TS.

CHU HONG MU

C S V PHNG PHP
SINH HC PHN T

NH XUT BN I HC S PHM

Li ni u.................................................................................................................................4
Chng 1: H GENE..............................................................................................................5
1. KHI NIM H GENE (GENOME) ..................................................................5
2. AXIT NUCLEIC ..................................................................................................9
3. ADN V TI BN ADN ..................................................................................11
4. ARN V C CH PHIN M .........................................................................19
THO LUN ...........................................................................................................23
Chng 2: C IM CU TRC GENE CA SINH VT PROKARYOT V
EUKARYOT ...........................................................................................................25
1. C IM CU TRC GENE PROKARYOT ..........................................25
2. CU TRC PHN ON GENE EUKARYOT .........................................25
3. MT S TRNH T ADN................................................................................27
THO LUN ...........................................................................................................32
Chng 3: MI LIN H GIA ADN, ARN, PROTEIN................................................33
1. THNG TIN DI TRUYN V MT M DI TRUYN .................................33
2. PROTEIN ...........................................................................................................39
3. QU TRNH SINH TNG HP PROTEIN .....................................................42
4. IU HO BIU HIN GENE ........................................................................45
THO LUN ......................................................................................................................49
Chng 4: ENZYME S DNG TRONG K THUT SINH HC PHN T ............50
1. ENYME GII HN (RESTRICTION ENZYME HOC ENZYME CT HN
CH (RESTRICTION ENDONUCLEASE - RE) .............................................50
2. CC NHM ENZYME KHC.........................................................................57
3. ENZYME NUCLEASE......................................................................................60
THO LUN ......................................................................................................................61
Chng 5: XC NH MI LIN QUAN GIA PROTEIN
V C TNH
SINH VT...............................................................................................................62
1. MT S LOI PROTEIN CHC NNG........................................................62
2. QUY TRNH XC NH CH TH PHN T PROTEIN ..............................66
3. K THUT PHN TCH PROTEIN .................................................................67
4. K THUT PHN TCH ENZYME..................................................................74
5. NGHIN CU RIPS BNG WESTERN BLOT ..............................................77
THO LUN ......................................................................................................................78
Chng 6: K THUT SINH HC PHN T TRONG PHN TCH H GENE SINH
VT..........................................................................................................................79
1. PHNG PHP TCH CHIT ADN..............................................................79
2. LAI PHN T ...................................................................................................83
3. XC NH TRNH T NUCLEOTIT CA ADN ..........................................85
4. RFLP TECHNOLOGY (K thut phn tch hin tng a hnh ca di cc
phn on ADN).................................................................................................87
2

THO LUN ......................................................................................................................91

Chng 7: PHN NG CHUI POLIMERASE
(POLIMERASE CHAIN
REACTION - PCR)................................................................................................92
1. PHN NG CHUI POLIMERASE (PCR) ....................................................92
2. PHN TCH TNH A HNH CA ADN C NHN BN NGU
NHIN (RANDQM AMPLIFIED PQLIMORPHIC DAN - RAPD) ................99
4. PHN TCH TNH A HNH CHIU DI CC PHN ON ADN C
NHN BN C CHN LC .........................................................................106
THO LUN .........................................................................................................108
Chng 8: T BO CH V VECTOR ..........................................................................109
1. T BO CH ..................................................................................................109
2. VECTOR ..........................................................................................................111
3. PHNG PHP CHUYN VECTOR TI T HP VO T BO CH ..122
THO LUN .........................................................................................................125
Chng 9: PHN LP GENE TCH DNG PHN T V BIU HIN GENE......126
1. PHN LP GENE ...........................................................................................126
2. TCH DNG GENE .......................................................................................127
3. BIU HIN GENE...........................................................................................139
THO LUN .........................................................................................................151
Ti liu tham kho chnh .....................................................................................152
nhng cng trnh ca tc gi cng cc cng tc vin cng b.....................155
C S V PHNG PHP SINH HC PHN T .....................................................162

Li ni u
Sinh hc phn t hin i ang pht trin mnh v tr thnh nng ct
ca Cng ngh sinh hc. Vit Nam, thnh tu nghin cu Sinh hc phn t v
p dng k thut phn t trong nghin cu Khoa hc s sngv Cng ngh sinh
hc c nhiu ng gg trong vic chm sc, bo v sc kho con ngi;
trong nh gi ti nguyn sinh vt; trong chn ging v sn xut nng lm ng
nghip.
Sinh hc phn t l mn Sinh hc hin i, c ging dy nhiu
trng i hc v ang c nhng ng gp nht nh trong o to lp ngi c
tri thc v Cng ngh sinh hc gp phn vo s nghip Cng nghip ho, hin
i ho C s v phng php Sinh hc phn t c bin son t nhiu ti
liu, bi ging v cng trnh nghim cu mi v Sinh hc phn t hin i ca
cc tc gi trong v ngoi nc, nhm cung cp nhng kin thc c bn v
nguyn l v ng dng ca Sinh hc phn t, lm ti liu cho nghin cu, ging
dy v hc tp mn ny trng i hc.
Cun C s v phng php Sinh hc phn t c cu trc bi 9
chng:

Chng 1. H gene
Chng 2. c im cu trc gene ca sinh vt Prokaryot v Eukryot
Chng 3. Mi lin h gia ADN, ARN, Protein
Chng 4. Enzyme s dng trong k thut sinh hc phn t
Chng 5. Xc nh mi lin quan gia protein v c tnh sinh vt
Chng 6. K thut sinh hc phn t trong tch h gene sinh vt
Chng 7. Phn ng chui polimerase (PCR)
Chng 8. T bo ch v Vector
Chng 9. Phn lp gene, tch dng phn t v biu hin gene.
Tc gi trn trng cm n PGS.TS. Nguyn Trng Lng, TS. Lng Th
Hng Vn c v gp cho bn tho, xin cm n nhng ng gp ca Hi
ng nghim thu nh gi gio trnh ca trng i hc S phm - i hc Thi
Nguyn v cm n cc kin ng gp qu bu ca ng o cc nh khoa hc.
Trong qu trnh bin son chc chn c nhng sai st, tc gi rt mong
nhn c nhng gp ca bn c. Mi ng gp xin gi v Khoa Sinh - K
thut Nng nghip trng i hc S phm - i hc Thi Nguyn.

Tc gi
4

Chng 1: H GENE
Tm tt: S ra i ca sinh hc phn t c nh du bng thi im m
Oatsn v Cric (1953) pht hin ra cu trc ADN. Tri qua hn 40 nm (1953 2000) sinh hc phn t t c nhng thnh tu v i m nh cao ca s
pht trin ny l nhng khm ph bn cht sinh hc ca s sng cp phn
t v xy dng cc k thut sinh hc phn t ng dng vo thc tin. Geneomics
v Proteomics l vn ang c c bit quan tm hin nay m c s ca
cc lnh vc ny l nhng pht hin v cu trc v chc nng ca axit nucleic,
v c im ca genome nhn, genome ti th, genome lp th. Nhng im khc
nhau v cu trc v chc nng ca cc h gene cho php ng dng vo thc t
chn ging v nghin cu ngi. Cng vi cu trc ADN v ARN cn c c
im ca qu trnh ti bn ADN v phin m cng c quan tm, v n l c s
ca nhng k thut sinh hc phn t - cc thao tc ADN v ARN. Ni dung c
bn ca chng cp n nhng c s ca sinh hc phn t.
Ni dung ca chng gm 4 vn c bn: (1). Khi nim h gene; (2). Axit
nucleic; (3). ADN v ti bn ADN, (4). ARN v c ch phn m.

1. KHI NIM H GENE (GENOME)

Qu trnh sinh trng v pht trin ca sinh vt tri qua rt nhiu giai
on v tt c qu trnh u ph thuc vo s iu khin ca cc gene. Cu
trc, chc nng ca t bo c quyt nh trc tip bi protein, l sn phm
cui cng ca s biu hin gene. Qu trnh th hin hot ng ca gene qua
protein, b nh hng rt ln bi cc yu t ngoi cnh nh nh sng, ch
dinh dng, s cng sinh v s tng tc ca cc gene trong h gene ca t bo.
Nh vy, gia cc thnh phn ca h gene trong t bo sng c s tng tc vi
nhau v c mi quan h vi cc yu t ca mi trng. Trong t bo, bn cnh
genome trong nhn, h thng di truyn cn phn b trong lc lp, ti th v
plasmid (vi khun). Cc c quan t ny cng tham gia vo qu trnh sinh tng
hp protein ca t bo. T bo l n v cu trc v chc nng c bn ca cc c
th sng, t bo cn l n v ca s di truyn.
H gene l ton h cc gene trong t bo ca c th sinh vt, h gene ha
ton b thng tin di truyn c trng cho tng loi, cho tng c th trong loi.
sinh vt Prokaryot h gene gm ton b cc gene trong t bo; cn Eukaryot
5

h gene gm ton b cc gene trong t bo n bi (n). T bo n bi c mt

h gene, cc sinh vt hoc t bo lng bi c hai h gene, sinh vt a bi c
nhiu h gene. H gene ca sinh vt Eukaryot bao gm h gene nhn (genome
nhn), h gene ti th (genome ti th), h gene lc lp (genome lc lp). H gene
Prokaryot ch c mt thnh phn gm cc on ADN khng ging nhau; cn
h gene Eukaryot c ba thnh phn ADN khng ging nhau: Cc on ADN
lp li nhiu (chim khong 25%), cc on ADN lp li t (30%) v cc on
ADN khng lp li (45%).
1.1. H gene nhn
H gene nhn l genome ln nht trong t bo xt v mt khi lng cng
nh s lng gene m ho. ADN nhn c sp xp gn trong nhim sc th
trong s lin kt vi protein cha histone v khng cha histone. ADN c vai
tr m ho thng tin di truyn, cn protein th bo v v tham gia iu khin s
sao chp, phin m c chnh xc. Qu trnh c bn trong pht trin ng, thc
vt l s nhn i vt cht di truyn v phn u cho cc t bo con khi t bo
phn chia. T bo thc vt cha mt lng ln ADN, khi lng ny thay i
theo loi. Arabidosis thaliana c lng ADN nh nht (0.07 picogram). Allium
cepa c lng ADN nhiu hn 33,5 pg/ haploid genome (h gene n bi).
1.2. H gene lc lp
Cho n nm 1962, ngi ta mi khm ph ra ADN v ribosom c trong
lc lp. Nh vy, lc lp hoc trong lp th cha tt c b my cn thit t
mnh biu hin hot ng gene. H gene lc lp l ton b cc gene trong lc lp
hay ton b lng thng tin di truyn cha trong ADN ca lc lp.
Lng ADN trong lc lp rt ln, chim ti 15% tng lng ADN c th
thc vt, ribosom chim 60% lng ribosom ca t bo, ADN lc lp c cu
to bi cc phn t c cu trc mch vng, c trng lng phn t 83 - 128 x
106, trong gn 85% phn t ADN l mch n.
ADN lc lp lm khun phin m tng hp mARN chloroplast. y cc
phn t mARN ch c khong 20 nucleotit, chng thc hin s tng hp protein
ti ch trong chloroplast. Ribosom trong chloroplast l 70S bao gm 30S v
50S. Sau khi tng hp xong protein c vn chuyn n ni m lc lp cn
thit.
V c bn h thng di truyn trong lc lp tng t vi h thng di truyn
ca sinh vt Prokaryot. Do vy c nhiu cuc tranh lun, c phi vt liu di
truyn ca sinh vt tin nhn l ngun gc ca ADN lc lp? Ngoi ra ADN ca
lc lp khng cha mt s gene c trng cho nhng sinh vt c nhn tht. iu
ny tm thy trong ADN thuc l v ng. Ribosom ca lc lp cng ging
6

ribosom ca sinh vt Procariot: 70S c cu to bi 2 tiu phn 50S v 30S.

Ribosom ca E. coli v lc lp thc vt c c tnh min dch ging nhau.
Thng qua vic nghin cu sn phm biu hin gene l protein, ngi ta
pht hin gia h gene lc lp v h gene nhn trong t bo thc vt c mi
quan h cht ch.
- Genome ca lc lp khng c kh nng tng hp tt c cc protein c
trong chng.
- Phn ln polipeptid ca lc lp c tng hp do genome trong nhn t
bo.
- Nhng polipeptid ny c chuyn vo lc lp theo c ch sau dch m
thc hin qua v lc lp.
- H thng di truyn lc lp iu khin tng hp cc protein cn cho s
pht trin ca chng vi s lng khong 100 polipeptid.
Bng 1.1. Kch thc mt s ADN lc lp (ct. ADN), ADN nhn v vi khun
Kch thc (bp)

Kch thc vng ( m)

1 200 x 103

Trc khun E-coli (chromosome)

3,8 x 106

Trng roi (Euglena) (ct. ADN)

1,4 x 105

44 - 44

Thuc l (Tabaco) (ct. ADN)

1,6 x 105

Ng (Maize) (ct. ADN)

1,36 x 105

43

u xanh (mungbean) (ct. ADN)

1,21 x 105

39

AND
Plasmid

- Polipeptid c m ho tng hp v ARN trong lc lp m nhim chc

nng ca c quan t lin quan n qu trnh quang hp. la ngi ta xc
nh c v tr ca cc gene quan trng iu khin qu trnh quang hp nh
rbcl, atpBE, pSbA, pstA, psbA. Cc gene ny ng vai tr quan trng trong qu
trnh sinh tng hp protein h thng quang ho II.
1.3. H gene ti th (Mitochondria Genome)
nghin cu h gene ti th iu quan trng u tin l thu c ADN ti
th. C th ly v d cy la theo Salech v cng s (1989), phng php phn
tch ADN trong ti th gm cc bc sau:
- Li tm vi tc chm loi b nhn, lc lp v cc thnh phn khc
ca t bo.
- Li tm vi tc nhanh tch ti th.
- Li tm vi tc chm ly nhn, lc lp v cc thnh phn khc.
7

- X l ADN- ase I phn gii cc cht ADN nhn dnh trong ti th.
- Lm sch ti th.
- Tch chit mtADN t ti th (mtADN).
Nh vy ngi ta c th thu c ADN ca mitochondria tinh sch phc
v cho cc cng tc nghin cu tip theo.
mtADN th hin mt s khc bit rt ln v mt kch thc v hnh dng.
i vi ng vt, mtADN c dng vng v kch thc xp x 15 - 20kb. Ngc
li mtADN ca thc vt c nhiu dng (vng.,thng va(cc dng khc), cng
nh c kch thc ln hn nhiu: cy bp ci l 200kb, hoc mt loi da l
2500 kb. Do vy genome ti th thc vt tng i ln v gm mt vi phn t
ADN. Lng mtADN trong thc vt chim khong 0,5 - 1% lng ADN ca t
bo nhng n ng vai tr sng cn cho s pht trin v sinh sn thc vt.
Chc nng ca mtADN ging nh ctADN. N c kh nng m hoatng
hp mt s lng nh pohlieptid nhng rt quan trng.,phc v cho nhng hot
ng ca chnh n.
Nhng sn phm chnh m mtADN iu khin tng hp l 3 tiu phn ca
cytochromoxydaza, 2 tiu phn ca phc cht cytochrom bc v mt s thnh
phn khc (Andre, 1991). Ngoi ra mtADN cn ng vai tr quan trng trong
hin tng bt dc c t bo cht (CMS). y l mt c tnh c khai thc
trong sn xut cc dng lai nhiu cy trng nh la, ng... Spruill v cng s
(1981) ch ra cy ng c s khc nhau gia mtADN trong t bo cy
thng v trong t bo cy c hin tng CMS. Khi phn tch sn phm protein
th nhng mtADN cy CMS c kh nng tng hp chui peptid 130000 MW,
nhng n mt kh nng tng hp chui polipeptid 21000 MW nguyn sinh
cht t bo cy bnh thng.
Mt khc ngi ta cn thy hin tng bt dc t bo cht cn lin quan
n nhng phn t ADN ging nh plasmid tm thy trong ti th.
Chng hn ng trong t bo cht ca cy CMS c mang hai phn t
AND mch thng vi kch thc 6,2 kb v 5,2 kb. Vic s dng code m ha
tng hp protein ca mtADN c nhng im cn lu : trong h thng hot
ng m di truyn thng thng code UGA cung cp tn hiu kt thc qu trnh
tng hp mt chui polipeptid, nhng ADN ca ti th n li l code ca
tryptophan. Ngc li ADN nhn code CGG m ho cho tryptophan th ti
th n li m ho arginin. Ngoi ra ngi ta pht hin mt dng mi ca
mARN trong genome ti th cy trng v n c gi l ARN - Editing. y l
mt vn th hin s tin ho ca thc vt bc cao, gip cho n c kh nng
8

thch nghi hn vi iu kin ngoi cnh.

2. AXIT NUCLEIC
2.1. Axit nucleic l vt cht di truyn
Axit nucleic bao gm axit deoxyribonucleie (ADN) hoc axit ribonucleic
(ARN) u p ng tiu chun ca vt cht di truyn. C rt nhiu bng chng
chng t ra axit nucleic l mt vt cht di truyn cp phn t.
Axit nucleic hp th tia t ngoi cc i bc sng 260nm v iu ny
ph hp mt cch chnh xc vi bc sng m tia t ngoi c th gy t bin
ti a t bo, trong khi protein bc sng 280nm.
- Nm 1928 F. Grifflth pht hin thy ni ph cu khun S, khun lc
nhn do c v bc polisaccharid (vi khun gy vim phi) ca vi khun
Diplococus pneumoniae lm chut cht khi tim vo c th chut. Trong khi
khun lc R (khun lc khng c v bc polisaccharid) khng gy c hi g.
Hn hp cc ph cu khun R cn sng v ph cu khun S cht th lm cho
chut b vim phi, cht v t mu ca chng phn lp c vi khun S sng.
Nh vy tc nhn no t ph cu khun bin np v lm cho ph
cu khun R thnh S. Ngi ta chng minh c rng: ADN tch t vi khun
S nu c a vo vi khun R th gy nn s bin i kiu R S, pht hin
ny c khng nh ADN l vt cht di truyn.
Nm 1944 O. T Avery, Mc Leod v Mc Carti chng minh c rng
tc nhn bin np l ADN. Ph cu khun S b x l bng protease hoc ARNaza th hot tnh bin np vn cn: nhng nu ph cu khun S b x l bi
ADN-aza th hot tnh bin np khng cn na.

Hnh 1.1. S chng minh vt cht di truyn l ARN

Nm 1957, H. Fraen Kel - Conrat v B. Singer cng b th nghim virut
m thuc l c li ARN v v protein, c hai dng a v b. Cc th nghim lp
rp li ARN ca dng ny vi protein ca dng kia v ngc li thnh cng
to ra virut c v v li thuc hai dng khc nhau. Sau em gy nhim vo
9

thuc l, kt qu cc virut con phn lp c t vt m u mang c v protein

v li ARN thuc cng mt dng l dng ca li ARN mang nhim ch khng
phi ca v protein. Nh vy thng tin di truyn c cha ng trong ARN
ch khng phi trong protein.
2.2. Axit nucleic virut, Prokaryot v Eukaryot
Virut l vt cht sng c cu to rt n gin, gm li l axit nucleic v
v l protein. Vt cht di truyn ca virut c hai loi: ADN v ARN. a s cc
loi virut u sng k sinh, khi xm nhp vo t bo vt ch (c th ton b virut
hay ch A. nucleic). Virut sng k sinh t bo Prokaryot c gi l phage hay
bacteriophage (thc khun th). Mt s virut c li l ARN ch yu k sinh
thc vt, cn cc virut k sinh ng vt v t bo Prokarvot a s c li l
ADN.
NST phi to ra mi trng vt l cho phn t ADN hot ng, tc
ng qua li vi cc h thng trao i cht trong t bo. H thng sp xp cch
t chc, b tr ADN trong NST nhng t chc sinh vt cha c nhn chnh
thc v sinh vt nhn chun c s khc nhau nh th no? Ngi ta s dng
ba hng chnh nghin cu t chc ADN trong cc i tng sinh vt khc
nhau, l vic s dng cc thit b v k thut nh knh hin vi in t, nhiu
x tia X, x l bi enzyme bin i cu trc NST.
i vi sinh vt Prokaryot, t chc ADN trong NST dng nucleoid
(vng nhn). Vng nhn cha ADN v ADN c gp v cun thnh nhiu
vng xon: ADN dng siu xon, tnh cht siu xon chu s kim sot ca
enzyme topoisomerase. V d E. coli ADN c kch thc 300 m khi co ngn
kch thc di 25 m v tip tc co ngn th kch thc ch cn 1,5 m. Cch
sp xp ny c pht hin nh enzyme ribonuclease v deoxyribonuclease.
Prokaryot cha ADN trn, chui kp, dng vng, ngoi ra vt cht di truyn cn
plasmid (chim l-2%). E. coli ch c 1 phn t ADN dng vng cha 30004000 gene.
i vi sinh vt Eukaryot, ADN trong t bo c c trong nhn, ti th v
lp th v ch yu ADN nm trn nhim sc th trong nhn t bo. ADN nhn
c dng mch kp, ch yu l B-ADN. ADN ti th v ADN lc lp c dng kp
vng. T chc ADN trong nhim sc th dng nucleosome (th nhn). Mi
nucleosome gm phn t ADN v protein (kim) c gi histon. Histon gm 8
phn t (octamer). Phn bit 5 loi histon vi t l gn nh nhau H1, H2A, H2B,
H3, H4. Trong c 2 phn t H3, 2 phn t H4 lin kt vi vng trung tm;
2 phn t H2A, 2 phn t H2B lin kt vng ngoi.
10

Cc nucleosome ni vi nhau bng mt on nucleotit (ADN) di 15 100 cp nucleotit. NST c cu trc xon theo nhiu bc: tp hp nhiu
nucleosome thnh si c bn c cu trc xon (100), si c bn tip tc xon
thnh solenoit (si nhim sc) c ng knh l: 250: solenoit cun xon ln
na to nn ng rng c ng knh 2000; ng rng ny cun xon mt ln
na thnh si chromatit c ng knh bng 6000.

3. ADN V TI BN ADN
3.1. c im cu trc ca phn t ADN
Hm lng ADN trong nhn t bo c trng cho tng loi v ph thuc
v s lng nhim sc th trong nhn. ADN l i phn t, c khi lng v
chiu di rt ln. sinh vt Prokaryot mi nhim sc th c thc nhiu phn t
ADN, cn sinh vt Eukaryt mi nhim sc th ch c mt phn t ADN.
Bng 1.2. Hm lng ADN ca mt s loi sinh vt
T bo lng bi (2n) T bo n bi (n)
Ngi

6,6 pg ADN

3,3 pg ADN.

Rui gim

2 pg ADN

1 pg ADN.

1 picogram (pg) ADN = 0,965. 109 bp = 6,1.1011 dalton = 29cm

Ngi ta phn bit cc dng ADN l B, A. Z, C, D. Cc dng AND
khc nhau bi chiu xon, chiu cao mt chu k xon, s cp nucleotit trong mt
chu k.

11

Bng 1.3. So snh c im ca mt s loi AND

Dng
AND

Chiu
xon

S cp
nucleotit
chu k

Gc xon
(0)

ng Chiu cao
Dng
knh mt chu k
thit in
()
xon

Phi

10

36

20

34

Trn

Phi

11

32,7

23

28

Trn

Tri

12

30

18

37,1

Phi

9,3

38,6

20

31

Phi

zigzac
Trn
Bt gic

Oatsn v Cric (1953) xy dng m hnh B-ADN (ph bin nht),

l chui xon kp gm 2 mch polinucleotit xon theo chiu t tri sang phi.
M hnh ca Oatsn v Crick l gii c nhng chc nng c bn ca ADN
l bo m cho vic ti sinh trong qu trnh t sao chp gian k v iu chnh
vic tng hp cc enzyme v cc protein. y l mc thi gian nh du s ra
i ca ngnh sinh hc phn t.
ADN v ARN u l nhng polime gm nhiu monomer ni vi nhau,
mi monomer gi l nucleotit ( ADN gi l deoxynucleotit, cn ARN l
ribonucleotit). Mi deoxynucleotit v ribonucleotit gm 3 thnh phn baz nit (
purin v pirimidin) ng pentose v axit photphoric.

Hnh 1.2. S cu to phn t ng deoxyribose v ribose

Cc baz purin (kch thc khong 7) gm Adeine (A); Guanine (G);
cn cc baz pirimidin (kch thc khong 5) gm Thymine (T); Cytosine (C);
Uracil (U).

12

Hnh 1.3. S cu trc ca cc loi baz nit

Hnh 1.4. S cu to cc deoxynucleotit

Kt qu nghin cu ca Chargaff v phn tch hm lng baz purin v
pirimidin trong ADN thy rng t l Adenine bng Thymine v t l Guanine
bng Cytosine (A = T; G = C).
ADN gm hai chui polinucleotil, mi mch polinucleotit c nhiu
nucleotit lin kt vi nhau bng mi lin kt phosphodieste. Cc baz i din
trn hai mch n lin kt vi nhau bng mi lin kt hydro theo nguyn tc b
sung (A - T; G - C). Hai mch polinucleotit ca ADN c chiu ngc nhau:
13

 5'P
3'OH
3'OH
5'P
Tnh c trng ca phn t ADN ph thuc vo 3 yu t: s lng, thnh
phn v trnh t sp xp cc nucleotit trong ADN, trong ph thuc nghim
ngt vo trnh t cc nucleotit.
Kch thc ca phn t ADN c tnh bng bp (base pair- cp baz)
hoc kb (kilobase - kb = 1000 bp), v tnh c hiu ca nucleotit l do baz, cho
nn khi ni n axit nucleic th baz thng c dng thay cho nucleotit.

Hnh 1.5. S chui polinucleotit ca B-ADN

3.2. Ti bn ADN
3.2.1. Cc kiu ti bn ADN
Ngi ta a ra 3 gi thuyt v cc kiu ti bn ADN:
Kiu bo ton: Chui ADN m gi nguyn, ADN mi c tng hp t
nguyn liu (cha c bng chng trong t nhin).
Kiu phn tn: ADN ban u t ra tng on nh, mi on nh lm
khun tng hp cc on nh khc. Cc on nh ni li vi nhau thnh
ADN.
Kiu bn bo tn: Meselson v Stahl chng minh 1958 E. coli s dng
ng v phng x N15.
Mathew Meselson v Franklin Stahl (M - California) tin hnh th
nghim nui E. Coli vi ngun N15, chng minh c c ch ti bn ADN.
3.2.2. H enzyme ti bn ADN
14

 Ba loi enzyme ADN-polimerase E. Coli

- ADN-poIimerase I: gi vai tr trong ti bn ADN, c th d sa cha.
- ADN-poIimerase II: xc nh s bt u tng hp ADN.
- ADN-poIimerase III: iu khin tng hp ADN, thc hin nhim v ch
yu gia tng chiu di ca mt si mi.
Ba loi enzyme ADN - polimerase Eukaryot
- ADN-polimerase (120.000 - 300.000 dalton) c chc nng ti bn
ADN ca nhn.
- ADN-polimerase (30.000 - 50.000 dalton) sa i ADN.
- ADN-polimerase (150.000 - 300.000 dalton) ti bn h gene ti th.
3.2.3. C ch ti bn ADN theo Okazaki -Ti bn na gin on
(Semidiscontinous replication )
3.2.3.1. Hin tng dui xon

Hin tng trn c s tham gia ca mt loi protein l SSB (Single Strand
Binding) bm vo si n ADN lun trng thi m xon, mi SSB bm vo 8
baz v mi chc ti bn c 250 SSB.
3.2.3.2. Khi u ti bn bng ARN mi (primer)
ARN polimerase hot ng tng hp on ARN mi ngn to ra u
3'OH t do, sau ADN polimerase III hot ng ti bn. i vi E. coli primer
c to ra bi enzyme primase.
3.2.3.3. Ko di, loi b mi v hnh thnh cc phn on Okazaki
Sau khi ARN mi c tng hp, th ADN-polimerase I hot ng loi
b mi nh ct t (5' - 3') v thay vo l mt on ADN mi (hnh thnh
phn on Okazaki). Mi phn on Okazaki c 1000 - 2500 baz. Cc phn
on Okazaki ni vi nhau bng ADN-ligase. Si ADN mi c tng hp
bng cch ni cc phn on okazaki c gi l si ra chm (lagging strand) l
si c tng hp khng lin tc (gin on); si kia c tng hp lin tc trn
khun ca si ADN c chiu 3' - 5'. C ch ti bn ADN c mt si c tng
hp lin tc v mt si c tng hp gin on c gi l ti bn na gin
on.
- Mch ADN mi c tng hp theo chiu 5' - 3', mt mch polinucleotit
c tng hp lin tc v mt mch c tng hp gin on- Mi bc ca
15

qu trnh ti bn u c s tham gia ca cc loi enzyme tng ng.

- Qu trnh lin kt vi cc nucleotit thc hin theo nguyn Ltc b sung
(A - T, G - C ).
- C s tham gia ca primer (ARN mi).
- Mi ADN con c to thnh u cha mt mch c v mt mch mi
(bn bo tn).

Hnh 1.6. Ti bn ADN theo Okazaki

3.2.4. Ti bn ADN ca virut
- Cc phage (virut k sinh vi khun) mang ADN 1 si hoc 2 si, virut
ARN c loi ARN 1 si v cng c th mang ARN 2 si.
- C ch ti bn ADN 1 si virut x 174 (nghin cu nhiu nht) k
sinh vi khun E. coli (hnh 1.7).

Hnh 1.7. Ti bn virut 174

16

Ch c si (+) c ti bn v ch c 1 trong 2 si trong RF lm khun

tng hp ra si (+).
3.2.5. Ti bn ADN t bo ca sinh vt Prokaryot
3.2.5.1. Ti bn ADN vng Prokaryot
E. coli, qu trnh ti bn xut pht t mt im ori (origine) c gi l
im xut pht v trin khai ra c hai pha. E. coli ch c mt im xut pht
sao chp ori nn c ADN thnh mt n v sao chp (replicon). Prokaryot
thng ch c mt replicon (hnh 1.8).

Hnh 1.8. n v ti bn (replicon) Prokaryot

3.2.5.2. Ti bn kiu ln ai thng (Rolling circle )
- E. coli F+ v F- tip hp. Yu t F c truyn t F+ sang F- v F- bin
thnh F+, ADN vng plasmid c ti bn theo kiu ln ai thng to thnh
ADN vng mi t bo nhn F-.

Hnh 1.9. Ti bn kiu ln ai thng (rolling circle)

3.2.6. Ti bn t bo ca sinh vt Eukaryot
sinh vt Eukaryot s sinh sn ca t bo l mt qu trnh sinh trng
phn nhn v phn bo mang tnh chu k, qu trnh c gi l chu k t bo
(cell cycle). Chu k t bo gm 4 pha:
Pha G1 (Gap 1) tip sau pha phn chia, nhim sc th tho xon tr v
17

dng si mnh v xy ra qu trnh tng hp cc cht cho s nhn i ADN v

din ra hng lot cc bin i dn n khi u cho s sao chp ADN.
Pha S (Synthesis): vt cht di truyn ca cht nhim sc c nhn i,
l qu trnh ti bn ADN, cc bng chng chng t rng sao chp ADN
ca sinh vt nhn chun cng din ra theo kiu na gin on nh sinh vt
nhn s. Cc enzyme cn thit cho s sao chp ADN ca sinh vt nhn chun
bao gm nhm ADN-polimerase c ADN polimerase , ADN-polimerase v
ADN-polimerase . Theo quan nim hin nay, cc sinh vt nhn chun bc
cao ni chung - polimerase c s dng sao chp ADN ca nhn, polimerase hot ng nh enzyme sa cha, -polimerase chuyn trch sao
chp ADN ti th.

Hnh 1.10. Cc s kin din ra mc phn t trong chu k t bo

M. Pha phn chia
G1: Pha sau phn chia, chun b tng hp cc cht, chun b tng hp
ADN
S: Pha tng hp trong din ra qu trnh ti bn ADN
G2: Pha trc khi phn chia t bo, trong din ra tng hp tubulin v
cubuin
1. Bt u phn chia; 2. Kt thc phn chia;3. Xut hin nhn t cm ng
tng hp ADN
sinh vt nhn chun, mi NST c nhiu n v sao chp (Replication
Unit) cn gi l replicon, mi replicon c mt im khi u v hai im kt
thc qu trnh sao chp. Cc NST ca sinh vt nhn chun bao gm ADN v
histon cng nm trong th nhn. Nhng bng chng cho thy cc gene m ho
histon sao m to ra cc bn sao tng hp s lng histon cn thit cho s
hnh thnh cc th nhn mi trong pha S.
18

Pha G2 (Gap 2): cc nhim sc th chun b cho nguyn phn. Mt s kin

quan trng trong G2 l ni tng hp protein tubulin, cubulin c trng hp ho
to ra cc vi ng ca b my thoi sc phn li cc NST trong nguyn phn.
Pha M (Mitosis): Din ra qu trnh nguyn phn ca t bo, c im c
bn ca nguyn phn l c nhng bin i ln v chc nng v cu trc ca t
bo.
Eukaryot c s lng ADN ln, ti bn ADN phc tp hn v tc
chm (50 nucleotit/giy). Ti bn ca Eukaryot c nhiu replicon v nhiu im
ori.
3.2.7. Sa sai trong ti bn
Hng sao chp ADN thc hin theo chiu t 5'-3' v hng sa cha sai
st trong ti bn li din ra theo chiu t 3'-5'. Enzyme ADN-polimerase I, III c
hot tnh ca exonuclease (enzyme c hot tnh ct nucleotit u t do ca
ADN), nu c nucleotit lp sai th ADN-polimerase s ct b nucleotit sai
theo hng 3'-5'.

Hnh 1.11. Ti bn Eukaryot (nhiu replicon)

Trong trng hp ADN trng thi khng ti bn th c xp x 20 loi
enzyme r sot dc cc NST tm ADN c bin i cu trc, 5 enzyme kim
sot s sai st ca lin kt ho tr, mt s enzyme khc pht hin sai st bt cp
gia cc baz khng b sung. Tng s c xp x 50 enzyme chuyn pht hin v
sa cc sai st trong ADN.

4. ARN V C CH PHIN M
4.1. Cu trc v chc nng ca ARN
4.1.1. Cc loi ARN
ARN thng tin (iARN; mARN) c cu to t mt mch
poliribonucleotit ph bin c t khong 600-1500 ribonucleotit. Phn t mARN
c mt b ba m u (AUG), sau n b ba quy nh axit amin th nht, th
hai... cui cng l b ba kt thc (UAA, UGA, UAG). Phn t mARN c chc
nng truyn t thng tin di truyn t ADN trong nhn t bo n t bo cht v
trc tip tham gia tng hp protein. Hm lng mARN rt t, ch chim vi %
19

ARN tng s trong t bo.

ARN vn chuyn - ARN ho tan (tARN; sARN): Cu to t mt mch
poliribonucleotit, c khong 75-100 ribonucleotit, khi lng phn t 26000
dalton. Hm lng tARN chim khong 10-20% ARN tng s. Phn t tARN
c cu trc xon to ra cc thy trn, mt thy mang b ba i m, mt thy
nhn bit enzyme gn vi axit amin mt u tARN. Mt u mt mang axit
amin kt thc l ACC, cn u mt kia l GGG. Chc nng ca tARN l kt hp
vi axit amin tng hp protein. Phn t tARN c 2 chc nng trong s tham
gia tng hp protein l tip nhn v lin kt.

Hnh 1.12. M hnh cu trc khng gian v cu trc phn t ca tARN

ARN ribosom chim phn ln trong t bo, khong 80% hm lng ARN
tng s. Cc phn t rARN cng vi protein cu to nn cc ribosome. Bn cht
ho hc ca ribosome l nucleoprotein, gn 36% protein v 64% rARN.
4.1.2. Cu trc ca ARN
Cc loi ARN u ch c mt chui poliribonucleotit, mi ribonucleotit
gm 3 thnh phn (H3PO4; C5H10O5; baz nit: A, U, G, C). Cc ribonucleotit
lin kt vi nhau bng mi lin kt phosphodieste, to thnh chui
poliribonucleotit, c chiu 5' 3'.
Tt c cc loi ARN u c tng hp t khun mu ca ADN v ch c
cu to si n. Phn t ARN ca virut l genome ca chng, c chc nng duy
tr v truyn t thng tin di truyn. Ring cc retrovirut mang ARN si kp.
4.2. C ch phin m
4.2.1. Phin m sinh vt nhn s
4.2.1.1. ARN-polimerase ca sinh vt nhn s
E. coli, ARN-polimerase c h s lng 11S - 13S v khi lng phn t
500.000 dalton; t enzyme ARN polimerase nhn t sigma ( ) c th kt hp
20

vi enzyme li khc.

