Chng 20.

DI TRUYN HC
VI SINH VT
Bin son: Phm Thnh H, Nguyn Ln Dng
20.1. M U
Mi n nhng nm 40 ca th k trc, cc i tng vi sinh vt mi
bt u c s dng vo nghin cu di truyn hc. Tuy ra i chm, nhng
cc pht minh quan trng c tnh cch mng trong sinh hc nh chng minh
trc tip ADN l vt cht di truyn, sao chp ADN, sinh tng hp protein, k
thut di truyn,... u thc hin trn cc i tng vi sinh vt. Do vy, c th
ni di truyn hc vi sinh vt ng vai tr cch mng ha, to ra nhng
bc tin nhy vt cho di truyn hc ni ring, hnh thnh Sinh hc phn t
v s pht trin Sinh hc hin i ni chung c v phng din l thuyt cng
nh ng dng thc tin.
Di truyn hc ca vi sinh vt gp phn ch yu cho s pht trin di
truyn hc phn t, m nh cao chi li l s ra i k thut ti t hp
ADN hay k thut di truyn (KTDT), lm bng n Cch mng Cng ngh
sinh hc.
20.1.1. Tnh cch mng ca Di truyn hc vi sinh vt
Nhn loi ang sng cui thp nin u tin ca th k 21 th k
Cng ngh sinh hc. Di truyn hc i su vo cc vn c bn ca s tn
ti v lu truyn s sng nn n gi mt v tr c bit quan trng, c ngi
v "Di truyn hc l tri tim ca Sinh hc", v khng t th nhiu, n lin quan
v chi phi bt k lnh vc no ca sinh hc, t cc c ch phn t ca s
sng cho n s tin ha ca ton b th gii sinh vt trn hnh tinh ca
chng ta.
Nhng pht minh ln vi s lng tng vt ca di truyn hc c tc
dng cch mng ha sinh hc, bin sinh hc t m t thnh thc nghim

chnh xc. Hi ngh Di truyn hc th gii Toronto (Canada) nm 1988

nu phng chm "Di truyn hc ha" Sinh hc.
Sut th k XX, cc nghin cu tp trung gii quyt vn gen dn n
Thi i gen, Cch mng di truyn, m nh cao l vic hon tt B gen
ngi vo nm 2003 v m ra Thi i sau B gen (Post-Genomeics Era).
Cc i tng vi sinh vt gp phn ch yu vo cc pht minh nn
tng khng nhng ca di truyn hc phn t, m ca sinh hc phn t v sinh
hc hin i ni chung. im qua lch s cc pht minh ca sinh hc phn t
s thy r vai tr cch mng ha ca cc i tng ny.
20.1.1.1. Gi thit mt gen mt enzym
Vo nm 1941, G. Beadle v E. Tatum tin hnh th nghim trn vi
nm Neurospora crassa. Cng trnh chng minh mi lin h gia gen v tnh
trng thng qua vic kim sot cc enzym protein v qua kim sot cc
phn ng sinh ha trong t bo. Gi thuyt ny m ra mt trang mi v mi
quan h chc nng gia gen v enzym trong con ng trao i cht ca c
th. Pht minh ny c ngha quan trng: l bc chuyn tip t di truyn
hc c in sang di truyn phn t. Chnh v vy, hai ng c nhn gii
thng Nobel vo nm 1958.
20.1.1.2. Cc chng minh trc tip rng ADN l vt cht di truyn
Ngay sau khi cc nh lut ca Mendel c pht hin li vo nm
1900, di truyn hc pht trin rt nhanh vi thuyt di truyn NST v nhiu
thnh tu khc nh gy t bin nhn to... Tuy nhin cho n 1940, vn
cha c mt bc tin trin no trong hiu bit bn cht ho hc ca vt liu
di truyn v cha hiu c bng cch no gen trn nhim sc th biu hin
ra tnh trng. Trong mt thi gian di, mc d c nhiu s liu gin tip cho
thy ADN l vt cht di truyn, nhng protein vn c coi l thnh phn
ch yu ca vt liu di truyn v n c cu trc phn t kh phc tp. Do vy,
cc chng minh trc tip trn cc Vi sinh vt c ngha quyt nh trong xc
nhn vai tr ca ADN.
a. Bin np: truyn thng tin di truyn nh ADN.
Nm 1928, Griffith pht hin hin tng bin np (transformation) vi
khun Diplococus pneumoniae (nay gi l Streptococus pneumoniae. Nm

1944, T.Avery, Mc Leod v Mc Carty xc nh r tc nhn gy bin np

l ADN. Hin tng bin np l bng chng trc tip u tin xc nhn rng
ADN mang thng tin di truyn.
b. S xm nhp ca ADN virut vo vi khun.
Nm 1952, A.Hershey v M.Chaz tin hnh th nghim vi
bacteriophage T2 (virut ca vi khun hay gi tt l phage) xm nhp vi khun
E. coli, chng minh trc tip rng ADN ca phage T2 xm nhp vo t bo
vi khun v sinh sn to ra th h phage mi mang tnh di truyn c kh nng
tip tc ly nhim cc vi khun khc. Kt lun: Vt cht di truyn ca phage
T2 l ADN.
Nm 1952 din ra nhiu cuc tranh lun si ni v vai tr ca ADN:
l vt cht di truyn. Sau , nm 1953 m hnh Waston v Crick t du
chm kt cho giai on nghi ng rng ADN c l vt liu di truyn hay
khng.
Cc chng minh trc tip nu trn l nhng tin quan trng cho
pht minh ra m hnh cu trc chui xon kp ADN ca Waston v Crick. N
to nn bc pht trin mi cho sinh hc dn n s hnh thnh sinh hc
phn t hin i.
20.1.1.3. Chi tit ha hc thuyt trung tm ca sinh hc phn t: ADN

ARN
Protein
a. Sao chp ADN
Trong nm 1955, A. Kornberg v ng s phn lp ADN polymeaz I t
vi khun E. coli. Sau khi cho enzym ny vo ng nghim mui cha ADN v
4 loi nucleotit triphotphat, hn hp ny c th tng hp mch ADN mi.
Nm 1958, trn i tng E. coli, M.Meselson v F. Stahl chng minh
ADN sao chp theo c ch bn bo tn: mi mch b m s lm khun cho
s tng hp mch ADN mi.
Cho n nay, ton b chi tit v sao chp ADN u c thc hin trn
E. coli.
b. Cc qu trnh phin m, dch m v iu ha sinh tng hp protein
Trn i tng E. coli:

 Nm 1961: S. Brenner, Fr. Jacob v M. Meselson khm ph mARN l

phn t mang thng tin di truyn ca ADN t nhn n b my sn xut
protein nm trong t bo cht. iu ny bc b gi thuyt ca Fr. Crick nu
ra nm 1958 cho rng ARN ca riboxom l phn t trung gian mang thng
tin di truyn t nhn sang t bo cht.
1961 1966: M di truyn c khm ph. (Gii Nobel)
Ngoi 2 pht minh trn, ton b cc qu trnh phin m, dch m, cu trc
ca riboxom u thc hin trc tin trn E. coli.
1962: Pht hin c ch iu ha sinh tng hp protein Operon
lactoz.(Gii Nobel)
1970: Pht hin enzym Reverse transcriptaz. (Gii Nobel)
20.1.1.4. Nhng ng gp c th ca cc i tng lm m hnh ch yu
Vic tm hiu nhng ng gp c th ca cc i tng lm m hnh
ch yu s gip hiu r mt cch h thng v c th hn v vai tr cch
mng trong cch mng ca di truyn Vi sinh vt.
a. Vi khun Escherichia coli (E. coli)
Cc ng gp ch yu: i tng ch yu c dng cho cc nghin
cu:
 Sao chp, phin m, dch m v ti t hp.
 t bin.
 iu ha biu hin gen (Gen regulation).
 K thut ADN ti t hp (recmbinant ADN technology).

ng

gp cho nghin
cu cc lnh
vc khc: Trao
i cht ca t
bo
(Cell
metabolism),
gen c ch v ngha (Nonsense suppressors), s tuyn tnh (Colinearity) gia

gen v polypeptit, cc Operon, s khng thuc da vo plasmid (Plasmidbazd drug resistance) v s vn chuyn tch cc (Active transport).
b. Nm men Saccharomyces cerevisiae (S. cerevisiae)

Vai tr ch yu cho cc nghin cu:

 Chu trnh t bo (Cell cycle): S xc nh cc gen kim sot phn bo
nh cc t bin nhy cm nhit (temperature-sensible mutants (cdc
mutants)) dn n m hnh rt tt cho nghin cu s phn bo.
 Tng tc gen (Gen interaction): nghin cu s c ch gen
(suppression). H thng plasmid lai kp (two-hybrid plasmid system) gip
tm cc tng tc gia cc protein nm men dn n cc bn tng
tc phc tp, m l khi u cho sinh hc cc h thng (systems biology).
 Di truyn hc ty th: nh cc t bin petite mt kh nng h hp
m pht hin cc gen ca ty th. Nh chng v cc t bin khc ca ty th
m phn tch chi
tit cu trc v
chc nng b gen
ty th.
 Di truyn
hc kiu bt cp
(Gentics of mating typ): Cc alen MAT nm men l cc gen kiu bt cp
u tin c xc nh cc c tnh mc phn t.
Nhng ng gp khc: Di truyn ca kha ng m (switching)
gia s tng trng kiu nm men (yeast-like) v si (filamentous); di truyn
hc s thoi ha (senescense).

c. Nm si Neurospora crassa:
Cc ng gp ch yu:
 Di truyn sinh ha v trao i cht.
 Di truyn hc ca gim phn.
 Di truyn ty th.
Cc ng gp khc: s a dng cc loi nm
v thch nghi (adaptation), di truyn t bo (cytogentics), cc gen kiu bt
cp, cc gen dung hp th d nhn (heterokaryon-compatibility gens).
20.1.1.5. K thut di truyn v s m u cch mng Cng ngh sinh hc
a. Enzym gii hn (restriction endonucleaz): Nm 1962, W.Arber ln
u tin chng minh rng c nhng enzym c bit hn ch (restriction) sinh
sn ca phage trong t bo E. coli. Nm 1968: Enzym ct gii hn u tin
c khm ph: EcoR I. n nm 1970, Hamilton Smith v cng s tch
c mt loi enzym ct gii hn mi t vi khun Haemophilus influenzae,
gi l Hind II.

Hammilton Smith, Nobel 1978 cng D.Nathans v W.Arber

Enzym ny l cng c u
tin trong k thut di truyn. (Gii
Nobel)

Stanley Cohen, Nobel 1986 cng Rita Levi-Montalcini

b. K thut ADN ti t hp: Nm 1972, nhm ca Paul Berg to nn

phn t ADN ti t hp in vitro u tin t ba ngun khc nhau: nguyn b
gen virut SV 40 gy ung th kh, mt phn b gen ca phage v cc gen
ca operon lactoz ca E. coli. Nm 1973, nhm Cohen, Helenski, Boyer

ln u tin thu nhn c cc sn phm c hot tnh t ADN ti t hp E.

coli (Gii Nobel)
c. Sn xut protein ti t hp: Nm 1977 c xem l nm m mn
cho k nguyn ca cng ngh sinh hc khi ln u tin hormon tng trng
ngi xomatostatin c sn xut thnh cng bng con ng ti t hp gen
vo vi khun.E. coli. Nm 1978, sn xut insulin ngi ln u tin nh
cng ngh gen.
d.Genomeics: S xc nh chnh xc trnh t (sequencing) tng
nucleotit ca ADN hay gi ngn l gii trnh t chui (sequencing) thnh
cng cc vi sinh vt v b gen ngi to nn khoa hc v b gen gi l
genomeics (genome - b gen) hay b gen hc. Genomeics gip xc nh
nhanh chng trnh t nucleotit ca ADN b gen v cc chc nng ca chng.
Nhng sinh vt u tin c hon tt gii trnh t
chui u tin cng l cc Vi sinh vt.
1995: Hai vi khun, cng l hai sinh vt c cu
to t bo u tin c hon tt gii trnh t chui
(sequencing) b gen l Haemophilus influenza v
Mycoplasma genitallum.
Vi khun Haemophilus
influenza

Thng 5/1996, nm men Saccharomyces

cerevisiae, sinh vt Nhn thc Eukaryota u tin
c gii trnh t chui hon tt.

Thng 9/1997: ng ti trnh t b gen hon chnh ca E. coli.

Ton b vic gii trnh t chui ADN tt c cc sinh vt, k c b gen
ngi u s dng cc cng c t cc i tng Vi sinh vt nh enzym ct
gii hn, vector plasmid to dng, NST nhn to ca nm men (YAC) v vi
khun (BAC).
20.1.1.6. S hp dn i vi cc nh vt l v ha hc
Di truyn hc hin i hnh thnh nhng phng php phn tch di
truyn (methodes of gentic analysis) cho php pht hin cc qu trnh sinh
hc ca tnh di truyn v bin d n cp phn t v nguyn t, tc nhng
phm tr nghin cu ch yu ca vt l v ha hc. Cc vi sinh vt nh vi
nm, vi khun v phage ng vai tr quyt nh trong vn ny. D

nhin, s nhn thc cc qu trnh di truyn cp phn t ch tr thnh

hin thc sau pht minh chui xon kp ADN. Nhiu ngi cho rng vai tr
hng u trong s hnh thnh di truyn hc phn t l vic s dng rng ri
cc phng php vt l v ha hc. Vt l v ha hc v ang ng vai tr
quyt nh trong cc nghin cu nhng c ch phc tp v mi quan h ca
b gen vi cc qu trnh sinh tng hp trong t bo. Tuy nhin ngha cn
bn ca cc quan nim di truyn phn t l s tng vt nng lc phn gii di
truyn (gentic resolving power) nh vic s dng cc vi sinh vt. V th c
th ni rng s pht trin cc quan nim di truyn phn t tr thnh hin thc
nh di truyn hc vi sinh vt.
nh gi vai tr ca cc nh vt l hc trong s pht trin ca di
truyn hc phn t, c th phn h thnh 2 nhm. Mt nhm tham gia trc
tip vo cc th nghim di truyn vi khun v bacteriophage (Delbruck,
Benzer, Fris, Levental,) v h thc hin nhng cng trnh rt th v, tuy
nhin h khng to ra phng php no mi trong phn tch di truyn
(gentic analysis). S tham gia ca h vo di truyn hc rt c li nh ngun
sng to ca t duy vt l. Nhm cc nh vt l khc tham gia vo di truyn
hc bng cc suy din tr tu v t c ng gp cho di truyn hc phn t.
Trn thc t, mm mng ca di truyn hc phn t cha ng ngay
trong hc thuyt v gen ca Mendel, trong cc nguyn l di truyn nhim sc
th ca Morgan.
Trong nhng nm 1940, mt giai on mi ca di truyn hc bt u.
Vo thi im ny c mt nhm cc nh khoa hc quan tm n bn cht ca
gen. Phn ln h l cc nh vt l hc, h khc cc nh di truyn hc c in
c v t duy ln nh hng. a s cc tn binh ny rt t bit v nhng
thnh tu di truyn hc trong vi thp nin trc , thm ch c sinh hc ni
chung. S quan tm ca h n sinh hc ch gii hn ch yu vo vn
thng tin di truyn c c s vt l nh th no?
Vic cc nh vt l hc tham gia gii quyt cc vn sinh hc th
khng phi iu l, v ngay Mendel xut thn cng l mt nh vt l hc. Tuy
nhin s cun ht cc nh vt l hc n vi di truyn hc vo nhng nm
1940 cn c nguyn nhn c bit. ng vo thi im ny, khi m gii khoa
hc ngng ph bin sinh lc lun, th nh vt l hc ni ting Niels Bohr,

c gii Nobel v thuyt c lng t (Quantic Mechanics), nu ra quan

im rng mt s qua trnh sinh hc c th khng gii thch trn vn bng
cc khi nim vt l c in. Sau khi xy dng thuyt c lng t, Bohr pht
trin cc khi nim tng qut hn. Nm 1932, ti hi ngh quc t v quang
hc, Bohr pht biu rng:S cng nhn tm quan trng rt ln ca cc
nguyn t cn bn trong cc chc nng ca cc sinh vt t n khng
gii thch mt cch ton din cc hin tng sinh hc. Vn l ch lm
th no khi phn tch cc hin tng t nhin khng b st mt s c im
rt quan trng hiu v s sng trn c s th nghim vt l.
Nm 1935, Max Delbrck, hc tr ca Bohr, trong bi bo V bn
cht cc t bin gen v cu trc gen gii thich rng, chnh Di tuyn hc
l lnh vc sinh hc, m trong nhng gii thch t cc quan im vt l v
ha hc dng nh khng theo quan nim m Bohr nu ra. ng ch ra
rng nu nh trong vt l cc s o, v cn bn, dn n o khng gian v
thi gian, cn khi nim cn bn ca di truyn hc - s khc nhau trong tnh
trng - chng l c th biu th trong nhng n v tuyt i. Theo Delbrck,
di truyn hc c tnh c lp v gen l phn t c tnh n nh cao trong mt
thi gian di khng ph thuc tc ng ca mi trng.
Nm 1945, ngay sau th chin th II, quyn sch nh Th no l s
sng ? (What is the life ?) c xut bn. Tc gi ca cun sch l E.
Schrdinger, mt trong nhng nh vt l hc ni ting xy dng thuyt c
hc lng t. Quyn sch thu ht c s ch ca cng chng, c bit
l cc nh vt l hc, v trong Schrdinger c v cho nghin cu sinh hc
tm cc quy lut vt l mi.
Bt u quyn sch, Schrdinger cho rng vic vt l v ha hc hin
i cha c kh nng gii thch cc qu trnh sng khng c ngha l khng
th gii thch nh cc khoa hc ny. ng cho rng chnh tnh di truyn l
ch cn c gii thch. iu lm cho Schrdinger ngc nhin l lm th
no cc gen nh b c th khng li nhng dao ng ca mi trng din ra
lin tc m nh hnh dng mi ca dng h Gabsburg c truyn qua nhiu
th k. ng cho rng nhim sc th l mt tinh th chu k (aperiodic
crystall), trn c s sp xp cc m di truyn (gnetic code) theo mt th t

chnh xc. Schrdinger cho rng trong cht sng c nhng quy lut vt l
cha bit n.
Cc quan im ca Bohr v Schrdinger c v nhiu nh vt l hc
chuyn sang nghin cu di truyn hc vi hoi bo lng mn l tm ra cc
quy lut vt l mi trong bn cht cu trc gen. n nm 1965, k nim 100
nm cng trnh ca Mendel, bn cht ca gen c sng t, nhng khng
tm ra quy lut vt l mi, ngoi chuyn t v ni cc lin kt hydro.
Tuy nhin, c th ni di truyn hc c tip thm mt ngun sinh lc
mi l cc nh vt l, ha hc vi cc cng c v t duy chnh xc thc
hin th nghim trn nhng i tng Vi sinh vt c nhiu u th. H bt u
cc nghin cu t nhng sinh vt n gin nht l cc phage, vi khun - l
cc sinh vt nhn s. M. Delbruck, mt nh vt l hc chuyn hon ton
sang nghin cu sinh hc, pht biu rng lnh vc nghin cu virut ca vi
khun l sn chi tuyt vi, ni m nhiu cu hi su xa c th ny sinh.
Thm vo ha hc trn pht trin ca mnh cung cp nhiu phng
php tinh vi mi xc nh nh gi cc kt qu th nghim.
20.2. U TH CA CC I TNG VI SINH VT
Trong nghin cu di truyn hc, cc i tng vi sinh vt c nhiu u
th hn hn cc ng vt thc vt bc cao.
20.2.1. Thi gian th h ngn, tc sinh sn nhanh
Trong iu kin thun li, t bo E.coli c th phn chia mt ln trong
20 pht, cn bacteriophage c th trong 30 - 40 pht to ra hng trm c th,
nm men c th phn chia t bo trong 2 gi. c im ca nghin cu di
truyn hc l theo di qua nhiu th h nn cc i tng vi sinh gip rt
ngn ng k thi gian th nghim. Nu so thi gian th h ca rui gim 2
tun, ca chut 2 thng, ca ngi 20 nm th cc vi sinh vt hn hn. Nu
nh di truyn hc phi ch 2 tun c mt th h rui gim, th i vi
E.coli, th nghim hm trc, qua ngy sau c th nh gi kt qu.
20.2.2. Tng vt s lng c th
Cc vi sinh vt n bo, mi t bo l mt c th. Nu dinh dng
cc vi sinh vt sinh sn nhanh to qun th c s lng c th ln c th
khong 1010 - 1012 t bo/ml. T bo E.coli c ng knh 1 m (micromet)

nu dinh dng v mi iu kin thun li khc th sau 44 gi c th to

ra mt lng sinh khi nng bng qu t. Rui gim l i tng thun tin
cho di truyn hc qun th cng ch c th t ti 105-106 c th. Nh s
lng ln c th ca vi sinh vt, c th pht hin cc s kin di truyn him
hoi (nh t bin hay cc dng ti t hp) vi tn s 108 - 1011.

Rui gim

Nh vy, s lng c th ln gip nng cao nng sut phn gii

(resolving power) di truyn, tc kh nng pht hin cc t bin v ti t hp
c tn s xut hin rt nh.
u th ny li c tng thm nh mi trng nui n gin, d nui
cy, d nhn ging, m iu kin nui cy khng cng knh, t tn din tch
hn so vi nui rui, nui chut v trng cy. Mi trng nui cy d kim
sot theo cng thc cht ch nh khi lm th nghim ha hc.
20.2.3. Cu to b gen n gin
Vi khun v virut c b gen l ADN trn d tin hnh th nghim trc
tip trn ADN cng nh chit tch tinh sch. S locus cng t hn so vi cc
sinh vt khc. Cc vi nm v vi to c th tn ti dng n bi (n) vi thi
gian di, nn cc gen ln c th biu hin ngay, m khi phi tin hnh lai
phn tch hay a v dng ng hp t ln. Tuy nhin, cc vi sinh vt k trn
c trng thi lng bi (2n) nn cng d dng thc hin phn tch ti t hp.
Cc tnh trng vi sinh vt cng n gin hn, xc nh di truyn cc
tnh trng ny t phc tp hn, nn d nghin cu. i vi cc tnh trng sinh
ha hay tnh khng th c th d dng s dng mi trng chn lc pht
hin.
20.2.4. D thu nhn cc t bin
Cc phn tch di truyn hc phn ln da vo nhng khc bit ca
dng bnh thng so vi t bin. Tn s t bin thc vt v ng vt

khong 105 107, kh thu nhn v cn thi gian di khong vi th h

khng nh ng l dng t bin. Nhiu t bin ng vt d gy cht nn
s lng t bin thu nhn c ng vt rt hn ch.
Cc t bin vi sinh vt c th thu nhn d dng, thm ch c tn s
xut hin thp 1010, m vic xc nhn dng t bin cng rt nhanh.
Nh u th ny m di truyn hc vi sinh vt pht trin rt nhanh hnh
thnh nn di truyn hc phn t v sinh hc phn t.
20.2.5. D nghin cu bng cc k thut vt l ha hc
a s cc vi sinh vt c cu to n bo nn qun th ca chng c
ng nht cao hn so vi cc t bo sinh vt a bo bc cao bt ngun t
nhiu loi m khc nhau. Cu to t bo vi sinh vt n gin, d chit tch,
tinh sch ADN. C th nui vi sinh vt ng nht (synchronous culture) tc
l a s cc t bo nhng giai on pht trin gn ging nhau. ng
nht cao ca vt liu th nghim to thun li cho vic s dng cc phng
php vt l ha hc trong nghin cu di truyn.
Do nhng u im k trn, vi vic s dng cc i tng vi sinh vt,
di truyn hc bc vo giai on nghin cu di truyn trong ng nghim
(in vitro).
Mc d cc vi sinh vt c nhng c im ring nhng chng vn tun
theo cc quy lut di truyn chung, cc kt qu thu c c th i chiu p
dng cho cc vi sinh vt bc cao.
20.3. CC C IM CA DI TRUYN HC VI SINH VT
20.3.1. Nghin cu trc tip cp t bo v cp phn t
Di truyn hc c in s dng cc m hnh i tng nh u H Lan
Pisum sativum, cy bp (ng) Zea mais, rui gim Drosophila melanogaster
v chut nht Mus musculus, l nhng sinh vt a bo m s biu hin tnh
trng phi tri qua mt qu trnh pht trin phc tp v km theo bit ha. V
d, t bo hng cu ng vt c v ch chuyn sn xut hemoglobin. D
nhin mi biu hin sng u lin quan n t bo, nhng cc quan st
nhng i tng trn l gin tip.
Trong khi , nhiu i tng vi sinh vt nh vi khun Escherichia
coli, nm men Saccharomyces cerevisiae l sinh vt n bo nn cc quan st

thc hin trc tip hn mc t bo. V d, trn mi trng ti thiu vi

lng mui hu c v glucoz cho trc, mt t bo E. coli c th tng hp tt
c cc hp cht cn thit cho s tng trng, sng st v sinh sn. Nu t bo
b t bin mt kh nng tng hp mt cht no nh mt loi axit amin th
n khng mc c trn mi trng ti thiu. Trn mi trng c b sung
thuc khng sinh th ch c cc t bo khng mi mc c.
Hn na, cc t bo vi sinh vt c nhu cu dinh dng n gin d
kim sot thnh phn mi trng nui, d nui cy, d nhn ging, d dng
thu nhn nhiu loi t bin phc v cho nghin cu trc tip v nhanh
chng cp t bo, phn t. Do vy, chng l nhng i tng l tng
cho nghin cu di truyn hc phn t.
20.3.2. Dng t bo
c im ca cc t bo vi sinh vt l rt nh b, phi nhn di knh
hin vi mi thy c. Do vy kh c th quan st tng t bo ring l, hn
na, bng cch ny cng khng ghi nhn c cc tnh trng bin dng, tnh
khng v nhiu tnh trng khc. Do vy, di truyn vi sinh vt khng nghin
cu tng t bo ring l m l dng ca t bo, tc tp hp ca nhiu t bo
bt ngun t mt t bo ban u nh sinh sn v tnh. Thng thng, t bo
vi sinh vt c cy ln mi trng thch c, ri tri u cc t bo nm
ri xa ra th mi t bo mc ln thnh mt cm ri gi l khun lc (colony).
Mi khun lc cng l mt dng t bo (clone) gm cc t bo bt ngun t
s phn chia ca mt t bo ban u.

Khun lc (colony) ca vi sinh vt

Mi khun lc d nhn thy bng mt thng, c ti thiu 107 (mi

triu) t bo. Nh vy, cc tnh trng vi sinh vt c ghi nhn qua mt
qun th gm hng trm triu, hng t t bo.

Dng t bo mang mt c tnh di truyn no gi l chng (strain).

V d: chng vi khun to nhiu vitamin B12 hay chng vi khun mt kh
nng tng hp mt axit amin no .
20.3.3. Cc tnh trng
Cc t bin vi sinh vt thng c pht hin theo s bin i cc
tnh trng sau:
a. Hnh thi: kch thc, hnh dng t bo hay khun lc, c mng
nhn hay khng, kh nng di ng...
b. Sinh ha: s hin din ca cc sc t, mu sc c trng.
c. Nui cy: nh kiu h hp, kiu dinh dng (khuyt dng auxotroph) hoc nhu cu i hi cc nhn t tng trng.
d. Tnh khng: nh khng thuc, khng phage, chu nhit...
e. Min dch: nh cc phn ng khng th, khng nguyn...
Cc t bin c th xut hin ngu nhin hay do gy to ra nh cc tc
nhn gy t bin. Mi gen c tn s t bin c trng.
Cc tnh trng vi sinh vt c k hiu bng 3 ch tt ting Anh hoc
i khi ch hoa u tin. Km theo k hiu cn thm du + hoc hoc ch
tt gii thch r thm tnh trng. V d: lac ch mt kh nng tng hp
lactoz; his+ - tng hp histidin; strS nhy cm (Sensible) vi Streptomycin,
strR khng (Resistant) vi Streptomycin.
ch hai gii tnh khc nhau khng dng hai k hiu v thay vo
l cc ch nh mt (+), mt (-) (mating typ) ( Chlamydomonas reinhardi),
hoc A, a ( Neurospora crassa) hay a v (vi nm).

