giao trinh di truyen hoc vsv va ung dung _ hoang trong phan, truong thi bich phuong

8/3/2019 Giao Trinh Di Truyen Hoc VSV Va Ung Dung _ Hoang Trong Phan, Truong Thi Bich Phuong

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Tai lieu chia se tai: wWw.SinhHoc.edu.vn


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HONG TRNG PHN (Ch bin)TRNG TH BCH PHNG

Gio trnhDI TRUYN hc VI SINHVT v NG DNG

NH XUT BN I HC HU

Hu - 2008



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Li ni un nay, di truyn hc ra i chmi hn mt trm nm song n

pht trin vi mt tc ht sc nhanh chng. c bit l, trong vng 50nm li y ktngy James Watson v Francis Crick khm ph ra cutrc phn tDNA, 25/4/1953. Shon thnh vic Gii m di truyn bihai nhm nghin cu ca Marshall Nirenberg v Gobind Khorana vo

thng 6 nm 1966, v sra i ca K thut di truyn vo gia thp nin1970 l hai skin ni bt nht ktsau khi Sinh hc phn t ra i. Spht trin cng vi nhng thnh tu tc ca di truyn hc trong thigian qua qu l v cng to ln!

gp phn i mi ni dung gio trnh Di truyn hc Vi sinh vt vng dng theo hng cp nht kin thc cng nhphng php dy vhc b mn bci hc, chng ti tham cu nhiu ti liu khc nhauv n lc bin son gio trnh trn tinh thn y. Chng ti hy vng rnggio trnh ny sp ngc phn no nhu cu ging dy v hc tp

ca ging vin v sinh vin, v cng c thsdng nhmt ti liu thamkho b ch cho gio vin Sinh hc cc trng THPT trong bi cnh imi gio dc hin nay.

Ni dung gio trnh gm Bi m u v 8 chng: Chng 1 giithiu cc c im ca di truyn hc vi sinh vt. Chng 2 - Csphnt ca tnh di truyn - trnh by khi qut vcu trc v tchc ca ccb gene vi sinh vt v cc cch truyn thng tin di truyn ch yu l sinh vt tin nhn (prokaryote). Chng 3 i su phn tch cc kha cnhca cc nguyn l iu ho biu hin gene vi khun. Chng 4 - Bin d

vi sinh vt - cp n cc qu trnh bin i ca vt cht di truyn cc vi sinh vt (t bin gene, sa cha DNA v cc yu tdi truyn vnng). Chng 5 t p trung vo lnh vc di truyn hc ca cc virus.Chng 6 trnh by cc nguyn l ca di truyn hc vi khun - tip hp,bin np v ti np. Chng 7 gii thiu nhng hiu bit mi c tnh chti cng vdi truyn vi nm v vi to. V chng 8 tp trung trnh bycc khi nim, phng php v thnh tu ca lnh vc cng ngh DNA tithp - to dng gene vi sinh vt, cng nhcc ng dng ca nguyn lkthut di truyn lin quan vi sinh vt trong vic to ra cc sinh vt bini gene (genetically modified organisms = GMOs) v phng thch chng



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vo mi trng.Cui mi chngu c cc phn Cu hi v Bi tp v Ti liu tham

khobn c tin n tp v tra cu. V, trong chng mc c th, ccthut ng khoa hc thng dngc sdng bng ting Anh hoc chthch trong ngoc n gip ngi hc ddng hn trong vic tip cnthng tin qua sch bo nc ngoi hoc internet.

Gio trnh Di truyn Vi sinh vt v ng dng do ThS. Hong TrngPhn v TS. Trng Th Bch Phng - cc ging vin ang cng tc tiKhoa Sinh hc cc trngi hc Sphm v i hc Khoa hc, ihc Hu- bin son, vi sphn cng nhsau:

ThS. Hong Trng Phn ch bin vi Bi mu v cc chng 1, 2,

3, 6, v 8; TS. Trng Th Bch Phng bin son cc chng 4, 5 v 7.Chng ti xin trn trng cm n Dn Gio dc i hc Hu titr cho vic bin son gio trnh trong khun kh ca Dn Gio dci hc mc B.