Hnh 1.13. Enzyme ARN -polimerase

Nhn t ng vai tr quan trng trong vic tng hp ARN, n gip cho
enzyme li c th khi u tng hp ARN ti nhng im c th - gi l im
khi u (promotor), nu thiu nhn t th qu trnh tng hp ARN s xy ra
im ngu nhin trn phn t ADN.
on nhn bit (recognition sequence) gm 35 cp baz nm trc im
khi u promotor. on ARN-polimerase nhn bit l on gm 7 nucleotit
c gi prinow, nm cch im phin m 5 - 6 baz. Hp prinow trn si i
khun (antitemplate) c cng thc chung l: 5' TAT phun AT purin 3'.
4.2.1.2. Cc giai on ca qu trnh phin m
Qu trnh phin m ca Prokaryot bao gm 3 bc: khi u, ko di v
kt thc.
1) ARN-polimerase li cng nhn t nhn bit v bm vo trnh t
khi ng (promotor). ADN polimerase trt dc gene (3' - 5') xc tc
bin tnh cc b 2 si ADN l ra si khun tng hp ARN.
2) Qu trnh tng hp ARN bt u v si ARN c sinh ra theo
chiu 5' - 3'.
3) on kt thc ra tn hiu cho ARN-polimerase dng phin m.
on kt thc gm 2 phn: phn c t l GC cao v mt loi baz T trn
si antitemplate.
4.2.1.3. c im
Sn phm ca qu trnh phin m l si n ARN.
ADN dn xon cc b, ch c 1 si lm khun tng hp ARN v c gi
l si c ngha (strand sense).
Nguyn liu l cc ribonucleotit triphotphat : ATP, GTP, CTP, UTP gi
chung l NTP, enzyme xc tc l ARN-polimerase.
ARN c tng hp theo chiu 5' - 3', enzyme ARN-polimerase di
chuyn theo chiu 3' - 5' trn si strand sense.
4.2.2. Phin m sinh vt Eukaryot
21

4.2.2.1. ARN polimerase

ARN-poIimerase I: enzyme khng b c ch bi a-amanitin (cht c ch
tng hp ARN), chuyn trch vic phin m cc gene tng hp rARN
(28S, 58S v 18S).
ARN-polimerase II: enzyme b c ch bi - amanitin, n cn cho tng
hp mARN.
ARN-polimerase III: enzyme b c ch bi - amanitin nng cao,
phin m tng hp cc tARN 4S, rARN 5S.
4.2.2.2. Cc on kim sot phin m
Hp TATA hoc hp Goldberg - Hogness v tr cch cp baz u tin
c phin m khong 30 cp baz v pha trc c coi l on khi u.
on kt thc cn nghin cu t, on kt thc khng di hn 21 baz, nm
men c trnh t l: TTTTATA.
4.2.2.3. Cc giai on ca qu trnh phin m Eukaryot
Giai on khi ng: chu s kim tra ca mt trnh t c bit l "hp
TATA" (TATA box) nm trc v tr bt u phin m khong 25 - 35
nucleotit. ARN-polimerase II hot ng phin m nh nhiu nhn t TF
c bn cht l protein (trascription factor) cho php khi ng s phin
m.
Giai on ko di: phn t ARN c tng hp t mch khun ADN.
Qu trnh ny nh nhn t TF IIS.
Giai on kt thc: phin m kt thc trc im gn ui poli A rt xa.
Kt thc phin m c lin quan n cu trc kp tc tip ngay sau trnh t
giu GC.
4.2.2.4. Din bin qu trnh phin m
1) Gn chp: khi pre mARN ang to ra di 20 - 30 baz th u 5'P ni
thm 7-methylguanosine lin kt 5'P - 5'P. Bn phin m u tin l tin mARN
gm c exon v intron.
2) Thm ui poli A: mt on ngn ca mARN b ct ra v cc baz
adenine ni vo thnh ui poli A.
3) Splicing: ct ri cc intron v ni cc exon nh phc hp
ribonucleoprotein (SnRNP) ca nhn, to cu trc khng gian thun tin cho cc
u exon gn nhau v xc tc phn ng ct ni hnh thnh ARN trng thnh.

22

Hnh 1.14. S cc giai on ca qu trnh phin m Eukayot

4.2.2.5. c im ca s phin m Eukaryot
ARN-polimerase II chu trch nhim tng hp mARN, cn 2 loi enzyme
khc tng hp rARN v tARN.
mARN, rARN v tARN cha thng tin dng bn sao ca mt gene.
Qu trnh phin m phc tp, u 5' ca mARN c gn thm 1 "chp"
(cap: chp, chp n) l 7-methylguanosine, cn cui mARN c "ui"
poliadenine di 100 - 200 A. Chp - Methylguanosine v ui poliA c
tc dng bo v ARN, ngoi ra ui poliA cn l tn hiu nhn bit m kt
thc trong qu trnh dch m.
Bn phin m u tin l tin mARN (premessager ARN-Pre-ARN) bao
gm c cc on intron v exon, sau sau cc intron b ct b to thnh
mARN trng thnh.

THO LUN
1. Phn bit cc khi nim: genome, genome nhn, genome ti th, genome
lp th.
2. c im v vt cht di truyn ca Virut, Prokaryot, Eukaryot.
3. Vn cu trc ca h gene ti th. Nhng ng dng nghin cu h gene ti
th cy trng v ngi.
4. c im cu trc v chc nng ca ADN v ARN.
5. C ch ti bn ca ADN. Ti bn ADN Virut, Prokaryot, Eukaryot. Ti
bn ADN v ti sao trong ti bn ADN li cn phi c primer? Ti sao
primer l mt trnh t ARN?
6. c im ca c ch phin m Prokaryot, Eukaryot. Vn phin m
23

ngc v ng dng trong thc tin.

24

Chng 2: C IM CU TRC GENE CA SINH VT

PROKARYOT V EUKARYOT
Tm tt: Nhng thao tc trn gene c da trn c s nhng hiu bit v c
im cu trc ca gene. Gene nhm sinh vt Prokaryot v nhm Eukaryot c
nhng c im cu trc c trng. Gene ca a s sinh vt Prokaryot c cu
trc lin tc, bao gm cc trnh t m ho axit amin; gene ca sinh vt Eukaryot
c cu trc gin on (gene phn mnh) gm cc intron v exon xen k nhau.
c im cu trc ny quyt nh s khc nhau gia qu trnh phin m
Prokaryot v Eukaryot. Cc trnh t nh promotor, operator, enhancer, yu t
di truyn vn ng (TGE), on xen (IS), trnh t lp li CEN, TEL c cu trc
v chc nng xc nh trong h thng di truyn ca t bo. Hin tng bin
tnh, hi tnh ca ADN, nhit chy Tm cng l c s ca mt s k thut di
truyn nh lai phn t, PCR, v mt s k thut khc.
Ni dung ca chng gm 3 vn c bn : (1). c im cu trc gene ca
sinh vt Prokaryot; (2). Cu trc phn on gene ca sinh vt Eukaryot; (3). Mt
s trnh t ADN.

1. C IM CU TRC GENE PROKARYOT

Gene ca a s sinh vt Prokaryol gm nhiu cistron (a cistron), trnh t
promotor, operator. Gene cu trc ca Prokaryot c cu trc lin tc, bao gm
nhng on m ho axit amin. Promotor l trnh t trn phn t ADN c i lc
vi enzyme ARN-polimerase, c chc nng khi ng qu trnh phin m.
Operator l trnh t c i lc vi protein c ch, nu operator gn vi protein c
ch s ngn cn hot ng ca ARN-polimerase v c ch qu trnh phin m.
Mi cistron cha thng tin ca mt loi chui polipeptit v cng l nhn t iu
khin qu trnh tng hp chui polipeptit thng qua qu trnh phin m v dch
m.

2. CU TRC PHN ON GENE EUKARYOT

2.1. Cu trc gene phn on (gene phn mnh)
Mt gene Eukaryot c cu trc bao gm on tng cng (enhancer)
on khi ng (promotor), intron v k nhau, cui cng l on kt thc. ho
(Noncoding Sequences) c gi l intron hay gi l on xen (Intervening
25

Sequences) (IS) xen vo v lm gin on cc on m ho (Coding Sequences)

c gi l exon.

Hnh 2.1. Cu trc gene phn on Eukaryot

Nhng gene c cu trc gm exo c gi l gene khm (mosaic gene).
Hin nay, cha r chc nng ca intron, tuy nhin c gi thuyt rng, intron c
vai tr nh l cc khong trng xen gia cc exon to thun li cho s ti t hp
gia cc exon.
Th nghim ca Pierre Chambon (Php) pht hin cu trc ca exon intron ca gene ovalbumine g. Ovalbumine l protein lng trng trng. gm
mt chui polipeptit c 386 axit amin (tp hp trong ng dn trng thi k g
mi trng).
Ngoi vic tch mARN ca ovalbumine, s dng enzyme sao m ngc
to ra cc bn sao ca mARN si n c gi l cADN v to thnh cADN si
kp. So snh ADN h gene nhn vi cADN khi gii trnh t cho php pht hin
ra intron v exon. Kt qu ovalbumine trng g c 7 intron, 8 exon.
2.2. Phng php pht hin cc on intron v exon
2.2.1. Phng php so snh trnh t gene v trnh t cADN
Bc 1:
1. Tch chit ADN h gene.
2. Phn lp gene bng k thut PCR hay enzyme ct hn ch.
3. To dng ADN ti t hp, nhn dng v tch dng gene.
4. Gii trnh t gene.
Bc 2:
1. Tch mARN trong t bo cht (hm lng mARN rt ln, mARN ca
ovalbumine chim ti 50% ARN trong cc t bo ny).
2. S dng enzyme sao m ngc tng hp cARN.
3. Gii trnh t cARN.
Bc 3: So snh trnh t ADN h gene vi trnh t cADN xc nh c
cc on intron v exon.
2.2.2. Phng php lai phn t
Pht hin intron v exon cn c tin hnh theo phng php lai phn t
26

v kim tra bng knh hin vi in t.

1. Tch ADN h gene v mARN t bo cht.
2. Phn lp gene.
3. Lai phn t gia ADN v mARN s xc nh c intron v exon.
Vn t ra l khi phin m tng hp mARN th cc on khng m ho
intron c c sao khng ? Ngi ta thy bn sao ca ADN c c exon v intron
chnh l tin mARN (Pre-mARN), cn gi l ARN khng ng nht trong
nhn (hn-ARN: heteroge nevusnucliar ARN). Loi hn-ARN ch c pht hin
sinh vt nhn chun.
ADN

sao m

hn-ARN
tin mARN

processing

mARN

ct ni

3. MT S TRNH T ADN
3.1. Promotor
Thc cht ca khi u phin m l quan h tng tc gia ARNpolimerase vi trnh t promotor. Khi ARN-polimease gn vo promotor th qu
trnh phin m c thc hin v kt qu s to phn t mARN.
Phn ln cc promotor E. coli v cn bn c cng mt cu trc theo s
sau:
TTGACA
-35 bp

mARN

TATAAT
-10 bp

+1

Nu baz u tin c phin m ra mARN (thng l A) c nh s

+1 v tt c cc baz pha 5' hay pha trc n khng c phin m c ghi l
(-). Ngay trc +l c 6 baz thng l trnh t TATAAT xung quanh -10, gi
l trnh t nht tr (consensus sequense), c th gi pribnow box. Xung quanh
trnh t -35 c trnh t nht tr TTGACA. C hai trnh t phi hp vi nhau cho
php ARN-polimerase gn vo promotor v qu trnh phin m v dch m bt
u.
Promotor Eukaryot nm pha trc im xut pht ca mARN. Hp
TATA, nh hng cho ARN-polimerase bt u phin m, nm khong di
27

30 bp (ng vt c v) v 6-120 bp (nm men). Trc hp TATA c hai trnh t

tng ng pha trc khong 40 bp l CCAAT v 110 bp l trnh t giu GC.
GGGCGGG
-110 bp

CCAAT
-40 bp

TATA
-30 bp

mARN
+1

S thay i hp TATA lm gim tc phin m. Hiu qu ca tt

phin m o bng s thay i tng baz trong promotor. Hp TATA v cc
trnh t pha trc phi c nhn bit bi cc protein iu ho, chnh cc
protein ny gn vi cc im nht nh trn chng v hot ho s phin m.
3.2. Enhancer (trnh t tng cng)
Enhancer l trnh t c tc ng cis (u pha) c chc nng lm tng tc
phin m, enhancer c hiu qu ngay c khi cch xa vi nghn cp baz v
chng hot ng bt k hng no, d l trc hay sau promotor.
Trnh t tham gia iu ho - cis c cu trc gm 2 phn i xng nhau:
AGGTCA

TGACCT

TCCAGT

ACTGGA

Enhancer c t chc gm mt dy cc trnh t c tc ng cis nhn

bin cc nhn t trans - nhn tprotein tham gia iu ho biu hin gene. Nhn
t trans gm 2 vng cu trc chc nng: vng gn trans vi ADN v vng tc
ng ln phin m. Cc nhn t trans c 4 kiu tc ng nh:
"Ngn tay km" (zine-finger)
"Xon-vng-xon" (hehx-turn-hehx)
"Xon-nt-xon" (hehx-loop-helix)
"Dy ko leucine" (leucine-zipper)
Gia trnh t cis v nhn t trans thng c cc tng tc c hiu do
nhng lin kt yu hnh thnh gia cc phn t ca nhn t trans vi cc baz
ca trnh t cis. Cc trnh t cis thng tip nhn protein iu ho (nhn t
trans) gm 2 tiu n v (dimer). Nhn t trans c th c 4 nhm cu trc, chng
lun gn ln cis.
iu ho bng cch bin i nhim sc cht hay ADN. Nhim sc th tn
ti nhng vng "nhy cm" tng ng vi cc gene hot ng. Cc vng nhy
cm ny d tip xc vi enzyme sao m.
Tm li, cc gene ca Eukaryot c hot ho bi hai trnh t ADN c
tc ng cis l promotor v enhancer. Chng c nhn bit cc nhn t protein
28

c Lc ng trans. Cc nhn t trans ny cho php ARN-polimerase khi ng

phin m v t tc phin m ti a.
3.3. Yu t di truyn vn ng (Transposable Genetic Element-TGE) Gene
nhy (Jumping genes -JG)
Gene nhy (Jumping genes) cn dc gi l yu t di truyn vn ng
(TGE) ln u tin c pht hin bi Barbara Mc Clintock khi nghin cu
ng vo nhng nm 40 ca th k XX v b c nhn gii thng Nobel
1983.
TGE l nhng on ADN c bit xen vo mt hoc mt s v tr trong h
gene, to nn nhng bin i di truyn.
Loi TGE n gin nht l cc on xen (Insertion Sequence - IS ). IS cn
thit cho qu trnh xen ADN vo NST, cho qu trnh chuyn TGE t v tr ny
sang v tr khc trong h gene. Cu trc ca IS ging nhau nhng sinh vt khc
nhau.
Ngi ta pht hin c E. coli mt on xen IS1 cha 720 bp (base
pair), nm gia on o ngc IR (24 bp)

Hnh 2.2. on xen IS1 E. coli

E. coli cn pht hin ra gene nhy mang tn Tn 1681 (Transposase)
gm 2 on xen IS1 hai u v 1 gene khc di 552 bp quy nh c t chu
nhit gy a chy.

Hnh 2.3. Gene nhy Tn 1681 E. coli

Gene nhy Tn E. coli c th chuyn v tr v xen vo mt nhim sc th
khc theo c ch c biu din bng m hnh hnh 2.4.
3.4. Bin tnh v hi tnh
Hai mch ADN lin kt vi nhau bng nhng lin kt hyro. Khi nhit
ln ti 80-950C th hai mch ADN tch nhau, c gi l hin tng bin tnh
ca ADN.
29

Nhit lm 2 mch ADN tch ri nhau c gi l im chy (melting

point). im chy c tnh gia ng cong OD hnh Sigma. im chy k
hiu Tm. ADN c hm lng G-C cao th im chy cao hn. ADN c 60% l
G- C th Tm = 950C.

Hnh 2.4. C ch xen ca gene nhy Tn 1681 E. coli

Khi nhit h xung v tr li bnh thng, th hai mch n li lin kt
vi nhau to thnh ADN mch kp. Hin tng ny c gi l hi tnh
(Renaturation).
3.5. Trnh t lp li
3.5.1. Khi nim
Hin tng trong cc phn t ADN c cc trnh t baz ngn c lp li
nhiu ln gi l trnh t lp li.
V d: ATAT GCGTCCATATCGCGCTATATGCGTAGC
S khng ng nht ca ADN Eukaryot th hin r khi thc hin phn
ng ti hp ADN (Reassociation).
ADN b ct nh bi enzyme gii hn.
ADN c bin tnh.
Hi tnh: Cc trnh t b sung d ti t hp vi nhau, t nhn bit c
cc trnh t lp li.

30

Hnh 2.5. ng cong Tm

Trnh t CEN
Cc trnh t lp li cao CEN l nm cc tm ng (Centromere) ca
nhim sc th. nm men Saccharomyces gm cc trnh t CEN.
- Trnh t u c 9 cp baz TCACATGAT ( u 5')
- Trnh t gia 80 - 90 bp, rt giu A - T (>90%)
- Trnh t 11 cp baz u 3': TGATTTCCGAA
Trnh t TEL (Telomere - u mt NST)
Trnh t TEL c tnh lp li cao, giu A v C: CCCCACACACCA (nm
men) v CCCTAA (ngi). u mt NST giu G gp li thnh nhng hnh kp
tc c cu trc bc IV. Cu trc ny bo v u mt NST c th b ct bi
nuclease, gi cho u mt khi mt cc trnh t m ho.
3.5.2.ng dng nghin cu trnh t nucleotit lp li
Gn y mt loi ch th phn t mi c gi l vi v tinh - microsatehtte
(Litt and Luti, 1989) hoc trnh t nucleotit lp li n gin - Simple sequence
repeat (Tautz 1989, Weber and May, 1989) c m t sau khi tin hnh
phn ng tng hp ADN nh polimeraza v in li trn gel poliacrylamit ngi
ta c th pht hin loi a hnh ny. Trnh t hai nucleotit lp li rt phong ph
trong b gene ca ngi cng nh ng thc vt.
Tanksley v cng s, 1993 khi nghin cu hin tng a hnh ca c( vi
v tinh la cho thy 2 nucleotit lp li (GA)n hoc (GT)n xut hin sau mi
mt on ADN di 150 Kb, tng s 2 nucleotit ny chng 3000. y l ngun
phn t ADN a hnh phong ph i vi vic lp bn gene loi cy trng
ny.
Yanagisawa v cng s (1994) nghin cu du vn ADN ca 47 ging
31

u tng thng qua cht d Oligonucleotit nhm pht hin trnh t ADN lp li
n gin v cho bit hin trng a hnh th hin r nht l khi dng
Oligolucleotit (AAT)6 lm on d ADN tt c ging u tng th nghim u
phn bit vi nhau bi on d . Khi s dng ba on d ADN khc nhau nh
(CT)8, (GAA)5 v (AAGG)4 th khng pht hin c cc bng ADN a hnh
t cc ging u tng thuc Subgeneus Soja (Glicine max v Glicine Soja), tri
li cc bng ADN a hnh xut hin trong s cc ging thuc Subgeneus,
Glicine. Kt qu nghin cu chng t G. max v G. Soja gn gi nhau v cu
trc b gene.

THO LUN
1. Phn bit cu trc ca gene sinh vt Prokaryot v Eukaryot. Vn v
c im ca gene sinh vt Eukaryot.
2. Phng php xc nh cc on intron v exon trong gene ca sinh vt
Eukaryot. Cho v d.
3. Phn bit khi nim v gene nhy v on xen. C ch xen ca gene nhy.
4. Trnh by cc khi nim: trnh t lp li, bin tnh v hi tnh.
ng dng ca cc k thut ny trong phn tch sinh hc phn t.

32

Chng 3: MI LIN H GIA ADN, ARN, PROTEIN

Tm tt: Thng tin cu trc ca phn t protein, tARN, rARN c m ho
trong gene bng trnh t cc nucleotit. Trnh t nucleotit trong gene m ho cho
chui polipeptit c xc lp bng thc nghim v th hin trong bng m di
truyn. M di truyn l m b ba nucleotit (codon), s thay i trong codon c
th dn n s thay i trong chui polipeptit v t bin tnh trng. Nhng
c im v cu trc v chc nng ca protein v mi lin h gia ADN, ARN,
protein v s iu ho biu hin gene cng l c s quan trng xy dng k
thut biu hin gene.
Ni dung ca chng gm 4 vn c bn: (1). Thng tin sinh hc v mt m
di truyn; (2). Protein; (3). Tng hp protein; (4). iu ho biu hin gene.

1. THNG TIN DI TRUYN V MT M DI TRUYN

Thng tin quy nh cu trc mt loi protein, mt loi tARN, rARN c
gi thng tin di truyn. Thng tin di truyn c cha ng v c m ho
trong phn t ADN bi trnh t cc nucleotit. Hin tng 3 nucleotit k tip
nhau trong gene quy nh mt axit amin trong chui polipeptit c gi l s m
ho b ba. Cn mt b ba c th xc nh cho mt axit amin c th ( v d:
UGU: serine) c gi l b ba m ho (codon - code). Phn t tARN c mt b
ba b sung vi b ba m sao ca mARN c gi l b ba i m (anticodon)
ADN cha thng tin di truyn, l thng tin quy nh cu trc mt loi
protein, rARN v tARN. Trnh t ca cc baz trong ADN quyt nh trnh t
ca axit amin trn protein tng ng.
Phn t ADN c cu to t 4 loi nucleotit, v protein li c cu to
t 20 loi axit amin, nu, mt nucleotit quy nh 1 loi axit amin s to ra 4 t
hp m; cn hai nucleotit quy nh 1 loi axit amin s to ra 42 = 16 t hp m
b 2, nh vy vn cn thiu 4 loi axit amin cha c xc nh bi cc m b
hai. V ba nucleotit quy nh 1 loi axit amin s to ra 43 = 64 t hp m b 3.
Ngi ta chng minh bng thc nghim: m di truyn l m b ba. i
tng l gene rII phage T4. Trong th nghim ngi ta to t bin mt (-) v
thm (+) mt cp baz trn ADN bin i ADN thnh phn axit amin ca
protein.
Gi s phn t mARN ch c thnh phn CAG v trong phn t protein
ch c mt loi axit amin:
33

mARN CAG CAG CAG CAG CAG

Protein a1

a1

a1

a1

a1

- Nu t bin lm mt C (-) v tr m s 2 trn mARN:

mARN CAG AGC AGC AGC AGC
protein a1 a2 a2 a2 a2
- Nu t bin (+) thm G vo gia b ba th 2:
mARN CAG AGC GAG CAG CAG CAG
protein a1 a2 a3 a1 a1 a1
Cc s liu thc nghim thu c trn th nghim rII thc khun T4
chng t lp lun trn l ng. Vy m di truyn l m b ba nucleotit.
1.1. Gii m di truyn
Nm 1961 M. Nirenberg s dng h thng v bo gii m di truyn.
H thng v bo c chit t E. coli c cha ribosome, ARN, enzyme
aminoacylsynthetase, mARN, cc axit amin v mt s ph gia khc. Phn ng
din ra trong vi pht ri dng li, nu b sung thm mARN th vic tng hp
li tip din.
Nu mARN nhn to ch ton uraxin th chui polipeptit c tng hp
ch c loi axit phenylalanine, nu mARN ch ton adenine th chui polipeptit
c tng hp ch c loi axit lisine; nu mARN ton cytosine th chui
polipeptit c tng hp ch c loi axit proline. T c th suy ra b ba UUU
quy nh axit amin phenylalanine; AAA : lisine; CCC : proline.
Bng phng php ngi ta tm ra 61 b ba m ho. Ba b ba cn
li l UAA, UAG, UGA lm nhim v nhn bit tn hiu kt thc qu trnh tng
hp chui polipeptit, cn gi l b ba v ngha (non sense) v chng khng quy
nh axit amin.
1.2. Cc c tnh ca m
Thng tin c c theo tng cm ba nucleotit mt cch lin tc khng ngt
qung.
- Thng tin c c theo mt chiu, bt u t mt im xc nh.
- M di truyn mang tnh ph bin (umversal).
- M di truyn mang tnh thoi ho (degenerate), tr 2 ngoi l AUG v
UGG.
- M di truyn mang nhng b ba khi u (AUG) u 5'. Cc b ba kt
thc UAG, UAA, UGA.

34

Bng mt m di truyn (m sao mARN)

U
U

UUU.

Phe

UCU

Ser

UAU Tir

UGU Cys

UUC

Phe.

UCC

Ser

UAC Tir

UGC Cys

UUA

Leu

UCA

Ser

UAA KT

UGA KT

UUG

Leu

UCG

Ser

UAG KT

UGG Trp

CUU

Leu

CCU

Pro

CAU His

CGU Arg

CUC

Leu

CCC

Pro

CAC His

CGC Arg

CUA

Leu

CCA

Pro

CAA Gin

CGA Arg

CUG

Leu

CCG

Pro

CAG Gin

CGG Arg

AUU Isoleu ACU

Thr

AAU Asn

AGU Ser

AUC Isoleu ACC

Thr

AAC Asn

AGC Ser

AUA Isoleu ACA

Thr

AAA Lis

AGA Arg

AUG

Met

ACG

Thr

AAG Lis

AGG Arg

GUU

Val

GCU

Ala

GAU Asp

GGU Gli

GUC

Val

GCC

Ala

GAC Asp

GGC Gli

GUA

Val

GCA

Ala

GAA Glu

GGA Gli

GUG

Val

GCG

Ala

GAG Glu

GGG Gli

M khi u AUG c 2 chc nng: va to ra s bt u dch m (m m

u) va m ho axit amin. (Mt vi trng hp m m u khng phi l AUG
m l UGU iu khin axit amin formyl methionine i vo u chui polipeptit).
M kt thc (codon v ngha - nonsense):
UAA (ochae)

Tn hiu kt thc

UGA (opal)

qu trnh tng hp protein

UAG (amber)
- Tnh linh hot
Trong t bo c nhng loi tARN c th ng thi c mt s b ba khc
nhau. Phn tch trnh t tARN cho thy rng mt s tARN c cu trc inosine
l mt trong cc baz ca cm m i (anticodon). c im ca inosine c
th hin hnh 3.1.
Phn tch trnh t cho thy u 5' ca codon i m (b tr vi baz th
ba ca cm m) khc vi 2 baz kia, c kh nng kt hp vi mt s baz trn
u 3' ca cm m. Hin tng ny cn c gi l tnh "linh hot" trong kt
cp baz v tnh linh hot l c gii hn:

35

Hnh 3.1. Cu trc ca inosine v guanine

Baz trn u 5' ca anticodon
Baz trn u 3' ca cm m
G kt cp vi

U hoc C

C kt cp vi

A kt cp vi

U kt cp vi

A hoc G

I (Inosin) kt cp vi

A, U hoc G

Hnh 3.2. Tnh linh hot ca m di truyn

1.3. t bin v m di truyn
Mi lin quan gia t bin v m di truyn c th hin 4 kiu t
bin c bn.
1.3.1. t bin nhm ngha
Mt cp baz b bin i dn n thay i cm m tng ng trn mARN
v lm thay i 1 axit amin trn polipeptit.

36

3' AGT CAA GGT TGC CAT 5'

1 2 3 4 5
t bin thay th b ba th 4 (G-C b thay th bi A-T)
3' AGT CAA GGT TAC CAT 5'
Thay i mARN
5' UCA GUU CCA AUG GUA 3' mARN
1.3.2. t bin v ngha
Bin i mt cp baz lm xut hin mt codon v ngha dn n xut
hin tn hiu kt thc chui polipeptit lm cho chui polipeptit c tng hp
ngn (t axit amin) hn bnh thng.
3' AGT CAA GAT TGC CAT 5'
1 2 3 4 5
t bin mt cp baz G-X b ba th 3
3' AGT CAA ATT GCC AT 5'
Xut hin m kt thc UAA mARN
5' UCA GUU UAA CGG UA 3'

mARN

1.3.3. t bin cp baz b ba quy nh axit amin 1m xut hin codon mi

(codon thoi ho) khng nh hng n chui polipeptit
Mt loi axit amin c th c quy nh bi mt vi b ba: v d GUU,
GUC, GUA, GUG cng quy nh axit amin valine.
Trng hp bnh thng:
3' AGT CAA GGT TGC CAT 5'
1 2 3 4 5
5' UCA GUU CCA ACG GUA 3'

mARN

Scrine - Valine - Proline - Threonie - Valine..

Protein

Trng hp t bin.

37

3' AGT CAA GGT TGC CAT 5'

1 2 3 4 5
t bin thay th b ba th 2
(A-T b thay th bi G-X)
3' AGT CAG GOT TAC CAT 5'
Thay i mARN 1m BD i GUU thnh GUC
5' UCA GUC CCA AUC GUA 3'

mARN

... Serine - Valine - proline - Threonie - Valine..... Protein

(GUU v GUC cng quy nh valine)
1.3.4. t bin dch khung
Hin tng mt hoc thm mt cp baz dn n t hp li cc codon v
lm thay i thnh phn axit amin ca chui polipeptit.
Trng hp bnh thng:
3' AAC ACA CAG GTT CAA AGG AGC 5'
Phin m

Gene

5' UUG UGU GUC CAA GUU UCC UCG 3'

Dch m

mARN

- Leucinne - Cysteine-Valine - Glicine - Valine - Tryptophane - Serine

Protein
Trng hp t bin dch khung:
3' AAC ACA CAG GTT CAA AGG AGC 5' Gene
t bin mt cp C-G b ba th 3
3' aAC acAn agg ttc aaa Gga gc 5' Gene t bin
Phin m
5' UUG UGU UCC AAG UUU CCU CG 3' mARN
Dch m
- Leucinne - Cysteine - Serine - Lisine - Tryptophane - Proline
Protein

38

Nh vy chui polipeptide c thnh phn axit amin:

- Leucinne - Cysteine-Valine - Glicine - Valine - Tryptophane - Serine
Bin i thnh:
- Leucinne - Cysteine - Serine - Lisine - Tryptophane - Proline.

2. PROTEIN
2.1. Chc nng ca protein trong t bo
Protein chim khi lng ln trong t bo v tham gia vo hu ht cc
qu trnh sinh hc.
Thnh phn cu to nn cc b phn ca t bo, quy nh nhng c im
v hnh thi v cu to ca t bo v c th.
Protein enzyme c kh nng xc tc ln v tnh c hiu cao.
Khng th: chng li cc tc nhn xm nhp vo t bo v c th. Tc
nhn gy min dch (khng nguyn) xm nhp vo c th ng vt c xng
sng s kch thch s sn sinh ra khng th.
Protein vn ng (myosin), cm ng.
2.2. Cu trc ca protein
Axit amin l n v cu to nn protein, c khong 20 loi axit amin cu
to nn cc phn t protein khc nhau. Axit amin c cu to chung nh sau:

Da vo din tch ca chng cc aminoacit c th chia lm 4 nhm:

Nhm I. Cc axit amin c tnh kim: lisine, argimne, histidinc. pH
ca t bo nhm amine (NH)2 nhnh bn b ion ho thnh NH3, nn
chng mang in tch dng.
Nhm II. Cc axit amin mang tnh axit, v nhnh bn b oxy ho thnh
COO-, bao gm: aspartic v glutamic.
Nhm III. Cc axit amin trung tnh k nc (khng mang in tch) nh:
alanine, valine, leucine... c nhnh bn mang cc nhm k nc.
Nhm IV. Cc axit amin trung tnh phn cc c nhnh bn nhm OH
to cc lin kt hyr a nc.
39

Cc axit amin lin kt vi nhau bng mi lin kt peptit.

Protein c cu trc theo nhiu bc:
Bc I: chui polipeptit c dng mch thng.
Bc II: chui polipeptit c dng mch xon .
Bc III: chui polipeptit c dng mch hnh cu.
Bc IV: t chc protein c nhiu chui polipeptit :

Hnh 3.4. Lin kt to chui polipeptit

Hnh 3.5. S cc bc cu trc ca protein

Mt phn t protein ch c mt chui polipeptit (cu trc bc I, II, III)
hoc gm nhiu chui polipeptit (cu trc bc IV).
40

41

Hnh 3.7. a, b: Cu trc bc II; c, d, e, g: Cu trc bc III; m. n, p, q: Cu trc

bc IV ca protein.(theo Phan Vn Chi, 2002)

3. QU TRNH SINH TNG HP PROTEIN

3.1. Cc yu t tham gia tng hp protein
3.1.1. Cc phn t ARN
Mi phn t mARN mang thng tin xc nh trnh t ca mt polipepetit,
thng tin ny c phin m t ADN.
S lng loi tARN bin ng theo loi t 30 - 40 loi (Prokaryot), 50
loi (Eukaryot), nhng cu trc ca cc loi tARN rt ging nhau.
Chc nng ca tARN c thc hin nh cc enzyme c bit l cc
aminoacyl-tARN-synthetase. C 20 loi aminoacyl-tARN-synthetase tng ng
vi 20 loi axit amin. Qu trnh gn axit amn vi tARN vi s tham gia ca
enzyme ny l qu trnh tiu tn nng lng v tri qua 2 bc:
Enzyme nhn bit v gn vi 1 axit amin c hiu
Enzyme + a.a + ATP enzyme - aminoacyl - AMP + P.P
Axit amin c chuyn t phc hp enzyme aminoacyl sang tARN tng ng.
Enzyme - aminoacyl - AMP + tARN tARN- aminoacyl + AMP + E
3.1.2. Ribosome (RBX)
42

Ribosome (RBX) c cu to t rARN v protein to thnh

ribonucleoprotein. RBX c 2 tiu phn (tiu n v); tiu n v ln cha 1 phn
t rARN ln v tiu n v nh cha 1 rARN nh. RBX Prokaryot v
Eukaryot c bn ging nhau.

Hnh 3.8. Ribosome

3.2. Cc giai on ca qu trnh tng hp protein
Qu trnh sinh tng hp protein trong t bo tri qua hai khu: phin m
v dch m (hnh 3.9). y ch trnh by khi qut cc giai on ca qu trnh
dch m.
3.2.1. Khi ng
Giai on c s tham gia ca nhiu nhn t protein - nhn t khi ng
(IF - Initiation Factor); Prokaryot c 3 nhn t IF, cn Eukaryot c 6 nhn
t IF.
Du hiu khi ng l codon AUG (m ho methyonine). C 2 loi tARN
mang a.a Met c kh nng kt hp vi codon AUG:
(1) tARNmet : kt hp vi Codon AUG gia mARN
(2) tARimet : kt hp vi AUG khi ng, gn a.a methyonine u tin ca
chui polipeptit.
Cc bc ca giai on khi ng:
Aminoacyl - tARN - synthetase chuyn bit (methionyl - tARN synthetase) gn 1 phn t met vo mt u ca tARNmet to thnh Met tARNimet.
Hnh thnh tiu n v nh ribosome - met - tARNimet - mARN vi s
tham gia ca nhn t khi ng (IF2 - Prokaryot ; IF4 - Eukaryot).
3.2.2. Ko di
Khi a.a met t vo v tr chui polipeptit bt du c tng hp.
43

Aminoacyl - tARN k tip n n xp vo ng v tr trn ribosome nh

mt nhn t ko di (EF). RBX c 2 v tr chuyn bit (hnh 3.6).

Hnh 3.9. S gn ca mARN vi cc tiu phn ribosom v s dch m ca

chng
A: tip nhn aminoacyl - tARN.
P: gi phc hp peptidyl - tARN hnh thnh chui polipeptit.
Qu trnh c lp li cho n khi xut hin du hiu kt thc dch m.
3.2.3. Kt thc
Khi nhn t (TF) kt thc nhn bit c du hiu kt thc (cc codon kt
44

thc: UAG, UAA, UGA), phc hp peptidyl - tARN lp tc tch thnh tARN t
do v chui polipepetit. RBX tch ra thnh 2 tiu n v v mARN c gii
phng.

Hnh 3.10. Dch m tng hp protem cc ribosom trn li ni cht

4. IU HO BIU HIN GENE

Ba thnh phn c bn ca s iu ho hot ng gene.
(1). Nhng tn hiu lm thay i biu hin gene.
(2). iu ho biu hin gene thc hin giai on no t sao chp n
dch m.
(3) C ch phn t ca s iu ho biu hin gene.
4.1. iu ho biu hin gene Prokaryot
Cc gene c phin m to ARN, c gi l gene cu trc, protein
c dch m t mARN c th enzyme hoc khng phi l enzyme. Trong s
cc protein khng phi enzyme c protein iu ho (regulatory prtotenin) chng
tng tc vi trnh t ADN c hiu kim sot phin m ca gene cu trc.
Gene iu khin tng hp protein iu ho gi l gene iu ho (regulatory
gene), pha trc nhm gene cu trc c trnh t protnotor, ni ARN-polimerase
nhn bit.

45

Hnhh 3.11. Mi quan h gia AND, ARN v protein

Thc cht ca khi u phin m l quan h tng tc gia ARN polimerase vi promotor. Khi ARN-polilmese gn vo promotor gene s to
phn t mARN.
4.1.1. Cu trc ca operon
S iu ha biu hin gene Prokaryot phn ln xy ra giai on phin
m. C ch iu ha thng qua operon. Mi operon bao gm: nhm gene cu
trc (cistron) cnh nhau v cc trnh t AND khc nhau tham gia iu ha.
Operon l n v phin m gm t nht 1 promotor (trnh t khi ng) v
mARN k cn m ha cho cc trnh t ca mt hay nhiu mch polipeptit.
Promotor c i lc vi ARN-polimerase, mt trnh t AND ni m protein c
ch (repressor protein) gn vo c gi l operator (im iu hnh), k tip l
cc gene cu trc (cistron). Prokaryot thng to ra mARN a gene
(policistronic), nhng mARN ca Eukaryot ch c 1 gene.
Kim sot m (Negative control): S gn ca repressor protein vo
operator lm cn tr s phin m ca tt c cc gene trong cng mt operon.
Kim sot dng (Positive control): Cc protein cn thit cho gene biu
hin gi l activator (cht hot ho), activator c th gn vi cc im khi ng
(initiator site) ca operon hay gn vi enhancer site (im tng cng) s kch
46

thch s phin m ca cc gene cu trc.

Cht gip cho gene phin m c gi l cht cm ng (inducer), cht c
tc dng ngc li gi l cht km hm. Cc gene cm ng (inducible gene)
thng thng tham gia vo cc phn ng thoi dng (kim sot m, cm
ng); cn cc gene c ch (repressible gene) thng tham gia vo cc phn ng
bin dng.
Kh nng c th xy ra 4 kiu t hp kim sot s phin m:
m, cm ng (negative, inducible control);
m, c ch (negative, repressive control),
Dng, cm ng (positive, inducible control);
Dng, c ch (positive, repressive control).
4.1.2. iu ho thoi dng: kim sot m, cm ng (negarive, inducible
control)
Cc cht dinh dng c phn hu to nng lng hoc to nguyn
liu cn thit cho qu trnh tng hp. C ch iu ho y l s c mt ca c
cht, dn ti tng hp cc enzyme phn hu. l operon cm ng m ho cho
cc enzyme ca con ng d ho.
in hnh cho kiu operon cm ng l operon lactose.
Tn hiu iu ho - ng lactose.
Khng c lactose gene m ho cc enzyme chuyn ho lactose khng biu
hin; Khi c lactose gene m ho cc enzyme chuyn ho lactose biu hin.
S iu ho xy ra hon ton mc phin m. C ch ny da vo s
tng tc ca protein iu ho (nhn t km hm - repressor) vi mt trnh t
ADN, promotor u 5' khng m ho ca cc gene cu trc. Repressor lin kt
vi operator nm gia promotor v gene cu trc. C 2 kh nng:
(1) Khng c lactose:
Khi mi trng khng c lactose m ch c glucose th repressor lin kt
vi operator v cc cistron khng tng hp mARN - lac.

