I H C THI

NGUYN

TRNG I HC NNG

LM

N N G V N H (Ch bin) - N G XUN B N H

NH XUT B N N N G N G H I P

I H C THI

NGUYN

TRNG I HC NNG

LM

N N G V N H (Ch bin)
NG XUN BNH

NH XUT BN NNG
H N I - 2008

NGHIP

Cng
tin pht

ngh

thng

tin v cng ngh sinh hc (CNSH)

trin hu ht cc quc gia trn th gii. N hiu

to ln ca CNSH
CNSH

l hai lnh

vc cng ngh

hc gi d bo sc

i vi ci to sinh gii, trong c bn thn con ngi.

u
mnh

Vai tr

ca

i vi pht trin kinh t v y hc, i vi an sinh x hi v h sinh thi trn

tri

t ang ngy cng mnh

trnh sng. N hiu

ln cng vi s tch ly nh anh

ca tri thc v cc

ch ng

qu

cu hi ln lin quan n pht trin cng ngh sinh hc ang t

ra:

. Lch s hnh thnh v pht trin ca cng ngh sinh hc?

2.

Cng ngh sinh hc l g? Cng ngh sinh hc bao gm nhng

hc v cng ngh
no?

ngnh

3.

Quan h gia CNSH

4.

Cc kin thc c bn ca

5.

Vai tr khm ph bn cht s sng v kh nng ci to sinh gii ca

6.

Vai tr ca cng ngh sinh hc i vi pht trin kinh t- x hi v ngc li

vai tr ca kinh t- x hi v chnh sch quc gia di vi pht trin
CNSH?

7.

Hin trng v d bo pht trin cng ngh sinh hc trn th gii v nc

8.

nh hng pht trin cng ngh sinh hc nc

vi cc ngnh khoa hc t nhin, khoa hc x hi

cng ngh sinh

khoa
khc?

hc?
CNSH?

ta?

ta?

Mt trong cc nhim v ca quyn sch ny l t ra nhng cu hi, ng thi cng

sinh vin v ngi c tm ra cu tr li cc cu hi mang tnh tng qut
Hin nay, nn kinh t ton cu ang tri qua nhng thch
s pht trin nng ca cc nn kinh t, nht l Trung
nc ng Nam trong c Vit Nam,
lng. Kh nng suy kit cc ngun
30 nm ti. Cc quc

trn.

thc ngy cng ln. l

Quc, An , Brasi,

Nga v cc

i hi s gia tng khai thc v s dng

than , du m, kh t l nhn tin trong

gia ng dn nh Trung

Quc,

nng
khong

An , Nga ang dn tr

thnh

cc nn kinh t ln vi mc tiu dng nng lng v sn xut kh thi nh knh tng

nh M hin nay. Tnh hnh c th y nhanh

qu trnh khng

mi trng ton cu, c th dn n chin tranh v nhng khong

cc ngun

nng lng.

tn ti? Pht

Vn mi trng

trin hay khng

pht

trin?

trin no c xem l bn vng trn phm

CNSH

n u trong gii quyt cc vn

nh hng
rt nhiu

v xu th pht

sinh thi tr thnh

Pht trin phi

khong
khng

nng lng

gian sinh tn v

vn tn ti hay

nh th no? Gii php

vi ton cu v cho tng quc gia? Vai tr

khng
pht
ca

trn?

trin ca cng ngh sinh hc trong

vo vic tr li cho cc cu hi trn

tng lai ph

thuc

y.

Vai tr ca CNSH

trong gii quyt cc vn d an sinh x hi, vn nng lng

ti

sinh v sinh thi bn vng l rt to ln. Cc nh khoa hc tr v sinh vin trong lnh
sinh hc - nng nghip,

bn cnh vic phi c nhng kin thc chuyn

mn su, phi

tm nhn rng ln hn, bao qut hn v nh hng tt hn trong hot ng thc

Cun

"Cng

ngh

sinh hc i cng"

kin thc c bn v CN SH

nhm gp phn

v vai tr ca CNSH

Gio trnh c phn cng vit nh

t n

Ng Xun Bnh - tham gia vit cc chng

lo v l i .

ngh sinh hc l mt lnh

mi hng ngy nn quyn

hon
thin.
Chng

vc khoa

sch ny kh trnh

ti xin trn trng cm

mi.

lo.

hc rng

khi

ln, ang pht

c thiu

st, mong

n.
Nhm tc gi

cc

sau:

Nng Vnh - Ch bin, vit cc chng

V cng

cung cp cho hc vin

trong giai on

tin.

i vi thc tin, ng thi gi mt

cc nh hng v gii php pht trin ca CNSH

vc

trin

bn c gp

Chng

KHI NIM, LCH s V BIN CHNG PHT TRIN

CNG N G H SINH

CA

H C

1.1. K H I N I M C N G N G H S I N H H C V Q U A N H C A C N G N G H S I N H
H C V I C C N G N H K H O A H C T N H I N V X H I K H C
C n g n g h sinh hc (CNSH) l tp hp c c n g n h khoa hc - c n g n g h v s sng,
bao g m sinh hc p h n t, k thut gen, c n g n g h t b o , c n g n g h v i sinh, c n g n g h
protein v enzym..., n g h i n cu v khai t h c c c q u trnh sinh hc, hot n g sng ca
v i sinh vt, t b o n g v thc vt, c t h sng v m phng c c q u trnh sinh hc
quy m sn x u t c n g nghip.
H i n nay C N S H bao g m c c l o i c n g n g h v k thut ch lc n h c n g n g h
gen, c n g ngh v i sinh vt, c n g n g h t b o , c n g n g h enzym, sinh hc p h n t, sinh
hoa hc, l sinh hc, i u k h i n hc, sinh t i n hc, genom hc (genomics), protein hc
(proteomics), nano sinh hc... T u y v y , C N S H k h n g ch n thun l khoa hc sinh
hc, n l s k t hp gia c c n g u y n l sinh hc v i c n g n g h i p sinh hc.
CNSH l s k t hp gia c c n g u y n l khoa hc v c n g n g h trong m t h thng
trong c c c t h sng, m hoc t b o , c quan t, c c p h n t hoc q u trnh sng
c khai t h c s d n g v o sn x u t quy m c n g nghip.
M i t hp sn x u t C N S H bao g m trong n c c y u t k h c nhau:
- C c n g u y n l khoa hc v c n g ngh.
- H thng c c t h i t b hoc d y c h u y n c n g n g h ( c c t h i t b n g h i n cu khoa
hc v k i m sot cht lng, c c thit b sn x u t n h c c bioreactor, h t h n g ln men
vi sinh vt, h i u k h i n t n g ) .
- Cc h thng sng c c mc t chc k h c nhau t p h n t, c u trc trn p h n
t, t b o quan, t b o , m , c quan, c t h sng, qun th....
- Cc q u t r n h c h u y n i vt cht v n n g lng n h m to ra c c sn p h m mong
m u n (v d," sn x u t c c cht hot tnh sinh hc n h h o r m o n sinh trng, intereron,
k h n g sinh, insulin) trn quy m c n g nghip.
S ra i v p h t t r i n ca C N S H c quan h cht c h v i s p h t t r i n ca n h i u
n g n h khoa hc v c n g ngh k h c nhau. C c m i quan h c t h h i n c c s
1.1 v 1.2.

Tch ly tri thc ton

hc.vt l, hoa hc,
cc khoa hc v tri t,
v khng gian...

Cuc cch mng cng

nghip ln 1,2 v 3:
Cng nghip in t,
ch to my, thng tin,
tin hc, vt liu...

Cng ngh sinh hc

Cng nghip sinh hc

S LI.

D bo

Tch t tri thc v

phng tin nghin cu
khoa hc sinh hc v
khoa hc t nhin khc

Cuc cch mng cng nghip

sinh hc the k XXI

Cng ngh sinh hc ra i v pht trin nh mt tt yu lch

Quan h gia c c n g n h khoa hc t h h i n quan h b i n chng gia c c h n h thc

v n n g k h c nhau ca v t cht v quan h gia v n n g x h i v p h t t r i n khoa hc
c n g ngh. H i u b i t c c c m i quan h logic , c h n g ta m i c c n c t chc
h thng khoa hc v x y dng c c c h n g t r n h n g h i n cu p h t t r i n p h h p .
V n n g sinh hc l t n g hoa ca n h i u h n h thc v n n g k h c nhau ca v t cht
v l k t q u ca v n n g v t l, hoa hc, v n n g v tr n i c h u n g . K h i l o i n g i c
k h n n g thao t c d i t r u y n mc p h n t v tr t h n h " n g s n g t o m i " trong sinh
g i i th v n n g sinh hc c n x y ra trong m i quan h n g y c n g cht c h v i v n
ng x h i .
V t n h cht phc tp ca v n n g sinh hc, n n C N S H c h c t h h n h t h n h v
p h t t r i n k h i c c n g n h khoa hc k h c t n q u t r n h t c h l y t r i thc v p h n g
t i n mc " t i hn" n o .
C N S H n g y nay a n g n g t r n n n tng ca c c khoa h c t n h i n k h c nhau v
t h n h tu ca c c cuc c c h m n g c n g n g h i p .
D o v y , m t n h C N S H chc chn p h i c t r i thc khoa hc t n h i n v k i n thc
c n g ngh. M t quc gia m u n p h t t r i n c n g n g h i p C N S H v n g chc p h i k t hp
c s p h t t r i n c c khoa hc sinh hc v i c n g n g h t i n hc, i n t, v t l i u m i v
phi c t m n h n t o n c u v t m n h n quc gia v k i n h t , sinh t h i v m i t r n g .
1.2. CC NGNH KHOA HC V s SNG V Mi QUAN H BIN CHNG
M i quan h gia c c khoa hc v s sng v c c n g n h khoa hc t n h i n k h c v i
C N S H c m t v n tt trn s* 1.2. S n y c n g t h h i n quan h gia t r i t hc
c c khoa hc x h i n h c h n h tr, lch s, k i n h t v i khoa hc c n g n g h n i chung v
v i C N S H n i r i n g . T r n s n y , n g n h khoa hc c p h n t h n h 3 l o i h n h hay 3
thang bc k h c nhau trong m i quan h v i thc t i n :
- C c n g n h khoa hc c bn;
6

- C c n g n h khoa hc c n g ngh: l sn p h m ca khoa hc c bn k t hp v i c n g

n g h i p v k i n h t ;
- C c n g n h khoa hc ng dng: l sn p h m ca 2 n g n h t r n , n ch ra l n h vc
i sng m trong c c n g n h khoa hc v c n g n g h c ng d n g .

Cng ngh
sinh hc

Cc ngnh khoa hc
v s sng

Cc ngnh khoa
hc c bn

Cc ngnh khoa
hc - cng ngh

CNSH ng
dng

Di truyn hc

K thut di
truyn

1. CNSH y hc:
- Cng ngh
vacxin v ch
phm dc
2. CNSH nong
nghip:
- Cng ngh vi
nhn ging
thc vt
- Cng ngh
nhn bn ng
vt
- Cng ngh to
ging mi
3. CNSH mi
trng-sinh thi
v nng lng ti
sinh

Sinh phn t
Sinh hoa hc
Sinh l hc
Ton sinh hc
iu khin hc
sinh hc
Genom hc
Protein hc
T bo hc
M phi hc
Sinh thi hc
Bnh hc
Min dch hc

Cng ngh t
bo
Cng ngh
erym v
protein
Cng ngh vi
sinh
Cng ngh ln
men
Cng ngh
vacxin v ch
phm dc

Cc ngnh khoa
hc cng ngh ^

S 1.2. Quan h bin chng gia cc ngnh

Khoa hc Cng ngh

Cng ngh in
t
Cng ngh lazer
Nng lng
nguyn t
Cng ngh vt
liu
T ng hoa
Cng ngh ch
to my

Cc ngnh
khoa hc t
nhin khc

Cc ngnh khoa
hc c bn

- Ton hc
- Vt l hc
- Hoa hc
- Tin hc
- Thin vn
hc
- Cc ngnh
khoa hc v
tri t.

Cng ngh
thng tin
Cng ngh nano

khoa hc cng

ngh

TRIT HC

KINH T HC

KHOA HC CHNH TR V LCH s

- Th gii quan: Quan h gia

vt cht v tinh thn, vt cht v
vn ng, vt cht v nng lng,
tinh thn l mt thuc tnh ca vt
cht c t chc cao. Lun v con
ngi nh mt sn phm ca t
nhin v d bo vai tr ca CNSH
i vi tin hoa ca chnh con
ngi.
- Php bin chng: Cc quy lut
ph bin ca vn ng vt
cht.vn ng x hi v t duy.
Tng hoa cc mi quan h tng"
tc gia cc s vt v hin tng
trong v tr. Vn ng sinh hc v
vn ng v tr.
- Nhn thc lun: Cc phng
php nhn thc c bn. Thc
nghim khoa hc v thc tin
khch quan l tiu chun ca
chn l. Vai tr ca khoa hc
trong vic pht hin cc quy lut
vn ng, t gii thch v d
bo pht trin CNSH trn nn d
liu hin c. Quan im h thng
trong xem xt s vt.

- Cc quy lut pht trin x hi,

quan h gia ngi vi ngui v
x hi loi ngi vi thin nhin.
- Quan h gia ch x hi,
tnh tin phong ca ch x
hi v pht trin khoa hc cng
ngh. Lut php v chnh sch
pht trin tin tin quyt nh
thnh tu pht trin KHCN v
gio dc cc quc gia.
- Tc ng ca khoa hc
cng ngh ln con ngi:
Phng tin sn xut v
phng tin sng - mi trng
sng - nng sut lao ng - cc
phng tin nhn thc ca con
ngi. CNSH v tc ng ca
n ln bn thn con ngi: tim
nng tr tu v kh nng sng
to, sc khoe, tui th...
- Vai tr quyt nh ca KHCN
i vi vic gii quyt cc vn
pht trin kinh t-x hi.
- Quan im lch s trong nhn
thc lun khoa hc.

- Quan h gia kinh t

v khoa hc cng ngh.
- n by kinh t i
vi pht trin khoa hc
cng ngh.
- n by khoa hc
cng ngh i vi pht
trin kinh t.
- D bo kinh t, th
trng, d bo tng lai
quy nh nh hng
pht trin KHCN cc
quc gia. D bo th
trng Nng nghip-Y
dc, d bo mi
trng sinh thi quy
nh nh hng nghin
cu CNSH.
- D bo vai tr quyt
nh ca CNSH oi vi
pht trin nng- lm ng nghip - y dc v
mi trng sinh thi.
Chnh sch quc gia v
pht trin CNSH.

1.2.1. C c n g n h k h o a hc c b n v s sng
- Di truyn hc

Sinh hoa hc

- Sinh hc p h n t

Bnh hc

- T b o hc

Phng sinh hc

- M p h i hc

i u k h i n hc sinh hc

- Sinh l hc

T o n sinh hc

- M i n dch hc v c c khoa hc k h c

G e n o m hc

- Sinh thi hc

Protein hc

- D i t r u y n hc l thuyt

Nano sinh hc
Vv...

C c n g n h khoa hc c bn v s sng m i nht h i n nay c t h n i l genom hc

(genomics) bao g m c u trc, t chc v chc n n g ca t n g gen, n h m gen, h t h n g

gen, c c h i u h n h gen. R i n g genom n g i c cha n k h o n g 30.000 gen k h c

nhau, m i gen l i c c c h i u h n h phc tp v n h i u c h i u . M i gen l i cha trong
n h n g t r m n h n g n g h n c c nucleotid v i c c t r n h t c t h . M i gen v n h m
gen l i k m theo n h i u k h i n phc tp trong m i quan h v i c c giai o n pht
t r i n k h c nhau ca t b o , ca c t h v m i trng sng. D o t n h phc tp n h v y n n
genomics chc chn s p h i tr t h n h m t khoa hc c l p . T n g t n h v y s l
protein hc, t o n sinh hc, i u k h i n hc sinh hc v nano sinh hc... s d n tr t h n h
c c n g n h hc c h u y n su.
1.2.2. C c n g n h k h o a h c c n g n g h v s s n g - C n g n g h s i n h h c
S p h t t r i n ca c c khoa
n g n h Khoa

hc - Cng

hc c bn v s sng v s d n n s ra i ca c c

ngh ca C N S H , trong bao g m :

- K ngh d i t r u y n (genetic engineering).

- C n g ngh t b o (cell biotechnology).
- C n g n g h enzym v protein (protein and enzym technology).
- C n g n g h v i sinh (microbiotechnology).
- C n g n g h l n men ( e r m e n t a t i o n technology).
- C n g n g h vacxin v c h p h m dc (vacxin and biopharmaceutical technology)
v c c khoa hc c n g n g h k h c .
1.2.3. C c n g n h c n g n g h s i n h hc n g d n g
S p h t t r i n ca c c khoa hc - c n g ngh sinh hc s to ra s b n g n ca c c
lnh vc k h c nhau ca khoa hc CNSH

ng dng,

bao g m :

- CNSH y hc: c n g n g h vacxin v c h p h m dc, l i u p h p gen,...

- C N S H N n g L m N g n g h i p : c n g n g h v i n h n g i n g thc v t , c n g n g h
n h n bn n g v t , C N S H to g i n g m i , C N S H bo v sc khoe v d i n h d n g c y
trng, vt n u i .
- CNSH M i trng - Sinh thi
- CNSH v n n g lng sinh hc
- CNSH v v t l i u m i
1.2.4. C c n g n h k h o a hc c n g n g h t n h i n
S p h t t r i n ca c c n g n h khoa hc t n h i n n h t o n hc, v t l hc, hoa hc, tin
hc, t h i n v n hc v c c n g n h khoa hc v tri t to ra h n g lot c c n g n h Khoa
hc - C n g n g h ( K H C N ) t n h i n nh:
- C n g ngh c h to m y
- C n g n g h i n t
- C n g n g h lazer

- N n g lng n g u y n t
- C n g n g h vt l i u
- T n g hoa
- C n g ngh t h n g t i n
- C n g ngh nano v n h i u n g n h k h c
C c n g n h K H C N n y to ra c c cuc c c h m n g c n g n g h i p , l m b i n i t n
gc c c p h n g t i n sn xut v p h n g t i n nhn thc ca loi n g i , c n g n h l m
thay i ton b i sng x h i . C h n g to ra nhng c n g c k h n g t h t h i u i v i s
p h t t r i n ca c c n g n h khoa hc v s sng v to t i n cho s ra i ca C N S H
c u i t h k th X X .
D o v y , m u n p h t t r i n C N S H p h i h i u r n n tng, c c bc i lch s k h n g t h
t h i u c, xu h n g p h t t r i n ca CNSH trong m i quan h b i n chng v i k i n h t x h i . C n g ngh sinh hc l q u trnh tng hp v c h u y n hoa c c t r i thc v k thut
v s sng t h n h c n g nghip sinh hc. Cng

nghip

sinh

hc

l q u t r n h sn x u t

h n g lot, quy m l n c c sn p h m sinh hc bao g m c c hot cht sinh hc, v t l i u

sinh hc, sinh k h i t b o n g , thc vt v v i sinh vt ( c n g n g h i p ln men), c c c h
p h m sinh hc ( k h n g sinh, c c enzym, protein, peptid hot t n h n h i n t e r e r o n , i n s u l i n ,
hormon sinh trng v.v...), c c vacxin v thuc cha bnh v c c c t h sng ( h n g t r m
t r i u cy trng, vt n u i ) .
Phi ni t h m rng s p h t t r i n ca K H C N n i chung v C N S H n i r i n g k h n g t h
t c h r i s p h t t r i n ca t tng trit hc v c h n h tr. N g y nay k h n g m t khoa hc
gia c h n c h n h n o c t h ng n g o i k i n h t , c h n h tr v lch s p h t t r i n ca q u c gia.
Khoa hc - C n g ngh , a n g v s l n b y v i nht i v i v n m i n h n h n l o i
v t i n b ca m i d n tc. K h o a hc - C n g ngh c n l g i i p h p duy n h t cu v n
n h n l o i a n g ri v o cuc khng khong m i trng sinh t h i v n n g l n g . M t
k h c , s p h t t r i n ca m i m t n g n h khoa hc c n g n g h m i u c n n n h n g
quyt s c h mnh m v i t m n h n rng l n ca c c l n h t quc gia.
S tr tr trong p h t t r i n khoa hc - c n g ngh v g i o dc ca m t d n tc trc
ht thuc v t r c h n h i m ca lnh o cao nht ca quc gia . T chc h thng,
nhng c ch, c h n h s c h qun l khoa hc, c n g n g h ca nc ta h i n nay h t sc lc
hu, t h i u c n c khoa hc v t m n h n c h i n lc. l n g u y n n h n quan t r n g l m
khoa hc nc n h a n g b h n g ht, t h i u tnh k tha, t n h lch s trong s p h t t r i n
i ln. N a n g b t h i u ht tnh h thng, tnh n n g n g , t n h mc tiu v n h h n g
c h i n lc.
Nc ta v hu ht c c nc a n g p h t t r i n h i n nay c h u , c h u Phi v M L a
tinh u n g h o n n , lc h u do i sau m t l n trong cuc c c h m n g c n g n g h i p l n
th nht v l n th hai. D o v y , c h n g ta p h i tranh t h c h i k h n g tt h u trong
cuc c c h mng c n g nghip sinh hc l n n y .
10

1.3. L C H S C N G N G H S I N H H C V V N B N C H T C A s S N G
1.3.1. T m t t c c g i a i o n p h t t r i n c a c n g n g h s i n h h c v c c s n
c h n h ca

phm

CNSH

a) C N S H h n h t h n h v ng d n g trc k h i c k h i n i m C N S H
T t h i xa x a , con n g i bit:
- Sn x u t r u , bia, c c c h p h m sa, m t s l o i nc g i i k h t .
- Sn x u t c c axit v dung m i h u c.
- C n g n g h i p k h n g sinh v i c c n i ln men h n g t r m n g n lt.
b) G i a i o n t 1 9 6 0 -

1975

- C n g n g h t b o thc vt (nui cy m ) bt u sn x u t h n g t r i u c y g i n g
sch bnh trong i u k i n n h n to.
- Sn x u t axit amin, axit glutamic, c c enzym, k h n g sinh bng c n g n g h i p l n
men sinh k h i k h n g l . Sn x u t c c cht b sung dinh d n g cho n g i , c h n n u i ,
n g u y n l i u c n g n g h i p dc v cht t y ra.
c) G i a i o n t sau

1975

- H n h t h n h k thut A D N ti t hp, k h i n i m C N S H v c n g n g h i p sinh hc tr

nn ph bin.
- Thuc cha b n h v p h n g bnh cho n g u i v gia s c : insulin, i n t e r e r o n , vacxin...
to ra n h c c k thut ti t hp A D N v c h u y n gen
- C c cht i u t i t sinh trng, p h t t r i n v sinh sn c y trng, v t n u i c b i t l
hormon sinh t r n g v c c cht hot tnh sinh hc k h c .
- C c b kt v p h n g p h p chn o n bnh.
- C y t r u y n hp t v c c k thut n h n ging v n h n bn n g vt.
- Cht b sung thc n gia s c : vitamin, axit amin, sinh k h i t b o , n m men.
- N h n g i n g c y sch bnh quy m l n .
1.3.2. N h n g p h t m i n h c n b n c a n h n l o i t r o n g l n h v c s i n h h c d n n s
ra i ca
1.3.2.1.

CNSH
Pht

minh

ra t bo, qu trnh phn

bo v phn

hoa c

quan

- N m 1665 Robert Hooke quan st thy t b o sng d i k n h h i n v i v a ra

khi

nim t bo

"Cell".

- Anton Van Leuvven Hoek (1632-1723), thit k knh h i n v i khuch i c 270 l n ,

ln u tin quan st thy v i khun, t b o tinh trng trong tinh dch n g i v ng vt.
- Matthias Schleiden v Theodore Schvvann x n g hc thuyt c bn ca sinh hc
g i l Hc thuyt

t bo nm

1838:

li

+ M i c t h sng c c u to b i m t hoc n h i u t b o .
+ T b o l n v c u t r c v chc n n g c bn ca c t h sng, l h n h thc n h
nht ca s sng.
+ T b o ch c to ra t t b o t n t i trc .
- Oscar H e r t w i g (1875) chng m i n h bng quan st trn k n h h i n v i r n g s t h thai
l do s hp nht ca n h n tinh t r n g v n h n trng.
- Hermann p, Schneider F. A . v Butschli o . m t c h n h x c q u t r n h p h n chia
t b o , n m 1883, W i l h e l m Roux l n u tin l g i i v s g i m p h n trong p h n b o
g i m n h i m c quan sinh dc.
1.3.2.2. Pht minh s pht trin ca c th sng l kt qu ca s phn hoa ca
t bo
C u i t h k X V I I v u t h k X V I I I , n g i ta ngh r n g trong t i n h t r n g cha
ng ton b n g i trng t h n h d i dng thu n h . S p h t t r i n c coi n h s p h n g
i n g i n cua h n h n h n . C c h h n h t h n h c c con vt t p h i t r t h n h i tng
tranh l u n khoa hc trong sut t h k X V I I I cho n c u i t h k X I X . C hai t r n g
p h i i l p nhau:
i v i nhng n g i theo thuyt t i n to, con vt m i h n h t h n h h o n t o n v i y
c c c qan c m t dng thu n h trong trng (hoc trong t i n h t r n g ) , i v i
nhng n g i ng h thuyt "vi ng vt" (khong t h i k 1651 - 1700). N h T n h i n
hc T h y S Charles Bonnet (1720 - 1793) quan st c h i n tng t r i n h sn con r p
(trong t h n g h i m , m t con rp ci c c l p m t c c h c n t h n sinh ra 95 r p con m
k h n g cn rp c), t n g i ta k t l u n rng nhng con v t m i t n t i h o n
chnh trong trng ca con vt c i .
i v i nhng n g i theo thuyt

biu sinh: trng ban u k h n g c m t d u v t t

chc n o v p h i c h n h t h n h d n d n bng q u t r n h x y d n g c s n g g p
"ging" ca c cha v m . N h n g n g i theo thuyt b i u sinh n h B u f f o n v Maupertuis
lu rng c cha v m u g p phn h n h t h n h con v t m i (s quan st con la chng
t i u ) .
Pht trin phi

v phn

hoa t bo tr thnh

mt khoa

hc trong t r n h t hot

n g ca t h n g t i n d i t r u y n v i u k h i n hot hoa ca gen trong q u t r n h p h t t r i n c

th c ngha quan trng. Khoa hc v i u k h i n hc sinh hc ra i sau n y p h t t r i n
song song v i c c t h n h tu v h i u h n h hot hoa ca gen.
1.3.2.3. Pht minh qu trnh tin hoa ca sinh gii v vt liu mang thng tn di
truyn
N m 1859, Charles D a r w i n - Cha ca hc thuyt t i n h a trong sinh hc - x u t
bn cun " N g u n gc ca c c loi to ra b i chn lc t n h i n " . Trong l n u t i n
a ra quan i m lch s ca p h t t r i n sinh g i i v l g i i s t i n hoa ca c c loi:
- S t i n h a trong sinh g i i x y ra n h d i t r u y n , b i n d v c h n l c t n h i n .

12

- M i m t loi u c t h c quan h m t t h i t v i c c loi k h c b i c n g c m t t

tin chung.
T i p sau , Gregor Mendel cha ca d i t r u y n hc p h t h i n c c quy l u t c
bn u tin ca d i truyn:
- C c tnh trng c d i truyn t t h h n y sang t h h kia theo c c quy lut thng k.
- T n h trng c x c nh b i c c y u t d i t r u y n (sau n y n g i ta g i l gen).
Oskar H e r t w i n g v Edouard Strasburger, trong nhng n m 1880 p h t h i n n h n
t b o l trung t m ca d i t r u y n . Cho t i m v t l i u d i t r u y n t v i k h u n cht v o v i
khun sng. Cht h a hc c h a b i t (c g i l y u t G r i f f i t h ) t m t n i v i k h u n g y
bnh b git cht t h m nhp v o m t n i v i k h u n sng k h n g c k h n n g g y bnh.
K t qu n i v i k h u n v n k h n g c k h n n g g y b n h n y c c h u y n h a t h n h
chng g y bnh. Sau n y , Osvvald A v e r y v n g n g h i p (1944) chng m i n h c vt
l i u d i truyn do G r i f f i t h t m ra hay
deoxyribonucleic ( A D N ) .

" y u t G r i f f i t h " c h n h l m t p h n t axit

1.3.2.4. Pht minh vai tr ca ADN i vi s sng

Ba c trng c bn ca s sng l sinh sn, trao i cht v t i n hoa. T n h cht ca
A D N quy nh c ba c t r n g c bn n y .
James Watson v Francis Crick (1953) p h t m i n h c u t r c si k p ca A D N . Si
x o n k p h n h t h n h theo n h lut Watson v Crick, trong : m t b a z trn m t si
A D N l u n l u n t n g n g hoc bt cp v i m t b a z t n g ng si kia. A d e n i n ch
g h p cp v i T h y m i n v Cytosin ch g h p cp v i Guanin. H q u ca nh lut g h p cp
n y l hai m c h ca v n g x o n k p b sung cho nhau n h c h a k h a v k h a . K h i hai
si t c h r i nhau, m i si n y l i c t h quyt nh v i c x y d n g m t m c h m i b
sung cho n d n n s n h n i ca A D N . N h v y c u t r c v t l ca p h n t A D N
g i i thch m t gen c t h t sao c h p c h n h x c n h t h n o m i l n t b o p h n chia.
- Cng v o n m 1953, G. G a m o w cho bit d i t r u y n tnh trng p h i t h h i n qua
tng hp c c protein trong t b o .
- Han Gobind Khorana v Marshall Nirenberg (1966 -1967) p h t m i n h m d i t r u y n .
T y " n h l u t t n g n g " gia c c n h m chp ba nucleotid (triplet hay c n c g i
l codons) ca A D N v c c axit amin ca c c protein c l m s n g t h o n t o n .
Trong :
+ M h n h hai si x o n k p ca A D N l g i i k h n n g sinh sn bng n h n i ca
A D N l c s l g i i h i n tng d i t r u y n tnh trng, sinh sn sinh g i i .
+ Trong q u t r n h sinh sn ca p h n t v t b o l i x y ra m t s sai st d n n
t b i n v ti t hp gen to ra t i n cho b i n d v t i n hoa.
1.3.2.5. Tng hp gen, k thut ADN ti t hp v thao tc di truyn mc phn
t v t bo
- N m 1960, Jacob. F. v M o n o J. ( g i i t h n g N o b e l n m 1964) l n u tin
p h t m i n h k h i n i m o p e r o n . Operon hay n h m chc n n g l n h m c c gen lin quan
13

v i nhau theo c c chc n n g h a hc nht nh trong m t c h u i c c p h n ng sinh h a .

Operon g m c :
Cc gen cu trc (structural genes): quy i n h c u t r c bc n h t hay t r n h t axit
amin ca enzym hoc protein.
Gen iu chnh (regulatory genes): i u k h i n hot hoa ca c c gen c u t r c .
P h t m i n h n y m ra c c n g h i n cu n g y c n g su v h i u h n h hot h a ca
gen trong t b o . Jacob F . v M o n o J. cng p h t m i n h vai t r ca A R N t h n g t i n
( m A R N ) trong c c h hot n g ca gen: gen ( A D N ) quy n h t r n h t nucleotid ca
A R N t h n g t i n , t i p n trnh t nucleotid trong A R N t h n g t i n l i quy n h t r n h t axit
amin trong p h n t protein. T n h c th ca protein l i quy n h c u t r c v m i q u
trnh hoa hc x y ra trong c t h sng.
N h v y , n h n l o i p h t m i n h c c c n g o n c b n trong d n g c h y ca t h n g
tin d i truyn: D N A - A R N - protein.
- N m 1967, A r t h u r Kornberg v cng s t n g hp c A D N ca m t virus
trong p h n g t h n g h i m .
- N m 1969, R. Edwards l n u tin t h thai t h n h c n g m t trng n g i t h t i n h
ngoi c th.
- N m 1970, Khorana H . G t h n h c n g t n g hp n h n to m t gen. C n g n m
n y , Hamiton v Smith t c h chit c enzym ct g i i h n Haemophilus

infuenzae

v x c nh c rng enzym n y ch ct A D N o n c t r n h t nucleotid c t h .

- Paul Berg v c n g s (1972) l nhng n g i u t i n s d n g e n z y m ct m
n h i m sc t h m c h v n g ca E. c o l i v ci v o m t A D N n g o i l a i ( A D N ca virus
g y bnh ung t h n g i ) , to ra p h n t A D N ti t hp u t i n . C n g t r n h n y c
nhn g i i t h n g Nobel n m 1980.
- N m 1973. S.'Cohen v H . Boyer h o n chnh p h n g p h p c h u y n gen n g o i l a i
v o c c v i k h u n , m u t h i i k thut d i t r u y n . N h v y , v i c n h n nhanh m t gen
v i s lng tuy c t h thc h i n c t h n g qua c h u y n gen v o v i k h u n ( k thut
n y g i l n h n d n g gen - gene cloning).
1.3.2.6. Pht minh mi v vai tr ca cc ARN
Sidney A l t m a n v Thomas Cech c trao g i i N o b e l n m 1989 do p h t h i n ra r n g
A R N c k h n n g hot n g n h m t cht x c tc (catalyst) v c c enzym c b n c h t
A R N c g i c c R i b o z y m . P h t m i n h n y m ra quan n i m h o n t o n m i v vai t r
ca A R N . Quan i m trc cho rng A R N ch n g vai t r trung gian gia A D N v
protein. C c n g h i n cu sau cho t h y A R N c t h t x c t c t sao c h p v t n g
hp c c p h n t A R N k h c . K h m p h d n n tng cho r n g A R N l v t l i u
d i t r u y n u tin trn tri t . H i n nay khoa hc k h n g n h r n g ribosomal R N A
x c tc to lin k t peptid gia c c axit amin trong q u trnh dch m . P h t h i n sau
14

c n cho t h y A R N k h n g ch n h cht x c t c , m c n n g vai t r quan trng h n na

trong i u k h i n b i u h i n gen:
- M t s lng l n c c p h n t A R N n h k h n g n g vai tr n h m d i truyn m lin
k t v i protein to ra cc phc hp g i l ribonucleoprotein. C c

ribonucleoprotein

nh

hng trc tip ln c c q u trnh p h i n m (transcription), dch m (translation), n h n bn

A D N v c u trc n h i m sc t h (v d g y x o n si n h i m sc th).
- T r o n g nhng n m u ca thp k 1980, n g i ta p h t h i n t h y c c p h n t
A R N n h (di k h o n g 100 nucleotid, sau n y c g i l c c A R N i ngha) trong v i
k h u n Escherichia

coli c t h b m v o c c trnh t t n g n g trong m A R N v c c h

phin m ( M i z u n o v cs, 1984; Nordstrom v Wagner, 1994). N g y nay, b i t khong

25 trng hp trnh t A R N i ngha (antisense A R N ) , c k h n n g t r i t tiu hot hoa
ca c c m A R N v i u t i t q u trnh dch m trong E.coli

(Gottesman, 2004). S i u

hoa q u trnh dch m b i antisense A R N c n g x y ra trong sinh v t n h n chun n h

c m i n h hoa n m 1993 cho t h y vai tr ca antisense A R N quy n h s p h t t r i n ca
tuyn trng Caenorhabditis

elegans

(Lee v cs, 1993; W i g h t m a n v cs, 1993).

H i n tng g y bt hot gen b i A R N i ngha, c k c h thc l n sau c

p h t h i n c c c t h sng n h n thc (antisense A R N ) .
Pht m i n h gn y nht, c coi l p h t m i n h l n nht ca khoa hc c u i t h k
X X l pht m i n h vai t r ca c c A R N ngn i v i i u k h i n hot hoa gen c c c t h
sng n h n thc. C c A R N ngn c k c h thc v o k h o n g 21-27

nucleotids g i l

s i A R N (short R N A ) hoc m i A R N (micro R N A ) c k h n n g b m v o c c trnh t tng

ng v i n trn c c si A R N (c t h l m A R N hoc A R N ca virus, c c l o i A R N
t h m nhp v o c t h v t b o theo c c con n g k h c nhau), l m cho c c si A R N
n y tr t h n h si k p m t o n ngn. C c enzym c t h (ch m i t h y c c t b o
n h n thc) c k h n n g n h n b i t c c trnh t k p ngn v ct si A R N l m i , g y
bt hot gen.
S bt hot ca gen c t h x y ra hai mc k h c nhau: B t hot gen giai o n
phin m (transcriptional gene silencing TGS) do methyl hoa c c t r n h t gen (Matzke
v cs,

1989;

Wassenegger v cs,

1994) v sau p h i n m (post-transcriptional gene

silencing PTGS) (Napoly v cs,1990; V a n der K r o l v cs, 1990; Smith v cs,1990; de

Carvalho v cs, 1992; van Blokland v cs, 1994). H i n tng bt hot gen sau p h i n m
(PTGS) c p h t h i n thc vt, n m men Neurospora

carassa,

tuyn trng,

r u i d m v n h i u n g vt k h c , k c con n g i . H a i n h khoa hc M Andrevv Fire

v Craig M e l l o c trao g i i t h n g Nobel n m 2006 do c c n g l n p h t m i n h ra
c c h g y bt hot gen b i c c A R N ngn m 2 n g g i l A R N i ( A R N i v i t tt t ting
A n h R N A i n t e r e r e n c e ) . C n g ngh A R N i h i n nay c c c n h khoa hc v c n g
nghip sinh hc g i l c n g n g h y sc m n h , c k h n n g can t h i p h i u q u v o
n h i u l n h vc sinh hc, n n g nghip v y hc.
15

1.3.3. M t v i n g d n g c a

CNSH

- N m 1977, h n g Genetech to ra v i k h u n b i n i gen sn x u t c h o r m o n

n g i - somatostatin phc v mc c h c n g nghip. N m 1978 h n g n y l i sn x u t
c insulin bng k thut t n g t.
- N m 1980, h n g Biogen sn x u t c i n t e r e r o n ca bch c u n g i bng k
thut d i truyn. I n t e r e r o n n g i l m t hp cht t h i n n h i n k h n g virus c k h n n g
l m t n g sc k h n g bnh n g i v c tnh k h n g ung th.
- N m 1984, m t tr s sinh u tin p h n g t h n g h i m ca c p h t t r i n t m t
p h i c n g lnh n h i u t h n g .
- N m 1985, n g i ta thu c k t q u tuyt v i k h i i u tr b n h ung t h bng
inteleukin-2, m t protein c sn x u t trn quy m c n g n g h i p n h c c v i k h u n
c h u y n gen.
- N m 1986, s. Willedson t h n g b o thc h i n t h n h c n g s sinh sn v t n h ca
cu c i .
- N m 1990, l n u tin n g i ta thc h i n ca i u tr b n h do gen t r n m t b n h
n h n gi 4 t u i b t h i u n n g m i n dch. P h n g p h p cha bnh bng l i u p h p gen
t ra y t r i n vng v i vic a v o c c t b o ca c t h n g i b n h m t gen b n h
t h n g cha bnh do t h i u gen n y .
- H i n nay, c c c h n g trnh n g h i n cu l n mang t m c t o n n h n l o i ra i
v a n g c t r i n khai, nh:
- N m 1991, D n Quc t v genom n g i (human genom project) bt u c
t r i n khai v i mc tiu n n m 2005 c t o n b trnh t genom ca n g i v i k h o n g
3.000 t r i u cp nucleotid trong giai o n 15 n m . N m 1994, m t n h m n g h i n cu
P h p p h n g t h n g h i m G i - n - t h n g ( G n t h o n ) c n g b bn u tin v h gen
n g i v p h b i n bn cho t o n t h g i i . V n n m 2004 t r n 21.000 gen
n g i c m hoa (c trnh t);
- C h n g trnh genom ca m t s c c sinh v t quan trng;
- C h n g trnh chc n n g genom (functional genomics) v i s tham gia ca n h i u
p h n g p h p c n g n g h m i n h so s n h Genom (comperative genomics), protein hc
(proteomics), c n g n g h nano sinh hc...
N h v y , n c u i thin n i n k X X sau c n g n g u y n n h n l o i chng n h n g x c
nh c bn cht sinh hoa ca gen m c n t c h c gen, c h u y n n t c t h sng
n y sang c t h sng k h c m t c c h k h d d n g , c c t r n h t nucleotid ca h gen
v a n g tng bc k h m p h chc n n g ca tng gen, ca h thng gen v hc c c h i u
k h i n c hot n g ca c h n g . T h k X X I s l t h k ca k thut d i t r u y n v
CNSH s n g to ra nhng dng thc sng m i , c c thit b v t l i u m i , c c p h n g thc
h i n i ca y hc tr l i u v l i ch ca n h n l o i .
16

1.4. K H M P H B N C H T C A s S N G V V A I T R C H O C A G E N
T t h i t i n s n h n l o i b m nh b i nhng c u h i g n n h v n h hng:
1. S sng do u m c ?
2. T h n o l s sng? C h t sng k h c v i cht k h n g sng c h n o ?
3. T i sao sinh g i i l i a dng v hi hoa n n h vy?
4. Y u t n o q u y t nh s t n t i , v n n g v p h t t r i n ca sinh gii?
5. Con n g i c t h c i to sinh g i i k h n g v bng c c h n o ?
T t c nhng c u h i gn n h k h n g c c u tr l i cho n k h i p h t h i n ra gen,
p h n t A D N v hot tnh sinh hc ca n .
C h n g ta c th p h n bit cht sng v cht k h n g sng bng 3 c tnh c bn di y:
- K h n n g sinh sn
- K h n n g trao i cht
- K h n n g d i t r u y n , b i n d v t i n hoa
Trong , nhng thuc tnh ca p h n t A D N quy n h c c c t n h c n bn trn
y ca c t h sng.
1.4.1. Khm ph bn cht ca s sng v kh nng ci to sinh gii bng cng ngh
sinh hc
Gen v c u t r c ca gen quy nh m i c tnh ca c t h sng v tnh a dng
phong p h ca h sinh t h i . M u n c i to sinh g i i phi t c n g trc t i p v o gen.
Sau n h n g p h t m i n h ca M e n d e l v M o r g a n , n g i ta k h n g n h : M i c
t n h ca c t h sng do gen quy nh. N h n g ch m i n n m 1944 n g i ta m i x c
n h c gen c h n h l c c p h n t deoxyribonucleic acid ( A D N ) . n n m

1953,

Watson v C r i c k p h t m i n h c u t r c h n h si g m hai c h u i x o n k p t n g n g
ca A D N . M i si A D N l m t p o l y m e r c to b i c c t r n h t sp x p k h c nhau
ca c c nucleotid. C b n l o i deoxyribonucleotid: adenin, guanin, cytosin v t h i m i n .
C ba nucleotid l i to ra m t t hp chp ba (triplet) hay c n g i l n v m d i
t r u y n (codon), b n nucleotid v i t hp chp ba s to ra 4 = 64 codon k h c nhau.
3

C h i u d i ca gen t h n g r t k h c nhau. N u t n h m t gen t r u n g b n h c t o b i

k h o n g 900 n u c l e o t i d = 300 codons. Theo l t h u y t v i 64 codon s to ra 64.300 c c
p h n t A D N hay c c gen k h c nhau.
V i 7 nt nhc, n h n l o i to ra h n g triu bi ca. V i 32 c h ci, c c n h v n
vit ra h n g t r i u c u n s c h . V i 64 codon c t h s n g to ra hng h sa s c c c u t r c
a d n g v c n g v t n ca p h n t A D N . i u g i i t h c h t i sao t h g i i sng ca ta
l i phong p h , a d n g v tuyt v i n n h v y . H n g t r i u c c loi sinh vt trn tri
t , trn n m t n g i h u n h k h n g ai ging ht ai. H y n h n k v o m t n g i , m t
n g i bn y u chng hn, c bit bao n h i u m u sc, bao n h i u c u t r c , bao n h i u tr
t u , n g n t v t m h n trong .