Chng ti xin by t lng cm n c bitn PGS. TS. Phm ThnhH- Trngi hc Khoa hc Tnhin, i hc Quc Gia Tp. H ChMinh dy cngc bn tho v cho nhiu kin qu bu.

Do kh nng cn hn ch , chc chn gio trnh cn nhiu thiu st.Chng ti rt mong nhn c s ph bnh v ch bo ca cc ng

nghip v bn c gio trnh c hon chnh hn trong ln in sau.Xin trn trng cm n!

Hu, ngy 10 thng 5 nm 2006Cc tc gi,

HONG TRNG PHN

TRNG THBCH PHNG



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Bi mu

Di truyn hc Vi sinh vt v Cch mngCng ngh Sinh hc

I. Sra i v pht trin ca di truyn hc v cng ngh DNAti t hp

S ra i v pht trin ca di truyn hc gn lin vi tn tui caGregor Mendel nm 1865 v tri qua cc giai on sau y.

1. Sra i v pht trin ca di truyn Mendel

Tu H Lan (Pisum sativum), vi tng vphng php nghin cu c o, nm 1865Gregor Mendel (Hnh 1) pht hin ra cc quylut di truyn csu tin v qua suy ra s tnti tt yu ca cc n vi truyn c th - nhn tdi truyn (genetic factor) - quy nh cc tnh trngc truyn t th h ny sang th h khc m sauny gi l gene. Tuy nhin, gii khoa hc ngthi khng hiu v do khng thnh gi tm

vc vi ca pht minh ny.

Hnh 1G. Mendel

Mi n nm 1900, ba nh thc vt hc l Carl Correns (Germany),Hugo de Vries (Netherlands) v Erich von Tschermak (Austria) c lpnhau khm ph li cc quy lut di truyn ca Mendel. V di truyn hcchnh thc ra i ty m ngi sng l p l Mendel.

2. Sra i v pht trin ca thuyt di truyn nhim sc th

T 1910, Thomas Hunt Morgan (Hnh 2) cngvi ba cng s l Alfred H.Sturtevant, CalvinBridges v Herman J. Muller xy dng thnhcng thuyt di truyn nhim sc th (chromosometheory of inheritance) da trn i tng nghincu l rui gim Drosophila melanogaster. Hcthuyt ny xc nhn rng gene l n v cscatnh di truyn nm trn nhim sc th ( trongnhn); trn cc gene sp xp theo ng thng

to thnh nhm lin kt. Nhng ng gp ng k ca cc mn xutsc ca Morgan l: xy dng bn di truyn (Sturtevant 1913), ch racch xc nh cc kiu hnh gii tnh rui gim (Bridges 1916) v pht

Hnh 2 T.H.Mor an



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trin phng php gy t bin bng tia X (Muller 1927). Vi ng gp toln Morgan c trao gii Nobel nm 1933 v Muller nm 1946.

Nm 1931, Barbara McClintock (Hnh 3) vHarriet Creighton thu c bng chng vt l trctip v ti t hp ng. Sau , hin tng nycng c C. Stern quan st Drosophila. Nhvy ti t hp c thc pht hin c v mt vtl ln di truyn ng vt cng nh thc vt.n 1944, McClintock pht hin cc yu t ditruyn vn ng (transposable genetic elements),v b c trao gii Nobel nm 1983 v khmph ny. Hnh 3 B.McClintock

3. Sra i v pht trin ca di truyn hc phn tS ra i ca di truyn hc phn t(molecular genetics) gn lin vi

cc khm ph v DNA (deoxyribonucleic acid) t gia th k XX trn itng nghin cu ch yu l cc vi sinh vt. Tuy nhin, trc FriedrichMiescher (1869) khm ph ra mt hn h p trong nhn t bo gi lnuclein m thnh phn chnh ca n sau ny c bit l DNA.