Hnh 3.12. Mi trng khng c lactose repressor lin kt vi operator v cc

47

cistron khng phin m

(2) C lactose:

Hnh 3.13. Mi trng c lactose inductor gn vi repressor v cc cistron

phin m
Khi mi trng c lactose, gi l nhn t cm ng (inductor). Inductor
gn vi repressor dn n s thay i cu trc khng gian ca repressor lm cho
repressor khng cn i lc vi operator dn n qu trnh phin m ca cc
cistron bt u v cc enzyme chuyn ho lactose c tng hp.
4.1.3. iu ho bin dng: m, c ch (negative, repressive control)
Bin dng (anabolism) l qu trnh tng hp nn cc cht cn thit cho
t bo (nh cc axit amin), l operon km hm lin quan n con ng ng
ho - in hnh l operon - triptophan.
Qu trnh tng hp tryptophan bt u t tin cht chorismique axit, tri
qua 5 giai on k tip do 5 enzyme xc tc. H thng tng hp amino axit
tryptophan E. coli do operon kim sot m, c ch.
Khi mi trng khng c tryptophan t bo tng hp tryptophan nh tch
cc sn xut enzyme chu trch nhim tng hp tryptophan. Mi trng c
tryptophan th enzyme khng c sn xut na.
Nhn t km hm repressor khng c i lc vi operator. Axit amin c
trng kt hp vi repressor s lm thay i cu hnh ca repressor.
Khi mi trng c tryptophan, repressor kt hp vi tryptophan gn ln
operator lm cho cc gene cu trc ca operon khng phin m.
Mi trng thiu triptophan, repressor khng gn c vo operator cc
gene cu trc ca operon phin m.
4.2. iu ho biu hin gene Eukaryot
B gene ca Eukaryot c kch thc ln, phn t ADN c nn trong
nhn t bo. C ch iu ho biu hin gene c th xy ra 5-6 mc khc
48

nhau v s iu ho biu hin gene Eukaryot c mt s c im c bn sau:

Prokaryot, cc gene iu ho v promotor nm gn nhau; cn
Eukaryot cc gene iu ho t khi nm gn cc promotor do chng kim sot.
Cc enhancer l nhng trnh t cng nm trn mt phn t vi cc
promotor, c th c hng trm bp pha trc hoc sau m chng kch thch.
Trnh t iu ho 5' pha trc promotor ca Eukaryot c th di ti hng chc
kb. C nhiu kiu iu ho dng cc nhn t tc ng trans l cc protein.
S iu ho hot ng gene Prokaryot phn ln p li t nhng tn
hiu bn ngoi; cn Eukaryot ch yu l tn hiu bn trong.
Promotor Eukaryot nm pha trc im xut pht ca mARN. Hp
TATA, nh hng cho ARN-polimerase bt u phin m. S thay i hp
TATA lm gim tc phin m. Hiu qu ca tc phin m o bng s thay
i tng baz trong promotor. Hp TATA v cc trnh t pha trc phi c
nhn bit bi cc protein iu ho, chnh cc protein ny gn vi cc im nht
nh trn chng v hot ho s phin m.
Enhancer l trnh t c tc ng cis (u pha) c chc nng lm tng tc
phin m ng k t cc promotor, enhancer c hiu qu ngay c khi cch xa
vi nghn cp baz v chng hot ng bt k hng no, d l trc hay sau
promotor.
Tm li, cc gene ca Eukaryot c hot ho bi hai trnh t ADN c
tc ng cis l promotor v enhancer. Chng c nhn bit cc nhn t protein
c tc ng trans. Cc nhn t trans ny cho php ARN-polimerase khi ng
phin m v t tc phin m ti a.
THO LUN
1. Trnh by v mt m di truyn.
2. t bin gene v mt m di truyn.
3. Mi lin quan gia protein v tnh chng chu.
4. c im cu trc ca protein. Qu trnh tng hp protein.
5. Phng php phn tch cu trc protein v c im cu trc ca protein.
6. iu ho biu hin gene v ng dng trong k thut di truyn.

49

Chng 4: ENZYME S DNG TRONG K THUT SINH HC

PHN T
Tm tt: Enzyme l nhng protein hot tnh sinh hc v da vo loi phn ng
xc tc m trong sinh hc phn t c th chia enzyme thnh 6 nhm khc nhau.
Trong cc nhm enzyme gii hn, enzyme polimerase, enzyme ni, cc
enzyme nuclease c quan tm nghin cu v ng dng trong cc lnh vc ca
sinh hc phn t v k thut di truyn. Enzyme c s dng trong cc k thut
sinh hc phn t nh RFLP, PCR, tch dng phn t.........
Ni dung ca chng gm 3 vn c bn: (1). Enzyme gii hn, (2). cc
nhm enzyme khc; (3). Enzyme nuctease.
H thng enzyme c th chia thnh 6 nhm tu theo loi hnh phn ng
m cc enzyme ny xc tc. Nhm enzyme nuclease c tc dng ct v ph hu
ADN, nhm ADN-ligase c chc nng ni cc trnh t ADN li. nhm enzyme
ADN-polimerase tham gia tng hp chui polinucleotit, nhm enzyme ADNpolimerase sa cha c chc nng sa cha nhng sai st trong phn t ADN,
nhm enzyme topoisommerase tc dng m xon ADN, nhm enzyme phin m
ngc reverse-transcriptase xc tc cho qu trnh phin m ngc t ARN thnh
cADN. Cc nhm enzyme trn c vai tr ht sc quan trng trong cc thao tc
vi ADN v trong cng ngh ADN ti t hp. Trong s c cc nhm enzyme
gii hn, enzyme sa cha ADN, ADN-polimease, ADN-ligase....

1. ENYME GII HN (RESTRICTION ENZYME HOC ENZYME CT

HN CH (RESTRICTION ENDONUCLEASE - RE)
1.1. Khi nim
Nhm enzyme nuclease gm DN-ase v RN-ase c kh nng b gy lin
kt phosphodiester kt qu l phn hu phn t ADN hoc ARN.
Nhm enzyme ADN-ase gm 2 loi: Exonuclease (enzyme ct ADN
ngoi bo) c tc dng ct tng cp nucleotit t hai u ca on ADN i dn
vo bn trong (hnh 4.1). Enzyme exonuclease c th ct ta tng cp nucleotit
c hai mch (ct kp) hoc ct tng nucleotit trn mt si ADN (ct n).
Endonuclease (enzyme ct ADN ni bo) c tc dng ct ri ADN ti nhng
vng xc nh gm vi cp nucleotit nm cnh nhau nh v trong phn t ADN,
to thnh nhng phn on ADN nh hn. Enzyme endonuclease c th ct n
50

hoc ct kp (hnh 4.1).

Hnh 4.1. c im ct ca exonuclease v endonuclease

Mt loi enzyme endonuclease c th nhn bit v tr nht nh bn trong
phn t ADN v ct c hiu, ct mt ln ti mt im . Nhng kt lun trn
c pht hin trong thp k 60 ca th k XX. Trong nhng th nghim vi
khun ngi ta pht hin mt nhm cc enzyme c th nhn bit v phn ct
phn t ADN ca phag khi ADN ngoi lai ny bo v t bo ch xm nhp.
Cc enzyme ny bo v t bo ch khi b nhim phag - l cc enzyme gii
hn.
Enzyme ct hn ch (enzyme gii hn) l loi enzyme nuclease c kh
nng nhn bit c im ct v ct ti im xc nh. Tuy nhin vn t ra
l, liu enzyme gii hn ct ADN ca chnh t bo ch? Nhng nghin cu cho
thy cc enzyme gii hn phn hu bt k ADN ngoi lai no xm nhp vo t
bo; trong t bo h thng enzyme sa i (methylase) sa i ADN t bo ch
bng cch methyl ho cc baz xc nh ca on trnh t nhn bit, nh vy
ngn cn enzyme gii hn ct vo ADN ca t bo ch.
Nhng enzyme ny l i din ca mt trong cc nhm enzyme quan trng nht
51

dng trong thao tc ADN.

1.2. Cc nhm enzyme gii hn
Cc enzyme gii hn c ba nhm I, II, III v cc enzyme gii hn c
dng ph bin ngy nay thuc nhm II. Enzyme gii hn nhm II c c ch tc
ng n gin nht, chng l cc nuclease, v v chng ct ti mt v tr nm bn
trong si ADN v khng phn hu ADN t hai u, nn gi l endonuclease.
Bng 4.1. c im ca cc loi enzyme gii hn
c im

Nhm I

Nhm II

Nhm III

im ct

Xa im nhn bit Nm trong im

hn 1000 bp
nhn bit

Nm ngoi im
nhn bit nhn bit

Kh nng methy ha gc
Adenine

Khng

iu kin ct

ATP,Mg++, Mg++
S-AdoMet

Mg++ hoc Mn++

Mg++, S-AdoMet

Cu trc ca enzyme (s
chui polipeptide)

Khc nhau

Ging nhau

Khc nhau

Hnh 4.2. Qu trnh methyl ho bi enzyme methylase

Cch gi tn cc enzyme gii hn da trn cc quy c quc t. Tn chi
v tn loi ca sinh vt, m tm thy enzyme, c dng t cho phn
52

u ca tn enzyme, phn gm ch th nht ca tn chi v hai ch u ca

tn loi. Chng hn enzyme tch chit c t mt ni ca Escherichia coli th
t tn l Eco, cn enzyme tch t Bacillus amiloliquefaciens th gi l Bam...
Hai loi enzyme c s dng rng ri c tch ra t cc vi khun ni trn l
EcoRI v BamHI.
V d: EcoRI: RE tch t vi khun E. coli, dng R, nhm I.
HindIII: RE tch t vi khun Heamophilus influenzae, dng d, nhm III.
Enzyme gii hn c tnh c hiu, mi enzyme c th nhn bit mt on
trnh t c th cc baz trn ADN, on nhn bit ph bin nht c chiu di t
4 - 6 bp. Nu mt trnh t ADN c 4 bp th c 44 = 256 bp lp li mt ln, trnh
t c 5 bp th c 45 = 1024 bp lp li, trnh t c 6 bp th c 46 = 4096 bp mi
lp li. Chng hn, mt enzyme nhn bit trnh t c 4 bp s to ra on ngn
hn so vi trnh t c 6 bp.
Loi hnh ca cc on ADN do mt enzyme c th to ra tu thuc vo
trnh t nhn bit v vo v tr ca im ct trong trnh t ny. Nh chng ta
thy, chiu di ca on ny ph thuc vo tn s lp li ca trnh t nhn bit.
im ct thc t ca enzyme quyt nh hnh dng ca u b ct. Hnh dng
ny rt quan trng i vi vic thao tc tip ADN. C hai kiu hnh dng cc
on c th sinh ra: (1) Cc on u bng (blunt ends); (2) Cc on u dnh
(so le) vi 2 kiu: on c u 3' nh ra; v on c u 5' nh ra.

Hnh 4.3. Cc kiu ct ca RE nhm II

Cc enzyme nh PstI v EcoRI to ra cc on ADN c u dnh, chng
c th bt cp baz vi trnh t b sung cng do chnh enzyme to ra. Nh
vy, bng cch ct hai mu ADN khc nhau bi cng mt enzyme v trn cc
53

on vi nhau c th to ra ADN ti t hp. Bng cch ny cc on ADN

ngun khc nhau c th ni li vi nhau nu chng c cc u dnh do enzyme
gii hn sinh ra. Khi cc vng b tr ghp vi nhau th b khung photphodiester
c gn li bng ADN ligase.
Trong thc nghim, ngi ta trn mt lng thch hp enzyme vi ADN
caanf nghin cu trong dung dch m, cho phn ng din ra 370C. Hot tnh
enzymc c do bng n v tng ng vi lng enzyme cn thit mt
microgam ADN trong mt gi 370C.
1.3. Lp bn ct gii hn
Phn ln cc on ADN u c cc im nhn bit i vi nhiu enzyme
gii hn khc nhauv nu bit c v tr tng i ca mt s im ny th c
th lp c bn cc im gii hn. Trong k thut ny, ngi ta s dng cc
enzyme gii hn ct mt on ADN, sau in di trn agrose, chp nh
phn tch c kch thc cc on ADN b ct. T cc kt qu phn tch c th
xc nh c v tr tng i ca cc im c ct trn phn t ADN.

Hnh 4.4. Kiu ct ca mt s loi enzyme

Gi s chng ta mun lp bn cc im ct ca cc enzyme BamHI,
EcoRI v Psd i vi on ADN c chiu di 15kb, kt qu thu c nh sau:
Nu ch s dng ring l mt loi enzyme th on ADN di 15 kb s b
ct thnh 2 on c kch thc khc nhau.
BamHI ct on ADN thnh 2 on vi kch thc 14 kb v 1 kb
EcoRI ct on ADN thnh 2 on vi kch thc 12 kb v 3 kb
PstI ct on ADN thnh 2 on vi kch thc 8 kb v 7 kb
54

Nu s dng phi hp 2 hoc 3 loi enzyme th on ADN b ct thnh 3

hoc 4 on nh:
BamHI + EcoRI ct on ADN thnh 3 on vi kch thc 11 kb, 3 kb v 1 kb.
BamHI + PstI ct on ADN thnh 3 on vi kch thc 8 kb, 6 kb v 1 kb.
EcoRI + PstI ct on ADN thnh 3 on vi kch thc 7 kb, 5 kb v 3 kb.
BamHI + EcoRI+PstI ct on ADN thnh 4 on vi kch thc 6kb, 5kb, 3kb,
1kb.

Hnh 4.5. Cc enzyme BamHI, EcoRI v PstI ct trnh t 15kb thnh 2 phn
on ADN c kch thc khc nhau
Vic pht hin cc enzyme gii hn l bc t ph quan trng trong lnh
vc sinh hc phn t ni chung cng nh cng ngh ti t hp AND ni ring.
Thc cht nhng enzyme dng trong cng ngh ADN ti t hp thuc enzyme
gii hn nhm II. Chng phn ct khu vc bn trong phn t ADN ti mt trnh
t nucleotit nht nh v to ra hn hp (qun th) cc on ADN vi kch thc
khc nhau. Cho n ngy nay ngi ta tch c hn 1200 enzyme gii hn
nhm II t cc c th procariot. Trn 70 enzyme gii hn hin ang c bn
sn trn th trng. Cc RE c th nhn bit nhng trnh t ngn ca ADN v
ct ng vo v tr phn chia phn t ADN thnh nhng on c di c
trng. Cc RE khc nhau th c on nhn bit vi trnh t baz c trng khc
nhau. S m t cc v tr ct khc nhau ca mt phn t ADN c gi l bn
ct hn ch. Nhim sc th ca Eukaryot cha phn t ADN gm nhiu gene
v n c th b ct bi enzyme ct hn ch thnh nhiu on nh, chnh v vy
c th s dng enzyme gii hn trong ng dng lp bn ct hn ch.

55

Hnh 4.8. in di cc phn on ADN b ct bi s phi hp cc enzyme

a. BamHI + EcoRI

b. BamHI + PstI

c EcoRI + PstI

d. BamHI + EcoRI+PstI

Quy trnh lp bn ct hn ch theo quy trnh di y:

1. Nghin cu a dng kiu hnh trong qun th.
2. Tch chit phn t ADN.
3. S dng enzyme RE.
4. in di trn gene agarose.
Phn on ct bi RE-A

ADN chun

Phn on ct bi RE-B

Hnh 4.7. S cc bng in di ca cc mu th nghim v ADN chun

Ngoi ng dng thit lp bn ct gii hn ngi ta cn s dng
enzyme gii hn kt hp vi enzyme ni to vector ti t hp (hnh 4.8).

56

Hnh 4.8. S dng enzyme gii hn v enzyme ni to ADN ti t hp

2. CC NHM ENZYME KHC

2.1. Polimerase
Cc polimerase tham gia vo qu trnh ti bn ADN v phin m tng hp
ARN v c s dng nhiu trong k thut di truyn. Cc pohmeasase gm 2
nhm: (1) ADN-polimerase xc tc tng hp chui polinucleotit theo chiu t 5'
n 3'. Taq polimerase (enzyme tch t vi khun sui nc nng) c kh nng
chu nhit, s dng trong phn ng chui PCR. Enzyme phin m ngc
(Reverse Transcriptase: RT-ase) xc tc tng hp CADN t khun ARN; (2)
Cc ARN-polimerase tham gia tng hp ARN. Ngoi cc enzyme trn cn c
nhiu enzyme tham gia sa cha nhng sai st trn phn t ADN.
2.1.1. Taq polimerase
Taq polimerase c tch chit t mt loi vi khun Thermus aquaticus
sui nc nng. Taq polimerase c s dng trong PCR v RAPD tng hp
on ADN trong iu kin c dung dch m, Mg+ primers. Hin nay cc nh
khoa hc c th phn lp gene xc nh enzyme polimerase, tch dng v biu
hin gene ny. Chnh v vy bng cng ngh ADN ti t hp c th to ra lng
ln enzyme Taq polimerase s dng trong k thut di truyn.
2.1.2. ADN-polimerase I
Enzyme ADN-polimerase I, ngoi chc nng polimerase, cn c hot tnh
57

ca exonuclease 5' 3' v 3' 5'. Enzyme ny xc tc phn ng thay th si,

trong phn ng chc nng exonuclease 5' 3' phn hu si khng lm
khun trong khi polimerase tng hp bn sao mi. Chc nng exonuclease
5'3' ca ADN polimerase I c th c loi b bng cch ct enzyme to ra
on Klenow. on ny vn duy tr cc hot tnh polimerase v exonuclease
3' 5'. on Klenow c s dng khi cn sao chp mt phn t ADN mch
n.
2.1.3. Enzyme phin m ngc
Enzyme phin m ngc c pht hin t 1970 c chc nng tng hp
ADN t ARN. Trong t nhin, enzyme ny do Retrovirus sn sinh phin m
ngc t h gene ARN ca chng thc hin qu trnh xm nhp vo h gene
ca t bo ch. Reverse Transcriptase c s dng trong k thut to cADN
bng phn ng PCR ngc (RT-PCR). Hin nay ngi ta s dng cc loi
enzyme phin m ngc nh: Avian Myeloblastosis virus reverse transcriptase
(AMV-RT) v Moloney Murine Leukemia virus reverse transcriptase (M-MLVRT), trc y AMV-RT phi tch chit t virus Myeloblastosis gia cm, cn
M-MLV-RT t virus gy bnh Leukemia chut. Cc enzyme ny l sn phm
ca gene pol ca virus v hin nay cc enzyme ny nhn c t cng ngh
ADN ti t hp.
Enzyme phin m ngc l mt ADN-polimerase ph thuc ARN, v vy
n sn sinh ra si ADN t si khun ARN. N khng c hot tnh exonuclease
km theo. Enzyme ny ch yu c s dng sao chp cc phn t mARN
khi chun b cADN (ADN b tr hoc ADN bn sao) tch dng, mc d n
cng c tc ng trn khun ADN.
2.1.4. T4 ADN-polimerase
Enzyme T4 ADN-polimerase c ngun gc t phage T4 k sinh trong t
bo vi khun. Hot tnh ca enzyme ny ging nh hot tnh on Klenow v
c s dng trong tng hp mu d c phng x cao. Bi v hot tnh
exonuclease 3'-5' mnh hn T4 ADN-polimerase.
2.1.5. Terminal transferase
Enzyme Terminal transferase c li trch t tuyn c b, c chc nng
xc tc phn ng gn cc deoxynucleotit vo u 3'-OH t do ca ADN.
Terminal transferase c s dng khi thm ui polinucleotit to u so le
cho ADN trong k thut to dng.
2.1.6. Enzyme Alkaline phosphatase
Enzyme Alkaline phosphatase c phn lp t E. coli hay t rut b.
Chc nng ca enzyme ny l loi b nhm 5' phossphate ca ADN, ARN v
58

cc nucleotit t do. Enzyme Alkaline phosphatase s dng loi b

5'phossphate ca ADN v ARN trc kho nh du phng x bng 32P; s dng
trong k thut to dng, khi vector c "m" bi RE v enzyme lm mt 5'
phosphate vector khng th ni li c.
2.1.7. Enzyme T4 polinucleotit kinase
Enzyme T4 polinucleotit kinase c ngun gc t phagene T4 k sinh trong
E. coli. Chc nng ca enzyme T4 polinucleotit kinase ngc li vi Alkaline
phosphatase, xc tc cho phn ng thm vo nhm phosphate u 5' ca ADN
hay ARN. T4 polinucleotit kinase s dng nh du phng x u 5' ca
ADN trong k thut xc nh trnh t ADN v phosphoryl ho cc trnh t ADN
khng c nhm phosphate u 5' chun b cho phn ng ni ca k thut to
dng.
2.2. Enzyme ni ADN-ligase
Enzyme ni ADN-ligase xc tc s hnh thnh lin kt ni hai on ADN
v ARN.
ADN-ligase l mt enzyme ni quan trng trong t bo v chc nng ca
n l sa cha cc mi lin kt photphodiester b t gy mt cch ngu nhin
hoc trong ti bn ADN hay ti t hp ADN. ADN-ligase c s dng trong k
thut to dng v thng c kt hp vi cc enzyme Alkaline phosphatase v
polinucleotit kinase. Nhm enzyme ligase c s dng nhiu nht trong cc th
nghim vi ADN v ARN ch yu gm E. coli ADN-ligase, T4 ADN-ligase v
T4 ARN-ligase.
2.2.1. E. coli ADN-1igase
Enzyme E. coli ADN-ligase c tch chit t E. coli, xc tc cho cc
phn ng ni hai trnh t ADN c u dnh (so le).

Hnh 4.9. Enzyme E. coli xc tc phn ng ni hai trnh t c u dnh

2.2.2. T4 ADN - ligase
Enzyme T4 ADN-ligase chit t phage T4 k sinh E. coli, xc tc cho
phn ng ni hai trnh t c u bng, c s dng nhiu trong k thut to
59

dng.
2.2.3. T4 ARN-ligase
Enzyme T4 ARN-ligase chit t phage T4 k sinh E. coli, xc tc cho
phn ng ni hai trnh t ARN bng lin kt phosphodiester. Enzyme T4 ARNligase thng c s dng nh du phng x u 3' ca ARN trong k
thut sn xut mu d (probe).
Nh vy, enzyme ligase c hiu qu trong s gn cc khe h ca cc trnh
t c u dnh hoc u bng vi nhau trong nhng iu kin thch hp. Nhit
thch hp l 370C, nhng n c th hot ng nhit thp hn nhiu
(4 150C).

3. ENZYME NUCLEASE
Enzyme nuclease l nhm enzyme phn ct ADN (ADNse) v ARN
(ARNse)
3.1. DN-ase I
Enzyme DN-ase I c kh nng ct lin kt ngay sau mt baz pyrimidine
trn ADN mch n hay mch kp. Trong thc nghim DN-ase I c s dng
loi ADN trong ch phm ARN v protein, to im t trong k thut nh
du mu d.
3.2. Nuclease S1
Enzyme nuclease S1 tch chit t Aspergillus oryzae c tc dng phn
gii ADN mch n, mch kp. Enzyme nuclease S1 c s dng phn tch
c im cu trc phn t lai ADN - ARN, loi b u so le to u bng,
loi b cc nt vng trn ARN trong phn ng phin m ngc to cADN.
3.3. Exonuclease III
Ezyme Exonuclease III chit t E. coli xc tc phn ng phn gii tun t
cc nucleotit t u 3'- OH t do ca ADN theo chiu t 3'5' to ra nhng
vng mch n trn ADN mch kp. Enzyme Exonuclease III c ng dng to
cu trc mch n ca mt vng trn phn t ADN, to t bin mt on trn
ADN khi phi hp vi nuclease S
3.4. RN-ase A
Enzyme RN-ase A c hot tnh rt mnh, c th chu ng c nhit
0
90 C trong 60 pht. Enzyme RN-ase A c kh nng ct lin kt phosphodiester
ngay sau mt baz pyrimidine ca ARN. Enzyme RN-ase A c s dng
loi b ARN trong ch phm ADN hoc protein, loi b cc on khng bt cp
60

trn ARN ca phn t li ARN -ADN.

3.5. RN-ase H
Enzyme RN-ase H c kh nng loi b ARN trong phn t lai ARN ADN v thng c s dng loi ARN sau phn ng phin m ngc
tip tc tng hp mch th hai ca CANAto ADN mch kp.
THO LUN
1. Th no l enzyme ct v enzyme ct hn ch? Cc nhm enzyme gii
hn.
2. Vn c im ca cc loi enzyme dng trong k thut sinh hc phn
t.
3. Trnh by cc kiu ct ca enzyme gii hn, cho v d. Nu nhng ng
dng c bn ca RE.
4. Th no l bn ct gii hn? cho v d minh ho.
5. Nhm enzyme nhn ADN v ni ADN.
6. Nghin cu ch th sinh hc phn t bng phng php s dng cc loi
enzyme ct hn ch.

61

Chng 5: XC NH MI LIN QUAN GIA PROTEIN V

C TNH SINH VT
Tm tt: Protein l sn phm biu hin gene, protein biu hin thnh tnh trng
v c tnh sinh vt. Trong t bo c nhiu loi protein chc nng quan trng
v nhng hiu bit v c im ca nhng protein ny l c s cho nhng ng
dng vo cng tc chn ging v bo v sc kho con ngi. Nhng loi protein
cp trong chng ny l protein lin quan n tnh chng chu ca sinh vt
v protein c ch hot ng ribosome. Xc nh mt lin quan gia protein v
tnh trng, c tnh sinh vt lp thnh cc ch th protein cho c tnh v tnh
trng. Cc ch th protein c th l ch th hm lng protein, enzyme, phn t
protein hay ph isozyme v xc nh cc loi ch th ny c thc hin theo
quy trnh k thut nht nh. Hm lng protem, ng v hot enzyme lin
quan n p sut thm thu trong t bo v quan h vi tnh chng chu ca
sinh vt. C nhiu c tnh v tnh trng ca sinh vt c quan tm nghin cu
hin nay l tnh chng chu ca sinh vt v vic tm ra cc ch th protein cho
c tnh ny c ngha cao trong chn to ging.
Ni dung ca chng gm 6 vn c bn: (1). Mt s loi protein chc nng;
(2). Quy trnh xc nh ch th protein; (3). K thut phn tch protein; (4). K
thut phn tch enzyme; (5). K thut phn tch ng, (6). Nghin cu RIPs
bng Westem blot.

1. MT S LOI PROTEIN CHC NNG

1.1. Protein lin quan n tnh chng chu
Kh nng chng chu ca sinh vt ni chung v ca thc vt ni ring lin
quan n cu trc v chc nng ca cc thnh phn trong t bo. Cc vng t
bo v cc qu trnh xy ra trong mi vng lin quan n kh nng chu hn,
nng, mn, phn ca thc vt (hnh 5.1).

62

Hnh 5.1. Chc nng ca cc vng t bo khi mi trng thiu nc (theo

Bohnert,Jensen, 1996 v inh Th Phng, 2001)

Hnh 5.2. Hot ng ca cc gene lin quan n s mt nc v s thay i p

sut thm thu (theo Bray, 1993 v Trn Th Phng Lin, 1999).
1.1.1. Protein sc nhit (HSP)
HSP c hu ht cc loi thc vt nh: la m, mch, la go, ng, u
hnh, ti.... chng chim khong 1% protein tng s trong l ca cc loi thc
vt ny. HSP cn tm thy vi khun v ng vt bc cao. HSP c tng hp
khi t bo gp iu kin cc oan nh hn, nhit cao, mui cao.
63

HSP c th chia lm 6 nhm da trn c s khi lng phn t khc

nhau: MW 110, 90, 70, 60, 20, 8,5 kDa. Trong s c nhm HSP70 v HSP60
c nhiu i din ca cht mi gii phn t (gi l chaperonin). HSP8,5 hoa
(ubiquytin) c chc nng bo v cho t bo nhng khng phi l mi gii phn
t. Ubiquytin c hot tnh protease vi chc nng phn gii cc protein khng
c hot tnh enzyme, ngn chn hin tng gy c t bo ca nhng protein
ny. Ubiquytin c MW rt thp, t chu nh hng ca nhit nn ubiquytin c
vai tr t sa cha ca t bo khi gp yu t cc oan, c bit l nhit .
1.1.2. Mi gii phn t (MGPT)
Theo Ellis (1997) MGPT l mt nhm gm nhiu loi protein khc nhau,
nhng chng u c chc nng tham gia to cu trc khng gian ng cho
protein trong t bo. Chc nng ca MGPT th hin nhng ni dung sau:
- Gi n nh chui polipeptit khi ang tng hp v to cu trc khng
gian ng cho chng.
- To li cu trc khng gian ca protein sau khi vn chuyn qua mng t
bo.
- Lp gip cc chui polipeptit vo phc to cu trc bc 4.
- MGPT c tng cng nh qu trnh tng hp trong iu kin cc
oan do nhu cu cp thit ca t bo.
Hot ng ca MGPT c thc hin nh vic s dng nng lng ATP.
MGPT c nghin cu u tin thc vt v lin quan n enzyme Ribulose
bisphotphat cacboxylase/oxygenelase (RUBISCO). RUBISCO xc tc cc phn
ng tng hp cht hu c nh s dng CO2 t do trong quang hp. RUBISCO
c cu trc bc 4 gm t 4- 8 tiu n v ln (MW52) v 8 tiu n v nh
(MW14 kDa). Mt loi protein c hiu gn vi nhng tiu n v chnh to
phc RUBISCO-binding protein (RBP) v RBP l MGPT.
MGPT thc vt tham gia to cu trc khng gian ng cho nhng phn
t protein mi c tng hp, chuyn protein qua mng, duy tr cu trc c
hiu ca protein, ngn chn s hu hoi protein cha to cu trc ng, khi u
s phn gii protein b bin tnh.
MGPT c th phn thnh mt s nhm ch yu sau:
HSP70 l nhm MGPT c hot tnh ATP-ase v c tnh bo th cao.
Trong iu kin cc oan HSP70 c th ngn chn s co cm v kt t
protein.
HSP60 (chaporonin) l loi protein cu to t cc tiu n v c khi
lng 60 kDa v cng c hot tnh ATP-ase. Chng c cu to 2 vng,
64

mi vng c t 7-8 tiu n v. t bo Prokaryot, chaperonin ging vi

protein tm thy ti th, lc lp v c gi l GroEL, GroES. Trong t
bo Eukaryot tm thy mt loi chaperonin trong t bo cht l CCT.
Nghin cu v CCT thc vt cn t c cp. Tuy nhin gn y c
nhng cng b u tin ca Nng Vn Hi v cng s (1998), Trn Th
Phng Lin v cng s (1999) v kt qu phn lp v tch c dng
gene CCT t cy u tng.
1.1.3. HSP100, HSP90, SHSP
Cc nhm HSP100, HSP90, sHSP c tnh bo th cao v c hot tnh
ATP-ase. Phn ln cc protein ny c sinh ra khi.gp nhit cao. Chc nng
ca HSP100, HSP90, SHSP l ngn chn hin tng co cm ca protein v ti
hot ho cc phn t protein bin tnh.
1.1.4. LEA (Late embryogenesis abundant protein)
LEA l loi protein c tng hp vi s lng ln trong giai on cui
ca qu trnh hnh thnh phi. LEA c vai tr quan trng i vi kh nng chu
kh hn ca ht. Cu trc phn t ca LEA ni bt l giu axit amin a nc,
khng cha xystein, tryptophan, c vng xon v c kh nng chu nhit.
Chng thc hin cc chc nng c lp ion, bo v mng, hu protein bin tnh
v iu chnh p sut thm thu.
LEA c chia lm 6 nhm: nhm 1-D19; nhm 2-D11 (dehydrin); nhm 3-D7;
nhm 4- D113; nhm 5-D29; nhm 6-D95. Nhiu gene LEA c nghin
cu, phn lp v xc nh chc nng. Khi gp hin tng mt nc mARN ca
n c tng hp v dn tr thnh mt lng ln gm nhiu loi mARN khc
nhau trong ht chn, mARN b phn gii ht khi ht ny mm. Mc phin m
ca gene LEA c iu khin bi ABA v mt nc ca t bo.
1.2. Protein bt hot ribosome (Ribosome inactivating proteins - RIPs)
Protein bt hot ribosome (ribosome inactivating proteins - RIPs) l
nhng c t (toxin) c hot tnh rRNA N-glicosidase v cc c tnh sinh dc hc rt a dng. C ch tc ng ca RIPs l phn ct cu ni Nglicosidic ca adenine c hiu trn vng Sarcin/Ricin Loop (cn c gi l
SRL hay R/S Domain) ca rRNA (v tr A4324 trn rRNA 28S gan chut).
Kt qu l ribosome khng gn kt c vi EF-2 v GTP, lm c ch qu trnh
sinh tng hp protein.
RIPs c phn loi theo cu trc phn t ca chng theo hai hoc ba tipe
khc nhau: (1) RIPs tipe I ch c mt chui polipeptit A (i khi dng glicosyl
ho) vi hot tnh RIPs N-glicosidase; (2) RIPs tipe II c hai chui: polipeptit A
lin kt vi chui plipeptit B (l mt phn t c hot tnh ging lectin) bng mt
65

hoc hn cc cu ni disulfide; (3) RIPs tipe III ging tipe I, bao gm mt chui
polipeptit, nhng li ch c hot tnh sau khi c phn ct on tn hiu bng
mt loi protease c hiu.
S lng RIPs tipe I (khi lng phn t khong 30kDa) nhiu hn RIPs
tipe II v III. Tuy nhin mt s RIPs khng th phn loi theo cc tipe k trn.
RIPs c pht hin hn 50 loi thuc 13 h thc vt, bao gm c cy
mt v hai l mm. RIPs khng ch thy h Bu b (Cucurbitaceae) vi rt
nhiu loi l cc cy thuc qu nh Trichosanthes, Luffa, m cn thy rt
nhiu cc h thc vt khc nh Cm chng (Caryophylaceae), Thu du
(Euphorbiaceae), Ho tho (Poaceae), H u (Fabaceae), h Hoa phn
(Nygtaginaceae)... RIPs khng ch tm thy thc vt, m cn thy mt s
loi vi khun ng rut. S c mt ca RIPs rt nhiu c th sinh vt khc
nhau a n mt gi nh v kh nng tn ti ca loi protein tng t th
gii ng vt.

2. QUY TRNH XC NH CH TH PHN T PROTEIN

Protein l sn phm ca s biu hin gene. Protein biu hin tnh trng
ca c th. Nghin cu xc nh c s protein ca tnh trng hnh thi, cu to,
sinh l, ho sinh ca c th sinh vt c quan tm mt s kha cnh c th nh
hm lng protein, cc tiu phn protein vi kch thc phn t khc nhau. Quy
trnh xc nh ch th protein cho mt tnh trng no c th c thc hin
theo cc bc sau:
Bc 1. Nghin cu c tnh hnh thi, sinh l
Sng lc, phn lp cc c th theo kt qu nh gi da trn kiu hnh.
Nhng c th mang tnh trng quan tm c th c to ra do lai ging hoc t
bin thc nghim, sng lc trong qun th t nhin v cng c th do x l, nh
gi trong iu kin nhn to. Hin nay ngi ta quan tm ch yu cc tnh trng
chung chu thc vt (kh nng chu nhit: nng, lnh; mn; hn; phn; kh
nng chu su, khng bnh), cc bnh do vim hoc ung th ng vt.
Bc 2. Xc nh ch th protein
Ch th hm lng protein
- Chit protein giai on sinh trng, pht trin quan tm ca c th.
- Phn tch hm lng protein.
- Xc nh mi lin quan n tnh trng.
66

 Ch th phn t protein
- Chit protein dng cho in di, tch chit phn on protein.
- Phn tch thnh phn in di.
- Phn tch a hnh protein.
- Gii trnh t axit amin ca protein.
Western blot
- Chit protein dng cho in di.
- Tch protein bng in di trn gel poliacrylamide.
- Chuyn protein t gel sang mng (PVDF) nh in trng.
Ch th enzyme
- Chit enzyme.
- Phn tch nh lng v nh tnh hot enzyme.
- Phn tch in di entyme.

