh chn to ging o cc giai
don sm cua qu trnh t th ho}c lai
li
) Gen cn chuyn chn vo mt phn
cua NST m ti vng ny cc gen
thung khng hot dng ho}c c s
thay di trong trnh t cua don DNA
duc chn vo.
S th hin ay ra nhung khng
thch hp d diu khin cc d}c
tnh mong mun.
uc pht hin trong qu trnh dnh gi
trn dng boi cc nh chn to ging hay
nng hc
6) Gen cn chuyn chn vo mt gen
sn c vn diu khin s pht trin
thc vt mt cch rt nhy cam. Iu
ny s anh huong dn cc d}c dim
nng hc v nng sut.
Cy trng trong trung hp ny
c th c tt ca cc d}c tnh mong
mun nhung cng c kha nng
lm anh huong (giam) nng sut.
uc pht hin trong qu trnh dnh gi
trn dng boi cc nh chn to ging hay
nng hc
hay nng dn.
Gene Silencing



During transformation, one gene copy may insert into a
chromosome, as in the picture above, or several gene copies
may insert, as in the picture below. Events where multiple gene
copies have inserted are suspected to have a higher probability
of gene silencing.
Hiu qua kinh t hi cua cc linh vc khc nhau cua
cng ngh sinh hc
Eng ngh tG b#o ^ ^
Eh.n gimng b]ng ch_
thg phbn tP
t t
Vi nhbn gimng 00 00
=hbn b-n *inh h.c t t
EhG phum th[ y 3 3
Ec lvnh "c 1hc 4 4
Sinh vt bin di gen Sinh vt bin di gen
(K thut di truyn) (K thut di truyn)
199 199
ource! "ayer#Tasch, $%%& ource! "ayer#Tasch, $%%&
Th h cy trng bin di gen th nht d thuong
mi ha:
1. Ng khng su
2. u tuong khng su
3. Ng khng thuc dit co
. u tuong khng thuc dit co
. Ng khng su khng thuc dit co
6. u tuong khng su khng thuc dit co
7. Cai du, khng su, khng thuc dit co
8. Bng khng su, khng thuc dit co
MT S THNH TU BIN NP GEN THC VT
MT S THNH TU BIN NP GEN THC VT
Cy du tuong chuyn gen
+ Xhng *bu 5`t8
+ Xhng *bu bnh 5in*ect )e*i*tance8
+ ;-p phFn l#m giKm l%&ng thumc t):
*bu cFn *P d7ng 5bKo " mi t)%Sng "#
giKm chi ph *Kn 6uWt8
+ \hay $Vi th#nh phFn a6t bUo
+ '#m thay $Vi th#nh phFn "# gi t)g
dinh d%wng
Cy ng chuyn gen
+ Khng bnh
+ Khng su bnh (Bt)
+ Khng mt sau thu hoch
(CM, serpin)
+ Chn sm
+ Rt ngn thi gian trng trt
+ Khng thuc dit co
MT S THNH TU BIN NP GEN THC VT
Cy c chua chuyn gen:
+ Gen ko di thi gian chn
+ Lm chm qu trnh chn nhn qua
+ Tng cung cht lung qua v ko
di thi gian bao quan sau thu hoch
+ Gen khng bnh virt: Khng vi virt
CMV
+ Gp phn giam lung thuc tr su su dng
trong qu trnh trng trt
Cy c chua chuyn gen khng virt
(bn tri) trong khi cy khng duc
chuyn gen khng mn cam vi virt
CMV - Cucumber mosaic Virus - khi
trng trn dng rung (bn phai)
MT S THNH TU BIN NP GEN THC VT
Cy bng chuyn gen khng su Bt
+ Mang gen khng su Bt
+ Gp phn hn ch su dng thuc tr su
Bng chuyn gen Bt khng su bnh (phai) v bng
khng chuyn gen mn cam vi su bnh (tri)
MT S THNH TU BIN NP GEN THC VT
Ging la duc chuyn gen tng
hp -carotene (ging la vng).
Sau qu trnh tiu ho, -carotene
duc chuyn ho thnh vitamin A.
Khoang 120 triu tre em trn th gii b|
cc ri lon do thiu vitamin A. Ging
la vng mang li ~nim hy vng trong
vic bao v khoang 1 dn 2 triu bnh
nhn b| cc ri lon do thiu vitamin A.
MT S THNH TU BIN NP GEN THC VT MT S THNH TU BIN NP GEN THC VT
Cy du du (2arica $a$aya L.) duc chuyn gen khng
bnh virt (tri) v cy di chng (phai)
C kha nng khng virt CMV
uc dnh gi l gp phn vo vic phc hi nn
cng nghip san ut du du o Hawaii.
MT S THNH TU BIN NP GEN THC VT

Khng cc diu kin bt li phi

sinh hc
x
Khng m}n
x
Ch|u hn
x
Ch|u lnh

Khng cc diu kin bt li sinh

hc
x
Khng su
x
Khng virus
x
Khng nm
x
Khng bnh vi khun

Bt dc dc nhn

Khng thuc dit co

Tng thi gian bao quan

Tng dinh dung

Tng cung cht lung

- Thay di thnh phn du
- Tng cung cht lung g

San ut hot cht sinh hc

Cc d}c tnh dang duc nghin cu
Khng su, bnh Khng su, bnh
Cai tin cht Cai tin cht
lung, tng cung lung, tng cung
hm lung hm lung
vitamin, vi lung, vitamin, vi lung,
ait amin khng ait amin khng
thay th.. thay th..
Chng ch|u Chng ch|u
cc diu kin cc diu kin
bt li phi sinh bt li phi sinh
hc, m}n, hn, hc, m}n, hn,
lnh. lnh.
Khng thuc dit Khng thuc dit
co co
Nng sut cao Nng sut cao
Sourse: Norman Borglag, 200
C
C
'y bi(n )*i gen th( +, $-
'y bi(n )*i gen th( +, $-