V mi quan h gia gene v protein, t 1902 Archibald Garrod quanghin cu bnh alcaptonuria ngi gi rng y l mt tnh trngln Mendel, c th lin quan ti s sai hng mt enzyme. Bng cc th

nghim gy t bin cc gene lin quan n cc con ng sinh ha trnnm mc Neurospora, nm 1941 George Beadle v E.L.Tatum (Hnh 4)xc nhn mi gene kim sot s tng hp mt enzyme c th. Chnhgithuyt mt gene-mt enzyme (one gene-one enzyme hypothesis) ni tingny mng cho s ra i ca di truyn ha-sinh, v hai ng ctrao gii Nobel cng vi Joshua Lederberg nm 1958. V sau, gi thuytny c chnh xc ha l mt gene xc nh ch mt chui polypeptid -cu trc scp ca cc protein, trong c cc enzyme.

Hnh 4 Beadle, Tatum, Jacob v Monod (t tri sang)

Vy bn cht ca gene l g? Nm 1944, Oswald Avery (Hnh 5) v



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cc cng s l MacLeod v McCarty bng th nghim bin np in vitrochng minh rng DNA l vt cht mang thng tin di truyn. Nm 1949,Erwin Chargaff cng b cc kt quu tin v thnh phn ha hc caDNA mt s loi.

Hnh 5 O.T. Avery, MacLeod v McCarty (t tri sang)

Vic nghin cu cu trc phn t DNA c bt u t 1951 vi ccdn liu nhiu x tia X ca Rosalind Franklin v Maurice Wilkins (Hnh6). Cc s liu ha hc v vt l ny l csm t James Watson vFrancis Crick (Hnh 7) xy dng thnh cng m hnh cu trc phn tDNA nm 1953, cn gi l chui xon kp (double helix). Pht minh vi ny mra k nguyn mi cho s pht trin ca di truyn hc v sinhhc ni chung. Vi pht minh , Watson v Crick cng vi Wilkins c

trao gii Nobel nm 1962 . K t sau l s ra i ca hng lot cccng trnh nghin cu trong lnh vc sinh hc phn t, ng k l vic giim di truyn c hon tt vo thng 6 nm 1966 bi hai nhm nghincu ca M. Nirenberg v H. Khorana (gii Nobel nm 1968).

Hnh 6 R.Franklin (tri), M.Wilkins. Hnh 7 J.D.Watson (tri) v F.H.C.Crick

4. Sra i v pht trin ca cng ngh DNA ti thpC th ni, nn tng ca cng ngh DNA ti t hp (recombinant

DNA technology) c thnh lp t 1972 khi Paul Berg (Hnh 8) to ra

phn t DNA ti t hp u tin trong ng nghim (recombinant DNA in



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vitro). Mt nm sau Herbert Boyer v Stanley Cohen (Hnh 8) ln u tins dng plasmid to dng DNA. Lnh vc ng dng mi ny ca sinhhc phn t to ra mt cuc cch mng mi trong sinh hc. ng gpng k trong lnh vc ny l khm ph v cc enzyme gii hn(restriction enzyme) t 1961-1969 ca Werner Arber, Daniel Nathans vHamilton Smith (gii Nobel 1978; Hnh 8); xut cc phng php xcnh trnh t base trong cc nucleic acid nm 1977 bi P.Berg, W.Gilbertv Frederick Sanger (gii Nobel ha hc 1980; Hnh 8); s khm ph racc gene phn on (split gene) nm 1977 bi Phillip Sharp v RichardRobert (gii Nobel 1993; Hnh 8); s pht minh ra phng php PCR( polymerase chain reaction) ca Kary B.Mullis nm 1985 (Hnh 8) vphng php gy t bin nh hng (site-directed mutagenesis) ca

Michael Smith t 1978-1982 (gii Nobel ha hc 1993)...