Hnh 5.3. S phn tch xc nh ch th protein

3. K THUT PHN TCH PROTEIN

3.1. Tch chit protein
Protein c tnh tan trong nc v mui, do vy ngi ta chit protein bng
mt s loi dch chit c pH khc nhau kt hp vi phng php li tm thu
67

dch chit. Sau y trnh by cch chit protein s dng cho nh lng v in
di protein.
3.1.1. Tch chit protein d tr trong ht thc vt
Protein d tr trong ht c th c chit bng cc loi dch chit nh
nc ct, NaCl, dung dch m phosphat xitrat pH = 10... i vi cc loi ht c
du th trc khi chit protein cn phi loi lipit, v d k thut loi loi lipit
ht u tng hoc u xanh: Ht u bc v, b phn phi mm sau nghin
nh. Bt nhn c gi 40C. Loi lipit bng petrolium ete qua m 40C. Li
tm 14.000 vng/pht loi dch li tm. Lp li mt vi ln na. Bt loi
lipit gi 40C.
3.1.1.1. Chit protein dng phn tch nh lng hoc in di
Bt loi lipit c s dng chit protein tng s bng dung dch NaCl
1M pH = 7.0, chit bng nc ct pH = 7.0 hoc chit bng m photphat
pH=10. Bt loi lipit c chit bng dung dch m qua m 40C, trong
24 gi. Ngy hm sau li tm 14.000 vng/pht trong 30 pht thu dch pha trn.
Dch li tm thu c cha protein tng s bao gm albumin v globulin. Trong
nh lng protein c th tin hnh lp li nhiu ln; cn trong phn tch in di
th c th thc hin mt ln. Dung dch protein c gi -200C.
3.1.1.2. Chit phn on protein cho phn tch in di
Tch chit v phn on protein theo phng php Osborne v Klimenko.
C th ly v d cy h u: Ht c nghin nh thnh bt mn, loi lipit
bng ete petroleum sau tin hnh phn on. Protein c chit bng dung
dch NaCl 1M v nc ct.
Theo cch th nht, bt loi lipit c chit bng NaCl 1M sau li
tm 14000 vng/pht trong 20 pht. Dch li tm cha albumin v globulin tng
s (phn on I). Cn li tm cha tinh bt v tp cht c loi b. Tip theo
albumin v globulin tng s c phn on bng cch thm tch, sau li tm.
Dch li tm thu c cha phn ln albumin (phn on II). Cn li tm l cc
protein khng tan trong nc m tan trong NaCl. Phn ny c ho vo NaCl
1M (phn on III).
Theo cch th hai, protein c chit bng nc ct sau li tm. Dch li
tm cha phn ln albumin v mt phn globulin (phn on IV).Cn li tm
c chit tip bng NaCl 1M v li tm ly dch (phn on VII) Phn on IV
c x l tip bng thm tch. Dch li tm cha phn ln albumin (phn on
V). Cn li tm cha phn ln globulin tan trong NaCl 1M (phn on VI).
3.1.2. Tch chit protein trong m ng vt
Chit protein t m ng vt n gin hn thc vt v c tin hnh
68

tng t nh thc vt. Protein trong m ng vt c chit nh mui hoc

nc v bng li tm.
3.2. nh lng protein
3.1.1. Phn tch hm lng nit tng s bng phng php Keldjal
Nguyn tc : Tt c cc dng nit c trong c th hay trong cc m c
gi l nh nit tng s. Nit c trong thnh phn axit amin ca protein l nit
protein. Nit khng c trong thnh phn protein nh mui v c, axit nitric... l
nit phi protein. Nit tng s = nit protein + nit phi protein. Oxi ho nguyn
liu bng H2SO4 c mt cht xc tc v nhit cao, kt qu ca vic cng ph
mu ny l m trong thnh phn cht hu c chuyn thnh (NH4)2SO4, di tc
dng kim mnh, nng NaOH, sc NH3 bay ln gp nc to thnh NH4OH,
NH4OH c H2SO4 th, Sau chun lng NH3 c H2SO4 bng NaOH.
Kt qu phn tch s thu c hm lng ma nitg s bao gm c mi
nittein v nit phi protein. Do vy cn tin hnh phn tch hm lng nit phi
protein xc nh hm lng nit protein m chng ta quan tm trong nh gi
cht lng sn phm sinh hc.
Xc nh hm lng nit phi protein c th c xc nh theo phng
php ca Barstein trn my phn tch m t ng Vapodest ca hng Gerhardt
v my Kjeldahl.
Hm lng nh pitotein = Hm lng nit tng s - Hm lng nitow phi
protein
3.1.2. Phn tch hm lng protein tan tng s
Nguyn tc chung l s dng cc thuc th nh Folin, Bradford, Gordan...
vi s hp th nhng bc sng khc nhau trn my quang ph Uvis ng
thi vi s phn tch mu th nghim l s dng protein chun bit trc hm
lng tnh hm lng protein ca mu th nghim.
Phng php nh lng protein tan tng s theo phng php Lowry.
C s ca phng php l s hnh thnh phc cht biur. Phc ny tham
gia vi thuc kh Folin-ciocalteu cho mu c trng. Cng mu t l vi
hm lng protein. o cng mu ca hn hp trong ng nghim trn my
so mu quang in bc sng = 750nm. Sau khi o c mt quang th
em i chiu vi th chun tm lng protein tng ng. Th nghim
xy dng th chun th dch phn tch l dung dch ca mt loi protein c th
( y l Albumim) bit trc hm lng.
Phng php s dng thuc th Bradford
Thuc th Bradford gm: Coomasie Brilliant Blue G, ethanol, H3PO4,
69

nc ct; protein chun.

Cc bc tin hnh:
- Pha thuc th Bradford.
- S dng protein chun v thuc th Bradford xy dng ng chun
v o trn my quang ph bc sng 595nm.
- Cn c vo ng chun xy dng c s xc nh c hm lng
protein ca mu th nghim.
V d:
T kt qu o trn my quang ph, s dng my vi tnh theo chng trnh
excel lp ng hi quy tuyn tnh y = ax +b (y : mt quang; x : hm lng
protein).
Cn c vo th chun y = 1,039. x + 0,0031 s tnh c hm lng
protein ca mu th nghim.
Gi s khi o mu th nghim vi thuc kh Folin-Ciocalteu trn my
quang ph bc sng 750nm c tr s y = 0,234 th thay vo phng trnh y
= 1,039. x + 0,0031 ta c hm lng protein : x = (0,234 - 0,0031)/1,039 =
0,22 mg/ml.
Tuy nhin, khi phn tch trn my quang ph bi phng php Bradford
hoc Lowry th cn c vo cc s liu lp ng chun vi albumin nguyn cht
my t ng lp phng trnh v tnh ton cho ta kt qu v hm lng protein.
3.3. Phn tch thnh phn axit amin
Xc nh hm lng tng loi axit amin trong mu th nghim bng
phng php sc k trn giy hoc hin nay thng s dng my phn tch axit
amin t ng Biochrom 20 ca hng Pharmacia Biotech hoc trn my HPAmino - Quant (Amino quant Series II - Operotor Handbook). S dng OPA
(ortho-phthaladehyde) to dn xut i vi axit amin bc I v FMOC (9Fluoreryl methyl chlorofarmate) i vi cc axit amin bc II. Mu c x l
theo phng php thy phn 1050C trong 24 gi.
Tu theo mc ch nghin cu m c th xc nh hm lng axit amin t
do hoc lin kt. Trong mc ch nghin cu tm mi lin quan gia hm lng
ca mt loi axit amin no vi c tnh chng chu hoc vi tnh trng cht
lng (tnh thm ca ht thc vt chng hn) th c th xc nh hm lng axit
amin t do trong t bo. Cn nu mc ch nghin cu l nh gi cht lng
protein ca sn phm th xc nh hm lng axit amin lin kt. Cn c vo hm
lng protein v hm lng t loi axit amin xc nh c thnh phn axit
amin trong protein. Thnh phn axit amin trong protein c tnh theo gam axit
70

amin/100g protein.
3.4. K thut in di protein
Cc loi protein c khi lng phn t khc nhau, do vy chng c tc
di chuyn khc nhau trong in trng, v th c th tch c cc loi protein
nh phng php in di. T kt qu phn tch thnh phn in di m ngi ta
c th t c nhng mc ch nghin cu khc nhau. K thut in di protein
bao gm phng php in di mt chiu v in di hai chiu. Cc k thut ny
cho php phn tch hn hp phc h protein thnh nhng tiu phn khc nhau
da trn nguyn tc tc dch chuyn khc nhau theo khi lng phn t ca
cc tiu phn . Nh k thut in di mt chiu ngi ta c th tch c cc
tiu phn protein trong protein tng s tan trong dung dch NaCl 1M v protein
tan trong nc. Da vo cc bng in di c th so snh thnh phn in di gia
cc mu nghin cu, xc nh tnh a dng sinh hc trn c s hin tng a
hnh protein. Bng phng php in di protein SDS-PAGE ngi ta pht
hin nhng sai khc v cc bng in di kch thc phn t 10-16kDa gia cc
th t bin 5 ging la khc nhau, xc nh c s khc nhau v thnh phn
v hm lng protein chit t l cy 10 ngy tui ca 12 th t bin u tng.
Cc kt qu nghin cu v thnh phn in di protein d tr trong ht u tng
v trong ht ny mm, nghin cu thnh phn din di enzym protease trong ht
ny mm ca cc ging du tng chu hn khc nhau cho thy mi lin quan
gia s thay i ph in di vi kh nng chng chu ca ging u tng ny.
K thut in di protein hai chiu gip cho cc nh sinh hc phn t phn tch
cc tiu phn protein) nh tnh v nh lng cc loi pohpeptide. K thut in
di cn c ng dng xc nh cc th t bin, xc nh s a hnh protein.
V mt l thuyt k thut in di hai chiu c th pht hin hai loi t bin: (1)
t bin do c s thay th mt hay nhiu nucleotit, mt cp nucleotit; (2) t
bin do s bin i trnh t nucleotit. Ngoi ra: k thut in di protein 2 chiu
trn gel poliacrylamid cn c ng dng trong vic nghin cu biu hin gene
giai on pht trin sm ca cy. xc nh c nhiu ch th protein lin quan
n cc tnh trng hnh thi khc ca cy trng... K thut in di protein c th
theo cc bc sau:

Khi thc hin phn tch in di protein, trc ht ngi ta ch to bn gel

71

in di protein (v d gel poliacrylamid). Tra dch cha protein bin tnh vo

"ging" trn bn gel poliacrylamid, t hai u mp gel ln din trng, chy
in di vi hiu in th khc nhau trong 1,5 - 2 gi. Gel c c nh bng
tricloaxetic, ngm vo dung dch nhum mu coosmasie blue R-250. Sn phm
in di protein c chp nh phn tch.
Thit b in di bao gm: my in di v b ngun (hnh 5.4). My in di
c th l in i ng hoc in di ngang.
Gel s dng in di protein hoc phn tch in di
poliacrylamid cha SDS hay khng c SDS. Ngoi ra c
gel agarose...
Bng 5.1. Thnh phn ca gel poliacrylamid
30%
Thnh phn
% gel
H2O (ml)
Degassed
nhn bn gel
acylamid/Bis
(ml)

cc enzyme c th l
th l gel thch agar,

Gel buffer
(ml)

SDS (ml)

Gel c mu

4%

Gel phn tch 12%

protein

6,1

1,3

2,5

0,1

3,4

4,0

2,5

0,1

K thut ch gel in di protein bao gm cc bc: (1) Lp rp khun gel;

(2) to gel; (3) gel.

Hnh 5.4. Thit b phn tch in di protein

Phn tch hnh nh in di protein cho kt qu thng k cc bng in di
ca cc i tng nghin cu, t xc nh c tnh a hnh protein ca cc
i tng nghin cu. Da vo thang protein chun s xc nh c cc bng
72

in di protein c trng v tm c mi lin quan gia tiu phn protein vi

c tnh quan tm nghin cu. Cng vic tip theo l phn tch tiu phn protein
t bn gel v thu nhn tiu phn protein bng nhng thao tc khc nhau. Tiu
phn protein c em phn tch khi ph xc nh cu trc v tiu phn
protein cng c xc nh thnh phn v trnh t axit amin ca tiu phn
protein . Ngoi k thut in di mt chiu ngi ta c th s dng phng
php in di hai chiu v thu nhn tiu phn protein cng theo nguyn tc trn.

Hnh 5.5. Hnh nh ph in di protein chit bng nc ct

(M. marker. 1. M ging gc; 2. MX10; 3. MXU21; 4. MXE01: 5. MX1608, 6.
MX1103; 7. Tiu ging gc; 8. TXU21; 9. TX16012. ( : bng mt i : bng
xut hin mi )

Hnh 5.6. S phn tch tiu phn protein phn tch t bn gel in di
(theo Phan Vn Chi, 2002)

73

4. K THUT PHN TCH ENZYME

Phn tch enzyme ni chung theo hai cch tip cn, l xc nh hot
enzyme v phn tch ph enzyme. C th tin hnh xc nh hot ca enzyme
thng qua nh lng hoc nh tnh. l xc nh lng c cht b phn gii
trong mt n v thi gian, hoc xc nh thi gian phn gii trong cng lng
c cht, o vng phn gii.... Kt qu xc nh hot enzyme s cho thy mi
quan h gia hot ca loi enzyme no vi c tnh nghin cu. Di y
gii thiu mt vi phng php xc nh hot ca enzyme.
thc vt, khi ht ny mm th enzyme hot ng mnh. Hot ca
enzyme tng dn n s bin i cc cht d tr. Mc hot ho cc enzyme
ph thuc vo tnh cht v thnh phn ho hc ca ht. i vi cc cy h u
cht d tr ch yu l protein cho nn hot tnh protease mnh hn cc enzyme
khc. Kt qu l protein b phn gii thnh cc axit amin, cc axit amin c th
c s dng tng hp nn cc protein th cp cu trc nn cht nguyn sinh
ca phi ht sinh trng v cy non, cng c th kt hp vi NH3 to nn cc
amt, c bit l s ny mm ca ht trong ti. Ngoi protein, trong ht u xanh
cn c tinh bt, do hot tnh - amylase cy h u cng kh cao. S tng
hot - amylase s lm tng hm lng ng do lm tng p sut thm
thu v tng kh nng gi nc. Ngi ta nghin cu thy - amylase, hm
lng ng v p sut thm thu c mi tng quan thun. Ngi ta nghin
cu thy rng c tnh ho sinh v sinh hc phn t ca cy u tng c kh
nng chu hn, chu nng c lin quan n vai tr ca a-amylase v protease.
Ht sau khi c ngm nc vi nhit thch hp v O2 th cc
enzyme bt u hot ng phn gii cc cht hu c. Qu trnh phn gii protein
v tinh bt din ra rt phc tp.
Enzyme -amylase thuc nhm hyrolaza c trong nc bt, ht ho
tho ny mm, ty tng, trong nm mc, vi khun. Enzyme -amylase phn gii
cc lin kt 1,4- glucozit gia chui mch polisacarit, to thnh cc extrin
phn t thp. Do di tc dng ca enzyme ny dung dch tinh bt nhanh
chng bt mu vi dung dch iot v b gim nht mnh. Ion canxi c tc dng
lm bn cu trc khng gian ca phn t enzyme. Enzyme - amylase tng
i bn vi nhit hn cc amylase khc nhng li km bn vi axit.
Protease cng thuc nhm hydrolaza phn gii cc lin kt peptit gia
chui mch polipeptit nn cn gi l endo peptit hydrolaza. Protease ng vai
tr quan trng trong c th sng. Chng khng ch tham gia vo phn gii
protein d tr trong ht nhm cung cp cc axit amin cho thi k pht trin u
tin ca cy m chng cn tham gia vo qu trnh x l phn t protein v hot
74

ho cc enzyme khc. Ngoi ra, protease cn tham gia phn gii cc protein l
hoc b bin tnh khi gp iu kin cc oan nh lnh, hn, mn,... Protease
trong ht c th c tng hp t trc dng tin cht v tn ti song song vi
protein d tr, cng c th c tng hp mi trong qu trnh ht ny mm. S
c mt ca protease trong ht d tr protein ca ht ang ny mm l bng
chng v s tham gia ca chng vo qu trnh phn gii protein, cc sn phm
peptit trong cc bo quan . Qu trnh phn gii protein d tr ca cc cy h
u u ging nhau. Khi protein b phn gii ht nhng khng bo ln c
hnh thnh trong cc t bo d tr. Cc axit amin c to ra trong qu trnh ny
c s dng sinh tng hp cc protein mi trong mm non. Qu trnh bin
i protein di xc tc ca protease din ra u tin l : protein d tr dng
nguyn thu b endopeptidaza c hiu ct mt s mi lin kt trn b mt ca
phn t globulin theo c ch "kho ko". Sau c s thay i cu trc khng
gian ca globulin v lm cho chng tr thnh c cht d phn gii bi
endopeptidaza khc v cc exopeptidaza c sn trong protein d tr trong ht,
to thnh cc polipeptit, oligopeptit ngn hn. Tip theo, cc sn phm ny b
aminopeptidaza phn gii n axit amin.
nh lng hot enzyme - amylase theo phng php Heinkel c
thc hin theo nguyn tc l: xc nh lng tinh bt b phn gii da trn c s
xc nh mc gim cng mu ca hn hp phn ng vi dung dch iot. n
v hot enzyme l lng enzyme c kh nng phn gii 1 mg tinh bt sau 30
pht 300C.
Da vo tnh cht ho tan trong dung mi hu c, chit enzyme aamylase bng dung dch m photphat xitrat pH = 6,8. em cn mu v nghin
nh trng dung dch m photphat xitrat, li tm lnh 12000 vng/pht trong 15
pht thu dch cha enzyme - amylase cho vo tube nha lm th nghim.
Th nghim c tin hnh ng th nghim, ng kim tra, ng i
chng. Trong ng th nghim, cho 0,25ml tinh bt 1% vo 0,25ml NaCl 0,1%
tip tc cho thm 0,5ml dung dch m photphat xitrat pH = 6,8 v 0,25ml dch
cha enzyme, sau lc u v trong t m 300C trong 30 pht. Sau khi t
m ly ra cho thm 1,25ml dung dch axit sunfosalisihc 20%, lc u. Ly 1ml
t hn hp trn cho thm 9ml iot long 150 ln lc u 10 pht ri o trn
my quang ph UV Visible Spectrophotometers bc sng 560 nm.
ng kim tra: tng t ng th nghim ch khc l cho 1,25ml dung dch
axit sunfosalisilic 20% vo trc sau mi cho dch cha enzyme v trong
t m 30 pht. ng i chng: tng t nh ng th nghim ch khc l thay dch
cha enzyme bng nc ct.
75

Kt qu: ly kt qu ng kim tra tr i ng th nghim nhn vi pha

long, tm c s mg tinh bt b thu phn bi dch chit cha enzyme amylase trong 30 pht 300C.
Cho hn dch (thch agar, tinh bt, nc) vo bnh nn, un cch thu cho
tan thch, vo a petri dy 4mm, ngui, c l ng knh (d-mm). Nh
100 l dch chit cha enzyme vo mi l, t lnh qua m enzyme
khuch tn, chuyn sang t m 300C trong 24 gi. Nhum lugol trong 5 pht
v trng bng NaCl 1N. o ng knh vng phn gii (D-mm). Hot tnh
enzyme a-amylase c xc nh bng: D - d (mm).
Xc nh hot enzyme protease theo phng php anson ci tin theo
nguyn tc: dng cazein lm c cht; sau dit enzyme v kt ta protein cha
b thu phn bng axit tricloaxetic. Xc nh hot phn gii protein ca
enzyme trn c s nh lng sn phm c to thnh trong phn ng bng
phn ng mu vi thuc th Folin-ciocalteau. Cn mu v nghin nh trong
dung dch m photphat pH = 6,5. Li tm 12000 vng/pht trong 15 pht 40C.
Dch thu c s dng lm th nghim nh sau:
ng th nghim: cho 1ml c cht t 300C trong 10 pht trn vi 0,5ml
dch cha enzyme protease t 300C trong 10 pht, sau cho thm 2,5ml
tricloraxetic 5%, lc u 30 pht 300C. Lc ly 0,5ml dch lc v cho thm
2ml Na2CO3 6%, lc u ri cho 0,5ml thuc th Foling- Ciocalteu. hn hp
trn 30 pht ri o trn my quang ph UV Visible Spectrophotometers bc
sng 720nm.
ng kim tra: sau khi cho 1ml dung dch c cht, cho ngay 2,5ml dung
dch axit tricloaxetic 5%, lc u ri mi cho 0,5ml dung dch cha enzyme
protease. Cc bc tip theo lm tng t nh ng th nghim. ng i chng:
lm tng t nh trn ch thay dung dch cha enzyme bng nc ct.
Cho hn dch (thch agar, cazein, nc) vo bnh nn, un cch thu cho
tan thch, vo a petri dy 4mm, ngui, c l ng knh (d-mm). Nh
100 l dch chit cha enzyme vo mi l, t lnh qua m enzyme
khuch tn, chuyn sang t m 300C trong 24 gi. Nhum lugol trong 5 pht
v trng bng NaCl 1N. o ng knh vng phn gii (D-mm). Hot tnh
protease c xc nh bng: D-d (mm).
Phn tch isozyme vi mc tiu l pht hin hot tnh ca mt loi
enzyme no . Tuy nhin trong phn tch di truyn, ngi ta cn c vo kt qu
phn tch ph isozyme m nh gi c s a dng sinh hc ca cc loi sinh
vt nghin cu v xc nh c mi quan h v di truyn mc sai khc gia
76

cc i tng nghin cu.

i a s cc enzyme c bn cht l protein, cho nn thng thng ngi
ta phn tch ph isozyme bng in di trn gel acrylamid. Cn c vo hot ng
ca loi enzyme m ngi ta trn thm c cht ca enzyme vo gel.
Trong iu kin nhit thch hp sau khi chy in di m enzyme cn phn
tch s hot ng tng tc vi c cht. Cn c vo kt qu nhum gel m pht
hin ra ph isozyme ca loi enzyme . Cng c th s dng mng
nitrocellulose pht hin hot ng ca enzyme.

5. NGHIN CU RIPS BNG WESTERN BLOT

Western blot l k thut pht hin cc tiu phn protein bng khng th
nh du. Western blot bao gm cc bc c bn sau:
Bc 1: Tch protein bng in di trn gel poliacrylamid.
Bc 2: Chuyn protein t gel sang mng (PVDF) nh in trng.
Bc 3: Nhum mng bng khng th c hiu.
Bc 4: Nhum mng bng cng hp (khng th khng khng th gn
enzyme).
Bc 5: B sung c cht cho enzyme chuyn ha c th nhn thy vt
protein phn ng vi khng th c hiu trn mng.
Enzyme s chuyn ha c cht thnh hp cht c mu c th nhn thy
trn mng. Nh vy ch vt protein no phn ng vi khng th th mi hin th
trn mng.

Hnh 5.7. S phn tch Western blot

Nu xt v cu trc bc mt v cc tnh cht ho sinh th cc protein bt
77

hot ribosome (RIPs) tipe I l mt h protein tng i ng nht, kim tnh,

kh bn vng. Tuy nhin chng vn rt khc nhau v c tnh enzyme v hot
tnh sinh hc. Do c cc c tnh sinh - dc hc cng nh kh nng ng dng
rt a dng, cho nn vic tm kim nhng loi RIPs mi c nhng tnh cht l
th vn ang tip din rt nhiu phng th nghim trn th gii. nc ta tuy
cha nhiu nhng bt u c nhng cng b v loi protein ny t ngun
thc vt. Di y l kt qu kho st s phn b ca RIPs tipe I bng k thut
Western blot trn mt s i tng thc vt (thuc h Cucurbitaceae) nh: cy
Trichosanthes sp. Bac Kan 8-98 thu thp Bc Kn, cy Hng qua (Mnh bt Coccinia cordifolia Cugn.), Gc (Momordica conchinchinensis), Da chut
(Cucumis sativus L.), Mp ng (Momordica charantia), Thin hoa phn (r
ca Trichosanthes kirilowii Maxim).
tin hnh vic kho st v xc nh RIPs, protein tng s t cc mu
r, ht c chit rt, dch chit protein c lc qua mng milipore 0,2 m.
Protein c ta li bng acetone lnh theo t l 1:1 (w/v) 40C. Cc bc in
di protein bng SDS-PAGE (12,5% acrylamide) c tin hnh theo cc phng
php ca Laemmli, sau thc hin phng php Western blot.
Phn ng cho gia khng th khng Trichobakin ti t hp vi cc mu
protein trn l rt c hiu v ch c vng 23 - 30 kDa.
THO LUN
1. Trnh by c im v vai tr ca mt s protein lin quan n tnh chu
nhit thc vt.
2. Protein bt hot ribosome RIPs, phng php kho st RIPs v ng dng
ca nhng nghin cu ny.
3. Xc nh mi lin quan gia protein v tnh trng c th tin hnh theo
quy trnh no?
4. Phng php phn tch ch th phn t protein c phn tch bng in
di.

78

Chng 6
K THUT SINH HC PHN T TRONG PHN TCH H
GENE SINH VT
Tm tt: Sinh hc phn t l nng ct ca cng ngh sinh hc. Phn tch sinh
hc phn t c bt u t vic thu nhn dch chit ADN t t bo sng
sch thc hin cc phn tch tip theo. Tu theo vt liu nghin cu m c
phng php tch chit ADN ph hp. Dung dch ADN sau khi kim tra hm
lng v sch c s dng phn tch theo nhng mc ch khc nhau
nh Southem blot, Northem blot, xc nh trnh t ADN, PCR, RFLP, AFLP,...
Ni dung ca chng gm 5 vn c bn: (1). Phng php tch chit ADN,
(2). Lai phn t, (3). Xc ih trnh t nucleotit, (4). K thut RFLP, (5). Trnh t
nucteotit lp li.
Thnh tu ca sinh hc hin i v nhng hiu bit v di truyn hc phn
t lm c s cho vic nghin cu, xy dng cc k thut sinh hc hin i.
Nhng k thut sinh hc phn t ng dng trong phn tch genome ca sinh vt
c cng b trong cc tp ch khoa hc v cc sch chuyn kho.

1. PHNG PHP TCH CHIT ADN

Mi nghin cu v ng dng sinh hc phn t u bt u bng vic thu
nhn mt lng axit nucleic, tinh sch tin hnh cc th nghim tip theo.
iu quan tm hng u l cc k thut tch chit axit nucleic l thu nhn cc
phn t trng thi nguyn vn khng b phn hu bi cc tc nhn c hc hoc
ho hc.
C th ly v d cc bc tch ADN t mm ht u di dy:

79

Hnh 6.1. S quy trnh tch chit ADN t mm ht u tng v u xanh

T v d c th trn ta thy quy trnh tch chit ADN gm ba bc c
bn sau:
Bc 1: Ph mng t bo v mng nhn (t bo Eukaryot). Thng thng
ngi ta nghin t bo, m trong hn hp cht ty (SDS, sarcosyl) v proteinase
K. Hn hp ny ph v mng t bo v mng nhn, gii phng ADN ra mi
trng ng thi phn hu cc protein.
Bc 2: Loi b cc thnh phn khng mong mun, ch yu l protein
bng li tm.
Bc 3: Ta axit nucleic trong ethanol hoc trong isopropanol thu nhn
c axit nucleic di dng c c.
Sau khi thu nhn axit nucleic dng sch, ngi ta tin hnh phn tch
nh tnh v nh lng chng bng mt s phng php nh o mt quang,
in i, siu li tm, sc k (hnh 6.2 v 6.3).

80

Hnh 6.2. Ph hp th ADN bc sng 260 nm

in di ADN l k thut c s dng kim tra cht lng ca ch
phm ADN tch t t bo sng. ng thi kt qu in di cn cho php d on
c hm lng ADN trong dung dch tch chit. Phng php in di dc s
dng kim tra sn phm PCR, gii trnh t ADN...

Hnh 6.3. Cu trc ca agarose (theo Nng Vn Hi, 2002)

Gel agarose c s dng phn tch in di ADN. Agarose dng bt
dc pha vi dung dch TBE hoc TAE v tu theo mc ch phn tch ADN
m pha gel vi nng thch hp.
K thut in di ADN bao gm cc bc (hnh 6.4):

81

Hnh 6.4. Cc bc to gel in di agarose

(1, 2) To khun ga agarose; (3) ga trn khun ga ;
(4) Tra dung dch ADN ; (5) Chy in di.
Sn phm in di ADN c nhum bi EtBr v chp nh di nh sng
n cc tm.
(1) Nhum gel bng EtBr ; (2) Soi gel trn nh sng n cc tm pht
hin s c mt ca ADN ; (3) Chp nh phn tch.

82

Hnh 6.5. Hnh nh in di ADN tch t mm u tng

1. ADN chun
2, 3, 4, 5, 6, 7, 8: ADN tch t mm u tng

Hnh 6.6. Cu trc ca EtBr (theo Nng Vn Hi, 2002)

2. LAI PHN T
Mc ch ca k thut ny l pht hin trnh t nucleotit c th, nghin
cu cu trc ADN h gene (gencomic ADN). Phn t ADN b phn ct bi
enzyme gii hn thnh nhng phn on ADN ring bit. Bng phng php
in di cc phn on ADN ny vi nhng kch thc khc nhau c tch ra
(separate) trn gel thch hoc hoc poliacrylamit v c chuyn sang mng lai
phn t. Qu trnh lai phn t xy ra gia phn t ADN v on d ADN c gn
nhn phng x theo quy tc lin kt b tr. Cc on ADN c th c pht
hin theo phng php thm tch ca Southern v phng php lai phn t vi
on d ADN c trnh t nucleotit tng ng. Phng php t ghi phng x
(autoradiogrophy) c th nhn bit v tr on d ADN trn mng lai phn t. T
khi E.M Southern thuc Trng i hc tng hp Edinburgh pht hin ra k
thut ny (1975) gi l k thut phn tch mng thm truyn Southern (Southern
blot analisis) gi tt l Southern, n c ng dng rng ri trong cc lnh
vc nghin cu sinh hc phn t v di truyn. Khi ng dng k thut Southern
i vi ARN th gi l Northern, cn vi protein th c gi l Western. Hin
nay c nhiu ci tin v phng php, hnh thnh nhiu k thut phi hp nh
Southwestern.
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2.1. Phng php Southern blot

S dng mt trnh t ADN bit trc lai vi on ADN cn tm. Cc
bc tin hnh c th thc hin theo cc bc sau:
- Ct ADN bi enzyme gii hn thnh cc on ADN nh c th dch
chuyn trn gel agasore.
- Bin tnh cc si ADN kp trn gel agarose bng phng php x l vi
kim.
- Chuyn ADN bin tnh sang mng nylon hoc nitrocellulose.
- Lai vi trnh t ADN c nh du bng phng x hoc hunh
quang.

Hnh 6.7. S m t phng php Southern blot

Phng php Southern blot cho php xc nh c trnh t on ADN
cn tm, c th pht hin c t bin mt on hoc thm on khi so snh c
th t bin vi c th bnh thng.
2.2. Phng php Northern blot
Phng php Northern blot tng t nh phng php Southern blt,
nhng mc tiu ca phng php Northern blot l nghin cu cu trc ca
mARN. l k thut lai gia mARN vi trnh t ADN bit trc, nh du
phng x.
(1) Tch chit ARN tng s v bin tnh.
(2). ARN c chuyn sang mng v lai vi trnh t ADN c hiu.
(3). V tr v s lng cc on ca ARN lai vi ADN c pht hin trn
phim phng x.
Phng php Northern blot cho php nghin cu cu trc ARN, pht hin
t bin.

84

3. XC NH TRNH T NUCLEOTIT CA ADN

C nhiu k thut xc nh trnh t cc i baz trong mt chui ADN:
- K thut in i phn on nh du u cui cho php xc nh trnh t
chui ADN si n.
- Xc nh trnh t nucleotit thng qua trnh t amino axit da vo cc b
ba m ho, tuy nhin s m ca cc amino axit thng c mt s nucleotit trng
lp hoc mt s b ba cng quy nh mt loi amino axit, cho nn trnh t ADN
thu c ch l tng i.
- Xc nh trc tip thng qua phn ng kt thc u cui. Nguyn tc
chnh l to ADN si n, m phn t sau c cng trnh t v ch di hn phn
t trc mt nucleotit mt u cui.
Gene t bo tin nhn c trnh t lin tc, cn gene sinh vt nhn thc
c th gin on. on nucleotit m ho protein c gi l exon, cn on
khng m ho gi l intron.
3.1. Phng php ho hc ca Maxam-Gilbert
Nguyn tc ca phng php ho hc ca Maxam-Gilbert l s dng
phng php nh du phng x (P32), x l ho hc ph hu 1 nucleotit to
ra hng lot cc on ADN c kch thc khc nhau.

Cc on DNA b ph hu 1 nucleotit in di trn gel poliacrylamid

Hnh 6.8. S minh ho phng php Maxam-Gilbert
Cc khu trong phng php Maxam-Gilbert :
(1). ADN si kp bin tnh thnh dng si n.
(2). nh du phng x (P32) u 5'.
(3). S dng 4 loi ho cht khc nhau, mi loi ho cht ch ph hu mt
loi nucleotit nht nh v tin hnh 4 phn ng c lp, kt qa to ra cc on
ADN kch thc khc nhau.
85

(4). Sn phm ca 4 phn ng c tin hnh in di trn gel

poliacrylamid. Trnh t nucleotit c c t di ln theo chiu t 5'3'.
3.2. Phng php Sanger
Nguyn tc ca phng php ny l s dng deoxynucleotit khng c
nhm OH v tr 3' do vy trong qu trnh ko di chui polinucleotit enzyme
polimerase gn chng vo chui polinucleotit th qu trnh tng hp b dng li.
Kt qu to ra cc on ADN c kch thc hn km nhau mt nucleotit, trn c
s xc nh c trnh t nucleotit.

Hnh 6.9. Deoxynucleotit mt oxy v tr C 3' ca phn t ng

Cc khu ca phng php Sanger:
(1) Bin tnh ADN si kp thnh ADN 2 si n.
(2). Mi tip hp vi ADN si n.
(3). Phn ng tng hp chui polinucleotit gm: ADN si khun, primer,
ADN polimerase v dNTP. S gn dNTP mt nhm OH v tr 3' vo chui
polinucleotit lm qu trnh tng hp b dng li.
(4). in di trn gel poliacrylamid s xc nh c trnh t ca on
ADN.

Hnh 6.10. S xc nh trnh t nucleotit theo Sanger

86

4. RFLP TECHNOLOGY (K thut phn tch hin tng a hnh ca

di cc phn on ADN)
Trong nhng nm gn y k thut phn lp gene dn thc y c bn
trong lnh vc th v nht l vic s dng cc on ADN phn lp nh cc ch
th phn t. K thut phn tch hin a hnh ca di cc phn on ADN l
mt k thut mi m y ha hn lm thay i hon ton mt s lnh vc trong
i truyn v chn ging.
Thng tin di truyn c cha trong cc chui ADN ca cc nhim sc
th nhn v b gene ca cc c quan t. Cy trng c kh nng sao chp phn t
ADN vi ch chnh xc cao v mau l song c nhiu tc ng t nhin v
nhn to gy ra bin d trong trnh t ADN. Nhng bin i v cp baz nit c
th xut hin hoc do bin i chui ADN trn phm vi rng ln nh l kt qu
ca s o on, chuyn on v khuyton hoc do chuyn ch trong nhim
sc th. C mt s cch xc nh bin d ca chui ADN, l xc nh trnh t
ADN v so snh tng on; s dng enzyme ct hn ch ct ADN thnh
nhng phn on c chiu di khc nhau.

Hnh 6.11. Thit b t ng xc nh trinh t ADN mao qun

Bin i trnh t nucleotit ti mt locus dn n xut hin bng ADN mi
trn gel thch hoc poliacrylamid. Hin tng ny c gi l a hnh ca
di cc phn on ADN. Hin tng a hnh di phn on ADN l cc ch
th phn t. Bng cch s dng cc ch th phn t (ch th phn t RFLP) cc
nh sinh hc phn t v di truyn chn ging c th thit lp cc bn gene
lin kt di truyn v nh v cc gene c tm quan trng v kinh t.
K thut RFLP c tin hnh theo cc bc sau:
87

(1). Tch ADN t m sng.