17

N h v y , tt c tnh a dng phong p h , t t c nhng v p t h i n t h n ca s sng

u do gen v c u trc ca gen quy nh. T r t t ca c c nucleotid quy n h c u t r c c
th ca gen. S lng a dng ca gen quy nh tnh a d n g ca sinh g i i .
1.4.2. Vai tr ca gen c th hin nh th no trong t bo sng?
C u trc c th ca gen l i quy nh c u trc c t h ca A R N t h n g t i n . n lt
A R N t h n g t i n l i quy nh c u trc c th ca c c protein. M i codon trong p h n t
A D N s thch ng v i m t p h n t axit amin trong m c h ca protein. T r t t ca 64
codon trong A D N s quy nh trt t sp x p ca cc axit a m i n ( g m trn 20 axit a m i n
k h c nhau) trong p h n t protein.
N g y nay n g i ta x c nh c trnh t c u trc nucleotid ca h n g t r m n g n
gen v trnh t axit amin trong p h n t ca h n g chc n g h n protein. T r o n g s c c
protein c tinh ch, c khong 90% protein l c c enzym, k h o n g 10% protein c n
l i l c c protein c u trc (thit k n n c c t chc m n g t b o sng v c t h sng) v
c c cht hot tnh sinh hc k h c nhau n h c c hormon, insulin, c c i n t e r e r o n . . . N h v y
trnh t axit amin c th l i quy nh tnh c t h ca c c enzym, trong m i m t
phn ng sinh hoa hc hoc m i bc trong q u trnh d n t r u y n i n t trong h thng
m n g t b o u c quy nh b i m t enzym hoc m t protein c u t r c . H n g n g h n
c c c h u i phn ng sinh h a hc trong m i t b o u do c c e n z y m hoc h thng
enzym quy nh. X t cho c n g gen v c c sn p h m trc t i p ca gen ( c c protein) quy
nh m i c u trc v q u trnh x y ra trong c t h sng.
Gen v h thng
c nm
trng

trong
sng,

mi

gen, enzym
quan

h bin

v h thng
chng

trong gen l cha khoa

phc

enzym,

protein

tp gia

v cc t chc

chng

ca vn ng sinh

vi nhau

sng,

v vi

tt
mi

hc.

N m 1955, l n u tin trt t ca c c nucleotid trong p h n t A D N ca m t con v i

k h u n c x c nh g m khong 1,8 t r i u cp nucleotid. N g y nay, n g i ta
khng nh trong m i v i k h u n ( t b o t i n n h n prokaryote) c c gen c sp x p trn
m t si A D N k h p kn. T r o n g trc k h u n n g r u t v i k c h thc k h o n g 1 - 2

um c

cha m t si A D N c di khong l m m g m t r n b n t r i u cp nucleotid hay t n g

n g v i khong trn 3000 gen k h c nhau. T r o n g c c sinh v t bc cao hem, trong t b o
eukaryotes, c c gen c sp x p theo trt t nht n h t r n si n h i m sc t h . T r o n g
m t t b o ca n g i c cha khong 30.000 gen k h c nhau.
M i gen trong t b o sng u c x p t theo m t trt t c quy n h v
hot hoa ca n c i u k h i n m t c c h c h n h x c . Thay i v tr ca gen, ct bt gen
hoc t h m c c gen, t h m c h thay i v tr ca ch m t o n n g n A D N hoc m t
nucleotid trong si A D N ca n h i m sc t h u c t h d n n n h n g b i n i phc t p
trong c th sng. H i n nay vic c h u y n gen v o c t h sng c n ph thuc n h i u v o
c c y u t n g u n h i n , nht l nh v ca gen l trn n h i m sc t h . n h v gen k h n g
ph hp cho b i u h i n gen d n n h i u q u c h u y n gen thp.
18

H i n nay c h n g trnh g i i m h gen (genom) v chc n n g h gen n g i v

a n g l m t c h n g trnh n g h i n cu l n nht t h k v i s tham gia ca c c siu c n g
k i n h t. C h n g trnh n y nhm v o vic x c n h c u t r c , t r n h t v chc n n g ca t t
c c c gen n g i .
Vi khong
ra bit bao nhiu
nhiu

loi sinh

mt triu cc gen khc

cc ti t hp khc
vt mi

nhau

nhau

tn ti trong
ca gen trong

t nhin,

s c th sng

to

t bo v s sng to ra

bao

na?

Trong thc t lng c c gen trong t b o ca m i loi c g i i hn b i s lng

n h i m sc t h nht nh v i c c trnh t k h n g h i m ngt ca c c gen trong m i n h i m sc
th. T i sao vy? T n h hp l trong t chc v hot n g ca h gen l c m t khoa hc
y b n. Cc khoa hc n h d i truyn hc, sinh hc p h n t i u k h i n hc sinh hc... s
phi l g i i cc b n . H i n t i c h n g ta c bit m i m t enzym (sn p h m ca gen)
cng ch c th hot n g trong h thng c c enzym, bi m t sn p h m sinh hoa t h n g
thng phi do nhiu enzym n g thi to ra (v d tng hp ra m t axit amin, p h n
g i i mt p h n t n g .. cn r t nhiu enzym - hay h enzym tham gia).
S sng n m trong trt t c u trc v i u h n h phc tp cht c h v i t n h k lut v
tnh t chc n g h i m ngt. T r o n g gen n m trong q u trnh i u k h i n v t i u k h i n ,
chng hn gen tng hp galactosidase ch hot n g k h i t h m n g lacto v o m i
trng n u i v i k h u n . K h i n g lacto b p h n g i i , c c gen hot n g c h m l i v sau
ngng hot n g n u sn p h m c u i c n g ca enzym b tch l y n mc b o hoa
hoc k h i trong m i t r n g lacto b p h n g i i h o n t o n . N h v y gen ch c t h t n t i
trong nhng t hp gen hp l v i s k i m sot ca m i t r n g sng v t i u chnh.
Kh nng sng to ra cc t hp gen mi l v cng v tn. Hng
chuyn

gen mi s c to ra. Nhng

lot cc c th

sng

qu trnh chn lc li v cng kht khe. Tin

hoa

l mt qu trnh o thi y bi kch t n s hon m hin ti ca s

sng.

K h n n g n h n i ca p h n t A D N , sinh sn v t i n hoa - l c tnh c bn

ca s sng. Chi p h i c p h n t A D N n h n l o i s tr t h n h "ch t h " ca s t i n hoa
sinh hc trn tri t . K h n n g n h n i ca A D N l i q u y t n h k h n n g n h n i ca
n h i m sc t h v k h n n g p h n chia ca t b o . N h v y m c hai q u trnh sinh sn:
sinh sn v tnh ( n h n bn t b o hoc c t h sng m i g i n g v i t b o v c t h m ) .
Sinh sn hu tnh c k t hp t b o sinh sn v genom ca hai c t h b v m . M t t
b o v i k h u n trong i u k i n p h hp s p h n chia ( n h n i ) c k h o n g 20 p h t m t
l n . Trong m t n i ln men v i sinh vt v i q u trnh ln men k o d i khong 3 n g y = 72
gi, m t t b o v i k h u n c th c n h n ln theo tnh t o n l thuyt t h n h 2

2 1 6

t bo

m i . N h vy m c c n g nghip ln men v i sinh vt v i c c n i ln men k h n g l h n g

t r i u lt sn x u t thuc k h n g sinh, m c h n h , bia r u , vacxin ti t hp... M t c y
phong lan n u i c y trong ng n g h i m c m i t h n g c t h n h n ln g p n m l n , m t
n m c t h n h n c trn m t t r i u c y phong lan con t m t c y ban u .
Q u trnh n h n g i n g n h v y l k t q u ca m t k h n n g c m t k h n g hai ca
sinh vt sng - k h n n g sinh sn ca p h n t A D N v t b o . N h n g ch m i n n m
19

1962 c tnh trn y ca t b o thc v t m i c ng d n g l n u t i n v o c n g

nghip n h n g i n g nhanh i n vitro.
Q u trnh n h n i ca p h n t A D N - to ra m t p h n t con g i n g h t p h n t m
ban u l bn cht ca h i n tng d i t r u y n . M c d v y , trong q u t r n h sao c h p v
n h n ln h n g t r i u l n , k h n g t h k h n g c sai st d n n s sai k h c trong c u t r c
p h n t ca gen, hoc thay i sp x p ca gen trn si A D N v trn n h i m sc t h . . .
d n n h i n tng b i n d . H i n tng b i n d t h n g t h n g x y ra t n s 10" - 10"
5

trong i u k i n t n h i n . Trong i u k i n t h n g h i m , k h i c tc n g ca c c c h t g y
t b i n hoc p h n g x , hoc trong n u i c y t b o trn k h i m n g t b o b p h n huy,
t n s b i n d c t h ln t i 10' hoc h n , ngha l c 100 c t h m i s x u t h i n c
2

m t c t h b i n d . N g y nay, k h n n g g y t b i n c h n g i n g n g y c n g c t n h
nh h n g v h i u q u cao. H n g lot c c chng v i sinh v t c n g n g h i p (sn x u t
k h n g sinh, axit glutamic...), c c ging c y trng m i c to ra bng t b i n .
d l nn tng ca chn

lc t nhin

v tin hoa ca sinh

Bin

gii.

1.5. CI TO DI TRUYN CC c TH SNG TRN c s SINH HC PHN

T V K T H U T G E N
T h i t k c c u trc v v tr k h n g gian ca gen, i u k h i n c hot hoa ca
n , con n g i c t h to ra h n g t r i u c c hp cht t h i n n h i n theo m u n , c i to c
sinh g i i v sa cha c c c khuyt tt d i t r u y n c h n h b n t h n con n g i . N h n g
CNSH n g y nay m i ch i c nhng bc u t i n trong cuc t r n g c h i n h c i to
vt cht sng. C c p h n g thc c b n trong c i to t h n g t i n d i t r u y n c t r n h b y
t m tt d i y :
/ . Thay i trnh t nucleotid

ca

gen

- Thay i trnh t gen c u t r c : T r n h t nucleotid trn gen thay i s l m thay i

t h n g t i n trn m A R N , d n n thay i hot t n h ca enzym v c c protein do gen n y
m hoa;
- Thay i c u trc ca gen i u h n h . C c gen i u h n h thay i d n n thay i
q u trnh dch m t c c gen c u trc d n n s thay i sinh tng hp protein v
lng: t n g hoc g i m , t h m c h l m ngng sinh tng hp.
2. Thay i v tr ca gen trong

genom

Thay i v tr ca gen trong genom: ti t hp trnh t ca c c gen t r n n h i m sc

t h v trong genom.
- Chuyn gen t v tr n y sang v tr kia trong p h m v i n h i m sc t h ;
- Chuyn gen gia c c n h i m sc t h trong c n g m t t b o : do trao i c h o
(crossing over), d i chuyn ca c c gen nhy (transposons).
3. Kim sot v thit k h iu khin hot hoa

gen

Thay i mc hot hoa ca gen quan t m , tnh c t h v v tr k h n g gian trong

b i u h i n ca gen.
20

4. Chuyn

gen t loi ny sang loi kia

a m t c tnh d i t r u y n m i t loi n y sang loi kia. V d gen k h n g su Btt o x i n t v i k h u n Bacillus

thurigensis

v o thc v t .

5. Lai hu tnh v v tnh to ra cc t hp gen

mi

D u n g hp t b o trn, lai t b o cht, n g h i n cu c c h p h n t ca u t h lai to

ra c c ti t hp h gen v genom ca b v m con l a i .
. Gy ra t bin gen v nhin cu kh nng gy t bin theo nh

hng

X u t h i n c c p h n g p h p g y t b i n m i r t hu ch cho n g h i n cu chc n n g
ca

gen

v i u k h i n hot hoa

gen:

t b i n bng ci gen

( A D N insertional

mutagenesis), k c h hot c c gen nhy (transposons) to ra c c t b i n a dng n g

thi d k i m sot v nhn b i t .
Bng

LI.

Cc kiu thay i vt liu di truyn v tc ng ca n i vi trao i

v cc c tnh sinh hc khc
nhau

cht

Cc kiu thay i vt liu di truyn

Kt qu nhn c

Thay i trnh t nucleotid trong gen cu trc

(do t bin, do ci gen l vo nhim sc th,
do gen nhy ci vo nhim sc th cc v tr
khc nhau trong qu trnh pht trin c th)

- Thay i cu trc v chc nng c th ca gen:

- Thay i trnh t sp xp ca nucleotid trong mARN v
trnh t axit amin trong protein.
- Thay i cu trc v gy bin tnh protein, lm thay i
hay lm mt hot tnh enzym.
- Thay i qu trnh sinh hoa v hnh thi ca c th
sng.

Thay i h iu khin ca gert

Thay i mc hot hoa, hoc mi trng hot hoa, hoc

cc iu kin hot hoa ca gen. Hu qu c th dn
n nh sau:
- Qu trnh nhn bn ADN v sinh tng hp mARN
khng th kim sot c. D vy, sinh tng hp mt s
protein v cc hot cht c th c tng cng hoc
suy gim hoc b trit tiu.
- Qu trnh phn bo b mt kim sot dn n ung th.

Thay i v tr ca gen trn ADN v nhim sc

th do t gy, o on nhim sc th, dch
chuyn gen, trao i cho.

Khng lm bin tnh gen, nhng c th lm thay i hot

hoa ca gen do v tr khng gian ca cc gen trong si
ADN thay i

Chuyn gen gia cc loi thng qua k thut

ADN ti t hp

- To ra cc c th sng bin i di truyn (GMO);

- C th sng c th tng hp c protein, enzym,
hoc cc polypeptid mi.
- C th gy bt hot cc gen v trit tiu cc hot cht
khng mong mun.
- C th gy t bin gen do gen chuyn ci vo nhim
sc th mt cch ngu nhin.
Ghi ch: (i vi t bo tin nhn (procaryote): nhim
sc th l mt si ADN mch vng gm vi nghn gen.
i vi t bo nhn thc (eukaryote): nhiu si ADN v
gen sp xp theo trt t trn nhim sc th).
21

1.6. M T S L N H v c NG D N G C A C N S H (Bng
ST

Lnh vc cng ngh v sn phm

1.2)
c trng c bn

CNSH thc vt

1.1

Cng ngh vi nhn ging: Nhn nhanh cc cy

trng c gi tr kinh t cao bng cng ngh nui cy
t bo nh hoa, cy cnh, cy n qu, cy dc
liu, cy lm nghip nhtch..., cy c du nh c
du...

1.2

Lm sch bnh: Lm sch bnh cc vt liu nhn Rt cn i vi nhn ging sch bnh v trao
ging bng nui cy nh sinh trng (kt hp vi i ngun gen.
cc k thut lm sch cc tc nhn gy bnh bng
hoa hc hoc x l nhit). K thut ny p dng ch
yu i vi cc ging u vit sinh sn bng con
ng v tnh nh cy n qu c mi, du ty, nho,
khoai ty, ma...

1.3

- Cn trong bo tn cc cy trng sinh sn

Bo tn ngun gen:
- Bo tn ngun gen nhit thp nh nit lng (- bng con ng v tnh, cc loi cy c c
v cy sinh sn bng thn r...
196C)
- Hoc bo tn thng qua phng thc sinh trng
chm trong mi trng nhn to

1.4

Cn phi:
- Cu phi trong cc k thut lai xa
- Cu phi a bi th (cu phi tam bi th cy n
qu c mi khi lai gia cy t bi v cy nh bi, cu
phi tam bi cc ht lp, ht nh...)
- Cu phi F1 khi lai to gia cc ging nho khng ht
- Cu phi khi ni nh ca ht km pht trin nh
trong nhn ging phong lan

1.5

Cng ngh n bi:

- Rt hiu qu i vi ging la, ng, la m,
Nhanh chng to ra cc dng thun, ging thun la mch. c ng dng rt thnh cng
chng ng dng trong to ging mi v to ging u Trung Quc, Triu Tin...
th lai (nui cy bao phn, ht phn, nui cy
non...)

1.6

t bin t bo soma:
Chn to cc t bin t bo soma. Rt hiu qu
trong to ging khng ht, dng bt dc c, nng
cao kh nng khng bnh v cc cht dit c, kh
nng chu hn, chu lnh
kh nng sn xut cc
hot cht sinh hc...

1.7

Cng ngh mang tnh t ph c th to ra

Thao tc di truyn v chuyn gen:
- Lai t bo trn to ging a bi th: to ging tam cc cuc cch mng trong cc lnh vc sinh
bi th khng ht bng dung hp gia t bo nh bi hc - nng nghip v y hc
v n bi, dung hp cc t bo nh bi vi nhau to
dng t bi th...
- To ging cy trng chuyn gen khng su, bnh
khng cht dit c, nng cao cht lng nng sn,
sn sinh dc cht

22

- H s nhn ging rt cao c bit l nhn

ging t t bo m so phi ha v phi v
tinh; p ng yu cu sn xut quy m ln m
cc k thut thng thng khng p ng c.
- Nhn nhanh cc kiu gen, hoc ging u vit.

- To cc con lai xa khc loi khng c kh

nng to ht c sc sng
- To ging khng ht (cam qut, bi,
chanh...)
- Cu phi lai gia cc ging nho khng ht
- Nhn nhanh cc ging phong lan u vit...

- Chn ging bt dc trong sn xut ht lai.

- Chn ging cy n qu khng ht (citrus).
- Chn to c nhiu ging chui, ma
mi...
- Nng cao cht lng protein...

STT

Lnh vc cng ngh v sn phm

c trng c bn

1.8

- Gip cho vic chn ging mc phn t

Marker phn t h tr chn ging:
Chn ging vi s h tr ca cc k thut sinh hc thng qua cc ch th phn t v cc gen
khng su bnh, gen cht lng, cc c
phn t (s dng cc k thut marker phn t)
tnh nng sut...

CNSH ng vt

2.1

Th tinh trong ng nghim, th tinh nhn to, cy - Nhn nhanh cc ging lai tt.
truyn hp t hoc phi

2.2

Liu php hormon lm tng s trng rng, kt hp - Nhn nhanh cc kiu gen v ging lai u
vi th tinh nhn to, cy truyn hp t nhn vit.
ging u vit.

2.3

To ng vt chuyn gen nng cao sn lng - Cc ng vt chuyn gen nh ln, g,

sa, tht, khng bnh, sn xut mt s sinh dc chut, th, b, cu, c...
qu nh protein trong sa, nc tiu, mu...

2.4

Thc n b sung chn nui (cc cht iu tit sinh Nng cao kh nng khng bnh, tng nng
trng, khng sinh chn nui, inteieron, vitamines, sut, t l nc trong tht, rt ngn chu trnh
protein n bo...
chn nui.

CNSH Y dc

3.1

Khng th n dng/s dng trong chn on bnh. - Sn xut bng cng ngh t bo lai
V d, cc bnh ng sinh dc, ung th, cc bnh (hybridoma)
virus nh vim gan B...

3.2

Mu d DNA (DNA probes): dng trong chn on - Sn xut bng cng ngh gen
bnh, VD: bnh kala-aser, bnh bun ng, st rt,...

3.3

Vacxin ti t hp (vim gan B, vacxin phng bnh - Sn xut bng cng ngh gen
virus gia sc ln, th, tru) b... nh l mm long
mng.

3.4

Cc loi thuc cha bnh c gi tr nh insulin, - Sn xut ch yu bng cc vi khun

intereron, hormon sinh trng ngi v b, chuyn gen
erythropoietin,...

3.5

Liu php gen (tr liu gen) cha cc bnh di truyn.

3.6

Sinh con theo gii tnh chn lc bng cch tch - C th dn n mt cn bng gii tnh trong
ring cc tinh trng mang nhim sc th X v Y v dn s
th tinh t bo trng bng 1 loi tinh trng-

3.7

Xc nh ph h hoc ti phm, xc nh m lit s - Xc nh chnh xc quan h h hng, dng

cha r ngun gc thng qua phn tch ADN ca h. tc bng phn tch ADN mu, t bo (tinh
trng), si tc...

CNSH Cng nghip

4.1

sx cc hp cht hoa hc nh cn, axit lactic, axit - Sn xut bi cc vi sinh vt ch yu l vi

citric, axit gluconic, aceton, glycerin...
khun

4.2

sx khng sinh: penicillin, streptomycin, erythromycin, - Sn xut bi cc loi nm, actinomycetes,

mitomycin, cycloheximide...
vi khun

4.3

sx cc enzym: amilase, protease, lipase...

- a cc gen bnh thng vo c th thay th

chc nng ca cc gen bnh bng nhiu k
thut khc nhau ang c nghin cu ci tin.

- Sn xut bi vi khun, nm s dng

trong cng nghip ty ra, dt, thuc l,
sa... trong cha bnh

23

ST

Lnh vc cng ngh v sn phm

c trng c bn

4.4

Ch bin mt s cht nn thnh cc hp cht c gi - Sn xut bng cc vi sinh vt hoc bng ki

tr kinh t v s dng cao hn nh s x cc hornn thut enzym c nh trong cc ermenters
steroids t sterols, sorbose t sorbitol...

4.5

sx cc protein n bo t vi khun, nm men, nm, - y l ngun sinh khi vi sinh giu protein
to, dng trong thc phm v thc n chn nui nh v axit amin khng thay th...
thc n b sung.

4.6

Cht t, nguyn liu v nhin liu ti sinh nh l - Sn xut bng cng ngh ln men vi sinh
vt biogas t phn chung v cht thi hu
cn bioethanol, biogas, biodiesel...
c, t sinh khi ma ng, sn, khoai lang,
cy c du, t dong to bin...

4.7

Thu hi mt s khong cht c gi tr nh vng, - Sn xut t cc sinh vt, ch yu l vi

khun c kh nng hp th v tch ly mt s
uranium, titan...
khong cht mt cch c th trong m t
bo ca chng.

4.8

C nh enzym s dng nhiu ln trong ch bin

mt s sn phm cng nghip.

u vit hn s dng c t bo vi sinh vt

nguyn vn trong ch bin cc cht hoc
lm sch mi trng

4.9

Thay i cu trc bc nht ca mt s protein/erym

bng cng ngh gen v cng ngh protein lm cho
chng hiu qu hn, bn vng hn vi mi trng;
thay i tnh c th ca enzym i vi mt s c
cht. V d thnh cng trong sn xut T4 lysozym,
trypcin, subtilisin, lactate dehydrogenase,...

- S dng computer thit k protein theo

hng c li hn. Cc nh khoa hc c bit
quan tm thay i enzym rubisco, mt enzym
ch lc trong quang hp enzym ny c i
lc mnh hn vi C 0 v km i lc vi 0
(lm gim quang h hp v tng quang hp)
2

4.10

sx cc vi sinh vt hoc t bo c kh nng sn sinh Thay i h iu hnh ca gen lm tng

cc enzym hoc nhiu hn cc protein hu ch quy cng biu hin ca cc gen m hoa cc
protein/enzym hu ch.
m ln.

5.

CNSH mi trng sinh thi

5.1

Phn gii cc cht thi sinh hot hu c ca con - Pht trin cc chng vi sinh hoc h vi sinh
ngi v cht thi th, x l chung tri chn vt hu hiu (EM), hoc cc enzym trong x
nui,...
l mi trng.

5.2

X l c t mi trng cng nghip v nng nghip - S dng cc vi sinh vt chuyn gen hoc
(hoa cht c t cng nghip, thuc tr su bnh, cc enzym c nh.
cht dit c, d lng phn bn trong ngun nc.)

5.3

X l cc ngun nhim t du m v cc sn - Cc chng Pseudomonas

phm t du m...

5.4

sx cc ch phm sinh hc kim sot su bnh (cc

loi virus, vi khun, nm, cn trng hu ch kim
sot cc tc nhn gy bnh, tr su)
To cc ging cy trng khng su bnh, cht dit
c lm gim s dng thuc hoa hc.

5.5

Sn xut cht do sinh hc, d tiu bin sau s - Sn xut cht do t cc sinh vt, cc
dng thay th cht do tng hp hoa hc kh polymer sinh hc
phn huy

5.6

Tng cng c nh C0 t kh quyn ng thi - Nng cao kh nng quang hp ca cy

tng gii phng dng kh 0 bng thm xanh thc trng v thc vt quang hp bin.
vt v cc sinh vt quang hp
- M rng thm xanh thc vt v nui trng
sinh vt quang hp bin
2

24

putida

- Gim thiu cc hoa cht nng nghip c

hi cho mi trng v sc khoe con ngi.
- Sn xut cc thuc su bnh sinh hc, cc
ging cy trng khng su bnh...

Chng

CNG NGH T BAO THC

VT

2.1. T B O T I N N H N V T B O N H N T H C
Cc c t h sng c t h l n b o hoc a b o . T t c c c c t h sng c c u trc t
b o c chia l m 2 n h m c h n h :
- Procaryotes - t b o t i n n h n
- Eucaryotes - t b o n h n thc
T b o t i n n h n x u t h i n khong 3.500 t r i u n m v trc. C c c t h sng t i n
n h n ch y u thuc n h m v i k h u n (bacteria).
T b o n h n thc bao g m protests, n m , c y xanh, n g v t v n g i .
Bng

2.1. S khc

nhau

c bn gia t bo tin nhn

T bo tin nhn
ng knh trung bnh 0,5-5nm
(micromet)
n bo hoc dng si
Si DNA mch vng nm trong t
Vt liu di
bao cht, khng c nhim sc
truyn
th, khng c nhn t bo v
hch nhn (nucleolus).
- Ribosome nh hn (70S)
- Khng c s tham gia ca
Sinh tng hp
endoplsmic reticulm
protein
- Cm ng mnh vi khng sinh,
v d streptomycin, kanamycin
c tnh
Kch thc t
bo
Hnh dng

Tiu bo t
(organeiles)

- Rt t tiu bo t (organelle).
- Khng c tiu bo t c mng
hai lp.

Mng cng v c cha

Thnh t bo polysaccharid v axit amin.
Murein l hp cht chnh to
bn ca mng
- Xy ra mesosome trong t
bo vi khun, tr vi khun xanh
H hp
lc (blue green) h hp xy ra
mng t bo cht.

v t bo nhn

tht

T bo nhn thc
- C dung tch 1000 - 10.000 ln ln hn so vi t bo
tin nhn. ng knh c th ti 40 um hoc hn
n bo, hnh si hoc a bo
Si ADN lin kt vi protein v ARN to nn nhim sc
th bn trong nhn te bo, trong nhn t bo c hch
nhn.
- Ribosome ln hn (80S)
- Ribosome nh vo h thng mng endoplasmic
reticulum.
- Rt nhiu tiu bo t, cc tiu bo t c bao bc
bi mng hai lp (v d: ty th, lc lp, nhn t bo).
Nhiu tiu bo t c bo bc bi mt lp mng (v
d: golgi, lysosome, khng bo, microbody,
endoplasmic reticulum).
Thnh t bo thc vt v nm rt cng v c cha cc
polysaccharid. Celluiose l thnh phn c bn to
cng ca mng t bo thc vt, trong khi chitin to
bn ca t bo nm.
H hp hiu kh xy ra ty th.

Quang hp

- Khng c lc lp
- Quang hp xy ra mng t
bo.

- Xy ra lc lp.
- Lc lp c cha cc lp mng, cc mng to ra cc
lamelle, cc lamelle xp lp to thnh cc ht gi l
grana

C nh nit

Mt s t bo c kh nng c
nh nit

Khng c kh nng c nh nit

25

2.2. N H N G C T R U N G c B N C A T B O S N G V C C H N G P H T
T R I N C BN CA CNG N G H T BO THC V T
T t c t h g i i sng a dng, phong p h v sinh n g tuyt v i trn tri t m
c h n g ta quan st t h y u c x y dng t c c t b o sng. T b o sng mang trong
n nhng k h n n g k d i u , l:
2.2.1. Kh nng phn bo v phn hoa
T b o sng c k h n n g p h n chia v p h n hoa. T b o thc vt c k h n n g p h n
chia, p h n hoa c quan v ti sinh t h n h cy trong i u k i n n u i c y i n vitro. B n g c c h
b i n i gen, cc n h khoa hc k h m p h hem 100 gen tham gia v o chu k p h n chia
t b o trn c s n g h i n cu m h n h n m men. M t trong s c c gen l cdc28, gen
n y c g i l gen k h i u v i n k i m sot bc quan trng u tin trong chu k
p h n chia t b o .
Chu k t b o bao g m m t lot c c s k i n x y ra theo t r n h t nht n h , trong
s sinh trng d n n s p h n chia m t t b o t h n h hai t b o con. N h n g t b o
ngng p h n chia s k h n g tham gia trong chu trnh t b o na. C c giai o n ca chu
trnh t b o c b i u h i n s b n g m cc pha sau:
G, - s - G - M
2

Gp Pha sinh trng sau p h n b o ;

S: Pha n h n i A D N ;
G : Pha sinh trng tip sau k h i A D N c n h n i ;
2

M : Pha p h n chia n g u y n n h i m (mitosis).

giai o n ny n h i m sc th chia u v hai pha v t i p theo t b o cht chia l m 2.
Pha M l i c chia l m 4 pha n h g m prophase, metaphase, a n a p h s e v telophase.
2.2.1.1. Cc gen iu khin chu k t bo
i u k h i n chu k t b o l c c y u t g y c h n M P F (maturing promoting factor),
bao g m n h m protein cyclins v Cdks (cyclin depent kinase). C c protein n y l m vai
tr k c h hot c c bc ca chu k t b o , to ra s c h u y n pha trong chu k t b o t G!
sang s, hoc t G sang M . M t trong c c protein l P53 - protein c chc n n g
2

phong toa chu k p h n b o nu n h A D N b h h i . N u s h h i l n g h i m trng, P53

c t h g y ra cht t b o . H m lng P53 c t n g ln trong c c t b o b h h i . N h
v y , t b o c t h i gian sa cha c c sai st trong k h i P53 phong toa hoc l m n g n g
chu k p h n bo. t b i n x y ra protein P53 l t b i n t h n g x u y n nht d n n
ung th. M t v d l h i chng L i a u m e n i , k h i P53 c sai st v d i t r u y n - d n n t n
s ung t h cao.
M t protein quan trng k h c l P27- m t protein c t h gn v i c y c l i n v Cdk l m
n g n g c h u y n t pha G, sang pha s. N g h i n cu gn y cho t h y rng c h n o n ung
th phi c thc h i n bng xc nh h m lng P27. K h i P27 g i m b o trc c t h
xy ra ung th.
26

Ba n h khoa hc g m Timothy Hunt, Paul Nurse, thuc Q u N g h i n Cu U n g T h

H o n g Gia A n h v Leland (Lee) Hartwell thuc T r u n g T m N g h i n Cu U n g T h Fred
Hutchinson, Seattle, M , nhn c g i i t h n g N o b e l y hc n m 2001 do nhng
n g h i n cu sut sc v p h t m i n h h i u h n h q u trnh p h n b o .
Pht m i n h ca h cho ta h i u t h m v nhng sai st mc p h n t v mc d i t
b o (su/?- cellular) c t h d n n ung t h n h t h n o .
Hartwell p h t m i n h ra c c Gen

chu

k phn

Division Circle genes) n m men Saccharomyces

chia

cerevisea.

t bo

Paul Nurse p h t m i n h

cc gen chu k p h n chia t b o m t loi n m men k h c l

pumle.

(CDC genes - Cell

Schiiosaccharomyces

Cc gen chu k p h n chia t b o C D C m hoa c c proteins k i m sot chu k

phn b o . Cdk v c c c y c l i n c pht h i n 2 l o i n m men trn. M t vi gen m

hoa cc p h n t Cdk v c y c l i n c t h c chc n n g ca genes ung t h (Oncogene, hoc
p h i hp v i c c sn p h m ca c c gen c c h ung t h (v d P53 v R b ) trong chu k t
bo).
Hartvvell cng a ra n g u y n tc " i m k i m sot - checkpoint" r t quan trng
trong n g h i n cu chu k t b o . Nurse cho bit c c Cdk i u h n h c c bc ca chu k
t b o bng phn ng phosphoryl hoa (phosphorylation) hai chiu m t s proteins (bao
g m c bn t h n c c Cdk) bng t h m hoc bt p h n t phosphate v o protein.
H u n cho bit cyclins gn v i c c p h n t Cdk to ra c c t hp Cdk - cyclins
(CDKs), do vy c h n g k h n g ch i u k h i n hot hoa ca Cdk m c n l m thun t i n
vic la chn t h m phosphate v o p h n t protein. C D K s n g vai tr i u k h i n c c
t r m k i m sot q u trnh p h n b o . C c t r m k i m sot k h c nhau v c c gen i u k h i n
cc t r m k i m sot n y c p h t h i n hai loi n m men v c c loi n h n thc
bc cao k h c , trong c n g vt c v.
H u n p h t m i n h ra c c protein cyclins p h n g i i theo chu k trong chu k t b o S p h n g i i ca protein c xem l m t c tnh n g vai tr quan trng k i m sot chu
k t bo.
H u n cng t m t h y cyclins c c c t h sng k h c nhau v cho b i t cyclins l c c
protein bo t h trong q u trnh t i n hoa.
Cc C D K s gen i u k i n q u trnh c h u y n dch c c pha trong chu k t b o n h
chuyn pha G sang M ( G / M ) , chuyn pha metaphase sang anaphase, G sang s, c c s
2

k i n lin quan n hng h c v chnh sa A D N . C c t r m k i m sot n y s k h n g cho

p h p t b o c h u y n t pha n sang pha kia n u pha trc c h a h o n t h n h .
V d : c c gen ng c c t r m k i m sot s k h n g cho p h p t b o c h u y n sang
giai o n M n u tng hp A D N c h a c sa cha, hoc ch cho p h p bt u pha
anaphase k h i t t c c c n h i m sc t h (NST) n m trn m t phang n g x c h o ca
t b o v c c kinetochores ca NST phi b m v o si thoi v sc.

27

2.2.1.2.

Chu k t bo v ng dng

trong thc

tin

* C h n o n v t i n t i n g n nga bnh ung t h

9
N h n g thay i trong chu k t b o c t h d n n c c b i n n g hoc s k h n g n
nh ca NST l n g u y n n h n g y bnh ung th. C c gen m hoa c c protein c h a khoa
trong i u k h i n p h n b o cng c h n h l c c gen ung t h (oncogenes). C c sn p h m cua
Cdk v cyclins gen c t h t n g tc v i c c sn p h m ca gen k m h m ung t h n h gen
P53 (tumor suppressor genes). t b i n gen P53 l n g u y n n h n g y ra t r n 5 0 % t r n g
hp ung t h n g i . N h n g hng h c c c gen i u k h i n chu t r n h p h n b o u c
th d n t i ung th. V d , c y c l i n D I (sn p h m ca m t gen ung t h - oncogen) c
b i u h i n vt mc b n h t h n g trong hu ht c c trng hp ung t h p h i . N h n g con
chut k h n g c c y c l i n D I t ra h o n t o n k h n g v i ung th. V i c t h i u v n g hot
ng

ca

cc

gen

hoa

c c cht k m

hm

phn

bo

n h P53

gen

Rb

(retinoblastoma) cng c t h d n n ung th. Gen P53 c g i l n g i bo v ca

genom (Guardian hoc Watchman).
Do vy vic pht minh ra cc gen ch o iu khin

chu k phn

m ra nhng trin vng mi m i vi chn on v cha tr ng

bo chc chn

th.

* C c ng dng quan trng k h c

N h n g h i n cu c c quy lut i u k h i n p h n chia t b o n g i ta c t h ng d n g
v o nhiu lnh vc k h c nhau ca i sng:
- N h n bn t b o ;
- Sn xut sinh k h i t b o n h protein n b o , sinh k h i dc l i u ;
- T o ra h n g lot bn copy ca m t c y u v i t hay l n h n b n c y t r n g quy m
c n g nghip. y l n n tng ca C n g n g h v i n h n g i n g (micropropagation);
- K h n n g p h n hoa ca t b o sinh d n g ( t b o soma) t h n h p h i trong i u k i n
in vitro l c s khoa hc ca c n g n g h sn x u t ht n h n to t t b o .
2.2.2. Kh nng trao i cht
T b o l n h m y sinh hc c k h n n g sn x u t ra h n g v n c c l o i hp c h t h u
c k h c nhau. K h n n g trao i cht ca t b o l c s ca c n g n g h i p sn x u t c c
cht hot tnh sinh hc ng dng trong n n g n g h i p v y hc (bng 3). V d :
- N u i c y t b o n h n s m trong bioreactors;
- Sn x u t taxol cha bnh ung t h bng n u i c y m t h n g ;
- Sn x u t vacxin v c c dc cht quan trng bng c n g n g h A D N ti t hp.
H i n nay, m t h n g ng dng m i ca k thut d i t r u y n a n g c c h : l
canh tc p h n t. C y trng hoc t b o tr t h n h bioreactor sn x u t ra c c c h p h m
sinh hc k h c nhau.

28

Bng

2.2. Mt s cc sn phm

vt v cng

dng

Cng dng

Cht

ST
1
2
3
4
5
6
7
8
9
10

th cp thc

Cha ung th
Cha ung th
Tng n hi thnh mch
Cha ri lon tim mch
Cha st rt
Thuc an thn
Hng thm
Thuc tr su
Hng v
Cha st rt

Vinblastine
Taxol
Ajmalicine
Digitalis
Quinine
Codeine
Jasmine
Pyrethrine
Spearmit
Artemysilin

2.2.3. T b o l k h o l u t r t h n g t i n v l n v t i n hoa
T h n g t i n d i t r u y n c lu g i trong c c p h n t A D N ca t b o . Trong c c t
b o t i n n h n (prokaryote): n h i m sc t h l m t si A D N m c h v n g g m vi n g h n
gen. Bn cnh , trong c c t b o t i n n h n c n c c c plasmid l c c p h n t A D N
m c h v n g n h k h n g cha c c gen thit y u i v i trao i cht ca t b o . Trong c c
t b o n h n thc (eukaryote) t h n g t i n d i t r u y n ch y u c lu g i trn n h i m sc
th, trong n h n t b o . N g o i ra, ty t h v lc l p cng mang gen trn m t p h n t A D N
v n g k h p kn, ging n h n h i m sc t h t b o t i n n h n . S lng gen trong t b o v i
k h u n l khong vi n g h n gen trong k h i n g i c t nht l 30.000 gen.
2.2.3.1. T bo l n v sng nh nht bo tn kh nng ti sinh thnh c th
hon

chnh
- N h n g b i n i d i t r u y n mc t b o l c s to ra c c c t h sng m i l m

t i n p h t t r i n c n g n g h t b o to ging.
- T b o l vt l i u t i n hoa, cung c p vt l i u d i t r u y n m i cho qun t h , qun x
v c k h n n g g y ra n h n g b i n i ca h sinh thi.
Chuyn gen v o t b o sng trong i u k i n ng n g h i m to ra c c c t h b i n
i d i truyn ( G M O - Genic m o d i f i e d organism). V d , gen c t tr su ca v i k h u n
Bacillus

thuringiensis

(gen Bt) c chuyn v o t b o n g n u i c y i n vitro, t c c t

bo mang gen Bt n g i ta ti sinh t h n h c y v to ra c c d n g n g Bt k h n g su. C c

d n g n g Bt n y c k h n n g th phn t do hoc lai to v i c c d n g , ging n g c
ngha k i n h t k h c to ra v vn c c ging, d n g mang gen ca G M O ban u v c
t h c nhng tc n g n h sinh thi {xem chi tit vGMO

chng

3).

2.2.3.2. T bo l n v thao tc di truyn v l tin ca cng cuc to ging

Thao tc d i t r u y n mc t b o l m t b i n p h p ch o trong to ging m i trong
t n g lai. Thao tc d i truyn s bao g m c c k thut d i y :
- K thut gen (ci gen, chuyn gen...).
- t b i n , b i n d t b o soma.

29

- L a i g h p t b o trn, lai xa, cu p h i v b i n np c quan t ( n h i m sc t h v n i

b o quan).
N h n g t i m n n g ca t b o thc vt c p h n tch t r n y cho t h y nhng
h n g ng d n g ca C n g n g h t b o l r t a d n g v a n g c t n g bc c h u y n
hoa v o thc t i n p h t t r i n n n g nghip, y hc, c i t h i n m i t r n g sinh t h i . T u y theo
mc tiu p h t t r i n ca n n k i n h t ca m i quc gia v nhu cu ca t h t r n g n g i ta
n g h i n cu pht t r i n c c c n g n g h p h hp.
2.2.4. T bo thc vt cn l nh my nng lng mt tri - nn tng cho pht trin
C n g n g h s i n h hc x a n h v c c c n g n g h sch
T b o thc vt c k h n n g k d i u : h p th n n g lng m t t r i v t o ra c c hp
cht hu c. N h v o q u trnh quang hp, trn tri t to ra:
- K h o n g 170 t t n c c cht hu c / n m .
- L n g thc v thc n k h c n u i d n g trn 8 t n g u i , h n g chc t n g v t v
v i sinh vt tri t .
- D n g kh: T t h n k h C 0

v nc, d i n h s n g m t t r i t o ra c c c h t h u c

v d n g k h ( 0 ) .
2

- H p th t h n k h ( C 0 ) : C c ging c y trng n n g sut cao c t h h p thu 70 t n

2

C 0 / h a . C c c y C4 c k h n n g h p t h C 0
2

v k h n n g quang hp cao h n c y C3.