To n bo Chlamydomonas

20.3.4. Cu trc b gen

ADN hin din trong b gen (Genome) ca tt c cc loi t bo t vi
khun n ngi. Gia cc sinh vt nhn s Prokaryota v nhn chun
Eukaryota c s khc nhau ng k v kch thc, thnh phn cu to v t
chc ca ADN trong t bo.
B gen ca vi khun E. coli v a s cc sinh vt nhn s l mt phn t
ADN c dng vng trn kn. Khi nim nhim sc th hin nay c dng
cho c vi khun, nn ni nhim sc th vi khun ta hiu l si ADN. Ty th
v lc lp cng c ADN ring, m cu trc b gen cng tng t vi khun.
a phn ADN ca Eukaryota nh nm si, nm men, vi to c t
chc thnh nhiu nhim sc th trong nhn t bo. Mi nhim sc th cha 1
phn t ADN thng mch kp km theo mt s protein nh histon. Cc nhim
sc th c s lng v hnh dng c trng cho t bo ca mi loi sinh vt
nhn chun.
Cc virut c b gen rt a dng: ADN mch n hoc kp, ARN mch
n hoc kp, nhng ch mt loi phn t.
20.3.5. Kiu sinh sn
Cc nm si, nm men, vi to c cc qu trnh sinh sn v tnh v hu
tnh v cn bn ging cc sinh vt bc cao, thc hin qua nguyn phn v
gim phn.
Cc vi khun khng c cc c ch nguyn phn v gim phn, phn
chia t bo theo c ch trc phn, nhng cng c sinh sn v tnh v s trao
i thng tin di truyn tng t sinh sn hu tnh c gi l qu trnh cn
hu tnh.
Cc virut ch sinh sn trong t bo ch sng vi nhiu c ch khc
nhau ph thuc kiu virut.
20.3.6. Vn bin d vi sinh vt
Mi n nm 40, G. Beadle v E.Tatum ln u tin s dng vi nm
Neurospora crassa vo nghin cu di truyn hc. Vic s dng chm tr cc
i tng vi sinh vt vo nghin cu di truyn, mt phn ln do vn bin
d. Trong khi thc vt, ng vt s di truyn cc tnh tp nhim (do
luyn tp hay b nhim trong i sng c th) c khng nh l khng

c th vi sinh vt vn c hiu ngc li: chng bin i trc tip di

tc ng mi trng. Nhiu hin tng thc t to cm gic cc vi sinh vt
bin i di tc ng trc tip ca mi trng. V d, hin tng cc vi sinh
vt nhn thuc hay cc cht c. Lc u thuc khng sinh s dng vi liu
thp, dn dn cc vi sinh vt nhn thuc nn phi s dng liu cao. Trc
y mt quan nim ph bin l cc vi sinh vt bin i theo nhng quy lut di
truyn khc. C th ni vn bin d vi sinh vt l thnh tr cui cng ca
ch ngha Lamack (cng nhn s di truyn tnh tp nhim).

Max Delbruck, Nobel 1969 cng

vi A.D.Hershey v S. Lurila

Tuy nhin mt s nh di truyn hc cho rng bin d vi sinh vt cng

tun theo quy lut di truyn chung. Hin tng quen thuc c gii thch l
phn ln t bo nhy cm b dit, mt s t b t bin khng thuc cn sng
st nh sinh sn nhanh to qun th mi vi kiu gen khng thuc nn gy
cm gic nhn thuc.
Tip theo nhiu th nghim xc ng chng minh rng bin d vi sinh
vt cng xut hin do cc t bin ngu nhin. l th nghim dao ng
(fluctuation test) ca P.Luria v M.Delbruck (1943) v c bit l phng
php in hay ng du ca E. Lederberg (1952).
Cho n nay, thc t cho thy cc d liu nghin cu di truyn phn t
t ADN, ARN, protein v cc qu trnh khc thu c t cc i tng vi
sinh vt hon ton c th p dng cho sinh vt bc cao. Hn th na, nu
thiu nhng nghin cu mc n gin hn trn cc i tng ny th
kh hiu c nhng c ch phc tp hn rt nhiu sinh vt bc cao.

20.4. CC NG DNG THC T

Di truyn hc l c s khoa hc ca chn ging. Cc thnh tu ca di
truyn hc c ng dng sm, nhanh v nhiu hn c cho n nay l trong
chn ging. Bc u, chn ging t bin c ng dng v c nhng
thnh tu ngon mc lm c s cho s pht trin ca cng nghip ln men.
Bc pht trin tip theo c tnh cht cch mng l ng dng cng ngh gen
dn n s bng n ca Cng ngh sinh hc, m th k 21 c mang danh.
20.4.1. Chn ging t bin
Cc thnh tu ca Cng nghip vi sinh vt khng th tch ri vi
nhng tin b nhy vt trong chn lc cc chng c nng sut cao. Cc
phng php chn lc t bin c ng dng vo chn ging lm tng nng
sut to cc sn phm, lm bin i bn cht ho hc cc cht v khc phc
mt s nhc im ca chng c dng trong sn xut cng nghip.
Phng php chn ging ny c cc c im:
Thu nhn kt qu rt nhanh.
Ch nh gi sn phm thu c, khng quan tm cc bin i sinh l, sinh
ho.

Vi nm Penoicillium chrysogenum

Qu trnh chn ging cc chng Penicillium chrysogenum M c th

ly lm v d in hnh cho phng php ny. T dng ban u c sn lng
60mg/l, chn cc t bin ngu nhin c dng 150mg/l, v sau s dng
cc t bin nhn c do tc ng tia X v UV. Vic chn lc theo nguyn
tc: ly dng c sn lng cao nht em gy t bin ri chn chng c nng

sut cao hn. Qua nhiu bc trung gian cui cng cho n nay nhn c
dng E.15.1 c sn lng 7000mg/l. Trong bng 20.2 khng nu mt thnh
tu quan trng l to chng khng sn sinh ra sc t c, phi tn km nhiu
cho vic loi b sc t. Vic nhn cc t bin khng tng hp sc t lm
gim ng k chi ph sn xut v cc dng ny c s dng nng cao
nng sut trong qu trnh chn ging tip theo.
Phng php chn ging t bin c s dng to cc chng sn
sinh ra nhiu axit amin (nh glutamic axit) hay cc nucleotit. Ngoi ra cn c
th nhn cc t bin lin quan n c ch iu ho trao i cht:
Cc t bin c cu (constitutive mutants): to sn phm khng cn cht
cm ng (inducer).
Cc t bin khng c ch ngc l cc t bin to sn phm nhiu m
khng b c ch bi sn phm cui (end product repression).
Phng php ny em li nhng thnh tch ngon mc, gp phn
tch cc cho s pht trin ca cng nghip ln men vi sinh nh:
Chng Penicillium chrysogenum ban u c nng sut 60 mg/l v nay t
60000 mg/l, hn chng ban u n 1.000 ln.
Chng Corynebacterium glutamicum to glutamic axit t hn 200g/l (ban
u 20g/l).

Vi khun Corynebacterium glutamicum

Chng Serratia marcescens sinh ra biotin t 600mg/l.

Vi khun Serratia
marcescens

Riboflavin (vitamin B2) l sn phm thng mi c tng hp bng

phng php ln men v phng php ho hc. Siu sn xut riboflavin do 2
chng vi nm Eremothecium ashbyii v Ashbya gossypii vi sn lng hn
20 g/l, gp 40000 ln nhu cu ca n.

Eremothecium ashbyii
trn mi trng

sinh vitamin B2 ngay

Sn xut vitamin B12 trong cng nghip do cc chng vi khun

Propionibacterium shermanii hoc Pseudomonas denitrificans. Cc chng
t bin sn sinh mt lng sn phm ln hn rt nhiu so vi nhu cu ca t
bo, thm ch ln hn nhiu ln khi lng kh ca n. Pseudomonas
denitrificans sinh ra vitamin B12 gp 100000 ln nhu cu ca n.

20.4.2. Cuc Cch

mng cng ngh sinh
hc
Cng ngh sinh
hc
(Biotechnology,
CNSH) l mt thut ng
khoa hc do k s ngi
Hungary l Karl Ereky
nu ra vo nm 1917
ch qu trnh nui ln vi quy m ln bng thc n l c ci ng ln men
nh cc vi sinh vt. Sau vo nm 1961, tp ch khoa hc JouARNl of
Microbiological and Biochemical Engineering and Technology (Tp ch k
C u trc c a vitamin B12

thut v cng ngh vi sinh sinh ha) c i tn thnh Biotechnology and

Bioengineering (Cng ngh sinh hc v k thut sinh hc). Tuy nhin,
thut ng ny t c nhc n trong hn 50 nm v ch c s dng rng
ri sau pht minh ra K thut di truyn-Gentic engineering (KTDT) vo u
nhng nm 70 ca th k trc, cho nn c lc ngi ta coi l s "bng
n" ca CNSH.
Trc thp k 70, CNSH c
hiu l s ln men cng nghip
(industrial fermentation) vi sinh vt
to thng phm. Trong nhng thp k
60 v 70, cng ngh ln men pht trin
thnh mt ngnh cng nghip ln trn
th gii vi doanh s gn trm t
USD/nm. S hon thin cc trang thit
b tt c cc khu v s kim sot, iu
CNSH thu khi u
khin phn ng ca t bo trnh cao
lm tng sn lng ng k. Tuy nhin, khu quyt nh lm gim ng k
gi thnh l nng sut chng ging. Phng php chn ging c in c
thc hin vi nhng chng phn lp t thin nhin v sau gy t bin
bng tia t ngoi, tia phng x hay bng ha cht.

u thp nin 1970, CNSH chuyn sang mt giai on mi cao hn

hn v cht nh KTDT. Cc k thut mi cho php to ging trc tip nhanh
hn, tn dng ngun gen t nhiu sinh vt khc nhau to cc chng sn
lng cao, m t tn cng sc phn lp v gy t bin nh trc y.
Nh kh nng vt gii hn tin ha ca KTDT, cc vi sinh vt, cc t bo
ng thc vt c th c s dng nh cc nh my sinh hc (biological
factories) sn xut hng lot cc protein ngi nh insulin, hormone tng
trng, interferon, Cc ng vt v thc vt c th tr thnh bioreactor t
nhin to cc sn phm t gen l a vo, khng cn lai to v chn lc cc
bin d bng lai trong loi nh trc y. Ngoi ra, KTDT ng thi cung
cp cc phng php tr liu v chn on mi cha tr bnh ngi. Cn
nhn mnh rng, KTDT l cng ngh cao, gm nhiu cng on phc tp v
n thc s to nn mt cuc cch mng.
S ra i ca Cch mng sinh hc mi lm cho thut ng Cng ngh
sinh hc tr nn thng dng vo na sau nhng nm 70. Trc 1973, ngi
ta thng dng cc t Vi sinh cng nghip, Cng ngh ln men, K thut
sinh ho... Cng ngh sinh hc l mt thut ng rt t, bao hm trong
n tt c nhng tn gi v cc lnh vc ng dng trc y v vi ni
dung mi. N phn nh nhng thnh tu ht sc to ln ca s pht trin sinh
hc trong nhiu thp nin trc . Cch mng sinh hc mi cao hn hn v
cht so vi Cch mng xanh vo nhng nm 60.
T thu s khai thi c La M-Hi Lp, khoa hc l mt h thng nht.
n thi Phc hng, s pht trin khoa hc dn n s tch bit thnh cc
ngnh v chuyn ngnh. iu ny cng th hin r trong sinh hc cho n
nay. S xut hin ca cch mng mi to ra cc lnh vc a ngnh v hp
nht cc ng dng sinh hc cp t bo v phn t, ng thi gn lin
vi cng ngh, di tn gi chung l CNSH v c coi nh "quyn lc gh
gm nht m loi ngi ginh uc k t sau thnh tu phn hch nguyn t
" hay" Cha t ca th k 21".
Kh k ht nhng thnh tu v cng to ln ca CNSH n mc m th
k 21 c mang danh l th k cng ngh sinh hc. y ch nu tip mt
s ng dng ch yu lin quan n cc i tng vi sinh vt.

20.4.3. Gii m cc b gen ca vi sinh vt

Ngay t nm 1995, Cr. Venter hon tt vic gii k t chui ca 2
loi vi khun Haemophilus influenzae (1830121bp) v Mycoplasma
genitatum (0,58Mb). Hin nay, s vi sinh vt c gii k t chui b gen
xong l hn 200 loi. Ngoi ra, nhiu loi quan trng khc gm:
Streptomyces coelicolor (9,05Mb 1996), Bacillus subtilis (4,21Mb 1997),
Agrobacterium tumefaciens (,67Mb 2001), Xanthomonas campestris
(5,08Mb 2002) v Corynebacterium glutamicum (3,31Mb 2003) bit
c chi tit v b gen.
S hiu bit genomeics vi sinh vt c ngha ht sc quan trng v
mt khoa hc c bn cng nh ng dng, m d n nu sau l mt v d v
nghin cu i su vo bn cht s sng.
T nm 2001, d n t bo vi sinh vt (Microbial Cell Project MCP)
c thc hin vi bn mc tiu: 1) Xc nh cc protein vi sinh t hp thnh
cc b my protein thc hin nhiu qu trnh ni bo quan trng cho s
sng ; 2) nh gi c tnh mi trng ni bo v cc hiu qu ca n ln cc
protein v b my protein n thc hin cc chc nng ; 3) nh gi c tnh
phn b, s lng, cc lung protein v b my protein ni bo ; 4) Dng
in ton xy dng cc m hnh gen gen, gen protein, cc tng tc
protein protein v ho sinh ni bo.
20.4.4. Cng ngh protein ti t hp
Nhng th nghim to dng gen u tin c thc hin vi sinh vt.
Nh thao tc d dng li c cng ngh ln men pht trin mnh, cc vi sinh
vt cung cp rt nhiu sn phm quy m cng nghip do KTDT, t cc
protein, ADN, ARN n cc phn t nh.
T lu, cc nh khoa hc mong mun sn xut cc protein vi s lng ln.
Cng ngh gen cho php sn xut nhiu loi protein ti t hp (recombinant
protein r-protein) khc nhau khng nhng vi s lng ln m cn c cht
lng protein tt hn. Cng ngh r-protein tht s ang pht trin vi nhiu
thnh tu rt ngon mc.
Nhiu protein tr bnh c biu hin cc h thng vi khun, nm
men. Chng cn cho cha tr mt s bnh, nhng chit tch t c th th s

lng t. Cc r-protein u tin c sn xut nh cc vi sinh vt ch nh E.

coli hay nm men S. cerevisiae. C th l su trong by loi protein tr liu
vi tng doanh s khng di 15 t USD, m bng sng ch (patent) ht
hn (thng khong 15 20 nm), c nu trn bng 20.1.
Cc protein ti t hp c sn xut gm cc nhm ch yu:
Bng 20.1: Su loi r-protein tr liu do E. coli hay S. cerevisiae to ra
STT

Tn sn phm

Cc bnh c iu tr

Nm ht
hn patent

Insulin

Tiu ng

2001

Interferon alpha

Vim gan siu vi C, mn cc qua

ng tnh dc, ung th

2002

Interferon beta

X cng (Multiple sclerosis)

2003

Hormone tng trng

ngi

Thiu nng tng trng (Growth

defiency)

2003

Erythropoetin

Thiu mu

2004

G-CSF (Granulocyte-

Ha tr liu gim bch cu trung

2006

Colony Stimulating
Factor)

tnh (Chemotherapy-induced
neutropenia)

Cc protein tr liu nh mt s nu trn bng 20.1.

Cc enzym: ADNz I, alginat lyaz, phenylalanin amoni lyaz, alpha-1antitrypsin.
Cc khng th dng trong chn on v cha tr nhiu bnh.
20.4.5. To cc ging vi sinh vt nh k thut di truyn
E. coli, Saccharomyces cerevisiae l nhng i tng u tin c s
dng KTDT to ra cc chng sn xut cc r-protein ngi, tip l nhiu
vi sinh vt khc. Sau y l vi v d c th.
a. Thit k trao i cht (Metabolic Design)
KTDT can thip vo qu trnh trao i cht cp gen sn
xut cc cht phn t nh. Cc ng dng u tin ca metabolomics, cng

ngh gen iu khin trao i cht, thc hin vi sinh vt. Hai v d in hnh
u tin l to dng t bo E. coli sinh tng hp cc cht, m vn n khng
c kh nng ny, nh xanh indigo v sc t en melanin. S to dng mt
gen t Pseudomonas putida vo E.coli lm n c kh nng tng hp indigo
xanh trong mi trng c tryptophan. S to dng gen tyrosinaz cho E. coli
lm n bin tyrozin thnh dopaquinone v cht ny ngu bin thnh melanin
khi c khng kh.
Tip theo, cc sn phm trao i cht s cp v th cp c sn xut
t cc dng thit k nh cc chng s dng lactoz, chuyn ho xyloz, sn
xut etanol t pentoz, phn gii cc cht d sinh (xenobiotics),
b. Cng ngh b mt t bo nm men
Cc protein trn b mt t bo nm men cng c tng hp bn trong
t bo, nhng sau chng c a ra gn ln b mt t bo. Da vo s
hiu bit cc gen tng ng, cng ngh b mt t bo (cell surface
technology) ra i. S dng CN gen c nh protein ngoi lai trn b
mt t bo nhm thay i v ci thin chc nng ca t bo. Tim nng ng
dng a dng ca n gm: sn xut cc enzym, khng th, khng nguyn, th
th,....
Bc u, cc nh nghin cu thnh cng trong biu hin cc
enzym nh glycosyl hydrolse, glucozamylaz, lipaz,... PTN Cng ngh sinh
hc phn t ca i hc KHTN (TPHCM) to c chng nm men gn
protein GFP (green fluorescense protein protein pht hunh quang xanh lc
ca sa) v chng gn alpha-amylaz.
c. S can thip ca KTDT vo sn xut etanol nhin liu
Nm men S. cerevisiae v vi khun Zymomonas mobilis l 2 vi sinh vt
ch yu c kh nng s dng trong ln men etanol nhin liu. Nm men vn
gi vai tr ch yu, nhng n khng ln men xyloz, pentoz v mt s lai
ng khc. Do tm quan trng chin lc cp thit, hin ti v trong tng
lai c rt nhiu n lc c tp trung cho ci thin chng ging nhm s
dng tt hn ngun nguyn liu a dng v thun tin cho quy trnh cng
nghip. Cc nghin cu ch yu nhm thit k:
Cc chng s dng lactoz tn dng ph phm cng nghip sa.

 Cc chng vi sinh c kh nng chuyn ho xyloz m nm men khng ng

ho.
Cc chng nm men phn gii tinh bt ln men bt khi phi qua ng
ho.
Cc chng vi sinh vt sn xut etanol t pentoz.
Ngoi ra, vn quan trng khc l sn xut enzym cellulaz gi r
gi thnh etanol nhin liu sc cnh tranh vi xng du.
Chuyn ho sinh khi thc vt thnh etanol nhin liu l mt im
nng ca CNSH hin i.
d. Ci bin cc chng vi sinh vt sn xut vitamin
Riboflavin (Vitamin B2): Phng php mi s dng cc loi Candida hoc
chng Bacillus subtilus ti t hp cho sn lng 20 30g/l.
Tng hp cc tin cht ca vitamin: Gn y (1990) thnh cng trong
to dng cc gen cho s sinh tng hp carotenoit vng, cha -caroten t
Erwinia uredovora. Sau khi 4 gen sinh tng hp -caroten c chuyn vo
Z. mobilis v Agrobacterium tumefaciens, cc khun lc mu vng thu c
trn cc a thch. Cc th tip hp sn xut 220 350g -caroten trn gram
khi lng kh phaz n nh trong mi trng nui.
Sinh tng hp L-ascorbic axit c ci bin nh KTDT. L-ascorbic axit
(Vitamin C) c hin tng hp thng mi theo mt quy trnh t tin, bao
gm 1 giai on ln men Vi sinh vt v mt s giai on ha hc bt u vi
D-glucoz. Giai on cui cng trong qu trnh l s chuyn ho 2-keto-Lglucoznic axit (2-KLG) thnh L-ascorbic axit di xc tc l axit. Do ,
cch tt nht chuyn ho glucoz thnh 2-KLG l ch to mt Vi sinh vt
c mang tt c cc enzym cn thit. Vic chuyn ha D-glucoz thnh 2,5DKG bng Erwinia herbicola bao gm nhiu bc xc tc bi enzym, trong
khi s chuyn i 2,5-DKG thnh 2-KLG do Corynebacterium sp. i hi
ch mt bc. Chin lc n gin nht thit k mt sinh vt c kh nng
chuyn D-glucoz thnh 2-KLG l tch gen 2,5-DKG reductaz t
Corynebacterium sp. v cho biu hin trong Erwinia herbicola.
Cc t bo Erwinia c bin np gen 2,5-DKG reductaz c kh nng
chuyn ho trc tip D-glucoz thnh 2-KLG, v cc enzym ni bo ca

Erwini nm mng trong ca vi khun chuyn glucoz thnh 2,5-DKG v 2,5DKG reductaz xc tc chuyn 2,5-DKG thnh 2-KLG. Do , bng thao tc
gen, kh nng chuyn ho ca 2 Vi sinh vt rt khc nhau li c kt hp
thnh mt v do c kh nng to sn phm cui ca qu trnh chuyn ho
c thit k. Sinh vt loi ny rt hu ch nh l mt ngun 2-KLG cho sn
xut L-ascorbic axit, bng cch thay th 3 cng on u ca quy trnh
hin ang c s dng.
Thng qua t bin im nh hng bng oligonucleotit thu c
t bin 2,5-DKG reductaz c hot tnh cao hn khong 2 ln v bn vi
nhit hn dng enzym t nhin.
Tm li, KTDT th hin quyn lc to ln trong ci bin sinh gii v
nhiu mt, khng nhng thay i cc tnh trng khc nhau, m can thip c
n chi tit cc thnh phn v qu trnh trao i cht. Trn cc i tng vi
sinh vt, cng ngh gen c ng dng u tin, k thut a dng, hiu qu
hn c v hng lot sn phm xm nhp th trng t nm 1982.
20.5. BIN D VI SINH VT
Bin d v t bin c ngha quan trng trong nghin cu di truyn
hc. Cc t bin gm nhiu loi khc nhau v chng c thu nhn d dng
t cc vi sinh vt nh cc phng php pht hin chuyn bit. Cc t bin
gen bt ngun t nhng bin i phn t trn ADN. Con ngi c th s
dng cc tc nhn vt l v ha hc gy nn cc t bin nhn to hay cm
ng.
Cc nghin cu v hi bin gip hiu r hn v cc c ch trong gen
ca s dch m
Bin d vi sinh vt cng tun theo cc quy lut di truyn nh sinh
vt bc cao. N c nhiu loi v s lng rt ln, cung cp nguyn liu
thng xuyn cho qu trnh tin ha v nghin cu di truyn hc. Nhiu
phng php pht hin v chn lc cc t bin khc nhau c s dng
thu nhn cc t bin c tn s xut hin rt thp. Chng gp phn ng k
cho cc nghin cu di truyn. Cc thay i thm, mt hay thay th cc
nucleotit trn phn t ADN dn n cc bin i phn t, gy ra cc t bin
gen nh: lch khung, sai hay nhm ngha v im lng. Pht minh ra cc tc

nhn gy t bin vt l v ha hc lm tng ng k ngun t bin phc v

cho nghin cu v chn ging. Con ngi c th tng ngun bin d bng lai
to hay s dng cc tc nhn gy t bin.
20.5.1. i cng v bin d vi sinh vt
20.5.1.1. Bin d vi sinh vt cng tun theo quy lut di truyn chung
C nhiu phng php chng minh bin d vi sinh vt cng do cc
t bin. y ch nu 2 phng php ch yu: th nghim dao ng
(fluctuation test) v c bit l phng php in hay ng du ca E.
Lederberg (1952).
a. Nguyn l ca th nghim dao ng (fluctuation test)
Nm 1943, Luria v Delbrck tin hnh th nghim nh sau. Ly 20
ng nghim cho vo mi ng mt t mi trng nui (1ml) lng vi mt t t
bo vi khun nhy cm vi bacteriophage v cho chng sinh sn t 108 t
bo/ml. Ly dch nui 20 ng nui ring l v 20 ng vi s dch nui tng
t t bnh ln nui chung v dch tng ng cy ln cc hp Petri mi trng
c cha bacteriophage. Nu t bin l s kin ngu nhin th s lng th
t bin (mutants) t (2 hnh bn tri) hay nhiu (8 hnh bn phi) do xut
hin mun hay sm nh m t trn hnh 20.1.

Hnh 20.1: t bin l s kin ngu nhin: s lng th t bin ph thuc thi
im xut hin.
S lng th t bin (cc t bo t m) t (2 hnh bn tri) hay nhiu (8 hnh bn
phi) do xut hin mun hay sm trong dng t bo.

Kt qu cho thy c s dao ng (fluctuation) v s lng khun lc

khng phage mc ln t dch nui ca 20 ng nui ring l. C th: 11 ng
khng c khun lc khng, s cn li c 1, 1, 3, 5, 5, 6, 35, 64 v 107
khun lc/hp Petri. Trong khi , dch nui 20 ng t bnh ln nui chung t

c s dao ng t hp Petri ny sang hp khc v trong kong 14 26 khun

lc/hp Petri hay thuc khng sinh.
b. Phng php in hay ng du
Cc vi khun c cy mc ri tng khun lc trn mi trng c
dinh dng. Dng ming nhung (c nhiu lng mn) n nh ln b mt mi
trng thng ghi du cc khun lc nh cc lng mn, sau in ng cc v
tr khun lc trn mi trng c phage hay thuc khng sinh. Cc khun lc
t bin khng phage hay khng sinh mc ln c. Cn c v tr khun lc
khng mc bn sao tch cc t bin tng ng (hnh 20.2).

Hnh 20.2: Phng php in dng pht hin cc t bin.

Nh vy, phng php in cho bit pht hin cc t bin khng cc

nhn t bt li no y ca mi trng trong qun th vi khun t trc khi
cc t bo ca chng tip xc vi tc nhn ny. iu c ngha rng, v d,
streptomycin khng phi l nguyn nhn gy bin d khng thuc, m ch l
tc nhn chn lc gi li cc t bin xut hin trc khi tip xc vi
thuc. Cc t bin ny c kiu hnh gip cho vi khun thch nghi vi bin
i ca mi trng v nh sinh sn nhanh sau mt thi gian ngn qun th c
kiu hnh mi.
Cc kt qu nghin cu ny khng nh cc vi sinh vt cng tun theo
cc quy lut di truyn nh cc ng vt v thc vt. iu ny c ngha rt
ln cho s pht trin ca di truyn hc v sinh hc phn t. Do vy, gii
Nobel Y hc nm 1969 c trao P.Luria v M.Delbruck v ng gp ny.
20.5.1.2. Qu trnh t bin t nhin

t bin theo ngha rng ch cc bin i di truyn xy ra t ngt. T

xa xa, con ngi nhn thy nhiu t bin t nhin. Nhiu ging cy
trng v vt nui bt ngun t cc t bin.
t bin gen l t bin c hiu theo ngha hp, l ch nhng bin i
xy ra bn trong cu trc gen. Mi t bin gen dn n s thay i trnh t
nucleotit to ra cc alen khc nhau. t bin c th xy ra do bin i nhiu
nucleotit, c th do 1 nucleotit. t bin gen khng pht hin c khi quan
st t bo hc.
Bng 20.2: Tn s t bin ngu nhin ca mt s gen

Sinh vt

t bin

Tn
s

Cn c nh gi

Phage T2

Km hm tan r r+

1.10-8

t bin/1 sao chp

E.coli

Ln men lactoz
lac lac+

2.10-7

1 ln phn bo

leu- leu+

7.10-

t bin /1 t bo

arg+ argtrp+ trpara ara

+

Khng
Streptomycin
StrS StrR
Neurospora
crassa

cc t bo t bin /

adenin
ade- ade+

10

4.10

t bin /1 t bo
-9

6.10-8
2.10

t bin /1 t bo
t bin /1 t bo

-6

t bin /1 t bo
4.10-10
4.10-8

t bin/ 1 bo t v
tnh

Trong t nhin, d gi trong iu kin no, tt c cc gen u c t

bin, c gi l t bin t nhin hay ngu nhin (spontanous mutation).
Cc t bin t nhin thng xut hin rt t. Khi nim tn s t bin c
dng nh gi mc xut hin nhiu hay t t bin mt gen. C ngi
phn bit tn s (frequency) vi tc (rat) t bin.

Cc gen khc nhau ca cng 1 sinh vt c th c tn s t bin khc

nhau. Nhng tn s t bin t nhin i vi mi gen l mt s n nh.
Tn s t bin c nh gi theo cc cn c khc nhau nh: trn 1
ln sao chp, 1 ln phn bo hay trn 1 giao t v trn 1 t bo / 1 th h
(bng 20.2).
d hiu cc s trn c th tnh o ngc li. V d: E.coli t bin
t nhy cm vi streptomycin sang khng tc StrS StrR vi tn s 4.10-10
t bin tnh trn 1 t bo/1 th h. d hiu ta c th tnh ngc li tc
trong 10 t t bo ca mt th h c 4 t bin StrR (resistance) khng
streptomycin xut hin ngu nhin.
Tuy tn s t bin ca tng gen l rt thp, nhng tng cc t bin ca
nhiu gen l mt s ng k, c ngha quan trng cho tin ha.
t bin nh hng n mi tnh trng khc nhau ca sinh vt v tc
ng theo mi hng.
20.5.2: Cc loi t bin thng gp vi sinh vt
Vi sinh vt cng c cc loi t bin nh sinh vt bc cao. y ch
nu cc t bin thng gp vi sinh vt v c ngha quan trng i vi di
truyn phn t.
20.5.2.1. t bin gen hay t bin im
Bin i rt nh trn mt on ADN, thng lin quan n 1 nucleotit
hay 1 cp nucleotit.
a) t bin ng ngha (Samesense) cn gi l trung tnh (neutral) hay
im lng (silent), khi codon m ha cho mt axit amin b bin i, thng
baz th ba nn vn m ha cho axit amin (do tnh suy thoi ca m di
truyn.
b) t bin v ngha (Non-sense) khi codon m ha cho mt axit amin
bin thnh mt trong ba codon UAA, UAG v UGA l cc codon kt thc
khng m ha cho axit amin no.
c) t bin sai ngha (Mis-sense): Khi codon ca axit amin ny bin
thnh codon m ha cho axit amin khc, lm thay i axit amin tng ng
trn phn t protein.

d) t bin lch khung: S thm 1 baz hay lm mt 1 baz dn n cc

codon sai ngha hay v ngha so vi codon tng ng ban u t im bin
i v sau, s dch m b lch khung c tnh dy chuyn t b ba b sai.
20.5.2.2- t bin nhim sc th
Cc t bin nhim sc th hay cn gi l sai hnh nhim sc th xut
hin sinh vt nhn thc.
Bin i trn 1 nhim sc th: mt on, lp on, o on.
Bin i gia cc nhim sc th: chuyn on.
t bin b gen (genome mutation)
a bi th (Polyploidy) hiu theo ngha rng l s thay i s lng
nhim sc th gm: a bi th nguyn (Polyploidy hay Euploidy) (2n > 3n,
4n, ), a bi th lai cn gi d bi th (Alloploidy)(2n A + 2n B) v a bi
lch (Aneuploidy), hay a nhim:(v d: 2n + 1 hoc 2n 1).
20.5.2.3- Cc bin i vi cu trc
Cc thay i thnh phn nucleotit ca gen.
a) t bin thay th: Thay mt nucleotit ny bng nucleotit khc.
ng chuyn (Transition) khi pyrimidin c thay th bi pyrimidin
hay purin bi purin. V d: T thay cho C hoc ngc li.
o chuyn (Transversion) khi pyrimidin c thay th bi purin
hay purin bi pyrimidin.V d: T hay C thay cho A hoc G v ngc li.
b) Mt nucleotit (Deletion): Mt mt phn nucleotit ca gen.
c) t bin xen nucleotit (Insertion mutant): Thm 1 hay nhiu nucleotit
vo gen.
20.5.2.4- Cc t bin kiu hnh
a) Cc t bin hnh thi: Cc bin i nh hng n hnh dng, mu
sc v kch thc. V d: mt dng t bin khun lc mu Serratia
marcescen.