Hnh 8ACc nh khoa hc ot gii Nobel y hc lin quan k thut

gene. T tri sang: D.Nathans, H.Smith, W.Arber, P.Sharp v R.Robert.

Hnh 8B Cc nh khoa hc ot gii Nobel ha hc lin quan k thut

gene. T tri sang: H.Boyer, S.Cohen, P.Berg, W.Gilbert, F.Sanger vK.Mullis.

Cng vi nhng thnh tu ng dng ly k trong sn xut v i sngx hi, nh vic sn xut cc ch phm y-sinh hc bng cng ngh DNAti t h p, s dng liu php gene (gene therapy) trong iu tr bnh ditruyn, to cc ging sinh vt mi bng con ng bin i gene(genetically modified organisms = GMOs), dn b gene ngi (HumanGenome Project = HGP)... gy ra khng t hoi nghi, tranh ci xung quanhcc vn vo l sinh hc (bioethics) v an ton sinh hc (biosafety).



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II. Di truyn hc vi sinh vt vi cch mng cng ngh sinh hc

Cho n u thp nin 1940 cc vi sinh vt, bao gm cc vi khun v

virus ca chng v cc vi sinh vt nhn chun n bo nh nm men, nmmc... thc s trthnh cc i tng nghin cu chnh yu ca di truynhc. Ty hnh thnh cc lnh vc di truyn hc sinh-ho v di truynhc vi sinh vt, hai nn tng chnh cho s ra i ca di truyn hc phn t(1953) v cng ngh ADN ti t hp sau ny (1978).

y vi khun E. colic xem l mt sinh vt m hnh nht quntuyt vi ca di truyn hc hin i. N c s dng mt cch rng ritrong cc th nghim chng minh cc phng thc ti bn bn ca DNA(Meselson v Stahl 1958; John Cairns 1961; Okazaki 1969), phn tch ti

t hp v lp bn di truyn, nghin cu cu trc tinh vi v chc nngsinh ho ca gene (Benzer 1961; Yanofsky 1961); c chiu ho sinhtng hp protein (Jacob v Monod 1961) v.v. Nm men bia S.s cerevisiaecng sm c s dng lm m hnh cho cc nghin cu di truyn hceukaryote v ng dng rng ri trong cng ngh DNA ti t hp sau ny.

Vi s pht trin v cng nhanh chng ca di truyn hc trong vith p nin qua, c bit l s tin b ca cng ngh sinh hc(biotechnology) ni chung c nhng tc ng mnh m ln nhiungnh khoa hc v trn mi mt ca i sng, kinh t, chnh tr v x hi

phm vi ton cu. Di truyn hc ni chung v di truyn hc vi sinh vtni ring c hnh dung v tr trung tm v giao thoa vi sinh hc, hasinh hc, k ngh, y-dc, nng nghip, sinh thi hc, kinh t hc, lut, xhi hc v trit hc (Hnh 9).

Hnh 9: Tc ng ca di truyn hc (vi sinh vt) ln cc lnh vc khc nhau.



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Gio s danh d mn ha hc i hc Havard, F.H.Westheimer,bnh lun v sinh hc phn t nh sau:"Cuc cch mng tr tu vi nhtca 40 nm qua xy ra trong sinh hc. Liu c th tm ra mt ngino c hc ngy nay m khng hiu bit cht g v sinh hc phn t?"(Weaver v Hedrick 1997, tr.15).

Cc thnh tu t c nhng dng di truyn hc trong nng nghipl v cng to ln, gp phn to nn cuc cch mng mi vi s ra i cahng lot cc ging vt nui-cy trng c u th vt tri, cc sinh vtbin i gene (GMO) mang nhng c tnh hon ton mi l.