- N g o i ra, t bo thc vt c n c k h n n g p h n l y nc t h n h oxy v hydro n g u y n t

iu k i n nhit bnh thng. y c t h l loi nhin l i u sch v gi trong tng lai.
T m l i , t b o thc v t l n h m y n n g lng m t t r i , c h u y n hoa n n g lng n h
s n g t h n h n n g lng hoa hc v to ra d n g k h . D o v y , c c n g h i n c u t b o thc
vt s n g vai tr quyt nh i v i q u trnh x y dng v phc h i c c n g u n n n g
lng sng ca tri t .
N g y nay, c n g n g h sinh hc n g y c n g c t i m n n g t r i thc v p h n g t i n c n g
ngh .tc n g v o k h n n g quang hp ca t h m thc v t v tham gia v o q u t r n h
n n g lng v m i trng t o n cu.
2.3. MT S HNG KHAI THC CC C TRUNG c BN CA T BO
TRONG TO GING
B i bn cht ca khoa hc - c n g n g h l khai t h c n h n g t i m n n g t h i n n h i n v
cuc sng con n g i . C h n g ta n g h i n cu nhng t i m n n g to l n ca t b o sng n h m
x c nh nhng k h n n g ng dng ca c c t i m n n g . D i y ch g i i t h i u m t s
ng dng trc m t trong n n g nghip m i n h hoa.
2.3.1. Xy dng h thng ti sinh nng cao hiu qu chuyn gen trong to ging
T h n h c n g ca k thut gen to ging c y trng ph thuc v o ba y u t c bn
n h thit k hp l gen v h i u h n h ca gen, k thut c h u y n v l p g h p gen v o v
30

tr t h c h hp trong n h i m sc t h v h gen ca t b o v ph thuc v o h thng n u i

c y v ti sinh t b o t h n h c y h o n chnh. V v y , s d n g c c g i n g c k h n n g ti
sinh cao i n vitro l c s k h n g t h t h i u ca c c thao t c c h u y n gen.
T h n h tu n i bt trong lnh vc n y l h t h n g ti sinh c y l a t n u i c y t b o
p h i hoa. T h n g qua chuyn gen v o h thng t b o c k h n n g ti sinh cao n y , vi
n g h n d n g la c h u y n gen v i c c tnh trng k h c nhau c to ra.
n g v m t s c y trng k h c , n g i ta c h a t h c h u y n c c gen m o n g m u n v o
c c d n g ging c gi tr t h n g m i m t c c h trc t i p do c c g i n g n y k h n g c hoc
c k h n n g ti sinh r t y u trong n u i c y i n vitro. V v y , trc ht n g i ta c h u y n
gen vo h thng t b o n u i c y ti sinh m n h . Sau k h i to ra c y mang gen m i , n g i
ta phi thc h i n p h p lai ngc (back-cross) to ra d n g thun c g i tr t h n g m i .
2.3.2. K thut n bi to ging mi v dng thun
G i tr ca c y n b i trong c c n g h i n cu d i t r u y n v c h n g i n g c n g i
ta bit n t lu. K t k h i Blakeslee (1921) m t c y n b i t n h i n u tin
Datura

stramonium,

c y n b i t n h i n c t m t h y r t n h i u l o i c y k h c

nhau. Tuy v y c c c y n b i t n h i n x u t h i n m t c c h n g u n h i n v i t n sut r t

thp k h n g t h p ng y u cu ca n g h i n cu v c h n g i n g . L n u tin trn t h
g i i , hai n h khoa hc A n Guha v Maheshvvari (1967), t h n h c n g trong vic to
ra h n g lot cy n b i t n u i c y bao p h n i n vitro c y c Datura

innoxia.

Ngay sau

, cy n b i c to ra bng n u i c y bao p h n h n g l o t c c l o i c y trng

k h c . Cc n h khoa hc t m ra c c y u t quan trng nh h n g n t h n h c n g ca
q u trnh n u i c y n h nh h n g ca k i u gen, giai o n p h t t r i n ca ht p h n v c c
i u k i n m i trng n u i c y .
N g o i n u i c y bao p h n , c c n h khoa hc c n c n h n g t h n h c n g r t l n trong
vic n u i cy n o n c h a t h tinh, n u i c y ht p h n t c h r i . K thut to c y n b i i n
vitro t h n g qua k c h t h c h t i u b o t hoc i b o t p h t t r i n t h n h c y trong n u i c y
ht phn, n o n cho p h p nhanh c h n g to ra h n g lot c y em b i phc v c lc cho
mc ch n g h i n cu d i t r u y n v to ging c y trng.
Trong k thut n b i , vt l i u ban u cho q u trnh n u i c y i n vitro g m :
- Bao phn, ht p h n t c h r i , c m hoa ( p h n g p h p n y t h n g c p dng cho
nhng loi c hoa n h ) .
- P h i non. K h i lai xa hai loi la m c h Hordeum

vulgare

v H. bulbosum

v i nhau,

sau q u trnh th phn p h i n b i c h n h t h n h do n h i m sc t h ca

bulbosum

H.

b l o i tr, n h n g n i nh ca p h i n b i k h n g p h t t r i n . S dng

p h n g p h p n u i cy p h i cu c c c p h i n y k h i b cht v to ra h n g lot c y
n b i (Jensen, 1977).
- T h tinh g i . y l q u trnh th phn n h n g sau k h n g x y ra s t h tinh.
M c d v y , t b o trng v n c k c h thch p h t t r i n t h n h c y n b i . Hess v
31

Wagner (1974) t i n h n h t h p h n i n vitro gia Mimulus

luteus

vi

Torenia/ournieri.

K t q u h to c c y n b i .
- N o n c h a th tinh.
Trong s c c vt l i u k trn th bao phn, ht p h n t c h r i v n o n c h a t h t i n h l
nhng ngun n g u y n l i u quan trng v t h n g c s d n g h n c to c y em b i .
K t t h n h c n g ca Guha v Meheshinari, c c c y n b i ca h n 247 loi thuc
88 chi v 34 h thc ht k n c to ra r n u i c y bao p h n v ht p h n ( B a j a j ,
1990) trong k h i t h n h c n g ca n u i c y n o n c n h n c h m t s l o i c y t r n g
n h i mch, la m , thuc l, c c i n g , n g .
2.3.3. Th tinh nhn to, nui cy v ti sinh t bo trng, t bo hp t, t bo ni
n h non t h n h cy trong i u k i n in vitro
Quy trnh th tinh k p thc vt: k h i n o n c t h p h n , t b o p h n hoa c m t
n h n sinh d n g s to t h n h ng p h n . N h n ca m t t i n h t I n tri theo n g p h n s
th tinh v i t b o trng I n v to ra t b o hp t 2n. N h n ca t i n h t t h 2 s t h t i n h
v i t b o trung t m ca n o n 2n v s to t h n h t b o n i n h 3n.
C c n h khoa hc t i V i n Thc vt i c n g c l n u t i n thc h i n v i c t h
tinh n h n to cho t b o trng h o n t o n trong i u k i n i n v i t r o v s p h t t r i n ca c c
sn p h m sau t h tinh c quan st t h n g x u y n d i k n h h i n v i . T b o trng
t c h r i sau k h i t h tinh to t h n h p h i v ti sinh t h n h c y h o n c h n h trong ng
n g h i m . T b o trung t m ca ti p h i (2n n h i m sc th) sau k h i t h t i n h v i n h n ca
t b o ht phn ti sinh i n vitro t h n h n i nh tam b i c c c y hoa tho n h l a m ,
la mch, n g v la nc... N h n g t h n h c n g n y a n g m ra k h n n g thao t c d i
truyn d i k n h h i n v i n h m to ging m i , trong c c c g i n g tam b i c y trng.
2.3.4. Cng ngh t bo to ging cy n qu khng ht, cht lng cao v cy lm
nghip u t h lai t h n g qua nui cy n i n h t a m b i
H i n t i c ba p h n g p h p c n g ngh t b o a n g c p d n g r n g ri to
ging u t h lai k h n g ht v ging u t h lai tam b i t h k h n g ht.
2.3.4.1. Phng php nui cy v ti sinh cy t ni nh tam bi
P h n g p h p c s dng k h p h b i n trong to ging c y n q u k h n g ht
cam q u t b i , c h u i , t o ty... v to ging c y rng u t h l a i tam b i . P h n g p h p
n u i cy n i nh ca ht non a n g c ng d n g v i c y n q u c m i t o g i n g
cam q u t k h n g ht, sch bnh.
2.3.4.2. Cu phi
H i n tng q u k h n g ht c t h do p h i sau k h i h n h t h n h k h n g c t i p tc
pht trin m tiu b i n i . H i n tng n y c p h t h i n c c g i n g nho k h n g ht.
c c ging n y , ht non p h t t r i n n giai o n nht nh r i d n g l i v b tiu b i n .
P h n g p h p m i to ging k h n g ht l lai hai ging k h n g ht v i nhau, ht l a i trc
k h i b teo i c t c h ra n u i c y i n vitro ti sinh t h n h c y . y l k thut n u i
32

c y m cu p h i ht lai i n vitro. T i Israel, trong c h n g t r n h to g i n g nho, m i

n m lai to v n u i c y khong 30.000 p h i non, to ra 30.000 c y con lai F l g i n g nho
k h n g ht k h o n g h i m n h m chn ra nhng con l a i F l u v i t nht. N h n g con lai
u v i t sau s c n h n nhanh bng p h n g p h p v t n h phc v sn x u t .
2.3.4.3. To ging a bi th4n
P h n g p h p c bn to d n g t b i t h l" x l colchicine t b o v m nh b i
i n v i v o hoc i n vitro hay bng p h n g p h p dung hp c c t b o t r n n h b i v i nhau.
C y ging a b i t h 4n sau k h i to c bng x l colchicine s c lai v i ging
nh b i 2n to g i n g tam b i k h n g ht 3n (cy n qu) hoc l g i n g tam b i t h u
t h lai c n n g sut cao ( c c g i n g d u t m ) .
C n g ngh t b o v s n g g p vai tr n g y c n g cao trong c h n to ging c y
trng. N h n g t i n b m i y trong l n h vc n u i c y t b o v m t c h r i n h ht
phn, bao phn, t b o trng v p h i non, n i n h tam b i , t b o tht l... cho p h p c n g
ngh t b o t h m nhp v o t t c c c lnh vc to g i n g k h c nhau. l:
- T o ging u t h l a i ;
- T o c c dng t b i n v b i n i gen ( G M O ) ;
- T o cc dng tam b i t h u t h lai k h n g ht c y n q u v c c c y l y l, l y
g v l y si;
- T o d n g t b o t b i n hoc b i n i gen c k h n n g sn sinh c c dc cht
quy m c n g nghip.
N h i m v ca c c n h khoa hc l la c h n c c c n g ngh t i n t i n nht p dng
v o p h t t r i n n n g nghip, bo v m i trng v sc khoe con n g i nc ta.
2.4. PHT MINH RA CCNG DNG KHC NHAU CA K THUT NUI CY
M V T B O
2.4.1. K thut nui cy nh sinh trng to ging sch bnh v nhn ging nhanh
- N m 1949, Limmasets v Cornuet p h t h i n r n g virus p h n b k h n g n g
nht trn c y v t h n g k h n g t h y c virus v n g n h sinh t r n g .
- N m 1952, M o r e l v M a r t i n to ra cy sch bnh virus ca 6 ging khoai ty t
nui cy nh sinh trng. N g y nay, k thut ny v i m t s c i t i n tr t h n h p h n g
p h p l o i tr bnh virus c d n g rng ri i v i nhiu loi c y trng k h c nhau.
- N m 1952, M o r e l v M a r t i n l n u t i n thc h i n v i g h p i n vitro t h n h c n g .
K thut v i g h p sau c ng d n g r n g ri trong to n g u n g i n g sch b n h
virus v t n g t virus n h i u c y t r n g n h n g i n g bng p h n g p h p v t n h k h c
nhau, c b i t l to g i n g c y n q u sch bnh.
- N m 1960, M o r e l thc h i n bc ngot c c h m n g trong s d n g k thut n u i
c y n h sinh trng trong n h n nhanh c c l o i a lan Cymbidium,

m u c n g nghip

v i n h n g i n g thc vt.
33

2.4.2. K t h u t n u i c y , t i s i n h t b o t r n v l a i t b o s o m a
- N m 1960, Cocking l n u t i n s d n g enzym p h n g i i t h n h t b o v to ra
s lng l n t b o trn. K thut n y sau c h o n t h i n t c h n u i t b o trn
n h i u c y trng k h c nhau.
- N m 1971, Takebe v cs., ti sinh c c y t t b o t r n m tht l (mesophill
cell) thuc l.
- N m 1972, Carlson v CS-, l n u tin thc h i n lai t b o soma gia c c l o i , t o
c c y t dung hp t b o trn ca 2 loi thuc l Nicotiana

glauca

v N.

langsdorfii.

- N m 1978, Melchers v cs., to c c y lai soma "c chua - thuc l " b n g l a i xa

t b o trn ca hai c y n y . n nay, vic ti sinh c y h o n chnh t t b o t r n hoc t
lai t b o t r n t h n h c n g n h i u loi thc vt.
2.4.3. K thut n bi v ng dng to dng thun, c nh u th lai
- N m 1964, Guha v Maheshwari l n u t i n t h n h c n g t r o n g t o c c y n
b i t n u i c y bao p h n ca c y c Datura.

K thut n y sau c n h i u t c g i

p h t t r i n v ng d n g r n g ri trong t o d n g n b i ( l x ) , d n g t h u n n h b i k p
( 2 x ) , c n h u t h l a i ( n u i c y bao p h n hoc h t p h n ca d n g l a i F l t o g i n g
thun mang t n h trng u t h l a i ) .
2.4.4. K thut nui cy t bo dch lng (suspenson culture) s dng trong sn
x u t cc c h t hot t n h sinh hc
- N m 1959, Tulecke v N i c k e l l t h n g h i m sn x u t sinh k h i m thc v t quy
m l n (134 lt) bng n u i c y c h m .
- N m 1977, Noguchi v cs., n u i c y t b o thuc l trong bioreactor d u n g t c h
l n 20.000 lt.
- N m 1978, Tabata v cs., n u i t b o c y thuc quy m c n g n g h i p phc v
sn x u t shikonin. H c h n lc c d n g t b o cho sn lng c c sn p h m t h c p
(shikonin) cao h n .
- N m 1985, Flores v Filner l n u tin sn x u t cht trao i t h c p t n h n n u i
r t Hyoscyamus

muticus.

N h n g r n y sn x u t n h i u hot c h t hyoscyamine h n

c y t n h i n .
H i n nay, c n g n g h n u i c y t b o v m (v d , m r ca n h n s m ) t r o n g
c c bioreactor d u n g t c h l n c t h n g m i hoa mc c n g n g h i p sn x u t
sinh d c .
2.4.5. To cc bin d dng t bo soma ng dng trong chn to ging v xc nh
c h c n n g c a c c gen
N m 1981, trn c s quan st c c b i n d x y ra r t p h b i n trong n u i c y m v
t b o v i p h b i n d v tn s b i n d cao, L a r k i n v Scowcroft a ra thut n g
34

" b i n d d n g soma" (somaclonal variation) ch c c thay i d i t r u y n t n h trng x y

ra do n u i c y m v t b o i n vitro. T c c d n g t b o hoc c y b i n d d i t r u y n n
n h c t h n h n nhanh, to ra c c d n g v g i n g t b i n c n n g sut, h m lng hot
cht h u ch cao, k h n g m t s c c i u k i n bt l i n h bnh, m n , hn,...
2.4.6. Cng ngh t bo ng dng trong chuyn gen vo cy trng
n nay c c n h khoa hc khng nh rng mc t h n h c n g ca c h u y n gen
vo c y trng ph thuc r t n h i u v o h thng n u i c y v ti sinh t b o t h n h c y i n
vitro sau c h u y n gen.
- N m 1974, Zaenen v cs., p h t h i n plasmid T i n g vai t r l y u t g y u
(crown gall) c y trng.
- N m 1977, Chilton v cs., chuyn thnh c n g T - D N A vo thc vt.
- N m 1979, M a r t o n v cs., x y dng quy trnh c h u y n gen v o t b o trn t h n g
qua Agrobacterium,

bng p h n g p h p n g n u i c y t b o t r n v

Agrobacterum.

- N m 1982, Krens c h u y n t h n h c n g A D N v o t b o trn.

- N m 1985, Fraley v cs., t h i t k vector plasmid T i l o i b c c gen c g y h i
s dng cho vic t h i t k vector chuyn gen v o thc vt. C n g trong n m , Horsch v
cs. chuyn gen v o m n h l bng Agrobacterium

tumeaciens

v ti sinh c y c h u y n

gen. A n v cs. (1985) p h t t r i n h thng hai vector cho c h u y n gen thc vt.
- N m 1987, K l e i n v cs. s dng s n g b n gen (particle gun) mang v i n trong
chuyn gen v ti sinh c cy b i u h i n gen chuyn.
- N m 1994, t h n g m i hoa ging c chua c h u y n gen 'Flavr-Savr'.
2.5. NUI CY M V T BO THC VT
2.5.1. M t s c c m i t r n g c b n t h n g d n g t r o n g n u i c y m
T h n h phn hoa hc ca m i trng n g vai tr q u y t n h i v i t h n h c n g ca
n u i c y t b o v m thc vt. M i loi c y , t h m c h m i k i u gen, c c k i u n u i c y
khc nhau ( n u i c y m so, h u y n p h t b o , t b o trn, bao p h n , ht phn...) c
nhng i h i k h c nhau v t h n h phn m i trng. K h i bt u n u i c y m m t loi
m i hoc m t g i n g m i , cn p h i la c h n cho i tng n g h i n cu m t l o i m i
trng c bn p h hp.
Cho n nay, c c n h khoa hc to ra s lng r t l n c c m i trng t h c h hp
v i tng i tng v mc tiu n g h i n cu. M t s m i trng c bn t h n g c s
dng trong n u i c y m v t b o thc vt, v t h n h phn hoa hc ca c h n g c t h
h i n bng d i y :

35

Bng

2.3. Mt s mi trng c bn thng c s dng trong

v t bo thc vt (n v khi lng
mgl)

nui

cy m

Heller
(1953)

Schenk
&
Hildebrandt
(1972)

Chu &
Cs
(1975)
N6

Nitsch &
Nitsch
(1967)

Knop
(1865)

2.500

2.500

2.830

125

250

134

463

200

500

1,400

440

150

75

200

166

MgS0 .7H 0

370

360

250

250

400

185

125

250

KH P0

170

400

125

250

NaH P0 H 0

16,5

150

125

NH H P0

300

FeS0 7H 0

27,8

27,8

15

27,8

27,8

Na EDTA

37,3

37,3

20

37,3

37,3

KI

0,83

0,75

0,75

0,01

1,0

0,8

H3BO3

6,2

1,5

1,6

10

MnS0 4H 0

22,3

10
(1 H 0)

0,1

10

3,3

25

ZnS0 7H 0

8,6

1,5

10

65

750

120

CuS0 5H 0
2

0,025

0,025

0,03

0,2

0,025

0,025

CoCI 6H 0

0,025

0,025

0,1

0,025

0,025

0,25

0,25

0,1

0,025

0,25

200

NaNOa

600

H S0

2,5

NiCI 6H 0

0,03

AICI3

0,03

30.000

20.000

20.000

20.000

30.000

20.00030.000-

Myo-lnositol

100

100

1.000

100

Nicotinic Acid

0,5

0,5

1,0

0,5

0,5

Pyridoxine. HCI

0,5

0,1

1,0

0,5 '

0,5

0,5

Thnh phn
KNO3
NH N0
4

(NH ) S0
4

Ca(N0 ) .4H 0
3

CaCI H 0
2

Murashige
& Skoog
(1962)
MS

White
(1963)

Gamborg
(1968)
B

1.900

80

1.650

KCI
4

Na M O .2H 0
2

Na S0
2

Fe (S0 )
2

FeCI 6H 0
3

FeC 0 H 5H 0
6

Sucrose
Glucose

36

30.000 90.000

Thnh phn

Murashige
& Skoog
(1962)
MS

VVhite
(1963)

Gamborg
(1968)
B5

0,1-1

0,1

10

Thiamine.HCI
Glycine
Ca-Pantothenate

Nitsch &
Nitsch
(1967)

Knop
(1865)

0,1-10

0,5

"

0,05

0,5

Cysteine.HCI

1
~

Glutamine

Chu &
Cs
(1975)
N6

Biotin

Folic Acid

Schenk
&
Heller
(1953) Hildebrandt
(1972)

Bng 2.4. Mi trng MS (Murashige

& Skoog
1962)
Murashige T. and Skoog F. 1962. A revised mediumor
rapid growth and
bio-assays
with tobacco tissue cultures. Physiologia
Plantarum.
15,
473-497.
Mui khong

Nng

a lng

Nng

(mg/l)

Vi lng

1.900

Manganese sulfate (MnS0 . 4H 0)

22,3

Ammonium nitrate (NH N0 )

1.650

Boric acid (H3BO3)

6,2

Calcium chloride (CaCI .H 0)

440

Colbalt chloride (CoCI .6H 0)

0,025

Magnesium sulate (MgS0 .7H 0)

2

370

Cupric Sulate (CuS0 .5H 0)

0,025

Potassium phosphate (KH P0 )

170

Ferrous sulate (FeS0 .7H 0)

27,8

Na EDTA.2H 0

37,2

Potassium lodine (KI)

0,83

Potasium nitrate (KN0 )

3

(mg/l)
4

Sodium molybdate (Na Mo0 .2H 0)

0,25

Zinc Sulate (ZnS0 .7H 0)

8,6

Cc cht hu c
Myo-inositol

100

Nicotinic acid

0,5

Pyridoxine.HCI

0,5

Thiamine.HCI

0,1

IAA

1-30 mg/l

Kinetin

0,04-10

Glycine (recrytallized)

2.0 g/l

Edamine

1,0 g/l

Sucrose

20 g/l

Agar

10 g/l
37

2.5.2. T h n h p h n hoa h c c a c c m i t r n g n u i c y m , t b o t h c v t
M i trng n u i c y m v t b o thc vt tuy r t a d n g n h n g u g m m t s
t h n h phn c bn sau:
- C c m u i k h o n g a lng v v i lng
- Cc vitamin
- C c amino axit
- N g u n c c b o n : m t s c c l o i n g
- C c cht i u hoa sinh trng
- C c cht hu c b sung: nc da, dch chit n m men, dch c h i t k h o a i t y , b t
chui kh...
- Cht l m thay i trng thi m i trng: c c l o i thch (agar).
T t c c c hp cht n y u tham gia v o m t hoc n h i u chc n n g trong s sinh
trng v p h n hoa ca thc v t n u i c y i n vitro.
C c n h khoa hc s d n g c c m i trng n u i c y r t k h c nhau. V i c la c h n
m i trng n u i c y v i t h n h phn hoa hc c t r n g ph thuc v o m t s y u t :
- i tng cy trng hoc m n u i c y k h c nhau c nhu c u k h c nhau v t h n h
phn m i trng.
- M c c h n g h i n cu hoc p h n g thc n u i c y k h c nhau ( n u i c y to m so
p h i hoa hoc p h i v tnh, n u i c y t b o t r n hoc dch l n g t b o , v i n h n ging...).
- T r n g thi m i trng k h c nhau (c, l n g , b n lng...).
- C c m i trng k h o n g c bn c s dng n h i u n h t trong n u i c y t b o thc
vt l M S (Murashige and Skoog, 1962), B5 (Gamborg cs. 1968), W h i t e (1963).

(xem

bng t h n h phn c c m i trng).

2.5.2.1.

Cc cht

khong

i v i c y trng, c c cht v c n g vai tr r t quan t r n g . V d , M g l m t phn

ca p h n t d i p lc, Ca l t h n h phn ca m n g t b o , N l t h n h phn quan t r n g ca
amino axit, vitamin, protein v c c axit nucleic. T n g t, Fe, Z n v M o c n g l t h n h
phn ca m t s enzym.
C c m i trng k h c nhau c h m lng v t h n h phn cht k h o n g k h c nhau, v
d t h n h phn v nng k h o n g ca m i trng W h i t e hoc K n o p k h n g h o n n ,
n h n g l i rt giu m i trng M S v B5.
M u i k h o n g l t h n h phn k h n g t h t h i u trong c c m i trng n u i c y m v t
b o thc vt:
- M u i k h o n g l c c vt l i u (ngun N , s, p...) cho s tng hp c c cht h u c .
Nit, lu h u n h , phospho l c c t h n h phn k h n g t h t h i u ca c c p h n t protein, c c
axit nucleic v nhiu cht hu c k h c . Canxi v axit boric c t m t h y c h y u
t h n h t b o , c bit l canxi c n h i m v quan trng g i p n n h m n g sinh hc.
38

- n g vai tr n h m t t h n h phn k h n g t h t h i u ca n h i u enzym (l c c cofactor): m a g i , k m , st... v n h i u n g u y n t v i lng l n h n g phn quan trng ca c c

enzym.
- C c l o n ca c c m u i hoa tan n g vai t r quan t r n g n n h p sut t h m t h u
ca m i t r n g v t b o , duy tr t h i n hoa ca thc v t . V d , K v c r t quan
t r n g trong i u hoa t n h t h m l c ca t b o , duy tr i n t h v tham gia hot hoa
n h i u enzym.
Trong m i trng, c c m u i k h o n g c chia t h n h c c n g u y n t v i lng v a
lng:
- C c n g u y n t a lng g m 6 n g u y n t : M g , Ca, p, s, N v K .
- C c n g u y n t v i lng g m : Fe, M n , Z n , Cu, M o , Co, B, , N i , C l , A I , . . .
V i c chia t h n h c c n g u y n t v i lng v a lng ch y u da trn nhu cu ca
thc vt i v i c c cht n y . N h u cu ca thc vt i v i c c n g u y n t a lng l l n
h n , v i nng > 0,5 m M . C c n g u y n t v i lng c s d n g trong m i trng
nng < 0,5 m M .
N h u cu ca c y i v i n g u y n t v i lng l r t t h p . D o v y nhng n g u y n t n y
cng c mt trong m i t r n g c c nng t n g ng. H u ht c c n g u y n t v i lng
s dng lng imol. M t s n g u y n t v i lng c nhu cu n h h n c t h thay t h d
d n g bng s l n tp n g u n h i n ca c h n g trong c c t h n h phn ca m i trng n h
agar, c c cht b sung n h nc da, dch chit n m men (yeast extract), c c m u i v
nc. T m quan trng ca m t s n g u y n t v i lng trong t h n h phn m i trng c n
cha c h i u m t c c h r r n g . Co, A I , N i . . . c t h c l i i v i thc v t n h n g cng
c th l k h n g cn thit. Trong thc t , hu ht c c n g u y n t v i lng ch c phn
k h o n g ca m u i (cation) l quan trng, c n vai tr c c anion c t h l k h n g cn thit.
lon SO^ d tha trong m i trng v ch y u p h t sinh t c c m u i M g S 0 , K S 0 .
7

Nhu cu ca thc vt i vi cc nguyn t a lng ln hn. Nguyn t a lng c

nng cao nht trong c c m i trng n u i c y m v t b o thc vt. N h n chung c
phn anion v cation ca c c n g u y n t a lng u quan trng i v i t b o thc vt.
V d n h K N O c K

v N O " l cn thit. Trong n h m c c n g u y n t a lng, c c

m u i c cha n i t c h y u dng k a l i nitrat, amonium hoc canxi nitrat.

C c n g u y n t a lng
+ Nit (N):
T h n h phn c h n h ca hu ht c c m i trng l n i t v c d i dng nitrat ( N O " )
hoc amonium ( N H 4 ) . C c m u i c d n g p h b i n l k a l i nitrat (KNO3), nitrat amon
( N H N 0 ) v canxi nitrat ( C a ( N 0 ) . 4 H 0 ) .
4

N h n g hp cht n y cung c p n i t v c

cho thc v t t n g hp c c p h n t cht hu c phc tp.

39

A m o n i u m c h y u c d tr r n h n g u n n i t h u c . N i t r a t c t h c v n
c h u y n theo m c h x y l e m n c c b phn ca c y , t i n s tham gia v o q u t r n h
n g hoa nit. Nitrat c t h c d tr k h n g b o v thc h i n chc n n g quan trng
trong vic i u chnh s t h m t h u v c n bng i o n ca c y t r n g .
S bin i nitrat:

Nitrat k h n g t h s d n g ngay l p tc sinh t n g h p c c

cht h u c phc tp m trc t i n c n p h i c k h t h n h amoniac. Phn n g d i n ra

n h sau:
NO" + 8H + 8e~ -> NH + 2 H 0 + OH"
+

Phn ng ny c thc hin qua 2 bc nh hai enzym: nitrat reductase v nitrit

reductase. Trc tin, nitrat c b i n i t h n h nitrit n h nitrat reductase. T i p theo,
nitrit b k h t h n h amoniac n h enzym nitrit reductase. S b i n i ca nitrat t h n h n i t r i t
d i n ra trong t b o cht. Trong h u ht c c c y trng, s k h nitrat c t h d i n ra c l
v n h c h i . S k h nitrat d i n ra mc n o p h thuc r t l n v o c c n h n t n h :
loi, t u i c y , nng nitrat. C t h , c c loi c y t h n g c k h n n g k h nitrat r t cao.
K h i nng nitrat t h p , s k h h u n h d i n ra r . N g c l i , n u n n g nitrat cao
th q u trnh n y d i n ra c l. C c cation k t hp v i nitrat c vai t r quan t r n g trong
vic h p thu nitrat. N u cation l K th hot n g ca enzym nitrat reductase r t h p
+

v nitrat s c v n c h u y n ln c c n h c h i ca c y . T r o n g t r n g h p cation l C a

2+

th s k h r l i d i n ra m n h h n .
S k h nitrit t h n h N H n h enzym nitrit reductase d i n ra trong l c y , trong
3

i n t c n thit cho phn ng n y c cung c p t s k h c c hp c h t st trong h

thng quang hp.
S kh nit cha trong cc hp cht: A m o n i u m v amoniac l n h n g c h t c i
v i thc vt ngay nng t h p . D o c h n g c n c c h u y n hoa tht nhanh t h n h
c c hp cht p h n t lng n h c cha N n h : asparagin, arginin, allantoin v betain.
Sinh tng hp glutamin v glutamat d i n ra c r v n h c h i l c c q u t r n h c b n
trong s c h u y n hoa amonium.
Bn cnh s k h c t n h ca amonium v amoniac, c c hp c h t cha n i t p h n t
lng t h p c n c m t s chc n n g k h c . Chc n n g quan t r n g n h t l cung c p N
trong c c lin k t h u c v n h m - N H c thc vt h p t h n h n g u n N h u c cho
2

q u trnh sinh tng hp c c amino axit v protein. C c hp c h t p h n t lng n h n y

c n c s dng l m cht mang cation n h M n , Cu v n c h u y n c c cation qua h
thng m c h d n trong c y . N g o i ra, c h n g c n n h m t kho d tr n i t d tha. N g c
l i v i con n g i v n g vt, thc vt k h n g t h b i t i t c c hp c h t n i t h u c n h
ur n h n g n h c c h n y cho p h p thc vt d tr c n i t h u c.
+ Phospho (P):
Phospho dng HPO4" c h p t h n h h thng r ca thc v t v n g c l i v i
nitrat, sunfat, n k h n g b k h . N c t h c m t trong thc vt d i dng p v c hoc
40

d n g hp cht este (R-O-P). N n g lng thu c k h i g i i p h n g m t n g u y n t p k h i

c c lin k t (cao n n g lng) l rt quan trng i v i q u trnh trao i cht ca t b o .
Axit

nucleic:

p l m t n g u y n t thit y u trong c u to ca A D N v A R N n i

c c om p h n t axit ribonucleic to t h n h i p h n t.
Phospholipid:

Phospholipid m n g sinh hc cng c cha m t lng l n p . Trong

nhng phospholipid n y , p (qua lin k t este) to n n cu n i gia diglyxerit v i m t

amin, axit amin hoc m t ru. Phospholipid c m t u h o nc, p h n t diglyxerit v
m t u k nc c cha PO^". C hai u c chc n n g quan trng trong vic g i n
nh m n g t b o . M n g t b o bao g m hai l p phospholipid n g h p l i to t h n h l p
m n g k p l i p i d . u a nc ca l p phospholipid quay ra n g o i h n g v p h a c c p h n
t nc trong k h i u i k nc l i quay v o p h a trong gia hai l p ca m n g t b o v
tng tc l n nhau.
Qu trnh

chuyn

hoa nng

lng:

Phospho d n g l i n k t este cao n n g (C-P)

rt quan trng i v i q u trnh chuyn hoa n n g lng v tng hp sinh hc thc vt.
n g vai tr quan trng h n na l c c lin k t cao n n g gia hai n g u y n t p n h trong
p h n t A T P (P-P = 30 kJ). N n g lng g i i p h n g trong sut q u trnh t h y p h n
glucoza, q u trnh o x i hoa, phosphoryl hoa hoc quang hp c s dng tng hp
A T P . Ngc l i n n g lng n y k h i cn l i c g i i p h n g ra qua phn ng t h y p h n
A T P t h n h A D P v p v c. V v y A T P c chuyn hoa v tng hp m i lin tc. M t
g m n h r a n g trong giai o n trao i cht m n h c t h tng hp 5g A T P / n g y .
Vng

d tr p (phosphat):

Trong t b o bao g m hai v n g d tr phosphat k h c

nhau. V n g trao i , ch y u phosphat dng este, n m trong t b o cht v ty t h .

V n g k h n g trao i , ch y u l dng p v c, n m trong k h n g b o . N u ngng cung
cp p cho c y , nng p v c trong k h n g b o ngay l p tc s g i m trong k h i v n g
trao i tc g i m c h m hom n h i u . K h i t n g c n g cung c p p , nng p cha trong
cc c quan ca t b o cng t n g theo, tuy n h i n k h i t n g q u mc b n h t h n g ti ch
c p v c trong k h n g b o t n g ln. V v y c t h n i , p d tha c d tr k h n g
b o d i dng p v c.
Cc enzym:

p v c c n g c k h n n g i u chnh t t trong n h i u q u t r n h trao i

cht ca thc v t . V y n n s p h n b p l cn t h i t i u chnh q u t r n h trao i

cht ca t b o . c chua, s g i m p v c k h n g b o trong t b o cht k c h t h c h
hot t n h ca e n z y m p h o s p h o r u c t o k i n a s e . E n z y m n y l e n z y m quan t r n g trong s
p h n g i i c c h t ca phn ng t h y p h n glyco v l m t n g s h h p ca t b o trong
q u t r n h c h n . C n g trong t h i gian n y , s t h i u ht p c t h l m c h m q u t r n h c h n
ca q u .
Trong q u t r n h tng hp t i n h bt ca lc l p , p c n g n g m t vai t r quan trng.
Ch v i nng t h p , p v c c t h g y c c h q u t r n h t n g hp t i n h bt. S d
n h vy l do ADP-gluco-pyrophosphorylase, enzym quan t r n g n h t trong q u t r n h
tng hp t i n h bt, b k m h m b i p v c v c k c h t h c h n h c c triossephosphat.
V t h , s c n bng gia c c hp cht c cha p l r t quan t r n g trong i u hoa t n g

41

hp t i n h bt lc l p . N g o i ra, p v c c n g tham gia q u t r n h n y theo m t con

n g k h c . C c p h n t v n c h u y n phosphat m n g t bao s m a n g p v c v o t
b o v c c triosephosphat ra n g o i t b o , l m n n g p v c trong l c l p t n g ,
triosephosphat g i m . i u n y l i t c n g n q u t r n h t n g h p t i n h b t theo c c h
t r n h b y t r n . Ribuloza biphosphat (RuBP), v i vai t r n h m t c h t n h n C 0 , l
2

hp cht quan t r n g trong s c n h C 0 . S ti t n g hp n y i h i p h i c c c

2

triosephosphat. K h i n n g p v c cao s k c h t h c h g i i p h n g c c triosephossphat

ra k h i lc l p , g y t h i u ht c c c h t n y , do l m k m h m q u t r n h c n h C 0 .
2

N g o i ra, phospho c n quan trng trong v i c i u k h i n hot n g ca n h i u e n z y m

k h c . N n g phospho t i u cho s sinh t r n g ca thc v t l 0,3 - 0,5 g / k g t r n g
lng k h . S t h i u p l m c y c h m l n , l c y c m u xanh t h m do t r o n g t h i g i a n
b t h i u p, s p h t t r i n ca l c h m h n s t n g hp d i p l c t n n l m t n g n n g
d i p lc t trong l c y .
+ L u h u n h (S):
L u h u n h n h SO4" c h p t h r c y v i tc c h m . G i n g n h nitrat, lu
h u n h p h i c k h trc k h i s dng sinh tng hp c c hp cht c cha l u h u n h
n h amino axit, protein v enzym. L u h u n h d n g c h a k h c k t hp trong c c
sulpholipid v c c polysaccharid.
S ng
hot hoa

hoa lu hunh:

gc

SO4"

Bc u tin trong q u t r n h n g hoa lu h u n h l s

n h enzym A T P sulfurylase. Phn ng n y t o ra adenosine

phosphosulfate (APS) v p v c. T i p theo l hai q u trnh hoa hc h o n t o n k h c

nhau. M t q u trnh k h n g d i n ra s k h lu h u y n h m t o l i n k t v i c c
polysaccharid trong sulpholipid. Trong q u t r n h t h hai, lu h u n h c k h t h n h
n h m -SH ( n h m thiol) v n h m s u l f u r y l ca APS c v n c h u y n t i gluthantione
(Glut-SH). Sau n h m -SH c v n c h u y n t i cho acetylserine v p h n t c h t h n h
acetat v cystein. Cystein l sn p h m b n u t i n trong q u t r n h n g hoa v l t i n
cht ca tt c c c hp cht hu c c cha lu h u n h trong thc v t , v d n h : protein,
co-enzym, c c hp cht trao i cht t h cp... Q u trnh n g hoa lu h u n h c h y u
d i n ra lc lp. K h i t h i u lu h u n h , sinh tng hp protein b k m h m , lng d i p lc
t trong l c y b g i m st.
Cc protein:

L u h u n h c m t trong c c protein c cha cystein v m e t h i o n i n . C

hai axit amin n y u l t i n cht ca tt c c c hp cht c cha lu h u n h trong thc

vt. L u h u n h , c u t hp t h n h ca n h i u coenzym v c c n h m prosthetic, c chc
n n g quan trng trong r t n h i u phn ng o x i hoa k h , c b i u d i n n h sau:
R-SH + HS-R - > R-S-S-R
Gc R c t h l phn c n l i ca p h n t cystein n h n g c n g c t h l tripeptid
gluthatione. Gluthatione tan c trong nc v do n n g vai tr n h m t h o x i
hoa k h lc l p v dch b o . Cu lu h u n h gia hai p h n t cystein r t quan t r n g
trong c u trc bc ba ca p h n t protein v hot n g ca enzym. N h m -SH, n h
42

cp trn c trong t h n h phn ca c c coenzym v APS, to t h n h m t phn ca n h m

chc n n g trong p h n t enzym.
Cc

metallothionein:

CC hp cht p h n t lng t h p c cha lu h u n h -

metallothionein, t h n g hay c t m thy trong thc vt. H u ht c c cht n y u c

cha cystein. c bit, c c k i m l o i nh: n g , cadimi v k m t h n g l i n k t trong
metallothionein. G n n h chc chn nhng p h n t protein n h n y tham gia v o s bi
tit c c ion k i m l o i trn k h i c h n g d tha, trc k h i lin k t v i n h m chc n n g -SH
ca c c enzym.
Lu

hunh

cha

b kh:

L u h u n h dng c h a b k h l t h n h phn c u to

sulpholipid, l phn t to t h n h c u trc ca m n g sinh hc. L u h u n h t h n g c m t

dng hp cht este ca lu h u n h v n g 6 cacbon, v d n h glucoza. Sulpholipid
c n h i u trn m n g thylakoid ca lc l p v tham gia v o q u n h v n c h u y n ion qua
m n g sinh hc. H n na, s c m t ca sulpholipid t r n m n g t b o chc chn lin
quan n k h n n g chu m u i ca thc vt. M i v c t r n g ca m t s loi nh: h n h ,
t i c h y u c lin quan n s c m t ca c c hp cht c cha lu h u n h d b i n i .
+ Kali (K):
Trong thc vt, K l m t cation c tnh l i n h n g cao, c mc t b o cng n h
+

trong q u trnh v n chuyn qua c c khong c c h d i trong m c h x y l e m hoc m c h libe.

Trong tt c c c n g u y n t , k a l i l n g u y n t c m t v i nng cao nht, t b o cht
t 100-200 m M , lc l p t 20-200 m M .
M u i k a l i c vai tr quan trng trong vic i u chnh t n h t h m ca t b o . i v i
s d n t b o c n g n h c c q u trnh k h c c i u chnh n h sc t r n g ca t b o , K

c vai tr n h m t l o n trung hoa c c lon v c v hu c hoa tan trong dung dch, n g

thi duy tr p H trong k h o n g 7 - 8 l p H t h c h hp cho hot n g ca h u ht c c enzym.
Vai tr ca K+ i vi cc enzym:

K cn t h i t cho hot n g ca n h i u enzym.

+

T r n 50 enzym ca thc v t hot n g v i s tham gia ca K hoc b k c h t h c h b i K .

+

S lin k t ca K

v i enzym g y ra s thay i c u h n h k h n g gian ca enzym, do

l m t n g i lc ca enzym v i c cht. K h i t h i u K

n g i ta t h y c s t n g nng

ng hoa tan v c c hp cht cha nit, k m theo s g i m n n g tinh bt, c h y u do

vai tr thit y u ca k a l i i v i vic i u k h i n hot n g enzym trong q u trnh trao
i cacbon. E n z y m ATPase m n g t b o cng chu s t c n g ca K . c y trng
+

t h n h , K cn cho q u trnh tng hp protein. K c n cn t h i t trong q u trnh dch m v

+

tng hp t R N A ribosome. S tng hp ribuloza biphosphat cacboxilase cng ph

thuc rt m n h v o nng K . y c n l ion cn t h i t cho hot n g v tng hp
+

enzym nitrat reductase.