Hnh 20.3. t bin khun lc mu Serratia marcescens

b) t bin sinh ha:

Cc t bin khuyt dng (Auxotrophe mutation) lm mt kh nng tng
hp cc cht.
Cc t bin c iu kin: Cc t bin c th khng c biu hin trong
nhng iu kin gii hn nht nh (restrictive condition) v c biu hin
trong cc iu kin cho php (permissive condition). V d, cc t bin nhy
cm vi nhit cao c biu hin nhit tng ng.
t bin khng: Cc bin i sinh ha gip khng li c cc tc nhn
bt li.
20.5.2.5. t bin v hi bin
a) t bin thun hay trc tip (Direct mutation): Bin i t kiu
hnh hoang di sang khc thng.
b) Hi bin (Reversion): t bin t kiu hnh t bin quay v kiu
hnh hoang di.
Hi bin tht hay t bin nghch. Bin i tr li y nh ban u.
V d: adenin guanin adenin

t bin thun

t bin nghch.

t bin c ch hay km hm (Supressor): t bin mt im khc. C

hai cng to kiu hnh gn nh hoang di.
- t bin km hm ngoi gen: Xy ra gen khc vi gen b t bin.
- t bin km hm trong gen: Xy ra nucleotit khc trong gen a
gen tr v trng thi to kiu hnh hoang di.
Ni chung, cc t bin l cc bin i rt a dng ca vt cht di
truyn v c nhiu tc ng khc nhau.
20.5.3- Cc phng php pht hin t bin

 nghin cu chi tit v cc t bin, cn c cc phng php pht

hin chng mt cch tng i y v chnh xc. y l cng vic kh
khn v a s t bin dng ln v nhiu dng kh pht hin.
S hon thin cc phng php pht hin t bin gp phn ng k
cho s pht trin ca di truyn hc v sinh hc.
20.5.3.1- Cc h thng chn lc t bin vi sinh vt
Mun pht hin cc t bin c hiu qu cn c h thng chn lc
tm thy cc t bin him hoi trong khi rt ln cc dng khng t bin.
Cc h thng chn lc t bin c nhiu v ph thuc vo cc t bin khc
nhau. Bng 20.3 nu cc cch pht hin 3 dng t bin.
Bng 20.3. C s pht hin cc kiu hnh t bin ca 3 loi t bin cn c kiu
hnh
Kiu gen

t bin c iu kin
(nhy cm nhit )
Nhit
thng

t bin khuyt dng

t bin khng

Nhit
cao

Khng b
sung

C b
sung

Khng tc
nhn

C tc
nhn

Kiu
hoang di

Bnh
thng

Bnh
thng

Tng
trng

Tng
trng

Tng
trng

Khng

t bin

Bnh
thng

t bin

Khng

Tng
trng

Tng
trng

Tng
trng

Khi nim lc phn gii (resolving power) c dng ch kh nng

pht hin cc t bin rt him. Lc phn gii cng ln khi pht hin c
cc t bin cng him. V d, cc t bin khng c phn gii cao v
khi cy s lng rt ln t bo ln mi trng chn lc c cha tc nhn th
phn ln t bo cht, ch s rt t t bo c t bin khng mc thnh
khun lc.
20.5.3.2- Phng php khng
vi khun, cc tc nhn chn lc thng l thuc v phage. Cc t
bin d dng c pht hin trn mi trng agar c thuc hay phage dng
cc khun lc c mc ln nh trn hnh 20.3.
20.5.3.3- Phng php lm giu chm: (Default enrichment method)

Vic pht hin cc t bin khuyt dng kh khn hn. Dung dch vi
khun pha long c cy ln b mt mi trng agar ti thiu mc ri
thnh khun lc. Mt lp mi trng tng t c ln trn, ph lp
mng. Hp Petri c cc khun lc bnh thng mc ln. Sau ,
ph ln thm mt lp mi trng dinh dng c b sung v tip cho cht
b sung khuch tn. Cc t bin khuyt dng s mc sau, khi c cht b
sung, nn khun lc nh hn do mc chm.
20.5.3.4- Phng php lm giu hn ch (Limited enrichment method)
y l dng n gin hn ca phng php lm giu chm. Cc vi
khun c cy trn mi trng ti thiu c mt t b sung. Trong iu kin
, cc t bin khuyt dng mc n khi ht cht dinh dng b sung th
dng, nn to khun lc nh. Cc vi khun bnh thng tip tc mc to
khun lc to.

20.5.3.5- Phng php lm giu nh penixilin

Phng php c p dng cho cc vi khun. Penixilin c tc ng
dit cc vi khun bnh thng khi phn chia. Cc vi khun c cho vo mi
trng ti thiu c penixilin. Cc vi khun ang tng trng b dit, ch cc t
bo t bin khng tng trng cn sng st. Sau , hn hp c cy ln
mi trng khng c penixilin th cc t bin khuyt dng mc ln vi t l
tng i cao hn (hnh 20.4).
T bo E. coli nui
trong mi trng
c gy t bin

Cy ln MT + leuxin

T bo E. coli nui
trong mi trng c
penicillin thiu leuxin

In qua MT leuxin

Hnh 20.4. Phng php penicillin chn lc m tnh

(v d chn leu)

Cc t bo E. coli c nui trn mi trng v gy t bin. Ra

sch t bo khi MT v chuyn sang MT c penicillin thiu leuxin (
leuxin). Sau cy t bo ln hp Petri cha MT c leuxin (MT + leuxin ),
cc t bo mc ln thnh khun lc. In cc khun lc sang hp Petri cha MT
ti thiu khng c leuxin. So snh 2 hp Petri xc nh c dng t bo t
bin: mc trn MT (+ leuxin ) v khng mc trn MT thiu leuxin (
leuxin )
20.5.3.6. Phng php lc
Phng php c s dng chn la cc t bin khuyt dng
nm si.
Dung dch cc bo t c nui trong mi trng dinh dng thiu
cht b sung. Cc t bin thiu cht b sung khng mc c, cc dng bnh
thng mc ra nhiu si. Khi lc qua mng lc si thy tinh, cc dng bnh
thng nhiu si b gi li, cc dng t bin i qua mng lc. Dung dch c
nhiu dng t bin c cy ln mi trng c cht b sung v kim tra tm
cc dng t bin.
20.5.3.7- Phng php in
Cc vi khun c cy mc ri tng khun lc trn mi trng c
dinh dng. Dng ming nhung (c nhiu lng mn) in ng cc v tr khun
lc trn mi trng ti thiu. Cc khun lc t bin khng mc ln c.
Cn c v tr khun lc khng mc bn sao tch cc t bin khuyt dng
(hnh 20.4 pha trn).
Ngoi cc phng php nu trn cn c cc phng php chuyn bit pht
hin nhiu loi t bin khc.

b
e
d

Hnh 20.5. Phng php in

a) Khc g bc ming nhung v trng. b) a gc trn mi trng y . c)
Ming nhung in dnh t bo tng ng cc khun lc. d) Mi trng hoc c
b sung cht cn thit. e) Mi trng ti thiu khng c c b sung

20.5.4- C ch phn t ca t bin

20.5.4.1- Cc bin i trn ADN
Tt c cc t bin u do nhng thay i trnh t nucleotit trn ADN.
Cc t bin c th xy ngu nhin (spontaneously) hay gy to (cm ng induced) bi cc tc nhn gy t bin (mutagens). Cc t bin c th do
thay i tng cp baz hay nhng trnh t di hn.
t bin im (Point mutation) l bin i trnh t ca mt cp baz.
t bin im c th do chuyn i ho hc baz ny thnh baz khc hoc
do bt cp sai trong sao chp. S bin i gm nhiu kiu:
S ng chuyn: thay cp GC bng AT v ngc li.
S o chuyn: thay AT bng TA v ngc li.
S bt cp sai (Baz mispairing) l s bt cp khng theo ng nguyn tc
ca m hnh Watson-Crick, m l adenin vi cytosin, thymine vi guanin.
Xen on (Insertion) l s thm vo mt on cp baz vo ADN. Tng i
on (Duplication) l mt dng c bit ca xen on.
Mt on (deletion) l s mt i mt trnh t ADN, m trnh t hai bn ni
li vi nhau tr trng hp mt u mt nhim sc th.

Transposon hay phn t di ng (transposable element) l trnh t

ADN c kh nng t xen vo (hay bn sao chnh n) v tr mi trn b gen
(genome), m khng cn c quan h g vi locus mc tiu. Xen on l kiu
t bin ph bin nht v do s di chuyn ca cc phn t di ng (ch. VI).
Phn ln t bin ngu nhin do s hin din ca cc baz bt thng
trn ADN. Ngoi ra, mt s baz b bin i (modified bazs) nh thng
gp hn c l 5-metylcytosin, c to ra do enzym metylaz thm nhm metyl
vo mt s cytosin nhng im c bit trn ADN.
20.5.4.2- Cc sai hng trong sao chp ADN
Cc t bin c th xy ra do sai lm khi sao chp ADN.
Mi baz tn ti 2 dng cu trc c gi l tautomer. V d, adenin
bnh thng mang nhm NH2 cung cp nguyn t hydro cho s bt cp b
sung vi dng keto (C = O - keto form) ca thymine. Khi c bin i
tautomer, adenin chuyn sang cu trc him l dng imino NH s bt cp b
sung vi cytosin. Thymine c th chuyn sang dng enol (COH) khng c
trong ADN bnh thng v bt cp vi guanin. Kh nng bt cp sai ca baz
vi tautomer khng ng c Watson v Crick nu ln.
S bt cp sai ny c th l cc t bin ng chuyn, trong purin
thay bng purin khc v pyrimidin thay bng pyrimidin khc.
Cc bin i trn, ngoi vic thay th cc nucleotit trn mch ADN cn
c th lm tng thm hay khuyt cc nucleotit gy nn cc kiu t bin nh
hng n sinh tng hp protein.
20.5.4.3- nh hng ca t bin gen n sinh tng hp protein
Sau khi tm hiu v dch m v m di truyn, cn lu n bin i
nh hng n ngha codon v v tr bin i u hay cui mch polypeptit.
a) t bin lch khung
Hai kiu t bin c hiu qu nng l thm baz (addition) v mt baz
(delection). Cc bin i ny thng lm enzym mt hot tnh. S thm 1
baz hay lm mt 1 baz dn n s dch m lch khung. T im bin i
v sau, t b ba b sai ci sai s ko di lin tc n cui mch polypeptit. V
d:
Bnh thng:

mARN: CCG GGA AGC AAU

Polypeptit: Pro Gly Ser Asn
t bin lch khung xen on (Frameshift - insertion)
mARN: CCG AGG AAG CAA
Polypeptit: Pro Arg Lys Gln
t bin lch khung mt on (Frameshift - deletion)
mARN: CCG GAA GCA AUG
Polypeptit: Pro Glu Asp Met
S tng hp mch polypeptit c th b kt thc sm nu s lch khung
dn n codon kt thc.
b) t bin thay th (Baz substitution)
t bin thay th baz nu l t bin sai ngha (mis-sense) s c hiu
qu thay i t axit amin ny thnh axit amin khc trong mch polypeptit,
cn nu l t bin v ngha (non-sense) hay t bin trung tnh (hay im
lng) s khng nh hng n mch polypeptit V d:
Bnh thng:
mARN: CCG GGA AGC AAU
Polypeptit: Pro Gly Ser Asn

Sai ngha (Missense):

mARN: CCG GCA AGC AAU
Polypeptit: Pro Val Ser Asn
V ngha (Nonsense)
mARN: CCG UGA AGC AAU
Polypeptit: Pro STOP
20.5.4.4- Sai hng ngu nhin
Ngoi cc sai hng trong sao chp, phn t ADN cn chu cc sai hng
ngu nhin c th dn n t bin. Hai kiu sai hng ngu nhin thng gp
l mt purin (depurination) v mt amin (desamination). Mt purin l kiu sai

hng thng hn, xy ra khi lin kt glycosidic gia C1 ca pentoz vi baz

b t v lm mt A hoc G.
S mt amin ca cytosin to ra uracil. Cc gc U khng c sa sai
s bt cp b sung vi A trong sao chp, gy ra ng chuyn G-CA-T.
Trong cc enzym sa sai, uracil ADN-glycosylaz nhn bit c hiu uracil
trn ADN v ct ri to l hng, sau c tng hp li ng theo mch b
sung.
Trn phn t ADN, mt s cytosin c metyl ha thnh 5-metyl
cytosin, cht ny mt nhm amin bin thnh thymine. Sai hng ny khng b
uracil-ADN-glycolaz pht hin nn khng c sa li. S chuyn C T do
mt amin thng xy ra cc im c 5-metyl cytosin. Kiu bin i ny c
c vi khun v t bo sinh vt bc cao.
20.5.5. t bin nhn to hay cm ng
Cc tc nhn lm tng tn s t bin cao hn mc t nhin c gi
l cc tc nhn gy t bin (mutagen). Cc tc nhn vt l nh phng x, tia
X, tia t ngoi gy t bin. Nhiu ha cht l tc nhn gy t bin nh cc
ng ng ca cc baz nitric, HNO2 (nitrous axit), cc cht alxyl ha
mch...Cc t bin loi ny c gi l t bin nhn to hay t bin cm
ng (induced mutation).
i vi cc vi sinh vt, cc tc nhn gy t bin ch yu l tia t ngoi
v mt s ha cht.
20.5.5.1-Tc ng ca tia t ngoi
Tia t ngoi c bc sng di (10-5 - 10-6 cm) nn kh to ion, c l ch
tc ng n nhng cht hp thu n trc tip. Trong t bo, cc cht hu c
c mch vng ch yu nh purin v pyrimidin hp thu trc tip tia t ngoi.
Mi lin quan cht ch gia tia t ngoi v cc cu phn ca ADN c
chng minh. ADN hp thu tia t ngoi mnh nht bc sng 2537, y
chnh l bc sng lm tng tn s t bin.

Cc thymin k
nhau

Hnh 20.6. Tc ng ca tia t ngoi to cc dimer thymine

Di tc ng ca tia t ngoi, cytosin gn thm phn t nc vo lin kt C

= C ca mch vng (hnh 3.6) v thymine b t lin kt C = C mch vng
ni 2 phn t thnh thymine dimer.
Ston v cc cng s nhn thy tn s t bin tng ln
Staphylococcus aureus khi mi trng nui chng c chiu tia UV trong
thi gian ngn trc khi cy vo. y l tc ng gin tip ca tia t ngoi.
C th khi hin din oxy, tia t ngoi to ra nhiu hn cc gc peroxit:
H* + O2 HO*2
HO*2 + H* H2O2
2HO*2 + H* H2O2 + O2
Cc peroxit l nhng phn t c phn ng mnh, chng d to cc t
bin.
Hin tng quang phc hi (photoreactivation) l mt c im trong
tc ng ca tia UV. Sau khi chiu tia t ngoi ln t bo, nu ngoi nh
sng, th cc sai hng phn ln c phc hi. nh sng c tc ng hot ha
enzym sa sai, ct t cc thymine dimer.
20.5.5.2- Cc tc nhn gy t bin ha cht
Ngay t u nhng nm 1930, Xakharov v Lobashov (Lin X) tin
hnh th nghim gy t bin bng ha cht, nhng hiu qu cha r. Vo

nhng nm 40, trong th chin th hai Anh, Auerbach v Robson chng

minh hi ngt nit v sulfua c kh nng gy t bin Drosophila (thi ny
c bn hi ngt sang nc Anh, nn h phi nghin cu tc ng sinh hc ca
cc cht c ny). V sau, nhiu nhm ha cht gy t bin c tm ra.
C nhiu ha cht gy bin d di truyn, n nay tm ra nhng ha cht
cho hiu qu t bin cao hn c phng x.
Cc ha cht gy t bin c c im l c th ch gy hiu qu t
bin i vi mt s lng t i tng.
V d: Streptomycin ch gy t bin to n bo v mt s vi sinh
vt, khng gy t bin trn nhiu i tng khc.
Cc tc nhn gy t bin ha hc c th chia thnh cc nhm sau:
Nhm 1: Cc cht c ch tng hp nitous baz trong cu trc ADN
nh coffein, etyl uretan...

Hnh 20.7. Thymine v 5-Bromuracil (dng keto)

Hnh 20.8. Adenin bt cp vi 5-Bromuracil v Guanin vi 5-Bromuracil

Hnh 20.9. Cng thc ca etyl metansulfonat (EMS)

 Nhm 2: Cc cht ng ng vi nitous baz nh coffein, 5-bromuracil

(hnh 20.7 v 20.8), cc cht gn ging vi nitous baz, nn n lm ADN
gn nhm khi tng hp.
Nhm 3: Cc cht alkyl ha lm t mch ADN nh etyl metansulfonat
(EMS) (hnh 20.9), metyl metansulfonat (MMS), etylen imine (EI),
nitrosoguanidin (NG),...
Nhm 4: Cc cht khc nh nhm oxy ha, kh.
Ngc vi sai hng sao chp, cc tc nhn gy t bin nh nitrous
axit v kh ngt nit (nit mustard) c th gy bin i trc tip trn ADN.
- Nhm 5: cc cht chm vo ADN
Nhm cc cht gm proflavin, mu acridin v cc cht c gi l ICR
(ICR compound) l nhng cht c phn t mt phng tng t cp baz.
Chng c th chm vo phn t ADN lm thm hoc mt baz. Chng
thng gy t bin lch khung do thm hay mt baz.
Tt c cc tc nhn gy t bin u l tc nhn gy ung th
(carcinogen), nhng cc tc nhn gy ung th khng phi u gy t bin.
Hin nay nhiu tc nhn gy t bin c s dng trong chn ging nhm
tng ngun bin d. Bn cnh vi nn nhim trn th gii ngi ta pht
hin nhiu tc nhn t bin ha hc mi xut hin trong mi trng.
20.5.6. Hi bin
Qu trnh t bin, ni chung, c tnh thun nghch, ngha l nu mt
gen A t bin thnh a (A > a) th, ngc li alen a cng c th t bin
quay li thnh A (a >A). Thng thng mt dng c gi l t bin khi
n mang kiu hnh khc vi dng hoang di. V d, rui gim hoang di c
bt t thin nhin vo phng th nghim c mt . Trong qu trnh nui xut
hin dng t bin mt trng. t bin t mt hoang di sang mt trng
gi l thun v t hoang di thnh t bin. Hi bin l trng hp t trng
thi t bin do bin d di truyn quay tr v kiu hnh hoang di nh t
bin t mt trng tr li thnh mt . Hi bin do t bin nghch (back
mutation) hoc do t bin c ch hay km hm (supression).
20.5.6.1. Cc t bin nghch

t bin nghch c c khi gen t bin c s bin i quay tr li c

y cu trc nh gen hoang di ban u. Trng hp ny kh xy ra v khi lai
tr li vi dng hoang di th th h con tt c u c kiu hnh hoang di. V
d:
t bin nghch: m > m+, khi lai vi dng hoang di cho th h con u
hoang di
t bin c ch: msu+ > msu, khi lai vi dng hoang di trong th h
con s c mt t kiu hnh t bin.
20.5.6.2- t bin c ch
t bin c ch (Suppressor mutation) l t bin c tc ng ngc
li hay km hm ca mt t bin khc. Cc t bin c ch c nhng tnh
cht sau:
- t bin c ch xy ra im khc vi t bin b c ch. Khi lai th
hi bin (revertant) vi dng hoang di s xut hin dng t bin b c ch do
ti t hp lm tch ri khng b km hm bi t bin c ch (hnh 20.10).
- t bin c ch c th xy ra trong cng mt gen, ngoi gen hoc
gen khc.
- Cc t bin c ch c th thc hin tc ng bng nhiu cch khc
nhau. V d, cc t bin c ch c th tc ng ln s phin m, dch m
hay nhng biu hin sinh l khc ca t bo.
t bin km hm thng gp hn, n c c do 1 t bin th hai lm
cho biu hin kiu hnh ca t bin khng biu hin ra c nn c kiu
hnh hoang di. t bin km hm c th xy ra ngay trn cu trc gen. V
d: t bin thun mt mt nucleotit, t bin km hm xy ra gn ch
thm vo mt nucleotit. t bin km hm c th do s b sung trong chu
trnh trao i cht. Sai hng do t bin th hai to sn phm b tr c t
bin th nht.
Vo nm 1962, S.Benzer v Chemp m t t bin c gi l t bin
amber locus rII ca phage T4. Chng c th tr v kiu hnh hoang di do
t bin khc (t bin c ch) b gen ca t bo ch. Cc t bin c ch
c pht hin nhiu gen khc ca phage T4 v E.coli. Cc nghin cu s
dng h thng gen- enzym cho thy, cc t bin amber dn n s kt thc

sm hn bnh thng s mc di ca mch polypeptit v trong cc t bo

ch cc on c u NH2 ca cc polypeptit tng ng c tng hp. Nh
t bin c ch, tng hp mch polypeptit c hi phc.
A.Haren, khi nghin cu s kim sot di truyn i vi tng hp
enzym photphataz kim E.coli, so snh thnh phn cc gc axit amin
trn phn t enzym ca dng hoang di v cc th hi bin trong gen m
ha cho enzym. Kt qu trn hnh 20.10 cho thy cc c ch i vi amber
lin quan n mt thay th nucleotit trong codon.
Trn c s cc s liu ny xc nh c codon - amber l UAG.
Sau , 2 codon chm dt khc c tm ra l ochre UAA v opal UGA.

Hnh 20.10. S thay th cc axit amin do cc t bin c ch i vi cc t bin

amber gen cu trc ca enzym photphataz kim

Cc bin d c ch c dng nghin cu su hn v c ch dch

m. V d, cc t bin nh hng ti anticodon ca tARN, lm thay i tnh
c hiu m ha ca n, c th to kh nng c ch t bin khc mc
phin m. Cc t bin c ch i vi codon v ngha (nonsens-suppressor)
thng xy ra trn cc tARN, m anticodon ca n c th b bin thnh
anticodon b sung vi codon kt thc do s thay th mt nucleotit. Cc
nonsens - suppressor nh vy thng l tri.
C th xy ra cc t bin c ch i vi cc t bin nhm ngha. V
d, tARNgly ca axit amin glycine c anticodon CCC thng bt cp vi
GGG (gly) trn mARN. S bin i t bin ca anticodon thnh CUC dn
n ch tARNgly t bin bt cp vi GAG (glutamic axit). Nh vy, nu nh

t bin thun mt gen cu trc no bin codon GGG (glycine) thnh

GAG (glutamic axit) ca t bin nhm ngha (missens mutation), th s c
ch i vi t bin ny c th do t bin ca tARNgly vi anticodon CUC
s gn glycine vo ch b t bin ( t bin nhm ngha l glutamic axit).
Cc t bin xy ra trn tARN c th l t bin c ch i vi cc t
bin lch khung.
S c ch mc phin m c th xy ra do cc t bin trn cc gen m ha
mt s protein ca riboxom.
20.6- DI TRUYN HC VI KHUN
Cc vi khun c qu trnh sinh sn cn hu tnh nn vn thc hin
c ti t hp di truyn. vi khun, thng tin di truyn c truyn mt
chiu t th cho sang th nhn v to ra hp t tng phn. Ti t hp vi
khun c th thc hin bng cc on ADN trn trong bin np, hay phage
trong ti np, nh giao np khi 2 t bo khc gii tnh gn vi nhau. Bn
di truyn vi khun c xy dng nh cc phng phc khc nhau: giao np
gin on hoc khng gin on, ti t hp hay dng mt on.

Plasmid l nhng ADN vng trn c th tn ti c lp hoc chn vo

ADN t bo ch c vai tr quan trng trong chuyn gen.
Transposition l s chuyn v gen gm IS, Tn v phage Mu. Cc phn
t di ng c th gy bin i s biu hin gen.
Vo nhng nm 1940, ti t hp vi khun E. coli c chng minh.
T n nay, vi khun E. coli tr thnh i tng m hnh cho di truyn
hc v sinh hc phn t. Nhng nghin cu trn i tng ny pht hin
cc c ch cn bn ca s sng cp phn t nh sao chp ADN, sinh
tng hp protein v iu ha biu hin gen. Ngoi ra, cc pht hin v cc

qu trnh di truyn c bit vi khun nh bin np, ti np, giao np,

transposition v plasmid c ngha quan trng cho s pht trin ca di truyn
hc phn t v gp phn xy dng nn k thut di truyn.Trong mt thi gian
di, cc nghin cu di truyn hc c tin hnh cc sinh vt nhn thc
Eukaryota, cn vi khun th cha v cho rng khng c sinh sn hu tnh.
Khc vi virut, vi khun v c vi khun (bacteria and archaea) l nhng t
bo sng thc hin cc chc nng cn bn ca t bo.
Cc sinh vt Prokaryota nh vi khun, vi khun lam cng c cc qu trnh
sinh sn tng ng sinh sn hu tnh, c gi l cn hu tnh
(parazxuality). S di truyn nh cc qu trnh cn hu tnh ny vi khun c
nhng c im:
S truyn thng tin mt chiu t t bo cho (donor) sang t bo nhn
(recipient).
S to thnh hp t tng phn (merozygote). Th cho (donor) ch
chuyn mt on ca b gen sang th nhn (recipient) nn ch lng bi
mt phn, cc phn khc n bi.
B gen thng ch l mt ADN trn nn ch c mt nhm lin kt
gen v ti t hp thc cht l gia hai phn t ADN.
20.6.1- Mt vi c im ca vi khun
20.6.1.1- Kiu hnh v kiu gen ca vi khun
Cc t bin c th tc ng n 5 loi kiu hnh:
Bin i t nguyn dng (prototrophy) sang khuyt dng
(auxotrophy) v ngc li. V d: mt kh nng tng hp mt cht trao i
ca chu trnh v hi bin li c kh nng tng hp cht .
S mt hay c c kh nng s dng cht dinh dng khc. V d:
t bin lm mt kh nng s dng ng lactoz.
Tnh nhy cm hay khng thuc nh nhy cm streptomycin t
bin thnh khng streptomycin.
Nhy cm vi phage thnh khng phage hoc ngc li. V d: t
bin trn th th b mt t bo lm khng vi s nhim phage.

 S mt i hoc c li cc thnh phn cu trc ca b mt t bo. V

d,mt loi Pneumococcus c v bao polyxacarit (polyxacarit capsule), trong
khi dng khc khng c v bao.
Cc k hiu dng biu hin kiu hnh v kiu gen c thng nht theo
nguyn tc:
K hiu kiu hnh gm 3 ch thng (ch u vit hoa) vi du pha
trn gc + hay nhm ch s hin din hay thiu tnh trng tng ng,
v s hay r tng ng ch tnh nhy cm (sensitive) hay khng
(resistance).
K hiu kiu gen c vit ch nghing, ch u khng vit hoa.
V d 1: T bo hoang di t tng hp c leuxin th kiu hnh c
vit Leu+. t bin khuyt dng mt kh nng tng hp leuxin c k hiu
kiu hnh Leu. Tng ng vi hai kiu hnh trn k hiu kiu gen l leu+ hay
leu v leu.
Nu cn nhiu hn mt gen to ra mt cht nht nh, sau 3 ch
nghing k hiu gen c thm ch nghing hoa. V d: leuA, leuB... l cc gen
cn thit cho tng hp leuxin khc nhau.
V d 2: Kiu hnh khng hoc nhy cm vi penixilin c vit l
Penr v Pens. Kiu gen tng ng l penr v pens.
Nu lng bi mt phn th vit thm gch nghing. V d, leu+/leu-.
20.6.1.2- Vi nt v sinh sn vi khun
T bo vi khun phn chia theo li trc phn. Phn t ADN gn trc
tip vo mng sinh cht. S sao chp ADN to ra hai bn sao gn
chung nhau trn mng sinh cht. Khi t bo ko di ra, cc bn sao
ADN tch xa nhau do phn mng gi chng ln dn ra. Kiu sinh sn
v tnh ny c gi lphn ihay ngt i (binary fission)
(hnh 20.11). T bo vi khun chia nhanh (20 pht trong iu kin tt)
hn rt nhiu so vi t bo Eukaryota (24-48 gi).