Trong y hc, l s ra i ca hng lot cc dc phm c snxut bng k thut di truyn dng cho iu tr bnh v ci bin tr thngminh ca con ngi; l cc phng php chn on v iu tr bnh mc phn t v.v. Nhng vn ny sc cp chng 8.

C th ni, s thnh cng ca d n b gene ngi (HGP) vo thng 4nm 2003 cho php chng ta ln u tin c c ton b trnh tkhong 3,2 t c p nucleotide trong b gene con ngi ( Homo sapiens).HGP l mt trong nhng k cng thm him vi nht trong lch s nhnloi (NHGRI 2005). Theo c tnh mi nht c cng b ngy21/10/2004 trn tp chNature, b gene chng ta cha s lng gene mha protein thp mt cch ng kinh ngc, khong 20.000 n 25.000 chkhng phi l 50.000 n 140.000 gene nh don ban u hoc 35.000theo don trong vi ba nm li y (NHGRI 2005).

Tuy nhin, nhng thch thc cho tng lai ca nghin cu khoa hcv cc b gene (genomics) i vi sinh hc, vn sc khe v x hicng c t ra (Collins v cs 2003) . S hon tt ca HGP t n khngc ngha l xong m ng hn l im khi u cho cng cuc nghincu thm ch cn hng th hn. Cc nh nghin cu hin giang cgng lm sng t mt s qu trnh phc tp nht ca sinh hc, l: mta b pht trin t mt t bo n l bng cch no, cc gene phi hpchc nng ca cc m v cquan nh th no, s tin nh bnh tt xy ra

nh th no v b no ngi lm vic ra sao (NHGRI 2005).

III. i cng v Genomics v mi lin quan gia n vi cclnh vc nghin cu khc

S tin b nhanh chng gn y ca sinh hc phn t v cng nghsinh hc (biotechnology), nh ni trn, l nh s pht trin mnh mca cc phng php v k thut mi trong sinh hc phn t nh: (i) Knhhin vi in t; (ii) Tch chit v phn tch nh tnh v nh lng thnucleic acid; (iii) Xc nh trnh t nucleic acid ca gene (bng phng



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php ho hc ca Maxam v Gilbert v bng phng php didesoxy caSanger); (iv) Lai phn t nucleic acid; (v) nh du ng v phng x vs dng cc mu d; (vi) PCR; (vii) To dng DNA ti t hp; (viii) Gyt bin nh hng; v.v.

Tuy nhin, chnh s kt hp tin hc v my tnh trong nghin cu sinhhc phn t dn ti s ra i ca hng lot cc lnh vc nghin cumi, l: Tin- sinh hc (bioinformatics) cho php thu th p, t chc vphn tch s lng ln cc s liu sinh hc nhs dng mng my tnh vcc ngun d liu (databases); Khoa hc v b gene hay B gen hc(Genomics) - phn tch ton b genome ca mt sinh vt c chn;DNAmicrochip technology - xc nh cc t bin trong cc gene; DNAmicroarray technology - nghin cu cch thc mt s lng ln cc gene

tng tc ln nhau v c ch mng li iu ha ca t bo kim sotng thi s lng cc k ln cc gene; v.v.

Di y l mt s khi nim cbn v Genomics v cc lnh vclin quan n k nguyn sau b gene (Post-genomic Era). y chnh lcnh ca mi v -OME v -OMICS hin c ph bin trn cc trang web(-OME and -OMICS Gateway):

http://www.nature.com/omics/index.html

http://www.genomicglossaries.com/content/gloss_cat.asp

Bn cnh s pht trin ca lnh vc genomics l s ra i ca khoahc v b protein (Proteomics) v nhiu lnh vc -omics khc, nh:Transcriptomics; Cellomics; Metabolomics; Ionomics v.v. Di ychng ta ch tm hiu v genomics v mt s vn lin quan lm sangt tc pht trin chng mt ca cc ngnh khoa hc mi m ny.