Ng ch|u hn
USA
C tm khng su
India, Philippines, Bangladesh
La khng su
China, Iran
La tng cung vitamin,
vi lung
Philippines, India, Bangladesh,
and Indonesia
Hoa hng anh
Japan
Thc phm c hm lung dinh dung cai tin
Source: Grant (2009)
La vng 2
i 34 ug beta-ca)oteneyg)am
i eJn 3> lFn *o "3i l[a "#ng
D
C chua tm
i e#m l%&ng anthocyanin cao hJn bInh th%Sng
0>z
Mt s ging cy chuyn gen khc
Hn k!ng lm c y
n c m t
Gene: lachrymatory factor
synthase
"i ng c#n s m$mu n%
c& m l ng car'ten'id
ca'
Genes: DET1,
LeETR4, ACC oxidase
Ng! ca' (ysine
Gene: ZLKR!D"
) c p m k!ng c a c t g*y d
ng% c& m l ng axit 'mega +%
axit 'leic ca'
"enes: Ara"#, $AD#
j+t *m nghirn cu t,o gimng cby
t)nng biGn $Vi gen Vit Aam

La bin di gen c hm lung vitamin A cao
".o th/0ng
".o 12
Tr3n Th4 15c 6o7, 8i9n :5a ;<1:
8=>
< 1
D
E
? 2 6 =
@ A
:
A
http://www.agi.gov.vn http://www.agi.gov.vn

pPAM-VP2 vector
Chn lc cc dng chuyn gen su
dng Imaging PAM
Phn tch PCR
Phn tch Southern
&7ng 3Po ao;RRia chuyn gen VP2 nui
trong diu kin in vitro
`Po t5m ao;RRia
1'y xoan ta "elia azedarach chuyBn gen 2$% v7 gene C1N- cDi thiEn
+hD nFng sinh tr/Gng v7 chHt l/Ing gJ
u du chuyn gen khng
virus dm vng PRSV
Chuyn gen, chn lc v ti sinh
in(ected
\)nng cc dYng chuyn gen t)ong nh# l%3i
Ti#vector
attB1 attB2 attB2 attB1
Gene
CP
Gene
CP
Phn lp gen CP ca virus P!" v
thi#t $# vect%r &i'plas(i)
a * c
) e
Plant regenerati%n thr%ugh (ultiple'sh%%ts +r%( ,yg%tic e(*ry%s in three
an elite "ietna(ese c%tt%n -Gossypium hirsutum ./0 cultivar .12133,
"453P an) C116/
+
a * c
)
)
!%(atic e(*ry%genesis an) plant regenerati%n %+ c%tt%n -Gossypium
hirsutum ./0 cultivar !!378/ -a'c0
e + g h
8=> +hKng s'u
L(u tM phi9n
mN! ch4u h.n
CC AC IM U VIT CUA PHNG PHP BIN NP GEN THC CC AC IM U VIT CUA PHNG PHP BIN NP GEN THC
VT SO VI CC PHNG PHP CHN TO GING TRUYEN THNG VT SO VI CC PHNG PHP CHN TO GING TRUYEN THNG
- Qu trnh bin np gen thc hin trong phng th nghim, khng ph thuc
nhiu vo thi gian, khng gian, ma v, thi tit, v.v...
- Cc gen duc chuyn c tnh d}c th cao hon nhiu. Cc gen tuong ng chnh
c vi cc tnh trng mong mun duc la chn dng cho qu trnh bin np,
loi bo duc cc tnh trng khng mong mun.
- Qu trnh chuyn mt gen mong mun vo mt c th din ra nhanh hon nhiu.
Chi cn sau mt th h, tnh trng mong mun duc biu hin v thu nhn, trong
khi cc phuong php truyn thng thung cn nhiu th h.
- Mc d ~linh dng cua cc gen duc chuyn ln hon nhiu. Nhiu gen cua loi
ny c th duc chuyn vo loi khc v biu hin thnh cc tnh trng hon ton
mi. Diu ny khng th thc hin duc khi su dng cc phuong php to ging
truyn thng
- Tng nng sut nng nghip v giam chi ph san ut. iu ny d}c bit
dng khi cc gen duc dng cho bin np vo thc vt thung l cc gen
lm tng nng sut, giam thi gian trng trt, tng kha nng trao di cht,
khng cc loi su bnh. Nh vy, thi gian, cng lao dng, vt tu ho cht
cn cho qu trnh san ut nng nghip s duc giam di nhiu dn dn s
giam chi ph san ut v hn ch nhim mi trung.
CC AC IM U VIT CUA PHNG PHP BIN NP GEN THC CC AC IM U VIT CUA PHNG PHP BIN NP GEN THC
VT SO VI CC PHNG PHP CHN TO GING TRUYEN THNG VT SO VI CC PHNG PHP CHN TO GING TRUYEN THNG