Hnh 20.11- Trc phn ngt i vi khun km theo tch 2 ADN con v 2 t bo

Qu trnh sao chp ADN c bt u t im xut pht oriC ko di

v hai pha song song vi qu trnh sao chp mng sinh cht, ni c im gn
vo ca ADN b gen, mc di tch 2 phn t ADN v 2 t bo con.
ADN ca E.coli cn 40 pht cho 1 vng sao chp tng ng vi tc
50.000 cp baz/pht. Ph thuc vo tc tng trng, thi gian phn chia
t bo trong khong t 18 n 60 pht. Nh vy cc t bo tng trng
nhanh, vng sao chp mi phi c bt u sm hn s phn bo trc
nh t bo con u tin.

20.6.2- Vi khun E.coli l i tng m hnh tt nht

Escherichia coli (E.coli) l vi khun c nghin cu k nht, rt nhiu
chng khc nhau c phn lp. Ba dng thng gp trong cc phng th
nghim di truyn l E.coli B (t bo ch cho cc phage dy T), E.coli C (t
bo ch cho phage mt mch nh X 174) v E.coli K12 (t bo ch ca
phage ). Do nhng thun tin trong nui cy, nhn ging, thu nhn cc t
bin v d phn tch cc s kin di truyn him hoi. n nay, n c coi l
i tng m hnh s mt ca Sinh hc phn t v cng ngh gen.
20.6.2.1. Cc d liu di truyn hc ca E. coli
Kch thc b gen (Genome size): 4,6 Mb
Nhim sc th: 1 phn t ADN vng trng
S lng gen: 4.000
Phn trm gen tng ng vi ngi: 8%
Kch thc trung bnh ca gen: 1 kb, khng c intron
Cc transposon: ty chng, ~ 60 bn sao/b gen
Kt thc gii k t chui: 1997
20.6.2.2- Cc phng php phn tch di truyn
Ngoi cc phng php lai phn tch ti t hp (recombination) v
b tr (complementation), c nhiu k thut bin i di truyn (Techniques
of Gentic Modification):
Gy t bin (Mutagensis):
 Ha cht v chiu x: t bin xoma ngu nhin.
 Dng transposon: xen on (Insertions) xoma ngu nhin.
Chuyn gen (Transgensis):
 Trn vector plasmid: t do hay chn vo (integratd).
 Trn vector phage: t do hay chn vo (integratd).
 Bin np: chn vo.
Lm im lng gen mc tiu (Targeted gen knockout):
 Alen khng (Null alen) trn vector: thay gen bng ti t hp.

 Alen c thit k (Engineered alen) trn vector: t bin im nh

hng (Site-directed mutagensis) bng thay gen.
20.6.2.3- E.coli l t bo ch cn bn ca k thut di truyn
Cc nghin cu trn E. coli lm c s cho s ra i ca k thut di
truyn. E. coli ng vai tr ch yu trong chuyn gen n cc sinh vt khc.
N l sinh vt chun to dng gen (cloning gens) ca bt k sinh vt no.
E. coli c coi l t bo vt ch n gin nht trong cng ngh gen. Vi
khun E. coli d dng chp nhn nhiu loi vector chuyn gen nh plasmid
hay phage qua bin np hay ti np.
Nhng protein ti t hp u tin nh insulin, xomatostatin v n nay
hng trm protein khc c to dng E. coli i vo sn xut cng
nghip vi th trng hng chc t USD/nm.
20.6.2.4. Cc ng gp ch yu ca E.coli
i tng ch yu cho cc nghin cu:
 Sao chp, phin m, dch m v ti t hp.
 t bin
 iu ha biu hin gen (Gen regulation).
 K thut ADN ti t hp (recmbinant ADN technology).
ng gp cho nghin cu cc lnh vc khc: Trao i cht ca t bo
(Cell metabolism), gen c ch v ngha (Nonsense suppressors), s tuyn
tnh (Colinearity) gia gen v polypeptit, cc Operon, s khng thuc da
vo plasmid (Plasmid-bazd drug resistance) v s vn chuyn tch cc
(Active transport).
20.6.2.5- Cc enzym v protein tham gia tng hp v ct nucleic axit
Sao chp ADN c nghin cu rt chi tit E. coli, m phn ch yu
nu chng II. y, mt s chi tit c b sung, c th l cc phc
hp enzym tch mch ADN m v tng hp cc mch con (bng 20.4). Hng
lot cc enzym v protein, m phn ln l cc phc hp protein a phn,
tham gia tng hp ADN, phin m to ARN. C t bo nhn s ln nhn
chun u c hot tnh ADN polymeaz a nng.

Mt s ADN polymeaz hot ng nh nhng enzym c lp, nhng s

khc (ch yu l replicaz) kt nhau thnh phc hp ln nhiu protein. Tiu
phn (subunit) tng hp ch l mt trong nhiu chc nng khc nhau ca
replicaz nh tho xon (unwinding), khi s tng hp mch mi,
Bng 20.4. Cc enzym tham gia sao chp ADN vi khun E. coli.

Enzym
1. ADN polymeaz III
2. ADN polymeaz I

Gen m ho

Chc nng

polC;
ADNE,Q,N,X;

Enzym polyme ho ch yu

polA-E; mutD.

Ct mi ARN, lp ch trng

polA
3. Helicaz

ADNB

Tho xon ch ba sao chp

4. Primaz

ADNG

To mi mch ADN mi

5. Protein gn im khi

Gn im khi s sao chp

s (Origin-binding
protein).

ADNA

(Ori); to thun li cho m

tch mch.

ssb

Ngn cc mch n tch

khng chp li.

ligA, ligB

Ni cc u h trn ADN

6. Protein cng mch SSB

(Single-strand binding
protein)
7. ADN ligaz

Bng 20.5- Cc enzym ADN polymeaz vi khun E. coli.

Enzym
1. ADN
polymeaz I

Gen m ho

Chc nng

polA;ADNE,Q,N,
X

Enzym phc hi ch yu. Ct mi ARN v

lp kn khong trng

2. ADN

holA-E; mutD.

Enzym phc hi ph (minor).

polB

Replicaz, polyme ho ch yu.

polC

Phc hi cp cu (SOS repair).

polymeaz II
3. ADN
polymeaz III

4. ADN
polymeaz IV

5. ADN

ADNB

polymeaz V

umuD2C

Phc hi cp cu (SOS repair).

E. coli tm ra v bit chc nng ca 5 loi ADN polymeaz (bng

20.5).
S phn hy nucleic axit cng i hi enzym c hiu:
Deoxyribonucleaz (ADNaz) l enzym ct cc lin kt trong phn t ADN.
N c th ct mch n hay kp. Gyraz l mt loi enzym topoixomraz ct
ADN lm tho xon.
Ribonucleaz (ARNaz) l cc enzym ct ARN, c th c hiu i vi ARN
mch n hay mch kp.
Cc nucleaz chia thnh 2 nhm:
Exonucleaz ct tng nucleotit mt t u mt ca mch polynucleotit;
chng c th c hiu i vi u mt 5 hay 3 ca ADN hay ARN.
Endonucleaz ct cc lin kt bn trong mch ADN; chng c th c
hiu i vi ARN hay ADN mch n hoc mch kp.
Cu trc v chc nng cc tiu phn ca ADN polymeaz III:
Tt c cc ADN polymeaz cng c nhng tnh cht cu trc chung
ging nhau. Replicaz ADN pol III l mt holoenzym 900 kD (kilodalton),
mt phc hp gm hn 10 phn t protein (hnh 6.12). c bit l protein

vng (-ring) lm mc (clamp) bao ADN gia trt v trc m mch

n ny khng b bong ra khi c chp.
Ngoi ra, ADN polymeaz phi c kh nng nhn bit 4 loi N nh cht
phn ng ph thuc vo ch c "c" trn mch khun.
Sao chp ADN E. coli din ra vi tc rt nhanh, c th t n
50.000 nucleotit/pht. Tht kh hnh dung mt qu trnh phc tp, c thc
hin chnh xc, m din ra vi tc nhanh nh vy, cha k n vic lm
sao cc N tp trung vi nng cao p ng kp thi cho nhu cu.

Hnh 20.12-. Replicaz ADN pol III l mt phc hp gm hn 10 protein

20.6.3-Bin np (Transformation)
20.6.3.1- Hin tng v iu kin
Hin tng bin np c Griffith pht hin vi khun Diplococus
pneumoniae (nay gi l Streptococus pneumoniae - ph cu khun gy sng
phi ng vt c v) vo nm 1928. Pht hin ny v cc nghin cu v c
ch bin np c ngha lch s cho s ra i ca Sinh hc phn t.
Vi khun ny c 2 dng khc nhau:
Dng SIII, gy bnh c v bao t bo (capsule) bng polysaccharid cn
tr bch cu ph v t bo. Dng ny to m mc (khun lc) lng (Smoothlng) trn mi trng agar.
Dng RII, khng gy bnh, khng c v bao, to m mc nhn (Roughnhn).

Th nghim tin hnh nh m t trn hnh 20.13.

Hnh 20.13. Hin tng bin np.

- Tim vi khun S sng gy bnh cho chut - chut cht.
- Tim vi khun R sng khng gy bnh - chut sng.
- Tim vi khun S b un cht cho chut - chut sng.
- Hn hp vi khun S b un cht trn vi vi khun R sng em tim cho chut - chut
cht. Tr ong xc chut cht c vi khun S v R.

Hin tng trn cho thy vi khun S khng th t sng li c sau khi
b un cht, nhng cc t bo cht ny truyn tnh gy bnh cho t bo R.
N c gi l bin np (transformation).Nm 1944, T.Avery, Mc Leod v
Mc Carty tin hnh th nghim xc nh r tc nhn gy bin np. Nu cc
t bo S b x l bng proteaz (enzym phn hy protein) hoc ARN-az
(enzym phn hy ARN) hot tnh bin np vn cn, chng t protein v
ARN khng phi l tc nhn gy bin np. Nhng nu t bo S cht b x l
bng ADN-az (enzym ch phn hy c hiu ADN) th hot tnh bin np
khng cn na, chng t ADN l nhn t bin np. Kt qu th nghim c th
tm tt nh sau:
ADN ca S + cc t bo R sng chut cht (c R + S)
Hin tng bin np l mt chng minh sinh ha xc nhn rng ADN
mang tn hiu di truyn.
Nh vy, bin np l hin tng truyn thng tin di truyn bng ADN.
Trong bin np, ADN trn t mt t bo vi khun (th cho) ny c truyn
sang t bo vi khun khc (th nhn). Bin np xy ra khi vi khun nhn
ADN ngoi lai v hp thu vo trong t bo. Khi t bo vi khun b v do b
tan (lysis), ADN vng trn ca chng thot ra mi trng thnh cc on
thng vi chiu di khc nhau, c kh nng gy bin np cho cc t bo
nhn khc.
Bin np c nghin cu k nht cc vi khun Streptococcus
pneumoniae, Bacillus subtilis, Haemophilus influenzae v mt s nhm vi
khun khc.
Hiu qu ca bin np ph thuc vo 3 yu t:
- Tnh dung np ca t bo nhn.
- Kch thc ca on ADN ngoi lai.
- Nng ca ADN
20.6.3.2- Tnh dung np ca t bo nhn
Mt iu quan trng ca bin np l t bo nhn phi c trng thi sinh
l c bit c gi l kh nng dung np hay kh np (competence). T bo
c kh nng hp thu ADN ngai lai v c bin np (transformable) gi l
kh np (competent) v y l tnh trng di truyn. Thm ch trong cc chi
(genra) bin np, ch mt s chng (strains) hay loi l c bin np. Tnh

kh np trong phn ln cc vi khun bin np t nhin (naturally

transformable) c kim sot (regulated) v cc protein c hiu tham gia
vo hp thu v tc ng n ADN. Cc protein c hiu kh np gm
protein gn ADN lin kt mng (membrane-associated ADN-binding
protein), autolysin vch t bo v cc nucleaz khc nhau. loi Bacillus
subtilis d bin np, cc t bo sn sinh v tit ra mt peptit nh trong qu
trnh tng trng v s tch ly nng cao ca peptit ny bin t bo thnh
kh np. Bacillus, ch 20% t bo bin thnh kh np v trng thi ny
trong vi gi. Trong khi , Streptomyces, 100% t bo c th thnh kh
np, nhng ch trong mt thi k ngn ca chu trnh t bo.
Bin np t nhin hiu qu cao c pht hin ch mt t loi vi khun nh
Acinobacter, Azotobacter, Bacillus, Streptococcus, Haemophilus, Neisseria
v Thermus. Ngc li, E. coli v nhiu loi vi khun kh bin np trong iu
kin t nhin.
Tuy nhin, c th gy ra s dung np bng x l ha cht hay to
nhng iu kin nht nh cho s tng trng ca t bo. Khi x l t bo E.
coli vi ion canxi nng cao, chng tr thnh kh np v bin np cc
plasmid thc hin c hiu qu.
Nhng t bo dung np trn b mt c cc nhn t dung np
(competence factor). Chng c tinh sch mt phn v nghin cu
nhiu loi vi khun. Streptococcus (trc y gi l Diplococcus)
pneumoniae tr thnh dung np c 30 n 80 im nhn trn t bo c kh
nng gn vi ADN mch kp hu nh ca bt k ngun no. Mt khc,
Haemophilus influenzae c mt s lng hn ch t 4 n 8 im nhn
(receptors), m nhng im nhn ny trc tin nhn bit ADN mch kp c
cc cp baz trnh t nh sau: 5AAGTGCGGTCA-3 c gi l im hp
th (uptake site). S kin l cc im hp th ny c bit chung ADN
ca Haemophilus (trn b gen c khong 600 im nh vy) v tng i
him ADN ca cc loi khc gii thch v sao Haemophilus ch bin np
gii hn vi cc vi khun trong loi.
Phn ln cc vi khun ch dung np trong mt giai on gii hn ca
chu trnh sng. Trong giai on dung np, t bo tng hp mt hay nhiu
protein c gi l cc nhn t dung np, chng bin i mng t bo

c th gn vi on ADN ngoi lai. Nh vy, cc im th th ch hin din

trong giai on dung hp.
20.6.3.3- S hp thu ADN
Trong bin np, vi khun kh np gn thun nghch vi ADN. Cc t
bo kh np c th gn ADN nhiu hn c 1000 ln so vi t bo khng kh
np.
iu kin quan trng th hai thc hin c bin np l ADN phi
c mch kp v on bin np phi c trng lng phn t ti thiu l
400.000 dalton (ln khong 1/200 b gen ca vi khun), d nhin y khng
phi l gii hn cao nht. S lng t bo c bin np (transformants - th
bin np) tng t l thun vi nng ca ADN cho n lc m cc im th
th (receptor sites) bo ha do cc on ADN gn vo (thng khong 10
on/t bo). Streptococcus pneumoniae mi t bo c th gn khong 10
phn t ADN mch kp di 10 15 kbp (kilobaz pairs)/phn t. ADN c
hp th vo t bo v b enzym ct lm gim trng lng phn t cn khong
8 kbp/phn t v mch n.
Mt ADN ti thiu c th pht hin bin np l 0,01 ng/ml, mt
con s thp n ni khng th pht hin bng phng php ha hc.
iu th v l trong bin np Haemophilus influenzae, on ADN
phi c chui k t 11 bp chuyn bit xy ra qu trnh gn khng o
ngc v thu nhn ADN. Chui ny c tm thy vi tn sut cao bt
thng b gen ca Haemophilus, b gen c gii k t chui hon
ton. Bng chng ny v vic mt s vi khun no c th bin np trong
mi trng t nhin lm c s cho gi thit l bin np khng ch xy ra
trong phng th nghim m cn ng vai tr quan trng trong vic truyn gen
cho th h sau trong t nhin. Bng cch tng cng trao i gen, nhng vi
khun kh bin t nhin tng tnh a dng v tnh thch ng.
20.6.3.4- on ngoi lai v on ni ti
Khi ADN mch kp xm nhp vo t bo, mt mch b phn hy. Bt
k on ADN no t t bo cho xm nhp t bo nhn, c gi l on
ngoi lai (exogenote), ADN nguyn vn ca t bo nhn gi l on ni ti
(endogenote). T bo vi khun nhn on ngoi lai s lng bi mt phn

b gen, c gi l hp t tng phn (merozygote). Tuy nhin, on ngoi

lai mch n khng bn vng v b phn hy nu khng c gn vo b gen
th nhn. Qu trnh trao i thng tin di truyn bng chuyn ch mt phn vt
liu di truyn t t bo ny sang t bo khc c gi l s giao np tng
phn (meromixis). C ngi cho rng on ngoi lai n mch c gn vi
protein (nh protein Rec-A E. coli), protein ny h tr tm vng b sung
trn on ni ti, lm t mch v gn on ngoi lai. Cc enzym s ct cc
u t do (ca c th cho v th nhn) v ligaz hn kn lm trng. Khi on
ngoi lai gn vo on ni ti th t bo khng cn l hp t tng phn
na.
Nu on ngoi lai cha mt alen vi on ni ti, th mch kp ti t
hp s c mt hay nhiu ch bt cp sai (mismatch baz pairs) v on ADN
ny c gi l heteroduplex. Nu cc t bo con nhn alen mi, sa sai
(mismatch repair) c thc hin bng cch ct on ni ti v dng mch
ngoi lai lm khun thay th. S gn on ngoi lai vo ADN t bo nhn
c thc hin nh ti t hp tng ng (xem sau).
Hai hoc nhiu hn cc gen lin kt cht c th nm trong on ADN
bin np. Nu s xm nhp ca hai hay nhiu gen vo on ni ti, th t bo
nhn s c ng bin np (cotransformed). Tn s ca ng bin np t l
nghch tng ng vi khong cch gia cc gen cng bin np.
20.6.3.5- C ch phn t
Din bin ca qu trnh bin np cp phn t c tm tt trn s
hnh 20.14. d hiu, cc gii thch da theo ADN ca cc dng vi
khun S v R trong th nghim ca Griffith. Qu trnh gm cc giai an ch
yu:
S phn hy ADN t bo cho: ADN t bo cho c th l ca t bo t
nhin b phn hy hoc trong th nghim b gy ch bng nhit cao hay tc
nhn ph v t bo.
ADN bm vo b mt t bo: Protein gn vo ADN.

 Thm nhp ca ADN: Si ADN mch kp ca dng vi khun S sau khi

chui qua mng t bo ca dng R th mt mch S s b nucleaz ca t bo ct,
cn li mt mch nguyn.
Bt cp (Synapsis)v ti t hp: Nh s h tr ca protein RecA ADN ca
th nhn R s bin tnh tch ri 2 mch 1 on d bt cp vi on ADN
th cho S va chui vo.

Hnh 20.14-. S din bin ca qu trnh bin np cp phn t

a. ADN bm vo. b. Thm nhp. c. Bt cp v ti t hp. d. T bo bin np

Sao chp: Sau khi bt cp to on lai R - S, phn t ADN sao chp to ra 2

si, mt si kp R-R v si kp khc c mang on ADN th nhn S-S. Kt
qu cui cng l an gen ca SIII chn vo b gen t bo nhn. Sau phn bo
th mt dng t bo nhn c ADN ngoi lai vo b gen - t bo c bin
np. T bo c bin np sinh sn to dng nhn RII mi.

20.6.4-Ti np (Transduction )
Vic tm ra bin np thc y nghin cu dn n pht minh ra ti
np, l hin tng truyn ADN qua trung gian virut t t bo cho n t bo
nhn. C hai kiu ti np: chung v chuyn bit.
Trong ti np (transduction), cc virut mang cc gen t t bo ny
sang t bo khc. chu trnh tan (lytic cycle), mt s bacteriophage gi
nhm ADN vi khun ch vo capsid. T bo b nhim bi cc virut nh vy
nhn on ADN ca vi khun A khc, ch khng phi ADN ca virut. Do
vy, ADN vi khun A ti t hp vi ADN nhim sc th ca t bo ch B v
bin i thnh phn di truyn (gentic composition).
20.6.4.1- Phage l nhn t chuyn gen
Th nghim c tin hnh trong ng hnh ch U, y ng c ngn
cch bng mng lc vi khun. Mng c l nh vi khun khng qua c,
nhng phage qua c (hnh 20.15). Nhnh A ca ng cha vi khun c kh
nng tng hp tryptophan (trp+), cn nhnh B nui cc vi khun mt kh
nng tng hp tryptophan (trp). Sau mt thi gian nui bn nhnh B xut
hin vi khun c kh nng tng hp tryptophan (trp+). Qua nhiu ln th
nghim vic phage ti gen trp+ t nhnh A sang nhnh B c chng minh.
Mng lc vi khun

Nhnh A
Vi khun
trp+

Nhnh B
Vi khu n

Hnh 20.15-. Th nghim chng minh c ti np do virut

20.6.4.2- Ti np chung (Genral transduction)

Ti np chung xy ra khi phage mang bt k gen no ca vi khun A

chuyn sang vi khun B.
Ti np chung (genralized transduction) c cc c im:
Thng do phage kiu P1 thc hin.
Bt k gen no ca vi khun cng u c ti np.
Ti np c c do gi nhm ADN ca t bo ch khi phage trng
thnh.
Cc th ti t hp n bi c to ra.
Do khng c s tng ng gia trnh t ADN trn cc phage ny vi
trnh t t bo ch, nn khng c im gn vo c hiu cho prophage. Bt
k gen no cng c ti np v u ca phage c th gi nhm vo mt on
ADN ca t bo ch. S ng ti np (cotransduction) l qu trnh ti np
ng thi 2 gen.
Qu trnh phage xm nhp vo vi khun v sinh sn c m t trn
hnh 20-16. u tin phage bm trn b mt ca vi khun, sau 4 pht bm
ADN ca n vo t bo, sau chng sinh sn v na gi sau th lm tan
vi khun v gii phng cc phage con mi.

ADN phage Nhim sc th vi khun A Capsid Phage mi ph v t bo, nhim VK

B
Hnh 20.16-. Qu trnh xm nhp ca phage v lm tan vi khun

Khi ADN ca phage xm nhp t bo vi khun A, chng ct ADN ca

vi khun A thnh nhiu on, ng thi ADN ca phage c sao chp thnh
nhiu phn t con v cc v capsid ca phage cng c to thnh. Sau
cc v capsid c lp rut ADN vo, ph v t bo vi khun ra ngoi v tip
tc xm nhp cc vi khun mi. Trong qu trnh lp rp khong 1-2% phage

v tnh mang on ADN ca vi khun c cha gen. Phage mang gen ca vi

khun A xm nhp vi khun B, qu trnh ti t hp xy ra lm gen A gn vo
b gen B.
20.6.4.3- Ti np chuyn bit (Special transduction)
Ti np chuyn bit hay hn ch (restricted transduction) l trng hp
ch mang mt vi gen nht nh, n c 4 c im:
Nhng gen c chuyn nm st ch prophage gn vo.
Ch prophage kiu thc hin.
Do kt qu s ct sai ca prophage khi tch khi nhim sc th ca t bo
ch.
Cc vi khun ti t hp c th lng bi mt phn.
V d: Phage ch mang gen gal (ng ha ng galactoz) t vi
khun ny chuyn sang vi khun khc.

Hnh 20.17: im gn ca phage vo b gen vi khun

im gn ca phage vo b gen ca vi khun nm gia 2 gen gal

(galactoz) v bio (tng hp biotin), tip theo xy ra qu trnh ti t hp
im chuyn bit (site-specific recombination) chn ADN ca phage vo
Nhim sc th vi khun (hnh 20.17). u ca phage ch c th cha mt
lng ADN gii hn, n ch ti np c gen gal hoc bio. Phage ti np
cc gen galactoz c gi l gal hay dg (d = defective: khuyt, g =
galactoz). Nu t bo gal c nhim bi dg (mang gen gal+), s rp phage
bin dng vo t bo ch s to lng bi mt phn. S ct sai ca phage
rt him nn ti np hn ch c tn s thp.
Tuy nhin, ti np tn s cao c th nhn c trong iu kin th
nghim. Nu t bo vi khun c gy nhim kp vi phage hoang di v
phage dg, phage hoang di c th h tr chc nng sai st phage bin
dng, v th h con s c c 2 kiu vi s lng bng nhau. Khi dch tan
c dng ti np, qu trnh c gi l ti np tn s cao.
Trong nhiu trng hp, do b gen bin dng dg khng gn c vo
b gen ca t bo ch (nn khng sao chp c). Sau mi ln phn bo, ch
mt trong 2 t bo c b gen ca phage bin dng; qu trnh ny c gi l
ti np sy (abortive transduction).
20.6.5- Giao np (Conjugation)
Vo nm 1946, J. Lederberg v E. Tatum chng minh c trao i vt
cht di truyn gia cc vi khun sng. S trao i ny gi l giao np hay tip
hp (conjugation). Giao np vi khun l s kt hp nht thi ca hai t bo
c kiu bt cp i nhau, c tip ni bng s chuyn mt phn vt cht di
truyn t t bo cho sang t bo nhn qua cu t bo cht, v sau cc t
bo tch nhau ra (exconjugants). Giao np i hi s tip xc trc tip gia 2
loi t bo c khi s bng ng giao np hay tnh mao (pilus), mt si ng
nh rt di do t bo cho (donor cell) to ra (hnh 20.18).

Hnh 20.18-. Hai t bo vi khun giao np qua cu t bo cht pilus

ADN c truyn qua mt cu t bo cht mng (a thin cytoplasmic

bridge) gi l ng giao np (conjugation tube). Kiu sao chp sigma () c
t bo vi khun s dng trong giao np truyn phn t ADN dng thng
sang t bo khc. Khi an ADN vo bn trong t bo nhn (recipient cell),
n thc hin ti t hp vi cc gen tng ng (homologous gens).
V thut ng, nhiu tc gi dng tip hp ch qu trnh ny, theo
chng ti, dng giao np tt hn, v phn nh c bn cht ca qu trnh,
nht qun vi bin np, ti np vi khun v khi nhm ln vi tip hp ca
nhim sc th.
20.6.5.1- Chng minh c hin tng lai vi khun
Vo nm 1946, J.Lederberg v E.Tatum s dng cc dng t
bin khuyt dng khc nhau E.coli chng minh c ti t hp gia cc
dng vi khun khc nhau. C th dng A c kiu gen metbiothr+leu+thi+ (c
kh nng tng hp threonin, leuxin v vitamin B1 (thiamin) v khng tng
hp c metionin v biotin), cn dng B th ngc li c kh nng tng
hp metionin v biotin vi kiu gen met+bio+thrleu thi. Trn A v B trong
ng nghim, sau cy ln mt mi trng ti thiu (Minimal medium MM). Cc khun lc mc trn mi trng ti thiu chng t c cc dng lai,
chng ch mc ln c nh s b p cho nhau cc nhu cu dinh dng.
Dng lai c kiu gen met+bio+thr+leu+thi+ mc c trn mi trng ti
thiu. Trong khi tng dng A hoc B ring l khng mc c trn MT ti
thiu (hnh 20.19).
Dng A = metbiothr+leu+thi+

dng B= met+bio+thrleuthi

Dng lai met+bio+thr+leu+thi+ mc thnh khun lc, mi dng ring l

khng mc thnh khun lc.

Hnh 20.19-. S lai vi khun (MM: mi trng ti thiu)

20.6.5.2- S phn ha gii tnh vi khun

Nm 1953, Hayes pht hin vi khun c cc dng khc nhau
tng t ging c v ci sinh vt bc cao. Cc dng c k hiu
F+ v F (t ch Fertility - hu th). F+ tng t ging c c sinh vt bc
cao, n truyn gen sang F. Tn s lai F+ x F khong 106 tc lai 1 triu t
bo s c 1 t bo lai.
a) Epixom v plasmid
Khi tip xc vi F+ mt thi gian t bo F tr thnh F+. V sau dng
Hfr (High frequency of recombination) c pht hin, dng ny c tn s lai
vi F cao hn F+ c th n 104 ln.
Khi F+ tip xc vi F mt thi gian, F bin thnh F+ do n nhn c
mt phn t di truyn gi l epixom. Epixom F+ l phn t di truyn ngoi
nhim sc th, c th tn ti hoc dng phn t ADN vng trn t sao chp
hoc gn vo phn t ADN ca t bo ch (v d nh phage ). Epixom F+
c gi l nhn t gii tnh (sex-factor).
Plasmid lc u c nh ngha l phn t ADN vng trn nh c kh
nng sao chp c lp vi nhim sc th t bo ch v khng c kh nng gn

vo nhim sc th t bo ch. Plasmid c th mang mt s gen khc nhau nh

khng thuc (v d, plasmid R khng nhiu thuc khng sinh),... Hin
nay plasmid c dng cho c hai ngha l epixom ln plasmid. Cc plasmid
c th tn ti c lp hoc gn vo b gen vi khun. V sau ngi ta pht hin
vi khun cn c nhiu plasmid khc. Bn cht di truyn ca cc dng F, F+
v Hfr c xc nh do cc plasmid nh sau:
F khng cha plasmid.
F+ cha plasmid dng t do khng gn vo b gen vi khun.
Hfr - plasmid c gn vo b gen vi khun (hnh 20.20).