1. Genomics

Vic gii thnh cng trnh t DNA ca b gene (genome) ngi vca hng lot cc sinh vt m hnh c tin hnh trong sut thp nin1990 v tip din cho n nay. [Cc kt qu ny c cng b rng ritrn nhiu trang web ni ting, v d: http://www.genome.gov/ ]. Chnhiu ny dn n s ra i ca mt lnh vc kh mi m gi l khoa hcvb gene (genomics).

Cc tri thc bt ngun tkhoa hc vb gene (genomics) cho phpchng ta khng nhng hiu su v chi tit v cc cch phn t ca ssng m cn to nn cuc cch mng tht s trong nng nghip, y-dchc v nhiu lnh vc k thut v cng ngh khc. N cng cung cp chochng ta nhiu cch tip cn mi nhm pht hin, bo tn v s dng tnha dng sinh hc. Bn cnh n cn thc y pht trin cc th h my



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tnh v phn mm mi da trn s m phng cch thc truyn tn hiuchnh xc v tinh vi ca cc t bo.

C th ni, s hiu bit chi tit v cu trc v chc nng ca b genengi v b gene cc sinh vt khc l nh cao ca cng ngh gene.

Genomics pht sinh ra mt khoa hc mi nghin cu ton b bgene bng cch xm nhp vo cc mn di truyn hc truyn thng nh ldi truyn hc qun th, s lng v phn t vi nhng cng ngh mitrong sinh hc phn t, phn tch DNA, tin sinh hc v cc h thng robottng ho (Hnh 1.10).

Hnh 1.10 Genomics mt mn hc rng ln xm nhp vao cc khu vc truynthng ca di truyn hc (phng theo cc Hnh 1.1 v 1.2 trong Liu 1998).

Ngun: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm



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Mt s lng ln cc phn mn ca genomics c th t hp li cung cp mt cch tip cn mnh m cho nghin cu s bin i di truynthch hp nh: Genomics cu trc (Structural genomics); Genomics chcnng(Functional genomics)- Genomics so snh (Comparative genomics);Genomics kt hp (Associative genomics); Genomics thng k (Statisticalgenomics) v.v.

1.1. Genomics cu trc (Structural genomics)

Genomics cu trc c gng hng ti xc nh ton b cc gene trongmt b gene, i khi gi l khm ph gene, v xc nh v tr ca chngtrn cc nhim sc th. Mc tiu ny t c bng cch phn tch trnh tcc gene ring l, cc on gene hoc ton b b gene.. Cc gene ring l

c xc nh t trnh t DNA thng qua cc chng trnh x l bngmy tnh (sophisticated computer algorithms). Cc chc nng sinh ho camt gene c suy din thng qua s so snh trnh t DNA vitnh tca cc gene c chc nng bit trong ngn hng d liu. Mt trong ccp dng ni bt nht ca genomics cu trc l nghin cu s bin i ditruyn thch nghi l phn tch cc locus tnh trng s lng(quantitativetrait loci = QTL) thng qua lp bn b gene (genome mapping). Tuynhin, mc ch ca cch ti p ny l nhm gii thch cu trc b gene(enomic structure) v s tng tc gene (gene interaction) mc b

gene hn l chc nng ca n, khng ging nh genomics chc nng.1.2. Genomics chc nng (Functional genomics)

Genomics chc nng i su tm hiu chc nng ca cc gene v cchthc chng xc nh cc kiu hnh. Mt trong nhng li th chnh cagenomics chc nng l s dng cc vi mng DNA (DNA microarray; cnggi l cc chp DNA = DNA chips) o s biu hin c th ca hngngn gene mt cch ng thi. DNA microarray cha hng ngn muDNA hoc cc trnh t oligonucleotide c in hoc tng hp trn mnglc nylon (nylon membrane filter) hoc slide knh hin vi trong mt kiu

chnh xc c bit v i din cho hng ngn gene trong b gene. Michm DNA i din cho mt gene duy nht m c dng o lngnh lng s biu hin ca mRNA (messenger RNA) bng ch em laivi mNA c nh du hunh quang (fluorescent labelled mRNA) (Hnh1.11).