Bn cnh vai tr trong hot n g ca nhiu enzym, K
ion v p H ca lc l p trong quang hp. K
dng H

c n i u chnh s c n bng

l lon trung hoa quan trng nht cho s a

qua m n g thylakoid. l o n n y cng c m t trong s to t h n h gradiep p H m n g

t b o cn t h i t cho q u trnh tng hp A T P . S t n g n n g K d n n s t n g c n g

+

q u trnh quang hp, h h p v hot n g ca enzym ribuloza biphosphat cacboxilase.

43

S dn ca t bo: S t n g k c h thc ca k h n g b o trung t m o n g t b o l m t

q u trnh quan trng trong s d n t b o . h n h t h n h k h n g b o , u t i n l s t n g
k c h thc l n ca t h n h t b o , t i p theo k h n n g t h m t h u ca k h n g b o t n g
ln. i u n y c t h t c n h s tch t K

g y ra s t n g m n h v t h t c h ca

k h n g b o do tnh t h m . G A v K d n g n h c t c d n g h tr nhau trong vai tr l m

+

t n g c h i u cao c y .
S cn bng

ion: K c vai tr quan trng cho vic duy tr c n bng l o n . N trung

+

hoa c c anion k m l i n h n g trong t b o cht v r t n h i u c c anion l i n h n g trong

m c h x y l e m , libe v k h n g b o . Trong q u trnh trao i nitrat, K c chc n n g c h y u
+

l v n c h u y n ion N O " qua nhng khong c c h d i trong m c h x y l e m hoc d t r trong

k h n g b o . Sau q u trnh k h nitrat trong l c y , lng K c n l i c s d n g tng
+

hp c c axit hu c trung hoa ion K . C c m u i k a l i ca c c axit h u c n h k a l i malat

+

c v n chuyn t i r , sau K c t h nhn ion nitrat t b o r v v n c h u y n c h n g

+

qua m c h x y l e m .
+ Canxi (Ca):
Ca l t h n h phn quan trng ca t h n h t b o v m n g t b o . S lng l n C a

2+

gn

trn t h n h t b o n g vai tr c h y u trong vic c n g c v n g chc cho t h n h t b o

v i u hoa c u trc m n g t b o . S v n c h u y n C a

2+

t n g o i qua m n g v o t b o

cht, trong phloem cng n h t t b o n y sang t b o k h c l r t h n c h .

lon Ca

2+

t do c m t trong t b o n n g r t t h p , k h o n g l | i M c t c d n g n g n

chn s k t ta p v c. D o h m lng l o n C a
tranh v i lon M g

2 +

2+

trong t b o t h p n n k h n g c s c n h

v v tr g n cation v t r n h l m bt hot enzym. l o n C a

d i c h u y n qua m n g t b o theo m t c h i u ( C a
v o c ) , do m bo c n n g ion C a

2+

2+

2+

ch c th

ch ra n g o i t b o c n h n g k h n g
n i b o t h p . c b i t trong c c t b o

l, c m t lng l n canxi lin k t v i c c k h n g b o . Canxi c n t h i t cho s t h i t l p

c n bng lon n h trung hoa c c anion h u c v v c. H u n h canxi d n g l i n k t to
m u i ocxalat. M c d hp cht n y k h tan n h n g n c vai tr duy tr n n g l o n C a

2+

thp trong lc l p v t b o cht. M u i canxiocxalat c n c chc n n g i u c h n h s

t h m t h u ca t b o . Canxi c vai tr quan trng trong q u t r n h n h n l n ca t b o v
r. N g o i ra s p h t t r i n ca ng p h n cng ph thuc v o canxi, y l q u t r n h c
nh h n g n h canxi ngoi b o . I A A tham gia v o q u trnh v n c h u y n canxi. C h t c
c h auxin n h T I B A cng c c h s p h n p h i C a

2+

trong thc v t l m x u t h i n s t h i u

ht canxi.
Vai tr ca Ca i vi thnh

t bo: Pectin l t h n h phn quan trng ca m n g l i n

k t gia c c b b o v i nhau v c p h n huy n h enzym polygalacturonase. T u y

n h i n , Ca c c h m n h hot n g ca polygalacturonase. H o t n g m n h ca e n z y m
n y c ghi nhn k h i t h i u Ca. N u nng Ca c th hu ht c c pectin s t n t i
d i dng m u i canxipectat. N h v y , t h n h t b o c k h n n g chng chu t t i v i
hot n g p h huy ca enzym polygalacturonase. S c m t ca l o n C a
quan trng trong vic n g n chn s x m n h i m n m .

44

2+

c n g c vai t r

lon Ca

2+

c tc n g l n n s n nh ca m n g t b o . S t h i u l o n C a

2+

s l m

t n g k h n n g thot ra n g o i m n g t b o ca c c hp cht p h n t lng n h . M n g t

b o c t h s b p h n huy h o n t o n k h i t h i u ht n g h i m trng lon C a . l o n C a
2+

2+

c kh

n n g l m n nh m n g t b o t h n g qua s t n g tc v i c c n h m phosphat, cacboxil

ca hp cht phospholipid v protein c m t trong m n g t b o .
'

Cc enzym:

K h c v i m a g i l n g u y n t t h a m gia v o q u t r n h h o t hoa ca r t

n h i u e n z y m , c a n x i ch c t c n g l n m t vi e n z y m n h : amilase v ATPase.
Canxi c h y u k c h t h c h c c e n z y m m n g t b o , m hot n g ca n h n g e n z y m
n y c quy n h n h c u t r c m n g . T u y n h i n , l o n C a

2 +

cng c tc dng km

h m m t s e n z y m ca t b o c h t . C a l m o d u l i n t r o n g t b o c k h n n g hot hoa
c c e n z y m n h phospholipase b n g c c h t o t h n h phc ca C a - c a l m o d u l i n v i
2+

enzym. N g o i -, n g i ta c n cho r n g c a l m o d u l i n c vai t r t r o n g v i c v n c h u y n

ion C a

ti khng bo.

2+

+ Magi (Mg):
Mg

2 +

l m t lon r t l i n h n g c k h n n g h n h t h n h phc v i c c n h m chc n n g

k h c nhau.
Vai tr ca Mg2+

i vi quang

hp: M g

2 +

l n g u y n t trung t m trong p h n t

chlorophyl ca h quang hp v n . Trong p h n t chlorophyl, c c photon c h p t h

to ra d n g i n t, t to ra A T P v N A D P H n g vai t r quan t r n g i v i c
nh C 0 . N u M g

2 +

c m t v i n n g t i u t h k h o n g 10-20% i o n M g

c c nh lc l p . N n g cao c c ion M g

2 +

v K

2 +

trong l

l c n t h i t duy tr p H

k h o n g 6,5 - 7,5 t r o n g l c l p v t b o c h t , tri v i k h n g b o p H c h v o k h o n g

5 - 6. Trong m t c h n g mc n o , p H x c n h c u t r c ca protein v enzym n n n
nh h n g n q u t r n h sinh tng hp protein v chc n n g ca lc l p .
Vai tr ca Mg2+

i vi hot tnh ca cc enzym:

Mg

2 +

l lon cn t h i t cho c u

trc bc ba ca n h i u phc enzym - c cht v n to ra dng c u t r c k h n g gian p h

hp gia enzym v c cht. M g
k h c nhau. M g

2 +

2 +

tham gia v o q u trnh tng hp protein v i n h i u cp

to t h n h cu n i gia c c d i n v ca ribosome. K h i t h i u

M g , c c d i n v s b t c h ra v q u trnh tng hp protein b ngng l i . S hot

2 +

ng ca c c enzym nh: A R N polymerase tham gia v o q u trnh sinh tng hp A R N

i h i p h i c m t M g , do t h i u M g
2 +

2 +

s k m h m sinh tng hp A R N . l c y ,

25% protein tng s n m trong lc l p , nu t h i u M g

2 +

th ngay l p tc c u t r c v chc

n n g ca lc l p b nh h n g .
Mg

2 +

c n quan trng trong hot n g ca enzym ribulose biphosphat cacboxilase.

y l m t enzym ph thuc n h i u v o p H v M g . L i n k t ca M g
2 +

t n g i lc v i c cht C 0

2 +

v i enzym l m

v V m a x .

Vai tr ca Mg2+ i vi chuyn

hoa nng

lng:

M g l m t cht k h n g t h t h i u

trong q u trnh chuyn ho n n g lng ca thc vt do vai tr quan trng ca n i v i

sinh tng hp A T P ( A D P + p v c = A T P ) , c b i t l lc l p . T r o n g q u t r n h n y ,
Mg

2 +

to t h n h c u n i gia enzym v A D P . N g o i ra, M g

2 +

c n c k h n n g to phc
45

v i A T P . E n z y m ATPase v n c h u y n c c n h m phosphoryl cao n n g , c u n g c p cho

protein hoc n g . M c d M g

2 +

c n h i u chc n n g n h v y n h n g h u n h n l i

t n t i d n g d tr trong k h n g b o . T i y , n n g vai t r n h m t i o n t r u n g hoa

v i c c anion h u c v v c trong v i c c n b n g i o n .
c n g u y n t v i lng (Fe, B, C l , Co, Cu, M n , M o , Zn...)
+ S t (Fe):
Trong c y , st c h y u c gn v i c c phc cht. H m lng F e , F e
2+

thp ( l o -

1 0

raM). H u h t thc vt ch h p thu F e . D o , F e

2+

3+

t do r t

3+

c n c k h t h n h F e

2+

b m t r trc k h i n c c h u y n v o trong t b o cht (ch m t s l o i c l h p t h u

st c h y u d i dng F e ) .
3+

Trong k h i v n c h u y n i xa, qua m c h x y l e m ca c y , st c h y u c d i c h u y n

d i dng hp cht st-cacbonhydrate ( dng Fe -citrate hay d n g phc h p st3+

peptid). Chc n n g c h n h ca st trng thc vt l to c c l i n k t st.

C c chc n n g c b n n h m t h thng o x i hoa k h t h u n nghch c b i u d i n
trong phn ng d i y :
Fe(H) <-> Fe(m) + e
Cc hemoprotein

(cc protein

cha st): C c hemoprotein c b i t n n h i u n h t

l c c cytochrome, c cha m t phc h st-porphyrin. C c cytocrome to t h n h m t

phn h thng o x i hoa trong c h u i t r u y n i n t l c l p v t y t h ca t b o thc v t .
Chc n n g ca c c cytocrome n h cht trung gian cho i n t, c n cho q u t r n h
k h nitrat t h n h nitrit n h enzym nitrat reductase trong q u t r n h n g hoa n i t .
Trong q u t r n h c nh n i t c y h u , c c cytocrome l trung gian trong c h u i
t r u y n ' i n t qua c c i n t c t r u y n i c u i c n g k h N t h n h N H .
2

C c catalase v peroxidase tham gia v o q u t r n h quang h h p , t h y p h n n g

v k h c ca hydrrogen peroxid, theo c n bng sau:
2H 0 - catalase -> 2H 0 + 0
2

Hydrogen peroxit c to ra trong q u trnh k h superoxit n h m trung hoa c c gc

superoxit. Hydrogen peroxit, n lt n l i c trung hoa n h enzym catalase.
C c peroxidase c r t n h i u trong t b o thc v t . T h n h t b o g n peroxidase x c
tc cho q u t r n h t r n g hp ca phenol v i l i g n i n . R cha lng l n peroxit v c v a i
tr h p thu st ca c y . L n g phenol d tha k h i t h i u st s c t i t ra n g o i .
Cc protein

st-lu

hunh:

N h m protein cha st t h hai l c c protein st-lu

h u n h . St c gn trong n h m t h i o l (-SH) ca cystein. F e r r i d o x i n l protein cha st

lu h u n h p h b i n nht v l cht mang trong c c phn ng t r u y n i n t c x c t c
b i nitrit reductase, sulphat reductase, q u trnh tng hp N A D P * trong quang hp v
k h n i t c thc h i n n h phc h nitrogenase. Ba l o i protein st - l u h u n h k h c
46

nhau, hot n g lin t i p nhau, u n m trong c h u i t r u y n i n t ca phc h

nitrogenase.
B n cnh hai n h m n y , thc v t c n c nhng enzym k h c c cha st. N g u y n t
n y cn cho c c phn ng o x i hoa k h v s nh v ca c c phc h p enzym - c cht.
St rt quan

trng

trong

sinh

tng

hp chlorophyl:

Trong l xanh 8 0 % st n m

trong l c l p . K h i t h i u st, t o n b st s t p trung l .

Trong l non, t h i u st s d n n s g i m nhanh nng chlorophyl do q u trnh
tng hp protein b n g n g l i . S lng ribosom c n g g i m m n h .
T h i u st r k o theo nhng thay i h n h t h i . S d i r g i m n h n g d i n tch v
s lng l n g r t n g .
+ Bo (B):
Bo c h p t h u b i r v c c h u y n t i c c b phn ca c y n h c c x y l e m .
c c m n g t b o , Bo c m t c h y u dng lin k t este. K h n g c bt c m t enzym
bit n o c cha hoc c hot hoa b i Bo.
C nhng ch d n cho b i t Bo c m t trn hoc b n trong c c m n g , c t h n h
h n g t i hot n g ca c c enzym lin k t m n g . C c chc n n g c h y u ca Bo l
ngoi b o , c p h t sinh trong q u trnh hoa g ca t h n h t b o v s p h n hoa x y l e m .
Thnh

t bo: N h n g lin k t este n g - Bo l m t phn c u t r c ca c c

hemicellulose trong t h n h t b o . H u h t Bo trong thc v t t n t i dng este trong

t h n h t b o ca c y . Y u c u v Bo i v i c y hai l m m cao h n so v i c y m t l
m m . C t h g i nh rng Bo, cng ging n h canxi, c chc n n g i u hoa q u trnh
tng hp t h n h t b o cng n h n nh c c t h n h phn ca t h n h v m n g t b o .
S t h i u h t Bo ngay l p tc c c h q u trnh p h t t r i n c h i u d i ca c c r s cp
v th cp. H n t h , Bo g p phn i u hoa q u trnh trao i phenol v tng hp l i g n i n .
+ ng (Cu):
n g l m t cation hoa tr 2 v c h p thu v o c y d i d n g C u
phc cht ca n . N u nng C u

2 +

2 +

hay d i dng

v phc n g t n g n g nhau, c y d n g n h a

ion ng t do h n .
Trong x y l e m v phloem, n g h u n h t n t i d i dng phc v c h y u l dng
m t phc amino a x i t - n g .
Trong t b o , n g c h y u l t h n h phn ca phc h enzym v r t quan trng
trong c c phn ng o x i hoa k h [ ( C u ) / ( C u ) ] c thc h i n n h nhng enzym n y .
2+

T h i u n g l p tc d n n s g i m hot ca c c enzym cha n g .

Vai tr ca ng i vi quang

hp: K h o n g 5 0 % n g trong lc l p c gn v i

plastocyanin, gia c h u i truyn i n t, gia quang h v quang h l i , c cha

n g u y n t n g trn p h n t. Trong trng hp t h i u n g , nng c c plastocyanin
s b g i m . Cng ging n h plastocyanin, c c plastoquinone n g v a i t r quan trng
trong t r u y n i n t gia quang h v quang h l i . K h i n g b t h i u , m n g l c l p s
47

t h i u 2 protein i u hoa

chuyn

n g ca

c c plastoquinone. t n g hp

cc

plastoquinone cn phi c enzym laccase, y l m t enzym cha n g v hot n g ca

n s b g i m ngay k h i t h i u n g . D o , h i n tng t h i u n g nhanh c h n g k o theo
h i n tng g i m quang hp.
Enzym

super

oxide

dismutase:

n g c n g v i k m l m t p h n ca e n z y m super

oxide dismutase (Cu-Zn.SOD), n g vai tr quan trng trong q u t r n h t r u n g hoa

gc

anion superoxide 0 " hot tnh m n h c to t h n h trong q u t r n h quang h h p . B n

cnh Cu-Zn.SOD, m t SOD cha mangan cng c trong t b o .
SOD hoa g i i c ca gc 0 ~ hot n g t h n h H 0
2

v O , n h bo v t b o
z

trc k h n n g p h huy ca gc n y . SOD c n g v i catalase phn ng n h sau:

0 + -> O2 (superoxide)
2

O2 + O2 + 2H -> SOD -> H 0

2

2H 0 -> catalase -> 2H 0 + 0

2

Superoxide c g i i c n h SOD v ngay sau g i i p h n g H 0

2

t h n h o x i v

nc n h catalase.
C c e n z y m SOD cha n g - k m c h y u c t m t h y c h t n n stroma ca
lc l p . T r o n g l non, 9 0 % S O D t p t r u n g lc l p v c h c 4 - 5 % t y t h . T h i u
n g s x y ra n h n g thay i trong c u t r c lc l p , i u n y t h h i n r chc n n g
bo v ca n g .
n g cng n g vai tr quan trng trong c h u i t r u y n i n t ca ty t h n h
cytochrome oxidase c cha 2 n g u y n t n g v 2 n g u y n t st.
+ Mangan (Mn):
Mangan t n t i trong thc v t dng ion M n

2 +

k h n g l i n k t , hoa tr hai v d n g

n y n c chuyn t r qua m c h x y l e m n c c phn k h c ca c y .

N g u y n t n y lin k t cht v i m t s l o i protein cha k i m l o i (metalloprotein),
hoc n h m t t h n h phn c u trc ca enzym hoc n h m t phn trong h o x i hoa k h
[Mn(II)/Mn(III)].
Phn

ng HUI: Mangan c hai chc n n g quan trng trong thc vt. l o n n y l i n

quan n phn ng ban u c g i l phn ng H i n ca quang h n , trong n c

c p h n l y t h n h o x i v c c photon, theo p h n g trnh sau:
2H 0 - 0 + 4H + 4e+

C g i thit cho rng 4 p h n t mangan l t h n h phn ca m t protein, x c t c cho

q u trnh quang p h n ly nc. C c electron c g i i p h n g ra t i p tc c h u y n t i m a g i
cha trong phc h 680, trung t m ca quang h l i .
48

Enzym

super

oxide

dismutase:

Cho n nay m i ch c m t vi l o i enzym cha

mangan c p h n l p . E n z y m c cha mangan quan trng nht l M n - S O D (xem phn

v o n g b i t t h m t h n g tin v SOD). Cng g i n g n h n g , n u t h i u mangan, s
x y ra c c thay i trong c u trc ca lc lp, t h h i n r nht h thng bo v ca
mangan.
+ Coban (Co):
Coban n g vai tr quan trng trong q u trnh c n h n i t r c y h u . Coban
l t h n h phn cn thit ca enzym cobalamin. Co ( I U ) l t h n h phn k i m l o i nh v
gia 4 n g u y n t nit trong c u trc porphyrin.
Rhizobium

Ba h thng enzym ca v i k h u n

c bit t i c cha Co. N g i ta thy rng c m i lin h gia nng Co

vi s c i n h nitrogen v s pht trin r c. Co c vai tr trong q u trnh tng hp

methyonine v i khun, tng hp ribonucleotid v enzym methymalonyl-coenzym A mutase, m t enzym cn thit cho s tng hp leghemoglobin.
K h n g ai bit chc rng l i u Co c g i vai tr g thc v t bc cao hay k h n g . Ch
c m t enzym ph thuc cobalamin c b i t t i l leucine -2,3-aminomutase
khoai ty. i v i thc v t bc thp, Co l y u t c n t h i t v c m t trong m t s c u
trc d i t b o v thylakoid lc l p .
+ Molybden (Mo):
M o l y b d e n ch y u t n t i dng M 0 O 4 " , m t dung dch g i n g n h nc. Trong
m i trng axit y u , l o n molybden tuy thuc v o axit c t h n h n hoc 2 proton
theo p h n g trnh sau:
MoO*- - HMoO; -> H2M0O4
Molybden c kh nng chuyn vn qua mch xylem v phloem nh lon Mo04~.
Nitrogenase: Mt s enzym s dng Mo nh mt co-factor. Hai loi enzym c
molybden c m t n h i u nht l nitrogenase v nitrate reductase.
Nitrogenase lin quan n q u trnh c nh nit trong n t sn r c y h u n h
vi khun

Rhizobium:

N + 8H + 8e~ -> 2NH3 + H

+

M o l y b d e n lin quan trc t i p n q u trnh k h N . P h n t nit c g n v i

2

n g u y n t m o l y b d e n trong phc h nitrogenase. M i p h n t n i t g n v i hai n g u y n t

molybden m c h n h c h n g l i l m t phn ca p h n t protein st - molybden. Sau q u
trnh hot hoa phc h enzym nitrogenase s dng A T P , phc h st - molybden thay
i c u trc ca n . Da trn s thay i hp l n y , q u t r n h k h N x y ra.
2

Nitrate

reductase:

Nitrate reductase k h nitrat t h n h n i t r i t trong q u t r n h n g hoa

nit t b o thc vt. Nitrate reductase cha p h n t heme-st v 2 n g u y n t

molybden. E n z y m n y x c tc cho q u trnh k h nitrat t h n h nitrit. S hot n g ca
49

nitrat reductase b g i m m n h k h i t h i u molybden n h n g c t h k h i phc nhanh c h n g

ngay k h i t h m molybden v o m i t r n g .
+ Km (Zn):
K m c h r h p thu d i dng Z n . T r o n g m c h x y l e m n c c h u y n v n
2 +

d i dng i o n Z n

hoc m u i k m ca m t axit h u c. N g u y n t n y l m t h p p h n

2 +

k i m l o i ca m t s enzym. N c t h l m t c o a c t o r c u t r c c n g n h cofactor i u
hoa ca phc h enzym.
Cc

enzym:

Thc vt c m t s l o i enzym cha k m , bao g m c

enzym

dehydrogen hoa r u trong v n g m p h n sinh ca c y .

T r o n g phc h enzym SOD, Z n lin k t v i Cu thay cho n g u y n t n i t t histidine
(xem t h m v SOD trong phn n g ) .
E n z y m cacbon anhydrase c n h C 0 , theo c n bng sau:
2

C0 + H 0 <-> Heo; + H

Phn ng ny gip thc vt c th d tr C0 di dng HCO3 mt cch thun

2

nghch. Sau k h i c h u y n t h n h C 0 , HCO3 c t h s d n g l m c c h t cho enzym

2

ribulose biphosphate cacboxilase. E n z y m n y c 6 t i u p h n d i n v , m i t i u p h n

c n g u y n t k m gn v o v c t h t m t h y trong lc l p v t b o c h t .
Tng

hp protein:

K m r t quan trng trong q u t r n h t n g h p protein. T h i u k m

g y nh h n g l n n q u trnh tng hp protein. S t p trung ribosom v s t c h l y

c c t i n cht ca protein n h c c amino axit v c c a m i n c n g n h hot n g ca A R N
polymerase c t h x y ra n h k m . D i i u k i n t h n g t h n g , A R N polymerase cha
2 n g u y n t Z n , to ra c u t r c i n h n h ca enzym. H n na, c m t m i quan h
nghch t n g ng gia nng Z n v hot n g ca A R N s e . N n g Z n t h p s l m
t n g hot n g ca A R N s e .
Tng

hp IAA: T h i u k m cng p h hng q u t r n h t n g h p i n d o l acetic a x i t trong

c y (Indol > Trytophan -> I n d o l Acetic A c i d ) .

K m n g vai t r quan trng trong tng hp tryptophan, m t t i n c h t ca I A A . V
d , t h i u k m trong n g c t h thay t h bng c c h b sung tryptophan.
+ Chlor (Cl):
Chlor c thc v t dao n g t 70 - 700 m M trong k g t r n g l n g k h (2000 -r
20000 mg/kg trng lng k h ) . Chlor c h p thu d i dng c r v r t c n g trong
c y . Chc n n g c h n h ca ion n y l i u hoa t h m t h u v b sung c c c h t mang.
Trong lc l p c cha h m lng chlor l n . N g i ta cho r n g chlor n g vai t r h t
sc quan trng trong quang hp.
C c l c l p ca rau c h n v t v c c i n g cha c h l o r n n g x p x l O O m M
n h n g trong l, c h c d i l O m M , cho t h y u t h r r n g ca c h l o r t p h p t r o n g
lc l p .
50

Nng

lc thm

thu:

l o n C l i u hoa s n g , m k h k h n g . T n h trng t h i u

chlor l m k h k h n g m m i , c t h g y tnh trng m t nc n g h i m trng.

Chlor r t quan trng trong i u hoa n n g lc t h m t h u ca c c k h n g b o v v i
c c q u trnh lin quan n sc trng.
S trao i nit: Chlor hot hoa enzym tng hp asparagin, m t enzym quan trng
trong q u trnh trao i nit. E n z y m n y chuyn hoa glutamin t h n h asparagin v axit
glutamic. Trong i u k i n c c r , tc ca phn ng t n g ln g p 7 l n . B i t h , chlor
thc h i n m t chc n n g quan trng trong q u trnh trao i n i t c c loi thc vt
s d n g asparagin n h cht mang.
2.5.2.2. Cc vitamin
T t c c c t b o c n u i c y u c k h n n g tng hp t t c c c l o i v i t a m i n c
bn n h n g t h n g l v i s lng d i mc y u cu. m c sc sinh trng t t p h i
b sung v o m i trng m t hay n h i u l o i v i t a m i n . C c v i t a m i n l r t c n t h i t cho c c
phn ng sinh hoa.
C c vitamin sau y c s dng p h b i n : inositol, thiamine HC1 ( B I ) , pyridoxine
HC1 (B), nicotinic axit, trong vitamin B I l k h n g t h t h i u v c s dng trong
hu ht nhng m i t r n g n u i c y m v t b o thc vt. Linsmaier v Skoog khng
nh vitamin B I l c n t h i t cho s sinh trng ca c y sau k h i n g h i n cu k lng v
s c mt ca n trong m i trng M S . ' C c tc g i k h c cng k h n g nh vai tr r t
quan trng ca B I trong n u i c y m .
Inositol t h n g c n i n n h l m t v i t a m i n k c h t h c h m t c c h tch cc i
v i s sinh trng v p h t t r i n ca thc vt, mc d n k h n g p h i l v i t a m i n cn t h i t
trong m i trng hp. C c v i t a m i n k h c , c b i t l nicotinic axit (vitamin B3), canxi
pantothenate (vitamin B5) v biotin cng c s d n g n n g cao sc sinh trng ca
m n u i cy.
Anh hng ca cc vitamin ln s pht trin ca t bo nui cy in vitro cc loi
khc nhau l k h c nhau hoc t h m c h c n c h i ( g y c ) .
2.5.2.3. Cc cht b sung vo mi trng nui cy m
Nc

da

C n g b u tin v s dng nc da trong n u i c y m thuc v V a n Overbeek v

cng s ( V a n Overbeek cs, 1941, 1942). Sau , tc d n g tch cc ca nc da trong
m i trng n u i c y m , t b o thc v t c n h i u t c g i g h i nhn. Nc da
c x c n h l r t giu c c hp cht hu c, cht k h o n g v cht k c h t h c h sinh
trng (George, 1993; George, 1996). Nc da c s d n g k c h t h c h p h n h a
v n h n nhanh c h i n h i u loi c y . Nc da t h n g c l y t q u ca c c ging v
c y chn lc s d n g ti hoc sau bo qun. Nc da c m t s c n g ty hoa cht
b n d i d n g n g chai sau c h b i n v bo qun. T h n g t h n g nc da c x l
l o i tr c c protein, sau c lc qua m n g l c k h t r n g trc k h i bo qun

51

l n h . T n d protein trong nc da k h n g g y nh h n g n sinh t r u n g ca m hoc

t b o n u i c y , n h n g c t h d n t i k t ta dung dch k h i bo q u n l n h . C h t cn co
t h c lc b hoc l n g d i y b n h r i gn b phn cn.
Nc da t h n g c s dng nng t 5 - 2 0 % .
Bt

chui

B t c h u i k h hoc bt n g h i n t q u c h u i xanh c s d n g trong n u i c y m

m t s c y trng n h phong lan. H m lng s dng v o k h o n g 40g b t k h / 1 . tranh
n g cc, cu b sung bt v o dung dch m t c c h t t r i k h u y u . N c c h i t t
khong 100 g tht q u c h u i xanh c n g c s dng b sung v o m t lt m i t r n g .
Mt s hn hp dinh dng hu c phc tp

khc

Nc ct c chua, dch chit khoai ty nghin, dch chit m c h nha, dch c h i t n m

men (yeast extract), casein t h y p h n (casein hydrolysate) c n g c s d n g l m
t n g s p h t t r i n ca m so hay c quan n u i c y .
Agar
i v i n u i c y tnh, n u s dng m i trng l n g , m c t h b c h m v s cht v
t h i u x y . t r n h t n h trng n y , m i trng n u i c y c l m c l i b n g agar m t l o i t i n h bt c c h t rong b i n v m c c y t r n b m t ca m i t r n g .
Agar t h n g c s dng nng 0,6 n 1 % .
2.5.2.4. Ngun cacbon (C)
n g sucrose (saccharose) l n g u n cacbon c h y u v c s d n g t h n g
x u y n trong hu ht c c m i trng n u i c y m , k c k h i m u n u i c y l c c c h i
xanh c k h n n g quang hp. K h i k h t r n g , n g sucrose b t h y p h n m t phn,
thun l i h n cho c y h p t h . T r o n g m t s t r n g hp, v d n u i c y m c y m t l
m m , n g glucose t ra tt h n so v i sucrose. M thc v t c k h n n g h p thu m t
s n g k h c n h maltose, galatose, lactose, mannose, t h m c h t i n h bt, n h n g c c
l o i n g n y hu n h r t t c s d n g trong n u i c y t b o v m thc v t .
2.5.2.5. Cc cht iu hoa sinh trng
Bn cnh c c cht cung cp dinh d n g cho m n u i c y , v i c b sung m t hoc
n h i u cht i u hoa sinh trng n h auxin, c y t o k i n i n v gibberellin l r t c n t h i t
k c h thch s sinh trng, p h t t r i n v p h n hoa c quan, cung c p sc sng t t cho m
v c c t chc. T u y v y , y u cu i v i nhng cht n y thay i tuy theo l o i thc vt,
l o i m , h m lng cht i u hoa sinh trng n i sinh ca c h n g . C c cht i u hoa sinh
trng thc vt c chia t h n h c c n h m c h n h sau y :
Nhm

cc auxin

(Bng

2.5)

C c auxin c t h l auxin t n h i n hoc tng hp, t h n g c d n g trong n u i c y

m v t b o k c h thch s p h n b o v sinh trng ca m so, c b i t l 2,4-D, to
p h i v tnh, to r,...

52

C c auxin lin quan t i di ca thn, t , c h i c h n h , r... i v i n u i c y m ,

auxin c s d n g cho vic p h n chia t b o v p h n h a r . N h n g auxin d n g rng
ri trong n u i c y m l I B A (3-indolebutiric axit), I A A (3-indole acetic axit), N A A
(napthaleneacetic
axit), 2,4-D
(2,4-D-Dichlorophenoxiacetic
axit)
v
2,4,5-T
(trichlorophenoxiacetic axit). Trong s c c auxin, I B A v N A A ch y u s dng cho
m i trng ra r v p h i hp v i c y t o k i n i n s d n g cho m i trng ra c h i . 2,4-D v
2,4,5-T r t c h i u q u i v i m i trng to v p h t t r i n callus. A u x i n t h n g h a tan
trong etanol hoc N a O H pha l o n g .
V a i tr ca c c cht thuc n h m auxin c k h i q u t d i y :
- K c h thch p h n chia v k o d i t b o .
- C h i n h cung c p auxin g y ra c c h sinh trng ca c h i b n . u t h c h i n h
lm c c h sinh trng ca c h i n c h . N u ngt b c h i n h s d n n s p h t c h i
n c h . N u thay t h vai tr ca c h i n h ( b ngt b ) bng m t l p cht keo c cha
I A A th c h i n c h v n b c c h sinh trng. C c h c c h ca c h i n h lin quan n
m t cht i u hoa sinh trng k h c k h c l ethylene. A u x i n ( I A A ) k c h t h c h c h i b n
sn sinh ra ethylen l m c c h sinh trng ca c h i n h .
- I A A n g vai tr k c h t h c h s p h n hoa ca c c m d n ( x y l e m v phloem).
- A u x i n k c h t h c h s mc r c n h g i m v k c h t h c h s p h t sinh c h i ph trong
n u i cy m .
- A u x i n c c c nh h n g k h c nhau i v i s r n g l, qu, s u qu, s p h t
trin v c h n ca qu, s ra hoa trong m i quan h v i i u k i n m i trng.
- T o v n h n nhanh m so (callus).
- K c h thch to c h i bt nh ( nng thp).
- T o p h i soma (2,4-D).
Nhm

cc cytokinin

(Bng

2.6)

Cytokinin lin quan t i s p h n chia t b o , p h n h a c h i . . . T r o n g m i trng n u i

cy m , c y t o k i n i n cn cho s p h n chia t b o v p h n h a c h i t m so hoc t cc c
quan, gy to phi v tnh, tng cng pht sinh chi ph. Cc l o i cytokinin thng c
dng l: tenyladenine; kinetin (6-furfurolaminopurine); B A (6-benzylaminopurine); 2-ip
(isopentenul-adenine). Cytokinin hoa tan trong dung dch H a pha long.
Chc n n g ch y u ca c c c y t o k i n i n c k h i q u t n h sau:
- K c h t h c h p h n chia t b o .
- T o v n h n callus.
- K c h t h c h p h t sinh c h i trong n u i c y m .
- K c h thch p h t sinh c h i n c h v k m h m nh h n g u t h ca c h i n h .
- L m t n g d i n tch p h i n l do k c h thch s l n ln ca t b o .
- C t h l m t n g s m ca k h k h n g m t s loi.
- T o c h i bt n h ( nng cao).
- c c h s h n h t h n h r .
- c c h s k o d i c h i .
- c c h q u trnh gi (hoa vng v rng) l, kch thch to d i p lc.

53

Gibberelin

(Bng

2.7)

T r o n g s h n 20 cht thuc n h m gibberellin, G A l c h t c s d n g n h i u hem

c trong thc t i n . G A k c h t h c h k o d i c h i v ny m m ca p h i v t n h . So VI
auxin v c y t o k i n i n , gibberellin h i m k h i c d n g . G A c t n h hoa tan trong n c .
3

Gibberellin c c c chc n n g c bn sau:

- C c m p h n sinh tr, a n g sinh trng, c c p h i non, t b o u r , q u non, ht
c h a c h n hoc a n g ny m m u c cha n h i u gibberellic axit.
- K c h thch k o d i c h i o t n g c n g p h n b o v k o d i t b o , v d k o d i
thn v n g la sau k h i phun G A , k o d i t t h n . C c c y l n t h n g b t h i u
gibberellin.
3

- P h ng ht g i n g hoc c ging, v d p h n g khoai ty sau thu hoch.

- K i m sot s ra hoa ca c c c y 2 n m t u i . N m u t h n m m n m i m , sau m a
n g m m hoa k o d i t r t nhanh v p h n hoa hoa.
- c c h s h n h t h n h r bt i n h .
- K c h t h c h sinh tng hp ca a-amylase ht c y n g cc n y m m , g i p t i u hoa
c c cht d tr trong n i nh n u i m m c y .
- C c cht c c h tng hp k c h t h c h q u trnh to c ( t h n c, t h n h n h v c ) .
- K c h thch s ny m m ca p h n hoa v sinh t r n g ca n g p h n .
- C t h g y to q u k h n g ht hoc l m t n g k c h thc q u nho k h n g ht.
- C t h l m c h m s hoa gi l v q u c y c m i .
Abscisic

axit (ABA)

(Bng

2.8)

Trong n u i c y m v t b o , A B A c t c d n g to p h i v t n h , k c h t h c h s c h i
ca p h i , k c h t h c h s p h t sinh c h i n h i u loi thc v t . C c t c d n g c b n ca
A B A l:
- T h a m gia v o s rng l, hoa, q u hu ht c c c y t r n g v g y ra s nt q u .
- A B A t h n g c sn sinh k h i c c c y u t c c h c y t r n g n h m t nc v
nhit t h p n g b n g .
- T h a m gia v o s n g ngh, k o di t h i gian n g ngh v l m c h m s n y m m
ca ht.
- c c h s k o di t h n v c s d n g k i m sot s k o d i t h n c n h . G y ra
s n g k h khng.
Ethylene
C c chc n n g c bn ca ethylene:
- G y gi hoa l, k c h t h c h s rng l v q u .
- L m c h n qu.
- Sinh tng hp ethylene c t n g c n g k h i q u a n g chi, c y a n g b n g , l o
hoa, t n t h n g c g i i v b n h i m bnh.
- i u k h i n s c h n ca m t s l o i qu.

54

- Ethylene k m h m s ra hoa ca a s c y . T u y v y , s ra hoa ca x o i , da, m t

s c y c n h l i c k c h thch b i ethylene.
- K c h t h c h n hoa, k c h thch s lo hoa ca hoa v l.
C c bng d i y m t c c c tnh c bn ca c c n h m cht i u hoa sinh
trng, c c h chun b hoa cht v bo qun ca c h n g (tham k h o Sigma - A l d r i c h :
http://www.sigmaaldrich.com/Area o f Interest/Life Science/
Plant Biotechnology/Tissue Culture Protocols/Growth Regulators.html
Bng

Tn cc cht

p-Chlorophenoxiacetic acid
(4-CPA)

2.5 Cc cht thuc

nhm

Auxin

Phn t lng v
Chun b dung dch, bo qun v s dng
tng ng
S
Bo
Bo
Phn
Nng
Pha
Kh
t
M =
Dung mi
qun
qun
s dung
long
trng
kh
lng
lng
mg/l
(mg/l)
186,6

5,36

EtOH

2-8C

CA

0,1-10,0

2,4-Dichlorophenoxi-acetic acid

221

4,53

2-8C

CA

0,01-6,0

2,4-Dichlorophenoxi-acetic acid
Sodium salt

243

4,12

Nc

2-8C

CA

0,01-6,0

lndole-3-acetic acid Free acid (IAA) 175,2

5,71

EtOH/1 N
NaOH

Nc

-0c

-0c

CA/F 0,01-3,0

lndole-3-acetic acid Sodium salt

197,2

5,07

Nc

Nc

2-8C

-0c

CA/F 0,01-3,0

lndole-3-acetic acid methyl ester

189,2

5,29

2-8C

2-8C

lndole-3-acetyl-L-aspartic acid

290,3

3,45

0.5N
NaOH

Nc

-0c

-0c

0,01-5,0

lndole-3-butyric acid (IBA)

203,2

4,90

EtOH/1 N
NaOH

Nc 2-8C

-0c

CA/F 0,1-10,0

lndole-3-butyric acid Potassium

salt (K-IBA)

241,3

4,14

Nc

2-8C

-0c

CA/F 0,1-10,0

Alpha-Naphthalene-acetic acid
Free acid (NAA)

186,2

5,37

IN NaOH Nc

2-8C

CA

0,1-10,0

Beta-Naphthoxi-acetic acid Free

acid (NOA)

202,2

4,95

IN NaOH

Nc

2-8C

CA

0,1-10,0

Phenylacetic acid (PAA)

136,2

7,34

EtOH

2-8C CA/F 0,1-50,0

Picloram

241,5

4,14

DMSO

2-8C

CA 0,01-10,0

2,4,5-Trichlorophenoxi acetic acid

(2,4,5-T)

255,5

3,91

EtOH

2-8C

CA

0,01-5,0

2,3,5-Triiodobenzoic acid Free acid 499,8

(TI BA)

2,00

IN NaOH

Nc

-0c

-0c

0,05-5,0

K hiu: CA :Kh trng cng vi cc cht khc trong mi trng.F : Kh trng bng lc qua phin lc.
CA/F: Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t
nhiu, c th b p bng cch b sung hoc kh ng bng lc qua phin lc.

55

Bng

2.6. Cc ch

thuc

Phn t lng
v tng ng
Tn ha cht

nhm

cytokinin

Chun b dung dch, bo qun v s dng

Pha Bo
long qun

Bo
qun
lng

Kh
trng

Nng
s dung
(mg/l)

1.0 HCI

Nc

2-8C

CA

50-250

5,43

Nc

2-8C

CA

50-250

225,3

4,44

IN NaOH

Nc

2-8C

CA/F

0,1-5,0

261,7

3,82

Nc

2-8C

CA/F

0,1-5,0

6-Benzylaminopurine (BA hoc BAP) 225,3

4,44

2-8C

CA/F

0,1-5,0

N-Benzyl-9-(2tetrahydropyranyl)adenine (BPA)

309,4

3,23

EtOH

-0c

-0c

CA/F

0,1-5,0

N-(2-Chloro-4-pyridyl)-N'-phenylurea
(4-CPPU)

247,7

4,04

DMSO

2-8C

2-8C

0,001-1,0

6-(gamma,gammaDimethylallylamino) purine (2iF^

203,2

4,92

IN NaOH

Nc -0c

-0c

CA/F 1,0-30,0

6-(Y, Y-Dimethylallylamino) purine

(2P)

203,2

4,92

IN NaOH Nc -0c

-0c

CA/F 1,0-30,0

1,3-Diphenylurea (DPU)

212,3

4,71

Kinetin

215,2

4,65

Kinetin

215,2

Kinetin
Kinetin Hydrochloride

Phn
t
lng

S
M =
img/l

Dung mi

135,1

7,40

Adenine hemisulate Hemisulate salt 184,2

6-Benzylaminopurine (BA)
6-Benzylaminopurine H ydrochloride

Adenine Free baz

IN NaOH Nc

DMSO

2-8C

0,1-1,0

IN NaOH Nc -0c

-0c

CA/F

0,1-5,0

4,65

IN NaOH Nc -0c

-0c

CA/F

0,1-5,0

215,2

4,65

IN NaOH Nc -0c

-0c

CA/F

0,1-5,0

251,7

3,97

Nc

-0c

-0c

CA/F

0,1-5,0

1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea 220,2

4,54

DMSO

2-8C

CA/F 0,001-0,05

Trans-Zeatin Free baz

219,2

4,56

IN NaOH Nc -0c

-0c

CA/F 0,01-5,0

Zeatin

219,2

4,56

IN NaOH Nc -0c

-0c

CA/F 0,01-5,0

Trans-Zeatin Hydrochloride

255,7

3,91

-0c

-0c

CA/F 0,01-5,0

Trans-Zeatin riboside

351,4

2,85

IN NaOH Nc -0c

-0c

Nc

RT

0,01-5,0

K hiu: CA = coautoclavable (kh trng cng vi cc cht khc trong mi trng).