Plasmid

T bo F+ Plasmid gn vi Nhim sc th T bo Hfr

Hnh 20.20-. Qu trnh chn ADN plasmid vo ADN Nhim sc th t bo vi khun

b) Cc nhn t F v tnh np (sexduction)

S ct ri nhn t F t nhim sc th ca dng Hfr nhiu khi khng
chnh xc v lc mt on b gen ca vi khun thay th mt phn ca F.
Trong trng hp ny, nhn t F c to thnh v n c kh nng chuyn
gen ca vi khun mt cch c lp, nhng vi cc tnh trng ca t bo cho.
Hin tng ny c gi l tnh np (sexduction), c ngha l s chuyn gen
km theo nhn t gii tnh. Nh tnh np c th nhn c cc th lng bi
tng phn (merodiploid) theo cc gen c gn vo F+. Do vy, c th
nghin cu mi tng quan alen v cc hiu qu do s gia tng liu lng
gen vi khun.

20.6.5.3- Ti t hp
Mun xy ra ti t hp th 2 dng vi khun phi tip xc vi nhau (F+ x
F- ) hoc (Hfr x F-). Dng t bo mang nhn t F+ c coi l t bo c v

c kh nng to protein pilin, t protein ny to ng giao np l pilus. S co

li ca pilus ang ni hai t bo lm chng tip xc k nhau. T bo F c
coi l ci (female). Sau khi giao np t bo F tr thnh F+.
Vic chuyn gen ch thc hin khi plasmid gn vo b gen ca vi
khun. Trong qu trnh chuyn vt cht di truyn sang F th ADN ca t bo
ch sao chp v mch mi c ori i u v F cui (hnh 20.21).
Qu
+

trnh chuyn ADN t F sang F c th b ngt qung.

Hfr X F Sao chp ADN truyn cho F Hfr F ti t hp

Hnh 20-21. S truyn ADN t th cho sang th nhn

Cc gen A, B, C c chuyn mt chiu t Hfr sang F. an gen th
cho bt cp vi cc gen tng ng ca ADN t bo F v din ra trao i
cho to t bo F ti t hp (hnh 20.22).
Dng Hfr c tn s lai cao hn nhiu v plasmid nm sn trong b
gen. Cn F+ phi qua giai on plasmid gn vo b gen ri mi chuyn gen.
Trong iu kin th nghim 37oC, nguyn b gen ca t bo E. coli
c chuyn sang t bo nhn trong vng 90 pht. Thng s giao np b
ngt gia chng do cc pilus b gy, nn t khi b gen c chuyn nguyn
vn vo t bo nhn. Lc t bo F vn l F. Bng cch ngt qung giao
np bn di truyn ca E.coli c xy dng v c dng 1 vng trn.

Hnh 20.22- on ADN t th cho trao i cho vi ADN th nhn to F ti t

hp

20.6.6- Lp bn di truyn nhim sc th ca vi khun

20.6.6.1- Giao np ngt qung (Interrupted conjugation)
Khi trn dch t bo Hfr vi F, s tip hp c th b ngt qung vo
thi im ty bng rung lc mnh. Mu c pha long nhanh v cy ln
mi trng chn lc. Ngoi ra, dng Hfr phi c gen nh du hay khuyt
dng chng khng mc c trn mi trng chn lc v ch c cc
dng ti t hp mc ln. S truyn ADN ca Hfr sang F c tnh phn cc,
cc gen u sang trc v ln lt cc gen khc theo sau. Nh ngt qung
c th xc nh c thi gian m mt gen c truyn sang F v khong
cch gia chng.
V d, dng Hfr c mang cc gen nguyn dng (prototrophic) nh
du a , b+, c+ c trn vi dng F mang cc alen khuyt dng a,b,c. S
+

tip hp c ngt qung vi khong cch 5 pht v pha long cy ln mi

trng chn lc (v d, mi trng ti thiu). Kt qu nh sau:
Thi gian (pht)

Cc th ti t
hp

a b+ c

10

a b+ c+

15

a+b+ c+

Trnh t gen dng Hfr l b+ c+ a+ ; b cch u mt truyn sang t

hn 5 pht; c t hn 10 pht ; a t hn 15 pht. y n v thi gian l
pht.
20.6.6.2- Giao np khng ngt qung (Uninterrupted conjugation)
Nu khng rung mnh ngt qung nhn to, cu t bo cht gia hai
gen cng gn ori ( u ca ADN th cho), xc sut c chuyn sang t bo
nhn cng ln. Tn s ca cc gen xut hin dng ti t hp trong th nhn,
c pht hin trn mi trng chn lc, t l nghch vi khong cch so vi
gen nh du u. Cc gen c nhiu dng ti t hp xut hin th cng nm
gn pha u c chuyn sang t bo nhn.
Khong cch gia cc gen c th tnh bng 3 loi n v: n v thi
gian, n v ti t hp v n v cp baz.

V d: Nu mt pht giao np tng ng 20 n v ti t hp

E.coli, v nguyn nhim sc th( ADN) c chuyn sang trong 100 pht, th
tng chiu di l 2000 n v ti t hp. Nu ADN c 107 cp nucleotit, th 1
n v ti t hp bng 107/2000 = 5000 bp.
20.6.6.3- Lp bn bng ti t hp
c im ca ti t hp vi khun l ch mt phn b gen c
chuyn sang t bo nhn. Mt hay nhiu gen c th c gn vo nhim sc
th t bo nhn nh giao np v ph thuc di on truyn sang.
Exogenote phi c gn vo nhim sc th nhn th mi c sao chp v
chia v cc t bo trong dng. Ch mt on ngn ADN thng c gn nh
bin np v ti np. Nh vy, nu hai gen cng bin np (cotransformation)
th hai locus phi lin kt cht. Tng t nh vy, hai gen cng c ti np
vo phage s gn nhau. Mc lin kt gia cc gen c chc nng khc
nhau (intercistronic) hay gia cc t bin bn trong gen chc nng
(intracistronic) c th nh gi t cc kt qu lai chuyn bit.
h thng hp t mt phn (merozygote), exogenote mun gn vo
endogenote phi xy ra 2 trao i cho hai u v nu ch c 1 trao i cho
th c hai sn phm nhn c u mt cn bng.
Trong cc h thng ny, tng c th tham gia ti t hp khng bit
c, nn tn s ti t hp khng th tnh nh rui gim (trn tng s c
th). Do , tn s ti t hp c trnh by tng i theo mt s tiu chun
chung cho tt c cc t hp lai. V d, s lng cc c th ti t hp nguyn
dng c to ra khi lai hai dng t bin c so snh vi kt qu khi lai
dng hoang di vi mi loi t bin. Tuy nhin c nhiu sai lch v cn
chnh l.
Xc nh trnh t gen vi sinh vt thng phc tp v c th xy ra
hin tng nhiu m cc b (localized negative interference). Phng php
xc nh trnh t gen thng dng l lai 3 im hon i (three - factor
reciprocall crosses).
20.6.6.4- Bn di truyn ca Escherichia coli

Hnh 20.23-. Bn di truyn E. coli dng K12 (theo Brock Microbiology 11,
2006)
OriC - im khi s sao chp; RE restriction endonucleaz Nost1.(xem thm gii thch
pha trn

Sau y l bn di truyn lin kt gen vng trn ca nhim sc th

E. coli dng K12 (hnh 20.23). B gen ca E. coli c gii k t chui
(sequencing) nm 1997, n gm 4.639.221 bp (cp baz) vi khang 4288
ORF (khung c m). Vng ngoi cng ghi k hiu mt s gen, m mt
trong cc s t 0 n 100 ch v tr tnh theo s pht gen c chuyn qua.
Cc s vng trong cng ch s kilo cp baz (kbp) ti cc im nhn bit
(recognition sites) ca enzym ct hn ch (RE restriction endonucleaz) c
tn Not 1. Cc mi tn ch im khi s ca cc dng Hfr.
20.6.7-Plasmid
Trong phn ni v qu trnh giao np (conjugation) hay tip hp trn
cp n plasmid, l nhng ADN vng trn nh hin din nhiu vi
khun. Plasmid l nhn t di truyn sao chp c lp vi nhim sc th
(Nhim sc th) ca t bo ch. Khng ging virut, plasmid khng c dng
ngoi bo (extracellular form) v tn ti trong t bo n gin l ADN t do
v in hnh l dng vng trn. Plasmid v ADN Nhim sc th ca t bo c
vi im khc nhau c bn: plasmid ch mang nhng gen khng thit yu
(nhng thng rt c ch), cn nhng gen thit yu nm trn Nhim sc th.

Plasmid di chuyn gia cc t bo trong qu trnh giao np

(conjugation), tc s tip xc gia hai loi t bo.
Cc kiu plasmid khc nhau c phn lai da vo cc gen m chng
mang. Mt s plasmid, cc nhn t trao i cht (metabolic factors) mang cc
gen m ha cho cc chc nng trao i cht khc thng (unusual metabolic
functions) nh phn hy du m do trn du.
n nay bit hng ngn plasmid khc nhau. Trn thc t, ngi ta
phn lp hn 300 plasmid khc nhau trong t nhin ch t dng E. coli duy
nht. Nhiu plasmid c s dng lm cng c chuyn gen trong k thut di
truyn.
20.6.7.1-Tnh cht vt l v s sao chp plasmid
Hu ht cc plasmid bit u c ADN mch kp, mch vng. Tuy
nhin vn c nhiu plasmid mch thng. Cc plasmid t nhin c kch thc
rt khc nhau, t 1 kbp n 1 megabp. Plasmid in hnh l mt phn t
ADN vng trn, mch kp nh cha n 5 % kch thc Nhim sc th (hnh
20.24). Phn ln nhng plasmid ADN c phn lp t t bo c dng siu
xon, dng nh gn nht c th tn ti trong t bo.
Enzym lin quan n vic sao chp plasmid trn thc t l nhng
enzym bnh thng trong t bo. V th nhng gen ca plasmid ch yu lin
quan n s kim sot qu trnh khi s sao chp v vi s phn b nhng
plasmid c sao chp vo cc t bo con.

Hnh 20.24-. Hnh hin vi in t cc plasmid vng trn

Tng t, nhng plasmid khc nhau hin din trong t bo vi s

lng khc nhau, gi l s bn sao (copy number). Mt s plasmid ch c 1-3
bn sao trong t bo, trong khi nhng plasmid khc li c n 100 bn sao.
S lng bn sao c kim sot bi nhng gen trn plasmid v bi s tng
tc gen gia t bo ch v plasmid. Phn ln cc plasmid vi khun Gram
m sao chp tng t nh Nhim sc th vi khun. Vic ny thc hin t
im khi s sao chp (Ori) v chp theo hai hng quanh vng plasmid, to
ra dng trung gian theta (). Tuy nhin mt s plasmid khng sao chp theo
hai hng. Do kch thch nh ca ADN plasmid, ton b qu trnh sao chp
xy ra rt nhanh, c l bng 1/10 tng thi gian din ra chu k phn bo.
Hu ht plasmid ca vi khun Gr+ sao chp theo c ch vng xoay
tng t nh phage x174. C ch ny s to ra mch n trung gian, v do
nhng plasmid ny thnh thong c xem nh plasmid ADN mch n.
Phn ln ADN mch thng sao chp dng c ch lin quan n mt protein
gn vi u 5 ca mi mch m c dng trong tng hp mi ADN.

20.6.7.2- Tnh khng tng hp (Incompatibility) v chnh sa (curing)

Mt s vi khun c th cha vi dng plasmid khc nhau. V d,
Borrelia burgdorferi (nhn t gy bnh Lyme) cha 17 plasmid mch thng
v vng. Kh nng hai plasmid khc nhau, m mi mt sao chp trong cng
mt t bo, c kim sot bi gen ca cc plasmid tham gia iu ha sao
chp ADN. Khi mt plasmid c chuyn vo trong t bo c sn mt
plasmid khc, thng th plasmid th hai c th khng tn ti c v mt i
trong qu trnh sao chp t bo sau . Nu iu ny xy ra, hai plasmid trn
c gi l khng tng hp (incompatible). Mt s nhm khng tng hp
(Incompatibility - Inc) c ghi nhn. Cc plasmid thuc cng mt nhm
khng tng hp s loi tr nhau khi sao chp trong cng t bo nhng c
th cng tn ti vi plasmid ca nhm khc. Plasmid cng nhm khng
tng hp c chung c ch iu ha sao chp, do chng c h hng vi
nhau. V vy, d t bo vi khun c th cha nhiu loi plasmid khc nhau,
th mi loi vn khc bit nhau v mt di truyn.
Mt s plasmid, gi l epixom, c th chn vo nhim sc th, v s
sao chp ca chng chu s kim sot ca Nhim sc th. Trng hp ny rt
ging vi virut m b gen ca chng c th chn vo b gen ca t bo ch.
Plasmid thnh thong c th b loi b khi t bo ch bng nhiu x l
khc nhau. Vic loi b ny, gi l chnh sa (curing), l kt qu ca c ch
sao chp plasmid m khng c s c ch song song i vi Nhim sc th
ca t bo ch. Trong phn bo, plasmid b loi b mt. S chnh sa c th
xy ra t pht, nhng c th tng nhanh ng k bng nhiu cch x l khc
nhau.
20.6.7.3- S chuyn plasmid t t bo ny sang t bo khc
C ch chnh ca vic chuyn plasmid t t bo ny sang t bo khc
l giao np, m chc nng thc hin c m ha bi chnh mt s plasmid.
Giao np l mt qu trnh sao chp ADN v plasmid ca t bo cho v truyn
bn sao ca Nhim sc th cng bn sao plasmid sang t bo nhn.
Hnh 20.25 cho thy bn gen ca plasmid F, mt plasmid c
nghin cu rt k ca Escherichia coli. Khang 25 gen trn F plasmid, bao
gm cc gen sao chp ADN, cc yu t thc hin chc nng ca mt epixom

v cui cng l mt vng cung ADN ln, gi l vng tra, cha nhng gen
gip n t di chuyn t t bo ny sang t bo khc.

Hnh 20.25-. S gen ca plasmid F ca E. coli.

Con s bn trong cho bit kch thc ca plasmid (chnh xc l 99.159

bp). Vng mu xanh sm di cha nhng gen chu trch nhim chnh
sao chp v tch plasmid F trong t bo pht trin bnh thng. Vng xanh
sng, vng tra, cha nhng gen chu trch nhim chuyn gen. Chui ori T l
im khi u ca qu trnh chuyn plasmid trong tip hp. Mi tn cho bit
hng chuyn. (vng tra s c chuyn qua cui cng). Nhng vng mu
vng l nhng yu t c th hon i v tr, ni m c th xy ra s hp nht
vo nhng yu t tng ng trn nhim sc th vi khun v dn n s hnh
thnh dng Hfr.
(Theo Brock Microbiology 11, 2006)

Plasmid iu khin s truyn gen ca chnh n bi s tip xc ca hai

t bo gi l c kh nng giao np. Khng phi tt c plasmid u c kh
nng tip hp. Kh nng truyn trong tip hp c kim sot bi nhng gen
trong plasmid gi l vng tra. Vng tra cha nhng gen m ha protein thc
hin chc nng bt cp. S hin din ca vng tra plasmid c th dn n
kt qu quan trng khc nu plasmid chn vo nhim sc th. Trong trng
hp ny, plasmid c th huy ng s chuyn ADN nhim sc th t t bo
ny sang t bo khc.
Mt vi plasmid tip hp t Pseudomonas c bin t bo ch ln.
iu ny c ngha l chng c th c truyn sang rt nhiu vi khun Gr+
khc. Nhng plasmid nh th c th chuyn thng tin di truyn gia nhng

sinh vt gn nhau. Nhng plasmid tip hp c th chuyn gia vi khun Grv Gr+, gia vi khun v t bo thc vt v gia vi khun v nm. Thm ch
nu plasmid khng th sao chp c lp trong vt ch mi, s chuyn
plasmid t n c th c ngha tin ha quan trng nu gen t plasmid ni
li vi b gen ca vt ch mi.
20.6.7.4 - Vi loi plasmid khc v ngha sinh hc ca chng
Mc d plasmid khng mang nhng gen thit yu i vi vt ch,
nhng n c th nh hng ng k n kiu hnh ca t bo. Chng hn, kh
nng ca Rhizobium tng tc vi thc vt v hnh thnh nt sn r c nh
m l nh nhng plasmid thc hin. Nhng plasmid khc mang li c im
bin dng c bit t bo, nh s phn hy cht gy nhim c hi.
Thc ra, plasmid dng nh l mt b my chnh to ra nhng tnh cht
ring bit ca vi khun, v trong nhiu trng hp, cn xut khu (export)
nhng c tnh ny bi s chuyn gen theo chiu ngang. Hn ch duy nht
i vi nhng kiu gen ny c trong plasmid l chng khng can thip vo s
sao chp ca chnh mnh hoc vo s tn ti ca vt ch.
a) Plasmid khng
Trong s nhng nhm plasmid ph bin nht v c nghin cu k
nht l cc plasmid R, c kh nng khng khng sinh v nhiu cht c ch
sinh trng khc. Plasmid R ln u tin c pht hin ti Nht dng vi
khun rut; l nhng vi khun c tnh khng i vi nhiu lai khng
sinh (a khng) v k t , chng c pht hin trn khp th gii. Bn
cht gy nhim ca plasmid tip hp R cho php chng lan rng nhanh chng
trong qun th t bo gy ra mt vn nn ln trong y hc lm sng l s xut
hin nhanh cc vi khun khng thuc.
Mt plasmid R c th mang nhiu gen khng. Chng hn, plasmid
R100 c 94,3 kbp (hnh 20.25), mang nhng gen m ha cho s khng
sulfonamid, streptomycin v spectinomycin, fusidic axit, chloramphenicol,
tetracylin v c th mt s gen khng thy ngn (mercury). Plasmid R100 c
th c chuyn gia cc vi khun ng rut thuc chi (Genra) Escherichia,
Klebsiella, Proteus, Salmonella v Shigella, c th chuyn vo vi khun

Gram m Pseudomonas. Nhng plasmid R khc c gen khng khng sinh

cng c ghi nhn.

Hnh 20.26- Plasmid R100: RTF l vng cc gen tra chuyn gen ; r-determinant l vng
cc gen khng (mer - thy ngn, sul - sulfamid, str - streptomycin,cml chloramphenicol, tet tetracyclin). (Theo Brock Microbiology 11, 2006)

b) Plasmid m ha cht c v nhng c tnh khc

mt s vi khun gy bnh, mt s tnh cht ny c mang trn
plasmid. Th d, dng E. coli gy bnh ng rut c tnh cht ny bi kh
nng sinh sn thnh tp on trong rut non v sn xut mt cht c gy cc
triu chng ca bnh tiu chy. S hnh thnh tp on i hi s hin din
ca mt protein b mt t bo gi l yu t khng nguyn to qun th (yu t
khng nguyn hnh thnh tp on), c m ha bi mt plasmid. Protein
ny gip t bo c kh nng gn vi t bo biu m ca rut. t nht hai cht
c E. coli gy bnh ng rut c m ha bi mt plasmid: hemolysin
lm tan t bo hng cu v enterotoxin gy tit qu nhiu nc v mui vo
rut v do vy l cht c gy tiu chy.
c) Bacteriocin
Nhiu vi khun sn xut protein c ch hoc git cht t bo cc loi
h hng gn, thm ch ngay c nhng dng khc nhau ca cng mt loi.
Nhng tc nhn ny, c gi l bacteriocin phn bit vi cht khng

sinh, c ph tc ng hp hn khng sinh. Cc gen m ha bacteriocin v

nhng protein ch bin (processing) v vn chuyn chng (to tnh min dch
cho sinh vt sn sinh ra n) thng nm trn plasmid hay transposon.
Bacteriocin c t tn theo loi to ra n nh E. coli c colicin, m ha
bi Colplasmid; Bacillus subtilis to ra subtilisin,...
Plasmid Col ca E. coli m ha nhiu colicin khc nhau. Colicin c
gii phng t mt t bo gn vi nhng th th (receptors) c hiu trn
mng t bo d cm nhim (susceptible). Colicin git t bo bng cch ngn
cn chc nng quan trng no ca t bo.
Bacteriocin hoc tc nhn tng t bacteriocin ca vi khun Gram
dng th hi khc colicin nhng cng thng c m ha bi plasmid, mt
s thm ch cn c gi tr thng mi. Chng hn, vi khun lactic to ra
bacteriocin Nisin A c ch mnh s tng trng ca mt lot vi khun Gr+ v
c dng lm cht bo qun trong cng ngh thc phm.
20.6.8- S chuyn v (Transposition)
Cc phn t chuyn v (transposable elements) hay phn t di ng
(mobile elements) l nhng trnh t ADN c kh nng di chuyn t v tr ny
n v tr khc (mc tiu) ca b gen (cng b gen hay khc). S di chuyn
ADN ny c gi l transposition (s chuyn v).
Ti t hp (Recombination) trong chuyn v (transposition) l s kt
ni (junction) gia cc u mt ca phn t chuyn v vi cc u mt b ct
h ca ADN mc tiu (target site). Khc vi ti t hp tng ng
(homologous recombination), transposition khng i hi s tng ng
(homologous sequence) gia cc phn t di ng v ADN mc tiu, v n
c lp vi chc nng c m ha bi recA, v n s dng enzym c hiu
l Transposaz.
y l hin tng rt ph bin trong thin nhin, cc transposon c
tm thy vi khun, nm, thc vt v ng vt (bng 20.6).

Bng 20.6- Cc phn t chuyn v c Prokaryotae v Eukaryota

Prokaryota

Eukaryota

Trnh t xen on: IS

Retrotransposon:

Transposon: Tn nh Tn3, Tn5, Tn10,...

Nm men (Yeast): Ty

Virut: Mu

Rui gim: copia, P

Transposon (ADN):
Ng: h thng Ac-Ds
SINE: ngi (h Alu)
Retrovirut: Rous sarcoma, HIV

Chng c chc nng nh cc vector quan trng thc hin bin i di

truyn. Cc xen on, mt on v cu trc li b gen thng km theo s di
chuyn ca cc transposon. S di chuyn ny lm sai hng chc nng bnh
thng ca gen. Chng hot ha hay lm bt hot cc gen bng cch xen
on vo k bn hay vo gia on gen. Cc transposon IS, l cc phn t
lp on (repetitive elements), cn to cc vng tng ng ri trong b gen,
nh cc h thng ti t hp tng ng c th tc ng.
Transposition gm cc kiu chnh:
IS: trnh t xen on (insertion sequence) l cc transposon ngn.
Transposon (k hiu Tn): Cc phn t di ng di (khong 5000 bp) c
cha mt hoc vi gen. N thc hin:
+ Transposition khng sao chp (nonreplicative): Phn t chuyn v
c ct khi ADN cho (donor) v c gn vo phn t ADN mc tiu.
Thuc loi ny c Tn10, Tn5, Tn7.
+ Transposition sao chp (replicative): ADN c nhn i v bn sao
c xen vo v tr mi to cointergrat (cng gn) v c Tn3, Mu.
Retrotransposition: S di chuyn qua trung gian ARN, nh reverse
transcriptaz to thnh cADN, v s xen on cADN vo v tr mi.

20.6.8.1- Cc trnh t xen on IS

Cc transposon n gin nht l cc trnh t xen on (insertion
sequence) v c k hiu bi tip u ng IS km s th t nh IS4.

Cc phn t IS l cc cu phn bnh thng ca ADN vi khun v

plasmid. Dng E.coli chun thng cha vi bn sao (<10) ca bt k mt
trong cc IS chung thng gp. m t s xen on vo im c bit, k
hiu 2 chm kp c s dng nh :: IS1 ch IS1 xen vo phage .
Cc IS l nhng n v t tr, mi mt trong chng m ha cho ch mt
protein cn thit cho s chuyn v bn thn chng. Trnh t ca mi loi IS
c khc nhau, nhng trong t chc cu to c nhiu tnh cht chung. gia
c gen transposaz tnp, hai u c lp on o ngc (IR Inverted
repeats). S xen on ca IS tiu im (target) c minh ha trn hnh
20.27. Cu trc transposon v s chn vo ADN mc tiu nu trn hnh
20.28.
Cc u mt ca transposon c trnh t lp li o ngc (inverted
repeat - IR). Trong v d ny, tiu im c 5 bp. Cc u mt ca transposon
gm cc lp on o ngc 9 bp c nh s t 1 n 9.

DNA m c tiu

IR

IR

Hnh 20.27. Cu trc IS v s chn vo ADN mc tiu.

hai u ca IS lun c hai trnh t lp on trc tip (direct repeat)

ngn. Cc trnh t ny dao ng ty transposon, nhng c nh i vi mi
loi IS. Chiu di ca phn ln lp on trc tip l 9, chng xc nh cc
u mt ca transposon.

M c tiu

Hnh 20.28- S cu trc ca transposon c cc IS, cc lp on o ngc IR

(inverted repeat) v to lp on trc tip hai u ADN im mc tiu

20.6.8.2- Transposon khng sao chp v sao chp

S chuyn v ADN theo 2 kiu khc nhau (sao chp v khng sao
chp hay bo tn (conservative)) v c nhng tnh cht chung (hnh 20.29).
S din bin transposition sao chp (transposon Tn3) c nu trn cc
hnh sau: ct v ni (hnh 20.30 ), s cng gn v phn tch.

Hnh 20.29- S v transposition sao chp v khng sao chp

ADN cho

S ni gia Tn v
trnh t mc tiu
(ligation)

ADN nhn
Hnh 20.30. S transposition sao chp (Tn3)

Tip theo l s cng gn (Cointegrat Formation) 2 loi ADN (hnh

20.31).

Hnh 20.31. S cng gn (Cointegrat) ca 2 loi ADN

Cui cng l s phn tch (Resolution) ra 2 loi ADN (hnh 20.32).

Hnh 20.32. S phn tch ra 2 loi ADN

Qu trnh transposition c nhng c im sau y:

 C hai u ca phn t mang gn nh mt trnh t c nh hng o

ngc nhau.
Cc transposon m ha t nht mt protein l transposaz. Transposaz gn
c hiu v ct cc trnh t cui thc hin chuyn v.
Cc transposon to bn sao ngn ( 12bp) ca ADN im mc tiu trong
transposition. Chiu di ca trnh t ny c trng v khng i i vi mt
phn t nht nh v c to ra nh s ct so le (staggered cleavage) ca
ADN mc tiu nh transposaz.
C ch transposition c nghin cu chi tit phage Mu v c th
tm tt nh sau:
S nhn bit v bt cp 2 u mt ca transposon nh transposaz hnh
thnh cu trc chuyn bit protein - ADN. S hnh thnh phc hp c
kch thch bi cc protein HU v IHF l cc nhn t tham gia ti t hp
im chuyn bit Mu transposaz, ging nh Int protein, c 2 vng gn c
lp.
S ct khc (nicking) do transposaz to ra 3-OH mi u ca Mu.
Ct tiu im nh transposaz to u mt so le 5 - P di 5 bp.
S ni u 5 - P ca tiu im vi 3-OH ca transposon to cu trc
trung gian chuyn mch (strand - transfer intermediate).
Tn10 c l s dng c ch tng t thc hin transposition khng
sao chp hay bo tn (conservative transposition) (hnh 20.33 a, b, c v d).
20.6.8.3- S chuyn v ngc (Retrotransposition)
Nhm cc transposon, c pht hin nm men (Ty) v Drosophila
(copia) v c lin quan n retrovirut, di chuyn n v tr mi qua trung gian
ARN. Transposon ca nm men Ty, ging vi retrovirut, c lp on cui
di (long terminal repeat) c gi l trnh t nm 2 pha on m ha.
Cc protein c m ha, c trnh t tng ng vi integraz ca retrovirut
v reverse transcriptaz, rt cn thit cho transposition.
Phng thc phin m ngc ca cc phn t di ng tng t nh
ca retrovirut. C s tng t v cu trc gia retrovirut v cc
retrotransposon nm men (Ty) v Drosophila (copia).

S chuyn v ngc to bn sao ca phn t v tr mi, trong khi

phn t cho ban u vn gi nguyn cu trc khng bin i. Do vy,
retrotransposition to nn mt t t on v cc ti cu trc (rearrangement)
ca b gen t bo ch. Nhng bin i ca b gen lin quan vi
retrotransposition s dn n vic lm ngng hay hot ha cc gen, m mt
s c th gy ung th.
Cc transposon gp phn vo s tin ha ca plasmid, v vi
chng c cho thy cc plasmid R c c tnh khng khng sinh thng
qua cc transposon.

a.
b.

i mc t

ADN cho

DNA nh n

c.
d.
Hnh 20.33. Transposition khng sao chp hay bo tn (conservative transposition)
Mi tn ch cc im ct v sau ni li theo trt t mi.

20.6.8.4-Gy t bin bng transposon

Cc ng dng ca transposon bao gm:
Gy t bin (mutagensis) bng transposition.

Hnh 20.34.

Transposon

Tn10 chn vo
chc nng,

A lm mt
nhng khng
tetra.

Cng c cho k thut di truyn in vivo (in vivo Gentic Engineering).