(2). S dng cc enzyme ct hn ch ct ADN thnh cc on c kch
thc khc nhau.
(3). in di trn gel agarose.
(4). So snh cc on ADN gia cc i tng nghin cu.
4.1. Bn RFLP
4.1.1. Nguyn l
Nguyn l ca k thut ny da trn s khc bit t nhin ca cc chui
nucleotit trong phn t ADN v khi phn t ADN b ct thnh nhiu on nh
bi enzyme ct hn ch th cc on ADN c th khc nhau v kch thc hay
chiu di. S khc nhau ny c th c khai thc phn tch s phn li ca
cc on nhim sc th khi lai to.
Cc phn t ADN c kch thc nh nh ADN lc lp c th s dng
enzyme (RE) gii hn lp bn RFLP, tuy nhin kh nng ng dng ADN
lc lp trong to ging rt hn ch, v hu ht cc gene c ngha kinh t quan
trng li nm trong nhn. Phn tch RFLP c th ng dng vi ADN nhn,
nhng rt phc tp v ADN nhn c kch thc ln v hm lng nhiu. Khi s
dng RE i vi ADN th hng triu phn on ADN c to thnh.
4.1.2. Th vin ch th RFLP
Ngi ta dng nhng on nhim sc th lm ch th nh du nhn
bit cc phn on ct hn ch. p dng phng php lai ADN - ADN c tnh
c hiu cao bi vic s dng cc on ADN mu nhn bit cc phn on
ADN nhn t hn hp cc on ADN to thnh bi vic x l RE. Tp hp cc
on ADN mu c gi l th vin RFLP. on ADN mu c di 2-5 kb
trng thi tinh khit v c s dng lai phn t. Cc on ADN tinh khit
ngi ta cn phi thc hin k thut nhn gene (gene cloning). K thut nhn
on ADN c th c hin nh sau: on ADN c gn vo plasmit ri bin
np vo vi khun. Vi khun sinh sn dn n on ADN c nhn ln to
nhiu bn sao on ADN. Bng k thut nui cy v phn lp plasmit t vi
khun s to ra mt s lng ln cc on ADN dng lm mu cho lai ADN.
Quy trnh xc nh RFLP c thc hin theo cc bc sau : (1) Tch
chit ADN nhn ; (2). X l ADN vi enzyme ct gii hn ; (3). in di sn phn
ct bi RE ; (4). Bin tnh ADN thnh dng si n; (5). Chuyn ln mng lai
nitrocellulose (Southern blot) on nADN mu lai vi on ADN tng ng
theo nguyn tc b sung gia cc cp baz. Ngi ta s dng k thut pht
quang ho hc (Enhanced Chemical Luminesence = ECL) hoc nh du phng
x xc nh kt qu lai bng phim m bn hoc bng phng x t ghi.
88

4.1.3. Thit lp bn di truyn RFLP

Ngi ta tin hnh lai hu tnh c F1 v cho F1 t th cho F2. Khi F1
gim phn hnh thnh giao t th cc nhim sc th c s ti t hp thng qua
trao i cho. Trao i cho dn n ti t hp l c s cho vic thit lp bn
di truyn truyn thng (bn lin kt). K thut RFLP cho php quan st trc
tip cc phn t ch th ADN trn on nhim sc th. Cc ch th RFLP phn li
hon ton chnh xc nh cc ch th gene v tun theo cc nh lut Menen mt
cch nghim ngt. V th bn RFLP c thit lp theo nguyn tc nh i
vi cc ch th truyn thng. Quy trnh thit lp bn di truyn bng ch th
RFLP c thc hin theo cc bc sau:
(1) Chn cp b m cng loi xa nhau v mt di truyn to F1. Tch ADN
v s dng RE x l ADN. Sng lc tnh a dng v cp b m no c mc
a dng thch hp c chn lm cp lai.
(2). To qun th lp bn . Cho cp b m lai vi nhau c F1, cho F1
t phi c F2 hoc c th lai ngc. Mt qun th F2 hoc lai ngc c 50 c
th th c th xy dng bn di truyn chi tit.
(3). nh gi RFLP trong qun th. Tch ADN tng c th v tin hnh k
thut RFLP. Qu trnh lp bn c tin hnh theo 3 qu trnh sau:
- Sng lc RFLP: Mu c th vi cp b m pht hin ra loi
enzyme gii hn thch hp to ra s a dng RFLP r rt nht gia hai c th b
m.
- Chn cp ch th 1enzyme: Chn cp ch th /enzyme thch hp, l
loi enzyme ct ADN. C th tin hnh nh sau: tch ADN cy b, cy m v
tng cy con lp bn . S dng tng loi enzyme ct, in di, thm truyn
ln mng lai (mi enzyme dng mt b mng lai ring), chn cp ch th /
enzyme th hin s a dng v kiu gene s dng lp bn .
- nh gi mc lin kt: Khi so snh hai ch th thu c hai kt qu,
(1). Chng c xu hng cng phn li v hai ch th c mi lin kt. V d: F2 d
hp v ch th 1 v d hp v ch th 2 trong khi b m ng hp v c hai ch
th. (2). S phn b tnh ng hp hay d hp ca hai ch th l khng theo quy
lut m hon ton ngu nhin, nh vy chng khng lin kt. S dng nhiu ch
th s xc nh c mc lin kt khc nhau ca tng ch th v nhm ch th.
4.2. ng dng ca RFLP
Vic pht trin k thut phn tnh hin tng a hnh ca di cc phn
on ADN m rng cnh ca i ti pht hin, theo di v iu khin s bin
d di truyn nhiu loi cy trng m trc y khng th lm c (Tanksley
89

v cng s, 1989). Nhng nghin cu gn y v la (Mc Couch v cng s,

1988), c chua (Pettersun v cng s, 1988, 1990, 1991), u tng (Keim v
cng s, 1990), la m (Sharp v cng s, 1989), Ng (Burr v cng s, 1988,
Grant v cng s 1989, Beavis v cng s. 1991, Stuber v cng s, 1992)
chng t rng vic lp bn cc ch th phn t RFLP l mt phng php c
sc mnh ln lao trong vic xc nh cc locus iu khin cc tnh trng s
lng - cc c tnh c tm quan trng v mt nng nghip.
Ngi ta cng xc nh nhiu tnh trng n gene cy la thng qua lin
kt vi cc ch th phn t RFLP (Mackill v cng s, 1993, Ronald v cng s,
1992, Mc Couch v Tanksley, 1991, Yu v cng s, 1991, Yoshimura v cng
s, 1992). K thut phn tch hin tng a hnh ca di cc phn on ADN
cho php pht hin hin tng a hnh ca b gene thng qua so snh cc bn
phn ct ADN gia cc c th khc nhau. Cc ch th phn t RFLP mang tnh
cht ring bit, ng tri v khng gy hi. Cc ch th phn t ny khng chu
tc ng bi iu kin mi trng (k c s thay i v ma v v v tr a l v
giai on pht trin ca cy. Vic phn tch hin tng a hnh ca di cc
phn on ADN c tin hnh trn phn t ADN tch chit t l non, trnh
vic phi chm sc cy cn non ti lc trng thnh.
Ngi ta p dng cc ch th phn t RFLP vo cng tc chn ging
gin tip, lm rt ngn qu trnh chn lc cc gene qu him, cc gene c tm
quan trng v kinh t. Bi l cc ch th phn t RFLP c th lin kt cht ch
vi cc gene trn v vn c duy tr trong qu trnh phn li. V vy ngi ta c
th theo di v chn lc cc gene thng qua cc ch th phn t .
N. R. Apuyru v cng s (1988) xc nh hin tng a hnh ca
di cc phn on ADN ca hai ging u tng hoang di Minsoy v Norin 1
bng cch s dng cc dng ADN chn lc ngu nhin lm on d ADN. Cc
tc gi nhn thy rng mt trong s nm on d th nghim bc l tnh
a hnh. Hn na s bng a hnh xut hin l do c s sp xp li phn t
ADN. P. Knh v cng s (1989) kho st 58 ging u tng chn lc t
Geneus Glicine v Subgeneus Soja bng 17 ch th phn t RFLP nh gi
mc a dng phn t, tnh hu hiu ca cc ch th phn t c th hin t
nht trong s cc ging u tng dng trng v n c biu hin nhiu nht
gia cc ging thuc cc loi u tng khc nhau nh Glicine max (L) Merr,
G. soja Sieb v Zucc.
Cc ch th phn t RFLP c p dng cho vic lp bn gene, nghin
cu a dng sinh hc, phn loi pht sinh chng loi cho n iu khin vt nui
cy trng. N l mt cng c c lc trong cng tc lai to v chn ging thc
90

vt. ng thi vic xc lp cc ch th phn t RFLP m ra mt hng mi l

phn lp cc gene m vn cha bit sn phm ca n.
THO LUN
1. Th no l lai phn t? Phn bit phng php Southern blot, Northern
blot. Nhng ng dng ca cc phng php ny.
2. Vn xc nh trnh t ADN. Nguyn tc v phng php.
3. Vn xc nh ch th RFLP v bn RFLP.

91

Chng 7
PHN NG CHUI POLIMERASE(POLIMERASE CHAIN
REACTION - PCR)
Tm tt: K thut phn ng chui dng enzyme polimerase (Polimerase chain
reaction = PCR hay PCR Technology) ln u tin c Mullis v cng s m
t nm 1986 v cng chnh do s pht minh ny m Mullis xng ng nhn
gii thng Nobel v Y hc v Sinh l hc nm 1993. Phn ng chui
polimerase (PCR) l k thut c bn v rt quan trng trong Sinh hc phn t.
Phn ng PCR gm 3 giai on chnh: (1). Bin tnh ADN mu; (2). Tip hp
mi, (3). Tng hp on ADN mi. PCR cng nh cc k thut sinh hc phn t
khc cn phi c ADN tinh sch v chu nh hng ca nhiu yu t. Hin nay
c nhiu phin bn ca PCR nh RT-PCR, RAPD, AFLP... PCR v cc k
thut ci tin t PCR c nhiu ng dng trong thc tin, nh: sn xut mu d,
khuch i s lng cc on ADN v ARN, nh lng bng PCR, phn tch
t bin, phn tch ADN a hnh, phn lp gene v tch dng gene, phn tch
sinh vt chuyn gene, xc nh quan h di truyn...
Ni dung ca chng gm 3 vn c bn: (1). Phn ng chui polimerase
(PCR); (2). Phn tch tnh a hnh ca ADN c nhn bn ngu nhin
(Random Amplified Polimorphic DNA - RAPD); (3). Phn tch tnh a hnh
chiu di cc phn on ADN c nhn bn c chn lc (Amplifed Fragment
Length Potimorphism - AFLP).

1. PHN NG CHUI POLIMERASE (PCR)

1. Nguyn tc
Phn ng chui polimerase l mt k thut n gin, cho php khuch i
invitro mt trnh t ADN ln hng triu ln trong vi gi. iu ny rt thun li
cho vic pht hin nhng trnh t him trong cc b gene (t bin im, ti t
hp, retrovirus) t nhng on ADN ngn c phn lp hoc t mt lng rt
nh ly ra c khuch i cho cc th nghim sinh hc, y hc v nng nghip.
ng thi n gip cho cc nh sinh hc phn t nghin cu cu trc trnh t
ADN trong b gene ca cc c thsinh vt khc nhau.
Phn ng chui PCR c thc hin khi c ADN mu, 2 on mi (mi
on mi di 18-30 baz), Taq polimerase (ensyme chu nhit c tch t vi
92

khun Thermus aquaticus):,bn loi deoxyronucleotit triphotphate ( dATP,

dTTP, dGTP, dCTP), dung dch m v con Mg2+ Phn ng chui PCR c
thc hin trn thit b nhn ADN (my PCR).

Hnh 7.1. Thit b nhn ADN (My PCR)

K thut PCR l mt phng php hon ton mi trong vic nghin cu
v phn tch h gen. S dng k thut PCR c th to ra mt s lng ln cc
bn sao ca on ADN cn tng hp m khng phi tch v nhn dng. Thc
cht PCR l mt phng php invitro cho php nhn bn nhanh mt on ADN
no m ch cn mt khi lng mu ban du hn ch. Cc thnh phn c bn
ca mt phn ng PCR gm: ADN khun, hai on mi xc nh cc im
bt u tng hp ADN, Taq polimerase, hn hp bn tin cht deoxynucleotit,
dung dch m cha mt s cation ho tr 1, ion Mg2+ v dung mi (nc kh
ion kh trng).
Enzyme Taq polimerase l enzyme chu nhit, c tch t vi khun
sui nc nng Thermus aquaticus. Enzyme Taq c trng lng phn t l 94
kDa v khng mt hot tnh nhit cao trong giai on gy bin tnh ADN.
Hot tnh ca enzyme Taq gim 50% sau 150 pht nhit 92,50C; sau 40 pht
nhit 950C v sau 5-6 pht nhit 970C. Nu nhit tng ln ti 950C
trong 20 giy bin tnh ADN kp thnh si n th hot tnh enzyme Taq cn
li 65% sau 50 chu k phn ng.
Nng enzyme Taq ti u cho phn ng PCR l 0,5-2,5 n v, nhng
nu nng enzyme qu cao s lm gim hiu sut xc tc ca phn ng. Ngoi
ra, nng Mg2+ v dNTP cng nh hng n hot tnh ca enzyme. Hin nay,
c nhiu loi polimerase chu nhit khc c chc nng ring bit v hon thin
hn nh Tth polimerase tch chit t Thermus thermophilus, c kh nng hot
ng nh mt enzyme phin m ngc khi c mt ca ARN khun v ion Mg2+.
ADN khun (template)
ADN khun l vt liu khi u cho phn ng PCR nn i hi c tinh
93

sch cao. ADN khun c th l si n hoc si i ca chui ADN c bit

trc trnh t hai u thit k mi. Thng thng, nng ca ADN khun
c a vo phn ng PCR khong 10-500ng.
on mi (primer)
Cc on mi thc cht l cc oligonucleotit di khong 4-10 baz (i
vi mi ngu nhin) hoc khong 12-24 baz (i vi mi c trng). Nng
mi thch hp tin hnh phn ng PCR l t 0,1-0,5 M. Lng mi a vo
phn ng PCR phi ph hp vi lng ADN cn tng hp (thng 106 phn t
ADN cn 108 phn t mi). Nu nng mi qu thp th mi s ht trc khi
s chu k kt thc, cn nng mi qu cao th s d lm tng sn phm khng
c hiu.
Cc on mi cn c tnh c th lm tng tnh c hiu ca phn ng.
Mi phi c thit k sao cho khng c b tr ln nhau, s lng 4 loi baz
nit trong mi nn xp x nhau, lng G-C gia hai mi phi bng nhau, trnh
nhng vng trnh t khng bnh thng nh polipurine hoc poliprimidine hay
cc trnh t lp.
Cc nucleotit (dNTPs)
dNTPs l hn hp ca bn loi deoxyribonucleotit (dATP, dTTP, dGTP,
dCTP) lm nguyn liu cho phn ng tng hp ADN. Tu thuc vo mc ch
nghin cu, cc nh khoa hc cn c th s dng mt s nucleotit c thay
i nh gn thm biotin hoc digoxygenin... Nng phn ng ca cc dNTP
thng dng vo khong 200mM mi loi, tuy nhin nng dNTP thp (10100mM) Taq ADN polimerase hot ng chnh xc hn. Hn na, nng ti
u ca chng ph thuc vo rt nhiu yu t nh: Nng Mg2+ nng cht
mi, di ca sn phm c khuch i s chu k ca phn ng.
Thnh phn ca dung dch m ph thuc vo loi enzyme polimerase
c s dng. Trong dung dch m quan trng nht l ion Mg2+ lm tng nhit
nng chy (Tm) ca ADN mch i, to ra phc cht tan vi dNTPs hnh
thnh c cht m enzyme polimerase c th nhn ra, iu ny rt cn thit cho
qu trnh lin kt ca cc dNTP. Nng Mg2+ trong hn hp phn ng cui
cng bin i t 0,5-5,5mM (nng ny c th thay i khi cn thit). Nng
ln Mg2+ qu thp s lm gim kh nng tng hp ca enzyme polimerase,
cn nu nng qu cao s to ra nhng phn on khng c hiu.
Nhn chung, nng MgCl2 c nh hng nhiu ti hiu qu v tnh c
th ca phn ng. Ngoi Mg2+ cn mt s cht khc trong dung dch m nh
AMSO, DMSO, formamide c thm vo nh cc cht ph gia nhm to ra
cc sn phm PCR c kch thc ln.
94

Mi mt chu k PCR gm 3 giai on c bn c nhit khc nhau:

(1) Giai on tch chui ADN: ADN b bin tnh t dng si kp thnh
dng si n nhit 94-950C trong khong thi gian rt ngn (khong 1-1,5
pht).

(2). Giai on gn mi: Nhit phn ng c h thp xung 400- 650C

cho php hai on mi gn vo ADN mu v tr tng ng theo nguyn tc
b sung (A - T, G - C) hai u ADN cn nhn.

(3) Giai on ko di: nhit 720C ADN Taq polimerase hot ng

ko di on ADN t on mi v hai on ADN mi c tng hp theo
nguyn tc b sung t hai u on ADN khun ban u.

Sau mt chu k gm ba giai on nh trn, mt on ADN khun c

nhn ln thnh hai, cc on ADN c nhn bn trong mi chu k li c coi
l ADN khun cho chu k nhn bn tip theo. Chnh v vy sau k chu k nhn
bn s to ra 2K on ADN ging on ADN khun ban u.

95

K thut phn ng di truyn dng polimerase l k thut tng i n

gin v phn tch mt cch nhanh chng s bin d di truyn mc phn t
ADN trong phm vi qun th v gia cc c th (Innis v cng s, 1990). PCR
l mt cng c hu hiu cho vic phn tch genome ca sinh vt, v n c kh
nng to ra mt lng ln cc trnh t ADN c hiu t bt k c th no. PCR
c s dng vo nhiu mc ch khc nhau nh xc nh trnh t trc tip ca
ADN c nhn bn, thit k cc trnh t ADN mi, nhn bn qun th ADN
lm mu lai, xc nh cc trnh t c hiu t cADN hay th vin gene, phn
tch tin ho v s a dng ADN ca qun th sinh vt hay gia cc c th. Hin
nay PCR c xem l phng php nhanh, chnh xc v tng i n gin
nh gi cy chuyn gene, phn tch mt cch nhanh chng s bin d di truyn
cp phn t ADN trong phm vi qun th v gia cc c th.

Hnh 7.2. S phn ng chui polimerase (PCR)

S hn ch ln nht ca k thut ny l phi c mt trnh t ADN nht
96

nh m t ngi ta thit k v tng hp cc on mi (on khi u cho

tng hp ADN). Tuy vy sau ny cc nh sinh hc phn t ci tin k thut
ny m khng cn thit phi bit trc trnh t chui ADN. John Welsh v
Michael Mc Clelland s dng k thut PCR vi cc on mi ngu nhin
trong vic xc nh in du genome. K thut PCR c ng dng pht hin
nhanh cc t bin im v phn tch ADN a hnh. PCR cn c ng dng
phn lp gene, phn tch sinh vt chuyn gene. Vit Nam cc nh khoa hc
thnh cng trong vic phn lp cc gene lin quan n kh nng chng chu
cy trng, nh gene chu hn cy la (L Trn Bnh v cng s, 1998), gene
chu nng u tng (Trn Phng Lin v cng s, 1999),... v hng lot cc
gene c tch v gii trnh t nucleotit.

Hnh 7.3. Hnh nh in di sn phm PCR (inh Duy Khng, 2002)

1.2. Phn ng RT-PCR (PCR ngc)
1.2.1. Nguyn l
Thc cht ca RT-PCR l phn ng nhn mt trnh t t khun ARN,
gm 2 giai on: (1) Tng hp trnh t ADN 2 si t si ARN lm khun; (2)
Trnh t ADN 2 si li lm khun thc hin phn ng PCR.
Phn ng bin i ARN thnh ADN phi nh enzyme phin m ngc
(Reverse-trancriptase) c gi l giai on chuyn ngc (RT) thc hin
nhit 500C - 550C trong 30 pht. Sau khi c sn phm ADN 2 si th phn
ng tip theo l PCR thng thng gm 3 giai on. RT-PCR c s dng
nhn ARN ca retrovirut hoc mARN tch t t bo cht.
1.2.2. Tin hnh phn ng RT- PCR
- Giai on 1 (RT): 1 chu k, thc hin sao chp ngc t ARN si n
sang ADN si kp nhit 500C- 550C, trong 30 pht.
- Giai on 2 (PCR): (1). Bin tnh nhit 940-950C /1pht; (2).Tip
hp mi 40-650C; (3). Tng hp ADN 720C/1 pht.
1.3. Kim tra sn phm PCR
Sn phm PCR cng vi ADN marker c kim tra bng phng php
in i trn gel agarose 0,8-2%. Tin hnh chp nh trn nh sng n UV
xc nh s c mt ca on ADN c nhn bi PCR.
97

Hnh 7.4. Hnh nh in di sn phm RT-PCR (inh Duy Khng, 2002)

1.4. Nhng yu t nh hng n phn ng PCR
Kt qu ca phn ng PCR chu nh hng ca cc yu t nh: ADN
khun, Taq polimerase, primer v nhit nng chy ca mi, ton Mg++,
nguyn liu l cc dNTP, nc, dung dch m,...
1.5. Cc lnh vc ng dng ca phn ng PCR
Phn ng chui trng hp PCR, t khi c Kary Mullis xut hon
thin, c nhng ng dng ht sc rng ri trong nhiu lnh vc khc nhau, c
trong khoa hc cng ngh v trong i sng x hi.
Trong nghin cu, k thut PCR gip ch hu hiu cho vic xc nh trnh
t nucleotit ca cc on ADN c nhm c th s dng PCR pht hin t
bin, PCR cho php phn tch lin kt gene t nhng t bo ring l, gip nghin
cu qu trnh tin ho mc phn t, thm ch phc hi nhng gene tn ti
cch y hng chc triu nm.
Trong chn ging vt nui v cy trng, c bit i vi ng vt v thc
vt bc cao, vn trc y vic la chn cp cha m lm ging l rt kh khn.
cn nhiu nm, th nay k thut PCR cho php thc hin ch trong vi ngy, thm
ch vi gi.
Trong y hc PCR c th chn on chnh xc cc bnh nhim trng t
virut n vi khun v cc bnh do nm, k c HIV-AIDS, chn on sm v
theo di iu tr ung th...
Trong t vn di truyn y hc PCR cho php chn on nhanh, chnh xc
cc bnh di truyn k c chn on trc sinh gii tnh v cc d tt bm sinh c
th c t khi thai mi c 8 tun tui v iu quan trng l khng cn chc i,
ch cn ly mt git mu ngoi vi ca m lm mu th nghim.
Ngy nay k thut PCR l khng th thiu trong khoa hc hnh s, gip
chn on nhanh, chnh xc th phm ch t mt vt mu kh, mt si tc hoc
mt sinh phm no m th phm li trn hin trng. Ngoi ra k thut
ny cn cho php xc nh chnh xc quan h huyt thng cha - con, ng chu... cng ch trong vi gi.
98

Trong bo v mi trng, vic xc nh mc nhim sinh hc c th

thc hin rt hiu qu v nhanh chng bng k thut PCR.
Nhng ng dng thc tin a dng ca phn ng chui trng hp PCR
tht v cng tn. Nu nh nm 1985 mi c 3 cng trnh c cng b v PCR
th 5 nm sau k thut ny c s dng trong hng nghn phng th nghim
trn khp th gii. Ngay nc ta, k thut PCR cng v ang c dng
trong nhiu trng i hc, vin nghin cu v ngy cng tr nn ph bin.
Phn ng chui trng hp tht s a li mt cuc cch mng trong lnh vc
ng dng thc t ca di truyn hc phn t.
Tm li, nhng ng dng c bn ca PCR c th tm tt nh sau:
- Sn xut mu d.
- Khuch i s lng cc on ADN v ARN.
- nh lng bng PCR.
- Phn tch t bin.
- Phn tch ADN a hnh.
- Phn lp gene v tch dng gene.
- Phn tch sinh vt chuyn gene.

2. PHN TCH TNH A HNH CA ADN C NHN BN NGU

NHIN (RANDQM AMPLIFIED PQLIMORPHIC DAN - RAPD)
Williams v cng s (1990) xc nh hin tng a hnh ca phn t
ADN da trn phn ng khuch i phn on ADN ngu nhin bng cc on
mi c trnh t ADN tu . K thut ny c gi l k thut khuch i ngu
nhin cc on ADN a hnh (RAPD technology).
Hin tng a hnh cc on ADN khuch i ngu nhin xut hin l do
c s bin i trnh t nucleotit ti v tr cc on mi lin kt (chng hn t
bin im) hoc do bin i cc nhim sc th trong khu vc gene khuch i
(lng on, mt on). Phng php ny n gin v mt k thut, tin hnh
nhanh chng v ch cn mt lng rt t phn t ADN (nanogram). Cc on
ADN a hnh c khuch i l mt loi ch th phn t c th m chng c
th pht hin hin tng a hnh gia cc c th khc nhau mc phn t
ADN.
Cc mi c trnh t nucleotit ngn (10 nucleotit) lin kt b tr vi nhiu
locus khc nhau v c kh nng khuch i nhiu on ADN khc nhau (t 2
99

n 10 on ADN khc nhau) trong mt phn ng dy truyn dng enzym

polimeraza. K thut khuch i ngu nhin cc on ADN a hnh c s
dng rng ri trong nhng nm gn y phn tch di truyn cc h thng sinh
hc. K thut ny t ra rt hu ch c bit trong cc trng hp ni m khng
c sn cc ch th ng enzyme (isozyme) hoc cc nhn t nh du RFLP.

Hnh 7.5. S phng php RAPD

(theo CIMMYT, 1997 v inh Th Phng, 2001)

Cc yu t cn thit tin hnh phn ng RAPD bao gm: (1) ADN

khun mu (DNA template) vi nng 5-50ng trong 25 l; (2) on mi
(primer): ch s dng mt mi l mt oligonucleotit c trt t nucleotit ngu
nhin v c chiu di khong 10 nucleotit, trong C+G chim 50-80%;
(3)ADN-polimeraza (Taq polimeraza): hot ng ca Taq polimeraza ph thuc
vo Mg2+, nng dNTP, pH, nhit bin tnh ADN; (4) Bn loi
deoxynucleotit triphotphat (dNTP): ATP, TTP, GTP: CTP; (5) Ion Mg2+.
100

Phn ng RAPD c tin hnh qua cc giai on ging nh PCR:

- Giai on bin tnh ADN: nhit 950C trong khong 30-60 giy lm
cho hai mch khun ADN tch nhau.
- Giai on tip hp mi: Khi nhit h xung 32-400C mi tip hp v
bm vo si ADN khun.
- Giai on tng hp: Nhit c nng ln 720C th cc on mi
bt cp vi cc mch n s c ko di vi s tham gia ca Taq polimeraza.
Sau mt chu k gm 3 giai on nh trn, mt on ADN khun c
nhn ln thnh hai, cc on ADN c nhn bn trong mi chu k li c coi
l ADN khun cho chu k nhn bn tip theo. Vy sau k chu k nhn bn s to
ra 2k cc on ADN ging on ADN khun ban u. RAPD c th thc hin t
40-45 chu k.
K thut ny khc vi k thut PCR ch s dng cc mi ngu nhin, s
chu k nhiu hn v giai on 2 nhit h thp xung 32-360C, khng i
hi thng tin v trnh t ADN c nhn bn.
Da trn s xut hin hay bin mt ca cc phn on ADN khi in di
sn phm RAPD c quan st thy cc c th khc nhau v c nh gi
theo quy c 1 = xut hin v 0 = khng xut hin. Mt bng gm cc gi tr 0
v 1 c thit lp t cc c th nghin cu s cho php tnh ra h s tng
ng di truyn ca cc cp theo Nei v Li.
Trong qu trnh thit lp mi quan h gia cc loi hay nhm loi, mt s
tc gi (Apostol v CS, 1993) xy dng k thut phn nhm thng qua cc
biu RAPD (RAPD Lot). Thc cht k thut ny gm 3 bc:
Bc 1: So snh tng cp i tng trong nghin cu bng cch tnh ton
khong cch quan h gia chng.
Bc 2: Lp mt ma trn gm tt c nhng gi tr tnh ton c trc
.
Bc 3: Gii ma trn v biu din thnh mt biu c trng.
Ngy nay, cc nh nghin cu thit lp c phn mm my tnh t
ng v nn biu mi quan h hay tng ng di truyn ca cc i tng
nghin cu sau khi nhp d liu v cc phn on c nhn bn ca cc c th.
NTSYS pc version 2.0 (Applied Biostatistics lc., USA., 1998) l tn ca mt
chng trnh thuc kiu trn lp ra biu hnh cy Biu hnh cy thu
c s th hin mc gn nhau ca cc c th cho php nh gi c mi
quan h di truyn gia cc c th c nghin cu. Chng trnh ny cho php
gim bt thi gian nghin cu v c chnh xc cao nn n l mt phn mm
101

c hiu qu trong vic phn tch k thut RAPD.

i vi a s thc vt, cc on mi di khong 10 nucleotit vi 60% GC
s to ra 2-10 sn phm c nhn bn. Hin tng a hnh cc on ADN c
nhn bn ngu nhin xut hin l do c s bin i trnh t nucleotit ti v tr cc
on mi lin kt. V vy cc a hnh thng c nhn ra do c mt hay vng
mt ca mt sn phm nhn bn t mt locut. Phng php ny n gin, nhanh
chng, ch cn mt lng nh (nanogram) phn t ADN c ngun gc t h
gen. Cc on ADN a hnh c nhn bn l mt loi ch th phn t c th
m chng ta c th pht hin hin tng a hnh gia cc c th khc nhau
mc phn t ADN. K thut RAPD c s dng rng ri trong nhng nm
gn y phn tch di truyn h thng sinh hc. N l phng php hiu qu
trong vic xc nh kiu gen, phn tch qun th v ngun gc loi, nghin cu
di truyn v lp bn di truyn.
Ngi ta p dng thnh cng k thut khuch i ngu nhin cc on
ADN a hnh vo nhiu lnh vc khc nhau, nh lp bn gen, in du vn
ADN, chn ging...
Lp bn gene (Gene Mapping)
Cc nhn t RAPD cng phn li theo quy lut Mendel v c th c s
dng nh cc gene nh du di truyn trong vic thit lp cc bn phn t
(Williams v cng s, 1990; Foolad v cng s, 1993). So vi k thut phn tch
hin tng a hnh di cc phn on ADN (RFLF Technology) th k thut
ny c mt s u im ni bt l: c th pht hin hin tng a hnh trong phn
t ADN trn mt phm vi rng ln hn; tnh n gin v nhanh gn; cn t
lng ADN. K thut ny c p dng thnh cng vo nhiu loi cy trng
khc nhau thit lp cc bn di truyn phn t nh Brassica (Quyros v
cng s 1991), Arabidopsis thaliana (Reiter v cng s, 1992) c chua (Klein
Lankhorst v cng s, 1991; Foolad v cng s, 1993)...
In du vn ADN(ADN Fingerprinting) xc nh bn ging mc
phn t v tnh a dng sinh hc.

102

Hnh 7.6. Hnh nh in di sn phm RAPD u tng v u xanh

(1. ADN marker, 2, 3, 4, 5, 6, 7, 8: Sn phm RAPD)

Hnh 7.7. Quy trnh s dng k thut phn tch RAPD

Vic d dng pht hin hin tng a hnh trong phn t ADN bng cch
103

s dng tc nhn t RAPD lm cho k thut ny tr hp dn trong nghin cu

nhn dng cc c th, chn on cc bnh di truyn, phn loi thc vt v tin
ho mc phn t, cc mi quan h pht sinh chng loi trong gii tnh.
Trong phm vi gii thc vt k thut khuch i ngu nhin cc on ADN a
hnh c s dng nhn dng cc ging cy trng (cy hoa l - Hu v
Quyros, 1991; cy cn ty - Yang v Quyros, 1993), pht hin mi quan h pht
sinh chng loi (bo hoa du - Van Coppennolle v cng s, 1993; Scroch v
cng s, 1992; ng cc - Dweikat v cng s, 1993), nh gi ging v s a
dng di truyn trong tp on ging cy trng (la m - Vierling v Nguyen,
1992; Caetano - Anolles v cng s, 1991). Zheng v cng s (1991) khi s
dng hai on mi khc nhau pht hin cc ch th phn t RAPD trong tp
on 19 ging la v ch ra tnh hu hiu ln lao ca cc ch th phn t trong
vic nhn dng ging.

Hnh 7.8. Hnh nh in di sn phm RAPD lc (a) v la cn (b)

Fukuoka v cng s (1992) xc nh cc on ADN a hnh c
khuch i ngu nhin 16 ging la khc nhau v cho rng chng khc bit
nhau bi t nht l mt bng ADN a hnh v phn b trong ba nhm la khc
nhau tng ng vi cc loi ph Japonica, Javanica v Indica. L. X. Yu v H. T.
Nguyen (1993) nghin cu s bin d di truyn ca cc ging la cn thng
qua s dng ti 42 on d ADN khc nhau. Cc tc gi thu c 260 on
gene khuch i khc nhau, trong s th 208 on gene (tc l 80%) th hin
s a hnh v xc nh mi quan h di truyn gia cc ging la .
David. J. Mackill (1995) cng bng k thut ny thnh cng xc nh
s a dng di truyn ca 134 ging la c ngun gc khc nhau v ch ra rng
s bin d di truyn ca cc nhn t RAPD l rt quan trng b sung thm cho
vic lp bn gene v in du vn ADN.
Dn nhn gene (tagging Genes)
K thut khuch i ngu nhin cc on ADN a hnh cng c s
104

dng mt cch thnh cng nh du (dn nhn) cc gene iu khin cc tnh

trng quan trng ca cy trng, chng hn nh gene quy nh tnh chng chu
bnh m vi khun cy c chua (Martin v cng s, 1991), gene quy nh kh
nng chng bnh giun trn cy c chua (Klein - Lankhorst v cng s, 1991),
gene chng bnh g st.
K thut RAPD cn c ng dng trong chn ging. Vic xc nh cc
ch th RAPD c trng. lm c s cho chn ging gin tip. Cn c vo sn
phm in di RAPD ngi ta c th thit lp bng thng k s c mt cc phn
on ADN c nhn (s 1 : c mt on ADN; s 0: on ADN khng c
nhn). Trn c s xc nh dc h s ng dng di truyn gia cc i
tng nghin cu, lp s ph h (cy pht sinh). T kt qu s xc nh
c s sai khc v b gene ca cc i tng nghin cu.
Bng 7.1. Cc phn on ADN c nhn bn ngu nhin trong phn ng
RAPD
Cc on
mi

RA 31

c lng
kch thc
(kb)

on
c
nhn

8,0
7,0
6,0
5,1
5,0
4,9
4,8
2,5
2,0
1,9
1,5
1,49
1,48
1,43
1,40
1,38
1,35
1,33
1,2
0,98
0,83
0,60

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
17
20
21
22

Cc dng t bin v ging gc

Ging
gc ML81 ML10 ML61 ML70 ML59 ML48
1
1
0
0
0
1
1
1
1
0
0
0
1
1
1
1
0
0
0
1
1
1
1
0
0
0
1
1
0
0
1
1
1
0
0
1
1
1
1
1
1
1
0
0
1
1
1
0
0
0
0
1
0
1
0
1
0
0
1
0
1
0
0
0
0
1
0
1
0
0
1
1
1
1
1
1
1
0
0
0
1
0
0
0
0
0
0
1
0
1
1
1
1
1
0
1
0
0
0
0
1
0
1
0
0
0
0
1
0
1
0
0
0
0
1
0
1
0
0
0
1
1
1
0
1
1
0
1
0
1
0
0
1
0
0
1
0
1
1
0
0
0
1
0
1
0
0
0
1
0
0
0
1
1

105

Bng 7.2. H s ng dng di truyn ca cc dng u tng

Dng B
v G.gc

Ging gc
Lng Sn

G.Gc

1.0000000

ML81

0.7954545

1.000000

ML10

0.5000000

0.5681818

1.000000

ML61

0.5681818

0.7272727

ML70

0.5909091

0.4772727

ML59

0.7954545

0.7727273

0.704545 1.000000
0
0.772727 0.522727 1.000000
3
3
0
0.431818 0.690909 0.522727 1.000000

ML48

0.7500000

0.8636364

ML81

ML10

ML61

ML70

ML59

ML48

0.477272 0.691818 0.522727 0.818181 1.00000

2
3
8
00

Hnh 7.9. S ph h ca cc dng u tng t bin chn lc t ging gc

Cc v tinh cng nh ch th phn t khc nh a hnh di cc phn
on ADN (RFLP), cc on ADN a hnh khuch i ngu nhin (RAPD)
c s dng rng ri thit lp bn phn t chi tit v nh v cc gene
iu khin cc tnh trng gi tr cy u tng (Keim v cng s, 1990; Diers
v cng s, 1992; Yu v cng s, 1994). Shoemaker and Specht (1995) thng
bo sp nhp cc nhm lin kt c in vi cc nhm lin kt di truyn phn t
cy u tng bng cch s dng 20 1ocus c in, 8 1ocus ch th phn t
RAPD v 110 1ocus ch th phn t RFLP.

4. PHN TCH TNH A HNH CHIU DI CC PHN ON ADN

C NHN BN C CHN LC
(Amplified Fragment Length Polimorphism - AFLP)
Zabeau v Vos (1993), Vos v cng s (1995) a ra v m t mt k
thut mi v in du vn ADN. l k thut khuch i chn lc cc on ADN
a hnh (AFLP - Technology). K thut ny da trn c s siu sao chp chn
106

lc cc on ADN a hnh t h gene. K thut khuch i c chn lc cc on

ADN hin nay l mt k thut c sc mnh ln v y tim nng v in du vn
ADN. N to ra mt cch nhanh chng nht, hiu qu nht mt bn di truyn
dy c cc ch th phn t nhm chn lc cc tnh trng mong mun thng qua
chn ging nh cc ch th phn t ng thi l mt cng c l tng i vi
vic xc nh v nh gi cy trng mc phn t ADN. Cc nhn t AFLP
l nhng ch th phn t, nhanh chng c p dng vo vic nghin cu di
truyn, lp bn gene, dn nhn gene quan trng nhiu loi thc vt khc
nhau nh khoai ty (Ballvoro v cng s, 1995; Folkertsma v cng s, 1996),
la mch (Becker v cng s, 1995). K thut phn tch ADN a hnh c chn
lc (hay l k thut khuch i chn lc cc on ADN a hnh - AFLP) gm 3
bc c bn sau y:
(1) Phn ct phn t ADN bng enzyme gii hn tip theo l gn kt cht
g (adapter).
(2) Phn ng chui s dng enzyme polimerase khuch i cc on
ADN.
(3) Phn tch cc on ADN trn gel poliacrylamid.
Do c s phi hp cc loi enzyme, gii hn khc nhau vi nucleotit chn
lc ca cht mi nn k thut sinh hc phn t hin i ny cho php to ra
nhiu cc ch th phn t hn so vi cc phng php trc .
Bng cc nhn t RFLP v AFLP K. Meksem v cng s (1995) trong
chin lc phn lp cc din chng chu bnh sng mai (Phytophthora
Infestans) cy khoai ty thnh cng thit lp bn gene c phn gii
cao v vng ph cn locus R1 trn nhim sc th s 5. Cc tc gi gp phn
chn lc mt cch gin tip cc ging khoai ty c kh nng chng bnh sng
mai thng qua chn ging nh cc ch th phn t (Marker Assisted Selection MAS).
D. J. Mackill (1996) s dng cc ch th phn t AFLP, RAPD v trnh
t nucleotit lp li n gin (Microsatellite) nghin cu mc a hnh trn
14 ging la khc nhau v cho rng cc ch th phn t AFLP v trnh t
nucleotit lp li n gin c th c xem nh l cc ch th phn t b sung cho
vic lp bn gene nhiu loi cy trng ni chung cng nh cy la ni
ring, ng thi cc ch th phn t ny cng t ra thch hp cho vic nghin cu
bn cc locus iu khin tnh trng s lng thng qua con lai bt ngun t 2
loi ph Japonica v Indica. Ngoi ra cc ch th phn t AFLP tr nn cn thit
i vi vic lp bn gene c cht lng cao ni m cn phi lm bo ho mt
107

on nhim sc th c hiu vi nhiu s lng cc ch th phn t (Thomas v

cng s, 1995).
S khm ph ra cc i phn t axit nucleic v protein - c s vt cht ch
yu ca s sng v nghin cu ng dng vo thc tin bng cc k thut hin i
trn c s s pht trin ca sinh hc phn t. Trong cng ngh sinh hc thc vt.
vic phn loi genome trong t bo thc vt v mi quan h gia chng lm
sng t vai tr vt liu di truyn thc vt, nhng hiu bit v axit nucleic v h
gene trong t bo thc vt l nn tng cho cc k thut sinh hc phn t.
Cc k thut sinh hc phn t c ng dng trong phn tch genome thc
vt. Trong cc k thut sinh hc phn t ng dng vo chn to ging cy trng
th cc k thut PCR v RAPD c th c ng dng rng ri v, hiu qu trong
iu kin Vit Nam. Bng k thut RAPD, da vo sn phm in di lp bng
thng k cc phn on ADN c nhn bn, t thit lp biu so snh s
khc nhau mc phn t gia cc dng v cc ging vi nhau. Nhng kt qu
bc u v ng dng k thut PCR v RAPD cho php khng nh hiu qu ca
s kt hp phng php chn ging truyn thng vi nhng phng php sinh
hc phn t hin di trong to ging cy trng, vt nui.

THO LUN
1. Trnh by c s, nguyn tc ca phn ng chui PCR v nhng ng dng
ca PCR.
2. PCR trong phn tch h gene ca sinh vt v trong cng tc chn ging.
3. RAPD l g? Phng php xc nh du chun RAPD v quan h di
truyn gia cc i tng nghin cu bng phng php RAPD.
4. Th no l AFLP? Cc bc ca k thut AFLP. ng dng ca AFLP.