F = ilter sterilize: Kh ng bng lc qua phin lc.
CA/F: Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t
nhiu, c th b p bng cch b sung hoc kh trng bng lc qua phin lc.

56

Bng

2.7. Nhm

cc ch

iu hoa sinh trng

Phn t lng
v tng ng
Tn ha cht

Phn t
lng

S iM

khc

Chun b dung dch, bo qun v s dng

Dung mi

Pha
long

Bo
qun

Bo
qun
lng

Kh
trng

Nng
s dung
(mg/l)

1mg/l

C.)-cis,trans-Abscisic acid
(ABA)

264,3

3,78

IN NaOH

Nc

-0c

-0c

CA/F

0,1-10,0

Ancymidol

256,3

3,90

DMSO

2-8C

-0c

CA/F

1,0-10,0

Chlorocholine chloride (CCC)

158,1

6,33

Nc

2-8C

p to 500

3,6-Dichloro-o-anisic acid
(Dicamba)

221,0

4,52

EtOH/Nc

2-8C

2-8C

0,01-10,0

Gibberellic acid (GA )

346,4

2,89

EtOH

2-8C

CA/F

0,01-5,0

Gibberellic acid Potassium

sau (K-GA3)

384,5

2,60

Nc

2-8C

-0c

CA/F

0,01-5,0

Gibberellin A Free acid (GA ) 332,4

3,01

EtOH

-0c

-0c

0,01-5,0

(*.)-Jasmonic acid

210,3

4,76

EtOH

2-8C

-0c

0,01-100,0

Phloroglucinol

126,1

7,93

Nc

2-8C

CA/F

> 162

N-(Phosphonomethyl)glycine
(Gyphosate)

169,1

5,91

IN NaOH

Nc

2-8C

Succinic acid 2,2dimethylhydrazide

160,2

6,24

Nc

2-8C

2-8C

CA/F

0,1-10,0

K hiu: CA = coautoclavable (Kh ng cng vi cc cht khc trong mi trng).

F = filter sterilize: Kh trng bng lc qua phin lc.
CA/F: Kh trng cng vi cc thnh phn khc ca mi trng nhng c kh nng b mt hot tnh t
nhiu, c th b p bng cch b sung hoc kh trng bng lc qua phin lc.
Bng

2.8. Gii thiu v nhm

ca mi nhm

cht iu hoa sinh trng chnh v chc nng

trong nui cy m v t bo thc vt

Nhm auxin

lndole-3-acetic acid (IAA)

lndole-3-butyric acid (IBA)

1-naphthaleneacetic acid (NAA)

2,4-dichlorophenoxi-acetic acid (2.4D)

(2,4,5-T)

Picloram

Dicamba

p-chlorophenoxi-acetic acid (CPA)

Phenylacetic acid (PAA)

Nhm cytokinin

B
1

Kinetin

6-Bezylamino-purine (BAP)

Zeatin (Z)

chung

Chc nng chung ca nhm

- Phn chia t bo
- To v nhn callus
- To r bt nh ( nng cao)
- To chi bt nh ( nng thp)
- Tao phi soma (2,4-D)
- c ch chi nch

Chc nng chung ca nhm

- Phn chia t bo
- To v nhn callus

57

Chc nng chung ca nhm

Nhm cytokinin

Zeatinriboside (ZR)

Isopentenyladenosine (iPA)

Isopentenyladenine (iP)

Thidiazuron (TDZ)

N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)

- Kch thch bt chi nch

- To chi bt nh ( nng cao)
- c ch s hnh thnh r
- c ch s ko di chi
- c ch qu trnh gi (hoa vng) l
Chc nng chung ca nhm

Nhm gibberellin

Gibberellic acid (GA )

Gibberllin 1 (GA,)

Gibberellin 4 (GA)

Gibberellin 7 (GA )

- Ko di chi
- Ph ng ht ging
- c ch s hnh thnh r bt nh
- Cc cht c ch tng hp kch thch qu trnh to
c (thn c, thn hnh v c)
Chc nng chung ca nhm

Nhm cc cht HST khc

Ethylene

- Gy gi hoa l
- Lm chn qu

Abscisic acid

- S chn ca th phi
- Kch thch s hnh thnh thn hnh v thn c
- Thc y s pht trin ca tnh trng ng

Nhm polyamine

Putrescin

Spermidine

- Kch thch s t hnh thnh r

- Kch thch s hnh thnh chi
- y mnh s pht sinh th phi

Spermine

Jasmonic acid (Ja)

Methyl jasmonate (MeJa)

- Kch thch s hnh thnh thn c v thn hnh

- y nhanh s hnh thnh nh sinh trng.

2.5.3. n g d n g c a c n g n g h n u i c y m v t b o
2.5.3.1. Nhn ging sch bnh bng k thut nui cy m
Nui cy nh sinh trng nhn ging sch

bnh

Limmasets v Cornuet (1949) p h t h i n rng, c c c y n h i m b n h virus, virus

p h n b k h n g n g nht t r n c y v t h n g k h n g t h y c h n g v n g n h sinh t r n g .
Pht h i n l c s M o r e l v M a r t i n (1952) chng m i n h g i t h u y t t r n b n g c c h
to c cy sch bnh virus t 6 g i n g khoai t y qua n u i c y n h sinh t r n g .
N m 1960, M o r e l thc h i n bc ngot k h i p d n g t h n h c n g k thut n y
trong n h n nhanh c c loi a lan Cymbidium

t h n g qua protocorm. Sau , v i c p h t

h i n ra c y t o k i n i n v m i trng n u i c y m c i t i n (Murashige v Skoog, 1962) t o

sc sng m i ng d n g n u i c y n h sinh trng trong n h n g i n g t h n g m i
thc vt.
K t q u n u i c y n h sinh trng ph thuc v o vt l i u k h i u , n g u n gc v
k c h thc ca m u . t c h i u q u cao, cn l y m u n u i c y t c h i a n g sinh
trng m n h (Gupta v cs., 1981) hoc c h i ca c y m i g h p (Jones v cs., 1985). N u i
cy n h sinh trng cy non d d n g h n c y trng t h n h , t l ra r trong t r n g hp
58

n y t 83%, trong k h i v i c y trng t h n h ch t 6 3 % (Vieitez v cs, 1985). i u

k i n n u i c y , t h i i m l y m u cng nh h n g r t l n n k t q u ti sinh c y t n h
sinh t r n g . M t s loi c u t h c h i n h m n h , n u i c y n h sinh t r n g t c h i
n h d d n g h n t c h i n c h , i v i m t s loi k h c l i thu c k t q u ngc l i .
K c h thc m u n u i c y c n g l n , t l ti sinh v sng st ca m u c n g cao, tuy
n h i n m u c n g n h th k h n n g sch bnh virus l i cao h n . D o v y , k c h thc m u
n u i c y cn phi x c n h bng thc n g h i m i v i m i loi. M u n u i c y n h nht
ch c c h p sinh trng v 2 - 3 m m l s l l t n g to g i n g sch bnh do m
p h n sinh n h n m c h p n h c h i , l trung t m hot n g sinh trng, p h n hoa v
pht t r i n ca thc vt. Ngay d i m p h n sinh n y l c c m m l. i k h i k c h thc
m u l n h n v n m bo sch bnh v u s ( V i n e v Jones, 1969), song m t s trng
hp k h c l i i h i m u n h h n (Hunter v cs, 1984).
N u i cy n h sinh trng l k thut n u i c y m c ng d n g r n g ri nht
trong n n g nghip v n g h l m v n v i c c u i m c h n h sau:
- L o i tr b n h virus v t n g t virus.
- Bo qun q u gen i n vitro v trao i n g u n gen an t o n gia c c quc gia.
- N h n nhanh h n g lot, n g nht v i s lng l n c y ging t m t c y m ban u .
2.5.3.2. Ghp mt in vitro
Ghp chi

nh

M t trong c c p h n g p h p quan trng to c y n q u c m i sch bnh l chn

lc cy con t p h i v t n h (phi c ngun gc t t b o p h i t m ) . T u y n h i n , nhng
cy n y c chu k sinh trng di (6-7 n m ) trc k h i bc v o giai o n sinh sn. D o
vy, h i n nay v i g h p l k thut to c y sch bnh c s d n g t h n h c n g n h i u
p h n g th n g h i m . Murashige v cng s (1972) d n g v i g h p n h sinh trng to
vt l i u sch bnh c y n q u c m i , c y con ti sinh sch c c t c n h n g y bnh
virus v tng t virus. N m 1975, Navarro v cng s h o n t h i n quy trnh v i g h p
c h i n h c y c m i i n vitro. Bng k thut n y , khong 90 l o i c y c m i k h c nhau
c l m sch b n h (Navarro v cs, 1988). K thut v i g h p l o i tr c h n g lot
bnh k h i n g u n gen c y c m i , v d :
- V i r u s g y bnh tristeza, psorosis
- Stubborn spiroplasma
- Exocortis v i r o i d
- T c n h n g y bnh chy g m , cristacortis, impietratura
K thut vi ghp chi bao gm cc bc

sau:

- Chun b gc g h p trong ng n g h i m : C y gc g h p t h n g d n g trong v i g h p l

Troyer

citrange

hoc m t vi gc g h p k h c c k h n n g t n g hp cao v i m t g h p .

59

H t d n g l m gc g h p c gieo trong m i trng l n g v t r n g , cho c y n y m m

trong t i 10 - 14 n g y , sau thc h i n v i g h p .
- Chun b m t g h p trong ng n g h i m : C h n g i n g t t c n p h t t r i n sn x u t v
c y n g g i n g cn n h n . T c y n y , c h n l y o n c n h non b n h t , k h t r n g r o i
n u i cy i n vitro. K h i t h n non bt m m , thao t c t c h l y c h i n h ( g m n h sinh
trng v 1-3 m m l non t c y m ) . Thao tc tch c h i n h t h n g c thc h i n
d i k n h h i n v i soi n i v c h i n h c k c h thc r t n h ( 0 . 1 - 0.2 m i n ) .
- C y g h p c h i n h ln o n t h n non ca gc g h p trong n g n g h i m .
- Sau 4-6 tun n u i d i n h s n g , c h i n h bt m m v c l tht, c h u y n c y g h p
ra t .
H i n nay p h n g p h p v i g h p c h i n h a n g c s d n g r n g ri t o c c
d n g Citrus

sch bnh phc v cho n h n ging t h n g m i .

Vi ghp kt hp vi x l nhit
Quy

hoc hoa

cht

trnh:

- Gc g h p c chun b t ht n u i c y i n vitro t r n m i t r n g c b n M S c 1%
agar. Troyer

citrance

t h n g c d n g l m gc g h p , c n gc g h p Etrog

citron

d n g g h p c c l o i chanh.
- C y ging n h i m bnh d n g l m m t g h p c c h u n b n h sau: b l, g i
cy nhit 3 2 c t 12-15 n g y . Sau , t c h n h sinh t r n g k c h thc 0,1-0,2 m m
t c h i m i h n h t h n h v v i g h p theo k i u c h T l n n g c t r n gc g h p .
- N u i c y sau v i g h p trn m i trng M S c 100 mg/1 m y o - i n o s i t o l , n g 4%,
thiamin HC1 0,2 mg/1, pyridoxine HC1 l m g / 1 , nicotinic l m g / 1 , c b i t s d n g n n g
n g sucrose, c c v i t a m i n B cao v i h m lng B A , I A A v G A

k h c nhau kch

thch bt c h i t n h sinh trng v i g h p . T l v i g h p t h n h c n g l 6 0 - 7 0 % v cy

c h u y n ra t sng x p x 90%.
C h n g trnh v i g h p to g i n g sch b n h c y c m i c t r i n k h a i h u ht
c c nc.
2.5.3.3. Cng nghip nui cy m thc vt nhn ging sch bnh
N h n g i n g sch bnh bng n u i c y m ( c n c g i l v i n h n g i n g ) tr
t h n h m t trong nhng p h n g thc quan trng nht n h n nhanh i v i n h i u l o i
c y trng k h c nhau. T k h i bt u x u t h i n c n g n g h i p v i n h n g i n g v o u n h n g
n m 70 ca t h k X X s lng c c p h n g t h n g h i m v i n h n g i n g t h n g p h m
t n g ln nhanh c h n g . S t o n cu h a ca c n g n g h i p v i n h n g i n g d i n ra r t t c h
cc trong nhng n m 80 ca t h k X X . S m rng ca c n g n g h i p v i n h n g i n g
k h n g ch g p phn t n g n n g sut c y trng m c n t h c y v i c c n g n g h h a c c
m t h n g n n g nghip.
C c loi c y trng a n g c v i n h n ging trn th t r n g t h g i i l:

60

- C c loi hoa, c y cnh c bit l phong lan, a lan. C c chi hoa lan c c y m v
s dng cho hoa ct, hoa chu thng p h m g m : Dendrobium, rachis, Aranda,
Ascocenda, Catleya, Cymbidium, Cypripedium, Oncidium, Mokara, Phalaenopsis, Vanda.
- C c c y d n g h n h , g m c c loi n h L i l i u m , L a y n,....
- C c loi c y c c n h gng, khoai ty.
- C c loi c y n q u n h c h u i , d u ty, u , mt...
- C c l o i c y trng k h c n h da, c y c du, m a , c c l o i c y l m n g h i p n h
bch n , keo l a i , h n g , tch...
- Cc d n g b m sn x u t ht l a i k h n h n v g i bng con n g h u tnh n h
cc d n g hoa, rau bt t h c.
Nhng

u im v nhc im ca cng ngh vi nhn

u im ca vi nhn

ging

ging

- a ra sn phm nhanh hn: T m t c y u v i t bt k u c t h to ra m t qun

th c n g u cao v i s lng k h n g hn ch, phc v sn x u t t h n g m i , d
cy l d hp v m t d i t r u y n .
- Nhn nhanh

vi h s nhn ging cao: Trong hu ht c c trng hp, c n g ngh v i

n h n ging p ng tc n h n nhanh cao, t c y trong v n g 1-2 n m c t h to

thnh hng triu cy.
- Sn phm

cy ging ng nht: V i n h n ging v c bn l c n g n g h n h n d n g .

N to ra qun t h c n g u cao d x u t p h t t c y m c k i u gen d hp hay

ng hp.
- Tit kim khng gian: V h thng sn x u t h o n t o n trong p h n g t h n g h i m ,
k h n g ph thuc v o t h i t i t v c c vt l i u k h i u c k c h thc n h . M t c y to
ra trn m t n v d i n tch l n h n r t n h i u so v i sn x u t trn n g rung v trong
n h l n h the p h n g p h p t r u y n thng.
- Nng cao cht lng cy ging: N u i c y m l m t p h n g p h p hu h i u l o i
tr virus, n m k h u n k h i c c c y ging n h i m bnh. C y ging sch bnh to ra
bng cy m t h n g t n g n n g sut 15-30% so v i ging gc.
- Kh nng tip th sn phm tt hn v nhanh hn: CC dng sn p h m k h c nhau
c th to ra t h thng v i nhn, g i n g n h c y con i n vitro (trong n g n g h i m ) hoc
trong bu t . C c c y g i n g c t h c b n dng c y , c b i hay l t h n c.
- Li th v vn chuyn:

Cc cy con kch thc nh c t h v n chuyn i xa d d n g

v thun l i , ng thi c y con to ra trong i u k i n v trng c x c nhn l sch bnh.

Do vy, bo m an ton, p ng c c quy nh v v sinh thc vt quc t.
- Sn xut quanh nm: Q u trnh sn x u t c t h t i n h n h v o bt k t h i gian n o ,
k h n g ph thuc m a v.
Nhc

im

ca vi nhn

- Hn ch v chng

ging

loi sn phm:

Trong i u k i n k thut h i n nay, k h n g phi

tt c c y trng u c n h n ging t h n g p h m bng v i n h n g i n g . N h i u c y trng

c gi tr k i n h t hoc q u h i m v n cha t h nhn. nhanh p ng nhu cu t h n g

61

m i hoc bo q u n n g u n gen. N h i u v n l thuyt lin quan n n u i c y v ti sinh

t b o thc vt i n vitro v n c h a c g i i p .
- Chi ph sn xut cao: V i n h n ging i h i n h i u lao n g k thut t h n h tho.
D o , gi t h n h sn p h m c n k h cao so v i c c p h n g p h p t r u y n t h n g n h chiet,
g h p v n h n ging bng ht.
- Hin tng sn phm b bin i kiu hnh: C y con n u i c y m c t h sai k h c
v i c y m ban u do h i n tng b i n d t b o soma. K t q u l c y con k h n g g i
c c c c tnh q u ca c y . m . T l b i n d t h n g t h p giai o n u n h n g i n g ,
n h n g sau c c h i u h n g t n g ln k h i n u i c y k o d i v t n g h m l n g c c c h t
k c h thch sinh trng. H i n tng b i n d n y cn c lu khc phc n h m m bo
sn x u t h n g t r i u c y g i n g n g nht v m t d i t r u y n .
2.5.3.4. Sn xut cc ch hot tnh sinh hc t thc vt quy m ln bng
bioreactor
Cc cht hot tnh sinh hc t thc

vt

H i n nay, n g i ta x c nh vi chc n g h n cht t h c p . C c c h t c hot tnh

sinh hc thc vt t h n g chia l m ba n h m :
- C c cht t h cp.
- C c peptid v protein hot tnh sinh hc.
- Cc sn p h m m i k h c .
V a i tr ca c c cht t h c p i v i i sng c y t r n g c h a c n g h i n cu k
n h n g n i chung c h n g thng c vai tr trong bo v thc v t . C c c h t n y c tch
l y k h n g b o ca t b o thc vt. R t n h i u cht t h c p l n g u n dc l i u c gi tr
t h n g m i cao.
N g i ta chia c c cht t h c p t h n h c c n h m c t h h i n b n g sau:
Bng

2.9. Cc cht th cp thc

Dng sn phm th cp
Nhm cht c cha nit:
Amin
Alkaloid
Cc axit amin khng phi protein (NPAAs)
Cyanogenic glycoside
Glucosinolate
Nhm cht khng c cha nit:
Monoterpene
Sesquiterpene
Diterpene
Triterpene, steroid, saponin
Tetraterpene
Polyketide
Polyacetylene
Flavonoid
Phenylpropanoid

62

vt bc

cao

S lng cc cht trong mi nhm

100
12.000
400
100
100
1.000
3.000
1.000
4.000
350
750
1.000
2.000
500

Sn xut cc cht c hot tnh sinh hc t thc vt bng nui cy

Thc t , m t s c y dc l i u q u r t k h trng hoc cho h m lng cht c gi tr*

cao trong sinh k h i q u thp n n c c n h khoa hc n g h i n cu ng d n g c n g n g h
n u i c y m t b o thc vt n h m t c n g c sn x u t c c cht hot t n h s n h hc.
C n g n g h n y bao g m c c bc sau:
- Chn lc loi thc vt hoc ging cy trng sn xut cc cht c gi tr thng m i .
- N u i c y t b o , m hoc c quan tch r i i n vitro to sinh k h i t b o .
- G y t b i n hoc c h n d n g t b o soma c k h n n g to sinh k h i l n , c h m
lng hot cht cao.
- Chit rt, thu h i c c cht hot t n h t sinh k h i .
Cho n nay, ch c m t s t c c q u trnh c n g n g h c ng d n g sn x u t quy
m pilot c n g nghip.
H u ht cc bioreactor trong n u i c y t b o thc v t c n g h i n cu c i t i n t c c
l o i n i ln men s d n g trong n u i c y t b o v i sinh.
Bng 2.10. Mt s loi bioreactor c s dng trong nui cy t bo thc vt
Phng thc nui cy

Loi bnh
Bnh c trc khuy-Stirred tank
Nui cy t bo dch lng
Bnh ln trn-Rotating drum
Bnh thi kh t di: Air-lift
Bnh c trc khuy-Stirred tank
Bnh c trc khuy ci tin-Modified stirred tank
Nui cy r tc
Bnh thi kh t di: Air-lift
Bnh nh git-Droplet reactor
Bnh sng m-Mist reactor
Nui cy c quan v t bo Bnh c trc khuy-Stirred tank
phi hoa
Bnh thi kh t di: Air-lift

Quy m (lt)
1- 75.000
2-1.000
1-1.500
1-20
1-14
1-13
2-500
1-35
1-10
0,6-6

Bn cnh vic t n g quy m sn xut (tng kch thc ca bioreactor), v n t ra l

phi chn d n g t b o c k h n n g to sinh k h i l n v h m lng hot cht cao trong
sinh k h i .
Bng

2.11. Cc hot cht c sn xut vi hm lng cao bi nhng

nui cy chn lc (Curts,
1999)

Tn hot cht
Anthocyanin
Anthraquinone
Berberine
Jalrorrhizine
Rosmarinic axit
Shikonin
stevioside
Theanine(Caffeine antagonist)
Vanillin

dng t bo

T l hot cht trong sinh khi kh (%)

20
18,7
10,5
10,1
36
21,2
36,4
22,3
8,0

63

Bng

2.12. Cc hot cht c sn xut quy m ln bng bioreactor

trng

Tn hot cht

and Wink,

Euphorbia millii

Berberine

Coptis aponica,
Thalictrum minus
Beta vulgaris

cy

2001)
Tc gi sn xut

Loi cy dc liu

Anthocyanin

Betacyanin

(Wldi

mt s loi

Nippon Paint (Nht)

Mitsui Petrochemical Industries (Nht)
Nippon Shinyaku (Nht)

Echinacea
polysaccharid

E. purpurea, E. angustiolia

Divers (c)

Gingseng saponin

Panax gingseng

Nitto Denko (Nht)

Rosmarinic acid

Coleus blumei

Nattermann (c)

Sanguinarine

Papaver somnierum

Shikonin

Lithospermum erythrorhizon

Vipon Research Labs (USA)

Mitsui Petrochemical Industries (Nht)

Taxol

Taxus breviolia, T. chinensis,

Taxus sp.

Nippon Oil (Nht), ESCA genetics (USA),

Phton Catlytic (USA)

Vanilin

Vanilla planiolia

ESCAgenetics (USA)

M c d cc n h khoa hc t c m t s t h n h c n g trong sn x u t dc cht

n h n g c n g ngh bioreactor v n c n phi tip tc c i t i n n n g cao n n g sut, n gin
hoa thit b g i m gi thnh sn xut. H i n t i bioreactor ch c s d n g sn xut
cc cht c gi tr cao, t t i n hoc c c hp cht k h n g t h sn x u t b n g con ng
k h c . Gn y m t hng pht trin m i g i l canh t c p h n t c h n h t h n h , trong
cy trng c xem n h m t l o i bioreactor sn x u t c c cht q u h i m . Bc u,
cc gen lin quan n dc l i u hoc c h p h m c n g n g h i p n h vacxin, k h n g th,
enzym... t n g i , n g vt, v i sinh vt c chuyn v o thc vt, to c y trng chuyn
gen c k h tng to ra sinh dc trn n g rung hoc trong n h k n h .
2.5.3.5.

K thut

n bi in vitro trong

cng tc ging

cy

trng

K thut to c y n b i i n vitro: t h n g qua trong n u i c y ht p h n v n o n cho

p h p to ra nhanh c h n g h n g lot c y n b i hoc n b i k p t c c t b o sinh dc
c v ci, phc v c lc cho mc c h n g h i n cu d i t r u y n v to g i n g c y trng.
N m 1964, l n u tin trn t h g i i , hai n h khoa hc A n Guha v Maheshwari
t h n h c n g trong vic to c y n b i t n u i c y bao p h n i n vitro c y c
innoxia.

Datura

Ngay sau , c y n b i c to ra bng n u i c y bao p h n h n g lot

c y trng k h c nhau. H a i p h n g p h p c bn ca k thut n b i h i n nay l:

- N u i c y bao p h n hay t i u b o t t c h r i hay c n g i l p h n g p h p t r i n h sinh
c trong ng n g h i m ( i n vitro androgenesis).
- N u i c y t b o trng c h a th tinh hay c n g i l p h n g p h p t r i n h sinh c i
trong ng n g h i m ( i n v i tro gynogenesis).
V t l i u ban u cho q u trnh n u i c y i n vitro to c y n b i t h n g l:
- Bao p h n , ht phn t c h r i , c m hoa ( p h n g p h p n y hay c p d n g cho
nhng loi c hoa n h ) .

64

- Cu p h i sau lai xa. K h i l a i xa gia hai loi l a m c h Hordeum

bulbosum,

vulgare

v H.

sau q u trnh th phn to ra n h i u p h i n b i do n h i m sc t h ca t b o

sinh dc loi H. bulbosum

b l o i tr. D o n i n h ca p h i n b i k h n g p h t t r i n n n

n g i ta p h i s d n g p h n g p h p n u i c y t b o cu nhng p h i n y v to ra
h n g lot c y n b i (Jensen, 1977).
- T h tinh g i . y l q u trnh th p h n n h n g k h n g x y ra s t h t i n h . M c d
vy, t b o trng v n c k c h t h c h p h t t r i n t h n h c y n b i . Hess v Wagner
(1974) t i n h n h th phn i n vitro gia Mimulus

luteus

v i Torenia ournieri

v k t

qu l to c c y n b i .
- N u i c y n o n c h a th t i n h .
Trong s c c vt l i u trn, bao phn, ht p h n t c h r i v n o n c h a t h tinh l
nhng ngun n g u y n l i u quan trng, c s dng p h b i n h n to c y n b i .
T i Trung Q u c , c n g n g h n b i c t r i n khai c h thng trn quy m l n
v c nh h n g c h i n lc r r n g trong to g i n g m i . H n m t n g h n c s n u i c y
bao phn hot n g trn t o n quc t nhng n m 1970, k t q u to c trn 100
ging la m i trong m t t h i gian ngn. Trong , g i n g la nc v l a m m i to ra
t k thut n b i m rng sn x u t trn d i n t c h vi t r i u ha. T i T r i u T i n , k
thut n u i c y bao p h n to ra 42 g i n g l a m i (Sasson,1993; Jain cs., 1997). u t h
ca c c p h n g p h p n u i c y n y l t t c c c c y to t h n h u c n g u n gc t t i u
b o t hoc i b o t, v v y c y con nhn c s l c y n b i hoc c y nh b i
ng hp t v i c c cp n h i m sc t h t n g n g h o n t o n g i n g nhau (tr trng hp
t bin). C y nh b i thu c l do q u trnh nh b i hoa t n h i n ca ht p h n n b i
trong n u i c y hoc x l a b i hoa bng thc n g h i m .
2.5.3.6. K thut chn to bin d t bo soma v kh nng ng dng
B i n d t b o soma - m t h i n tng chung v p h b i n x y ra trong n u i c y m i n
vitro. C n c v o quy l u t ca V a v i l p , trong nhng i u k i n m i trng ging nhau c
th quan st t h y c c b i n d d i t r u y n ging nhau c c loi g n nhau v p h n l o i . S
l i u ca h n 30 n m qua cho t h y m v t b o t c h r i , n u i c y t r n m i trng n h n
to l ngun k h n g c g i i h n ca c c b i n d d i t r u y n . L a r k i n v Scowcroft (1981)
a ra k h i n i m b i n d t b o soma l m tn g i chung cho c c l o i b i n d n y .
Ngun

gc ca cc bin d t bo

Nhng

thay i di truyn xy ra trc khi nui cy m i n vitro:

soma

Trong q u trnh p h t t r i n c t h , p h n hoa v gi hoa ca m v t b o m t s c c

thay i d i t r u y n x y ra v c tch l y trong c c t b o soma (D'amato, 1985).
N h k thut n u i c y m , cy c ti sinh t t b o soma s l c c t b i n . C c n h
khoa hc a ra k h i n i m automutagenesis - t t b i n ( t b i n x y ra k h n g do c c
tc n h n t b n n g o i g y n n ) . De Vries (1901) - n g i k h i x n g hc thuyt v t
b i n cho r n g c y b t b i n do c ti sinh t ht gi ( c n h i u t b i n t i m n t
trc trong ht g i ) . Ba m i n m sau, Nawacin chng m i n h t b i n ht gi l do c c

65

cht tham gia q u trnh trao i cht v c c sn p h m c n b c trong h t g y n n . C c

cht n y c t h l: hp cht cha lu h u n h , amin, amino axit, a m i d , aldehyde, alkloide,
phenol, quinone, axit nucleic v c c sn p h m k h c . N g u y n n h n g y t b i n c the
do c u trc b n h t h n g ca ht v t b o b p h v - c c e n z y m t i p x c v i c c cht v
to ra sn p h m t b i n .
Tuy theo p h n g thc n u i c y t b o soma, n g i ta p h n b i t c c l o i b i n d d i
y:
- B i n d d n g t b o m so (callusoclonal variation): k h i c y b i n d ti sinh t m
so (callus).
- B i n d d n g t b o trn (protoclonal variation)-khi c y b i n d ti sinh t t b o
trn.
N g o i ra Evans v cng s (1984) c n a ra k h i n i m " b i n d giao t " (gametoclonal variation) k h i c y c ti sinh t c c t b o sinh dc (gamete). N h n g b i n d di
truyn x y ra v c p h t h i n trong q u trnh n u i c y i n v i t r o g i chung l t b i n t
bo dng.
Nhng

thay i di truyn xy ra trong qu trnh in

vitro:

M t ngun b i n d t b o soma quan trng l thay i trong b m y d i t r u y n x y ra

k h i n u i cy to m so v p h n hoa c quan i n vitro. n nay c c n h khoa hc pht
h i n hai n g u y n n h n c bn d n n c c b i n d ( t b i n ) x y ra trong t b o n u i cy
i n vitro:
- T b o n u i chu tc n g trc t i p ca c c cht hoa hc trong m i t r n g n u i
cy.
- Trong i u k i n i n vitro, c c gen d i n g (transposon) hay c n g i l c c gen nhy
hot n g r t m n h , c c gen n h y c p h t h i n l n u t i n n g n m 1963. N g i
p h t m i n h ra gen n h y (transposon) l M c C l i n t o c r , b c g i i t h n g N o b e l n m
1983 n h p h t m i n h n y . n nay, khoa hc x c n h c c c gen n h y t n t i
hu ht c c c y trng. H o t n g ca c h n g p h thuc v o k i u gen v i u k i n m i
trng. Trong c y m , c c gen n y d i c h u y n tch cc, n c t h ci v o b t k gen no
trn n h i m sc t h v g y ra t n s t b i n r t cao.
Chroqui v Bercetch (1985) cho b i t a u x i n g y ra a b i hoa b n trong t b o n u i
c y (endopolyploidization). Oono (1982) n u i c y m so t ht l a v ti sinh c y t
m so cho bit B A P c nng 30 mg/1 to ra t n s t b i n l n g p 50 l n so v i
B A P nng 2 mg/L M c t b i n t b o soma l n n mc t n s t b i n do c c
cht t b i n g y ra cng k h n g cao h n ( L a r k i n and Scowcroff, 1981).
Orton (1984) cho b i t trong t h i gian n u i c y trn m i t r n g d i n h d n g h gen
ca c c t b o thc vt t r i qua q u trnh c i t nhanh c h n g v p h t h i n n h n g
thay i s lng, c u trc n h i m sc t h , t b i n gen. T n s t b i n gen n h i u k h i
rt cao ( l o -10"') tnh theo locus trn c y . Thay i d i t r u y n p h b i n n h t x y ra trong
2

t b o n u i c y l a b i t h . Sau k h i m c n u i cy, n h n t b o c t h t r i qua q u

66

trnh n i p h n (endoreduplication) l m s lng n h i m sc t h trong t b o t n g ln g p

i hoc h n na n h n g k h n g x y ra p h n chia t b o . K t q u s lng n h i m sc t h
ca t b o t n g ln. Q u trnh n i p h n ca n h i m sc t h trc k h i p h n b o cho
p h p n g i ta nhn c cy lng b i n g hp (dihaploid) t ht p h n n b i trong
n u i c y bao phn. Ph rng c c b i n i n h i m sc t h c quan st t h y n h i u
loi c y trng k h c nhau (Murashige v Nakano, 1967; Sacristan v Melchers,

1960;

Sunderland 1973; N i s h i v Mitsuoka, 1969). N g h i n cu t b o hc cho t h y 10% s

loi p h n hoa c quan k h n g k m theo h i n tng n i p h n n h i m sc t h , 9 0 % s loi
p h n hoa c quan c k m theo n i p h n n h i m sc t h .
t b i n t b o soma x y ra c c gen trong t b o cht c chng m i n h bng
phng p h p tch A D N ty t h v lc l p n h enzym ct (Kemble R . T cs, 1984). t
bin t b o soma ng dng v o chn ging h i u q u n h i u c y trng n h m a , khoai
ty, c chua, thuc l, l a v la m . Petunia

n h n c g i n g m i g i l

Velevetrose, g i n g m a Q47 ( L a r k i n and Scrowroft, 1981).

2.5.3.7. K thut nui cy t bo trn (protoplast) v thao tc di truyn mc t bo
Tch nui t bo trn
K h c v i t b o v i sinh v t b o n g vt, t b o thc v t c t h n h t b o cng c
to ra bi nhiu l o i polyme k h c nhau. T h n h t b o c chc n n g c hc, to khung
x n g t b o v h m n g lin k t gia c c t b o v i nhau. T h n h phn hoa hc ca t
b o rt phc tp. Cellulose l t h n h phn ch y u ca m n g t b o thc vt. C n g thc
hoa hc tng q u t ca cellulose l ( C H 0 ) . P h n t cellulose c h n h si d i , t h m c h
6

1 5

rt di v i s lin k t ca h n g n g h n c c p h n t n g n v i nhau. N g o i cellulose

cn c hemicellulose, pectin, l i g n i n , m t s cht b o v cht k h o n g , pectin c n n g
vai tr quan trng lin k t gia c c t b o v i nhau.
T b o trn (protoplast) l t b o c l o i b m n g t b o ( m t l p polyme bao
bc t b o ) v m n g lin k t gia c c t b o . C c enzym n g vai tr c h y u trong p h n
huy m n g t b o v to ra t b o t r n l cellulase v pectinase. T b o sau k h i b m t l p
m n g cng s c d n g h n h cu d i p sut t h m t h u p h hp ca m i trng.
T b o trn c t h c t c h ra t c c m hoc c quan k h c nhau c y n h l, r ,
m so n u i c y i n vitro ( h n h 5 - t c h n u i t b o t r n t m l c i du).
u t h ca k thut t c h v n u i c y t b o t r n l t b o k h n g c m n g cng,
trng thi n b o , m t t b o thu c trn n v t h t c h m i trng c t h r t
cao (t l o t b o / 1 m i m i trng). T b o trn m t s c y trng c k h n n g ti sinh
rt mnh, v d t b o m tht l thuc l, c i du... Bng thao t c d i t r u y n t b o
trn c t h d d n g to ra c c t b o b i n i gen. T b o v i k i u gen b i n i s c
6

bo t n k h i ti sinh t b o t h n h c y h o n chnh.
Mt s ng dng
- Lai

ca k thut

t bo

trn

xa:

N g i ta c t h d d n g lai t b o trn ca c c c y k h c loi v i nhau: khoai ty v i

c chua, cam ba l v i cam ngt v to ra con lai k h c loi.
67

- Lai t bo cht to ra cc dng ng ng bt

dc:

P h n g p h p to d n g n g n g bt dc c t m tt n h sau:
- T o qun t h t b o trn t c c d n g cng c bt dc ( d n g B t i m n n g ) VI te
b o cht bnh t h n g .
- T o qun t h t b o trn t c c d n g bt dc c t b o cht ( d n g C M S ) v i t
b o cht bt dc.
- P h n h n ca d n g C M S bng c h i u x hoc hoa cht.
- Dung hp (hay lai) hai loi t bo trn v i nhau v ti sinh t b o lai thnh cy.
- Chn lc c c c y t t b o lai mang ton b gen n h n ca d n g B v t b o cht
ca d n g CMS.
Cy chn lc bt dc c s l d n g A n g n g bt dc c ca d n g B , g i n g ht
nhau v gen n h n , d n g A ch k h c d n g B tnh trng bt dc c t b o c h t . N h n
d n g c y lai t h n g qua th p h n c y n y bng phn ca d n g B to ra d n g A . D n g
B t i m n n g tr t h n h d n g B cng c (maintainer) ca d n g A . P h n g p h p n y cho
p h p nhanh c h n g to ra c c d n g thun n g n g bt dc phc v cho t o g i n g u
t h lai. N h n g h i n nay k thut n y c h a c ng d n g v i n h i u c y k i n h t quan
trng n h la, n g . . .
- Chuyn

np cc c quan

t:

Trong q u trnh dung hp t b o trn gia hai loi hoc hai g i n g s x y ra chuyn
np c c c quan t (ti t hp t b o cht). Trong t b o cht thc v t c r t n h i u c
quan t n h lc lp, ty th... v i nhiu c tnh d i t r u y n quan t r n g n h k h n n g k h n g
bnh, tnh trng bt dc hoc hu dc, gen quang hp... L a i t b o c h t c t h t o ra cc
d n g t b o hoc d n g ging c y trng c nhng c t n h d i t r u y n t b o c h t m i .
- To ra cc ti t hp mi ca

ADN:

Trong q u trnh dung hp t b o c t h to ra c c ti t hp m i ca n h i m sc t h

hoc A D N . Trong q u trnh lai t b o k h i x l bng p h n g x , n h i m sc t h ca n h n
c th b t g y m t c c h n g u n h i n , dung hp t b o t r n c t h c n t o ra ti t hp
A D N gia cc loi hoc c c t b i n gen k h c nhau.
- ng dng trong nghin cu c bn
T b o trn c ng dng x c n h chc n n g ca t b o cht v c quan t
t h n g qua p h n tch con l a i t b o cht.
Thao tc di

truyn:

V t b o trn k h n g c m n g cng n n r t thun t i n cho n h i u thao tc d i t r u y n

k h c nhau n h c h u y n gen hoc n h i m sc t h , t i u b o t, n h n t b o bng c c v i thao
tc (vi t i m vt l i u d i t r u y n v o t b o ) , xung i n . . . Thao tc d i t r u y n t r n t b o t r n
m t s loi c k h n n g ti sinh i n vitro m n h n h c i du, thuc l, khoai ty... c t h
g p phn to ra h n g lot c c ging m i chuyn gen trong t n g l a i .

68

- Dung hp t bo trn to cc dng a bi

th:

D u n g h p 2 l o i t b o t r n n h b i (2n) c t h d d n g t o ra c c t h t b i
(2n + 2 n = 4n). D u n g hp t bao t r n nh b i v i t b o t r n n b i to ra c c t h tam
b i (2n + I n = 3n).

Ct l trong mi trng PCM, dng

enzym tch t bo trn

Ti sinh callus trn

mi trng

Chi ti sinh

Ly tm thu c
cc t bo trn

Phn chia t bo

c o n

Nui t bo trn trong

mi trng

in vitro

Hnh 2.1. Tch nui t bo trn t m l v ti sinh cy (ci du)

69

Chng

C S SINH H C PHN T CA K T H U T D I T R U Y N

3.1. C C N G U Y N L C U T R C H O A H C C A A D N
N m 1953, James Watson v Francis Crick c n g b m h n h p h n t A D N g o m
hai si xon k p , si n y l tng n g b tr ca si kia. M h n h to ra l c s
khoa hc g i i thch c nhng s thn k ca c c h i n tng sinh hc n h k h n n g
sinh sn bng n h n i , k h n n g d i truyn, b i n d v t i n hoa, t n h a d n g v phong
ph tuyt v i ca sinh g i i .
M i si A D N l m t p o l y m e r c to b i b n p h n t d e o x y r i b o n u c l e o t i d k h c
nhau g i tt l nucleotid l Adenin ( A ) , T h y m i n ( T ) , G u a n i n ( G ) v C y t o s i n (C).
T r n h t ca c c nucleotid trong si A D N quy nh tnh a d n g ca c c gen.
P h n t A D N g m 2 si x o n k p , trong i d i n v i nucleotid A t r n si ny
l u n l T trn si kia v ngc l i v i d i n v i G trn si n y s l c t r n si k i a v
ngc l i . Hai nucleotid i d i n n h v y g i l t n g n g v t o ra m t c p nucleotid
(hay m t cp baz) (s 3.1.). V d n h cp T hay cp C G . n v o l n hoc
k n h thc ca c c p h n t A D N c tnh bng s lng cp b a z , c 1000 c p b a z s
g i l kb (kilobaz).
C c b a z t r o n g c c n u c l e o t d (Adenn

v Thymin,

Guanin

v Cytosir)

c k h nng

bt cp tng n g v i nhau tng i m t b i lc lin k t h y r y u . L c l i n k t y u

n y cho p h p 2 si A D N c t h d d n g t c h i (trong t r n g hp t n g hp si A D N
m i hoc sao m gen). A d e n i n trn si n y bt cp hay lin k t v i T h y m i n t r n si kia
v ngc l i bi hai lin k t hydro. Guanin v Cytosin trn hai si c n g lin k t v i nhau
bng ba lin k t hydro. K h n n g lin k t tng i ca c c b a z n h t r n quy nh
tnh i xng trong c u trc ca hai si A D N (s 3.2).
L u t i x n g n y t r o n g c u t r c p h n t c a A D N l p h b i n trong sinh g i i .
N quy nh nhng thuc tnh quan trng nht ca s sng: kh

nng

sinh

sn

v di

truyn.
- Kh nng sinh sn: K h n n g n h n bn ca p h n t A D N to ra c c p h n t A D N
m i c c u trc ging ht v i p h n t A D N m . D i tc n g ca c c enzym, c t h l
enzym helicase l m hai si A D N m x o n k p v t c h i to ra hai si k h u n . D i t c
n g ca c c enzym A D N - polymerase, c c nucleotid t do trong t b o s bt cp v i
cc baz tng n g trn 2 si k h u n to ra si A D N k p m i t n g n g v i si k h u n
ban u . K t q u l t m t p h n t A D N x o n k p to hai p h n t A D N g i n g h t nhau.

70

Si A
S 3.1. Nguyn

si B
l cu trc hai si ca phn t ca

ADN

S 3.2. Lin kt hydro gia 2 nucleotid tng ng trong phn tADN:

mi
nucleotid nm trn si ca ADN lin kt vi Nucleotid
tng ng trn si kia ca
phn tADN.
K hiu: T: Thymin, A: Adenin, C: Ctosin, G: Guanin.