Nhn t trung gian khng khng sinh (mediator of antibiotic
resistance)
y cp ch yu n gy t bin nh transposon (Transposon
Mutagensis). S chn transposon vo gia gen s dn n t bin lm mt
chc nng bnh thng ca gen (hnh 20.34). Transposon cung cp mt
phng tin d dng to cc t bin trn Nhim sc th. Mt thun li
cho gy t bin bng transposon l n c cha 1 gen khng khng sinh, m
gen ny dng lm marker (du chun) nhn bit transposon c chn
vao. Trc tin cc dng mang gen khng khng sinh c phn lp t mi
trng giu dinh dng, ni cc t bin khuyt dng tng trng to
khun lc. Sau chng c th c sng lc trn mi trng gii hn c
cung cp cc cht tng trng no xc nh cht no cn thit cho t
bin.
Cc transposon cng hu ch cho s chn 1 maker gen khuyt dng
(auxotrophic) vo trong dng sinh vt hoang di. Thng th cc t bin
khuyt dng kh phn lp trc tip. Nhng nu marker khuyt dng c
a vo cng transposon vi marker khng khng sinh, th c th qua chn
dng khng khng sinh m xc nh marker khuyt dng. Hai transposon
thng c s dng trong gy t bin l Tn5 (khng neomycin v
kanamycin) v Tn10 (cha 1 marker khng tetracycline).

Integron l cc transposon c th thu nhn v biu hin cc gen t

nhiu ngun khc nhau. Tuy nhin, khc vi cc transposon c chn vo
mt cch ngu nhin, cc integron chn vo c tnh chn lc cao (high
selective) trong cc im xen on (insertion site) ca chng, thng th
c chn vo trong plasmid.
Integron cha gen m ha cho protein c gi l integraz, cn thit cho
s ti t hp im chuyn bit (site-specific recombination). B gen ca
phage (lambda) chn vo Nhim sc th ca E. coli mt v tr c bit nh
hat tnh ca enzym integraz . Integron cng cha trnh t ADN c bit cho
php integraz chn cc nhm gen gi l cassette vo Nhim sc th, m trn
c promoter cho php cc gen cassette mi c biu hin.
20.7. DI TRUYN HC VIRUT
Virut c cu to n gin v rt nh nn c pht hin mun hn.
Vic cc nh vt l hc khi chuyn sang lnh vc sinh hc s dng cc
virut vo nghin cu di truyn hc cho thy r tm quan trng ca cc i
tng ny. Di truyn hc virut gp phn ng k cho s pht trin ca
sinh hc phn t. S hiu bit chi tit v cc virut khng nhng gip con
ngi bit r cc c nguyn gy nhiu bnh him ngho (ung th, AIDS,...),
m cn c th bin chng thnh cc cng c chuyn gen c li cho con ngi
20.7.1. Sinh hc ca virut
Virut c pht hin vo nm 1892, khi nh khoa hc Nga Ivanovski
(hnh 4.1) nhn thy virut m thuc l (tabacco mosaic virut - TMV) qua
c mng lc ngn vi khun.Nm 1935, W.M Stanley pht hin cc virut c
th to thnh tinh th. Cn cc sinh vt khc, t bo d n gin nht cng
khng th to thnh tinh th c. c nhng bn ci rng virut c c
coi l sinh vt hay khng. n nay, chng c coi l nhng thc th sng
cha c cu to t bo. Cc virut hay cc virion l nhng dng sinh vt c
cu to n gin nht. Xt t gc phn loi hc, sinh gii c th chia
thnh hai nhm ln: cc sinh vt c cu to t bo v virut.
20.7.1.1. c im chung ca virut
Vo nhng nm 1950, s quan st trc tip bng knh hin vi in t
cho thy virut c rt nhiu im khc vi khun. Trc tin cn k n c

tnh rt nh b, cu to n gin ca virut. Virut nh nht c ng knh ch

20 nm, nh hn c riboxom.
Ngoi ra, virut khc vi cc sinh vt c cu to t bo nhiu im:
Virut l cc th ni k sinh bt buc, khng c cu to t bo. Chng pht
trin v sinh sn ch trong cc t bo sng ca vt ch chuyn bit.
Virut s dng b my sinh tng hp ca t bo ch sinh sn v thng
ph hy t bo ch phng thch th h virut con tip tc gy nhim cc vt
ch mi.
Virut c b gen ch gm mt loi nucleic axit (ADN hoc ARN), trong khi
cc t bo c c hai loi, c bao trong v protein. Phn ln virut gm 1
lai nucleic axit v mt t lai protein.
Chng khng c h thng sinh tng hp protein ring (khng c riboxom);
khng c h thng trao i cht ring nn khng bin dng nng lng
(khng phn hy thc n to ATP ), khng thc hin s ln men, h hp
v quang hp.
Virut khng to mng lipit ring, mc d mt s virut c mng bao
(enveloppe) c to ra bng bin i mng ca t bo ch trc khi thot ra
khi t bo ch.
Virut khng b tc ng bi cc thuc khng sinh v khng c cu trc vch
t bo v b my sinh ha riboxom nh t bo vi khun.
Virut khng c khung sn t bo (cytoskeleton) hoc phng tin di ng
ngoi s khuch tn.
Virut khng tng trng theo ngha l tng khi lng, khi virut hnh
thnh n khng tng kch thc. Hnh dng c trng ca virut c xc nh
bi v protein.
Virut hnh thnh trn vn c gi l virion, b gen ca n c gi
trong v protein v bn ngoi c th c mng bao.

20.7.1.2. Cu to virut
Virut c b gen rt a dng. B gen (Genome) ca virut c th l ADN
mch kp (double strand - dsADN), ADN mch n (single strand - ssADN),
ARN mch kp (double strand - dsARN), hay ARN mch n (single strand ssARN). Cc virut c ADN hoc ARN mch n cn phn bit thnh 2 dng
dng hay cng (+) v m hay tr ():
Virut mch dng (Plus (positive)-strand virut): virut vi b gen ARN hay
ADN m b gen c cng trnh t nucleotit nh mARN ca virut.
Virut mch m (Minus (negative)-strand virut): virut vi b gen ARN m
mch ARN ny c trnh t nucleotit ngc chiu (b sung vi) vi mARN
ca virut.
B gen virut thng l mt phn t nucleic axit dng vng trn hay
thng. Virut nh nht c chng 4 gen, virut ln nht c chng vi trm gen.
V protein bc b gen c gi l capsid thng c th dng hnh
que, hnh ng xon, hnh a din hay phc tp. Nucleocapsid l phc hp
nucleic axit v cc protein ca mt virut. Cc capsid thng c to nn bi
mt s ln t hp cc phn t protein gm t loi, c gi l capxome. V
d, virut m thuc l c mt capsid hnh que di cng c to ra t hn
1000 capxomre (hnh 20.36).

Hnh 20.36. Virut m thuc l

a) S cu trc ; b) nh hin vi in t.

Cc Adenovirut, thng gy nhim ng h hp ca ng vt, c 252 phn

t protein tng t nhau xp li to capsid hnh a din vi 20 mt (hnh
20.37).

a)

b)

Hnh 20.37. Adenovirut c capsid u a din

a) S cu trc ; b) nh hin vi in t

Mt vi virut c cu trc ph h tr chng nhim vo t bo ch.

Virut Flu v nhiu virut ng vt c mng bao (envelope) pha ngoi capsid
(hnh 20.38). Bao ny bt ngun t mng ca t bo ch, nhng ngoi
photpholipit v protein ca t bo ch, chng cn c thm cc protein v
glycoprotein ngun gc virut.

Hnh 20.38. S cu trc ca virut c mng bao (enveloppe) vi cc gai

a) S cu trc ; b) nh hin vi in t

Cc virut nhim vi khun c capsid phc tp nht. Cc virut ca vi

khun c gi l bacteriophage (thc khun th - n vi khun) hay gi ngn
l phage. By phage u tin gy nhim E.coli c nghin cu mang tn T1,
T2, T3...T7 (T t ch typ). Cc phage T chn (T2, T4, T6) c cu trc rt ging
nhau. Capsid ca chng gm mt u a din (20 mt) bc cht di truyn.
Phn th hai l bao ui bng protein thnh ng di v phn th ba l si
ui di bm vo t bo vi khun khi gy nhim.
Tm li, virut c m t theo 4 tiu chun cn bn khc nhau:

 B gen (Genome) ch gm mt loi phn t l ADN hay ARN.

Nucleic axit b gen l mch n (single strand - ss) hay mch kp
(double strand - ds)
Hnh dng (shape) ca virion n gin hay phc tp.
Virion c hay khng mng bao (membrane hay envelop)
20.7.1.3. Sao chp ca cc virut
Cc ht virut (virut particle) hay virion l nhng vt k sinh ni bo bt
buc (obligate intracellular parasite). Chng ch biu hin cc gen ca chng
v sinh sn bn trong 1 t bo sng khc. Ph thuc vo loi t bo ch m
virut k sinh, ngi ta gi tn loi virut, v d: virut thc vt k sinh t bo
thc vt, virut ng vt k sinh t bo ng vt. Do c im ny, s sinh sn
ca virut khc hn vi s sinh sn ca t bo. im ni bc l virut to ra
hng trm hay hng ngn virion trong mi th h.
Do c s a dng nh nu trn, cc b gen ca virut c sao chp theo
nhiu con ng khc nhau:
Cc ADN mch kp > ADN mch kp: kiu bn bo tn.
Cc ARN mch kp > ARN mch kp: kiu bn bo tn nh ARN
replicaz do gen to enzym sao chp ny c m ha trong b gen ca virut
ARN.
Cc ADN hoc ARN mch n > chng lm khun (mch +) tng
hp mch b sung (mch ) thnh dng mch kp trung gian (+/), gi l
dng sao chp (replicative form), m t n ly mch lm khun to ra mch
n + ging b gen ban u ca virut (hnh 20.39).
ADN hoc ARN mch n: +
mch +
Dng sao chp

mch

B gen cho virion mi: mch +

Hnh 20.39. Sao chp ca ADN hoc ARN mch n qua trung gian mch kp

 Retrovirut ARN > nh enzym reverse transcriptaz tng hp cADN mch n t khun ARN virut > c-ADN kp > Chn vo b gen t
bo ch > to ra b gen ARN virut mi v mARN.
Ph thuc vo b gen ADN hay ARN mch kp hay n, mch dng
(+) hay m (), kiu sao chp b gen v tng hp mARN, cc virut c chia
thnh 7 lp (classes).
Mt cch sao chp c bit c t bo vi khun s dng trong tip hp
truyn phn t ADN dng thng sang t bo khc hoc c cc virut s
dng to cc b gen ca chng, mt khc lm t mch ca vng trn
xon kp, to u h 3OH v 5P kt thc. Helicaz v SSB protein chen
vo to ch 3 sao chp. S sao chp c thc hin khng cn mi (primer)
v mch 3OH sn sng cho vic ni di nh mch trc (leading strand)
nh ADN polymeaz I. ng thi vi sao chp mch trc, mch khun sau
dch chuyn. S dch chuyn ca mch sau gin on tng hp cc on
ngn Okazki nh bnh thng v u 5 mch khun dui thng ra (hnh
20.40). S sao chp c hnh ging ch sigma () ca Hi Lp, nn c gi l
sao chp sigma ( replication). Kiu sao chp ny cn c gi l vng
trn quay (rolling-circle replication), v mch khun trn gia khng b t
v quay trn lm khun cho mch trc. S sao chp kiu ny c th lp li
vi ln to ra si ADN di, lp li nhiu ln b gen thng ca virut, c gi
l concatemer. Enzym endonucleaz ct nhng im khc nhau trn mi
mch ca ADN to ra cc on c b gen vi hai u dnh. B gen thng
ny s to thnh vng nh bt cp b sung cc u dnh.

Hnh 20.40. Sao chp (sigma) hay vng trn xoay

(ng m di ch mch khun c, cc on ngn ch mch mi c tng hp.
Pha trn tng hp xong 1 b gen, pha di c 2 b gen ca virut)

20.7.1.4. S t rp (Self-assembly) ca cc virion

Cc gen ca virut s dng cc enzym, cht dinh dng, riboxom v cc
ngun khc ca t bo ch to ra nhiu bn sao ca b gen v cc protein
ca cc cu phn khc (capsid, c, bao ui...). Khi cc sn phm ring l
tch , chng t rp (self-assembley) nhau thnh s lng ln cc virion ri
ph v t bo tm cc t bo ch mi.
S rp nucleic axit ca virut vi cc protein ca capsid hnh thnh
virion mi din ra t ng tng t cc mch polypeptit t gn vi nhau
to nn cc protein c cu trc bc bn. Cc cu phn c gn vi nhau
bng lin kt yu nn khng cn enzym.
20.7.1. 5. Tnh c hiu vi t bo ch
Mi kiu virut c th nhim v k sinh ch mt bin gii hn ca
t bo c gi l bin ch (host range). Cc virut nhn bit t bo ch
theo nguyn tc "ng kha v cha kha" cc protein bn ngoi ca virion lp
va cc im nhn (receptor site) trn b mt t bo. Mt s virut c bin
ch rng xm nhp vo vi loi. Cc virut bnh di c th nhim nhiu
loi c v gm gm nhm, ch v ngi. Bin c th rt hp nh nhiu
phage ch nhim vi khun E.coli.
20.7.2. Bacteriaphage Virut ca vi khun
Cc phage l virut ca vi khun c pht hin t nm 1915. Vo
nhng nm 40, chng c s dng cho cc nghin cu sinh hc phn t.
Chng l nhng virut c nghin cu k nht, mc d mt s t chng c
cu to phc tp.
By phage u tin gy nhim E.coli c nghin cu mang tn T1, T2,
T3...T7 (T t ch typ). Cc phage T chn (T2, T4, T6) c cu trc rt ging
nhau (hnh 20.41). Capsid ca chng gm mt u a din (20 mt) bc cht
di truyn. Phn th hai l bao ui bng protein thnh ng di v phn th ba
l si ui di bm vo t bo vi khun khi gy nhim.

Phage nhn bit t bo ch thng qua s gn kt c hiu gia cc

protein capsid v cc protein th th trn t bo ch. Khi xm nhim, cc si
ui bm ln b mt v bao ui co ngn li bm b gen virut vo bn trong
t bo. Cc virut c th sinh sn trc tip v git ch t bo ch, hoc sinh sn
chm hn bng chn nucleic axit ca chng vo b gen t bo ch.

Hnh 20.41. Bacteriophage T4 chn

Cc nghin cu phage k sinh t bo E.coli pht hin rng chng c

2 c ch sinh sn: chu trnh tan (lytic cycle) v chu trnh tim tan (lysogenic
cycle).
20.7.2.1 Chu trnh tan (Lytic cycle)
Trong chu trnh tan, t bo nhim virut b tan phng thch th h phage
con. B gen nucleic axit ca phage chi phi b my sinh tng hp ca t bo
ch qua 2 giai on: sm v mun. B gen virut cha trnh t promoter mnh
thu ht ARN polymeaz ca t bo ch.
Trong giai on sm, cc gen phage k cn promoter c phin m.
Cc sn phm ca gen sm thng gm cc protein lm dng phin m ca
t bo ch v kch thch sao chp b gen (genome) ca phage.

Hnh 20.42. Chu trnh tan ca phage T4

a) Bm vo ; b) Xm nhp ; c) Sinh tng hp cc cu phn nh t bo ch ; d) Trng
thnh: cc virion c t rp ; e) Lm tan t bo, cc virion thot ra

giai on mun, cc gen mun ca phage m ha cho protein v v

enzym gy tan t bo ch dn n phng tch cc virion con.
Cc bacteriophage lm cht t bo ch gi l c (virulent) v chng
sinh sn theo chu trnh tan (hnh 20.42). Chu trnh bt u khi si ui ca
phage T4 gn vo cc im nhn (receptor sites) trn b ngoi ca t bo
E.coli. ng ui co li to l thng xuyn vch t bo v bm ADN vo
trong t bo tng t nh dng ng tim (syringe) bm thuc. Capsid rng
ca phage cn li bn ngoi t bo.
Khi ADN trn xm nhp vo trong t bo, cc phage khc nhau c
th s dng cc cch khc nhau sn sinh ra th h con. Sau khi b nhim,
t bo E.coli nhanh chng bt u phin m v dch m cc gen ca virut.
Phage T4 c khong 100 gen v phn ln c bit r. Mt trong nhng
enzym c to ra u tin ct ADN ca t bo ch.

Ni chung, ADN ca phage c phin m u tin nh ADN

polymeaz ca t bo ch to ra mARN sm. Cc mARN mun hn c th
c tng hp bi ARN polymeaz ca phage hoc ARN polymeaz ca vi
khun b bin i phin m cc gen ca phage. Cc mARN mun c
dch m to cc loi protein enzym iu ha v cu trc. Cc protein iu
ha ca phage kim sot s phin m ni tip ca cc gen (kiu iu ha
ni tip).
Khi ADN ca t bo ch b phn hy, b gen ca phage kim sot ton
b hot ng ca t bo to cc cu phn ca n. Cc nucleotit c dng
sao chp ADN ca phage ra hng trm bn sao. Cc protein ca capsid
c tng hp thnh 3 phn ring: u a din, ng ui v cc si ui; ri
chng t rp li vi nhau thnh virion con (hnh 20.42 - vng trn pha di
gc phi). Phage hon tt chu trnh khi enzym Lysozym c to ra tiu
ha vch t bo. T bo vi khun b v, 100 n 200 virion thot ra v chng
c th lp li chu trnh mi.
Ton b chu trnh t lc phage tip xc b mt t bo n tan din ra
trong khong 20 - 30 pht 370C. Trong thi gian s lng phage T4 tng
hn c trm ln, cng khong thi gian s lng t bo E.coli mc nhanh
nht cng ch tng gp i. Nu nh mt ht T4 ln thm t bo E.coli mc
trn mi trng rn trong hp petri th sau xut hin vt tan (plaque) do
cc t bo vi khun b ph v. Trn thc t, c th o c cc ht phage
trong dung dch bng cch pha long dch ri trn vi dch t bo vi khun v
cng cy ln mi trng rn trong hp petri v sau tnh s vt tan suy ra
mt phage.

Hnh 20.43. DNA ca phage lamda dng thng

hoc vng trn

Phage dng si (filamentous phage) nh M.13 c kh nng xm nhp

vch t bo v lc enzym ca t bo ch tiu hy v protein lm nucleic
axit thot ra.
phage lamda (), s sao chp theta ( ) hay sao chp mt xy ra
rt sm trong chu trnh tan lm tng nhanh cc khun cho phin m v sao
chp tip. V sau, sao chp vng trn xoay m bo cc b gen gi vo
u ca phage th h con. Cc b gen ca phage cng c ct ra t
concatemer, nhng khc vi phage T4, s ct c thc hin mt trnh t
chuyn bit di 12N gi l im cos ( t ch cohesive site - im c kt ) do
terminaz hay h thng Ter (Ter system - nhn bit trnh t c hiu). S ct
bi Ter i hi hai im cos hoc mt im cos v u mt c kt t do (1/2
cos) hin din trn phn t ADN ca concatemer. Nh trnh t cos 2 u
mt m ADN b gen ca phage c th dng thng hoc vng trn v c
th chuyn i qua li (hnh 20.43).
B gen ca phage c bin i c chiu di trong khong 79-106%
ca b gen phage bnh thng khi b ct bi h thng Ter v c rp vo

u ca phage. y l tnh cht quan trng ca phage c s dng trong

k thut ti t hp ADN (chng VII).
C mt s c ch khc nhau gi ADN vo v. phage T4 ca E.coli,
s sao chp vng trn xoay ca mch ADN kp to ra mt si ADN mch
kp di gm mt dy cc concatemer ni tip nhau. Cc concatemer b ct
nhng im khng chuyn bit. V kh nng cha ADN ca u ln hn
chiu di 1 b gen ca phage, nn mi on thng c ct ra thnh tng
concatemer vi trnh t gen khc nhau.

Hnh 20.44. Concatemer b ct thnh nhiu b gen virut vi s gen hoc di hn

mt t, nhng c th t gen khc nhau v khi cun li c th i u.

Cc on cui thng c on lp li nn c gi l s d tha cui

(terminal redundance). V mi on ca concatemer c ct nhng im
khc nhau nn chng to thnh cc b gen c th t khng ging nhau v c
th to vng trn i u (cyclically permuted) (hnh 20.44).
Sau khi phage t rp cc cu phn xong, protein phn hy gi l
Lyzozym c sn sinh ra lm tan t bo v gii phng cc phage con. Phn
ln cc phage c theo chu trnh tan va nu trn. Tuy nhin, c mt s ngoi
l, nh phage si (filamentous) M13 ca E.coli hu nh khng bao gi lm
cht hoc lm tan t bo.
Cc t bo vi khun ch v cc phage k sinh c s ng tin ha
(coevolution). Cc t bo vi khun c cc c ch bo v nh bin i mng t
bo phage khng bm vo c hoc cc enzym restriction endonucleaz

ct cc ADN ca phage. Phage cng bin i xm nhp c vo t bo vi

khun. C trng hp c hai cng tn ti trong chu trnh tim tan.
Phage c ph v t bo vi khun ch, nn c th dng chng tr bnh
do vi khun gy ra. Kh nng ny c chng minh rt sm, nhng cha
c ng dng do s gia tng thuc khng sinh. Nhng hin nay do s gia
tng cc vi khun ln thuc khng sinh, n c quan tm tr li.
20.7.2. 2. Chu trnh tim tan (Lysogenic cycle)
Trong chu trnh tim tan, phage khng lm cht t bo ch, m b gen
virut gn vo b gen t bo ch v cng tn ti nhiu th h. Vi khun b
phage xm nhim m khng tan gi l vi khun tim tan (lysogenic bacteria).
Cc virut c th sinh sn m khng lm cht t bo ch c gi l phage n
ha (temperat virut). Vi khun tim tan cha phn t ADN ca phage khng
lm tan, gi l prophage, chn vo nhim sc th ca n. Vi khun tim tan
ny c kh nng min dch chng s xm nhim ca phage mi t ngoi,
tuy c ch ny khng ng ngha vi min dch t bo. Trong mt s iu
kin, prophage cng c sao chp theo chu trnh sinh sn bnh thng ca
vi khun ch m khng gy hi cho vi khun. Mt s iu kin khc s hot
ha prophage, lm n khi s chu trnh tan dn n phng thch s ln phage
t do.
Chi tit chu trnh tan v tim tan c nghin cu phage (hnh
20.45).

C hai kiu chu trnh tim tan. Trong kiu chung cho a s nh phage
, ADN ca phage gn vo nhim sc th t bo ch. kiu khc m i
din l phage P1, ADN ca phage khng gn vo nhim sc th t bo ch,
m bng cch no sao chp ng thi nh plasmid. C hai dng u c
gi chung l prophage.

Hnh 20.45. Chu trnh tan v tim tan phage

Hnh 20.46: Chu trnh tan v tim tan phage

Chu trnh sng bt u khi phage gn vo b mt t bo E.coli v bm

ADN vo trong gy nhim. ADN ca phage sau khi c bm vo t bo to
vng trn v s tham gia vo mt trong 2 chu trnh. ADN ca phage c th
hoc tham gia vo chu trnh tim tan ca phage T4 hoc gn vo nhim sc
th ca vi khun nh ti t hp im chuyn bit (specific site) bc vo
chu trnh tim tan. prophage phn ln cc gen khng c hot tnh. Tuy
nhin c t nht 1 gen ca prophage lun hot ng: gen cI m ha cho
protein km hm (repressor protein) gi cho phn ln cc gen ca phage im
lng. Trong qu trnh sinh sn ca t bo, ADN ca prophage cng c sao
chp v chia u v cc t bo con nh ADN ca t bo. Mt t bo b nhim
c th nhanh chng sinh ra nhiu t bo vi khun cha prophage.
Qu trnh gn ADN ca phage thc hin qua 4 bc:
ADN mch thng c bm vo t bo; ADN ca phage dng vng
trn nh s bt cp cc on ui d tha.
Mt s gen sm ca phage phin m v tng hp mt s phn t
protein c ch (repressor) v enzym integraz (enzym xc tc vic gn vo b
gen t bo ch ). Repressor c ch s phin m cc gen ca phage.
ADN ca phage thng c gn vo im chuyn bit trn nhim
sc th thnh prophage nh enzym integraz.
Vi khun sng v sinh sn; prophage c sao chp cng vi nhim
sc th ca t bo ch.

i khi prophage c th tch khi ADN ca vi khun mt cch ngu

nhin hoc c th tch khi do tc ng ca cc nhn t mi trng nh
phng x hay ha cht. Qu trnh tch din ra ngc li vi gn vo.
Prophage c tch ri ra c lp tr thnh phage t do v bt u chu trnh
tan.
Nu vi khun tim tan b tc ng ph hy ADN , prophage s c li
nu tch khi nhim sc th, chuyn sang chu trnh tan to ra cc phage mi
thot khi t bo. Khi ADN ca vi khun b thng tn, proteaz (recA
protein) ca h thng sa sai SOS c hot ha. ADN ca prophage b c
ch, excisionaz (enzym ct ri) c tng hp v prophage tch khi nhim
sc th. Qu trnh c gi l cm ng ca prophage (prophage
induction). Nu tia UV ph hy ADN ca t bo ch lm cm ng prophage
th c gi l cm ng do UV. Khi t bo vi khun F khng tim tan giao
hp vi t bo Hfr tim tan, t bo nhn b cht do chu trnh tan ca phage.
Dng cm ng phage ny c gi l cm ng hp t (zygotic induction).
V t bo ch mang prophage trn nhim sc th ca mnh s c kh
nng b tan nu prophage tch ra nn c gi t bo tim tan. i khi mt t
gen ca prophage biu hin t bo tim tan gy bin i kiu hnh vi
khun, qu trnh c gi l chuyn ha tim tan (lysogenic conversion). Mt
s prophage c th to c t
20.7.2.3. Kim sot s phin m phage
Cc virut c chin lc iu ha s phin m rt phc tp, m y
ch nu tm tt c ch . Bacteriophage l phage trung ha (temperat), c
ngha l n c th i vo chu trnh tan hoc tim tan.
Khi t bo ch tng trng trn mi trng giu, prophage vn duy tr
tnh tim tan (lysogenic); nhng khi t bo ch suy yu, prophage tr thnh
sinh tan (lytic). y c mt cng tc di truyn (gentic switch) xc nh
hnh vi ca prophage.
C 2 protein iu ha, cI and Cro, cnh tranh nhau 2 v tr
operator/promoter trn ADN ca phage. Mt operator kim sot hot tnh cc
gen sinh tan; ci kia thuc chu trnh tim tan. Hai protein iu ha ny c tc
ng ngc nhau trn 2 operator. cI c ch operator/promoter sinh tan v

hot ha operator/promoter tim tan. Cro hot ha operator/promoter sinh tan

v c ch operator/promoter tim tan. Cc nng tng i ca cI v Cro
xc nh u ra (hnh 20.47).