108

Chng 8
T BO CH V VECTOR
Tm tt: Trong chng ny, chng ta s bn n kha cnh sinh hc ca vic
tch dng gene bao gm vic s dng cc phn t vector v h thng sng thch
hp, tc vt ch m vector c th c nhn ln. Cc loi t bo ch s
c m t u tin, tip theo l cc h thng vector v cc phng php dng
a ADN vo cc t bo. T bo ch v vector l hai i tng cc k quan
trng trong k thut tch dng. C hai loi t bo ch, l: T bo ch nhn
s, T bo ch nhn chun v E. coli l vt ch c s dng nhiu nht trong
k thut tch dng. Vector tch dng c phn bit vi vector chuyn gene
mt s c im c bn. C nhiu loi vector c s dng trong k thut di
truyn, nhng c s dng rng ri nht l plasmid.
Ni dung ca chng gm 3 vn chnh l: (1) T bo ch; (2). Vector, (3).
Phng php chuyn vector ti t hp vo t bo ch.

1. T BO CH
Loi t bo ch s dng trong mt ng dng c th ch yu ph thuc vo
mc ch ca th nghim tch dng. Nu mc ch l tch ra mt gene
phn tch cu trc th yu cu dy ch l mt h thng n gin, d s dng.
Nu mc ch l biu hin thng tin di truyn mt sinh vt nhn chun bc
cao, chng hn thc vt, th i hi phi c mt h thng c th hn. Thng
th mt vt ch ban u n gin c s dng tch mt on trnh t m
on ny sau c a vo mt h thng phc tp hn biu hin. Cc loi
t bo ch yu c nu trn bng 8.1.
1.1. Cc vt ch nhn s
Mt t bo ch l tng l mt t bo d nui cy v d nhn ging, d thu
nhn c nhiu loi vector. Vi khun E. coli p ng cc yu cu ny v c
s dng trong rt nhiu k thut tch dng. E. coli c nghin cu rt t m, rt
nhiu loi khc nhau c cc nh di truyn hc vi sinh vt phn lp khi
nghin cu cc c ch di truyn ca sinh vt nhn s ny. Cc nghin cu
cung cp nhng thng tin khoa hc gi tr lm c s cho k thut di truyn.

109

Bng 8.1. Cc loi t bo ch dng trong k thut di truyn

Vi khun Nhn s

Gram m

Escherichia coli

Gram dng

Bacillus subtilis
Streptomyces spp.

Nm

Nhn chun Vi sinh vt

C khun li

Thc vt Nhn chun T bo trn

saccharomces cerevisiae
Aspergillus nidulans
Cc loi khc nhau

T bo nguyn

Cc loi khc nhau

C c th

Cc loi khc nhau

ng vt Nhn chun T bo su b

Drosophiia melanogaster

T bo ng vt Cc loi khc nhau

T bo trng

Cc loi khc nhau

C c th

Cc loi khc nhau

E. coli l vi khun gram m c mt nhim sc th ring l nm trong mt

cu trc gi l vng nhn (nucleoit). Kch thc ca h gene vo khong 4 x 106
bp. Cc qu trnh biu hin gene (phin m v dch m) i i vi nhau, sinh ra
si mARN c tng hp mi v c s dng ngay cho qu trnh dch m. V
vy, E. coli c th c coi l mt trong nhng t bo vt ch n gin nht, cho
nn cc th nghim tch dng gene u s dng E. coli lm vt ch vi nhiu ni
khc nhau v mt di truyn v cho nhng ng dng c bit.
Ngoi E. coli, cc vi khun khc cng c dng lm vt ch cho cc th
nghim tch dng gene, chng hn: Bacillus, Pseudomonas v Streptomyces.
Cc loi vi khun ny c nhng hn ch nht nh i vi cc vt ch, l c
rt t vector thch hp s dng trong cc t bo v a ADN ti t hp vo
cc t bo c th gp kh khn, c bit kh khn khi di mt vi cc th
nghim tch dng ban u.
1.2. Cc vt ch nhn chun
Mt trong nhng nhc im s dng E. coli lm vt ch tch dng l
l sinh vt nhn s khng c mng nhn nh cc t bo nhn chun. Cc t
bo nhn chun c ph tn ti rt rng t cc vi sinh vt nh nm men v to
cho n cc t bo ca cc sinh vt a bo phc tp, nh con ngi chng hn.
Cc t bo vi sinh vt nhn chun c nhiu c tnh ca vi khun nh d nhn
ging, d to ra cc t bin. Trong khi cc sinh vt nhn chun bc cao gy ra
hng lot kh khn i hi phi c nhng cch gii quyt c hiu.
110

Nm men Saccharomyces cerevisiae l mt vi sinh vt nhn chun c

a thch nghin cu k thut di truyn. N c s dng nhiu th k trong
sn xut bnh m v bia, v c nghin cu rt tch cc. Sinh vt ny c th s
dng d dng phn tch di truyn hc kinh in, chng c nhiu loi t bo
t bin. H gene ca S. cerevisaae c khon 1,35 x 107; baz nit, nhiu hn
E.coli khong 3,5 ln. Cc nm khc cng c dng trong cc th nghim tch
dng gene l Aspergillus nidulans v Neurospora crassa.
Cc t bo thc vt v ng vt cng c th c s dng nh cc vt ch
trong cc th nghim thao tc gene. Cc t bo thc vt v ng vt khc thng
c nui cy trong cc mi trng thch hp v iu kin ny, s dng
trong cc th nghim thao tc gene so vi cc t bo trong c th.

2. VECTOR
2.1. Khi nim
thu nhn gene di dng tinh sch vi hm lng ln ngi ta phi
tch dng (clone) gene . Vic tch dng c bn l tin hnh gn trnh t ADN
vo vector. Vector cng l mt ADN dng vng, c kh nng xm nhp vo t
bo vi khun v mn t bo vi khun to ra nhng bn sao khc ging ht
vector ban u.

Hnh 8.1. S plasmid

Vector c cc ng dng ch yu sau:
- To dng nhm khuch i s lng bn sao mt trnh t ADN xc nh.
- To sinh vt chuyn gene (chuyn cc gene vo t bo hay c th).
- Sn xut ARN vi s lng ln t ADN c to dng.
- Sn xut protein vi s lng ln t ADN c to dng.
c tnh cn c ca mt vector:
111

- Vector c kh nng t sao chp tch cc trong t bo vt ch. c lp

vi s sao chp ca b gene vt ch.
- Vector c kch thc cng nh cng tt.
- Vector phi c pht hin r rng trong vt ch.
- Phi tn ti trong t bo ch qua nhiu th h.
- Vector ch tn ti mt v tr nhn bit cho mi enzyme.
Vector c s dng trong k thut tch dng phn t ADN i vi t bo
E. coli: (1) Plasmids; (2) Bacteriophage l, (3) Cosmids; (4) Bactenophage M13;
(5) Phagemids v mt s loi vector c bit khc nh: (i) Ti plasmid; (ii)
YACS; (iii) BACS; (iv) Retrovirus; (iiv) Baculovirus. (iiiv) Yeast 2 micron
plasmid.
Vic la chn s dng mt loi vector da vo kch thc ca on ADN
cn to dng v mc ch to dng.
Vector c s dng vi mc ch tch dng gene (vector tch dng) v
chuyn gene (vector chuyn gene).

Hnh 8.2. Plasmid pBR322 (a) v plasmid pUC18 (b)

(theo Nng Vn H, 2002)
2.2. Cc vector plasmid dng cho E. coli
C mt s c im quan trng m cc vect cn phi c, chng phi
nhng phn t ADN nh gip cho vic phn tch v bo qun chng d dng.
Phi c im khi u sao chp (origin of replication), cho ADN n c
nhn ln v do vy c duy tr trong qun th t bo khi sinh vt ch sinh
trng v phn chia. Cn c mt loi du chun, chn lc (selectable marker)
to iu kin cho vector c d dng pht hin v cng cn phi c t nht mt
im ct bi enzyme gii hn to iu kin cho ADN xen vo trong qu trnh
to ra cc th ti t hp. Cc plamid c cc c im ph hp vi yu cu trn
v chng c s dng thng xuyn nh cc vector trong cc th nghim tch
dng.
2.2.1. Plasmid
112

Nhiu loi plasmid c tm thy trong t nhin cc vi khun v trong

mt s nm men. Chng l nhng phn t AND mch vng tng i nh so
vi nhim sc th ca t bo vt ch. Cc plasmid tn ti ch yu trng thi
ngoi nhim sc th. Plasmid cos kh nng truyn cc tnh trng cho vt ch
(chng hn: tnh bn vng vi khng sinh), do vy gip cho vic la chn d
dng trong nhng iu kin xc nh. Cc gene bn vng vi khng sinh do
AND plasmid m ha thng c s dng trong vic thit k cc vector dng
cho k thut di truyn v chng cung cp mt phng tin thun li la chn
cc t bo c mang plasmid.
Cc plasmid c th phn chia thnh 2 nhm: tip hp v khng tip hp.
Cc plasmid tip hp c th t truyn t vi khun ny sang vi khun khc thng
qua qu trnh tip hp. Qu trnh ny i hi cc chc nng ca cc on c th
tra (transfer) v mob (mobilising) nm trn plasmid. Cc plasmid khng tip hp
th khng t truyn i c. Mt cch phn loi khc da trn s lng cc bn
sao ca plasmid c trong t bo ch. Cc plasmid c s bn sao thp c khuynh
hng kim sot cht ch vic sao chp ADN, thng ADN plasmid loi ny
phi sao chp bt buc cng vi s sao chp ADN nhim sc th ca vt ch.
Cc plasmid c s bn sao cao gi lo cc plasmid thoi mi, vic sao chp
ADN ca chng khng ph thuc vo s sao chp ADN nhim sc th ca t
bo ch. Ni chung, cc plasmid tip hp thng ln, c c ch kim sot cht
ch vic sao chp ADN v tn ti trong t bo vi s lng bn sao thp. Trong
khi , cc plasmid khng tip hp thng nh, sao chp ADN mt cch thoi
mi v tn ti trong t bo vi s bn sao cao.
2.2.2. Cc plasmid c bn dng tch dng
Trong k thut di truyn, cc plasmid gp trong t nhin thng c sa
i nhiu to ra cc vector c c im mong mun. t tn plasmid, ch
p c dng ch plasmid v tip theo thng l tn ca nhng ngi
phn lp hoc thit k plasmid. Cc con s cng c th c s dng phn
loi cc chng c th. Mt trong nhng plasmid c s dng rng ri l
pBR322, n c hon thin bi Francisco Bolivar v ng nghip. Vic thit
k pBR322 bao gm mt lot nhng thao tc c c nhng on ADN cn
thit cng ni vi nhau vi kt qu cui cng l mt ADN c 3 ngun gc.
Plasmid ny c tt c cc c tnh ca mt vector tt, chng hn nh: trng
lng phn t thp, c cc gene khng khng sinh, c im khi u sao chp,
v c mt s im ct ring bit bi cc enzyme gii hn.
C mt s plasmid trong nhm pBR mang cc c im khc nhau cht t.
Tuy nhin, plasmid bt ngun t pBR322 c s dng rng ri, v chng c
113

mt s c im u vit so vi plasmid ban u. V d: plasmid pAT153, l mt

dn xut mt on ca pBR322. Plasmid ny c to ra bng cc loi b 2
on ADN t pBR322 sau khi dng enzym gii hn Hael. Lng ADN b loi
b rt nh (705 cp baz), nhng hiu qu lm tng s bn sao ln 3 ln v
loi b cc on trnh t cn thit cho qu trnh huy ng (mobilisation). Nh
vy, pAT153 v mt s phng din l vector "tt hn" pBR322, v n c nhiu
bn sao hn trong t bo v n c mc bn vng sinh hc cao hn do mt
kh nng huy ng.

Hnh 8.3. Vector pBluescript (theo Nng Vn Hi, 2002)

S c mt ca 2 gene khng khng sinh (Apr v Tcr) cho php la chn
cc t bo c mang plasmid, v cc t bo s khng vi c ampicilin v
tetracylin. Mt u vit na l cc im ct gii hn duy nht nm trong cc gene
khng sinh cho php la chn c cc ADN tch dng, v do vy c gi l
bt hot do xen on (insertional inactivation), khi m on ADN xen vo lm
gin on on m ho ca gene khng v v vy lm thay i kiu hnh ca t
bo mang ADN ti t hp.
2.2.3. Cc vectorplasmid khc
Mc d cc plasmid pBR322 v pAT153 c s dng rng ri trong
nhiu ng dng ca tch dng gene, cn c cc vector plasmid khc cng c
s dng. Ni chung, chng dc thit k sao cho c c nhng c tnh c th,
khng thy c trong cc vector n gin. Chng c th mang nhng khi im
c hiu biu hin cc gene xen vo hoc chng c th c nhng u vit khc
nh to ra kh nng chn lc trc tip cc th ti t hp. Mt nhm cc vector
plasmid cng rt ph bin l cc plasmid thuc h PUC. Cc plasmid ny c
114

mt on ngn cha mt s cc im ring l b ct bi enzyme gii hn. on

ny gi l on a lin kt (polilinker) hoc im a tch dng (multiple cloning
site), chng rt hu hiu bi v c th la chn im ct mong mun xen
on ADN trong qu trnh to ra ADN ti t hp. Bn ca mt trong cc
vector pUC c cc im ct gii hn nm trong vng polilinker, cc plasmid
pUC cn c vng mang gene - blaceosidaza, gene ny m ho - peptit. on
trnh t ny c cha vng polilinker, v vic mt on ADN vo mt trong
nhng im tch dng s dn n mt - peptit khng hot ng chc nng.
Vic t c s cho mt phng php sng lc trc tip cc th ti t hp rt
hu hiu s dng c cht to mu X-gal. Mc d cc vector plasmid c nhiu
tnh cht hu hiu v chng rt quan trng cho vic thao tc gene, nhng chng
vn mang hng lot nhc im.

Hnh 8.4. To vector ti t hp (theo Nng Vn Hi, 2002)

2.3. Cc vector phage dng cho E. coli
Mc d cc vector da trn c s phage v nhiu phng din c hiu
hn cc vector plasmid, nhng v thc cht chng thc hin cng mt chc
nng, c ngha l chng hot ng nh cc phn t mang cc on ADN. C 2
loi phage( v M13) c hon thin tch cc cho cc mc ch tch dng.
2.3.1. Phage
Trong nhng nm 40, Max Delbruck v "nhm phage" do ng thnh lp
115

 t nn mng cho sinh hc phn t hin i bng cch nghin cu cc phage

l nhng "th n khun", v l cc virut ch nhn ln c bn trong vi
khun. V chng c kch thc rt nh nn ngi ta cn gi chng l ht
(particle).
V mt cu trc, cc phage chia thnh 3 nhm chnh: (1) khng ui;(2)
c u v ui, v (3) c lng. Vt cht di truyn c th l ADN mch n,
mch kp, hoc ARN. Thng thy l cc ADN mch kp (DdsDN) (doublestranded ADN). cc phage khng ui v c ui, h gene c bao bc trong
v protein gi l capsid (i khi c bit n nh v hoc u ca phage.
Trong cc phage, dsADN in hnh h gene l nhng h gene bao gm khong
50% ton b khi lng ca ht phage. Nh vy, phage l nhng h thng tng
i n gin nu so snh vi vi khun, v nguyn nhn ny chng c s dng
rt rng ri nh cc m hnh nghin cu s biu hin ca gene.

Hnh 8.5. Hnh nh phage

Cc phage c th phn thnh phage c (virulent) v phage n ho
(temperate), tu thuc vo chu trnh sng ca chng. Khi mt phage chui vo
trong mt t bo vi khun, n c th sinh ra nhiu phage v git cht t bo ,
l chu trnh sinh tan (litic) hoc n c th nhp vo nhim sc th v li
m khng git cht t bo, l chu trnh tim tan (lisogeneic). Phage c l
phage ch c chu trnh sinh tan. Phage n ho l phage c chu trnh tim tan,
nhng cng c th c phn ng sinh tan trong nhng iu kin thch hp.
H gene ca phage c chiu di 48,5kb, m ho cho 46 gene. Ton b
h gene c gii trnh t, v tt c cc im iu ho u c bit.
hai u ca h gene mch di c cc on ngn (12bp) si n, chng b tr cho
nhau. Cc on ny l nhng "u dnh", chng to iu kin cho h gene tr
thnh mch vng sau khi nhim vo vi khun. on ca h gene c sinh ra
bng cch gn cc u dnh gi l im cos (cos site).

116

Hnh 8.6. H gene ca Phage

Qu trnh xm nhim ca phage vo vi khun bt u bng vic phage
bm vo dinh dng im nhn trn b mt vi khun. Khi phage b hp th th
ADN c tim vo bn trong t bo v chu trnh sng c th bt u. H gene
tr thnh mch vng v phage bt u chu trnh sinh tan hoc tim tan tu thuc
vo mt loi yu t nh dinh dng v trng thi trao i cht ca t bo ch v
ph thuc vo tnh a nhim (s lng phage xm nhp vi khun trong qu trnh
hp th). Nu chu trnh tim tan bt u th h gene ca phagenhp vo nhim
sc th vt ch v chng c duy tr dng prophage. Sau , n sao chp
cng vi ADN nhim sc th v c chuyn ti cc t bo th h con trong mt
dng n nh. Nu chu trnh sinh tan bt u th mt lot cc s kin phin m
s din ra, phage hon ton lm ch t bo vt ch v sinh ra nhiu bn sao ca
h gene phage cng nh cc protein cu trc. Sau cc thnh phn ny c
lp rp li hay cn gi l bc gi to thnh cc phage trng thnh, sau
chng thot ra khi t bo ch khi t bo ny b tan.
xc nh s lng cc phage c trong dch tan, ngi ta pha long
nhiu ln dch phage v trn ln vi mt s lng d tha vi khun ch th ri
cy ln mt thch. Sau khi , vi khun s mc v to thnh ci gi l thm vi
khun. Phage mc trn thm ny s lm cho cc t bo b phage xm nhim tiu
tan, to nn cc vt tan (plaque) trn thm. Sau , ngi ta m s lng cc
vt tan v xc nh c s lng phage trong dch huyn ph ban u. Vt tan
c th c ly ra cho mc v phn tch tip. Phage c th nhn ln trong dch
lng bng cch cho nhim ging ang mc ca t bo ch v cho n khi cc
t bo b tan hon ton, s lng cc ht phage sinh ra ph thuc vo a
nhim v vo trng thi ca chu trnh sinh sn vi khun m chng nhim vo.
Khi ADN chui vo t bo th n chuyn sang phn t mch i gi l
dng sao chp (RF) (replicative from), n sao chp cho n khi c khong 1000
bn sao trong t bo. Ti thi im ny, vic sao chp ADN tr nn bt i xng
(asymmetric) v cc bn sao mch n ca h gene c sinh ra v thot khi t
bo nh cc ht M13. Vi khun khng b tan v vn cn sng trong sut qu
trnh ny, mc d qu trnh sinh sn v phn chia c chm hn so vi cc t bo
khng b xm nhim.
117

2.3.2. Cc vector da trn cs phage

Vic s dng phage lm vector tch dng ph thuc vo mt thc t l
khng phi tt c h gene ca u cn thit cho phage hot ng chc nng.
Do vy, c mt phm vi a ADN ngoi lai vo, mc d mt s i hi nht
nh cn phi c trong qu trnh hon thin cc vector tch dng da trn c s
phage . Mt l s sp xp cc gene trn h gene s quyt nh phn no cn
phi loi b i hoc cn phi c thay th a ADN ngoi lai vo. Vng
trung tm ca h gene nm gia cc v tr 20 v 35 trn bn , l mt on
ln c th b qua m khng cn c s sp xp li invitro mt cch phc tp i
vi h gene. Vng trung tm ny ch yu kim sot cc tnh cht tim tan ca
phage, v phn ln vng c th ct b m khng lm suy yu cc chc nng
cn thit cho chu trnh sinh tan. Hai l, phage kiu di ni chung c nhiu
im nhn bit ca cc enzyme gii hn thng c s dng trong k thut
tch dng. iu c th l mt kh khn ln, v n gii hn s la chn cc
im xen ADN vo. Trong thc t s tng i d dng la chn i vi
phage c s lng gii hn cc im b ct bi cc enzyme gii hn c th, v k
thut gy t bin in vitro c th c s dng sa i cc im cn li - tc
cc im khng cn thit. Nh vy, c th nhn c phage mang t hp mong
mun cc im nhn bit bi enzyme gii hn.

Hnh 8.7. Vector Phage

118

Mt trong cc nhc im ln ca cc vector l capsid ch c th bc

gi mt s lng ADN nht nh trong qu trnh lp rp phage, do vy, n gii
hn kch thc ca cc on ADN ngoi lai cn tch dng. Trong qu trnh bc
gi, cc ht phage c kh nng sng c th sinh ra vi chiu di ADN nm trong
khong 38kb n 52kb.
2.3.3. Cc vector da trn c s phage M13
C hai im trong vic gy nhim M13 c gi tr i vi k thut di
truyn. Mt l, dng sao chp RF rt ging vi plasmid, v vy c th tch chit
v thao tc bng cng nhng k thut nh nhau. u im th hai l ADN si
n sinh ra trong qu trnh nhim rt hu ch trong cc th nghim nh xc nh
trnh t theo phng php dng dideoxynucleotit. Ch ring v mt ny khin
cho M13 tr thnh mt vector c tim nng ht sc hp dn. Khc vi phage
M13 khng mang cc gene khng quan trng. H gene 6407 cp baz nm
vng gia cc gene. Vng ny c dng thit k mt lot cc vector
M13mp bng cch xen mt on polinker/lac Z - peptit vo . Vic ny
to iu kin cho vic s dng h thng sng lc X-gal pht hin cc th ti
t hp, chng hn nh trng hp vi cc plasmid pUC. Khi M13 mc trn
thm vi khun th cc vt tan xut hin do lm gim s sinh trng ca cc t
bo (chng khng b tan), v c th ly ra phn tch tip.

Hnh 8.8. Tch dng gene bi vector Phage hai si M13

(1). AND ca Vector M13RF b ct bi HindIII
(2). on AND c ct bi Hind III
(3). Ligase
119

(4). Bin np
(5). AND Ti bn
u vit th hai ca cc vector M13 l chng khng hot ng chc nng
mt cch y khi cc on ADN di c xen vo vector. Mc d v mt l
thuyt khng c mt gii hn no i vi kch thc ca cc on c th tch
dng, v cu trc capsid c xc nh bi kch thc hiu qu tch dng i
vi cc on ln hn 1,5kb. Trn thc t, khng phi l vn chnh v cc
vector M13 c s dng ch yu khi tch dng cc on ADN nh xc nh
trnh t. Trong ng dng ny, vic sn sinh ra ADN si n cng vi s d dng
trong vic tinh sch ADN t m t bo lm lu m nhng hn ch v kch
thc.
2.4. Cc vector khc
Cho n nay, chng ta mi tp trung vo cc plasmid v phage dng
trong cc vt ch E. coli, vi khun ny cho n nay vn l i din ch yu c
s dng trong phn ln cc cng ngh thao tc gene. Tuy nhin, cn tn ti cc
h thng vt ch / vector khc cng rt quan trng, mc d chng chuyn dng
hn. Trong phn ny, chng ti s gii thiu nhng c im ca cc vector vi
khun b sung v mt s vector c s dng trong cc sinh vt khc.
2.4 1. Cc vector plasmid / phage
Mt c tnh ca cc vector phagel ch k thut bc gi invitro c lp
v mt trnh t, khng i hi phi c cc im cos nm tch bit bi ADN vi
kch thc c th bc gi c (38-51kb). iu c s dng thit k
cc vector hnh thnh t cc on trnh t plasmid kt ni vi cc im cos ca
phage . Cc vector nh th gi l cc cosmid. Chng c kch thc nh (46kb) v v vy, c th cha cc on ADN tch ng di ti 47kb. V chng
khng c cc gene ca phage nn chng hot ng nh cc plasmid khi a vo
E. coli bng c ch bc gi/ nhim ca . Bi vy, cc vector cosmid cung cp
mt h thng ht sc l tng - mt phng php c bit v c hiu qu cao
a ADN ti t hp vo trong t bo ch, v kh nng tch dng ny ln hn
khong 2 ln so vi cc vector tt nht. Tuy nhin, chng cng c nhng
nhc im v thng th vic s dng cosmid thay cho cc vector phage lm
mt i s d dng trong s dng v x l cc trnh t tch dng ny.

120

Hnh 8.9. Vector Cosmid

Cc vector lai plasmid/ phage c hon thin khc phc nhng hn
ch v kch thc ca h thng tch dng M13, v n nay chng c s dng
rng ri cho cc ng dng nh gii trnh t ADN v sn xut cc mu d s
dng trong cc nghin cu v lai axit nucleic. Cc vector ny l nhng plasmid
quan trng, chng cha im khi u sao chp fl ca phage M13. Khi cc t
bo c cha plasmid ny b siu nhim bi phageth chng sn sinh ra cc bn
sao si n ca ADN plasmid v tit chng ra mi trng dng cc ht ging
M13. Cc vector, chng hn nh pEMBL9 hoc pBluescript c th tip nhn cc
on ADN di ti 10kb.
2.4.2. Cc vector dng trong cc t bo nhn chun
Khi xem xt cc t bo ch nhn chun th cc yu cu v vector tr nn
rt phc tp. Trong khi cc vi khun tng i n gin v mt di truyn th cc
t bo nhn chun li c nhiu nhim sc th nm trong nhn c v bao bc. V
cc sinh vt nhn chun ht sc a dng, nn l t nhin l cc vector c xu
hng c hiu cao v cn thit k ring bit cho cc mc ch c th.
Nhiu vector c hon thin s dng trong cc t bo nm men v
tu thuc vo tng ng dng c th m la chn vector. Cc plasmid episom
ca nm men (Yeps) c hon thin trn c s plasmid nm men thng gp
trong t nhin, chng c th t sao chp hoc ho nhp vo nhim sc th. Cc
plasmid ho nhp ca nm men (Yips) c thit k gn vo nhim sc th
bng cch tng t nh cc plasmid YEp, cn cc plasmid t sao chp ca nm
men (Yrps) vn trng thi c lp, khng ho nhp vo nhim sc th. Cc
plasmid c cha cc on trnh t trong vng tm ng ng nh cc nhim sc
th mini. Cui cng, cc nhim sc th nhn to ca nm men (YACs) l cc
vector nm men phc tp nht.
Chng c nhng vng ca tm ng v im mt, v c th c s dng
tch dng cc on ADN c kch thc rt ln, bi v th ti t hp c duy
tr ch yu nh mt nhim sc th nm men. Trn thc t, bn vng ca cc
121

YAC dng nh tng ln khi kch thc ca on tch dng tng ln, iu ny
khng ging vi nhiu h thng vector khc.
Bng 8.2. Mt s vector c th dng cho cc t bo ng vt v thc vt
Loi t
bo
Thc vt

Loi
vector

Loi h
gene

Th d

Plasmid

ADN

Plasmid Ti ca Agrobacterium tumefaciens

Virut

ADN

Virut khm ci hoa, Geminivirut

ARN

Virut khm thuc l

ADN

Cc loi vector plasmid khc nhau. Nhiu loi

l vector li c cha mt phn h gene ca
SV40

ADN

Baculovirut

Plasmid

ng vt Virut

Virut Papilloma
Virut Simian 40 (SV40)
Virut

ARN

Gene nhy ADN

Retrovirut
Cc phn t P trong Drosophilla melanogaster

Khi lm th nghim vi cc sinh vt nhn chun bc cao a bo, nh thc

vt v ng vt, cc vn a ADN ti t hp vo trong c th c khc bit
cht t so vi trng hp cc sinh vt nhn chun n bo nh nm men. Mc
tiu ca k thut di truyn cc sinh vt nhn chun bc cao bao gm:
(1) a ADN ti t hp vo cc t bo ng vt v thc vt trong m
nui cy nhm nghin cu c bn v s biu hin ca gene hoc nhm sn xut
cc protein hu ch.
(2). Bin i cu trc di truyn ca sinh vt v to ra mt vn chuyn
gene.
Cc vector dng cho cc t bo thc vt v ng vt c th c a vo
cc t bo trc tip bng cc k thut hoc chng c th c c ch xm nhp sinh
hc nu da trn cc virut hoc cc tc nhn xm nhim khc nh Agrobacteria.

3. PHNG PHP CHUYN VECTOR TI T HP VO T BO

CH
Vic thao tc vector v xen ADN to ra cc vector ti t hp c thc
122

hin trong ng nghim, v sau l a ADN ti t hp vo t bo ch nhn

ln. Hiu qu ca bc ny thng l yu t quyt nh s thnh cng ca k
thut tch dng. Cc phng php hin c ph thuc vo kiu loi ca h thng
vt ch/ vector v bao gm cc k thut n gin n nhng k thut rt phc tp
v c th.
3.1. Bin np v ly nhim
Cc k thut bin np v ly nhim l nhng phng php n gin nht
a ADN ti t hp vo t bo ch. Trong trng hp cc t bo E. coli th
bin np c ngha l a ADN plasmid vo trong t bo, cn ly nhim l a
ADN phage vo trong cc t bo. Bin np cn c s dng vi ngha rng
hn, c th l: a bt k ADN no vo bt k t bo no.
cho bin np E. coli c hiu qu cc t bo cn tr nn kh bin
(competent). t c iu ny, ngi ta ngm cc t bo trong dung dch
calcium chlorid lnh ng , khi , cc t bo tr nn kh bin theo mt cch
m cho n nay cha bit r nguyn nhn. Vic bin np cc t bo kh bin
c thc hin bng cch trn ADN plasmid vi cc t bo, ri trong 20
n 30 pht, sau to mt sc nhit ngn (2 pht 420C), lm nh vy ADN
s chui vo t bo. Cc t bo bin np thng c trong nc tht dinh
dng 370C trong vng 60 n 90 pht lm cho cc plasmid n nh v
biu hin cc tnh trng ca chng ra kiu hnh. Sau , cc t bo c a ln
mi trng chn lc nhn ln cc t bo c plasmid.
Trong qu trnh bin np, ch c mt phn rt nh t bo kh bin c
bin np. Mc d c nhc im rt ln, bin np vn l mt k thut quan
trng, n c th to ra ti 109 cc t bo bin np (tc cc th bin np) khi a
1gr ADN vo th nghim. Thng trn thc t vi 1 g ADN ngi ta to ra
c 106 n 107 t bo bin np.
Ly nhim l mt qu trnh tng t vi bin np, ch khc ch ADN
plasmid c thay th bng ADN phage. Qu trnh ny cng khng din ra vi
100% cc t bo, v v vy chng thng c thay th bng cc bc gi invitro
trong cc th nghim i hi phi a ADN ca phage vo bn trong cc t bo
E. coli.
3.2. Bc gi ADN ca phage invitro
Trong qu trnh sinh tan ca phage , ADN ca phage c sao chp
to thnh ci gi l chui khm (concatemere). l mt phn t ADN rt di
bao gm nhiu bn sao ca h gene c ni lin vi nhau. Khi cc ht phage
c lp rp th ADN c bc gi thnh cc capsid, qu trnh ny bao gm
vic ct ADN ti cc im cos bng enzyme endonucleaza do phage m ho.
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Cc ht phage trng thnh sau dc sinh ra, v thot ra ngoi khi cc t bo

tan, sau chng li c kh nng nhim vo cc t bo khc. Qu trnh ny
thng din ra invitro di s iu khin ca cc gene phage. Tuy nhin, cng
c th thc hin trong ng nghim.
bc gi invitro th cc thnh phn ca capsid , v endonucleaza phi
c trong ng nghim. Trn thc t, hai ni vi khun c s dng to ra dch
tan gi l dch chit bc gi (packaging extract). Mi ni mang t bin v mt
chc nng trong qu trnh pht sinh hnh thi ca phage). Nh vy, cc dch
chit bc gi s khng hot ng ring r. Khi trn hai ci vi nhau vi ADN ti
t hp khng hot ng khm (concatemer) trong nhng iu kin thch hp v
vi y cc thnh phn th cc ht phage s c sinh ra. Cc ht ny sau
c th dc s dng gy nhim cc t bo E. coli v to ra cc vt tan.
3.3. Cc phng php truyn ADN khc
Cc phng php dng a ADN vo cc t bo vi khun khng d p
dng cho cc loi t bo khc. H thng bc gi c th cho phage khng dng
c cho cc h thng khc, v vic hin np bng cc phng php thng
thng c th khng thc hin c hoc hiu qu rt thp. Tuy nhin, c
nhng phng php khc dng a AND vo cc t bo. Thng thng, cc
phng php ny c nhng i hi v k thut v km hiu qu so vi cc
phng php dng cho vi khun. Tuy nhin, ngi ta t c kt qu trong
mt s trng hp.
Vn kh khn nht trong vic a ADN vo cc t bo khng phi l
vi khun thng xy ra vi cc t bo thc vt. Cc t bo ng vt li c
bin np tng i d dng. Cn vic bin np cc t bo thc vt gp kh khn
v v t bo ca chng rt cng, ngn chn khng cho ADN chui vo. C th
khc phc vic ny bng cch to ra cc t bo trn (protoplasts) l nhng t bo
m thnh ca chng c loi b bng enzym. Sau , cc t bo trn c th
c bin np bng cc k thut nh m l bng in, khi mt cung c s
dng to ra cc l trn mng t bo m qua ADN c th xm nhp. Sau
cc t bo trn c th c ti sinh (to v mi). Ngoi ng dng ny, cc t bo
trn cn c vai tr quan trng trong vic to ra cc t bo lai thc vt khi dung
hp vi nhau. Mi loi phng php bin np khc c th a ADN vo bn
trong t bo l cc phng php vt l. Mt cch lm vic l s dng mt
ci kim rt mnh tim ADN trc tip vo trong nhn. K thut ny gi l vi
tim v c s dng thnh cng c i vi cc t bo ng vt v thc vt.
T bo c gi trn u mt ng thu tinh u ty, cn chic kim nhn c
dng m xuyn qua mng t bo. K thut ny i hi phi c mt my vi
124

thao tc c hc, mt knh hin vi v c bit phi c k nng thc hnh tinh vi.
Gn y ngi ta hon thin mt phng php t ra rt hu hiu
bin np t bo thc vt, gi l k thut bn ADN vo t bo. ADN c dng
bao bc cc ht vonfram cc nh gi l vi n. Khi bn, chng c tng tc
trn vin n ln l b phn c bn i do ngi thuc n chy. Ti mt u ca
"sng" c mt l nh chn vin n ln li nhng vn cho vi n chui qua. Khi
nhm bn vo t bo, cc vi n s a ADN vo trong t bo v trong mt s
trng hp bin np s thc hin thnh cng.

THO LUN
1. Nu cc loi t bo ch v u, nhc im trong k thut tch dng gene.
2. Plasmid l g? cc loi plasmid. Trnh by v cc vector plasmid dng cho
E. coli.
3. Trnh by v vector phage.
4. Cc vector dng trong cc t bo nhn chun.
5. Trnh by c im c bn ca mt s phng php chuyn vector ti t
hp vo t bo ch.
6. E. coli v s dng E. coli trong k thut di truyn.
7. Vector tch dng v vector biu hin gene.
8. Vn tch dng gene.
9. Vn chuyn gene v sinh vt chuyn gene.

125

Chng 9
PHN LP GENE TCH DNG PHN T
V BIU HIN GENE
Tm tt: Cng ngh ADN ti t hp bao gm cc k thut tch dng gene v
biu hin gene. Bt u t s phn lp gene v sau l to vector ti t hp,
bin np vo t bo ch, tch dng v xc nh trnh t gene. Protein l sn
phm biu hin gene. Qu trnh biu hin gene ngoi lai bt u t vic a
gene vo vector biu hin v bin np vo t bo ch. Vector biu hin gene
c thit k bao gm promotor mnh v iu khin c. E. coli l vi khun
c s dng rng ri trong cng ngh ADN ti t hp v s biu hin gene
thnh cng nht hin nay vn l E. coli. Phn lp, tch dng v biu hin
gene c minh ho bng v d v s phn lp, xc nh trnh t gene m ho
cho trichobakin-RIP v qu trnh biu hin gene ny.
Ni dung ca chng gm 3 vn chnh l: (1).Phn lp gene; (2). Tch
dng gene; (3). Biu hin gene.

1. PHN LP GENE
Phn lp gene hay phn lp on ADN theo mc ch c th c tin
hnh theo phng php s dng enzyme ct hn ch hoc bng phn ng chui
PCR. Phn lp gene mang ngha trong k thut gii trnh t ADN hay to dng
ADN ti t hp trong k thut chuyn gene.
1.1. Phn lp gene bng k thut ct hn ch
Enzyme gii hn (restrition enzyme) c kh nng ct ADN nhng im
c hiu. Vi c tnh ngi ta vn dng xc nh nhng on ADN
m ho cho cc tnh trng ca sinh vt v phn lp on ADN . K thut phn
lp gene nh enzyme gii hn c th gm cc bc sau:
(1). Tch chit v tinh sch ADN.
(2). Ct ADN bi enzyme gii hn.
(3). in di trn gel agarose.
(4). Da vo ADN marker xc nh c on ADN cn phn lp.
(5). Bin tnh ADN thnh 2 mch n ngay trn gel, ri chuyn ln mng
126

lai phn t. V tr cc on ADN c gi nguyn.

(6). ADN c nh trn mng c lai vi mu d c nh du phng x
Ra loi b cc mu d khng bt cp.
(7). Dng k thut phng x t ghi nh v cc phn t lai ADN- mu
d.
1.2. Phn lp gene bng k thut PCR
Phn ng chui polimerase c tin hnh vi cc primers c trnh t bit
trc. Cp primer gm primer xui (sens primer) v ngc (antisens primer)
c thit k theo trnh t gene t ngn hng gene. Cp mi chuyn bit ny
c bt cp b sung vi hai u ca mt trnh t ADN. S dng cp mi
chuyn bit cng vi ADN mu, enzyme, dNTP, Mg++, bufpher... trong thit b
nhn ADN (my PCR) th on gene t phn t ADN mu s c khuch i.
Phn lp gene bng PCR c th c tin hnh theo cc bc sau:
(1) Tch chit v tinh sch ADN.
(2). Thit k primers.
(3). Chn iu kin phn ng PCR.
(4). Nhn on ADN trong my PCR phn lp on gene theo mc
ch.

2. TCH DNG GENE

2.1. Mc ch
- To ra mt lng ln bn sao mt trnh t ADN xc nh.
- Chn lc mt th vin gene.
2.2. Cc bc ca k thut tch dng
- Chn v x l vector.
- X l ADN cn to dng.
- To vector ti t hp.
- Chuyn vector ti t hp vo t bo ch.
- Pht hin dng cn tm trong th vin gene.