- Kh nng phn bo: Kh nng nhn i ca ADN dn n kh nng phn chia ca

t b o . T m t t b o m to ra hai t b o con mang vt l i u d i t r u y n ging nhau.
M i p h n t n u c l e o t i d g m 3 t i u p h n : p h n t b a z (baz), p h n t n g
(deoxyribose) v m t p h n t phosphate. H a i nucleotid trn c n g m t si A D N lin k t
v i nhau qua cu phosphate (s 3.3).
71

[
c

H
c
Phosphate

Baz (base)

H
li

/
H

/ /
H
5*

ng (deoxyribose)

c .
4

1 /

\ l
c .
3

I
H

Lin kt vi phosphat ca
nucleotid tip theo
^

S t 3.3. Cu trc hoa hc ca phn

tnucleotid

3.2. DNG CHY CA THNG TIN DI TRUYN

Q u trnh c h u y n hoa t h n g t i n d i t r u y n g m hai d n g c h y d i y :
ADN -> ADN
T h n g t i n d i t r u y n p h n t A D N m c sao c h p l i theo l u t i x n g to ra
hai p h n t A D N m i c c u t r c t n g t nhau v g i n g h t v i p h n t A D N m (nu
k h n g x y ra t b i n ) . y l d n g chy t h n g t i n d i t r u y n t t b o m sang t b o
con trong q u trnh p h n b o v t t h h n y sang t h h k h c qua q u t r n h sinh sn.
ADN -> ARN -> Protein
A x i t deoxyribonucleotid ( A D N ) v axit ribonucleic ( A R N ) l hai l o i p o l y m e r quan
trng ca c t h sng. T r o n g k h i A D N c to b i b n deoxyribonucleotid k h c nhau:
Guanin, Adenin, Cytosin v T h y m i n t h A R N t h n g t h n g c h g m m t si v c c u
to b i b n

ribonucleotid

k h c nhau v i c c baz: A d e n i n , Guanin, Cytosin v U r a c i n .

T h n g t i n d i t r u y n ca t t c c c c t h sng t r n tri t u lu g i t h n g t i n trong

p h n t A D N g m hai si (tr m t vi loi virus) v q u t r n h sinh t n g hp A R N v
protein g i l q u trnh biu hin ca

gen.

ARN -> ADN

P h n t A D N c h n h t h n h t si A R N v i s x c t c ca enzym sao c h p ngc
(reverse transcriptase). H i n tng sao c h p ngc x y ra trong q u t r n h sinh sn ca
m t s virus c genom l A R N trong t b o sinh v t ch.
72

3.3. B I U H I N C A G E N
3.3.1. Q u t r n h p h i n m ( t r a n s c r i p t i o n )
Q u trnh sinh tng hp A R N trn k h u n A D N g i l q u trnh p h i n m
(transcription). Trong hai si x o n k p ca A D N ch c m t si n g vai t r si k h u n
quy nh trnh t c u trc ca A R N theo n g u y n tc t n g n g b tr. Q u trnh p h i n
m d i t r u y n ca gen c bt u t u 5' ca gen trong v n g promoter cho n k h i
gp i m k t t h c p h i n m (termination) u 3'. Q u trnh sao m c thc h i n v i
s tham gia ca c c ARN-polymerase.
i v i c c c t h sng n h n thc, sau k h i k t t h c p h i n m , si A R N c tng
hp trong n h n ca t b o c n phi t r i qua m t q u t r n h chnh sa:
Phn t A R N u tin c tng hp t A D N c th c g i l h n A R N (ting A n h
heterogeneous nuclear A R N - A R N n h n d hnh n h s 3.4) hay c n g i l A R N s cp
(primary R N A ) hoc l tin m A R N . Cu trc ca tin A R N g m cc o n trnh t c g i
l exon v intron (cc o n exon biu th bng c d u c h m m ) n h s 3.4.
Sau q u trnh p h i n m l q u trnh chnh sa h n A R N :
Q u trnh n y x y ra n h A R N splicing (ct v g h p n i ) . C c o n A R N c
phin m t introns s b ct i , c c o n c p h i n m t c c exons c g h p n i l i
vi nhau v b sung t h m m t o n l m t o n u i g m 4 p h n t adenin ( A A A A ) .
K t q u to ra p h n t m A R N ( A R N t h n g t i n ) . Trong t b o n h n thc, h n A R N c
to ra trong n h n , sau k h i chnh sa to t h n h m A R N v d i c h u y n ra n g o i t b o cht.
Q u trnh p h i n m v dch m c m t c c h n h n h d i y (S 3.4).
Gen
Genom

ADN

Prnmnter

)
Phin m

hp.ARN

v / \ / l

Y \ / \ A

Y \ / \

I A / N

Chnh sa
mARN

AAAA-3'

x A A / 1
Dch m

Protein

S 3.4. S biu hin gen theo Mieseld

R.L,

1999.
73

C c bc trong q u trnh dch m v p h i n m t b o t i n n h n ch g m 2 bc:

- Bc : m A R N c trc t i p to ra t A D N k h n g qua chnh sa
- Bc 2: Protein c tng hp trn si m A R N
C 5 i m k h c b i t c bn gia sinh tng hp A D N v A R N n h sau:
- Trong p h n t A R N k h n g c T h y m i n thay v o l U r a c i n ( U ) , n n trong k h i
h n h t h n h si A R N , i xng v i A d e n i n trn si A D N s l U r a c i n t r n si A R N .
- ARN-polymerase hot n g k h n g cn i m m i primer trn si A D N .
- T n g hp A D N lin quan trc t i p v i q u trnh p h n chia v sinh sn ca t b o .
K h i lng A D N trong t b o c n h n i h i n tng p h n chia t b o m i x y ra.
Trong k h i sinh tng hp A R N c lin quan trc t i p n c c q u t r n h trao i cht c a
t b o : to ra c c protein v enzym k h i n g c c q u trnh sinh hoa hc ca t b o .
- i m k h i u sinh tng hp A D N ( g i l origin o f replication) k h c v i i m
k h i u sinh tng hp A R N .
- Trong k h i sinh tng hp hai si A D N con x y ra n g t h i trn c hai si ca A D N
m . Sinh tng hp m A R N ch c thc h i n trn m t si A D N ca gen, g i l si k h u n
(template) hay l si c ngh a

(sense),

si c n l i k h n g c m hoa

(non-sense).

N u A R N s c tng hp trn m t si A D N tng n g v i si k h u n , n g i ta s

nhn c m t si A R N tng n g b tr, n h n g nghch ngha (antisense

ARN)

vi

si m A R N c tng hp t si k h u n . K h i hai si A R N nghch ngha gp nhau, c h n g

s to n n m t si A R N x o n k p l m cho m A R N m t k h n n g sinh tng hp protein.
N g i ta ng dng c tnh n y ca sinh tng hp A R N to ra c c gen nghch
ngha (antisense gene) n h m k h n g c h sinh tng hp m t enzym hoc m t hot cht
n o , v d , c c n h khoa hc M ng dng n g u y n l gen i ngha to ra
ging c chua chuyn gen c k h n n g k h n g c h sinh tng hp ethylen k m h m q u
trnh c h i t n h i n ca qu.
N h i u copy k h c nhau ca p h n t A R N c tng hp t ch m t si trong p h n t
A D N . S lng copy ca A R N c quy nh b i tn sut ca enzym A R N - polymerase
k h i n g q u trnh dch m , t h h i n mc hot hoa ca gen.
v i k h u n , m t n h m ( m t d y ) c c gen c t h c dch m c n g lc v to ra
m t si m A R N di. D y gen n y c g i l m t operon. H o t n g ca operon cho
p h p k i m sot c m t n h m gen c c n g chc n n g .
3.3.2. Qu trnh dch m (translation)
Q u trnh tng hp protein trn si m A R N c c t b o n h n thc (eukaryote) x y ra
t b o cht.
Sinh tng hp ca protein c x y ra trong t b o cht ca t b o v i s tham gia
ca m A R N , ribosome, c c t A R N ( A R N v n chuyn), c c axit a m i n t do, ATP,...
Sinh tng hp protein l q u trnh chuyn hoa t h n g t i n ( m d i truyn) t gen t h n h
c u trc v chc n n g c th ca protein. T r n h t axit a m i n ca protein l do trnh t

74

c c nucleotid trong gen quy nh. A R N t h n g t i n ( m A R N ) c cha c c codon, m i

codon g m ba nucleotid g i l triplet (cp ba). P h n t A R N v n c h u y n ( t A R N ) c c u
t r c 2 cc: M t u ca t A R N c n h p h n t axit amin, m t u k i a l b ba
(triplet) g i l anticodon. M i m t t A R N c mang m t axit a m i n v m t anticodon c
t h cho n . T r o n g q u trnh tng hp protein, c c anticodon ca t A R N s n h n b i t c c
codon t n g ng trn si m A R N v t m ra v tr k h n g gian t h c h hp cho n trn
m A R N v n g t h i c n g l v tr k h n g gian thch hp hay l trnh t t h c h hp ca axit
a m i n m n mang theo u kia ca t A R N . T n g s c 64 codon k h c nhau, trong
c 61 codon quy n h trnh t ca 20 axit amin, ba codon c n l i c g i l c c codon
k t t h c hay l c c codon v ngha ( k h n g ng v i m t acid amin n o trong p h n t
protein). T i i m codon v ngha trn m A R N q u trnh tng hp protein s d n g l i .
3.4. CC ENZYM THAM GIA VO QU TRNH SINH HOA CA ADN
C c enzym tham gia v o c c q u trnh trao i cht ca A D N c ngha r t quan
trng i v i k thut d i t r u y n . C c enzym n y c t h n g m i hoa v c g i l b
kt c c enzym ca sinh hc p h n t (xem chi tit c h n g 4).
3.5. IU KHIN BIU HIN CA GEN
i u k h i n hot n g ca h thng gen l m t trong c c b n l n nht trong t
n h i n v vic k h m p h ra n c t h l c h a khoa m ra c c b n k h c trong h i u
k h i n c c q u trnh sinh hc.
Cho n nay n h n l o i v n c h a h b i t n m t h thng i u k h i n n o l i tinh t,
c h n h x c , nhy b n n h c c h thng i u k h i n trong t b o v c t h sng, nht l
trong h thn k i n h trung n g ca con n g i . l nhng b m t ca to hoa t n t i
ngay trong m i c h n g ta m tr t u n h n l o i h i n v n c n a n g tng bc n g h i n cu.
N g h i n cu b i u h i n ca gen l n i dung hot n g c bn ca c h n g trnh chc
n n g genom. M u n t m h i u chc n n g ca m t gen cn x c n h r c c n i dung sau:
- C c A R N do gen n y m hoa trong q u trnh p h i n m gen (transcription).
- Protein c u t r c , hoc enzym sn p h m ca gen n y to ra trong q u trnh dch m
gen (translation).
- C c q u t r n h i u k h i n b i u h i n ca gen n h t h n o : gen n y c p h i n m
l o i t b o , m n o trong c t h v v o giai o n n o trong q u trnh p h t t r i n ca c
t h , c c i u k i n m i trng tc n g ln b i u h i n ca gen...
- V a i t r ca c c promoter hoc c c gen i u k h i n lin quan n b i u h i n ca
gen n y .
- C c promoters n g vai tr quyt nh i v i b i u h i n ca gen. H i n nay, h n g
lot c c promoter c t h c p h t h i n , v d , promoter c th m (tissue specific
promoter) quy n h m t gen gn v i promoter c th m s ch b i u h i n k h i n n m
m t n g ng. V d , gen c gn v i promoter c t h ht s ch b i u h i n trong ht
(seed specific promoter).

75

3.5.1. P h p b i n c h n g t r o n g n g h i n c u h t h n g s n g v v n i u k h i n h o t
hoa c a gen
Ci hay nht ca trit hc duy v t l p h p b i n chng. Quan trng nht ca p h p
b i n chng l p h n tch c u trc h thng, t m ra "gc" ca h t h n g v quy l u t v n
h n h ca n trong c c m i quan h n h i u chiu trong k h n g gian, t h i gian v n h n g
i u k i n c t h .
Sng c ngha l v n ng, h i u h n h phi chi p h i s v n n g k h n g n g n g ,
m t s c x y ra l tn h i u c c s c k h c bt u hoc dng l i . C c t chc sng v
c c q u trnh sinh hc l u n l u n t n t i trong q u trnh p h t t r i n lch s ca n : h n h
t h n h , p h t sinh, p h t t r i n v d i t vong, trong d n g c h y ca t h i gian v k h n g gian,
trong s lun h i gia p h n g i i v tng hp, gia b i n d v d i t r u y n , gia tr hoa v
lo hoa, gia vt cht v n n g lng, gia n h v q u n , gia sng v cht. M i m t n g y
c h n g vn t b o m i sinh ra v h n g v n t b o cht i trong c t h sng. M i n g y
c h n g t r i u sinh l i n h m i sinh ra trong k h i h n g t r i u c c sinh l i n h k h c b d i t vong.
S cht c h a phi l n g ct. S cht c h n y c t h l bt u ca s sinh n i k h c .
S t n g ln tr t h n h tn h i u b o trc s g i m xung, s cn k i t b o trc bt u ca
s to ra. l nhng x u h n g m ta c t h t m t h y trong h i u h n h ca gen v c c
chu trnh v n n g sng. T n h c n bng, n nh trong c c h t h n g sng v n l t h i t
y u i v i s sinh t n , n h n g s c n bng ch l t n g i v l u n trng t h i c n bng
n g . V n n g sng tht phc tp b i t bao. N x y ra trong m t k h n g gian v t h i
.gian rng l n , l i r t c t h trong c c i u k i n v b i cnh c t h ca m i m t p h n ng
sinh hoa.
P h p b i n chng trong k h i p h n tch h thng phc tp v l y k n h v y p h i m x
n , t m ra c c m t x c h v c c k h u c h a khoa ca s vt, k h n g b qua c i tng t h v
k h n g ri v o s v n vt v p h i n d i n . K h o a hc h i n nay t m t h y " u m i " ca
v n n g sng - m t cun ch r i nht trong t n h i n . u
chnh

l phn

tADN,

u ngun

ca dng chy s

mi ca vn ng

sinh

hc

sng.

3.5.2. Tnh phc tp ca h thng sng

C u trc ca h thng sng r t a dng v i v v n c c " l i n h k i n " l i t i . C c " l i n h
k i n " l i c c u trc t c c p h n t. V d , n g i c k h o n g 30.000 gen, v i s
lng ngn y c c p h n t m A R N v khong 30.000 c c protein c t h , b i t bao n h i u
t r i u siu c u trc, hay c c " l i n h k i n " k h c nhau s c to ra t t hp ca c c p h n
t ? D o v y , c h n g ta t h y nhng c u trc a dng v i nhng thuc tnh c b i t , v
d n h c c g i c quan k d i u n g i . K d i u nht c l l i m t . Ch v i m t lng
t ca n h s n g ri trn m t n g i c t h gy ra xung thn k i n h v p h n ng trong
n o b. Trong k h i c c thit b tinh v i nht ca n g n h vt l h i n i c n g c h a c k h

76

n n g ghi n h n m t n n g lng n h n h v y . C nhng n g i c t h c c suy ngh

ca n g i k h c k h i h gn nhau. C nhng n g i t h n g tho g n 100 ngoi ng.
N h n g "thit b" hay h thng c u trc p h n t n o to ra nhng thuc t n h c b i t
n h v y ca vt cht sng?
H thng

sng

c cc mc t chc

v iu khin

khc

nhau.

T n g ng v i c c

mc t chc k h c nhau ca h thng sng s l c c h t i u chnh v i u k h i n t h c h

hp (s 3.5).
Mc phn

t: Bt u l c u trc p h n t v i h n g vn c c hp cht v c v hu

c,trong c:
- C c monomer n h c c n g n , c c axit amin, c c nucleotid... S c m t ca
m i monomer trong t b o v i h m lng l bao n h i u , c tng hp k h i n o v u
u c i u chnh hp l.
- C c polymer ca s sng n h polysaccharid, c c proteins, c c lipids, c c p h n t
A R N v A D N . . . T r o n g quan trng nht l gen v i u k h i n hot hoa ca gen.
Mc trn phn

t: T i p n l cc t chc trn p h n t do t hp ca v s c c p h n

t to ra n h Operon, n h i m sc t h , h thng Cytochrom trong lc l p (cytochrom l

thit b d n t r u y n ca h n g t r i u c c d n g i n t l i t i trong b m y quang hp thc
vt) h thng m n g ca t bo... c i m c u trc mc trn p h n t c quy nh
b i c u trc c th ca c c p h n t do gen x c nh.
Mc t bo quan

(organell):

T r n na l c c t chc n i b o b n trong m i t b o :

n h n t b o , ty t h , lc lp... trong t b o cht c g i l t bo

quan.

T bo sng: C c t chc trn y c h a c t h g i l sng k h i n c h a to ra t b o

v i nhng c t r n g c l p nh: k h n n g sinh sn bng n h n i , k h n n g trao i
cht v i m i t r n g n g o i . V i r u s c t h coi l c t h sng (c k h n n g sinh sn) ch k h i
n c k sinh trong t b o ch.
Trn mc t chc t bo l m, c quan, c th sng, trn na l qun t h , qun x,
h sinh t h i , x h i loi n g i trong m i tng tc thng x u y n v n n g lng v vt
l i u v i t h g i i k h n g sng v t o n v tr.
n g v i m i mc t chc chc chn phi c c c mc i u k h i n v t i u k h i n
thch hp. C c n h khoa hc x c nh c rng t chc sng l u n l t chc "t n " .
C c p h n t "t n " ti t hp v i nhau to ra c c t chc trn p h n t. "T t chc"
v "t i u chnh" l p h n g thc v n n g t n t n n g lng nht trong m t h
thng phc tp n h v y .
i u n g n i y l h i u k h i n trong c t h sng l u n l h t i u chnh v
thch ng m t c c h tuyt v i v i m i trng n i t i v b n n g o i .
77

S 3.5. Cc mc khc nhau trong t chc sng t phn

(Theo N elson. D.L and CoxMM,
2000)
Phn

bo v phn

hoa t bo v c quan l cc thang

t n t bo

bc trong

h iu hnh

gen:

N u t b o l u n sinh sn bng n h n i m t c c h c h n h x c , n u t h n g t i n d i t r u y n
m i t b o sng trong c t h ca c h n g ta u c copy g i n g h t nhau, t h t i sao t
m t t b o p h i ban u , c h n g ta l i c m t c t h sng h o n c h n h v i n h n g c quan
chc n n g k h c b i t v b sung l n nhau m t c c h h i hoa v nhp n h n g n h v y . H
tiu hoa to ra v t l i u v n n g lng cho c t h hot n g g m m i n g , h m r n g , thc
qun, t u y n nc bt, d d y , gan, mt... c m g i c n g n n k h i no v t h m n k h i t h i u
cht. H h h p v i n h p t h cung c p o x y t i c c mao m c h ca hai l p h i v t i t h n
k h t c t h ra n g o i , r i c m g i c t h i u hi l m ta p h i t h s u v nhp t h c t n g
c n g k h i c t h v n n g m n h . H tun h o n v n t i v t l i u , d n g k h n m i t
b o sng v v n c h u y n c h t t h i , t h n k h ra n g o i . H thn k i n h ca c h n g ta i u p h i
hot n g sng, n h n g x c c m n g h thut v t h m m , tr n h v k h n n g s n g to,
t n h y u . P h n hoa c quan to ra s k h c b i t v s thng nht trong t o n c t h . i u
k h i n q u trnh p h n hoa ca t b o l c m t khoa hc v p h t t r i n c t h . T n h t h n g
nht v t n h " h i hoa" t u y t m trong s " p h n hoa" v p h n c n g chc n n g b n trong
c t h sng c n a n g n m trong b m t ca h i u k h i n ca t b o v c t h . i u

78

k h i n hot hoa ca gen l m t trong c c h i u k h i n sinh hc l t h nht a n g c

n g h i n cu k h m p h .
n nay, c c n h khoa hc t m t h y rt n h i u c c y u t i u chnh hot hoa ca
gen, trong quan trng nht l c c promoter c d n g g i l tissue specific promoter
(promoter c h u y n hoa m ) . C c promoter n y ch cho p h p m t gen b i u h i n m t m
hoc t b o n o m k h n g b i u h i n c c m hoc t b o k h c . l n g u y n n h n
c bn to ra s p h n hoa m v c quan.
3.5.3. H i u h n h gen
H thng gen, hoc n h m gen tham gia v o c n g m t chc n n g n o ca c t h
sng, v d c c gen tham gia p h n g i i n g trong t b o v i k h u n E. coli chng hn,
t h n g c chia l m hai l o i c h n h :
- C c gen i u h n h hoc gen i u chnh
- C c gen c u trc l c c gen tham gia trc t i p v o v i c m hoa c c protein c t h
Protein cng c p h n l o i t h n h :
- Cc enzym: cht x c tc ca m i phn ng sinh hoa trong t b o
- C c protein l c c cht k m h m hoc c c h b i u h i n ca gen
- C c protein c u t r c : protein c u trc t n hoc k t hp v i c c polymer sinh hc
k h c l c c vin gch hay l c c l i n h k i n c c u trc c t h x y dng n n c c t chc
trn p h n t v to ra c c t b o quan n h lc lp, ty t h , m n g t b o , v.v...
- Cc protein l k h n g t h to ra k h n n g m i n dch c t h v i c c tc n h n g y
bnh. C c protein to ra sc k h n g ca c t h sng i v i c c i u k i n bt l i ca
m i trng.
- C c protein l c c hormon i u tit c c q u trnh sinh l, sinh hoa, l sinh, t h m
c h c m x c ca c t h .
V c t h c c c protein l c c cht hot tnh tr t u v tr nh...
V i c c l o i protein n h trn, c h n g ta c t h d d n g n h n t h y vai tr ca protein
trong i u k h i n c c q u trnh sinh hc (xem chi t i t c h n g 6).
H thng

iu khin

biu hin ca gen mc phin

m:

- K i m sot bt u v k t t h c sinh tng hp A R N .

- K i m sot s lng v chng l o i m A R N c tng hp trong t b o .
Chng l o i hay p h a dng ca m A R N ph thuc v o c c y u t c h n h sau:
- T b o v m c t h : M t s m A R N c tng hp ra m i t b o sng, c h n g
cn cho c c q u trnh trao i cht thit y u ca m i t b o .
M t s m A R N c tnh c th: ch tng hp m hoc t b o n o , trong giai
o n p h t t r i n t h hoc x u t h i n d i tc n g ca c c y u t m i trng x c nh.

79

Ci bin

AR
N

y l q u trnh ch x y ra t b o n h n thc. Sau k h i si A R N n g u y n t h y c

tng hp ra t A D N , n s t r i qua q u trnh c i b i n (ct b bt hoc b sung t h m m t
s trnh t nucleotid nht nh) r i m i to ra si A R N thun thc g i l m A R N ( A R N
t h n g tin).
iu khin

sau phin

m:

- L q u trnh i u k h i n sinh tng hp protein trn si m A R N v i s tham gia ca

ribosome v n h i u y u t hoa hc v sinh hc k h c . Q u t r n h n y quy n h s lng
protein s c tng hp trn A R N t h n g t i n .
- G n y , c c n h khoa hc p h t m i n h m t c c h i u k h i n m i i v i hot
hoa ca gen t b o n h n thc g i l A R N i (Ting A n h : R N A i n t e r e r e n c e = R N A i ) .
y l c c h g y bt hot gen b i c c si A R N ngn ( s i R N A ) c c h i u d i t k h o n g
21-25 nucleotid. C c si A R N ngn c k h n n g bt cp v i c c A R N c c c o n trnh
t tng n g v i c h n g v l m cho m A R N b p h n huy.
iu khin

sau dch

m:

- Sau k h i protein c to ra, c h n g c t h c n c t i p tc c i b i n . V d , trong

trng hp protein l c c enzym, protein phi c k t hp v i c c y u t k h c n h c c
yu t ng

thi (cofactor)

hay coeniym

c c chc

nng

xc tc hoa hc

(xem

chi tit c h n g v enzym).

- C c gen i u k h i n g i vai tr n g hoc m c c gen c u t r c : k h i n o , u ,
trong h o n cnh n o m t n h m gen s hot hoa (sinh t n g hp A R N ) hay s n g n g
hot n g .
3.5.3.1. Cu trc h iu hnh ca gen
Operator:

l m t o n ca p h n t A D N c k h n n g t n g t c v i protein k i m c h

(repressor), do v y k i m sot sinh tng hp A R N t h n g t i n .

Leader

ARN:

si A R N n g u y n t h y c tng hp ra t A D N g m hai phn: Phn

leader A R N v phn A R N m hoa protein. Leader A R N l o n A R N c bt u t

nucleotid u tin v k t t h c i m bt u x y ra m hoa sinh t n g hp protein.
on

m hoa gen cu trc) ca ADN:

o n gen quy n h c u trc ca protein, c

cha m i truyn cho axit amin u tin k o di cho n axit a m i n c u i c n g ca si

polypeptid.
Promoter:

l m t o n ngn trn p h n t A D N c k h n n g t n g t c v i A R N -

polymerase. K h i A R N - polymerase b m v o promoter, sinh tng hp A R N bt u .

Operon:

v i k h u n , c t h c vi gen c n g n m trn m t p h n t A D N v c dch

m c n g m t lc, to ra m t si A R N d i . N h m c c gen g i l m t operon - c c gen

trong c n g m t operon b k i m sot b i c n g m t repressor. Operon c n g h i n cu
u tin l Lac-operon, gen ca operon n y tham gia v n c h u y n v p h n g i i n g
lactose (xem s 3.6).

80

Hng ca qu
trnh dch m

3""
lch

I
loi

IcL

S 3.6. T chc ca Lac-Operon

vi khun

E.coli

Trong s 3.6, Ba gen LacZ, LacY v LacA cng tham gia phn huy ng
lactose, ba gen n y x p theo trnh t n i t i p trn si A D N v to ra L c - Operon. LacOperon to ra m t si A R N t h n g t i n duy nht. p (Promoter) l o n A D N n i A R N polymerase b m v o bt u dch m . Operator (O) l o n A D N n i m protein k m
h m (repressor) b m v o c c h b i u h i n ca gen.
Q u trnh b i u h i n ca Lac-Operon bt u n h sau:
- P h n t n g Lactose b n v o cht k m h m (Repressor) v to ra t hp p h n t
Repressor + Lactose, repressor m t k h n n g b m v o operator v L c - operon c g i i
p h n g k h i cht k m h m .

81

- E n z y m A R N - polymerase b m v o promoter - o n k h i n g ca gen v bt u

q u trnh tng hp A R N t h n g t i n .
- Si A R N t h n g t i n c tng hp bt u t gen z n gen Y v k t t h c gen A .
3.5.3.2. Mt s phng thc iu khin hot hoa gen
i u k h i n b i u h i n c a gen b n g c h t k m h m ( r e p r e s s o r ) :
T n g hp A R N b k m h m b i m t protein g i l repressor. Protein n y b m v o
operator, m t v tr c th trong p h n t A D N i m bt u ca gen do n k i m sot
(xem s 3.7). K h i c c repressor c n b m trn si A D N , th ARN-polymerase c h a t h
bt u q u trnh sao m (sinh tng hp A R N t h n g t i n trn k h u n A D N ) . H o t hoa ca
gen do repressor k i m sot c n g h i n cu r t k v i k h u n . V d , q u trnh i u
k h i n hot hoa ca c c gen tham gia vo sinh tng hp c c enzym p h n g i i c c p h n t
ng: k h i trong m i trng sng ca v i k h u n x u t h i n c c p h n t n g , n g x m
nhp v o t b o , p h n t n g s tng tc v i repressor to ra m t t hp: repressor +
ng. T hp n y l m cho repressor m t k h n n g b m v o gen. D o , c c gen tng hp
c c enzym p h n g i i ng c g i i p h n g v bt u hot n g . C c m A R N s tham gia
vo q u trnh tng hp ra c c enzym p h n g i i n g . K h i c c enzym p h n huy ht cc
p h n t ng trong m i trng, repressor s k h n g c n b c c p h n t n g b m dnh
na, repressor s tr l i lin k t v i p h n t A D N v n g operator v l m cho q u trnh
tng hp m A R N b dng l i . N h v y , v i k h u n s ch bt u tng hp ra c c enzym
p h n g i i ng k h i trong m i trng c x u t h i n c c p h n t n g .

S 3.7. H iu hnh gen theo c ch km

82

hm

 i u k h i n h o t hoa c a gen b n g " t m n g n g " s i n h t n g h p m A R N :

T m n g n g l c c h k i m sot b i u h i n ca gen trong sinh tng hp A R N
t h n g t i n s t m n g n g k h i m t lng nht nh ca n c to ra. C c h t m
n g n g hot hoa gen c n g h i n cu k i v i c c gen tham gia v o q u trnh sinh
tng hp axit amin v i k h u n . C h n g ta u b i t c c axit a m i n l t h n h phn cu trc
k h n g t h t h i u ca protein. Trong t b o c n 20 l o i axit a m i n k h c nhau. H m
lng ca m i axit a m i n trong t b o phi g i mc c n thit cho hot n g sng ca t
b o v trong mc c n bng hp l v i c c amino axit k h c . Sinh tng hp m i axit amin
i h i t b o phi tiu t h ngun n n g lng v vt l i u k h n g n h . D o v y , vic tng
hp tng axit amin trong t b o p h i c i u chnh hp l t h n g qua i u chnh hot
hoa ca gen.
V d , q u trnh to tryptophan v i k h u n c k i m sot b i tryptophan operon,
A R N - Polymerase bt u sinh tng hp leader - A R N . o n leader - A R N c cha
t h n g t i n cho tng hp m t protein ngn g i l leader-protein, trong t h n h phn ca n
c tryptophan.
Bn trong p h n t A R N - leader c m t o n ngn g i l attenuator c vai tr tch
cc cho vic n g n g hot n g ca operon. K h i tryptophan c d trong t b o , ribosome
bt u tng hp leader protein. K h i leader protein x u t h i n , o n attenuator trn leader
A R N g p l i b i lin k t t n g n g b tr l m n g n g c h u y n n g ca A R N polymerase trn operon.
K t q u ch c m t o n A R N leader ngn c to ra. K h i lng tryptophan trong
t b o cn k i t , tng hp leader protein (c cha tryptophan) s n g n g codon
tryptophan. Ribosome d n g codon tryptophan ca leader- A R N l m cho si A R N
leader d u i ra v q u t r n h dch m operon t i p tc x y ra. Si m A R N t h n g t i n bt u
to ra c c enzym tham gia v o q u trnh tng hp tryptophan. K t q u m t lng
tryptophan m i s c to ra ngay sau . N h v y , k h i trong m i trng c d
tryptophan c c gen tham gia sinh tng hp cht n y s n g n g hot n g .
K i m s o t b i u h i n c a gen b n g " h o t hoa

gen":

P h n g thc i u k h i n n y p h b i n c c c t h bc cao. S lng c c gen c n g

tham gia v o m t q u trnh sng nht nh trong c t h g i l c c gen c n g n h m chc
n n g , c c gen trong c n g n h m c s i u chnh p h i hp hot n g m t c c h nhp
n h n g , n g t h i i m v n g v tr k h n g gian cn n chc n n g c t h ca m i gen.
sinh vt bc cao, c c gen c n g trong m t n h m chc n n g c t h n m c c v tr
k h c nhau trong n h i m sc t h v hot hoa ca c h n g c i u chnh b i m t vi
protein, c c protein n y t h n g l c c protein hot hoa - hay c n g i c c y u t k i m
sot dch m , c c protein n y s b m v o promoter p h a trc ca v n g sao m . Cc
protein n y c tnh c th cao v thng gp m t s l o i t b o c th, v d: t
83

b o gan, hoc thn. C c gen i u chnh c t h n m r t xa c c gen c u t r c . o n gen

t n g c n g (enhancers) c t h i u chnh hot n g ca c c gen n m c c h n h n g
n g h n nucleotid. Gen t n g c n g l o n m t protein c t h b m v o , sau l i l
c h cho c c protein k h c b m theo. Trong c c trng hp n h v y o n A D N p h i c u n
l i to ra c h lin k t c c protein. K t q u m t t hp protein - A D N x u t h i n l m c h
b m cho A R N - polymerase bt u sinh tng hp si A R N m i . N h v y , sinh t n g hp
A R N t m t gen cn n hot hoa ca m t n h m protein c t h , b m v o c c o n
A D N c t h trn gen i u chnh (s 3.8).
V n g m hoa ca gen hay l gen c u trc

B
v (B) l

C c o n Enhancer trn D N : ( A )
cc ch

Protein

b m cho c c protein i u chnh

bm vo on A v protein

bm vo on

B
B

on ADN

gp li to mt cu trc c th ADN

Vng m ha

Proten

Vng m hoa
]
Cu trc c th ny to ch bm cho ARN polymerase
tng hp

hot ng v bt u

mARN

Vng m hoa
n

S 3.8. iu hnh gen theo c ch "tng cng -

84

Enhance"

sinh

L m t h a y i h o t hoa c a gen b n g c c p h n g p h p d i t r u y n :

Promoter

- cha khoa

ca gen v h thng

gen:

Promoter c nh ngha l m t o n nucleotid ngn t r n si A D N , l n i enzym

A R N - polymerase b m v o v bt u k h i n g sinh tng hp A R N . V a i tr ca
promoter rt ging v i vai tr ca m t c n g tc i n : n g v m hot n g ca gen
hoc n h m gen. C c gen c u trc ging n h c c b n g n . C h n g ch p h t s n g k h i
n g m c h i n . Promoter quy nh u v k h i n o s x y ra b i u h i n ca gen, tng
t n h n g hoc m m c h i n . T n g ng v i c c protein k h c nhau hoc c c m t
b o c th k h c nhau c t h c c c promoter k h c nhau v c t h . M t s promoter c
b i u h i n c th c c m nht nh. V d , promoter c t h h r s ch cho p h p
b i u h i n gen r; c nhng promoter cho p h p b i u h i n gen m i ni, tr r;
promoter c th t h n , hoa, ht w . . . C nhng promoter k h n g c t h cho p h p
b i u h i n gen m i c quan. V i c t h i t k gen c u trc k m v i promoter c th c
ngha thc t i n quan trng.

Thit

k mi cc gen iu

khin:

C h n g ta bit gen c c h hot n g ca ribosome (ribosome inhibitor gen - R I G ) ,

k h i gen n y hot n g th b m y sinh tng hp protein ca t b o b t l i t . N g i ta
cng bit m t s c c promoter c t h ch k h i n g hot hoa gen c c m nht inh.
V d promoter c t h bao phn hoa (anther specific promoter = A S promoter).
Promoter n y ch k h i n g b i u h i n ca c c gen trong bao p h n . C c n h khoa hc
t h n h c n g trong vic t h i t k gen k m h m ribosome ( R I G ) lin k t v i promoter hot
hoa c th bao p h n v c h u y n c gen n y v o la. C y la mang t hp gen
k m h m ribosome ( R I G gen) v A S promoter v n c k h n n g sinh trng v p h t t r i n
bnh thng v gen c c h hot n g ca ribosome ch tng hp protein k m h m
ribosome trong bao p h n hoa. K h i hoa h n h t h n h , gen k h i n g (promoter ASP) trong
bao phn m i bt u hot n g , k t q u cht c c h hot hoa ca ribosome c sn
sinh v l m cho q u trnh to ra ht p h n b p h v. C y la tr n n bt dc c.

To t bin h iu khin

gen:

T o n b genom ca v i k h u n E. C o l i l m t si A D N v n g l n . N u c k o thng
ra, si A D N n y s c di g p 1.000 l n d i ca c h n h t b o . B n gen ca E.
Coli c chia l m 100 n v, bng p h n g p h p g y t b i n , n g i ta to ra t h
t b i n E.Coli m t k h n n g i u chnh sinh tng hp ca Lac-Operon. t b i n
n y repressor m t k h n n g b m v o operator d n n t n h trng operon n y hot n g
lin tc. Ngay c k h i trong m i trng h o n t o n k h n g c n g n h n g enzym (5galactozidase v n c tng hp v i h m lng r t l n gn bng 100% protein tng s
ca c v i k h u n .
N h v y , bng thit k m i c c gen i u k h i n (promoter), hoc g y t b i n c c
gen i u k h i n hot hoa ca h gen, n g i ta c t h to ra nhng c t h sng m t k h
n n g t i u chnh trao i cht v m t k h n n g thch nghi v i m i trng. M t s hot
85

cht ca c t h sng c t h c sn x u t ra v i s lng theo m u n . C c p h n g p h p

n y rt cn cho m t h n g n g h i n cu ng dng m i ca sinh hc, l canh t c p h n
t (Molecular Farming). N g i ta trng c c c y chuyn gen c t h i t k h i u k h i n
b i u h i n gen theo m u n sn x u t ra m t s hot cht sinh hc n h sn x u t c c
enzym c n g nghip, dc phm...

iu khin

hot hoa gen theo c chARN i

N
(R Ai)

y l c c h i u k h i n hot hoa gen t h n g qua s bt cp ca c c A R N n g n

( s i A R N c d i 21-27 nucleotid) v i c c m A R N c h (c o n trnh t t n g n g b
tr v i s i A R N ) v l m cho m A R N b p h n huy (b ct i b i c c enzym ct g i l Dicer
ch c c c c t h sng n h n thc). C c h A R N i c 2 n h khoa hc M , F e v
M i l o c n g b n m 1998. N m 2006, hai n h khoa hc n y c trao g i i t h n g
Nobel. C c lnh vc ng dng c bn ca c c h bt hot gen A R N i :
- P h hy c c m A R N t h n g t i n , t x c nh c chc n n g ca gen.
- T o sinh dc hoc ging m i v i ng dng R N A i : C c s i A R N v i sinh v t n h n
thc hot n g c k h n n g g y bt hot gen c c k sinh g y b n h n h virus, v i k h u n ,
n m , c n trng,... l m cho c h n g b tiu d i t hoc m t k h n n g g y h i . C c A R N ngn
a n g tr t h n h m t sinh dc tr l i u quan trng i v i b n h H I V - A I D v n h i u bnh
virus k h c .
- L m bt hot c c gen g y c t hoc l m t n g cht lng n n g sn ( l m g i m c c
cht b o giu cholesterol c y c du, l m m t c t gossypol ht b n g t h n g qua
g y bt hot gen tng hp gossypol trong ht, l m cho ht b n g v n g i u protein c t h
tr t h n h ngun thc n cho n g i v c h n n u i ) .
- C t h l m bt hot c c gen to ht to ging k h n g ht hc ht n h . . .
N g h i n c u chc n n g gen v n h m gen:
Trc k h i c n g ngh R N A i c p h t m i n h , c c n h khoa hc ng d n g c c c n g
ngh sau y x c nh chc n n g ca gen hoc n h m gen:
- G y to t b i n bng h a cht hoc c h i u x , p h n g p h p n y c ng dng
p h t m i n h ra h n g lot c c gen v i sinh vt v n g thc vt, trong c p h t m i n h
quan trng ca Jacob v M o n o v c c gen i u k h i n hot h a gen v i sinh v t .
- C n g ngh ci T - D N A v c c gen nhy v o n h i m sc t h l m cho gen c h b t
b i n . C t h t b i n b m t c tnh ban u , t x c n h chc n n g gen c h .
Tuy vy, c c c n g ngh trn c nhc i m chung l i h i t h i gian, t n h c h i u
cha cao, nht l tnh n g u n h i n ca c c p h n g p h p g y t b i n gen bng c c t c
n h n h a l v ci gen.
- C n g ngh R N A i ngha (antisense R N A ) l m bt hot gen c h .

86

C n g n g h R N A i ngha (antisense R N A ) l q u trnh to ra m t si A R N i

ngha v i si m A R N ca t b o . K h i si A R N i ngha to ra, n s bt cp v i si
m A R N t n g n g v i n theo quy lut bt cp ca 2 si c c u t r c t n g n g b tr,
to ra si A R N k p . D o , sinh tng hp protein t r n si m A R N s b c h l i . i u
x y ra do Ribosom v c c t A R N k h n g c k h n n g b m v o A R N si k p . Tuy vy,
q u trnh n y k h n g p h i bao g i cng l m m t chc n n g ca gen c h . Fire v cs
(1998) n g h i n cu t i p c n g ngh A R N i ngha bng c c h c n g lc a c 2 si
n g ngha (sense) v i ngha (antisense) v i si m A R N c h v o t u y n t r n g
c.elegans,

k t q u l m t n g k h n n g l m bt hot m A R N c h ln 10 l n so v i vic

ch a v o t u y n t r n g trong 2 si A R N . Bng c c h t i m c 2 si A R N (ng ngha

v i ngha) c n g lc v o t u y n t r n g to ra m t si i v si i n y c ngha
l m bt u q u trnh g y bt hot gen. K t q u n g h i n cu n y t h c y n g h i n cu
v R N A i v c c enzym ct (Dicer).
C c n h m p h n t A R N k h c n h a u c k h n n g g y b t h o t gen c p h t
hin:
Trong t b o , l o i A R N c n h i u nht l Ribosom A R N ( r A R N ) , t i p theo l t A R N
( A R N vn chuyn) v m A R N ( A R N t h n g t i n ) . N g o i ra n g i ta m i p h t h i n ra c c
A R N sau:
- A R N si k p ( d s A R N = double stranded R N A ) .
- H a i r p i n A R N ( h p A R N - A R N cp t c ) .
- A R N n h (small i n t e r e r i n g R N A = s i R N A , t i n g V i t s i A R N ) , khong

21-25

nucleotid.
- A R N siu n h (micro R N A , t i n g V i t m i A R N ) , khong 2 1 - 2 5

nucleotid.

C c e n z y m v A R N c n g h a q u a n t r n g i v i g y b t h o t gen b n g c c h
ARNi:
/ A R N si k p ( d s A R N ) : N h m A R N n y h n h t h n h do 2 si A R N t n g n g
i ngha bt cp v i nhau to t h n h (Agrawal v cs, 2003). C c si A R N k p di c
tm thy trong t b o v t h n g c n g u n gc t c c gen n h y (Schramke v A l l s h i r e
2004) hoc l virut (Rovere v cs, 2002) v i s sn sinh ra si A R N k p .
Si A R N k p d i c ct t h n h c c o n n h g i l s i A R N . H a m i l t o n

Baulcombe (1999) l nhng n g i u tin p h n l p c c c o n ngn n y , c h n g c

chiu d i t 2 1 - 25 cp b a z (bp).
2/ A R N cp tc ( h p A R N ) : A R N cp tc l m t dng A R N si k p n g i n c
ngun gc t m t si A R N di c 2 trnh t u si t n g n g v c t h bt cp v i
nhau to t h n h si k p , trong k h i phn gia cong l i (to ra dng cp tc) (Wesley v
cs, 2001).