Hnh 20.47. C ch iu ha phage lamda:

protein Cro hot ha sinh tan, c ch tim tan; protein cI hot ha tim tan, c ch sinh
tan.
(Lytic gens: cc gen tan; Lysogeny gens: cc gen tim tan)

Khi ADN ca phage i vo t bo k ch mi, con ng sinh tan v

tim tan bt u vi cng mt cch. C hai con ng ny u cn cho s
biu hin ca nhng gen rt sm v nhng gen sm. Nhng sau chng
tch ra: theo con ng sinh tan nu nhng gen mun c biu hin, hin
tng tim tan xy ra nu c s tng hp cht kim hm
Lambda ch c hai gen rt sm c phin m c lp nh ARN
polymeaz ca k ch:
Gen N m ha cho mt nhn t chng kt thc v hot ng ca nhn
t ny ti nhng v tr nut cho php s phin m tin hnh trong nhng gen
sm.
Gen cro c hai chc nng: lm ngn cn s tng hp cc cht kim
hm (hot ng ch yu nu chu trnh sinh tan c tin hnh) v lm tt s

biu hin ca nhng gen rt sm (nhng gen khng cn v sau trong chu
trnh sinh tan).
Cc gen sm gm 2 gen sao chp (cn cho s gy nhim sinh tan), 7 gen
ti t hp (mt vi gen lin quan n s ti t hp trong sut qu trnh gy
nhim sinh tan, 2 gen cn thit cho vic gn ADN ca phage lambda vo
nhim sc th vi khun trong hin tng tim tan) v 3 gen iu ha.
V th nhng gen sm p ng hai vn quan trng: mt s gen cn
cho phage i vo hin tng tim tan, nhng gen cn li lin quan ti nhng
kim sot khc ca chu trnh sinh tan.
Trng thi t nhin ca chui kim sot ny gii thch nhng nt c
trng sinh hc ca s tn ti th tim tan. Hin tng tim tan c n nh
v chui kim sot m bo khi mc cht km hm thch hp, c s biu hin
lin tc ca gen cI. Kt qu l OL (Operator left tri) v OR (Operator right phi) vn cn b chim gi v thi hn. Bng cch biu hin ton b chu trnh
sinh tan hot ng ny duy tr prophage dng khng hot ng.
Nhng vng kim sot phin m cung cp mt im p lc, ti im
ny vic i vo chu trnh ton vn c th c kim sot. Bng cch khng
cho ARN polymeaz n gn nhng promoter ny, mt protein km hm ngn
cn b gen ca phage i vo chu trnh sinh tan..
Protein km hm c m ha bi gen cI. t bin gen ny khng
th duy tr hin tng tim tan nhng n lun lun i vo chu trnh sinh tan.
Tin hnh tch s khi cc protein km hm, m t c im, ngi ta bit
ti sao protein km hm c th duy tr c trng thi tim tan v to kh nng
min dch cho th tim tan khng li s bi nhim bi nhng b gen ca
phage lambda mi.
Lambda c s la chn gia hai cch: hoc i vo hin tng tim tan
hoc bt u s ly nhim sinh tan. Hin tng tim tan c bt u bng s
hnh thnh chui duy tr t sinh lm ngn cn ton b chu trnh sinh tan (lytic
cascade) qua vic s dng p lc ti hai im. Chng trnh cho s hnh
thnh hin tng tim tan tin hnh qua mt vi s kin tng t nhau m
nhng s kin ny cn cho chu trnh sinh tan (lytic cascade) (s biu hin ca

gen sm qua s biu hin ca N l cn thit). Lm th no phage i vo

chu trnh sinh tan?
Gen cro c vai tr quan trng nh hng ln chu trnh sinh tan (gen
cro m ha cho mt cht km hm khc). Gen cro chu trch nhim ngn cn
s tng hp protein km hm, hot ng ny lm ngng kh nng hnh thnh
hin tng tim tan. Nhng t bin cro thng hnh thnh hin tng tim
tan hn l i vo con ng sinh tan. Cro c dng mt dimer nh hot ng
trong vng min dch. Cro c hai tc dng l ngn cn s tng hp cht km
hm v gim bt s biu hin ca nhng gen sm.
20.7.2.4. Ti t hp phage
Cc phage tuy c kch thc nh b phi nhn di knh hin vi in t
mi thy c. Nhng cc tnh trng ca phage c quan st da theo cc
vt tan (Plaque) hoc bin ch. Vt tan (Plaque) l vng tan hay s c ch
t bo gy ra bi s nhim virut ca cc t bo nhy cm. Phage T2 c dng
hoang di r+ to vt tan bnh thng, cn dng t bin r (rapid hoc k hiu
r) lm tan nhanh nn vt tan to.
Lai: T2 h+r x T2 hr+

T2 hr+, T2 h+r
Th h phage con: Cc dng cha m

T2 h+r+ , T2 hr
Cc dng ti t hp

DNA b m

DNA ti t hp

Hnh 20.48. S nhim kp hai

phage

Hnh 20.49. C ch ti t hp
gen

V bin ch c dng hoang di h+ (host) ch lm tan vi khun E.coli

dng B nhng khng lm tan dng B2, t bin h (hoc k hiu h) lm tan
cc vi khun E.coli c 2 dng B v B2. Dng phage T2 h+r lm tan c B v B2
vi vt tan nh c lai vi dng T2 hr+ lm tan ch B nhng vt tan to.

S nhim phage (hnh 20.48), c ch trao i cc an ADN (hnh

20.49) v s biu hin cc vt tan th hin trn hnh 20.50.

Hnh 20.50. Cc vt tan th hin ti t hp bacteriophage

S xut hin cc dng ti t hp T2 h+r+ v T2 hr chng t 2 dng
phage T2 lai vi nhau.
Ti t hp virut c cc c im:
Trong qu trnh lai virut c s tham gia ca c b gen nh
Eukaryota ch khng tng phn nh vi khun.
Qu trnh ti t hp c tnh qun th. ADN ca 2 dng phage xm
nhp vo t bo vi khun, chng sao chp ra hng trm bn sao v gia hng
trm ADN b gen ca phage xy ra ti t hp. Do iu ny, phage
thng thy hin tng nhiu m (negative interference) trong ti t hp: tn
s ti t hp i gia 2 locus ng l phi gim th li tng cao hn mc bnh
thng.
20.7.2.5. Locus rII ca phage T4.
Cc nghin cu chi tit v cc t bin rII ca Phage T4 lm sng t
hn v cu trc gen. Phage T4 dng hoang di r+ c kh nng nhim dng
thi hai ni E. coli B v K. Cc t bin rII ch nhim ni B nhng khng
nhim ni K. S.Benzer (1955) nhn c vi nghn t bin rII c ngun
gc c lp vi nhau. ng cho lai cc t bin vi nhau v cn c vo s
xut hin cc dng ti t hp hoang di r+ m lp bn cc im t bin
(mutation sites) (hnh 20.51).

Trc th nghim ca ng, rII c coi l mt locus. Th nghim cho

thy cc t bin xp thnh hai nhm rIIA v rIIB. Lai cc t bin rIIA X
rIIB s c r+, nhng lai rIIA X rIIA v rIIB X rIIB th kiu hnh t bin r.

Hnh 20.51 Bn cc t bin locus rII.

(Cc hnh tam gic m l cc im nng- hot spot)

Kt qu th nghim cho thy, gen c th phn chia nh v mt t bin.

Cc on rIIA v rIIB c gi l cistron, n v chc nng nh nht m
bo kh nng xm nhp ni K. Thut ng cistron thc cht l gen, ngy nay
n ch c tnh cht lch s, t c dng. Theo quan nim hin nay, rIIA v
rIIB l hai locus. Hai khi nim mi c nu ra l muton - n v t bin
v recon - n v ti t hp.
on gen nghin cu tm thy c 2000 im t bin, chng phn b
khng u nhau, c nhng im nng (hot spot) tp trung nhiu t bin
hn (hnh 4.16).
20.7.3. Cc virut Eukaryota
20.7.3.1. Cc virut thc vt

Cc virut thc vt l mt tai ha ln cho trng trt v n lm gim ng

k nng sut cy trng. Cc virut thc vt c th c truyn theo 2 ng:
Theo chiu ngang (Horizontal transmission) l s ly truyn t cy ny
sang cy khc.
Theo chiu dc (Vertical transmission) qua ng sinh sn cc virut t
cy m n th h con.
xm nhim t bo thc vt, virut phi xm nhp vch t bo v mng
sinh cht ca thc vt.
Cc cn trng c th l tc nhn truyn virut, khi chng ph v vch t
bo thc vt v a virut vo. Mt cch ly nhim virut khc l s tip xc
gia m b thng tn ca cy b nhim vi cy khng nhim.
S truyn theo chiu dc c th thng qua sinh sn v tnh hay hu
tnh. Khi vo trong t bo thc vt, cc virut c th pht tn bng cch di
chuyn theo cu cht lin bo (plasmodesmata), cc ch ni t bo cht gia
cc t bo. Cc virut gn vi cc protein c hiu gip chng bng cch no
i xuyn qua cc l hp ca plasmodesmata.
a) Cc virut ARN
Phn ln cc virut thc vt pht hin cho n nay, a s c b gen
ARN mch n kiu (+) v nhiu dng c capsid hnh que, k c virut m
thuc l (hnh 20.36 pha trn). Cc protein capxomr xp hnh xon.
b) Cc virut ADN
Cc virut thc vt c b gen l ADN rt him v ch gm 2 nhm:
Nhm th nht l cc cauliflower mosaic virut. Chng c b gen l
ADN mch kp nm trong v (capsule) a din (polyhedral).
Nhm th hai l cc geminivirut (gemini c ngha sinh i). Chng
c c tnh l cc capsid dnh thnh i, mi ci cha mt phn t ADN
mch n vng trn vi khong 2500 nucleotit. Cc b gen bt cp c th
tng t nhau mt s virut v khc nhau ng k s khc.
c) Cc viroid
Cc viroid l mt nhm tc nhn gy bnh thc vt, c kch thc nh
v cu trc n gin nh virut. Chng l nhng ARN mch n, trn, nh b

c chiu di ch vi trm (240 - 350) nucleotit. C s bt cp mnh gia cc

bn trong phn t ARN to cu trc kp. Cc viroid khng c bao trong v
protein, khng gn vo b gen t bo ch v sao chp khng qua trung gian
ADN. Mt s t bo thc vt c enzym sao chp ARN.
Cc phn t ARN ca viroid bng cch no ngn tr trao i cht ca
t bo v lm ngng tng trng ca c thc vt. Mt bnh viroid khc lm
hi ng k n sn xut hoa cc M. Cc viroid cng tc hi n khoai ty
v c chua.
Gn y pht hin rng trnh t nucleotit ca viroid ging vi trnh t ca
intron ca cc gen Eukaryota. Ngi ta cho rng c l cc viroid bng cch
no tc ng ln cc h thng iu ha kim sot cc gen ca t bo.
20.7.3.2. Cc virut ng vt
Chnh xc cc virut ng vt th hin y s a dng ca cc b
gen virut. Mt s virut ng cha ADN, v mt s ARN, mch n hay
kp nhng b gen ca chng rt nh b. Cc virut ng vt gy nhiu bnh
ng vt v ngi, mt s virut lin quan n c cc bnh ung th, AIDS...
Arbovirut xm nhim c cn trng v ng vt c xng. Cc virut truyn t
cc loi chn khp (arthropod), vector trung gian, n ng vt c xng
(vertebrat) qua vt t ca cn trng.
Cc virut ng vt xm nhp th bo theo 3 con ng khc nhau:
S ni nhp bo (Endocytosis) ca virion trn.
S ni nhp bo (Endocytosis) ca virut c mng bao (a membrane-encazd
virut)
S dung hp (Fusion) virut c mng bao vi mng t bo.
a) Cc nhm virut ng vt
S a dng rt ln ca cc virut ng vt th hin 15 - 20 h khc
nhau, cn c theo cc c tnh nh: kiu v cu trc ca nucleic axit, hnh
thi virion v cch xc nh th khng nguyn chung. Do s ph thuc ca
cc b gen virut vo mARN ca chng, cc virut ng vt c chia thnh 7
nhm.
b) Cc chu trnh sao chp ca virut ng vt

Cc chu trnh sao chp ca virut ng vt c nhiu im tng t vi

cc virut khc vi nhiu bin dng ng k. V d trng hp ca cm
(influenza virut) hay ca Paramyxovirut gm cc virut gy bnh si v quai
b. Cc virut cm v Paramyxovirut c b gen l ARN mt mch c gi
trong capsid xon do. Pha bn ngoi capsid c mng bao l tnh cht chung
ca nhiu nhm virut ng vt v phage (hnh 20.52).

Hnh 20.52. Virut cm

Bao mng gip virut xm nhp t bo ch. Khi virut tip xc vi t bo,
cc glycoprotein thi ra mng bao gn vo cc th th (receptor) trn mng
sinh cht (hnh 20.52). Qu trnh ny chuyn capsid c cha ARN vo t bo
cht v capsid b mt (bc v). Cc enzym ca virut tham gia sao chp
ARN ca b gen v to mARN, nhng b my ca t bo c s dng
tng hp protein ca virut. Cc capsid mi c lp rp bao cc b gen ca
virut v chng i mng sinh cht ca t bo mc chi ri thot ra khi t
bo. Bng cch ny cc capsid c mng bao chnh l mng sinh cht ca t
bo ch c v cc virion mi c th s dng cc bao mng ny ha nhp
vi mng ca t bo ch mi. Ton b chu trnh ny c gi l chu trnh
sinh sn (reproductive cycle), ch khng phi chu trnh tan v cc virut c
th thot ra bng mc chi m khng phi ph v t bo ch. ng ch
l chu trnh sinh sn ca cc virut loi ny din ra t bo cht ca t bo
v khng tc ng n nhn t bo.

Hnh 20.53. Chu trnh sinh sn ca virut cm (Influenza virut

Khng phi tt c cc mng ca virut u bt ngun t mng sinh cht.

V d, bao mng ca herpesvirut t mng nhn ca t bo ch. Cc b gen
ca herpesvirut l ADN mch kp , chng sinh sn bn trong nhn t bo, s
dng phi hp c enzym ca virut v t bo sao chp ADN v phin m.
Khi trong nhn ca t bo, ADN ca herpesvirut (tin virut) tng t
prophage vi khun. Provirut dng tim n c th gy mt s triu chng
bnh ngi. i khi cc stress vt l (nh tc ng nh sng mt tri mnh)
hay tnh cm c th lm cho provirut herpes bt u chu trnh sinh sn to
cc triu chng kh chu cho con ngi.
c) Cc retrovirut
Cc nh khoa hc nhn thy rng mt s virut c th gy ung th
ng vt v c ngi. Cc virut gy ung th gm: retrovirut, papovavirut,
adenovirut v nhm herpesvirut.
C l cc virut gy ung th quan trng nht l retrovirut, cc virut c b
gen ARN sinh sn qua trung gian ADN. Retrovirut HTLV-I gy bnh bch cu
t bo T (T-cell leucemia) v retrovirut HIV gy bnh AIDS lm suy yu h
thng min dch (hnh 20.54).Ni chung, retrovirut lin quan n nhng bnh
nguy him, kh cha tr nht hin nay ca nhn loi, k c ung th.

Tt c cc virut bin i t bo qua vic gn nucleic axit ca virut vo

ADN ca t bo ch. S gn ny c n nh (cc provirut khng bao gi b
ct ri ra nh prophage). i vi cc virut gy bnh ung th l ADN, s gn
vo l qu trnh trc tip. Cc retrovirut th khc, trc ht chng s dng
reverse transcriptaz t khun ARN to ra ADN. Sau ADN ca virut c
th c gn vo ADN b gen ca t bo ch v chng cng sao chp vi b
gen ny trong mi th h.

Hnh 20.54. Cu trc ca virut HIV (retrovirut).

Hnh 20.55 m t v gii thch chu trnh sinh sn retrovirut trong t

bo.
Hin nay, nhiu ngi cho rng cc virut c th tc ng n s biu
hin ca cc gen ung th. Thng cc virut gy ung th phi hp vi nhng
tc nhn khc trong gy bnh.
V ngun gc ca cc virut th nhiu ngi cho rng chng bt ngun
t cc on nucleic axit c gi li mt cch chuyn bit. Mt s virut
Eukaryota ging v cu trc v chc nng so vi mt s gen ca t bo ch
hn virut ca vi khun. B gen ca mt s virut ging cc phn t di truyn
ca t bo nh plasmid hay transposon.

Hnh 20.55. Chu trnh sinh sn ca retrovirut HIV gy bnh AIDS

20.7.4. Cc Prion
Hai bnh thoi ha no ngi l bnh kuru v Creutzfeldt-Jacob
(bnh tng t cu) c th gy nn do cc phn t protein gy nhim c
gi l prion (proteinaceous infectious particle). Cho n nay, s hiu bit v
chng cn rt t. Gn y, khi xut hin dch b in Anh, chng c nhc
ti nhiu v khng pht hin thy cc virut.
20.7.4.1. Lc s
Vo nhng nm 1960, T. Alper v J.S. Griffith nu gi thuyt rng mt
s bnh no nhng (spongiform encephalopathies) ly truyn do cc tc nhn
gy nhim ch gm cc protein. Thuyt ny gii thch vic pht hin tc nhn
b n gy bnh gi v Creutzfeldt-Jakob (scrapie and Creutzfeldt-Jakob
diseazs) khng tia t ngoi UV.
Francis Crick, ngi nu Hc thuyt trung tm ("Central dogma
of molecular biology") c bit quan tm n gi thuyt ca Griffith v n
mu thun vi hc thuyt ca ng.
Vo nm 1982, S.B. Prusiner (i hc California) thng bo tinh
sch tc nhn gy bnh gi nh v n ch gm protein c hiu. Prusiner

dng thut ng prion ch tc nhn gy bnh, l s kt hp 2 ch u ca cc

t proteinaceous (phn t protein) v infectious (gy nhim), cn ui -on th
tng t virion. Protein c hiu m prion to ra gi l PrP (vit tt ca cm
t "proteaz resistant protein" protein khng proteaz). Prusiner nhn gii
Nobel Sinh l hay Y hc (Nobel Prize in Physiology or Medicine) vo nm
1997 do cng trnh nghin cu cc prion.
20.7.4.2. Cu trc
S tn ti cc ng dng (Isoform). Protein m cc prion to ra
c thy khp c th, thm ch c ngi v ng vt khe mnh. Tuy
nhin, protein ca prion trong vt liu nhim c c tnh gi cun khc
(different folding pattern) v khng vi proteaz, enzym m trong c th c
th phn hy cc protein mt cch bnh thng. Dng protein bnh thng
gi l PrPC, dng gy nhim l PrPSc ( C bt ngun t ch 'cellular' (ca t
bo) hay gi chung ('common') l PrP, cn Sc t ch 'scrapie', l bnh prion
cu. Trong khi PrPC c xc nh cu trc tt (structurally well-defined),
th PrPSc phn tn rng v kh xc nh. PrP c th c gy cm ng gp
cun (induced to fold) thnh cc isoform xc nh tt (well-defined isoforms)
in vitro, v mi quan h vi dng gy bnh (pathogenic) in vivo cn cha r.
PrPC: PrPC l protein bnh thng trn mng t bo. N c 209 axit
amin s ( ngi), mt cu disulfite , 35-36kDa v ch yu l cu trc xon
alpha. PrPC d dng b phn hy bi proteinaz K v c th phng thch khi
b mt t bo in vitro bi enzym photphoinositit photpholipaz C (PI-PLC).
PrPSc

PrPSc: Dng ng hnh gy nhim (infectious isoform) ca PrPC l

c kh nng chuyn i PrPC protein bnh thng thnh isoform gy

nhim bng cch thay i cu hnh (conformation) ca n. Mc d cu trc 3

chiu (3D structure) ca PrPSc cha bit, y c s gia tng lng phin
beta (-sheet content) dng nhim bnh ca phn t thay th cc vng
bnh thng ca xon alpha (-helix). Cc t hp (aggregations) ca nhng
isoform bt thng c th hnh thnh cc si c cu trc giu amyloid
(highly structured amyloid fibers). Phn cui ca si tc ng nh l khun
cho cc phn t protein t do, n lm cho si mc ra. C nhng khc bit nh
trong trnh t axit amin ca vng hnh thnh prion (prion-forming regions)
dn n nhng tnh cht cu trc khc bit trn b mt ca cc si prion. Do

vy, ch cc phn t protein t do tng t trong trnh t axit amin vi

protein prion c th c tham gia vo si ang mc ra (growing fiber).
20.7.4.3. Chc nng
Hin nay chng minh c rng vai tr t bo bnh thng ca cc
protein prion l cht oxy ha (antioxitant) ph thuc Cu.
PrP v ghi nh di hng (long-term memory). C chng minh cho thy
PrP c th c chc nng bnh thng khi duy tr s ghi nh di hng.
cho thy chut thiu cc gen cho protein PrP bnh thng ca t bo c
hippocampal LTP bin i.
PrP v s ti sinh t bo gc (stem cell renewal). Bi bo nm 2006
cho thy rng s biu hin PrP t bo gc l cn thit cho s t ti sinh ty
xng ca sinh vt (organism's self-renewal of bon marrow). Cng trnh ny
cng ch ra rng tt c cc t bo gc to mu di hng (all long-term
hematopoietic stem cells) biu hin PrP trn b mt t bo ca chng v cc
m to mu (hematopoietic tissues) vi cc t bo gc khng PrP (PrP-null
stem cells) th hin s nhy cm gia tng i vi s suy kit t bo.
20.7.4.4. Bnh Prion
Tt c cc bnh theo gi thuyt do prion u tc ng ln cu trc ca
no hay cc m thn kinh khc v cho n nay tt c bnh u khng cha tr
c v lun gy cht. Ni chung, prion c th lin quan c hai l n v
nhim bnh l thuyt (theoretical unit of infection) hay protein c hiu (v
d, PrP) bin thnh tc nhn gy nhim (infective agent), d c hay khng
trng thi nhim bnh (infective stat).
Cc prion c gi nh l gy nhim v pht tn bng cch ti gp
cun (refolding) bt thng thnh cu trc c kh nng chuyn i (convert)
cc phn t bnh thng (normal molecules) ca protein thnh dng cu trc
bt thng. Tt c cc prion bit c gy ra s hnh thnh gp cun amyloid
(amyloid fold), m trong protein polyme ha thnh phc hp (aggregate)
gm cc phin beta (beta sheets) gi cht nhau. Cu trc bin dng ny rt n
nh v tch ly trong m nhim lm ph hy v gy cht t bo. S n nh
c ngha l cc prion khng cc tc nhn vt l v ha hc gy bin tnh

(denaturation) lm cho vic loi b v ngn chn cc phn t ny rt kh

khn.
Cc protein ca prion (PrP) rt ging nhau tt c cc ng vt c v.
Do nhng khc nhau nh ny trong PrP gia cc loi khc nhau, nn bnh do
prion c truyn t loi ny sang loi khc mt cch khng bnh thng.
Tuy nhin, bnh prion ngi bin dng bnh Creutzfeldt-Jakob c cho
rng gy nn bi prion gy nhim in hnh b v c truyn qua tht b
nhim (infected meat). Ngoi nhng bnh k trn, cn mt s bnh khc
ng vt v ngi do prion.
Mc d xc nh v bit r cc tnh cht chung ca prion, nhng
c ch ca s gy nhim v pht tn prion vn cn trong b n.
20.7.4.5. Cc gi thuyt v prion
Cho n nay c nhiu bn ci v bn cht ca prion. Cc gi thuyt ch
yu c tm tt nh sau:
Gi thuyt ch protein (Protein-only hypothesis). Theo gi thuyt
ny, protein c th sao chp khng dng nucleic axit.
Gi thuyt a cu phn (Multi-component hypothesis): cc prion gy
nhim c th gm nhiu cu phn ca t bo ch nh PrP, lipit, v cc phn
t polyanionic, hn l ch c PrPSc.
Gi thuyt virut (Viral hypothesis): thng 1/2007 cng b bi bo
cho thy 10% hoc t hn cc t bo nui cy b nhim prion c virut v cho
rng bnh do phn t thng tin c kh nng sao chp (a replicable
informational molecule) tng t nucleic axit gn vo PrP.
Gi thuyt nhim c kim loi nng (Heavy metal poisoning hypothesis):
s lng bt thng ca ng (Cu) v manganese (Mg) trong mi trng v
thc n chn nui c th gy ra bnh.
20.7.4. 6. Di truyn hc (Gentics)
Nhiu vn cn cha sng t. Gen cho protein bnh thng c
phn lp: PRNP gen. Mt s bnh prion c th c di truyn v trong mi
trng hp lin quan n cc t bin gen PRNP ny. Nhiu t bin khc
nhau ca PRNP c xc nh v cho rng cc t bin bng cch no
lm cho PrPC ngu bin (spontaneously change) thnh dng PrPSc bt thng.

20.7.4. 7. Cc prion nm men v nm khc

Protein ca prion c pht hin nm men Saccharomyces
cerevisiae t u nhng nm 1990. Sau , prion cng c tm thy
Podospora anserina. Cc pht hin protein c biu hin kiu prion (prion-typ
behavior) ny mt s nm (fungi) c ngha ht sc quan trng gip cho
s hiu bit v cc prion ng vt c v. Tuy nhin, cc prion nm khng
gy bnh trong t bo ch v thm ch cn c u th tin ha (evolutionary
advantage) thng qua dng di truyn da vo protein (form of protein-bazd
inheritance).
Sinh hc hin i pht hin hai dng c biu hin sng c bit: viroid
c nucleic axit, khng c protein v prion c protein, khng c nucleic axit.
20.8-DI TRUYN HC VI NM V VI TO
Cc vi sinh vt nhn thc Eukaryota nh vi nm, vi to c chu trnh
sng a dng v nhiu im ging vi cc Eukaryota bc cao. Cu to n
bo gip cho vic s dng c hiu qu cc phng php phn tch di truyn
vi sinh vt. Phn tch b bn gip nghin cu c chi tit tng sn phm
ca gim phn. Cc nghin cu di truyn vi nm tp trung 4 vn cn
bn: tnh khng phi hp, phn tch b bn, ti t hp nguyn phn v s
n bi ha. Neurospora crassa v Saccharomyces cerevisiae l nhng i
tng nghin cu quan trng ca di truyn. vi to Chlamydomonas
reinhardii c nghin cu chi tit hn c trong s cc vi to. Cc nghin
cu vi to lm sng t nhiu vn ca di truyn quang hp v s xc nh
cc sc t.
Trong s gn 50.000 loi vi nm, n nay nghin cu v di truyn
hc c khong 30 loi. Nhng vi sinh vt u tin c s dng vo
nghin cu Di truyn hc t nhng nm 30 ca th k trc chnh l cc vi
nm. l Neurospora v cc loi khc nhau ca chi Saccharomyces.
Saccharomyces cerevisiae l Sinh vt nhn thc (Eukaryota) n bo, cho
nn l m hnh rt tt cho nghin cu di truyn ca Sinh vt nhn thc v v
th cho nn c hiu bit chi tit hn c.
Cc vi to (Microalgae) cng c s lng loi rt ln (gn c 100.000)
v gi vai tr rt quan trng trong sinh quyn, l ngun cung cp oxy ch yu

trong nc. Tuy nhin ch mt s t to lc n bo c dng lm i tng

nghin cu ca di truyn hc, c bit l Chlamydomonas reinhardii.
Hai i tng tiu biu cho nghin cu di truyn hc l nm men
Saccharomyces cerevisiae v nm si Neurospora crassa. Chng c cc c
tnh u vit v va l vi sinh vt, m t bo c nhn thuc Sinh vt nhn thc
Eukaryota.
20.8.1-Nm men Saccharomyces cerevisiae
Nm men Saccharomyces cerevisiae (hnh 20.56) l mt loi thuc lp
Nm Ti (Ascomycetes), cn gi l men bnh m (baker yeast) hay men
ru. l loi nm men ch yu c dng trong cc qu trnh ln men
ru. V l Eukaryota n bo (kch thc khong 5 10m , c bit rt
chi tit v di truyn hc v sinh l hc, cho nn l i tng m hnh nghin
cu di truyn cc sinh vt nhn thc.

Hnh 20.56- Nm men S.cerevisiae di knh hin vi in t v knh hin vi quang

hc

a) Cc c tnh sinh hc
Cc nghin cu di truyn hc c tin hnh S. Cerevisiae t hn
70 nm nay. i tng ny kt hp trong n 2 tnh cht tuyt vi: l n bo
nn c th tin hnh th nghim nh vi khun, ng thi c nhng c tnh
ch yu in hnh ca Eukaryota v c ty th vi b gen ADN nh. Ging
vi vi khun, n c th nui trn mi trng dch th hay c v to khun
lc trn mi trng thch. C th nui t bo nm men quy m ln trong cc
ni ln men v d dng thu nhn sinh khi t bo.
Chu trnh sng ca nm men c nu ln trn hnh 20.57

Nm men c 2 dng t bo n bi (n) l a v c th tn ti c lp

nh sinh sn v tnh.qua nguyn phn (mitosis). Khi 2 dng a v gp nhau
bt cp, ri phi hp t bo v hp nhn to 1 t bo lng bi (2n). T bo
lng bi c th sinh sn v tnh v hng v y l dng thng s dng
trong sn xut. Trong nhng iu kin nht nh, t bo 2n sinh sn hu tnh
qua gim phn to nang c 4 bo t (2 a v 2 ), m s kt hp a v to t
bo 2n, lp lai chu trnh.

Hnh 20.57- Chu trnh sinh sn ca nm men S. cerevisiae

b) Cc d liu di truyn hc ca S. cerevisiae:

Kch thc b gen (Genome size): 12 Mb
Nhim sc th: n = 16
S lng gen: 6.000
Phn trm gen tng ng vi ngi: 25%
Kch thc trung bnh ca gen: 1,5 kb, 0,03 intron/gen
Cc transposon: mt t l nh ca ADN
Kt thc gii k t chui: 1996
c) Phn tch di truyn

Nm men c th tn ti dng n bi vi 2 kiu bt cp (mating typ)

l a v .Ngoi cc phng php lai phn tch ti t hp (recombination)
v b tr (complementation), c nhiu k thut bin i di truyn
(Techniques of Gentic Modification):


Gy t bin (Mutagensis):

Ha cht v chiu x: t bin xoma ngu nhin.

Dng transposon: xen on (Insertions) xoma ngu nhin.