127

Hnh 9.1. Quy trinh chung ca k thut tch dng gene

2.3. Th vin b gene
Th vin b gene l tp hp tt c cc trnh t ADN cu thnh b gene
c gn vo vector. V nguyn tc, th vin b gene c thit lp t bt c t
bo no ca sinh vt nghin cu.
Cc bc thit lp th vin b gene:
(1) Tch chit ADN.
(2). Ct ADN thnh nhng on c kch thc xc nh bng RF.
(3). Gn on cc ADN vo vector to vector ti t hp.
(4). Bin np vector ti t hp vo t bo ch.
(5). T bo ch nui cy trn mi trng c v hnh thnh nn cc dng.

128

Hnh 9.2. Tch dng ADN ti t hp

ng dng:
- Gii m thng tin di truyn cha trong b gene, c bit cu trc itron,
exon ca mt gene xc nh.
- To dng cc trnh t khng m ho nm cnh cc gene v ng vai tr
quyt nh trong s iu ho biu hin gene.
2.4. Phn lp, tch dng v xc nh trnh t gene chaperonin u tng
Mt v d v xc nh trnh t gene chaperonin lin quan n tnh chu
nng hn cy u tng Glicine max (L.) Merrill trn thit b t ng theo
129

nguyn tc ca phng php Sanger. Glicine max (L.) Merrill l loi cy trng
c v tr quan trng hng u trong cc loi cy h u. Ht u tng c hm
lng protein cao t 28% - 45%, d tan v cha hu ht cc loi axit amin, c
bit l cc loi axit amin khng thay th. Ngoi protein, ht u tng cn c
lipit v nhiu cht hu c khc. R u tng c nt sn cha vi khun c nh
m Rhizobium.
Cc dng u tng M48, M10 v M61 c to ra bng phng php
t bin thc nghim (Chu Hong Mu, 2001) c nh gi v chn lc qua
nhiu th h. Ngoi cc c im v nng sut v cht lng, tc gi cn quan
tm nghin cu mi lin quan gia kh nng chu hn vi c im ho sinh v
sinh hc phn t ca cc dng u tng t bin ny. Dng u tng t bin
chu hn ML61 c ngun gc t ging u tng Lng Sn v c to ra bng
t bin thc nghim vi cng thc x l phi hp (16 kr + 0,01%EI). Trn
Th Phng Lin (1999) nghin cu, phn lp v xc nh trnh t gene
chaperonin t ging u tng chu nng M103, genome ca cc dng u
tng t bin ML10, ML48 v ML61 c tn ti gene chaperonin v cu trc
ca n c im g khc vi gene chaperonin c nghin cu. Kt qu nhn
v tch dng gene chaperonin ca cc dng u tng t bin M48, M10 v
M61 v xc nh trnh t gene chaperonin ca dng u tng chu hn M61
c thc hin nhm mc ch trn. Pht hin gene chaperonin theo phng
php ca Kary Mullis v cng s (1985) vi vic s dng cp mi
Chap_N/chap_C thit k da trn trnh t ADN b sung (cDNA) ca ging u
tng Nht Bn.
Chap_N:
5_GCC ATA TGT CGG CAA TCG CGG CCC C
Chap_C:
5_CGG GAT CCC TAC CTC ACA GTT ACA GTT ACA ATA TCA TC
Chu trnh nhit phn ng: 950C : 1 pht; 550C : 1 pht; 720C : 1 pht 20
giy: 32 chu k; 720C : 8 pht; Kt thc 40C.
tin hnh tch dng gene chaperonin sn phm PCR c ct thnh
cc on nh hn bng SacI. Sau em sn phm ct gn vo vector
pBluescript KS (-) c m vng. ADN ti t hp c bin np vo chng
E. coIi-DH5 . Mi trng nui cy, cc phng php gn, to t bo kh bin,
bin np, tch ADN plasmid, kim tra on gene bng cch x l vi enzyme
gii hn dc tin hnh theo sch cm nang chn dng phn t ca Sambrook
v cng s 1989.
130

2.4.1. Kt qu tch chit v tinh sch ADN tng s

Mm 2-3 ngy tui ca cc dng du tng ML10, ML48 v ML61 c
chia nh t 0,5-1g v gi -700C. Khi tch chit, mm c nghin trong nit
lng v ADN tng s c chit ra bng dung dch m c cha 10 mM TrisHCl pH 8.0,1 mM EDTA, 100 mM NaCl, SDS 2,1%. S dng enzyme
proteinaza, hn hp phenol : chlorofoc : isoamylalcohol (25 : 24 : 1) loi
protein v cc hp cht khng mong mun, EDTA c tcdng c nh cc ion
Mg++ - l yu t cn thit cho s hot ng ca cc nucleaza, do ADN trong
dung dch khng b phn gii bi cc enzyme ny. Thc hin quy trnh tch
chit ADN nh m t [1], thu c dung dch c cha ADN. Dung dch
ADN thu c vn tn ti c ARN, loi b ARN bng cch x l vi ARNaza,
370C trong 2-3 gi.
Sau tin hnh kim tra sn phm bng in di trn gel agarose 0,8%.
Bn gel sau khi in di c nhum bng EtBr (ethidium bromide) trong 10-15
pht. Do cu trc ca ADN c cc lin kt hydro gia hai mch n nn khi
nhum, cc phn t EtBr s bm vo cc lin kt ny. Phn t EtBr c kh nng
pht quang di nh sng ca tia t ngoi (UV) nn c th pht hin bng vch
khi chiu bn gaeldi n UV.

Hnh 9.3. Kt qu in di ADN tng s trn gel agaroza 0,8%

2.4.2. Nhn gene chaperonin t 3 dng u tng bng k thut PCR
Chaperonin t bo cht l mt dng protein tn ti trong tt c cc t bo ng
vai tr quan trng trong vic to cu trc khng gian ng cho protein to khung
t bo l actin v tubulin. Chng cng ng thi thc hin nhiu chc nng khc
nh tham gia ti to cu trc khng gian ng cho protein b bin tnh khi gp
iu kin bt li. Cc tc gi s dng on mi c tng hp da vo trnh
t cDNA ca chaperonin t bo cht ging u tng chu lnh Nht Bn tin
hnh nhn gene chaperonin ca cc dng u tng t bin ML10, ML48 v
ML61 bngk thut pPC.

131

Hnh 9.4. in di sn phm PCR gene chaperonin ca 3 dng u tng

Sau 32 chu k phn ng, sn phm PCR c kim tra bng in di trn
gel agarose 0,8% v kt qu cho thy c ba dng u tng u thu c sn
phm PCR c hiu. Kch thc phn t (KTPT) ca sn phm PCR vo khong
1,6 kb. ng thi sn phm PCR nhn c c KTPT ging vi on tng ng
on cDNA ca gene chaperonin trong ging u tng Nht Bn. iu
chng t rng cc dng u tng nghin cu u c gene chaperonin. Nh vy,
trong trnh t ca n ch c nhng vng c dch m sang protein (exon), m
khng c nhng vng khng dch m (intron).
2.4.3. Phn tch gene chaperonin bng enzim gii hn
Theo cc nghin cu cng b, trnh t gene chaperonin c im nhn
bit cho mt s enzyme gii hn nh: SacI, AluI, SauI. Trong SacI ct trnh
t cDNA thnh 5 on c kch thc phn t tng ng l 260, 189, 198, 630 v
325 bp. Nh vy, so snh gene chaperonin ny vi cc trnh t cng b. Sn
phm ct c in di trn gel agaroza 1,2%.

Hnh 9.5. Ph in di ADN plasmid

Kt qu trn din di cho thy khng c s khc bit v v tr im ct
SacI cc dng u tng t bin so vi cc gene chaperonin c cng b
trc y. iu ny chng t sn phm PCR nhn c c th chnh l on
gene chaperonin. y l mt trong nhng bng chng khng nh sn phm
132

PCR thu c chnh l gene chaperonin c cng b. Tuy nhin c th

khng nh chc chn v gene ny cn tip tc nghin cu tch dng sn phm
PCR ca mt i din l dng t bin ML61 - dng c nh gi l c nng
sut cao, chn sm v kh nng chng chu hn tt tin hnh c trnh t
ADN ca on gene ny lm c s cho vic so snh vi trnh t ca gene
chaperonin c cng b trn ngn hng gene th gii.
2.4.4. Tch dng mt s on ADN ca gene chaperonin dng ML61
Nhm mc ch nghin cu c trnh t gene, cn phi gn mt s on
ADN ca gene chaperonin dng ML61 c ct bng enzyme SacI vo
vector pBluescript KS (-). Da vo cc c im ca vector pBluescript KS (-)
c cha gene lacZ v v tr ct ca SacI... ng thi trn gene chaperonin cng
cha v tr cc im ct c hiu ca SacI. V vy, s dng SacI x l sn
phm PCR ca dng u tng ML61 v m vng vector pBluescirpt KS (-).
Phn ng gn ca vector vi cc on ct ca sn phm PCR c tin hnh
160C trong vng 24 gi, s dng enzyme T4 ligaza. Sau , dch phn ng c
bin np vo chng E. coli - DH5 v cy tri trn mi trng chn lc c
ampixillin, X-gal v IPTG. Kt qu nhn c mt s khun lc trng v
khun lc xanh trn mi trng chn lc. Nhng khun lc trng c th mang
on ADN ngoi lai, cn khun lc xanh mang plasmid khng cha on gn
vo. kim tra kt qu ca qu trnh chn dng 9 khun lc mu trng (k hiu
cc khun lc t 1 n 9) v mt khun lc mu xanh (lm i chng) c s
dng tch chit ADN plasmid. Sau khi tch chit v lm sch, sn phm
ADN plasmit c kim tra trn gel agaroza 0,8%. Trn nh in di cho thy
c 9 khun lc u c ADN plasmid vi di ng in di cao hn i chng.
xc nh c nhng khun lc cn tm c gn on sn phm PCR ct bng
SacI, chn ADN plasmid t bn khun lc 1, 4, 7 v 8 tin hnh phn ng ct
kim tra bng enzyme SacI. Sn phm sau khi ct c kim tra bng in di
trn gel agaroza 0,8 %.
Kt qu trn nh in di dng s 8 gn c mt on ADN khong
0,6 kb; dng s 7 gn c on ADN khong 0,4 kb; dng s 4 gn dc
on ADN khong 0,2 kb. Ring sn phm ct ca dng s 1 cho thy c c 2
on ADN khong 0,6 kb v 0,2 kb c gn vo vector. Trong qu trnh tch
dng, s dng enzyme SacI ct gene chaperonin thnh 5 on. V SacI l
enzyme ct to u dnh nn sn phm ct ch cho c 3 on c u dnh
tng ng vi kch thc l 189, 198 v 630 bp.
Nh vy, r rng l dng s 8 gn c on tng ng c kch thc
l 630 bp. Dng s 4 th gn dc hoc l on 189 bp hoc 198 bp (v hai on
133

ny rt kh phn bit trn din di ). Cn dng s 1 gn c c hai on 630

bp v mt trong hai on ngn ni trn. i vi dng s 7 on gn c kch
thc khong 400 bp c th l tng ca hai on ngn 189 v 198 bp v cng c
th l on ln tp trong sn phm ct ngu nhin. c nhng kt lun chnh
xc v cc dng thu c, cn tin hnh c trnh t ADN.
2.4.5. Xc nh trnh t gene Chaperonin cy u tng
Vector tch dng TA cloning v cc ho cht cn thit ca hng
Invitrogen, vi khun E. coli chng DH5 . To dng phn t c tin hnh
theo phng php ca Sambrook v Russell. Xc nh trnh t gene chaporonin
c thc hin trn my c trnh t t ng ABI PRISM 3100 Avant Genetic
Analizer. Nhn on gene chaperonin t ADN tng s, tiu phn CTT ca
dng u tng t bin ML61. Sau , sn phm PCR c a vo vector TA
cloning, c bin np vo vi khun E. coli DH5 . Kim tra vector ti t hp
bng x l vi enzyme EcoRI thu c dng mang on gene mong mun
tin hnh c trnh t. kim tra kt qu c trnh t gene chaperonin, s dng
phn mm Sinh hc Clustalx so snh trnh t gene ny vi cc gene
chaperonin ca ging Cc Vng (cn gi l Cc Lc Ngn), mt ging a
phng c kh nng chu hn, ging M103 t bin c kh nng chu nng v
ging u tng chu rt Nht Bn - Bonminori.

134

135

Trnh t gene chaperonin ca dng u tng t bin ML61 c s sai

khc 41 v tr nucleotit so vi ging M103, 57 v tr nucleotit so vi ging Cc
Vng v 25 nucleotit so vi ging u tng Bonminori. S sai khc ny to
nn s thay i trnh t 9, 10 v 11 axit amin sai khc ca ML61 tng ng so
vi Bominori, M103 v Cc Vng.
Bng 9.1: Trnh t axit amin ca gene chaperonin ML61 sai khc so vi
Bonminori, Cc Vng v M103
STT
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

V tr
8
24
29
43
54
76
99
110
128
129
191
204
220
280
308
360
447
506
514

Bonminori
Gln (Q)
Glu (E)
Gli (G)
Thr(T)
Ile (1)
Val (V)
Ser (S)
His(H)
Glu
Pro (P)
Pro (P)
Val (V)
Leu (L)
Ser (S)
Ser (S)
Phe (F)
Asp (D)
Leu (L)
Thr(T)

Cc Vng
Gln (Q)
Asp (D)
Ala (A)
Thr(T)
Thr(T)
Gli (G)
Thr(T)
Leu (L)
Asp (D)
Ala (A)
Pro (P)
Ile(I)
Pro (P)
Gli (G)
Ser (S)
Val (V
Ala (A)
Leu (L)
Met (M)

M103
Gln (Q)
Asp (D)
Ala (A)
Thr(T)
Thr (I)
Val (V)
Thr (T)
His(H)
Asp
Pro (P)
Pro (P)
Val (V)
Leu (L)
Gli (G)
Ala (A)
Phe (F)
Ala (A)
Pro (P)
Thr (T)

MI61
His (H)
Glu (E)
Ala (A)
Ala (A)
Ile (I)
Val (V)
Thr (T)
Leu (L)
Asp
Ala (A)
Ala (A)
Val (V)
Leu (L)
Ser (S)
Ser (S)
Phe (F)
Ala (A)
Leu (L)
Thr (T)

Cc trnh t nucleotit sai khc cn li u do s dt bin im c tnh cht

ng chuyn (hon v ca cc nucleotit trong nhm vi nhau) m khng lm
thay i trnh t axit amin. Mt khc dng u tng MI61 cn c s sai khc
3 v tr axit amin 8Gln-His, 29Thr-Ala v 191Pro-Ala so vi c 3 ging so
snh trn. ML61 l mt dng u tng c x l t bin phi hp vi 16
kr + 0,01%EI, c th chnh vic x l phi hp tia v EI gy nn s t
bin trnh t nucleotit ca dng u tng ny.
136

Xem xt n 3 vng chc nng quan trng ca chaperonin nghin cu

nh gi trnh t nucleotit ca dng ML61 n vic to cu trc khng gian ca
chaperonin. Vng hot tnh ATPaza v vng gn vi ATP, y l hai vng bo
th ca gene. Vng hot tnh ATPaza vn gi dc tnh bo th, cn vng gn
vi ATP ML61 sai khc vi Bominori v tr 99Thr-Ser v M103 99Thr - Ile.
Cc Vng c trnh t vng ny ging ML61. Mt vng th 3 c kim tra l
vng gn c cht vi phc CCT, y l vng a hnh nht so vi cc vng chc
nng khc ca chaperonin tiu phn CCT . Trong vng ny ML61 sai khc vi
M103 v tr axit amin 280Gli-Ser, Cc Vng hai v tr 220Pro-Leu v
280Gli-Ser. Khng c s thay i ca vng ny so vi Bominori. Ngoi ra
nh gi kh nng chu hn ca ML61, trnh t gene chaperonin ny c xem
xt theo quy lut bin i cc axit amin trong cc enzim v kh nng chu lnh
sang nng. Da trn trnh t axit amin, dng ML61 cng c s bin i tng t
v tr axit amin 29 Gli-Ala, 99Ser-Thr (Bominori) ging nh Cc Vng v
M103. Hai ging u tng c nh gi l c kh nng chu hn.
2.5. Th vin cADN
Th vin ADN l tp hp cc bn sao cADN t tt c cc mARN ca t
bo. Khc vi th vin b gene, th vin cADN c thit lp t mt t bo xc
nh trong gene nghin cu biu hin thnh mARN.
Mc ch ca th vin cADN l nghin cu biu hin mt gene xc nh
cng vi nhng vn lin quan nh biu hin gene, mi tng tc gia cc
gene trong qu trnh sng.
Cc bc thit lp th vin cADN:
(1) Chn vector.
(2). Tch chit mARN.
(3). Tng hp cADN nh enzyme sao m ngc.
(4) To vector ti t hp gm plasmid v cADN.
(5) Bin np vector ti t hp vo t bo vi khun.
(6) Nui cy vi khun trong mi trng lng, sau chuyn sang c. Cc
dng vi khun s hnh thnh cc khun lc.
(7). Pht hin dng cn tm.

137

Hnh 9.6. To vector cADN ti t hp (theo Nng Vn Hi, 2002)

2.6. Pht hin dng t bo ch cha vector ti t hp
Vector v on ADN ngoi lai c trn ln vi nhau di tc dng ca
enzyme ni ADN-ligase. Sau hn hp vector v on ADN ngoi lai c
trn ln vi t bo ch thc hin bin np v c 3 kh nng c th xy ra: (1)
T bo ch khng c plasmid. (2) T bo ch nhn c plasmid khng c gene
ngoi lai xen vo; (3) T bo ch nhn dc vector ti t hp. Chnh v vy
trong k thut tch dng cn phi xc nh dc ng dng t bo ch cha
vector ti t hp. C th s dng 3 phng php c bn sau: lai axit nucleic, da
vo s biu hin kiu hnh, hin tng mt hot tnh do xen on.
(a) S dng phng php lai axit nucleic c hiu qu trong vic tch dng
gene mong mun t th vin ADN bng vic s dng mu d c nh du
phng x. C th thc hin theo cc bc: lm tan cc khun lc trn giy
nitrocellulo gii phng ADN, lai mu d lai vi ADN trn mng lai. Vic pht
hin cc phn t lai thng qua phng x t ghi.
(b) Pht hin khun lc cha plasmid ti t hp thng qua biu hin kiu
hnh. Cc vector c thit k mang trnh t iu ho ca gene lacZ, khi bin
np vo t bo ch, lm bin i t bo ch c kiu hnh Lac- thnh Lac+. Cc vi
khun c kiu hnh Lac+ c nhn bit nh s chuyn ho c cht X-gal lm
khun lc c mu xanh. Nu on ADN ngoi lai c gn xen vo trnh t iu
ho ca lacZ, lm gene khng hot ng, do vy khng thc hin c phn ng
bin i X-gal thnh mu xanh, v vy khun lc c mu trng. Nh vy ngi
ta c th nhn bit nhanh chng bng mt thng cc khun lc cha vector ti
t hp.
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(c) Hin tng lm mt hot tnh do xen on c s dng i vi cc

vector mang gene khng thuc ampiciline (hoc tetracycline), cha cc im
nhn bit ca cc enzyme ct hn ch. Plasmid c m vng di tc dng ca
mt RE m im ct ca n nm trn mt gene khng thuc no v on
ADN ngoi lai c xen vo v tr m vng trn plasmid, lm cho gene khng
thuc trn mt hot tnh. Hn hp trn c bin np vo t bo ch E. coli, t
bo ch c th c 3 loi kiu gene v kiu hnh: (1) T bo ch khng nhn c
vector s khng mc c trn mi trng c tetracyline (hoc ampiciline); (2)
T bo ch nhn c vector nhng gene ngoi lai khng gn c vo vector,
n s mc c trn mi trng c ampiciline, tetracycline; (3) T bo E. coli
nhn c vector ti t hp, gene amp b mt hot tnh do gene l xen vo, gene
khng tetracyline vn hot ng bnh thng. Do vy cc t bo ch mang
vector ti t hp khng mc c trn mi trng cha ampiciline, nhng vn
mc c trn mi trng cha tetracycline.

3. BIU HIN GENE

3.1. Khi nim
3.1.1. Cc h biu hin gene
Trong t bo sng, biu hin gene c hiu l qu trnh sinh tng hp
protein trong mi lin h: ADN ARN Protein v protein l sn phm biu
hin gene. Trong k thut di truyn, biu hin gene l hin tng protein ca
gene ngoi lai c tng hp trong t bo ch.
Hin nay c 4 h sau hay s dng biu hin gene nh Escherichia coli,
Bacillus subtilis, nm men (Saccharomyces cerevisiae, Pichia pastoris) v t
bo ng vt. Tuy nhin E. coli v nm men l hai h biu hin chnh hin ang
c s dng rng ri trong nghin cu v ng dng.
Cho n nay c th ni rng vn biu hin gene ngoi lai vn cn mang
tnh thc nghim i vi tng gene, cha c mt m hnh chung cho php biu
hin ti u i vi cc loi protein khc nhau trong mt biu hin. V vy, t
c mc biu hin cao i vi mt gene ngoi lai. trong nhiu trng hp
phi nghin cu thm d vi cc yu t khc nhau tm ra diu kin ti u.
Cc protein biu hin trong cc t bo khc ngun gc c th c chia
thnh 4 nhm chnh:
Nhm th nht bao gm cc peptit nh c di di 80 axit amin. y
l on peptit c biu hin d dng nht di dng protein dung hp (fusion
139

protein) v thng c biu hin trong t bo E. coli.

Nhm th hai l cc chui polipeptit thng c tit ra ngoi t bo (nh
enzyme, cytokin, hormon...) c kch thc trong khong 80-500 axit amin. Cc
protein thuc nhm ny c th biu hin c trong c 4 h biu hin.
Nhm th 3 gm cc protein tit v protein ng vai tr th th trn b
mt t bo c kch thc hn 500 axit amin.
Nhm th t bao gm tt c cc protein khng tit c kch thc ln hn
80 axit amin.
Hu ht cc protein u c th c biu hin trong t bo E. coli nu
protein khng qu nh hoc qu ln, hoc qu k nc, hoc cha qu nhiu
gc cystein. Cc protein nh v polipeptit thng khng bn vng trong t bo
ch nn mun biu hin tt trong E. coli th cn c gn vi mt on protein
khc. on protein ny s gip cho on peptit dc bn vng trong t bo E.
coli sau i c tng hp v hin nay n c cn c dng nh mt du nhn
bit tinh ch protein lai ny. S d cc protein c cha nhiu gc cystein v cu
disulfit khng c biu hin tt trong E. coli l do mi trng bn trong t bo
E. coli c tnh kh cao.
B. subtilis v nm men mc d khng phi l h c s dng rng ri
trong nghin cu biu hin gene ngoi lai nh E. coli nhng nhng h biu
hin ny cng c nhiu u im ng ch . in hnh l B. subtilis c kh nng
biu hin v tit rt tt protein c ngun gc t Prokaryot ra ngoi mi trng
nui cy cn nm men li c nhng u im quan trng ca c Eukaryot v
Prokaryot. Nm men sinh trng nhanh v c kh nng ch bin protein sau khi
dch m rt tt tr thnh dng protein hot ng m h biu hin gene vi
khun khng c. Hn na nm men c mc an ton sinh hc cao, c s
dng nhiu trong cng nghip dc liu v thc phm. V vy, nm men rt
c a chung biu hin cc gene ca Eukaryot v gene m ho cho protein
s dng trong thc phm v cha bnh.
H biu hin trong t bo ng vt c kh nng biu hin c tt c
protein ca 4 nhm nhng tn thi gian v qu trnh chn lc, nhn dng v
nui cy dng t bo phc tp hn nn h biu hin ny thng khng c s
dng rng ri.
Nhng c tnh sinh hc phn t iu chnh s biu hin gene th hin
(1) c im t nhin ca nhng trnh t promotor v terminator lin quan
n dch m; (2) mnh yu v v tr bm ca ribosome; (3) S phin bn ca
gene tch dng; (4) V tr tng hp protein trong t bo, (5) Hiu sut ca qu
140

trnh gii m trong t bo ch; (6) S bn vng ca phn t protein c m ho

bi gene tch dng trong t bo ch.
Mc biu hin gene ngoi lai (foreign gene) cn ph thuc vo s
nhn bit ca c th vt ch. Hin nay c ph rt rng cc c th biu hin
gene ngoi lai (c Prokaryot v Eukaryot) nhng a s cc sn phm c sn
xut bi cng ngh ADN ti t hp c tng hp E. coli.
3.1.2. Biu hin gene t nhng promotor mnh v iu khin c
Yu cu ti thiu vi mt h thng biu hin gene c hiu qu l s c
mt ca mt trnh t promotor mnh v c th iu khin c (strong and
regulatable promotor) nm pha trc gene tch dng.
Promotor mnh l mt promotor c i lc cao vi enzyme ARN
polimerase v s bo m cho trnh t nm sau promotor dc phin m vi tn
sut cao. Kh nng iu khin promotor cho php t bo ch kim sot quy
m phin m mt cch chnh xc. Promotor lac-operon c s dng rng ri
trong vic biu hin gene tch dng. Ngoi ra nhiu promotor c phn lp
t nhiu c th khc nhau s dng trong biu gene. Phng php ti u ho
s biu hin ca gene tch dng l gn gene vo plasmid di s kim sot
ca promotor phin m lin tc v mnh. Tuy nhin, s biu hin lin tc v
mc cao ca mt gene ngoi lai thng gy hi cho t bo ch v n gy ra
s thiu ht nng lng, mt cn bng trong t bo l suy gim nhng chc nng
thit yu ca t bo. ng thi mt s hay ton b cc plasmid cha gene tch
dng s b mt i sau mt s ln phn chia t bo. Nhng t bo khng cha cc
plasmid mang gene tch dng s phn chia mnh hn cc t bo cha plasmid
mang gene tch dng v cc t bo khng cha cc plasmid mang gene tch
dng s chim u th. iu ny cn tr qu trnh biu hin v sn xut cc
sn phm t gene tch dng. khc phc kh khn ny ngi ta c tng
mong mun kim sot c s phin m ca gene tchdng mc ch cho gene
ny biu hin mt giai on sinh trng ht nh ca t bo. S dng mt
promotor mnh v iu khin c s l gii php thc hin mong mun .
Promotor mnh v iu khin c ang s dng rng ri l promotor ca
operon lac v tryp ca E. conli Promotor lac c thit k v ch to invitro
cha:
- Vng -10 ca lac promotor.
- Vng -35 ca tryp promotor.
- Promotor bn tri (pL) ca bacteriophage
- Promotor gene 10 ca bacteriophage T7.
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Mi promotor ny tng tc vi cht c ch v nh c th tt hoc m

s phin m ca nhng gene tch dng. Mi promotor li dc nhn ra bi cu
trc ca ARN polimerase holoenzyme nh nhn t (nhn t bm vo li
ca ARN polimerase hnh thnh holoenzyme, nh hng cho enzyme bm
vo nhng vng promotor).
Promotor lac b c ch (tt) bi protein km hm lac khi mi trng thiu
lactose v c m bng cch b sung vo mi trng nui cy lactose hoc
isopropyl- -D-thiogalactopyranoside (IPTG). Lactose v IPTG bn vo
promotor lac v qu trnh phin m sy ra. Qu trnh phin m t promotor lac
cn c iu khin bi protein hot ho chuyn ho (catabolite activator
protein-CAP). Khi CAP bm vo promotor lac s lm tng i lc ca promotor
vi enzyme phin m.
Promotor tryp b c ch bi phc h protein km hm, phc h ny bm
vo operator tryp v ngn can qu trnh phin m. Promotor m bng vic loi
b tryptophan hoc thm vo mi trng axit 3-indoleacrylic.
Promotor pL c kim sot bi protein kim hm cI ca bacteriophage .
Promotor ca bacteriophage T7 i hi phi c enzyme polimerase T7 ARN
thc hin phin m.
3.2. K thut biu hin gene
Gene c biu hin l gene c phn lp v bit trnh t. Cc
gene ny c th c phn lp bng cc phng php khc nhau. Sau khi c
on gene mong mun chng s c biu hin trong cc h thch hp theo cc
bc cc cnh.
Bc 1: La chn h thch hp biu hin gene.
Bc 2: Da vo trnh t gene, thit k cc on mi nhn gene. Tng
hp mi.
Bc 3: Chun b ADN khng nhn gene.
Bc 4: Nhn gene bng PCR t ADN h gene ca vi khun hoc t ngn
hng cADN nhng tt nht l nhn gene trc tip t chnh plasmid mang gene
tng dng c trnh t gene .
Bc 5: a gene vo vector tch dng v bin np vo t bo E. coli
khuch i dng gene.
Bc 6: Tch plasmid t cc th bin np E. coli. Kim tra gene trong
vector tch dng bng enzym hn ch. Kim tra gene v c bit l chiu ca
gene trong vector biu hin. Gene cn biu hin phi nm ng chiu ca
prromotor.
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Bc 7: a gene vo vector biu hin v bin np vo t bo E. coli..

Bc 9: Bin np vector biu hin c mang gene vo t bo ch (E. coli
hoc nm men). Bt u t bc ny, cc thao tc biu hin gene cc chng
khc nhau l khc nhau.
Bc 10: Kim tra s biu hin ca gene.
3.2.1. La chn h biu hin
E. coli v Nm men l hai h c s dng nhiu trong k thut biu hin
gene. E. coli c dng biu hin rt nhiu gene c ngun gc c t
Prokaryot v Eukaryot. Nm men l c th c ch c s dng rt rng ri
sn xut nhiu loi protein ti t hp dng lm dc liu cha bnh v c
protein dng trong cng nghip thc phm. Cc gene khi c biu hin trong t
bo E. coli trnh c hin tng protein b glicosil ho, trong khi nm
men li c hin tng glicosil ha. T bo E.coli sinh trng nhanh, thun tin
cho cc thao tc sinh hc phn t nn E. coli cng thng c chn biu
hin gene dng cho nhng mc ch nghin cu khc nhau. Tuy nhin, vic biu
hin cc on gene ln trong t bo E. coli c nhng hn ch nh protein ti t
hp b tn ti dng khng tan, dng th vi v khng c tit ra ngoi mi
trng. nm men cc protein ti t hp ny thng c tit ra ngoi mi
trng rt hiu qu, c tinh sch cao v m bo hot tnh sinh hc ca
chng. Men c th biu hin v tit tt cc sn phm c ngun gc t ng vt,
thc vt bc cao, nm v vi khun ra ngoi mi trng.
3.2.2. Thit k mi nhn gene
Mi dng nhn gene bng PCR l mt cp mi (mi xui v ngc
c thit k bm vo vng tn cng 2 u ca gene cn nhn.
Trc khi thit k mi nhn gene, vic u tin quan trng l phi
kim tra trnh t gene, quyt nh on gene cn nhn a vo biu hin v
kim tra cc im ct ca enzyme hn ch trong on gene. tin cho vic a
gene vo vector biu hin, cc on mi nhn gene thng c thit k c cha
thm trnh t ca enzyme hn ch 2 u tn cng ca gene. Khi gene c th
c ct trc tip bng cc enzyme ny da vo vector biu hin. Trnh t
enzyme hn ch c a thm vo mi cng c la chn rt k cng. Trc
ht cc trnh t ny phi c mt trong vng a ni (polilinkers, multiple cloning
sites) ca vector biu hin. Tip theo, cc trnh t ny phi l trnh t rt him
gp trong gene cn biu hin (tt nht l trnh t ny khng c mt trong gene
cn biu hin). Trnh t enzyme c thm vo mi xui nn l trnh t enzyme
gii hn ng ngay u 5' ca vng a ni cn enzyme hn ch c thm vo
mi ngc l trnh t enzyme ng tn cng u 3' ca vng a ni. Nu 2 mi
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c thm 2 enzyme gii hn khc nhau th enzyme gii hn mi xui phi ng

trc cnzyme gii hn mi ngc trong vng a ni, tnh theo chiu promotor
m bo on gene a vo s nm xui chiu vi promotor.
Mi lun c thit k v c ghi theo chiu t 5' n 3' ca gene. Mi
xui c trnh t tng ng vi on trnh t ca si khng m ho (si
antisense). on trnh t tng ng thng c s dng mi mi m
bo kt cp c hiu l khong trn 15 nucleotit tr ln. Tn cng u 5' ca mi
nn d ra t nht mt nucleotit on gene nhn ln c trnh t c nguyn
vn. Nucleotit d ra u 5' khng nn l nucleotit c kh nng bt cp tng
ng tt vi nucleotit tn cng u 3' trn cng on mi, kch thc on
mi cng khng nn qu di. Ti thiu ch cn 18 nucleotit ca on mi bt cp
chnh xc vi on gene cn nhn l c. Tt nhin di ca on mi c th
thay i ph thuc vo t l cc gc A, T ti v tr trong on gene m on mi
cn bt gp.
Sau khi thit k c mi, vic quan trng l kim tra s dch m ca
on gene s c nhn ln bng 2 mi va thit k trong vector biu hin.
Khung c ca gene phi c m bo mt cch nghim ngt.
3.2.3. Chun b ADN khun nhn gene
nhn c gene bng PCR, nguyn liu u tin cn phi chun b
l ADN khun.
3.2.4. Nhn gene bng PCR
Trc ht, si ADN khun phi c bin tnh si ADN khun tr
thnh dng si n. Sau vic tng hp v nhn gene din ra vi khong 25 30 chu k. Sau khi kt thc cc chu k trn, mu c 750C trong khong 3
n 6 pht, sau gi mu 40C.
tin hnh nhn gene bng PCR, ngoi 2 on mi v ADN si khun
ra th phc hp mu c trn cn c nc kh ion v trng, 4 loi
deoxyribonucleotit, Taq DNA polimerase hoc Vent polimerase, m thch hp
cho Taq polimerase hoc Vent polimerase, MgCl2. Thng thng MgCl2 c sn
trong m nn c th khng cn phi b sung thm na. Nng MgCl2 trong
mu cng ln th cng lm tng kh nng bt cp khng c hiu ca mi. Taq
polimerase tng hp gene c d ra mt nucleotit A u 3' cn Vent polimerase
tng hp gene cho u bng. V vy, tu theo iu kin tch dng m chn Taq
hay Vent cho PCR. Nu sn phm PCR c a vo vector tch dng l u
bng th nn chy PCR bng Vent. Vent t gy ra sai st trong trnh t gene c
nhn hn Taq. Nu s dng mt s vector tch dng c mua t cc hng, cc
vector ny c thit k dng thng v c d nucleotit T c kh nng kt
144

cp tng ng vi nucleotit A d ra sn phm PCR th nn s dng Taq

polimerase.
Kim tra sn phm PCR trn gel agarose. ADN tch in m c kh nng
chuyn ng trong in trng theo chiu t cc m ti cc dng. Tc
chuyn ng ph thuc vo 2 yu t: dng tn ti ca ADN v kch thc ADN.
Cc ADN c kch thc ln s chuyn ng chm trong gel agarose cn ADN
c kch thc nh s di chuyn nhanh hn. CC ADN dng siu xon cng
chuyn ng nhanh hn ADN dng thng. T mt phm vi nht nh ph thuc
vo nng gel agarose, kch thc phn t tuyn tnh vi qung ng ch
chuyn ca ADN trn gel agarose. Thng cc on gene c kch thc t 300
n 10000bp c th c phn tch trn gel agarose 0,8%. on gene cng nh
th nng agarose cng phi tng ln v pht hin cc on gene nh ti vi
chc nucleotit hoc mt chui nh nucleotit ngi ta thng phi s dng gel
poliacrylamid. Cc on gene trn gel agarose c pht hin bng cch nhum
vi EtBr hoc bng nh du phng x. Thng thng nht l cc on gel c
nhum bng ethidium bromit v c pht hin bng n chiu tia UV.
xc nh c kch thc sn phm PCR, thng sn phm PCR c
chy song song vi on phc hp ADN chun bit kch thc.
3.2.5. a gene vo vector tch dng v bin np vo t bo E. coli
a gene vo vector tch dng
Sn phm PCR gn trc tip vi vector v sau c bin np vo t
bo E. coli DH5 chn lc. Plasmid dng cho tch dng dc s dng rng
ri l pCR2.1 v pUC118. Hai vector tch dng ny c kh nng cho s lng
bn sao ln trong t bo (200 bn sao/t bo), c kh nng ti cc on gene tt
v n nh trong t bo ch. a on gene vo vector tch dng bng u
bng, trc ht plasmid phi c ct trc tip bng enzyme hn ch u bng
c mt trong vng a ni ca vector tch dng. Sau khi c ct bng mt
enzyme, plasmid rt d b ng vng ngay c khi khng c DNA-ligase, v vy
vector c m vng phi c x l bng Alkaline photphatase. Enzyme
ny c kh nng loi gc phosphate u 5' ca ADN.
Sn phm PCR khng c gc phosphate. V vy, c th gn vi vector
b loi phosphate th sn phm PCR phi c gn thm gc phosphate nh
polinucleotit kanase. Nu sn phm PCR c nhn ln bng Taq polimerase th
trc khi gn phosphate, on ADN phi c lm bng u bng enzyme.
X l ADN bng enzyme hn ch
Phn ng ni ghp gene
145

Bin np sn phm lai vo t bo E. coli: C hai cch chnh bin np

sn phm lai vo t bo E. coli l phng php sc nhit v phng php xung
in.
3.2.6. Tch ADN plasmid v kim tra gene trong plasmid
Tch plasmid
Cc t bo E. coli mang plasmid sau khi nui cy n pha dng th c li
tm th li. Thnh v mng t bo c ph v bng kim v cht to sc cng
b mt. Sau ADN h gene v protein c ta li cng vi SDS bng mui
axetat kali v b loi b khi ADN plasmid bng cch li tm. ADN plasmid c
ta li bng cn.
Kim tra gene trong plasmid
Gene ngoi lai trong plasmid c kim tra s b da trn trnh t cc
enzyme hn ch c trong gene. Enzyme hn ch c chn ct kim tra l
enzyme c th cho ra nhng on ADN c kch thc thch hp c th pht hin
c trn gel agarose.
3.2.7. a gene vo vector biu hin
Cc plasmid tch dng mang on gene mong mun c ct hon ton
bng hai enzyme hn ch c thit k hai mi. Vector biu hin cng
c ct bng enzyme tng t. Sau vector v on gene c kch thc nh
mong mun c tinh sch t gel agarose v c ni li vi nhau bng DNAligase.
Nu on gene c a vo vector biu hin bng mt enzyme hn ch
th phi kim tra chiu ca gene trnh hin tng cc on gene nm ngc
chiu vi chiu phin m cc promotor.
3.2.8. Bin np vector biu hin vo t bo ch
Vic bin np vector vo t bo E. coli v t bo nm men l khc nhau.
Cc t bo E. coli thng c bin np bng phng php sc nhit (nh
c trnh by phn trn) cn cc t bo nm men c bin np bng phng
php xung in hoc bng mui lithium acetat nhng phng php xung in
cho hiu qu bin np tt hn v c s dng rng ri hn l phng php
dng mui.
3.2.9. Kim tra s biu hin ca gene
Bc quan trng kim tra s biu hin ca gene l nui cy cc th
bin np trong mi trng c cht cm ng promotor protein ti t hp c th
c tng hp. Tip theo, kim tra protein ti t hp ngi ta thng dng
phng php in di protein tng s trn gel acrylamit pht hin s b bng
protein ti t hp.
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3.3. Biu hin gene ngoi lai vi khun E. coli