87

3/ m i A R N c c t n h t n g t s i A R N (Bartel 2004; Nelson v cs, 2003). C 2 u

ngn (trong k h o n g 2 1 - 2 5

bp) v c s dng bt cp v p h h y si m A R N c

t h . m i A R N l c c A R N n i sinh v l m t h n h thc i u k h i n b i u h i n m i ca gen

trong q u trnh p h t t r i n .
4/ Dicer v RISC
Si d s A R N , h p A R N , m i A R N v s i A R N n c k h n g t h g y ra b t hot gen v
p h hy m A R N , c h n g cn c s tr g i p ca 2 enzym l dicer v RISC l m v i c n y .
E n z y m dicer c Bernstein v cs p h t m i n h ra l n u r u i d m (2001), l m t e n z y m
phc tp thuc n h m RNase n i (RNase m f a m i l y ) . E n z y m n y c c c n h i m v r t c
th l thc h i n chc n n g ct si d s A R N hoc h p A R N , to ra c c s i A R N . R I S C l m t
hp phn ca b m y R N A i . RISC l n u tin c p h t m i n h ra r u i d m b i
H a m m o n d v cs (2000). RISC c c u to t protein v A R N . Protein trong phc hp
c hot tnh ca ribonuclease enzym v i k h n n g ct A R N . RISC mang si s i A R N c
s t n g n g v i m A R N to ra o n g h p n i k p gia s i A R N v m A R N v sau
ct t m A R N ra l m hai.
C ch ARN:
E n z y m dicer ct si d s A R N hoc h p A R N d i t h n h c c o n n g n g i l s i A R N .
C c si ngn sau c t c h t h n h 2 si n v m t trong 2 si g h p v o RISC to
t h n h t hp s i A R N - R I S C . T hp n y n h m v o c c t r n h t t n g n g t r n si
m A R N . m A R N b ct b i RISC v sau c c c enzym endonuclease c trong t h n h p h n
ca R I S K s p h h y h o n t o n m A R N .
C ch hot ng ca ARNi (RNAi theo nguyn bn ting Anh)
Mt si RNA kp c chiu di khong vi trm bp
c ct bi e'nzym decer

Cc si siARN kp ngn t 21 - 23 bp
(Small Intrtenng RNA)

Enzym RNA induced silencing complex (RISK)

m xon siRNA kp. Si siRNA n trong t hp RISK bt cp vi
mARN on tng ng vi n v enzym RISK s ct mARN lm 2

Enzym RNA dependent ARN polymerase (RdRP)

Nhn siARN mi
(Theo Hutvagner v Zamore, 2002)

88

C c A R N n h v methyl ha promoter:
N g o i c c h g y bt hot gen t h n g qua p h n huy m A R N trn y , q u trnh l m
c m gen c t h x y ra mc gen v c g i l l m c m dch m gen Transcriptional
Gene Silencing - TGS). H i n tng n y c Wassenegger v cs p h t h i n l n u
thuc l n m 1994. H n h thc l m c m gen n y c g i l m e t h y l h a A D N d i tc
n g ca A R N ( R N A - Directed D N A methylation = R d D M ) ) . Q u trnh n y x y ra k h i
promoter ca gen b methyl h a v l m gen m t k h n n g p h i n m (tng hp m R N A ) .
3.6. T BIN GEN
Q u trnh n h n i ca si A D N t h n g x y ra m t c c h c h n h x c , t m t p h n t
A D N m to ra hai p h n t g i n g ht p h n t m . D o v y bo m k h n n g duy tr c c
c tnh d i t r u y n t t b o n sang t b o k i a v gia c c t h h trong q u trnh t i n
hoa. l tnh "bo th" ca gen.
M t k h c trong q u t r n h sinh tng hp ca A D N c t h x y ra n h n g sai st d n
n s thay i c u t r c hay l t r n h t c c nucleotid trong A D N d n n h i n tng
t b i n - hay c n g i l b i n d . Quan h b i n chng gia t n h " d i t r u y n " , t n h "bo
t h " v i k h nng thay i c u t r c ca n , t n h " t b i n " , t n h " b i n d " l c s cho
s t i n hoa.
N h n g n g u y n n h n ca q u trnh t b i n c r t n h i u ti l i u khng nh,
bao g m :
C c t c n h n v t l k h c n h a u : p h n g x , bc x ca m t t r i , tia nhit (nh
hng ca nhit cao t c n g ln t bo)...
C c t c n h n hoa h c k h c n h a u : c c cht g y t b i n n h n to nh: colchicine,
thuc bo v thc vt, c c cht d i t c, hoa cht c n g nghip k h c . . .
S g i hoa c a t b o v m . Trong q u trnh l o hoa ca m t b o v c t h , m t
s cht c v c c sn p h m t h cp tch l y c t h d n n s sai l c h trong n h n bn
A D N . H i n tng n y c t h quan h v i t l mc bnh ung t h cao n g i gi, hoc
b m l n t u i sinh con d dng v i t l cao h n .
K h i t b o v m t c h k h i c t h sng h o n c h n h v c n u i c y i n vitro
B i n d ca c c t b o trong i u k i n n u i c y g i l b i n d t b o soma
(somaclonal variation). T n s t b i n trong t b o n u i c y c t h t 10' - 10" , cao
2

hn c k h i s d n g m t s c c tc n h n g y t b i n .
N g y nay, n g i ta a n g tranh ci n h i u v n h n bn n g i bng con n g v tnh
hoc n h n bn t b o n g i cho mc c h sinh sn. V n nay, con n g i v n c h a t h
k i m sot c c c t b i n x y ra trong q u trnh n u i c y m v t b o n g i n g o i
c t h . D o v y q u trnh n h n bn c t h d n n nhng t b i n nguy h i m c h a lng
trc c.
S 3.9 cho ta t h y s thay i trong c u trc ca A D N c t h d n n nhng thay
i trnh t axit amin trong p h n t protein n h t h n o .

89

Si A D N ban u ( b n h thng)
CCT

G A G G A

Thay
ADN

x y ra t bin

Nhn i A D N

trong

P h n t b n h thng
ca t h h con
c

T G A

C C T G g G G A G

G A G

G G A C l C C T C

GGACTCCTC
m . A R N bnh

thng

c c

UG

Th t bin

ch m

A G G

C C U G

AG

m-ARN

I G G A G

Phin m

( ^ P T ^ G I ^ G U ^ )

Protein bnh thng

S 3.9. t bin xy ra trong phn tprotein

Pro

Gi

T h t b i n protein
(Theo Karl

Drica,1997)

K h i x y ra t b i n , cp nucleotid tng n g A - T trn hai si A D N i x n g c t h

c thay bng cp G-C, C-G hoc T - A . T r o n g t h d c t h s n , cp b a z A - T
trn si A D N b n h t h n g b t b i n t h n h T - A d n n m d i t r u y n t r n gen v
trn si m A R N b thay i , cp ba G A G trn si A R N b n h t h n g b thay i t h n h
G U G trn si A R N t b i n . K t q u trong p h n t protein t b i n , V a l i n c h i m v tr
ca Glutamin.
S thay t h i k h i ch m t axit a m i n trong protein c t h d n n l m m t chc
n n g hoc thay i hot tnh ca protein hoc enzym, d n n thay i trong c u t r c
hoc chc n n g ca t b o , c t h sng mang c c t b i n n h v y g i l c t h t b i n .
N u t b i n l m thay i chc n n g ca m t protein quan trng, c t h t b i n c t h
b cht hoc b bnh hoc b i n dng. i k h i x y ra c c t b i n c l i , l m c i t h i n

90

k h n n g sinh t n v sinh sn ca c t h t b i n . N h v y , c t h t b i n tr n n c u
t h h n trong q u n t h v m t bc t i n n h x y ra i v i s t i n hoa.
M c c h n h x c trong q u trnh n h n i ca p h n t A D N l r t n g ngc n h i n .
v i k h u n , t n s t b i n x y ra m t gen ch l m t phn t r i u , tc l ch c m t t
b o t b i n gen trong tng s m t t r i u t b o quan st. N g y nay n h gi t c
n g g y t b i n ca m t tc n h n hoa hc hoc v t l, v d : thuc tr su ln q u
t r n h t b i n c t h sng, n g i ta t h t c n h n l n v i k h u n v x c n h t n sut
t b i n ca v i k h u n .
T r n c s t n sut t b i n do thuc tr su g y ra v i k h u n n g i ta c t h d
b o mc nguy h i ca thuc su i v i con n g i v m i sinh. t b i n l
n g u y n n h n g y ra r t n h i u bnh l con n g i , trong c c c b n h ung th, c c
trng hp sinh con d dng. N g h i n cu t b i n l m t trong c c p h n g p h p c bn
x c nh chc n n g ca gen.
Si ADN

bnh

thng

Sthtcc nucleotit 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
TnnhtnucleotittrongADN
Trinh t axit amin trong protein

-A-A-G-6-C-C-A-C-C-A-G-C-C-G-A-G-A-A

Phe

Arg

Trp

Ser

Ala

Lu

t bin im lm i ngha ca codon:

No.10:A->G

TrnhtNuongADN A A-GGCCA-C C G-G C-G-C-T-C T-TTrinh t axit amin trong protein Phe

II

Arg

li

li

Trp

li

Pro

l i
Ma

Lu

A v tr 10, n u A thay t h bng G, prolin s thay t h serine trong p h n t protein.

N u c v tr 8 thay t h bng T, t b i n to s to ra codon v ngha-codon dng tng
hp protein (end codon).
No.8:C-T

Trnh t nucleotit trong ADN

-A-A-G-G-C-C-A-T-C-T

Trnh t axit amin trong protein

Phe

li

li
Arg

.
end

li

C-G-G-C-T-G-A-A-

_ J I

91

K h i c thay t h bng T v tr t h 8, codon t n g ng v i tryptophan b thay t h b i

codon v ngha A T C ( U A G trn m A R N ) . Q u trnh dch m d i t r u y n b n g n g l i , sinh
tng hp protein k h n g t i p tc c na.
C c tc n h n g y t b i n : B o c o ca T Chc N n g lng n g u y n t q u c t
( I A E A - International A t o m i c Energy Agency, 2006) cho b i t bng c c p h n g p h p g y
t b i n thc n g h i m cho n nay (2006) to c trn 2600 g i n g c y t r n g k h c
nhau thuc 180 loi thc vt. a s c c ging t b i n c to ra bng p h n g x
( c h i m 88% tng s ging t bin), trong tia Gamma n g vai t r c h y u , to ra
khong 6 4 % c c ging t b i n p h n g x . C c tc n h n hoa hc to ra k h o n g 10% c c
ging t b i n . G i n g la t b i n mang gen na l n s d l (semidwaft gene s d l l m t
gen l n ) l ngun gen quan trng trong to ging la. H u h t c c g i n g l a l n , n n g
sut cao, chu p h n b n v t h m canh mang gen n y .
3.7. GENOM V THNG TIN DI TRUYN
Genom l kho lu g i t h n g t i n d i t r u y n k h n g l trong m t t h t c h s i u n h .
N g i ta v genom ca c y la n h m t pho s c h c 12 c h n g , m i c h n g l m t
n h i m sc t h . M i c h n g cha h n g n g n c u chuyn, m i c u c h u y n c g i l
gen. M i c u chuyn c r t n h i u t, m i t c g i l m t codon. M i codon c v i t
b i ba ch ci, m i ch ci l m t baz. T t c c b n b a z k h c nhau: A d e n i n , T h y m i n ,
Cytosin v Guanin, to ra 64 t. Ta h y tng tng genom ca m i n g i g m k h o n g
3000 t r i u c h c i . M i n g i l m t kho s c h t h n k k h c b i t . B y t n g i l by t
c u chuyn n h i u k k h c nhau. H y c h i m n g n g s a d n g phong p h ca t h g i i
n y v h n g th n i m v u i sng k h n g c n g ca n h n thc s sng.
N m 1997, Fred Blattner v c c cng s t i T r n g i hc W i s c o n s i n h o n
t h n h vic g i i m trnh t nucleotid ca m t chng E. c o l i K 1 2 k h n g g y b n h g i l
chng M G 1655. K t q u sau 15 n m n g h i n cu h gen ca E . c o l i c c n h khoa hc
p h t h i n genom ca E.coli c cha tt c 4.639.221 cp b a z . H gen ca
subtilis

c cha 4100

Mycobacterium

protein v i 4.214.810 cp b a z . H gen

tuberculoss

bnh d d y Helicobacter

Bacilus

ca v i t r n g

lao

c 4000 gen v i tng s 4.411.529 cp b a z , v i k h u n g y

pylori

c t i t h i u 1590 protein trong h gen g m 1.667.867

cp baz.
C c p h n tch m y tnh trnh t A D N ca E.coli

x c nh c 4288 gene, trong

88% gen m hoa protein v c c A R N , 1 1 % gen c chc n n g i u k h i n , n h h n 1 %

cha c c trnh t A D N l p l i . K h o n g c c h trung b n h gia c c gen ca E.coli

khong

120 cp baz. Trong s 4288 gen, chc n n g c t h ca 3200 gen c x c n h .

T o n b h gen ca n m men s. cerevisiae

c 12.068.000 cp b a z v con s n y

n g i l khong 3 t cp baz, l n g p 250 l n genom n m men (Bng 3.1).

92

Bng

3.1. S lng ADN

trong

cc sinh

vt

Sinh vt

S cp baz
(triu)

Sinh vt

S cp baz
(triu)

Sinh vt

S cp baz
(triu)

Sinh vt

S cp baz
(triu)

Vi
khun

4,7

1.000

Cy
Arabidopsis

70.000

s v 40
(virus)

5,243

Nm
men

15

Ngi

2.800

Ng

15.000

X174

5,386

Giun
trn

80

Chut

3.000

K nhng

90.000

Rui
dm

155

Cy thuc
l

4.800

Loa kn

90.000

Bng 3.1 cho t h y trong n h i u trng hp lng A D N k h n g t h h i n s t n g quan

v i thang bc t i n hoa ca sinh g i i .
T r n h t A D N c c sinh v t k h c nhau c t h r t g i n g nhau. Trong thc t khong
10% c c gen ca n g i t n g t v i gen ca r u i d m v giun. T r n h t A D N ca n g i
v tinh tinh ging nhau 9 9 % , 50% gen n g i g i n g v i thc vt; H n g t r m gen ca
n g i d n g n h c n g u n gc t v i k h u n . Theo ngha r n g genom ca n g i c m t
s c i m n h virus. N g u y n n h n l do m t s q u trnh sinh hc c b n n g i c
th ging v i v i k h u n , thc v t v c c n g v t k h c . C c h gen tham gia q u trnh
quang hp v q u trnh d d n g to n n g lng ty t h c c loi thc v t k h c nhau s
rt ging nhau.
3.8. PHT HIN CC GEN QUAN TRNG LM c s CHO TO GING MI
C c p h n g p h p c n b n h i n nay p h t h i n c h c n n g c a gen:
- P h n g p h p l a i p h n tch k i u Mendel.
- P h n g p h p g y to t b i n l m p h v hoc thay i chc n n g ca gen.
Da trn k t q u p h n tch c c c t h mang t b i n (so s n h mc p h n t hoc lai
th t b i n v i i chng), n g i ta c t h x c nh c t n g quan gia gen v tnh
trng, gen l n hay t r i , tnh trng n gen hay a gen, n h m l i n k t ca gen hay v tr
ca gen trn n h i m sc t h .
- M t trong c c p h n g p h p h i u q u nht h i n nay x c nh chc n n g ca gen
l gy t b i n bng ci gen (xem phn k thut g y t b i n bng ci gen).
- P h n g p h p p h n tch s k h c bit d i t r u y n ( C h n g t r n h so s n h h gen Comparative Genomics) bng k thut sinh hc p h n t: V d , s k h c b i t gia c t h
khoe m n h v i n g i bnh, gia n g i t h i u n n g tr t u v i n g i t h n g minh...
- So s n h trnh t A D N gia n g i v i tinh tinh, gia n g i v i chut, gia n g vt
v i thc vt... p h t h i n s t n g n g v k h c b i t gia h gen v n h m tnh trng
( C h n g trnh G e n o m v C h n g trnh chc n n g ca Genom).
93

- So s n h t h v i n c A D N gia m t c t h v i i chng ca n , so s n h t h v i n
c A D N gia hai giai o n pht t r i n k h c nhau ca c n g m t c t h p h t h i n chc n n g
ca gen v thi i m b i u h i n ca gen trong m i chu k p h t t r i n ca c t h sng.
- P h n g p h p m i nht a n g c d n g p h b i n n g h i n c u chc n n g v b i u
h i n ca gen l A R N i (ting A n h l R N A interference, v i t tt R N A i ) . N g i ta to ra
c c trnh t A R N ngn t 21 - 25 khong nucleotid t b o c h t bng n h i u p h n g
p h p k h c nhau. C c A R N ngn s b n v o c c m A R N ca gen c h ( m A R N c m t
o n trnh t t n g n g v i A R N ngn), l m m A R N b ct l m i v m t chc n n g
tng hp protein. T h n g qua s m t protein v m t vi t n h trng k m theo, n g i ta c
t h x c nh c chc n n g ca gen.
P h v chc n n g ca gen i u k h i n k h n g ch l p h n g p h p n g h i n c u chc
n n g ca gen, m c n c ngha thc t i n to l n . P h n g p h p n y m a n g l i cuc
c c h m n g i v i c n g n g h i p sinh hc sn x u t c c cht hot t n h sinh h c n h k h n g
sinh, vitamin, axit amin, enzym, c c alcaloid v n h i u dc c h t k h c . C t h t b i n
k h n g c n k h n n g k i m sot c c q u trnh sinh hoa c l i cho n , n g c l i c h n g
phi sn sinh ra c c hot cht c l i cho con n g i .
Gen i u k h i n c t h c coi l c i n g u n ca c c d n g c h y sinh h c c h n g ? T
gen i u k h i n c h n g ta c t h l n ra c c m t x c h c n b n to n n p h p b i n chng ca
s sng c h n g ?
P h t h i n c c gen q u a n t r n g t r o n g t n h i n l c s t o r a c c c t h b i n i
di truyn:
C c p h n g p h p sinh hc p h n t g i p p h t h i n n g y c n g n h i u c c gen m i
c t h to ra cuc c c h m n g trong n n g n g h i p (xem c h i t i t c c c h n g sau):
- P h t h i n c c gen bo v thc v t chng s u b n h h i .
- P h t h i n ra c c gen k h n g thuc d i t c v c h u y n c c gen n y v o c y trng
g i m c h i p h thuc d i t c v t n g n n g sut.
- P h t h i n ra c c gen c h i sm: n g i ta t h n h c n g c h u y n gen c h i s m n y
v o m t s c y trng k c c y t h n g .
- P h t h i n c c gen k i m sot q u trnh c h i v cht lng ca q u n h c c gen k m
h m sinh tng hp ethylen: Q u ca c c g i n g mang gen k m h m c h n c t h t n t i
trn rung lu h n , d bo q u n v bo q u n c lu h n trong q u t r n h v n c h u y n .
- Pht h i n c c gen c h a khoa ca q u trnh quang hp: 3 gen n g vai t r c h o
trong chu trnh quang hp ca c y C4 c t c h ra v c h u y n v o c y C3. N h p h t
h i n n y , c c n h khoa hc hy vng to c c c g i n g la nc c t h t n g n n g sut
20 - 50 % so v i g i n g i chng.
C c c n g ty N a m T r i u T i n c n g b t h n h c n g c h u y n gen quang hp t v i
k h u n v o la l m t n g n n g sut ging la c h u y n gen ln 3 0 % so v i i chng
(www.whfreeman.com).
94

D k i n c c gen c n bn quyt nh n n g sut, cht lng, k h n n g chng chu su

b n h ca c y trng v v t n u i , c c gen sn x u t dc c h t q u s c p h t h i n . C c
p h t m i n h n y chc c h n s to ra cuc c c h m n g sinh hc trong k n g u y n m i n y .
3.9. KT QU BC AU TRONG NGHIN cu SINH HC PHN T PHC v
NNG NGHIP NC TA
V i t N a m , n g h i n cu sinh hc p h n t phc v n n g n g h i p c V i n D i
t r u y n N n g nghip t r i n khai m n h trn c y la. L n u tin nc ta, V i n to
c hai d n g bt dc c n h n c m ng nhit ( T G M S ) m i v hp tc v i c c
nh khoa hc nc n g o i n g h i n cu l p bn p h n t ca hai gen n y ( N g u y n V n
n g v cs, 1999; Phm N g c L n g v cs, 2002).
C c n h khoa hc V i t N a m cng chng m i n h c mc p h n t rng gen
k i m sot tnh trng T G M S ca d n g la T G M S - V N 1 l m t gen m i , n m trn vai ngn
ca n h i m sc t h s 2 ca l a . N k h n g ging v i gen t m s l thuc n h i m sc t h s 8
do tc g i Trung Quc p h t h i n n m 1995 v cng k h n g g i n g gen tms3(t) do c c tc
g i I R R I p h t h i n n m 1997.
C h n g ta c n g l p t r n h t c ch th p h n t lin k t cht v i gen tms4(t) (gen
k i m sot tnh trng T G M S ca d n g l a T G M S - V N 1 ) . Ch t h n y c lu g i t i
N g n h n g gen mang m s: A F 07954 v x c n h c t r n h t ch t h lin k t gen
TGMS-VN6.

Phi hp v i T r u n g t m k thut gen v C N S H quc t , C n b ca V i n p h n l p

c ba gen m hoa c c men helicase m x o n k p A D N t u H L a n v i c c trnh t
m hoa c d i 1,6 k b , 2,2 k b v 2,9 k b . T r n h t nucleotid ca ba o n gen n y
c x c nh v n g k bn q u y n t i N g n h n g gen t h g i i v i c c m s t n g ng
l Y 17186, A F 271892 v A F 271893 (Phm X u n H i v cs, 2001).
V i n s d n g c c p h n g p h p marker p h n t x c nh v tr, chc n n g ca
cc gen quan t m t r n n h i m sc t h la. P h n g p h p n y cho p h p n h gi nhanh,
chnh x c s c m t hoc k h n g c m t ca m t gen n o trong m t c t h hoc trong
d n g ging c y trng, vt n u i hoc chng v i sinh vt v c p d n g trong c h n to
ging m i v i tn g i l K thut Sinh hc p h n t h tr c h n g i n g .
Bng k thut n y x c nh c m t s gen k h n g bnh o n , bc l, r y n u ,
gen k h n g su v thuc d i t c, gen quy nh h n g t h m n h i u g i n g la, t s
dng c c gen n y l m gen ch th (marker genes) n h gi nhanh tp o n g i n g la
m i . H n g t r m d n g ging la c n h gi k h n n g chng chu bnh o n, bc
l... qua c c gen ch t h . P h n g p h p n y c n g i p x c n h c c gen c gi tr trong con
lai F l v c c t h h t i p theo n h m c h n ra c c c t n h mc p h n t n h t n h k h n g
bnh o n , r y n u , bc l...
P h n g p h p sinh hc p h n t c v i n s dng trong n g h i n cu bnh h i la
o n . s d n g k thut Pot2-repPCR x c nh c c chng n m o n V i t
N a m . Qua , x c nh c 88 chng o n m i n Bc v m i n Trung. C c chng

95

 o n n y s d n g n h gi c tnh ca c h n g v k h n n g k h n g ca t p o n
ging V i t N a m i v i tng chng. x c nh c c c g i n g k h n g o n g m :
n ; T m t h m , SH z2, C h i m Bc, T m xoan, D , Bt e n . C c g i n g n y c s
dng l p bn gen k h n g v t m c ch t h p h n t l i n k t v i gen k h n g . C c
ging la k h n g o n n i trn c s dng l m v t l i u k h i u cho l a i t o
a gen k h n g ca c h n g v o c c ging a n g c s d n g trong sn x u t n h n g b l y
n h i m cao. Sau d n g ch t h p h n t chn c c c t h v d n g l a k h n g . N h n g
d n g la k h n g c c c c tnh t t k h c s c a v o sn x u t .
Bng p h n g p h p t n g t c t h quy t n h i u gen k h n g s u b n h k h c nhau v o
m t ging n h s tr g i p ca ch th p h n t:
- C c d n g la quy t gen k h n g o n to ra c c g i n g sn x u t quan trng
trong nc n h CR 203, D T 10, D T 12, D T 13, D T 21 c quy t 3 gen k h n g o n
( P i - 1 , Pi-2, Pi-3).
- C c d n g la quy t gen k h n g bc l: K h a n g d n , Q5 v m t s d n g k h c mang
3 gen k h n g bc l (Xa-5, Xa-7, Xa-21).
- C c d n g la quy t gen k h n g r y n u : D T 2 1 , D T 1 0 , N p 415 mang 2 gen k h n g
ry n u (bph-2, bph-3).
V i n D i t r u y n N n g nghip n g h i n cu s d n g k thut c i gen (insertional
mutation) v o genom ca c c chng n m g y bnh c y n g cc v t o c nhiu
chng n m m t k h n n g g y bnh. C c gen b m t t n h trng do c i gen ( m t k h n n g
to c t quy i n h k h n n g g y bnh ca n m ) c x c i n h .

96

Chng

CNG NGH A D N TI T

HP

4.1. V I R U S , V I K H U N V K H N N G C H U Y N T I V T L I U D I T R U Y N
M t s v i k h u n v virus v i nhng c t r n g c bn sau y c s dng l m
vectors chuyn gen t t b o sang t b o , t c t h sng n y sang c t h sng k h c :
- v i k h u n , plasmid l m t si A D N m c h v n g c k c h thc n h h n n h i u so
v i A D N n h i m sc t h ca v i k h u n . Plasmid c t h c n h n bn m t c c h c l p
k h n g ph thuc v o q u t r n h n h n i ca n h i m sc t h .
- M t s t b o E. c o l i c mang plasmid v n g ( g i l y u t g i i t n h F hay l sex
factor). Y u t F c t h t n t i trong t b o trng t h i t do trong t b o cht hoc c
th dng g h p n i v i n h i m sc t h m c h v n g ca v i k h u n .
- Trong q u trnh t i p hp gia c c v i k h u n , y u t F ca v i k h u n c t h chuyn t
t b o mang F sang t k h n g mang F.
- Siu v i k h u n l m t l o i virus g y bnh v i k h u n . A D N ca siu v i k h u n c t h
l vector mang A D N t m t v i k h u n n y sang v i k h u n k h c .
- Trong q u trnh c h u y n gen, m t o n A D N n g o i l a i n o c t h g h p n i v i
A D N ca siu v i k h u n . Sau k h i t b o v i k h u n b ly n h i m b i s i u v i k h u n , A D N
siu v i k h u n s t h m nhp v o t b o m n k sinh v mang theo v o v i k h u n o n
A D N ngoi l a i .
- A D N trong m i t r n g c t h x m nhp v o t b o v i k h u n v g h p n i v o N S T
v i k h u n l m thay i genom v i k h u n n y .
4.1.1. Vi khun
V i k h u n thuc n h m sinh v t t b o t i n n h n . Trong chu t r n h tun h o n v t cht
v n n g lng ca h sinh t h i , v i k h u n l m t t h n h phn ht sc quan trng.
N h n g c t n h c b n ca v i k h u n c khai t h c s d n g trong r t n h i u lnh
vc i sng nh:
- C n g nghip sn x u t k h n g sinh, vitamin, axit amin,...
- Sn x u t vacxin ti t hp v c c cht hot tnh sinh hc k h c d n g trong y t .
- C n g nghip c h b i n thc p h m .
- C n g nghip v i sinh bo v thc vt.
- P h n b n sinh hc.
D o v i k h u n c c u t r c genom n g i n ch g m si A D N , chu k sinh sn ngn,
h s sinh sn cao v l tc n h n g y bnh t r u y n n h i m n g i , n g vt, thc v t n n
97

v i k h u n tr t h n h m t i tng n g h i n cu l t n g ca n g n h d i t r u y n hc v
c n g ngh sinh hc.
N h c n g h i n cu v i k h u n l n u tin con n g i p h t h i n ra b n c h t ca v t
l i u d i t r u y n l A D N , m d i t r u y n , c c t b i n sinh hoa hc, h i u k h i n ca gen,
k thut A D N ti t hp.
H i n nay, v i k h u n Agrobacterium

v n l vector h u h i u n h t trong thao t c c h u y n

gen cy trng. V v i k h u n n y mang hai c tnh c b n c lin quan n k thut gen

l kh nng

tip hp gia cc t bo v kh nng

hp th ADN

mt cch kh d dng

t m i trng.
D o v y , h i u b i t ca c h n g ta v c c h i n tng d i t r u y n v i k h u n s l k h n g
t h t h i u c trong n g h i n cu k thut gen v c n g n g h sinh hc.
4.1.1.1. Hin tng tip hp vi khun
T i p hp (confugation) l q u trnh t i p c n gia hai v i k h u n t n g hp trong
x y ra s v n c h u y n A D N t v i k h u n n y sang v i k h u n kia.
S t i p hp x y ra t h n g qua m t c u t r c ca v i k h u n g i l si t i p hp ( p i l i ) .
T i p hp l h i n tng l a i c h o n g i n x y ra g i i v i sinh v t . n g h i n cu h i n
tng n y v i k h u n E. c o l i l m t i tng l tng.
V i c t i p hp v chuyn gen E . c o l i c thc h i n b i m t plasmid A D N mch
v n g g i l y u t g i i tnh F.
4.1.1.2. Yu t'F v kh nng ti t hp v vn chuyn vt liu di truyn gia cc
t bo
Y u t F c t m t h y k h n g p h i m i t b o , trong t b o c mang y u t F
g i l F* v t b o k h n g c y u t F g i l F . Y u t F c cha k h o n g 100 gen. C c
gen n y quy nh m t s tnh trng quan trng ca plasmid, l:
- Plasmid F c t h t n h n bn;
- T bo F

c th tng hp yu t tip hp (pilus) t r n b m t t b o . C c y u t

t i p hp c dng h n h n h m t ng n h (c bn cht l protein) cho p h p c c t b o *

t i p n i v i cc t b o k h c (s t i p hp).
- Khi 2 loi t bo tip hp, yu t F c nhn bn v plasmid

F m i c n h n

bn s chuyn t t b o F * sang t b o F . Trong q u t r n h , t b o F * l u n bo t n

c y u t F. Q u trnh t i p hp x y ra nhanh c h n g , do v y plasmid F c t h lan rng
ra c c t b o k h c trong qun t h .
- Cc t bo F

b c ch tip hp vi nhau. D o , y u t F k h n g c c h u y n t

t b o F sang t b o F*
- i k h i t b o F mang trong genom ca n m t hoc vi t i u phn c k h n n g d i
c h u y n g i l c c trnh t ci xen ( g i tt l phn t IS - insertion sequence). C c phn
t IS n y l c c t i u phn dch c h u y n ca A D N (mobile segment), n c t h d i c h u y n
t v tr n y sang v tr k h c b n trong genom ca t b o c h , hoc t n h i m sc t h
98

(NST) n y , plasmid n y sang NST hoc plasmid k h c . S t n t i ca p h n t IS trong

plasmid hoc N S T cho p h p to ra c c v tr k h n n g x y ra trao i c h o gia c c
o n t n g n g (homologous). N h v y , plasmid A D N c t h c i v o N S T ca v i
khun.
K h i x y ra s c i t y u t F v o NST, y u t F c t h v n c h u y n c c gen t N S T
ca t b o c h sang t b o nhn, k t q u l to ra s c h u y n gen k p , c c gen ca t b o
ch cng v i y u t F c chuyn sang t b o nhn.
T r n thc t , h i n tng y u t F g h p v o N S T c h x y ra m t t l n h t b o
trong qun t h v i k h u n mang y u t F . S t t b o n y c t h c k h n n g c h u y n c c
allen ca N S T sang c c d n g v i k h u n k h c , n g i ta c t h n h n b i t c h i n tng
ny v n h v y p h t h i n ra h i n tng ti t hp gen v i k h u n v s c h u y n gen
trong q u trnh t i p hp. N g i ta c t h p h n l p c c c t b o t i trong qun t h c
yu t F c i trong N S T ca t b o v n h n c c t b o n y l n t h n h q u n t h n g nht
c F trong NST. Trong d n g t b o n y , m i l n t i p h p t b o u x y ra s c h u y n
allels ca N S T c n g v i y u t F sang t b o n h n v do t n sut ti t hp gen
d n g n y cao h n so v i c c qun t h ban u . V v y , n g i ta g i c c d n g trong
yu t F c c i trong N S T ca v i k h u n l d n g c t n sut t i t hp cao ( H - high
equency o f recombination) p h n bit c h n g v i c c qun t h F* t h n g t h n g trong
ch c m t v i t b o l H . D n g HT r t h i u q u trong vic c h u y n c c gen
markers ca NST.
- Y u t F c c i xen v o N S T i k h i l i t c h k h i N S T ca d n g HT v t r
nn c c plasmid c l p trong t b o cht n g t h i mang theo n l m t v i N S T ca t
bo ch. K h i plasmid F n y c g i l F b i n tnh ( m o d i f i e d F ) , k h i u l F . T h n g
qua tip hp ca v i k h u n , y u t F c c h u y n sang c c t b o F. N h v y , t b o
nhn c t h n g t h i mang hai b n copy ca c n g m t gen, m t b n copy trong N S T
ca v i k h u n v m t b n copy trong y u t F m n m i n h n c.
N h p h n tch t r n c h n g ta t h y rng c c c t h sng, d l r t n g i n n h c c
vi k h u n mang trong n nhng k h n n g k d i u :
Mt l: s t i p hp gia 2 t b o hoc 2 c t h sng trong x y ra s trao i v t
l i u d i truyn.
Hai l: tnh l i n h hot v k h n n g c i xen, g h p n i , t i t hp, d i c h u y n ca v t
l i u d i truyn, trong c tnh ca y u t F l m t t h h i n r n t . l y u t quan
trng ca a dng sinh hc trong t i n hoa.
4.1.2. Siu vi khun X (Phage lambda)
Siu v i k h u n X ( S V K X) c t h mang b n trong v protein ca n m t p h n t A D N
kch thc k h o n g 50kb. y c h n h l genom ca S V K X. o n gia ca si A D N ca
S V K X l o n k h n g nht thit phi c i v i t i b n v t i t h p ca S V K n y . D o
v y , n g i ta c t h d n g enzym ct g i i h n ct b o n gia n y i v thay v o
m t p h n t A D N l c k c h thc khong 10-15 k b v i n h n g t n h trng d i t r u y n x c
nh. K t q u to ra A D N (genom) ti t hp S V K n y .
99

C c siu v i k h u n c to ra theo c c h n c t h sn s n g g y l y n h i m VI
k h u n , t siu v i k h u n mang genom ti t hp c sinh sn g p n h i u l n .
4.1.3. Cc plasmd dng trong chuyn gen
v i k h u n , plasmid l m t si A D N m c h v n g c k c h thc n h h n n h i u so v i
A D N n h i m sc t h ca v i k h u n . Plasmid c t h c n h n bn m t c c h c l p
k h n g ph thuc v o q u trnh n h n i ca n h i m sc t h . T u y n h i n , s l n g tiu bn
plasmid trong qun t h v i k h u n l i h o n t o n ph thuc v o q u t r n h sinh sn ca v i
k h u n . N h n g s p h n b ca plasmid trong qun t h t b o ca m i l o i l k h ngu
n h i n . M t s t b o c t h mang plasmid, m t s k h n g c , v d n h t r n g hp
plasmid F E. c o l i . Plasmid c s dng theo c c c c h thc k h c nhau g h p v i
A D N (cc gen) ca c t h cho (donor) g i p cho v i c n h n d n g A D N n y v n g h i n cu
chc n n g ca n .
M t trong c c c trng quan trng ca plasmid v i k h u n l m t s c c plasmid
mang c c gen k h n g k h n g sinh. T r o n g t h i k m u ca k thut gen, c c plasmid
mang gen k h n g k h n g sinh c s dng n h i u h n c trong k thut gen b i gen
k h n g k h n g sinh trong plasmid cho p h p n g i ta d d n g t u y n c h n c c t b o
c h u y n gen mang gen k h n g hoc c c plasmid ti t hp gen.
Plasmid c t h t n h n bn trong t b o v i k h u n , trong m i t b o c t h c nhiu
p h i n bn plasmid. i k h i m t t b o c t h mang vi t r m plasmid. N h v y , plasmid
t h n g c d n g g h p c c A D N l (cc gen quan t m ) ca c t h cho v sau
n h n c c A D N n y ln h n g t r i u l n ( g i l n h n bn c l o n e - A D N ) .
M t s plasmid t n h i n c c c n h sinh hc p h n t t h i t k l i v t r n n cc
vector r t thch hp cho n g h i n cu c h u y n gen.
M t s c c plasmid s dng n h i u trong c h u y n gen:
Plasmid

pBR232

c c u trc n g i n , n g t h i mang hai gen k h n g k h n g sinh

l Tt v A m p . M i gen u c cha m t i m n h n b i t hoc i m p h n ct ca

m t enzym ct g i i hn nht nh. M t gen l c t h d d n g ci v o trong gen Tt
i m p h n ct l m cho gen n y b t b i n .
Tuy theo enzym ct m ta s dng, v tr ci ca gen l v o plasmid c t h k h c
nhau. K t qu c c t b o v i k h u n k h n g k h n g sinh Tetracycline b t b i n t r n n
c m ng v i k h n g sinh n y . Q u n t h c c v i k h u n t b i n gen Tt c k h i u l
A m p T e t (R = resistant, s = sensitive) l qun t h mang A D N ti t hp.
s

Plasmid

pUC:

p U C plasmid c mang gen ch th m u sc g i p ta c t h d d n g

nhn bit c c t b o m a n g plasmid n y . Trong plasmid c cha gen P-Galactosidase

{Lai).

N g i ta ci v o gen Laz m t o n A D N ngn g i l polylinker ( o n a lin

k t ) hay c n g i l o n c n h i u i m p h n ct ca c c enzym ct g i i h n k h c nhau.

T r n c s , t m ra 10 l o i enzym ct k h c nhau c k h n n g ct o n p o l y l i n k e r
c c i m nhn bit c th r i n g cho m i enzym. C c i m p h n ct n y r t t h u n l i

100

cho ci c c A D N l . Trong trng hp k h n g c ci t gen l , 2 t i u phn ca protein

c m hoa b i Lai s lin k t l i to ra enzym /?-Galactosidase c chc n n g b n h th
ng. t h hot tnh ca enzym n y n g i ta s d n g m t cht g i l X - G a l . N u t h m
X - G a l v o m i trng trong enzym /-Galactosidase hot hoa b n h t h n g , enzym
n y s p h n ct X - G a l v chuyn n t h n h cht m u xanh. Q u n lc t b o s c
n h u m m u xanh v d d n g p h n bit v i c c t b o k h c .
N u m t gen l c ci v o o n polylinker, c u trc ca enzym /?-Galactosidase
s b b i n i , k t q u enzym n y m t k h n n g c h u y n hoa X - G a l t h n h cht m u .
Trong trng hp n y qun lc t b o mang gen l (ci xen trong o n polylinker) s
k h n g c m u . Bng c c h n y n g i ta c t h c h n ra c c qun lc (colony) m u trng
k h n g A m p i c y l i n e (Amp) mang gen l cho c c n g h i n cu t i p theo.
Cc n h khoa hc c n g t h i t k c c plasmid mang n g t h i gen c h (gen quan
tm) v gen ch th m u gus c h u y n v o c y trng. C c m hoc t b o ca c t h
nhn gen c h u y n s c m u xanh, d p h n b i t .
Cc plasmid mang c c o n gen l c n g l n s c n g k h n g b n v n g . C c o n gen
di h n 20 k b s k h n g t h c h hp k h i n h n d n g bng c c plasmid v c h n g d d n g
tch k h i plasmid.
4.1.4. Cosmids
Cosmid l nhng vector c to ra n h lai g h p A D N ca S V K v i plasmid A D N .
Cosmid c t h c n h n bn theo 2 c c h : hoc l sinh sn n h c c plasmid hoc sinh
sn n h S V K . T u y n h i n cosmid c t h mang theo m t o n A D N l l n g p 3 l n
genom ca S V K .
Q u trnh to m t cosmid c d i n ra theo trnh t sau: trc ht, n g i ta ct b
hu ht A D N ca genom S V K ch g i l i m t o n ngn g i l cos site v o n n y cn
cho s t hp t n h i n ca S V K . G h p cos site v i plasmid to ra m t cosmid. T i p
theo d n g c c enzym ct g i i hn c k h n n g to ra c c u n h t n g n g cosmid
v n g t h i genom ca c t h cho t h n h c c o n .
Chn lc c c o n A D N c k c h thc 35 - 45kb t c t h cho g h p n i v i
cosmid A D N n h s tr g i p ca enzym ligase. Cosmid ti t hp c g h p v i c c
tiu phn k h c ca siu v i k h u n to ra m t siu v i k h u n ti t hp c k h n n g n h n
bn tip trong E . c o l i .
4.1.5. Siu vi khun n si
M t siu v i k h u n c genom ch l m t A D N n si c g i l siu v i k h u n n
si. T r o n g q u trnh ly bnh v i k h u n , si n A D N c c h u y n hoa t h n h si A D N
xon k p g i n g n h A D N genom ca c c c t h sng k h c v c k h n n g n h n bn
b n h t h n g n h c c siu v i k h u n k h c .

loi

4.2. C C E N Z Y M T H A M G I A V O Q U T R N H A D N T I T H P
Cc enzym tham gia v o c c q u trnh trao i cht ca A D N c ngha r t quan
trng i v i k thut d i truyn. C c enzym n y c t h n g m i hoa v c g i la b
kt cc enzym ca sinh hc p h n t. B kt n y chia l m ba n h m c bn sau:
- Cc enzym ct g i i hn c t h ca A D N .
- Ligase v kinase.
- A D N - polymerase v A R N - polymerase
4.2.1. Cc enzym ct gii hn c th ca ADN
Nm

1962,

W e m e r Arber v cng s x c nh c m t e n z y m n i b o

(endonuclease) c k h n n g p h n ct A D N ca virus g y bnh v i k h u n n h n g k h n g

p h n ct c c A D N ca k ch.
n nm
Haemophilus

1970,
infuenzae

H a m i t o n v Smith t c h chit c enzym ct g i i hn

v x c nh c rng enzym n y ch ct A D N o n c trnh

t nucleotid c t h .
H i n nay, n g i ta p h t h i n c khong 1200 c c e n z y m ct k h c nhau, trong
khong 100 enzym c t h n g m i hoa. C c enzym ct tr t h n h m t p h n g
t i n k h n g t h t h i u ca c c k thut sinh hc p h n t:
- P h n ct A D N v to ra c c p h n o n A D N , giai o n c t r n h t nucleotid c
th tt c c c c t h sng.
- S dng thit k c c v e c t c h u y n gen.
Cc enzym n i b o n g vai tr quan trng trong bo v v i k h u n trc s t n cng
ca c c siu v i k h u n . K h i A D N ca siu v i k h u n t h m n h p v o t b o v i khun,
c h n g b enzym ct n i b o ca v i k h u n p h n huy, do v y , v i k h u n t r n h b n h i m
bnh. C c n h khoa hc cng p h t h i n t h y rng A D N ca c c v i k h u n n y c k h
n n g " m i n dch" i v i c c enzym n i b o ca c h n h n . K h n n g " m i n dch" n y c
c l do q u trnh methyl hoa c c p h n t A D N ca v i k h u n . C c A D N c methyl
hoa ca v i k h u n s k h n g b endonuclease p h n huy.
K h n n g " m i n dch" ca v i k h u n lin quan v i c c hot t n h d i y ca enzym:
- E n z y m ct g i i hn (restriction endonuclease) ct A D N si k p c c i m c trnh
t c th. i m c t h trn A D N l m t o n trnh t ngn, x c n h ca c c
nucleotid.
- K h i enzym A D N - methylase l m methyl hoa c c b a z c h n h c c i m c th
trong h gen ca t b o ch, enzym ct g i i hn (restriction endonuclease) s k h n g
t h n h n bit i m c th v do v y s k h n g ct A D N c c i m c m e t h y l hoa.
Da trn s k h c bit ca c c enzym ct v i k h u n n g i ta chia ra m t s k i u
enzym k h c nhau. V d , k i u l i ca enzym ct g i i hn c s d n g trong d i t r u y n
p h n t b i c h n g c t h n h n b i t v p h n ct A D N i n v i t r o trong o n c t h c t
4-8 nucleotid.
N g i ta c t h ct m t p h n t A D N b t k k h i p h n t c c c o n t r n h t
n u c l e o t i d c t h m e n z y m ct c t h n h n b i t v k h i A D N k h n g b m e t h y l
102

hoa

( A D N t b o n h n thc v plasmid A D N c c c h n g v i k h u n m t k h n n g
methyl

hoa).