Chuyn gen (Transgensis):

Plasmid tch hp (integrative): xen on (Inserts) nh ti t hp tng ng

(homologous recombination).
Plasmid sao chp (replicative): C th sao chp t lp (autonomously) (Ori
sao chp ca plasmid 2 hay ARS).
Nhim sc th nhn to ca nm men: sao chp v phn ly nh mt nhim
sc th.
Vector con thoi (Shuttle vector): C th sao chp trong t bo nm men hay
E. coli.
Lm im lng gen mc tiu (Targeted gen knockout): Thay th gen (Gen
replacement): ti t hp tng ng thay alen hoang di bng alen khng
(null alen).
d) K thut di truyn

Hnh 20.58- Cu trc t bo nm men S.cerevisiae vi cc

c im ca t bo nhn thc

Cc thao tc gen (Gen manipulation) thc hin trn S. cerevisiae d

dng hn bt k sinh vt Eukaryota no khc. c bit n c nhim sc th
nhn to (Yeast artificial chromoxom - YAC) c trnh t sao chp ARS
(Autonomously replicative sequence), tm ng (centromere) v 2 telomere
(trnh t u mt nhim sc th). YAC tn ti c lp trong t bo nm men
nh mt nhim sc th v cc gen c chn vo n c di truyn nh
nhim sc th Mendel. YAC c kh nng mang on gADN ngoi lai di 1
2 Mb, nn thun tin cho to dng v gii k t chui (sequencing) nhng b
gen ln nh b gen ngi. Nm men S. cerevisiae c s dng rng ri lm
t bo ch trong KTDT.
Ngoi ra, mt s protein ti t hp (gc ng vt) nh interferon khi
biu hin trong t bo Prokaryotae nh E. coli th khng c hot tnh do thiu
bin i sau dch m, nhng khi cho biu hin trong t bo nm men th sn
phm c hot tnh.
e) Cc ng gp
Vai tr ch yu cho cc nghin cu:
Chu trnh t bo (Cell cycle): S xc nh cc gen kim sot phn bo nh
cc t bin nhy cm nhit (temperature-sensible mutants (cdc mutants))
dn n m hnh rt tt cho nghin cu s phn bo.
Tng tc gen (Gen interaction): nghin cu s c ch gen (suppression).
H thng plasmid lai kp (two-hybrid plasmid system) gip tm cc tng tc
gia cc protein nm men dn n cc bn tng tc phc tp, m l
khi u cho sinh hc cc h thng (systems biology).
Di truyn hc ty th: nh cc t bin petite mt kh nng h hp m
pht hin cc gen ca ty th. Nh chng v cc t bin khc ca ty th m
phn tch chi tit cu trc v chc nng b gen ty th.
Di truyn hc kiu bt cp (Gentics of mating typ): Cc alen MAT nm
men l cc gen kiu bt cp u tin c xc nh cc c tnh mc phn
t.

 ng gp khc: S di truyn ca kha dng m (switching) gia s tng

trng kiu nm men (yeast-like) v si (filamentous); di truyn hc s thoi
ha (senescense).
20.8.2- Nm si Neurospora crassa
a) Cc c tnh sinh hc
Ging vi vi khun v nm men, nm si ny c th nui trn mi
trng dch th hay c v to khun lc trn mi trng thch. N th hin
cc tnh cht in hnh ca Eukaryota ging nh nm men: b gen gm nhiu
nhim sc th, bo t ti c trng thi n bi (n) v hp t lng bi (2n),
sinh sn v tnh qua chu trnh nguyn phn (mitotic cycle), sinh sn hu tnh
qua gim phn v c ty th vi b gen ADN nh. Nm thng mc trn bnh
m c v c mu vng ti (hnh 20.59):

Hnh 20.59- Nm si Neurospora crassa di knh hin vi quang hc

Si nm phn on c cha nhiu nhn n bi. C hai kiu gii tnh

c iu khin bi mt cp alen A v a. Sinh sn hu tnh ch thc hin khi
c s kt hp ca cc t bo khc alen (hnh 20.60). Nh hnh v m t khi
bo t nh a ri trn tin qu th dng chai (protoperithecium). Cc c quan
sinh dc ci hay A ri ln tin qu th a th s th tinh s c xy ra. Lc
u hai nhn n bi A v a tn ti song song v chia ng thi. Sau xy
ra s hp nhn v tin qu th dng chai bin thnh qu th dng chai
(perithecium). Mi hp t lng bi 2n to thnh mt t bo ko di gi l
nang hay ti (ascus) v lp tc chia gim nhim c 4 nhn, m 4 nhn ny

li chia nguyn nhim mt ln na to thnh 8 nhn ca 8 bo t nang hay

bo t ti (ascospores).

Hnh 20.60. Chu trnh sng ca nm si Neurospora crassa

Mt c im c o ca Neurospora crassa l cc sn phm ca

gim phn, 8 nang bo t xp thng hng trong nang. Nh vy, qua phn tch
b bn (tetrade analysis) nghin cu hng lot cc c ch di truyn nh trao
i cho, gen chuyn bin (conversion), ti cu trc nhim sc th
(chromoxom rearrangement), s khng chia ly trong gim phn (meiotic
nondisjunction) v s kim sot di truyn i vi gim phn. Cc Nhim sc
th tuy nh, nhng c th nhn thy d dng.

N. crassa t k lc v tc tng trng trong cc loi nm: mi si

nm (hypha) mc di ra hn 10 cm/ngy. S tng trng nhanh kt hp
vi chu trnh n bi v kh nng mc trn cc mi trng nui cy c thnh
phn xc nh lm cho n thnh mt i tng rt thun tin cho nghin cu
di truyn ha sinh (biochemical gentics). Trn thc t, n ni ting vi s ra
i gi thuyt mt gen - mt enzym.
b) Cc d liu di truyn hc ca Neurospora crassa:
Kch thc b gen (Genome size): 43 Mb
Nhim sc th: 7 Nhim sc th thng (n = 7)
S lng gen: 10.000
Phn trm gen tng ng vi ngi: 6 %
Kch thc trung bnh ca gen: 1,7 kb, 1, 7 intron/gen
Cc transposon: rt him
Kt thc gii k t chui: 2003
c) Phn tch di truyn
Khi nui chung 2 loi si t nm c kiu bt cp i ngc nhau l A
v a d dng nhn c cc nang bo t lai. Mc d Neurospora crassa
khng nhn c cc si t lng bi in hnh (nhn 2n), nhng s phi
hp 2 loi t to ra th d nhn (heterokaryon), m trong 2 nhn n bi
khc nhau cng tn ti song song trong t bo cht. Nh vy c th tin hnh
phn tch ti t hp v b tr. Ngoi ra, c cc k thut bin i di truyn
sau:


Gy t bin (Mutagensis):

Ha cht v chiu x: t bin xoma ngu nhin.

Dng transposon: khng.


Chuyn gen (Transgensis):

Bin np qua trung gian plasmid (Plasmid-mediated transformation): xen

on ngu nhin (Random insertion).


Lm im lng gen mc tiu (Targeted gen knockout):

RIP: cc t bin GC > AT trong cc on tng i chuyn gen trc khi

lai.

 c ch (Quelling): bt hot xoma sau phin m cc gen c chuyn vo

(Xomatic posttranscriptional inactivation of transgens).
d) K thut di truyn:
Bin np u tin trn Eukaryota c thc hin Neurospora
crassa. Ngy nay, n d dng chp nhn nhiu vector plasmid mang cc gen
mong mun. Khng c plasmid no ti bn trong Neurospora, v nh vy cc
gen chuyn vo phi c chn vo nhim sc th mi c s di truyn n
nh.
e) Cc ng gp
Di truyn sinh ha v trao. i cht.
Di truyn hc ca gim phn.
Di truyn ty th.
ng gp khc: s a dng cc loi nm v thch nghi (adaptation), di
truyn t bo (cytogentics), cc gen kiu bt cp, cc gen phi hp th d
nhn (heterokaryon-compatibility gens).
20.8.3- Cc c im di truyn va vi nm
Chu trnh sng ca cc loi nm khc nhau rt a dng, nhng c th
xp vo 5 kiu cn bn khc nhau nh: 1. Sinh sn v tnh; 2. Th n bi; 3.
Th n bi vi giai on d nhn; 4. n bi - Lng bi ; 5. Lng bi.
Ngay trong cc nm sinh sn hu tnh cng c s dao ng t hon
ton n bi nh Neurospora n hon ton lng bi nh Saccharomyces
cerevisiae
i vi phn ln nm si, im c trng l nhiu nhn cng hin din
trong mt t bo. Cc nhn ny c th c kiu gen khc nhau. Trong trng
hp ny c gi l th d nhn (heterokaryon) tng t d hp t.
cc vi sinh vt ni chung, cc tnh trng hnh thi thng khng
nhiu, nhng c s a dng ln v nhiu loi phn ng ha hc khc nhau.
S ra i ca gi thuyt ni ting 1 gen - 1 enzym da trn cc t bin
sinh ha N. crassa. Cc mi trng chn lc c s dng thu thp cc
dng lai him hoi gia cc dng cha m n bi mang cc t bin khuyt
dng ln, khi sai hng trong chui phn ng sinh ha ca chng b sung

cho nhau. Ngoi ra tnh khng vi cc tc nhn bt li c s dng rng

ri lm tnh trng nh du trong cc nghin cu di truyn nm.
Ni chung, cc vi sinh vt nhn thc c u th c bit l s kt hp
gia cu to t bo v chu trnh sng nh sinh vt bc cao vi c th n bo
ca vi sinh vt. Nu nh trong nhng nm 70 ca th k trc, cc i tng
cn bn ca di truyn hc phn t l phage v vi khun, th nhng t nm 80
cho n nay cc Eukaryota n bo thay th dn.
Bn vn cn bn c ch trong nghin cu di truyn nm l:
tnh khng phi hp (incompatibility), phn tch b bn (tetrade analysis), ti
t hp nguyn phn (mitotic recombination), v s n bi ha
(haploidisation).
Nhiu nm si c qu th nhn thy c khng phi l vi nm, nhng
c th s dng cc phng php dng cho vi nm nghin cu.
20.8.3.1- Tnh khng phi hp nm
Khi nim tnh khng phi hp (incompatibility) nm c dng
ch kh nng kt hp vi nhau gia cc dng nm trong sinh sn hu tnh.
Cho n nay, gn 450 loi nm c nghin cu v cc kiu khng phi
hp. S khng phi hp c xc nh v mt di truyn. Theo kiu phi hp
th nm c phn lm 2 loi:
ng tn (Homothallic) l khi c s kt hp vi nhau gia cc t bo (hay
h si tmycelium) ging nhau trong sinh sn hu tnh. V d, t bo a kt
hp vi t bo a hay vi to dng lng bi (2n) tng ng aa hay .
D tn (Heterothallic) l kiu khi c s lai nhau gia 2 loi t bo khc
nhau nh a vi to dng d hp t lng bi a nh nm men. Cc nm
d tn c th chia thnh: lng cc (bipolar) v t cc (tetrapolar).
i din in hnh ca nm d tn lng cc (bipolar heterothallic) l
nm men S. cerevisiae (hnh 20.56). nm men ny, s hp bo (cytogamy)
v hp nhn (karyogamy) ch xy ra gia cc t bo (hay nang bo t
ascospore) c kiu bt cp (mating typ) khc nhau nh a v , v cc alen
khc nhau ca locus MAT. Do c s tham gia ca 2 alen, nn gi l lng
cc. Nm Neurospora crassa cng thuc kiu khng phi hp ny (hnh

20.59). Nm rm (Volvariella volvacea) v nm m (Thachicus bisporus)

cng thuc loi ny.
Kiu d tn t cc (tetrapolar heteropolic) c trng cho nhiu loi
nm m Basidiomycetes, m i din l Schizophyllum communae. Nm bo
ng (Pleurotus) v nm hng (Lentinula edodes) thuc kiu khng phi hp
ny. S xc nh di truyn khng phi hp cc loi nm ny do 2 gen A v
B. Mi gen c 2 alen hoc nhiu hn; thng l A1, A2 v B1, B2. S kt hp
gia cc dng n bi ch to dng hu th c kiu gen A1A2B1B2 tc bn
nhn t khc nhau (nn gi l t cc) nh trn bng 20.7.
Bng 20.7. S kt hp gia cc dng n bi c cc kiu khng phi hp khc
nhau nm d tn t cc

A1B1 A1B2 A2B1 A2B2

A1B1

A1B2

A2B1

A2B2 +

Du + ch cc t hp lai c vi nhau.

Podospora anserina, trong cc gii hn ca mi chng a l c 2

kiu bt cp + v . Qu trnh sinh sn hu tnh bnh thng ch c c
khi lai dng + v dng ging nhau theo alen ca gen t. Trng hp ny
c th gi l ng tn khc alen.
S a dng ca cc chu trnh sng v cc kiu khng phi hp
nm c nh hng n cc phng php phn tch di truyn (gentic
analysis). mt s nm sinh sn hu tnh thc hin trn c s d hp bo
(heterogamy) nh Neurospora crassa. nhng loi khc trn c s ng
hp bo (isogamy). Song song vi sinh sn hu tnh cn c chu trnh cn
hu tnh hon ton hay khng hon ton ph thuc vo loi nm. Chu
trnh cn hu tnh (parazxual cycle) l qu trnh kt hp v ti t hp gen
din ra trong nguyn phn ch khng phi gim phn, khng c s th tinh
nh sinh sn hu tnh.
20.8.3.3- Phn tch b bn nang khun (Nm ti)

Lp Nang khun hay Nm ti (Ascomycetes) c c im l khi cc t

bo lng bi chia gim nhim s to ra cc bo t nm trong mt nang.
Nm men bnh m, men ru S. cerevisiae v nm si N. crassa l nhng
nang khun c s dng rng ri trong nghin cu di truyn hc.
im c bit ca nm si N. crassa l cc nang bo t xp thng
hng trong nang nh lc cc nhim sc th xp trn mt phng xch o ca
k gia.

Chng xp theo mt trnh t nht nh phn nh c tin trnh

gim phn I v II (hnh 20.61).

nm men, bn nang bo t i din cho 4 nhim sc th ca gim
phn (xt mt cp nhim sc th tng ng) xp theo mt th t no , kh
bit trnh t. S dng my vi thao tc (micromanipulator), ngi ta c th
tch ri tng nang bo t mt ca mt nang nghin cu. Phng php
nghin cu trc tip cc sn phm ca mi ln gim phn c gi l phn
tch b bn (tetrade analysis).
Khi c s phn li ca mt cp alen th trong mi nang ca Neurospora
s c s phn b nang bo t 4: 4 nu s phn li xy ra gim phn I hoc s
phn b 2: 2: 2: 2 nu s phn li xy ra gim phn II.
a) S phn li gim phn I
Hnh 20.61 m t qu trnh lai mt dng N. crassa hoang di (c+) vi
mt dng t bin (c) c khun lc cm (colonial). Nu tch cc nang bo t
theo th t trong nang v chng mc ln thnh dng th s c th t
4c:4c+. S phn b 4:4 cho thy khng c trao i cho xy ra gia gen v
tm ng (centromere). Gen cng cch xa tm ng xc sut xy ra trao i
cho gia gen v tm ng cng ln. Do nu s phn trm nang 4c:4c+
cng ln, tc trao i cho t xy ra th gen c cng gn tm ng.
b) S phn li gim phn II
Trng hp b (hnh 20.61), khi c trao i cho xy ra gia gen v tm
ng. Sau ln chia th nht hai nhim sc th ch v em vn cn ging nhau.
n ln chia th hai mi c s khc nhau th hin s phn li v t l 2c:
2c+: 2c: 2c+ phn nh iu . Kt qu ny chng minh rng trao i cho
xy ra gia 4 nhim sc th.

Hnh 20.61- Phng thc to bo t nm si Neurospora crassa.

c) Lp bn di truyn bng phn tch b bn

i vi trng hp b bn sp theo th t nh Neurospora, s phn
trm cc nang ti t hp phn nh khong cch gia gen v tm ng. Cn
nh rng trao i cho ch lin quan n hai nhim sc th, nn ch c hai sn
phm ca gim phn cha nhim sc th ti t hp. Do tnh phn trm
cc nhim sc th ti t hp, ly phn trm nang ti t hp chia i.
Trng hp b bn khng theo th t nh nm men th vic tnh tn
s ti t hp c phc tp.

Hnh 20.62 Cc kiu b bn khc nhau:

Kiu PD parental dityp (khng trao i cho) Kiu RD recombination dityp hay
NPD nonparental dityp (c trao i cho) Kiu TT tetratyp (c trao i cho).

V d: Phn tch b bn t t hp lai hai gen AB x ab. S th tinh cho

nhn lng bi (AB/ab) v n chia gim nhim ngay. Nu khng c trao i
cho xy ra hoc trao i cho i xy ra trn cng hai nhim sc th th s
c cc b bn kiu cha m 2AB: 2ab c gi kiu i cha m (parental
dityp) (PD ) (hnh 20.62).
Nu trao i cho xy ra gia nhim sc th 2 vi 3 v 1 vi 4, s c
2Ab:2aB. Kiu ny c gi kiu i ti t hp (Recombinational Dityp RD) hay kiu i khng cha m (Nonparental Dityp - NPD). Kiu ny t nht
trong cc b bn c ti t hp.
Cc trng hp to ra mi nang bn loi bo t c kiu gen khc nhau
1AB: 1Ab: 1aB: 1ab, c gi kiu bn (tetratyp - TT).
d) Hin tng chuyn bin (conversion)

Chuyn bin (conversion) l hin tng lch khi s phn li bnh

thng 2: 2 ca cc b bn, khi c s di truyn ca 1 gen, c Lindegreen
pht hin nm 1949.
V d: D hp t ca nm men a+/+b to cc b bn bt thng nh: (a +) (a
+) (a b) (+ b) v (a +) (+ +) (+ b) (+ b) hoc (a +) (a b) (+ b) (+ b) v (a +)
(a +) (+ +) (+ b). S phn li bt thng c th lin quan n c 2 gen nh: (a
+) (a +) (a +) (+ b) hay (a +) (+ b) (+ b) (+ b).
Cc nghin cu tip theo cho thy chuyn bin (conversion) l hin
tng bnh thng cc gen khc nhau ca nm men vi tn s 1%. Hin
tng ny cng c m t cc loi nm khc nh Neurospora crassa,
Sordaria fimicola v n c ngha quan trng trong gii thch c ch ti t
hp.
20.8.3.4- Phn tch di truyn trong chu trnh cn hu tnh
Nhiu loi nm c si dinh dng (vegetative hyphae) kt hp vi
nhau, lm cho cc nhn n bi t cc dng cng chung trong t bo cht.
Cc th d nhn (heterocaryon) c to nn c th tn ti lu di nh N.
crassa. S to thnh cc th d nhn c s dng rng ri nghin cu s
tng tc gia cc gen, gia cc alen v gia cc gen ca nhn vi t bo
cht. Trong mt s trng hp, s so snh cc d hp t v d nhn cho thy
s khc nhau trong tng tc gia cc alen, c l do:
T l s lng nhn v tng ng cc alen trong th d nhn c th khc
nhau.
Cc alen ca cc gen mt nhn khng c ngn cch nh gia cc th
d nhn.
Cc nhn th d nhn i khi hp nhau to nn on lng bi. Hn
na, trong qu trnh chia nguyn phn tip theo, nhn lng bi c th chu
tc ng ca 2 qu trnh: n bi ha hoc ti t hp nguyn phn.
a) S n bi ha
S n bi ha (Haploidisation) din ra ngu nhin. N c th c
gy to c hiu qu bi cht nfluorphenylalanin.
Nu nh cc nhn trong nhiu nguyn phn b mt 1 nhim sc th (2n-1), th
nhn lch bi va xut hin tr nn khng n nh v tip tc mt cc nhim

sc th khc ca mt b n bi, cho n khi tr thnh nhn n bi (n) n

nh. Trong qu trnh nhim sc th b mt c lp nhau, cc gen ca cng
mt nhim sc th c s lin kt hon ton. Da vo c im ny c th xc
lp s lin kt gen da vo gen nh du trn mi nhim sc th. V d, nu
c lng bi d hp t (AB/ab N/n) khi n bi ha s c cc kiu gen:
(ABN), ABn, (abN) v (abn).
b) Ti t hp trong nguyn phn
Ti t hp nguyn phn (Mitotic recombination) l hin tng thng
gp nhiu sinh vt , khi xy ra trao i cho gia cc nhim sc th tng
ng trong nguyn phn (hnh 20.63).
Trong trng hp ny, khong cch ca gen nh du xa tm ng
nht, s ng hp t ha thng xy ra hn c (c coi l 100%) v s phn
b cc gen c tnh theo cng thc:
D

Nab
.100%
Nb

D biu th khong cch ca gen n tm ng.

Nb - tng s cc dng phn li, ng hp t theo b.
Nab - s cc dng phn li ng hp c a v b, nu nh b l gen nh du xa
tm ng nht.
Bn lin kt gen c xy dng bng ti t hp gim phn v ti t
hp nguyn phn (trong chu trnh cn hu tnh) c th t gen xp ging nhau
Aspergillus nidulans.

Hnh 20.63. Trao i cho nguyn phn (Mitotic crossing-over) on a-b.

a) Trong t d hp t theo cc gen lin kt , khi alen ln c (cn c xc nh v
tr) nm trn on tng ng c nh du.
b) Trong on tng ng khng nh du, trao i cho on gia gen v tm
ng a n s ng hp t ca cc gen xa im trao i mt na s nhn ca t bo
(th h con ca nhn ti t hp). Nh , c th s dng trao i cho nguyn phn
xc nh v tr gen mt vai ca nhim sc th so vi tm ng.

20.8.4- Di truyn cc To lc n bo
Cc to n bo (Microalgae) cng c dng lm i tng nghin
cu ca Di truyn hc, c bit l di truyn quang hp v di truyn ca bo
quan lc lp. So vi nm, cc vi to v ni chung l tt c thc vt, cc t
bin khuyt dng to lc n bo ch hn ch trong mt phm vi hp nh
mt kh nng tng hp arginin (arg) v mt s vitamin. Nguyn nhn ca
hin tng ny n nay cha r.

20.8.4.1. Vi to Chlamydomonas reinhardii

Loi vi to c s dng sm nht v nghin cu di truyn chi tit

hn c l Chlamydomonas reinhardii (hnh 20.43), n c chu trnh sinh sn
hu tnh. u th ca i tng ny l c th tin hnh lai v phn tch b bn
khng xp theo th t.
Roi

Hnh 20.64- Cu trc v hnh thi Chlamydomonas

a) c im sinh hc
Vi to Chl. reihardii c th nui cy trong mi trng dinh dng dch
th hay mi trng thch c nh cc vi sinh vt khc. Cc t bo mc thnh
khun lc trn mi trng thch. Vi to c th nui ngoi sng (nh sng mt
tri hay n hunh quang thng dng) hoc trong ti (phi b sung axetat
natri hoc glucoz).. Trong iu kin th nghim, c th nui cc t bo Chl.
reinhardii nhn cc t bo ng nht (synchronous culture), khi thay i
chu k 12 gi sng 12 gi ti u n. Theo di tng hp ADN cho thy ADN
ca lc lp tng hp vo gi th 5-6 ngoi sng, cn ADN ca nhn tng hp
khong gi th 16 - 18, sau chia t bo ng lot.
Chu trnh sng ca Chl. reihardii c nu trn hnh 20.65. Bnh
thng cc t bo dinh dng tn ti lu di trng thi n bi n vi 2 kiu
bt cp khc nhau mt+ (mating typ) v mt tng ng vi 2 gii tnh, m v
hnh thi th rt ging nhau. Cc t bo dinh dng nay c th to nang bo
t kn hay ti bo t (sporangia) v sinh sn v tnh to ra cc t bo con n
bi.
Khi c kch thch, nh nui trn mi trng dinh dng thiu nit,
cc t bo dinh dng chuyn thnh cc ng bo t (zoospores) mt+ v mt.
S kt hp v phi hp 2 loi t bo khc kiu bt cp mt+ v mt s to ra
hp t lng bi 2n. Hp t ny gp iu kin thun li s thc hin gim

phn to ra 4 t bo n bi n, m chng sinh sn v tnh to ra s lng ln

t bo dinh dng.
Chl. reinhardii c th nhn c nhiu t bin khc nhau, c bit l cc
t bin sc t khc vi mu khun lc xanh lc ca dng hoang di nh mu
cam, vng, trng kem, xanh lt,thun li cho nghin cu di truyn lc lp
v quang hp.

Hnh 20.65- Chu trnh sng ca vi to Chlamydomonas reinhardii

b) Cc gen ca lc lp Chlamydomonas reinhardii

S di truyn ca lc lp c nghin cu chi tit hn c vi to
Chlamydomonas reinhardii. T bo ca vi to ny c mt lc lp ln vi
ng knh trung bnh 5 micrometer cha 50 n 80 bn sao ca phn t
ADN vng trn di 196 kb.

Bng 20.8- Cc t bin trong nhm lin kt gen ca lc lp

Cc gen
1.ac1ac4

Kiu hnh t bin

1.Mt kh nng quang hp, cn b sung axetat.

2.tm1-tm3 tm9

2.Khng mc c 350C.

3.tm2

3.Mc 35oC khi c streptomycin.

4.til1 til5

4.Hnh thnh khun lc nh tt c cc loi MT.

5.ery1

5.Khng erytromycin nng 50 g/ml.

6.kan1

6.Khng kanamycin 50 g/ml.

7.spc1

7.Khng spectinomycin 50 g/ml.

8.spi1-spi5

8.Khng spiramycin 50 g/ml.

9.ole1-ole3

9.Khng oleodomycin 50 g/ml.

10.car-1
11.el 1
12.ery3-ery1

10.Khng carbamycin 50 g/ml.

11.Khng eleosin 50 g/ml.
12.Khng erythromycin, carbamycin, oleandomycin
spyramycin nng ty cc cht nu trn.

13.sm2-sm5

13.Khng streptomycin nng 500 g/ml.

14.sm3

14.Khng streptomycin nng 50 g/ml.

15.sm4

15.Ph thuc streptomycin.

(phi c streptomycin mi mc c).

Mt s t bin khng streptomycine c thu nhn v nhn thy

mt s c s di truyn trong nhn, s khc c s di truyn ngoi nhn.
Theo Sager (1975), Chlamydomonas reinhardii c cc t bin trong
nhm lin kt gen ca lc lp nh sau (bng 20.8):
Cc t bin c biu hin kiu hnh nh sau:

 Mt kh nng quang hp; mc c ngoi nh sng v trong ti cn b

sung ng kh l axetat.
Nhy cm vi nhit cao hoc thp.
Tnh khng vi thuc khng sinh hoc c nhu cu c cung cp thuc
khng sinh.
Tt c cc t bin trn c s di truyn theo mt cha m (uniparental
inheritance), c kiu bt cp mt+ (c th coi l dng m). iu ny lin quan
n s hnh thnh lc lp trong hp t, bng cch no ch nhn ADN t
lc lp mt+.
Nm 1954, R.Sager nghin cu cc t bin khng streptomycin
Chlamydomonas reinhardii t dng hoang di nhy cm sm-s. Mt s t
bin smr c s di truyn nhim sc th vi s phn li 1:1. Tuy nhin, mt s
t bin c s di truyn khc thng nh sau:
smr mt+

sms mt tt c th h con u smr vi t l 1mt+: 1mt

sms mt+

smr mt tt c th h con u sms vi t l 1mt+: 1mt

Nh vy, y khi c s hon i cha m trong lai, th h con u c

kiu hnh streptomycin ca mt+. S truyn th tnh trng ny c gi l s
di truyn theo mt cha m (uniparental inheritance). Sager coi mt+ nh dng
m v trng hp trn ging nh di truyn theo dng m. Cc gen kiu bt
cp mt c t l phn li ca gen trong nhn l 1: 1.
C. reinhardii, streptomycin c tc ng gy t bin. Cc t bin
nhn c do tc ng bi streptomycin u c s di truyn theo mt cha m
(bng 20.9).
c bit t bin smt4 cn gi l smd (streptomycin dependent) ch
mc khi trong mi trng nui c streptomycin.
Sager tin hnh th nghim chng minh s di truyn ca cc gen nu
trn lin quan n cpADN ca lc lp. Cc k thut dng ng v phng x
nh du N15 v li tm trn thang nng CsCl c s dng. CpADN ca
hai cha m em lai c nh du khc nhau: mt dng mang N14 nh, dng
kia mang N15 nng. T trng ca cpADN t cc t bo cha m nh vy tng
ng vi 1,69 v 1,70. Nh li tm trn thang nng CsCl c nhy cao nn
c th ghi nhn s khc nhau gia cc dng cha m v dng lai (bng 20.9).

Bng 20.9- T trng ca cpADN th h con t cc t hp lai Chlamydomonas

T hp lai T trng ca cpADN ca hp t (g/cm3 )

N14 mt+ x
N14 mt

1,69

N15 mt + x
N15mt

1,70

N15 mt + x
N14mt

1,70

N14 mt + x
N15mt

1,69

Trn thc t, cc kt qu ny cho thy cpADN ca cha m mt b mt

hoc b phn hy nh th no . S mt km theo mt cc t bin (v
d: sm) trn cha m mt.
Cc th nghim khc theo nguyn tc tng t c tin hnh vi vic
s dng cc on RFLP. Mi dng cha m c cpADN vi RFLP khc nhau.
Kt qu cng cho thy cpADN th h con nhn c t dng mt+.
Nh vy, c th kt lun rng cpADN ca Chlamydomonas c
truyn th t mt dng cha m (uniparental) v cc gen ca n cng c s
truyn th tng t.
c) Lp bn cc gen ca Chlamydomonas
Trong t hp lai mt+smr mtsms c khong 0,1% th h hp t
con mang c smr v sms. Cc hp t nh vy l hp t hai cha m
(biparental zygote) v c gi l cytohet (cytoplasmically heterozygote).
Tn s cc cytohet c th tng ln 40100% th h hp t con nu mt+
c chiu tia t ngoi trc khi lai.
Chlamydomonas, cc cytohet hay hp t hai cha m c dng lm
im xut pht cho tt c cc nghin cu v s phn li v ti t hp ca cc
gen lc lp. Trn c s nhiu t hp lai, R.Sager nu ra bn vng trn
ca cpADN vi cc gen tng ng.
20.8.4.2- Cc vi to sinh sn v tnh

Cc th nghim di truyn c tin hnh cc vi to sinh sn v tnh

nh: Clolla, Euglena, Scenedesmus. Do khng tin hnh lai c nn phn
tch di truyn ch gii hn thu nhn cc t bin. Cc t bin sc t
Clolla gp phn hiu su v c ch di truyn quang hp.
Cc nguyn sinh ng vt (Protozoa) l mt nhm ln gm hn 65.000
loi, trong c khong 10.000 loi k sinh. Di truyn hc ca nhm ny hu
nh cha c nghin cu.