Trong biu hin gene E. coli cn ch i vi cc protein c v tr
ngi ln nht l mt s protein ngoi lai rt c i vi E. coli (v d nh RIPs)
hoc protein c kh nng gn vo mng trong vi khun E. coli v ph hu phc
hp mng. Nh vy vic tng hp nhng protein ny chc chn b hn ch. Tuy
nhin, E. coli vn l mt chng ch c gi tr biu hin gene ngoi lai v
trong nhiu trng hp n vn l thng c a chung s dng. Khi biu hin
bt c mt gene ngoi lai no E. coli cn quan tm n vic la chn plasmid
v promolor ph hp cho qu trnh khi u phin m.
3.3.1. Cu trc vector biu hin E. coli
Vector dng biu hin gene Prokaryot c thit k y bao gm
tp hp tc nhn t di truyn c sp xp hp l nhm iu khin ti u c qu
trnh phin m, dch m tng hp nn protein c chc nng dng lm du
hiu chn lc. Gene thng c s dng trong vector biu hin l gene khng
cht khng sinh. Gene ny to iu kin thun li cho vic nhn bit cc dng t
bo mang plasmid. i vi plasmid t sao chp: mt trnh t khng th thiu
l tm sao chp. Tm sao chp quyt nh s lng bn copy ca plasmid trong
mt t bo. to iu kin thun li cho vic a cc on gene vo vector
biu hin theo ng chiu promotor. trn vector lun phi cha vng a ni
(Poilinker region) mang cc trnh t ca enzyme hn ch c tn s xut hin
thp trn cc gene cu trc.
3.3.1.1. Promotor
Cc promotor c kh nng biu hin gene cao c nhng c im chung
sau: (1) promotor mnh c kh nng to protein ngoi lai ln hn 10 n 30%
protein tng s ca t bo. (2) Promotor ch hot ng phin m gene mc
c s (phin m khi khng cm ng) vi mc nh ti a: (3) Promotor c
cm ng theo cch n gin nhng hiu qu cao. S cm ng bng nhit hoc
bng ho cht thng c s dng rng ri to ra lng ln protein mong
mun.
Operon lac l mt h iu ho hot ng ca gene Prokaryot cbit
n sm nht v promotor lac c s dng rng ri to protein ngoi lai.
Promotor t operon tryptophan ca E. coli c la chn biu hin
protein bn trong t bo. Promotor tryp c mt s u im: (1) l promotor
c nghin cu rt k; (2) l promotor mnh, cc gene ngoi lai c phin m
v dch m bi promotor tryp thng to ra lng protein ln; (3) mc biu hin
gene c bn (khi promotor khng c cm ng) tng i thp, v vy vic
biu hin protein ngoi lai s khng nh hng ti s sinh trng ca t bo v
147

lm cho plasmid tn ti n nh hn trong t bo; (4) vector biu hin s dng

promotor ny c th bin np vo hu ht cc chng vi khun E. coli, nh
ngi ta c th chn bt k chng E. coli no c c im mong mun biu
hin gene; (5) vic cm ng promotor l rt n gin k c khi s dng bnh lc
hoc ni ln men.
3.3.1.2. Terminator (vng kt thc phin m)
Qu trnh phin m c bt u t promotor, sau si mARN c
ko di n khi gp mt trnh t c hiu, ti qu trnh phin m b dng li.
Trnh t c hiu l vng kt thc phin m (terminator). C hai c ch
ngng phin m l c ch lm gim tc phin m v c ch dng khng c
hiu. V tr gim phin m (attenuator) mang c im ca mt terminator n
gin, c gn gia promotor v gene cu trc u tin trong mt operon. V
attenuator l yu t iu khin m (negative regulatory elements) nn thng
khng c s dng trong h biu hin.
Qu trnh ngng phin m khng c hiu c th xut hin trong bt k
mt qu trnh phin m no do c cha trnh t trn ARN thng tin ging vi
trnh t ca terminator mt cch ngu nhin. Cc yu t chng li s dng phin
m (antiterminator) cng c a vo vector biu hin chc chn on ARN
thng tin c tng hp c di y .
3.3.1.3. Peptit tn hiu (signal peptite)
Cc protein ngoi lai c tng hp trong t bo E. coli cn thit phi
c tit ra ngoi v: (1) E. coli tit rt t protein ra ngoi mi trng nn protein
ngoi lai nu c tit ra s c tinh sch cao. (2) Protein tit ra ngoi khoang
chu cht (perplasmid space) c kh nng hnh thnh cu trc bc 4 ng tr
thnh protein c hot tnh sinh hc. Qu trnh cun xon v hnh thnh cu
disulfit ca protein nm trong t bo thng b hn ch v dn n sai hng
trong cu trc protein. (3) Protein khi c tit ra ngoi s trnh b phn ct bi
mt s protease c trong t bo.
c tit ra khi mng trong t bo, cc chui polipeptit bt buc phi
c trnh t peptit tn hiu nm tn cng u N. Thng thng chui peptit tn
hiu c cu to gm 3 vng: vng tn cng u N, vng tn cng u C v
vng k nc. Mi vng c ti a 20 axit amin v ti thiu l 2 axit amin i vi
vng N, 8 axit amin i vi vng k nc v 6 axit amin i vi vng C.
3.3.1.4. im sao chp (Ori)
S lng bn sao ca plasmid trong t bo ph thuc vo nhn t khi
u s sao chp ADN ti mt v tr c hiu gi l tm sao chp (Ori). i vi
cc plasmid sao chp t do trong t bo ch, s lng bn sao t nht cng b
148

nh hng bi tc sinh trng ca t bo. Nhng plasmid a phin bn

thng b mt i qua cc th h t bo (10-5- 10-6 trn mt th h) khi t bo
mang plasmid c nui cy trong mi trng khng c p lc chn lc hoc
gene trn plasmid m ho cho protein gy c cho t bo.
3.3.1.5. Du chun chn lc
tin li cho vic nhn ra cc t bo bin np c mang plasmid, thng
thng ngi ta s dng mi trng nui cy m ch c t bo c tnh trng
chn lc nht nh mi c th sinh trng c. C mt cch gip d dng nhn
bit cc th bin np mang plasmid bin np l a cc on gene khng
khng sinh vo plasmid . S c mt ca plasmid trong t bo gip cho t bo
sng st trn mi trng c cht khng sinh. V vy, gene khng khng sinh c
mt hu ht cc vector tch dng hin ang c s dng nh du chun chn
lc.
3.3.1.6. Vng a ni (Polilinker region)
Vng a ni bao gm trnh t nhn bit c hiu ca cc enzyme hn ch
hay c s dng gip cho qu trnh a on gene ngoi lai vo vector biu
hin tr nn d dng. Hu ht trnh t ca cc enzyme hn ch trong vector biu
hin u c t theo ng khung c v nm sau on peptit tn hiu (Signal
peptit). tin cho vic ghp ni cc on gene cn biu hin, cc im nhn
bit ca cc enzyme hn ch trong vng da ni c thit k sao cho chng ch
tn ti nh nhng v tr duy nht trn ton b phn t plasmid.
3.3.2. Qu trnh tng hp protein ngoi lai
Qu trnh khi u dch m trn phn t ARN thng tin E. coli i hi
mt trnh t nhn bit c hiu Shine - Dalgarno (SD) b sung vi tn cng 3'
ca ARN ribosom 16S. l trnh t 5' - UAAGGAGG - 3' ng trc b ba
khi u. B ba khi u thng thng nht l AUG. C khong 8% b ba khi
u dch m GUG, ngc li trnh t UUG, AUU l trnh t khi u rt him
gp, thng ch c mt cc gene iu khin mt cch t pht (v d gene m
ho cho protein S20 ca ribosome, v nhn t khi u s 3). Khong cch t
SD n m khi u thng l 8 nucleotit m bo s khi u dch m ti
u nht. Tuy nhin, s khi u dch m ch nh hng r rt khi khong cch
gim xung di 4 nucleotit hoc di hn 14 nucleotit. Thng Prokaryot,
qu trnh phin m v dch m din ra rt gn nhau, v vy cu trc bc 2 ca
mARN c th bao quanh trnh t SD lm cho qu trnh biu hin gene b gim.
iu ny c gii quyt bng cch lm tng trnh t tng ng ca SD vi
trnh t 5' UAAGGAGG v tng s lng adenin trong vng khi u. Cu trc
ca on thm vo ca ARN thng tin (Dowstream Box - DB) c nh hng ti
149

khi u dch m c t vo u m khi u v b sung vi baz nit 14691483 ca rARN 16S. Cc DB c trnh t l 5' - AUGAAUCACAAAGUG 3'
ng vai tr ch yu nh nhn t tng cng dch m. Mc d khi a trnh t
DB vo u 5' ca gene m ho cho protein ti t hp c th lm thay i trnh
t axit amin ca chng. Trong t bo tc ko di cc chui polipeptit khng
xy ra vi tc nh nhau. E. coli cc b ba trong gene c mc s dng
khc nhau ph thuc vo s c mt ca cc ARN vn chuyn tng ng trong t
bo. V vy cc gene c biu hin yu c th l do gene c cha nhiu b ba
ch dc nhn bit bi mt s loi ARN vn chuyn him gp. Trong 6 b ba
m ho cho Leu UUA, UUG, CUU, CUC, CUA, CUG, th ch c CUG c
nhn bit tt v c biu hin cao bi ARN vn chuyn c th cho b ba ny
trong t bo E. coli. E. coli, chui peptit mi tng hp ri khi phc h ARN
thng tin v ribosom nh cc nhn t nhn bit m kt thc: RF1 nhn bit m
UAA v UAG cn RF2 nhn bit UAA, UGA. Ba m kt thc UAA, UAG v
UGA c hiu qu dng dch m khc nhau, trong b ba UAA c cc nhn
t kt thc dch m nhn bit tt nht.
3.3.3. Qu trnh sa i protein sau khi dch m
3.3.3.1. Qu trnh thu phn do protease
Vic biu hin protein ngoi lai trong t bo E. coli nhiu khi khng hiu
qu do cc protein ny b thu phn bi protease c trong t bo ch. C nhiu
bng chng cho thy qu trnh thu phn ny ph thuc vo nng lng. V vy,
cc th t bin nh hng ti protease ph thuc nng lng s m bo n
nh ca protein ngoi lai. Bn cnh vic nh hng protein tit ra ngoi
khoang chu cht hoc ra mi trng nui cy t bo nhit thp, cho gene
ngoi lai biu hin cng vi s biu hin ca phn t "bo mu ("chaperone").
Trong t bo cc protease ny c chc nng quan trng l thu phn cc protein
bt thng v proteinkhng hon thin.
3.3.3.2. Qu trnh tit protein
Mt trong nhng vn hay gp khi biu hin protein ni bo ca t
bo E. coli l chng thng to thnh th vi (inclusion bodies). Mc d biu
hin protein di dng th vi cng c mt s u im nht nh nhng protein
to ra thng b mt hot tnh sinh hc v qu trnh ti to li cu trc
(refolding) tng i phc tp v tn km. Hu ht protein c biu hin trong
E. coli u c thit k sau khi biu hin chng c chuyn ra khoang chu
cht (perplasmic space) ca t bo. Mt yu t khng th thiu protein c th
c chuyn qua mng trong ra ngoi khoang chu cht l on peptit tn hiu.
3.3.3.3. Hnh thnh cu trc khng gian ca protein
150

Protein sau khi c tng hp mun tr thnh protein c hot tnh sinh
hc th chng phi c cu trc khng gian ng. Qu trnh hnh thnh cu trc
khng gian ph thuc rt nhiu vo mi trng tbn trong v bn ngoi t bo.
Nu mi trng c cht gy bin tnh mnh nh ur, guanidin - HCl v cc tc
nhn kh th qu trnh hnh thnh cu trc khng gian khng th din ra thun
li.
S to thnh cu disulfit trong qu trnh cun xon ca protein c th din
ra bi oxi ho trong khng kh c s xc tc ca ion kim loi hoc phc hp oxi
ho kh ca S.
Mt v d v s biu hin gene m ho cho RIP E. coli. RIP c biu
hin bng h vector pMAL - C2. Vector biu hin protein bt hot ribosome tipe
I c thit k da trn c s casette vector pMAL - C2 (RIKEN DNA Bank
Japan). tin hnh, cp primer c cha cc im ct ca EcoRI v HindIII
c tng hp v s dng nhn on gene m ho cho ton b phn t RIP
thnh thc (bao gm 247 amino axit, bt u t Asp24 v kt thc bng Ala27).
on gene nhn c bng PCR sau x l vi EcoRI v HindIII c gn vo
vng ct gn a v (MCS) ca vector pMAL - C2 ng cc v tr ct ca cc
enzyme k trn.

THO LUN
1. Th no l s phn lp gen? Nu nguyn tc v quy trnh ca cc phng
php phn lp gen.
2. Phn bit khi nim th vin b gene v th vin cADN.
3. Biu hin gene l g?
4. Gii thch k thut biu hin gene.
5. Biu hin gene ngoi lai vi khun E. coli

151

Ti liu tham kho chnh

1.

L Trn Bnh v cng s. Cng ngh sinh hc thc vt trong ci tin

ging cy trng. NXB Nng nghip H.1997.

2.

L Trn Bnh, L Th Mui. Phn lp gene v chn dng chng chu vi

iu kin bt li cy la. NXB i hc Quc gia, 1998.

3.

L Trn Bnh, Phan Vn Chi, Nng Vn Hi, Trng Nam Hi, L Quang
Hun. p dng cc k thut phn t trong nghin cu ti nguyn sinh vt
Vit Nam. NXB Khoa hc v K thut, 2003.

4.

Phan Vn Chi v cng s. S dng mt s primer ngu nhin xc nh

rapds la DT-33 v Tm thm. Tp ch di truyn v ng dng, s 41997, 15-18.

5.

Phan Vn Chi. Khi ph trong nghin cu proteomics. Tp ch Cng ngh

sinh hc 1(1), 2003.

6.

H Hunh Thu Dng. Sinh hc phn t. NXB Gio dc H.1997

7.

Helena Curtis. Sinh hc. Ngi dch: Phan C Nhn. NXB Gio dc.
H.1995.

8.

L Th Thu Hin. Lun n tin s sinh hc, 2003.

9.

L Thanh Ho. Sinh hc phn t: Nguyn l v ng dng. Ti liu ging

dy sau di hc, 2003.

10.

Phm Thnh H. Di truyn hc. NXB Gio dc H. 1998

11.

K yu Hi ngh Cng ngh Sinh hc ton quc. NXB Khoa hc v k

thut H. 1999.

12.

K yu Hi ngh Cng ngh Sinh hc ton quc. NXB Khoa hc v k

thut H. 2003.

13.

inh Duy Khng. ng dng cc k thut min dch hc v SHPT trong

chn on bnh ngi. The Second German-Vietnamese Workshop on
Genetic Engineering and Bioinformatics, Bi ging. 2002.

14.

Trn Th Phng Lin. Lun n tin s Sinh hc. 1999.

15.

L nh Lng, Phan C Nhn. C s di truyn hc. NXB Gio dc.

1997.

16.

L nh Lng. K thut di truyn. NXB Khoa hc v K thut. 2001.

17.

Chu Hong Mu. Lun n tin s sinh hc. 2001.

18.

Chu Hong Mu. Di truyn hc. Bi ging. 2001.

152

19.

Chu Hong Mu, Nguyn Th Tm. Gio trnh Di truyn hc. 2004.

20.

Nhng vn nghin cu c bn trong sinh hc. NXB i hc Quc gia,

2000.

21.

Nhng vn nghin cu c bn trong khoa hc s sng. NXB khoa hc

k thut, 2003.

22.

inh Th Phng. Lun n tin s sinh hc, 2001.

23.

Nguyn Tin Thng. Gio trnh sinh ho hin i. NXB Gio dc. 1998.

24.

Nguyn Xun Th v cng s. S dng du chun RAPD nhn dng

mt s ging chui trng Vit nam. Tp ch di truyn v ng dng, s 31998, 20-25.

25.

Phan Hu Tn. Phn tch c im ca gene t bin lm mt ng thi

cc subinit a v b ca protein b -Congglicinin bng k thut PCR u
tng. Tp ch di truyn v ng dng, s 3-1997, 34-38.

26.

T khoa hc sinh hc phn t n cuc sng v chm sc sc kho. Bo

co khoa hc Hi ngh Sinh hc phn t v ho sinh ton quc, 2003.

27.

Nguyn Vn Uyn. Nhng phng php cng ngh sinh hc thc vt.
Nh xut bn Nng nghip H. 1995, tp I, 48-60.

28.

Nguyn Vn Uyn. Nhng phng php cng ngh sinh hc thc vt.
Nh xut bn Nng nghip H.1995, tp II, 1995, 56-63.

29.

Biotechnology in Agrialture and Forestry. Vol 10, 1990. Legumes and

Oilseed Crops Edited hy baiiaj Y.P.S NXB Spriger - Verlag.

30.

Phan Vn Chi. Stategy for protein purifcation and identiication. The

Second German-vietnamese Workshop on Genetic Engineenng and
Bioinfurmatics. Bi ging, 2002.

31.

Foolad M.R., Siva A. and Rodriguer L.R.. Applications of polimerase

chain reaction (PCR) to plant genome analisis. I: Tissue and organe
culture. Fundamental methods. Spnnger Verlag Berhn, Heidelberg. 1995,
281-298.

32.

Nng Vn Hi. Gene Cloning (Molecular Cloning/ Recombinant DNA)

Techniques. The Second German-Vietnamese Workshop on Genetic
Engineering and Bioinformatics, Bi ging, 2002.

33.

Jena K.K. et al. RFLP analisis ofrice (Oryza sativa L.) introgression lines.
Theor. Appl. Genet 1992, 608-616.

34.

Karuzina I.P. Gio trnh v c s di truyn hc. Ngi dch Trnh B

Hu. NXB Mir, 1985.
153

35.

Long Mao. Molecular Markers and Their Applications in Rice and Wheat.
Dissertation for a degree of Doctor of Philosophy, 1995, 70-78.

36.

Monna L. Determination of RAPD markers in rice and their conversion

into sequence tagged sites (STSS) and STS..Specirc primer. ADN
Research 1, 1994, 139-148.

37.

Soybean: Genetics, Molecular Biology and Biotechnology Edited by D. P.

S Verma and R. C Shoemaker, CAB INTEARNTIONAL, 1996.

38.

Tanksley S.D et al. RFLP mapping in plant breeding: new tools for an old
science. Biotechnology vol 7, 1989, 257-263.

39.

Vierling R., Nguyen H.T. Use of RAPD marker to determine the genetic
relationships of diploid wheat geneotipes. Theor Appl. Genet. 84,1992,
835-838.

40.

Weslh J., Mcclelland M. Fingerprinting genome using PCR with

arbitraryprimer. Axit nucleics Res. 18, 1991, 7213-7218.

41.

Yu J., Nguyen H.T. Genetic variation detected with RAPD markers

among upland and lowland rice cultivars (oryza sativa L.). Theor Appl.
Genet, 87, 1994, 668-672.

154

NHNG CNG TRNH CA TC GI

CNG CC CNG TC VIN CNG B
1.

Chu Hong Mu, Nng Th Man (1994). nh hng ca tia gamma v

ho cht ho hc nitrozometilure (NMU) n kh nng ny mm v kh
nng sng st ca mt s ging u tng Glicine max (L.) Merrill Bc
Thi. Thng bo khoa hc, S 2, Chuyn ngnh Sinh hc, Trng i hc
S phm Vit Bc, 23-27.

2.

Chu Hong Mu, Nng Th Man (1995). Hiu ng t bo di tc ng

ca tia gamma v ho cht gy t bin nitrozometilure cc ging u
tng Glicine max (L.) Merr. Thng bo khoa hc, S 1, Trng i hc
S phm - i hc Thi Nguyn, 62-66.

3.

Chu Hong Mu, Nng Th Man (1995). nh hng ca tia gamma v

ho cht ho hc nitrozometilure n tn s v bin d ca cc ging u
tng Glicinemax (L) Merrill. Thng bo khoa hc, S 2, Trng i hc
S phm - i hc Thi Nguyn.

4.

Chu Hong Mu, Nh Tin, Nng Th Man, o Vit Bc, L

Trn Bnh (1995). Kh nng phn ng kiu gene ca mt s ging u
xanh Vigna radirata (L) M1 v M2 i vi cc tc nhn t bin vt l ho hc. Thng bo khoa hc, S 3, Trng i hc S phm - i hc
Thi Nguyn.

5.

Chu Hong Mu, Nh Tin, Nng Th Man, o Vit Bc, L

Trn Bnh (1996). So snh nh hng ca cc tc nhn vt l, ho hc
ln qu trnh sinh trng, pht trin v bin d ca cc ging u xanh
Vigna radiata (L.) a phng M1. Tp ch Khoa hc v Cng ngh, S
1, i hc Thi Nguyn, 49-55.

6.

Chu Hong Mu, Nng Th Man (1996). Ph bin d hnh thi v gi tr

trung bnh mt s tnh trng s lng ca hai ging u xanh mc tiu
Bo Lc v m Ch n M1. Thng bo khoa hc, S 4, Trng i
hc S phm - i hc Thi Nguyn, 81-88.

7.

Chu Hong Mu, Nng Th Man (1997). Bin d v di truyn ca mt s

tnh trng s lng ca cc ging u tng M3. Tp ch Khoa hc v
Cng ngh, 1(2), i hc Thi nguyn, 28-32.

8.

Chu Hong Mu, on Bch Lin, Nng Th Man, o Vit Bc, L

Trn Bnh (1997), Hiu qu ca x l thun v phi hp cc tc nhn t
155

bin n cc ging u tng a phng M1. Tp ch Khoa hc v

Cng ngh, 2(3), i hc Thi nguyn, 25-29.
9.

Chu Hong Mu, Nng Th Man, o Vit Bc, L Trn Bnh (1997).
nh gi mt s dng t bin t hai ging u tng H4 v Cc vng
th h M4. Tp Ch sinh hc, 19(3), 53-56.

10.

Chu Hong Mu, on Bch Lin, Nng Th Man, L Trn Bnh

(1997). c im phn ng ca ging u tng a phng Lng Sn
M2. Tp ch di truyn v ng dng, S 3. 24-28.

11.

Chu Hong Mu (1997). Hiu qu gy bin d ging u tng Lng

Sn khi x l ring r v phi hp tia gamma vi cc ho cht
ethyleneimine v nitrosomethyl urea. Tp ch Di truyn v ng dng, S
4, 1-5.

12.

Chu Hong Mu, Nh Tin, Nng Th Man, o Vit Bc, L

Trn Bnh (1997). c im ca s pht sinh bin d th h th hai ca
hai ging u xanh a phng, min ni. K yu Annual Report, Vin
Cng ngh Sinh hc, Nh xut bn khoa hc k thut, 76-83.

13.

Chu Hong Mu (1997). To vt liu khi u trong chn ging u

tng bng phng php t bin thc nghim. ti NCKH cp c s

14.

Chu Hong Mu, on Bch Lin, Nh Tin, Nng Th Man

(1998). Gy bin i nhim sc th u tng v u xanh bng x l
ring r v phi hp tia gamma vi cc ho cht etilen imin v
nitrozometilure. Thng bo khoa hc, S 2, Trng i hc S phm i hc Thi Nguyn, 29-35.

15.

Chu Hong Mu, Nh Tin, Nng Th Man, o Vit Bc (1998).

Nghin cu hm lng protein, lipit v thnh phn axit amin ca mt s
dng u dng t bin M2. Tp ch Khoa hc v Cng ngh, 2(5), i
hc Thi Nguyn, 39-44.

16.

Chu Hong Mu (1998). Nghin cu l thuyt v k thut sinh hc phn

t hin i ng dng trong phng php t bin thc nghim, gp phn
chn to ging u tng v u xanh. Tp ch Khoa hc v Cng ngh,
4(7), i hc Thi Nguyn, 63-68.

17.

Chu Hong Mu, on Bch Lin, Nng Th Man, L Trn Bnh

(1998). Nghin cu h s di truyn v mt s ch tiu ho sinh nhm nh
gi cc dng t bin u tng Lng Sn. Tp ch Di truyn v ng
dng, S 1, 6-10.

18.

Chu Hong Mu, on Bch Lin, Nng Th Man (1998). c im

156

ca s pht sinh bin d ging u lng Lng Sn trong th h t

bin th ba. Tp ch Di truyn v ng dng, S 1, 28-34.
19.

Chu Hong Mu, Nh Tin, Nng Th Man (1998). c im cc

bin d s lng ca hai ging u xanh a phng th h t bin M3.
Tp ch Di truyn v ng dng, S 3, 40-45.

20.

Chu Hong Mu, Nng Th Man, o Vit Bc, L Trn Bnh (1998).
To bin d hai ging u tng Cc vng v H4 bng tc nhn vt l
ho hc. Tp ch Sinh hc. 20(2), 55-58.

21.

Chu Hong Mu, Nng Th Man, L Xun c, inh Th Phng, L

Trn Bnh (1999). ng dng k thut phn tch hin tng a hnh cc
phn on ADN c nhn bn ngu nhin (RAPD) vo vic nh gi b
gene ca cc dng u xanh t bin. Tp ch Sinh hc. 21(3), 15-23.

22.

Chu Hong Mu, Nng Th Man, L Trn Bnh (2000). nh gi mt

s tnh trng kinh t quan trng v kh nng chu hn ca cc dng u
tng (Glicine max (L.) Merrill) t bin. Tp ch Khoa hc v Cng
ngh, 1(13), i hc Thi Nguyn: 16-21.

23.

Chu Hong Mu, Nng Th Man, L Xun c, inh Th Phng, L

Trn Bnh (2000). nh gi geneom ca mt s dng u tng t bin
bng k thut phn tch tnh a dng ADN c nhn bn ngu nhin.
Tp ch Sinh hc, 22/(1). 21-26.

24.

Chu Hong Mu, Nng Th Man, Nguyn Huy Hong, Trn Th

Phng Lin, L Trn Bnh (2000). Nghin cu hin tng a hnh
protein ca cc dng u tng v u xanh t bin. K yu Annual
Report, NXB Khoa hc k thut H Ni.

25.

Chu Hong Mu, Nh Tin, Nng Th Man, L Trn Bnh (1996).

Nghin cu mt s bin d hnh thi v kinh t quan trng ca cc ging
u xanh Vigna Radiata(L.) cc th h t bin M1, M2. K yu Annual
Report, NXB Khoa hc k thut H ni.

26.

o Xun Tn, Nng Th Man, Chu Hong Mu (2000). nh gi mt

s dng u xanh t bin M6. Tp ch Khoa hc v Cng ngh, 3(15),
i hc Thi Nguyn, 46-49.

27.

Chu Hong Mu, Nng Th Man, L Trn Bnh (2001). Phn tch mt
s ch tiu ho sinh trong ht ca cc dng t bin u tng v u
xanh. Tp ch Khoa hc v Cng ngh, 1(17), i hc Thi Nguyn, 5963.

28.

Chu Hong Mu, Nguyn Phc Chnh, Hong Mai Phng, Nguyn
157

Lam in (2000). Phng th nghim Sinh hc hin i vi s pht trin

khoa hc v cng ngh phc v sn xut Nng - Lm nghip a phng.
Tp ch Khoa hc v Cng ngh, 1(17), i hc Thi Nguyn, 64-69.
29.

Th Dng, Chu Hong Mu, Nng Th Man (2000). Su tp v

nh gi mt s ging la cn a phng. Tp ch Khoa hc v Cng
ngh, 1(17), i hc Thi Nguyn, 13-16.

30.

Hong Mai Phng, Chu Hong Mu, Nng Th Man, Nguyn Th

Ngc Lan (2000). Nghin cu thnh phn in di protein d tr ht ca
mt s ging la cn v cc dng lc t bin. Tp ch khoa hc v Cng
ngh, 1(17), i hc Thi Nguyn, 26-29.

31.

Chu Hong Mu, Nng Th Man, L Trn Bnh (2001). nh gi nng

sut, cht lng ht v kh nng chu hn ca cc dng u xanh t
bin Vigna Radiata (L.) Willzeck. Tp ch Sinh hc, 23/(1).

32.

Chu Hong Mu (2000). Hm lng protein v c im b r ca cc

dng u tng t bin giai on ny mm v cy non. Thng bo
khoa hc, s 2, Trng i hc S phm - i hc Thi Nguyn, 26-31.

33.

Chu Hong Mu (2000). Nghin cu hin tng protein a hnh v mt

s ch tiu sinh ho gp phn chn lc cc dng u tng v u xanh
t bin. ti KHCN cp B, M s: B98-03-20.

34.

Chu Hong Mu (2000). Nghin cu phc v pht trin ngnh trng trt
khoa Sinh-K thut nng nghip, trng i hc S phm Thi Nguyn.
Hi tho cng tc chuyn giao khoa hc cng ngh Nng - Lm khu vc
Trung du min ni pha Bc. i hc Thi Nguyn, 43-44.

35.

Chu Hong Mu, Th Dng, Hong Mai Phng, Nng Th Man

(2001). Kh nng chu hn ca mt s ging la cn a phng giai
on cy m. Kt qu nghin cu v Sinh hc v Ging dy sinh hc,
NXB Khoa hc v K Thut, 149-156.

36.

Th Dng, Chu Hong Mu, Nng Th Man (2001). nh gi cht

lng ht ca mt s ging la cn a phng. Kt qu nghin cu v
Sinh hc v Ging dy sinh hc, NXB Khoa hc v K Thut, 35-39.

37.

Nguyn Th Hng, Chu Hong Mu (2001). Nghin cu c im

phn ng kiu gene ca mt s ging la np nng a phng giai
on cy m trong iu kin gy hn nhn to. Kt qu nghin cu v
Sinh hc v Ging dy sinh hc, NXB Khoa hc v K Thut,. 69-75.

38.

Nguyn Th Ngc Lan, Chu Hong Mu (2001). Nghin cu c im

hnh thi, cht lng ht v hm lng protein, chlorophyll ca ba ging
158

la cn a phng. Kt qu nghin cu v Sinh hc v Ging dy sinh

hc, NXB Khoa hc v K Thut 122-125.
39.

Chu Hong Mu, Hong Cao Nguyn, Nguyn Thu Hin, L Ngc
Cng (2001). Nghin cu c im hnh thi v mt s ch tiu sinh ho
ca cy c phng v (Pteris multifida Poir). Kt qu nghin cu v Sinh
hc v Ging dy sinh hc, NXB Khoa hc v K Thut. 161-163.

40.

Hong Mai Phng, Chu Hong Mu, Nguyn Thu Hin (2001).
Nghin cu c im hnh thi, ho sinh ht ca mt s dng lc trng ti
Lo Cai. Kt qu nghin cu v Sinh hc v Ging dy sinh hc, NXB
Khoa hc v K Thut, 173-176.

41.

Chu Hong Mu, Nng Th Man, L Trn Bnh (2001). nh gi nng

sut, cht lng ht v kh nng chu hn ca cc dng u xanh t
bin - Vigna radiata (L.) Wilzeck. Tp ch Sinh hc, 23(1). 54-60.

42.

Chu Hong Mu, Nng Th Man, L Trn Bnh (2001). Hm lng

axit amin trong ht ca cc dng u tng (Glicine max (L.) Merrill v
u xanh (Vigna radirata (L.) Wilczeck) t bin. Tp ch Sinh hc, 23(4),
57-61.

42

Chu Hong Mu, Nguyn Th Hi Yn, Nguyn Thu H (2002). Kt

qu phn tch c im hnh thi, khi lng v cht lng ht ca mt
s ging la cn su tp Thi nguyn v H Giang. Tp ch Khoa hc
v Cng ngh, 2(22), i hc Thi Nguyn, 62-67.

43.

Chu Hong Mu, Nguyn Th Sm (2002). nh gi mt s ging lc

Arachis hypogea (L.) trng ti Bc Giang. Tp ch Khoa hc v Cng
ngh, 3(23), i hc Thi Nguyn, 5-10.

44.

Chu Hong Mu, Thn Th M Ngc (2002). c im hnh thi, khi

lng v ho sinh ht ca mt s dng lc t bin. Tp ch Khoa hc v
Cng ngh, 3(23), i hc Thi Nguyn, 59-62.

45.

Chu Hong Mu, L Thanh Sn (2002). Kho st c im hnh thi,

cht lng ht v nhng bin ng hm lng protein giai on mm
ca mt s ging la cn a phng. Tp ch Khoa hc v cng ngh,
3(23), i hc Thi Nguyn, 68-76.

46.

Chu Hong Mu (2002), Nghin cu c im ho sinh v sinh hc phn

t ca mt s ging la cn a phng gp phn chn lc ging la cn
thch hp cho vng ni ng Bc Vit Nam. ti KH-CN cp B, m s
B2001-03-14.

47.

Nguyn Thu H, Chu Hong Mu, Nguyn Th Hi Yn, Th

159

Dng (2003). a dng sinh hc cy la cn min ni pha bc Vit

Nam. Nhng vn nghin cu c bn trong khoa hc s sng. NXB
Khoa hc v K thut, 86-89.
48.

Nguyn Th Kim Dung, Chu Hong Mu (2003). Cht lng ht ca

cc ging lc L05, L08, L12, L14, MD7. Nhng vn nghin cu c bn
trong khoa hc s sng. NXB Khoa hc v K thut, 306-308

49.

Nguyn V Thanh Thanh, Chu Hong Mu (2003). nh gi cht

lng ht ca mt s ging u xanh Vigna radiata (L.) Willzech. Nhng
vn nghin cu c bn trong khoa hc s sng. NXB Khoa hc v K
thut, 398-400.

50.

Chu Hong Mu, Nguyn Th Sm (2003). Nghin cu ph in di

protein v thnh phn axit amin trong protein lc ca mt s ging lc
(Arachis hypogaea L.). Nhng vn nghin cu c bn trong khoa hc
s sng. NXB Khoa hc v K thut, 348-351.

51.

Nguyn Thu Hin, Chu Hong Mu, Nguyn Th Sm (2003). Nghin

cu mt s c im ho sinh trong giai on ht ny mm v cy non
ca mt s ging lc (Arachis hypogaea L.). Nhng vn nghin cu c
bn trong khoa hc s sng. NXB Khoa hc v K thut, 315-318.

52.

Hong Th Thu Yn, Chu Hong Mu, Nghim Ngc Minh, Nng
Vn Hi, Trnh nh t (2003). Phn lp gene chaperonill cc dng
u tng t bin ML10, ML48 v ML61. Nhng vn nghin cu c
bn trong khoa hc s sng. NXB Khoa hc v K thut, 1073-1076.

53.

Chu Hong Mu (2004). Nghin cu v protein v thnh phn axit amin

trong protein ht ca mt s ging la cn a phng. Nhng vn
nghin cu c bn trong khoa hc s sng nh hng nng lm nghip
min ni. NXB Khoa hc v K thut, 517-520.

54.

Nguyn V Thanh Thanh, Chu Hong Mu (2004). Nghin cu thnh

phn in di protein d tr trong ht ca mt s ging u xanh. Nhng
vn nghin cu c bn trong khoa hc s sng nh hng nng - lm
nghip min ni. NXB Khoa hc v K thut, 622-625.

55.

Hong Th Thu Yn, Chu Hong Mu, Nguyn nh Cng, Trn

Th Phng Lin, Nng Vn Hi (2004). c im nng sinh hc, ho
sinh ht v trnh t gene chaperonin ca dng u tng t bin Ml61
c kh nng chu hn. Nhng vn nghin cu c bn trong khoa hc s
sng nh hng nng - lm nghip min ni. NXB Khoa hc v K thut,
733-737.
160

56.

Phm Th Thanh Nhn, Chu Hong Mu, V H (2004). Nghin cu

c im huyt hc, ho sinh mu v ph in di protein huyt thanh
SDS-PAGE ca mt s bnh nhn ung th ph. Nhng vn nghin
cu c bn, trong khoa hc s sng nh hng y dc hc. NXB Khoa
hc v K thut, 291-294.

57.

Phm Th Thu Nga, Chu Hong Mu, V Th Thu Thu (2004). Kt

qu nh gi v nghin cu tnh a dng di truyn ca mt s ging la
cn a phng su tp t hai tnh Bc Cn v Cao Bng. Hi ngh khoa
hc trng i hc Khoa hc T nhin - i hc Quc gia H Ni.

58.

Chu Hong Mu, Nguyn Th Kim Dung (2004). Nghin cu tnh a

hnh protein trong ht ca mt s ging lc (Arachis hypogaea L.) bng
k thut in di SDS-PAGE. Hi ngh khoa hc trng i hc Khoa hc
T nhin - i hc Quc gia H Ni.

161

Chu trch nhim xut bn:

Gim c INH NGC BO
Tng bin tp L A
Ngi nhn xt:
PGS.TS NGUYN TRNG LNG
TS. LNG TH HNG VN
Bin tp:
TRN TH THANH BNH
K thut vi tnh:
O PHNG DUYN
Trnh by ba:
PHM VIT QUANG

C S V PHNG PHP SINH HC PHN T

In 1000 cun, kh 17 x 24cm, ti Cng ti In Thi Nguyn.
Giy php xut bn s: 16 - 1845/XB - QLXB, k ngy 20/10/2004.
In xong v np lu chiu thng 7 nm 2005.

162