S 4.1. S ct c hiu ca eniym

ct gii

hn

4.2.2. Ligase v kinase

Hai enzym n y n g vai tr quan trng trong k thut A D N ti t hp. A D N - ligase
xc tc hnh thnh lin k t phosphodiester 5-3' gia 2 nucleotid trong c n g m t si trn
phn t A D N . K t q u n g u y n t C3 trn p h n t ng deoxyribose ca m t nucleotid s
lin kt v i p h n t C5 trn p h n t ng ca nucleotid tip theo qua p h n t phosphae.
Kinase l enzym l m phosphoryl hoa c cht bng to lin k t n g hoa tr gia
phosphate ca A T P v i p h n t c cht.
4.2.3. ADN - polymerase v ARN - polymerase
C c enzym n y tham gia vo q u trnh sinh tng hp A D N v A R N v i vic s dng
m t si A D N k h u n (template) v m i (primer) A D N hoc A R N . Trong k h i vic tng
hp A R N c t h bt u h o n ton m i .
T n g hp A D N v A R N bng enzym trong i u k i n i n vitro c ngha quan trng
trong sinh hc p h n t. V d, n h n c c trnh t A D N bng phn ng c h u i polymerase
(PCR). Phn ng PCR cn phi s dng A D N - polymerase b n v i nhit g i l Taq
A D N polymerase. E n z y m ny hot hoa t i u nhit 7 5 c . Trong trng hp A D N
c tng hp t m A R N i n vitro v i vic s dng enzym sao c h p ngc (reverse
transcriptase) s c g i l c A D N .
103

4.3. C N G N G H A D N T I T H P
4 . 3 . 1 . T h n o l m t A D N t i t h p
T A D N tng s c tch ra k h i m t c t h sng, n g i ta c t h ct A D N ra
t h n h n h i u p h n o n A D N k h c nhau. C c p h n o n t c h r i n y c t h cha m t
hoc vi gen.
o n A D N n y c t h c g h p n i v i m t A D N vector ( g i l A D N mang),
A D N vector mang theo n m t o n A D N m i g i l A D N ti t hp. A D N ti t hp
k t hp trong n c c p h n o n A D N hoc c c gen c n g u n gc k h c nhau.
A D N ti t hp hay l vector ti t hp sau c th c chuyn v o v i k h u n . V i
khun mang gen m i ny c th sinh sn rt nhanh to t h n h m t qun lc (colony) - mt
tp hp cc t b o c ngun gc t m t t b o ban u - v i h n g triu t b o mang gen.
M t colony hay qun lc t b o c n g mang c c A D N ti t hp g i n g nhau g i l
d n g A D N . V t A D N tng s ca m t t b o n g i ta c t h ct A D N t h n h v s cc
p h n o n A D N k h c nhau (c t h ln h n g chc n g h n p h n o n A D N ) , do v y sau
k h i chuyn c c o n gen n y v o v i k h u n t h n g qua c c vector n g i ta s n h n c
h n g lot (v s) c c colony ( d n g t b o ) mang c c gen k h c nhau. Bc t i p theo l
chn lc c c d n g t b o mang c c gen quan t m (gen c t h mang m t t n h trng d i
t r u y n x c nh).
4.3.2. ngha ca vic tch cc gen c th
- Cn cho vic x c nh trnh t nucleotid ca gen n y .
- Sau k h i trnh t ca gen c x c nh, c n c v o m d i t r u y n n g i ta c th
x c nh c gen n y m hoa sinh tng hp protein n o hoc b n trong gen n y c m y
o n intron v v tr ca intron b n trong gen. N h v y , x c n h c chc n n g ca
gen t h n g qua vic t m ra protein n o l sn p h m ca gen.
- So s n h trnh t A D N ca c c gen hoc c c allel k h c nhau ca c n g m t gen t
t m h i u q u trnh t i n hoa ca gen.
- Chc n n g ca gen quan t m c t h n g h i n cu bng c c h c h u y n gen n y v o t
b o hoc c t h sng k h c v n g h i n cu s thay i ca t b o hay c t h sng . N h
vy gen n g h i n cu c b i u h i n c t h c c h u y n gen v chc n n g ca gen c
th hin.
4.3.3. Cc bc to ADN ti t hp
4.3.3.1.

Tch chit

AD
N

Bc l tch A D N t t b o hoc t c t h sng quan t m v A D N vector. C c

p h n g p h p tch chit A D N t c c n g u n k h c nhau cng k h c nhau v c n h i u
ti l i u m t.
A D N tng s c t h c t c h ra t n h n t b o ca t b o n h n thc (eukaryote)
hoc t A D N ca lc l p hoc ty t h , hoc t n h i m sc t h ca t b o t i n n h n
(prokaryote). N h i m sc t h ca t b o t i n n h n l m t p h n t A D N m c h v n g .
104

A D N vector c t h l plasmid m c h v n g ca v i k h u n hoc l A D N ca siu v i

k h u n , v d A D N ca siu v i k h u n X.
4.3.3.2. Phn ct ADN
P h n ct A D N c thc h i n n h c c e n z y m ct g i i h n ( r e s t r i c t i o n e n z y m ) .
E n z y m ct g i i h n do v i k h u n t o ra c h n g l i s x m n h p ca c c s i u v i
k h u n . C c e n z y m ct hot n g n h n h n g c h i c k o ct A D N ca s i u v i k h u n
t h n h n h i u o n n h l m cho n m t k h n n g sinh sn t r o n g t b o v i k h u n . T u y
n h i n , c c e n z y m ct k h n g p h n ct A D N c c v t r tuy t h c h . C h n g c h p h n ct
A D N c c v n g c t r n h t n u c l e o t i d c t h m t h i . D o v y , A D N ca c c t b o
sng t n g i n ( n h virus) n phc t p n h t b o n g i u c cha m t s c c
t r n h t A D N n g n g i l i m p h n ct (restriction - e n z y m target site). C c e n z y m
ct p h n ct A D N c c i m p h n ct k h c nhau to ra v s c c p h n o n A D N
k h c nhau. C c p h n o n n y c t h c t c h ra t h n h n h n g v t ( b n g ) c t r n g
trong q u t r n h i n d i .
M t p h n t A D N c t h c nhng o n trnh t nucleotid c t h m trn o n
hai si A D N c trnh t nucleotid n h nhau n h n g chy ngc c h i u nhau. N h n g
trnh t nucleotid c t h n h v y c g i l c c palindrom. N h i u enzym ct g i i hn
nhn bit v ct p h n t A D N ra t h n h c c m n h n h t i v i tr ca palindrom.
V d: E n z y m ct E c o R I (t E.coli) c t h n h n b i t cp trnh t g m 6 nucleotid
bt k c t h sng n o d i y :
5" - G A A T T C - 3 '
3' - C T T A A G - 5 '
Trnh t n y g i l palindrom, v tr ct palindrom n y ca E c o R I n h sau:
5'-GAATTC-3'
_
3" CTTAAG-5'

.
^

5' - G A A T T C - 3'
y _

T o ra hai si c c c u n h sau
v

- - " G - 5'

Cc im ct nh trn c gi l cc u dnh bi n d dng bt cp vi cc on

A D N k h c c c c u d n h t n g n g . K t q u 2 o n A D N s g h p n i v i nhau v s
k t n i c thc h i n b i c c lin k t hydrogen gia c c nucleotid 2 si i xng.
Enzym E c o R I c s dng ct m t plasmid m c h v n g s to ra si plasmid
A D N thng v i 2 u d n h .

105

Hai u d n h n y rt d k t n i v i m t o n gen ( A D N ) c u n h t n g n g v i
n c tch ra t t b o n g i hoc t bt k y c t h sng n o k h c . K t q u s to ra
m t plasmid mang gen l . y l m t A D N ti t hp.
P h n g p h p to m t plasmid A D N ti t hp trong plasmid (vector) v si A D N
v d ca n g i , c ct b i c n g m t enzym l E c o R I c m t s 4.2: A D N
n g i c the to ra n h i u m n h A D N k h c nhau v i nhng a u d n h t n g n g v i u
dinh si plasmid. K t qu ta c t h thu c cc plasmid ti t hp k h c nhau. Trong
q u trnh g h p n i hai si A D N , s lin k t gia p h n t n g trn nucleotid n y v i
p h n t phosphate ca nucleotid k h c trn c n g m t si ca A D N ti t hp c n c s
xc tc ca m t enzym n g vai tr k h u n i g i l A D N - l i g a s e .
H i n nay n g i ta sn x u t t h n g m i h n g t r m c c enzym ct k h c nhau. M i
enzym ct nhn bit v ct m t trnh t A D N c th trn si A D N (bng 4.1).
Trong bng 4 . 1 , c t h d d n g nhn bit m t s enzym ct A D N k h n g to ra cc
u dnh, t i v tr ct c c enzym n y s to ra c c p h n o n A D N c u t.
Tuy n h i n , cc m n h A D N u t v n c t h g h p n i v i c c A D N k h c to ra
c c A D N ti t hp.
V i c ct A D N t h n h c c m n h n h c t h thc h i n c bng p h n g p h p c gii,
trong dung dch A D N c lc m n h v i s c m t ca c c m n h cng. M t si
A D N n h i m sc t h di c t h b t ra to t h n h c c o n c u t. C c o n n y cng
c th c n h n ln t h n h d n g A D N .
Nhim sc th ngi
Gen intereron

yj

NST v plasmid cng

b ct bi mt
enzym ct
^

Gen Intereron ghp

vi plasmid

Vi khun hp th
plasmid + gen
intereron

V Ghp
ni

Ghp ni

Gen intereron

Vi khun sinh sn to ra
cc vi khun mang gen mi

Tch chit
intereron

Nui cy vi khun sn xut interteron

S 4.2. To ADN ti t hp sn xut intereron ngi bng vi khun
106

G i i thch s 4.2: Cc bc to v i khun mang gen i n t e r e r o n ti t hp:

1.

Ct A D N ca n h i m sc th n g i c o n mang gen Interferon v A D N ca

Plasmid bng c n g mt enzym ct to ra c c p h n o n A D N c u d n h tng
ng.

2.

o n A D N mang gen Interferon ti t hp v i A D N ca Plasmid to ra Plasmid

ti t hp-mang gen Interferon.

3.

Chuyn plasmid ti t hp v o t b o v i k h u n to d n g v i k h u n mang gen

Interferon.

4.

N u i c y d n g v i k h u n mang gen I n t e r e r o n trong b n h n u i v trong e r m e n t o r

sn xut I n t e r e r o n t h n g mai.
Bng

4.1. V tr ct ADN

ca mt s enzym

N g u n gc

T n enzym
EcoR

c trng

Escherichia

coli

gii

hn

V t r ct c t h ca A D N

5' - G - A - A - T - T - C -C-T-T-A-A-G-

5'

t
EcoRII

E.coli

5' -G-C-C-T-G-G-C-C-G-G-A-C-C-G-

5'

t
Hindl

H.influenzae

5' - A - A - G - C - T - T -T-T-C-G-A-A-

5'

t
HaelII

H.

aegyptius
5'

-G-G-C-C-C-C-G-G- 5'
t

Hpall

H.paranfluenzae
5'

-C-C-G-G-G-G-C-C-

5'

t
Pst

Provdencia

stuartii
5'

-C-T-G-C-A-G-G-A-C-G-T-C-

5'

107

Smal

Serratia

marcescens
5'

-C-C-C-G-G-G-G-G-G-C-C-C-

5'

Bm!

Bacillus

amyolique/aciens
5'

-G-G-A-T-C-C-C-C-T-A-G-G-

5'

t
B.globiggi

Bgll

5'

-AG-A-T-C-T-T-C-T-A-G-A-

5'

t
Hindll

Haemophilus

influenzae

5' -G-T-Py-Pu-A-C-C-A-Pu-Py-T-G-

5'

4.3.3.3.

Ghp ni

ADN

T h n g thng, A D N ca c th cho (donor D N A ) v ADN

vector

c p h n ct b i

c n g m t enzym t h n h n h i u m n h A D N k h c nhau c u d n h . K h i c c m n h A D N
n y c h n hp v i nhau trong m t ng n g h i m , c h n g s to ra c c p h n t A D N ti
t hp. V m t enzym ct c t h p h n chia A D N ca m t c th cho t h n h r t n h i u cc
o n k h c nhau mang c c trnh t A D N (hoc c c gen) k h c nhau. K h i h n hp 2 l o i
A D N , trong dung dch c n h i u si plasmid mang u d n h t n g n g , n n n g i ta
c t h thu n h n c r t n h i u c c plasmid vector ti t hp mang c c gen k h c nhau
ca c th

cho.

C h rng trong q u trnh g h p n i gia c c o n A D N cho (donor) v A D N

plasmid cn c s tham gia ca enzym ligase. E n z y m n y n g vai t r g h p n i lin k t
n g - phosphate trn si A D N .
4.3.3.4. Nhn nhanh ADN ti t hp
Plasmid A D N ti t hp c t h c c h u y n m t c c h k h d d n g v o v i k h u n .
T r o n g t b o v i k h u n , c c plasmid c t h c n h n l n n h i u l n . V p l a s m i d ti t
hp mang c c A D N ca c t h cho, n n c c A D N n y s c n h n b n l n n h i u l n
c n g v i s n h n bn ca plasmid mang n . K t q u trong t b o ca v i k h u n c t h
c r t n h i u plasmid ti t hp. N h t b o v i k h u n c t h sinh sn theo c p s n h n ,
ch trong m t t h i gian ngn t m t t b o mang plasmid ti t hp c t h t o c

108

q u n lc g m n h i u t p h i n bn ging nhau ca A D N ti t hp mang o n gen quan

t m ca c t h cho.
H i n nay c n h i u l o i vector k h c nhau c s d n g n h n d n g gen, la c h n
vector c n ph thuc v o k c h thc ca o n A D N l c n n h n bn nhanh.
N h v y n g i ta c t h d n g c c A D N vector c n g u n gc k h c nhau ( A D N
vector c t h g i l A D N mang hay A D N mang chuyn) g h p v i c c m n h A D N
mang c c gen quan t m k h c nhau ca c t h cho.
t i n cho vic thao tc c h u y n gen, vector mang gen cn c m t s tiu chun
chung l:
- K c h thc phi n h .
- C k h n n g t n h n bn n h i u l n trong t b o v do n h n gen quan t m (gen
l) ln nhiu l n .
- C i m ct g i i hn t r n vector thch hp cho g h p n i v i gen l . N u m t
plasmid chi c m t i m ct ca enzym th s t h u n l i h n v ta c t h d d n g hem
trong nhn bit v tr g h p n i ca gen l .
- D d n g x c nh v p h t h i n A D N ti t hp.
4.4. NGHIN CU PHT HIN CC GEN V NG DNG K THUT ADN TI
T HP
Ngay sau k h i k thut A D N ti t hp ra i n g i ta u t ng dng k thut
ny trong lnh vc y t . T r o n g c n g b ca U y ban c h u u (1998) cho bit: "lnh vc
sc khoe, thc n, k i m sot bnh dch v bnh virus v t b o l n h m y " s l c c
hot ng c h a khoa ca khoa hc v c n g ngh. V i c p h n g v cha tr bnh v s
n g vai tr thit y u trong c i t h i n cht lng i sng. G i m gi c h m s c y t l r t
quan trng. N g i ta t n h rng n g E U R O chi cho t i m chng c t h tit k i m c
21-29 E U R O cha bnh. T h i trng vacxin t h g i i v o khong 3 t E U R / n m v t n g
trng ca th trng n y khong 9,7% n m . C c sn p h m C N S H c d k i n t n g
15% n m .
Cc sn p h m A D N ti t hp c gi tr trong y t l erythropoietin (EPO),
granulocyte colony stimulating factor (G-CSF ), a- I n t e r e r o n ( a - I F N ) , hormon sinh
trng n g i (human growth hormone ( h G H ) , insulin... G n y , khoa hc a n g s dng
c n g ngh A R N i sn x u t A R N ngn c k h n n g i u tr b n h H I V , k i m sot c c
bnh virus, bnh ung t h n g i .
4.4.1. a-Interferon ti t hp
I n t e r e r o n l c c protein v glycoprotein t n h i n do t b o sn sinh ra k h i c tc
n h n g y bnh x m nhp v o c th n g i .
a-Interferon c t h do t b o bch cu (leucocytes) hoc lymphoblasts n g i hoc
do c n g n g h A D N ti t hp sn xut ra. a-Interferon l protein 19 k D a c s dng
109

cha bnh n g sinh dc, v i m gan B m n (chronic hepatitis B ) , v i m gan k h n g A v

k h n g B (non-A, non-B Hepatitis), kaposi sarcoma lin quan v i A I D . . .
Cng

ngh

sn xut:

Chng E . C o l i c c h u y n gen v i plasmid mang gen a - I F N

n g i . Cc t b o E. C o l i c h u y n gen sn sinh ra Ct-IFN c thu hoch sau l n men.

Sau CC-IFN c tinh c h v i sch cao v i hot t n h c t h 200 t r i u n v/mg.
Sn p h m c k h n n g t h n g m i v t n n g sut k h o n g 15-25 mg/1 m i t r n g . G n
y v i s dng bioreactor dung tch 5 lt, trong m i t r n g n u i c y d u n g t c h 3,5 lt
sn x u t c 14 g a - I F N , x p x 4,0 g/lt. l k t q u p d n g c n g n g h n u i cy
v i mt t b o cao (high cell density cultivation- H C D C ) , n g u n cacbon d u y nht l
glucose v k c h thch sinh tng hp a - I F N bng y u t n h i t .
4.4.2.-Interferon ti t hp ngi
I n t e r e r o n l nhng sn p h m t v ca c t h . Phn ng ca c t h bt u bng
vic tng hp oe, p v y-Interferon. -Interferon do t b o c n g u n gc t T h y m u s tit ra
sau k h i c t h b n h i m bnh v i k h u n hay virus. -IFN c hot t n h c h n g virus, v i
t r n g , ung t h v m i n dch. y l protein s d n g cha c c b n h v i m k h p , vim
n g sinh dc, v i m da, ung t h t b o thn, b i u b , chng n h i m hc t c t n h . Phm
v i s dng y - I F N a n g m rng. T r n 200 gen c i u k h i n b i -IFN.
4.4.3. Sn xut bFGF (Basis FibrobIast Growth Factor)
N m 1980, Thomas p h t h i n cht b F G F c bn cht l m t protein do hypophysis
(tuyn y n n o ) tit ra. Sau h n g lot c c c n g t r n h n g h i n cu v b F G F ra i .
bFGF b v b F G F n g i ch k h c nhau 2 axit amin. T n m 1986, c c p h n g th
n g h i m n h i u nc trn t h g i i bt u n g h i n cu k thut A D N ti t hp sn
xut bFGF d n g trong y hc v m p h m . Gen tng hp b F G F c t c h ra t b hoc
n g i c thit k v chuyn v o E. c o l i . C h p h m b F G F c sn x u t t r n n i ln
men e r m e n t o r v i c c chng E. c o l i mang gen n y . M t e r m e n t o r 50 lt c t h sn xut
s lng bFGF cho c nc Trung Q u c . b F G f l m t protein c u t r c k h n gin,
n si, si ngn v i s lng 146 axit amin, n gen, c hot t n h sinh hc cao i v i
n g i v n g v t n n c ngha y hc v t h n g m i l n . V l n gen m hoa bFGF
n n vic to ra sn p h n A D N ti t hp v chuyn gen v o E. c o l i k h n g i n .
4.4.4. Vacxin ti t hp phng vim gan B - Hepatitis B virus
Hepatitis B l m t bnh dch nguy h i m . K h o n g hai t n g i h i n nay n h i m
virus n y nhng t h i i m n o . C b n t r i u n g i b v i m gan c p v t r n 350
t r i u mang bnh m n tnh trn t h g i i . K h o n g 15% s n g i mang virus m n t n h
(chronic carriers) c t h b x gan ( c i h o s i s gan) v thng d n n ung th. T chc Y

no

t t h g i i ( W H O ) k u g i nhp vacxin cho t i m chng m r n g . N h n g m t s nc

c h a l m c v g i vacxin cao.
V a c x i n ti t hp u tin c sn x u t n m 1986 v b n t r n th trng sau .
Cng

ngh

sn xut:

M t o n ca A D N d i 720 cp nucleotid (HBS) c tch

ra t virus g y bnh v i m gan B (Hepatitis B virus) c gn v i plasmid PBS+. o n

PBS/HBS c chn lc thit k vector b i u h i n . o n PBS/HBS c gn v o
p A O 815 vector n m men c cha gen A l c o h o l oxidase c m ng cao. T h i t k thu c
g i l expression cassette ( C ) - b n g b i u h i n . M t s c c n i ca chng n m men
Pichia pastoris GS115 mang t n 8 o n l p l i (copy) ca b n g b i u h i n C
c to ra v b i u h i n . N m men c h u y n HBS s sn sinh ra k h n g n g u y n H B s A g
Lng H B s A g sn sinh ra ph thuc v o s lng copy ca C trong m i d n g . c
i m ca vacxin ti t hp l:
V a c x i n k h n g p h i l virus v i m gan b l m y u m ch l m t o n gen ca
virus gy bnh n y . K h i t i m chng, gen HBS to ra t c c n i n m men ti t hp c
th gy biu h i n m i n dch v i virus v i m gan B. N n g sut thu c 50mg/l v sn
phm vacxin c gi t h n h thp. N g h i n cu c tnh ca vacxin ti t hp cho t h y
an ton cao.
4.4.5. K thut ADN ti t hp sn xut Kt chn on bnh (virus HIV)
Bnh suy g i m m i n dch ( A I D S ) do c c virus g y ra, ch y u l H I V I v H I V 2 . D
tnh c trn 30 t r i u n g i b n h i m H I V v g n 6 t r i u n g i mc A I D S . T r n 90%
s n g i b n h i m b n h c c nc a n g p h t t r i n . H i n t i , vic chn o n H I V / A I D S
gp k h k h n do t h i u p h n g t i n c h n o n bnh. H i n nay n h i u c n g ty v c c t
chc quc t c b n kt c h n o n c t h v r t n h y bng k thut A D N ti t hp
phn tch H I V v H I V 2 .
Chn o n H I V trong m u da trn k thut E L I S A t i n h n h n h sau: trong v i l
(microvvells) ca m y E L I S A c cha peptid tng hp ca virus hoc peptid ti t hp
ca virus m c k h n n g k c h t h c h p h n ng to k h n g t h (antibody) n g i bnh.
K h i nh huyt t n g v o c c v i l , k h n g t h (antibody) c trong huyt t n g bnh n h n
s phn ng v i c c peptid c c v i l . C c k h n g t h b gn v i c c peptid s phn ng
vi anti-human immunoglobulins v bt cp v i m t cht ch th - horseradiss peroxidase
(HRP). M c bt cp v i H R P t h h i n n h i m H I V . S d n g c c peptid ca c c
loi H I V k h c nhau l m t n g tnh c t h ca chn o n . B kt g m c c peptid to ra
bng k thut A D N ti t hp c n h y v tnh c th cao.
4.4.6. Chn on virus vim gan c (Hepatitis c virus - HCV)
N h i m hepatitis c virus thng lin quan t i x gan v t h n g n n ung t h gan.
K h o n g 1/2 s n g i n h i m virus c t h b v i m gan m n . D o n c 1-2% d n s t h
g i i b v i m n h i m m n tnh. Bn cnh dng m n tnh, c dng v i m cp. C h n o n
bnh virus H C V c c nc a n g p h t t r i n gp k h k h n do t h i u b kt chn o n .
IU

Cng

ngh:

C h n o n H C V da v o k thut E L I S A : Peptit ca virus H C V c

tng hp n h k thut A D N ti t hp. Ph v i l (microvvells) bng c c peptid ca virus.

C c peptid n y g y ra vic tng hp k h n g t h antibody n g i . K h n g t h antibody
huyt t n g s phn ng v i c c peptid trong v i l . K h n g n g u y n l i n k t l i phn
ng t i p v i anti-human i m m u n o g l o b i l i n v bt cp v i cht ch t h H R P . H R P l i n k t
cho bit mc n h i m H C V . P h n g p h p c nhy v t n h c t h cao.
4.5. K THUT ADN TI T HP PHC v CHUYN GEN VO CC CY
TRNG
n n m 2006, c y trng chuyn gen c sn x u t t h n g m i 22 q u c gia,
t d i n tch ton cu 102 t r i u ha, t n g 12 t r i u ha so v i n m 2005. u t n g , n g ,
b n g v c i du l 4 c y trng b i n i gen c h lc, c h i m h u ht d i n t c h c y chuyn
gen ton cu. C c t n h trng quan trng nht c c g i n g c y t r n g b i n i g n h i n nay
l k h n g cht d i t c v gen k h n g su. C c ging mang gen k h n g virus, n m bnh v
c c i u k i n bt l i k h c m i ch c trng trn d i n hp.
4.6. QUAN IM V CNG NGH GEN V GING CY TRNG CHUYN GEN
V i s ra i ca c n g n g h gen con n g i t h n h n g s n g t o m i . M c d
vy, cho n nay nhng sn p h m do C N S H s n g to ra c n r t k h i m t n so v i nhng
s n g to ca t h i n n h i n . Theo V i n s Dobzhanski th c n 1,0 t r i u l o i n g vt v
265,5 n g h n loi thc vt k h c nhau. M i loi l i c n c v s n h n g d n g thc t n ti
a dng na. T h i n n h i n to ra m t t h g i i sng cc k a dng, trong c nhng
sinh vt hu ch v nhng sinh vt g y bnh h i cho con n g i v sinh g i i , k c nhng
con n g i l k th ca loi n g i . N h n g t c n h n g y b n h h i cho m i t r n g v con
n g i c n a n g t i p tc h n h t h n h , v d n h c c virus m i a n g t i p tc x u t hin,
trong virus m i nguy h i m c t h c tng hp, c c i b i n ngay c c p h n g th
n g h i m . D o v y , c h n g ta lu nhng i m sau:
- N h n g s n g to m i ca C N S H s ch l m t b p h n trong t o n b s n g to ca
t h i n n h i n , mc d h i u q u ca n c h a t h l n g trc c.
- Ti n g u y n sinh vt trn tri t cn p h i c c o i l gc r ca s t n t i loi
n g i . N h n g s n g to ca d i t r u y n hc m i cn c x e m n h c n h , n h n g n ch
quyt k h n g t h l i l p v i nhng s n g to trc ca t h i n n h i n .
- V i c bo t n v k h a i t h c hp l ti n g u y n sinh v t , t r o n g c c c n g u n gen
h u c h , p h i l n n t n g v n g chc cho s n hoa k t t r i ca c n g n g h gen, da
t r n n n tng t h c c c t h c h u y n gen m i m a n g l i h i u q u k i n h t v s i n h t h i
bn vng.
- Sn p h m ging ca C N S H c t h l nhng sinh vt hu ch n h n g c n g c t h l
nhng sinh vt g y h i , p h v c n bng sinh thi, nhng sinh v t g y bnh dch v l v
k h sinh hc.
112

- V t h quan i m ca c h n g ta i v i c n g n g h gen s k h n g p h i ch l quan

i m m t c h i u , n thun l d n g tnh, m p h i l c c h n h n t o n d i n t n h i u p h a .
- S p h n h n h o n t o n i v i c c sn p h m ca c n g n g h gen a n g thnh h n h
h i n nay m t s n g i , nht l c h u A u s l k h n g c n g b n g , p h i n d i n , c tnh
nht t h i .
- N h n g n h n g quan i m thn t h n h hoa vai tr ca c n g n g h gen, coi t h n g
c n g n g h d i t r u y n t r u y n thng, bt c h p c c n g h i n cu v an t o n sinh hc l k h n g
t h c h p n h n c.
Con n g i a n g d n tr t h n h " n g s n g to m i " ra nhng d n g thc sng m i .
V i nhng t h n h tu ca k thut d i t r u y n con n g u i s vt qua c m t s bnh
tt nan y v s tr n n k h e m n h h n , sng lu h n , c tr n h v k h n n g s n g to b n
vng h n .
Trong t h k t h X X I n y C N S H s l m t c h i m i i v i s p h t t r i n c tnh
cht b n g n ca n n g nghip, y hc v s t i n hoa ca c h n h b n t h n con n g i n u
n h c h n g ta t r n h c c c m t tri ca n .
4.7. TH VIN ADN
N h trn ta p h n tch, m u n n g h i n cu genom v chc n n g gen ca m t c t h
trc tin n g u i ta p h i t c h A D N tng s t t b o . Sau , p h n o n v n h n d n g c c
p h n o n k h c nhau ca genom. K t q u n g i ta thu c m t b su tp l n t t c
cc d n g A D N ca t b o g i l t h v i n A D N ( A D N library)
4.7.1. Phn loi th vin ADN
P h n l o i t h b i n A D N da trn 2 c t r n g :
- L o i vector n o c s dng.
- Ngun A D N .
C c vector n h n d n g A D N (cloning vector) k h c nhau mang c c p h n o n A D N
k h c nhau. D o v y v i c la c h n c c vector ph thuc v o l n ca genom. Plasmid v
vector siu v i k h u n mang lng A D N n h , c h n g t h c h hp n h n d n g c c c t h
n g i n c genom n h . Cosmid c k h n n g g h p n i v i c c A D N l n h n , c c vector
n h Y A C S v B A C S c t h mang theo lng A D N l n nht.
T h v i n siu v i k h u n (phage library) l m t dch l n g siu v i k h u n (suspension o f
phagas). T h v i n plasmid hoc t h v i n cosmid l dch l n g v i k h u n (suspension o f
bacteria) hoc m u v i k h u n n u i c y trong n g n g h i m hoc a petri.
Th vin cADN:
T h v i n c A D N ( c A D N library) l tp hp c c p h n t c A D N c t n g hp t
A R N t h n g t i n v i s tham gia ca enzym sao c h p ngc (enzym n y c t c h ra t
retroviruses).

113

Bc m t A R N t h n g t i n c s dng l m si k h u n t n g hp m t A D N n
si. Sau A D N n si n y l i tr t h n h k h u n cho tng hp A D N si k p . V c A D N
c tng hp t m A R N n n c A D N k h n g c cha c c o n A D N i u k h i n hot hoa
gen v c c introns. i u c ngha rng c A D N t c t h t b o n h n thc c k h n n g
tng hp protein trong t b o v i k h u n .
T h v i n c A D N ch g m c c gen c b i u h i n trong q u t r n h d c h m t A D N
t h n h m A R N . m A R N c t h ch c tng hp c c t b o , c quan hoc m n h t nh,
thi i m x c nh.
T h v i n n y s k h n g cha c c gen i u k h i n hoc c c t r n h t m hoa

cc

introns.
Th vin

genomic:

T h v i n genomic cha t t c c c p h n o n ca genom, t h v i n n y s l n h n so

vi th vin c A D N
V d : T h v i n genom c t h c to ra bng n h n d n g gen b i s i u v i k h u n X
n h sau:
- Genom ca t b o v genom ca siu v i k h u n c ct o n b i c c e n z y m ct v
to ra v s c c o n p h n ct A D N k h c nhau ca genom.
- C c o n p h n ct ca A D N genom c g h p n i (hay c t h g i l l a i ) v i A D N
ca siu v i k h u n c u d n h t n g n g n h enzym ligase to t h n h v s c c A D N
siu v i k h u n lai hay l c c vectors mang c c o n A D N k h c nhau ca genom.
- C c vector (siu v i k h u n lai) mang genomic A D N c ly n h i m v o q u n t h vi
k h u n . M i v i k h u n s t i p n h n m t siu v i k h u n l a i . K t q u genom l a i ca siu vi
k h u n c n h n bn ln n h i u l n . M i m t k h u n lc v i k h u n s b l y n h i m bi
siu v i k h u n , to t h n h v s c c k h u n lc (clones), trong m i k h u n lc mang theo
d n g siu v i k h u n l a i (mang m t p h n o n ca A D N ) c n h n bn n h i u l n .
- T p hp t t c c c qun lc v i k h u n mang c c d n g s i u v i k h u n l a i k h c nhaum i d n g mang theo o n A D N genom ca t b o , to t h n h t p hp t t c c c d n g
siu v i k h u n lai mang c c o n genomic A D N ca t b o c g i l m t t h vin
genomic ca t b o .
4.7.2. Phng php pht hin dng mang gen quan tm
- Chun b sn m t o n A D N hoc A R N c b i t l c quan h v i gen quan
t m ( g i l A D N m i hoc A R N m i ) .
- T p hp siu v i k h u n l a i c c h u y n ln m n g l c nitrocellulose v l m cho
protein v ca c c siu v i k h u n b hoa tan v b ra sch ch c n l i A D N l a i t r n m n g
lc.
- L m cho c c si A D N k p m x o n v thu nhn c c A D N n si ( t c h c c A D N
k p t h n h c c A D N n si), A D N lai om si n y c b m d n h v o m n g ilter l c .

114

- M n g l c n y c n u i trong dung dch cha m t o n m i c n h d u

b i c h t p h n g x hoc cht p h t quang do n h u m m u l u o r e s c e n c e ( o n m i n y l
c A D N c n h n ln t m t m A R N t h n g t i n ) .
- o n m i s bt cp hoc lai v i bt k o n A D N n o trong s c c A D N ca t h
v i n genom m c trnh t tng n g v i n trn m n g l c .
- C n c v o v tr ca A D N lai c p h n g x trn m n g x c nh d n g ( k h u n lc)
mang g n bt cp v i c A D N m i , c h n lc k h u n lc v n h n ln thu nhn
d n g siu v i k h u n mang gen x c n h mong m u n .
V t h v i n genomic A D N c cha rt n h i u ( h n g t r m n h n g n g n c c o n
A D N k h c nhau ca t b o ) . D o v y , x c nh d n g mang gen quan t m trong t h
v i n n g i ta phi s d n g c c o n m i c t h . C hai l o i m i : l o i m i n h n bit
A D N v l o i m i n h n b i t protein.
4.7.3. Mi nhn bit ADN
P h n g p h p n y da trn bn cht t n h i n ca m t o n trnh t A D N n si cn
tm k i m v g h p n i v i m t o n A D N n si k h c c c c trnh t t n g n g v i n .
M i A D N n si s bt cp v i o n A D N n si t n g n g v i n trong t h v i n
genomic A D N . V o n m i c n h d u b i cht p h n g x hoc cht m u p h t
quang (Auorescence) n n n g i ta c t h d d n g p h t h i n ra i m c p h n t lai trn
m n g n h m t i m c p h n g x trn autoratiogam.
4.7.4. To ADN mi (probe)
N g u n A D N t n g i ta chn l m m i x c nh m t gen quan t m c t h l:
- c A D N : c A D N c t c h c h i t t m m gen quan t m b i u h i n : V s
lng m A R N trong m r t n h i u , do v y s l n g c A D N v s vector mang c c
c A D N k h c nhau c n g c t h a d n g . T u y n h i n , m t s t b o v m c t h ca
c t h , m t vi gen c t n g c n g b i u h i n v c h i m s l n g l n m A R N t h n g
t i n , v d , m A R N sn p h m ca gen P - G l o b i n . D o v y , m t s t b o c c o i l
ngun m A R N t t n h t t o c c c A D N m i t r o n g t m k i m c c gen quan t m trong
t h v i n genom.
Trong trng hp s dng c A D N m i x c nh m t gen trong h gen ta ch c t h
xc nh c v n g dch m ca gen quan t m , trong k h i c A D N k h n g t h bt cp
vi c c o n i u k h i n hoc c c o n introns ca gen n y . D o v y , ta cn t m k i m
thm ngun cc A D N m i khc.
- A D N m i c n g u n gc t c c gen t n g n g t c h ra t c c c t h sng c h
h n g gn v i i - t n g n g h i n cu. V d , m t gen n o c n h n d n g t
ascomycetes ca n m Neurospore c t h c s dng l m m i x c nh m t gen

115

tng n g n m podospora. P h n g p h p n y da trn t n h l p l i hay t n h b o t h ca

c c trnh t A D N trong q u trnh t i n hoa. Trong trng hp A D N m i v A D N ca gen
quan t m k h n g h o n ton ging nhau, c h n g ta v n c t h c c c o n t r n h t t n g
n g v v n c t h lai g h p v i nhau.
- A D N m i c t h c to ra k h i ta bit protein (sn p h m ca gen quan t m ) v
protein c x c nh trnh t. Da trn h i u b i t v m d i t r u y n ta c t h d
d n g thit k c trnh t A D N ca gen m hoa protein . T u y n h i n , m t axit amin
trong protein c t h t n g ng v i vi codon trong gen, do v y ta c t h t h i t k c
m t s trnh t A D N k h c nhau c k h n n g m hoa c u trc ca protein n y . trnh
k h k h n n y , n g i ta tch trnh t protein t h n h nhng o n n g n h n t x c nh
trnh t c c o n m i v i s lng nucleotid n h g i l c c oligonucleotids. T m t on
peptid c t h to ra m t b oligonucleotids g m nhng t r n h t nucleotid dao

ng

n h n g c k h n n g to ra c n g m t peptid.

4.8. D N GENOM NGI

D n Genom c nhiu nc v t chc quc t tham gia thc hin. M D n
Genom do B N n g lng (Department o f Energy) v V i n q u c gia v sc khoe
(National Insititute o f Healths) i u p h i thc h i n .
D n bt u t r i n khai t n m 1990, k o d i 13 n m k t t h c v o n m 2003 sm
h n 2 n m so v i d k i n ban u l 15 n m n h c c n g u n u t v t i n b m i v k
thut.
M c tiu ca d n
- X c nh t t c c c gen ca n g i v i s lng k h o n g 30.000 gen.
- X c nh trnh t nucleotids ca k h o n g 3 t cp b a z trong A D N n g i .
- L u tr t h n g t i n trong d l i u .
- C i t i n c c p h n g p h p p h n tch d l i u
- Chuyn giao c c c n g n g h lin quan cho c c c n g ty t n h n .
- G i i quyt c c v n t ra n h c c n g u y n tc o c, lut p h p , c c v n x
hi khc.

4.9. CC D N NGHIN cu CHC NNG H GEN (FUNCTIONAL GENOMICS)

V SO S N H C H C N N G H G E N ( C O M P A R A T I V E

GENOMICS)

- T o ra m t tp hp c c d n g C - A D N ca m t c t h sng t x c n h t r n h t
ca gen v n g h i n cu c c protein - sn p h m ca gen.

116

- X y dng c c p h n g p h p n g h i n cu chc n n g ca c c t r n h t k h n g m hoa

protein.
- P h t t r i n c n g n g h k h c nhau p h n tch b i u h i n gen.
- C i t h i n c c p h n g p h p g y h n g lot c c t b i n genom, ln m t chc n n g ,
thay i chc n n g ca gen hoc h gen, t x c n h chc n n g ca gen.
- C c c n g n g h p h n tch protein: c bit l s ra i ca thit b proteomics cho
p h p p h n tch v p h n bit h n g n g h n protein c n g m t l c .
- So s n h genom n g u i v i genom tinh tinh, k h . . . so s n h genom ca n g i bnh v i
n g i bnh thng, hoc ca n g i c nhng c tnh siu v i t (tr nh, t u i t h ,
t h n g m i n h siu vit...) v i n g i b n h t h n g hoc v i n g i c c c c t n h i lp.
Chc n n g ca c c gen v c c v n g A D N n g i c t h c l m s n g t n h n g h i n
cu tnh n g nht v s k h c b i t trong h gen ca c c loi sinh vt k h c nhau so v i
con n g i .
- n nay s so s n h n y c t h thc h i n c n h c c n lc phi t h n g ca n h i u
p h n g t h n g h i m t r n t o n cu trong vic c t o n b trnh t nucleotid ca c c h
genom

sau:

Cacnorhabditis

vi

khun

elegans,

E.coi,

nm

men

Saccharomyces

cerevisae,

giun

trn

r u i d m , chut t h n g h i m v n h i u c t h sng k h c . S so

snh n y to ra nhng t p h trong n g h i n cu t i n hoa v c c con n g trao i cht.

117