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Hnh 1.11 S dng cc DNA microarray trong phn tch s biu hin bit hoca cc gene (T Albelda v Sheppard 2000). Th nghim lai so snh lin quanti vic cch ly mRNA t hai mu ring bit (A). mRNA t mi mu c x lvi reverse transcriptase (B) v c nh du vi mt ch hunh quang ring(C). Hai cng c RNA anh du c trn ln, lai vi nhau cho DNAmicroarray c cha mt by gm hng ngn hoc hng chc ngn trnh t

DNA da trn b gene hoc hoc cc trnh t DNA b sung (cDNA), v ra sch(D). Microarray array ny c qut nh s dng mt my ghi hnh hunhquang chuyn dng (specialised fluorimage), v mu sc ca mi chm scxc nh (E). Trong v d ny, cc gene chc biu hin Mu A s c mu, cc gene chbiu hin Mu B s c mu xanh v cc gene y c biuhin ngang bng nhau trong c hai mu e cho mu vng. iu ny cho phpnh nghin cu xc nh cc gene c biu hin mt cch c bittong vicp ng vi vic x l hoc bnh,hoc cc gene c th cho m c biu hin mt m, ch khng phi cc m khc.



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Hnh 1.12

2.1.3. Genomics so snh (Comparative genomics)

Genomics so snh s dng thng tin t cc loi khc nhau v trgip

cho vic hiu bit t chc v s biu hin ca gene cng nh s sai khcv mt tin ho. N c li th v s bo tn cao ca gene v cu trcv chc nng (ngha l c sai khc nh ngang qua cc n v phn loi adng) v p dng nguyn l ny theo cch thc gia cc loi(interspecific) trong s tm kim cc gene chc nng v s t chc bgene ca chng. Genomic so snh cn tng cng nghin cu bng cchkim tra sa dng ca ccsinh vt m hnh (model organisms) m trong cc tnh trng sinh l, pht trin hoc sinh ho c sn sng nghin cu.

c bit l cc nghin cu genomics cc thc vt c hoa nh nhcy ci Brassica,Arabidopsis thaliana, vn c s dng rng ri nh lcc loi m hnh, m s liu trnh t ca b gene c gii xong ri.Mt trnh t b gene y ca cy dng (populus) chng bao lu nacng s c sn cho phn tch genomics so snh.

2. Xc nh trnh tDNA ca ton b cc b gene

Vic gii hon tt trnh t cc b gene ca nhiu loi quan trng v mhnh l mt thnh tu ng k ca genomics, vn cung cp cscho phntch so snh v cu trc v chc nng. Cc cu tr li cho cc cu hi



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chng hn nh: (1) s lng, snh khu v phn b ca cc gene tronggenome; (2) t chc ca gene v chc nng ca chng; (3) cc gene no lging nhau hoc c bo tn cao bng qua cc loi khc nhau; v (4) ccgene no chu trch nhim cho cc loi thch nghi v tin ho m c ththu nhn c by gi. Cc trnh t b gene y c xc nh chonm men bia Saccharomyces cerevisiae (5/1997), giun trnCaenorhabditis elegans (12/1998), rui gim Drosophila melanogaster(3/2000), thc vt c hoa hng nm Arabidopsis thaliana (12/2000), conngi (2/2001), v cn nhiu loi khc sos liu gii trnh t cng sp honthnh nh chut, la, ng, v.v.. Hin gic th xc nh bng th nghimtrnh t b gene y ca cc cy rng nh l cy dngPopulus (500triu cp base) hayEucalyptus (340-580 triu cp base).

S lng cc gene trong mt b gene l c gii hn v khng qu caonh c don trc y (c th, ch ~26,000 cc thc vt vng vt, trong khi ~6,000 nm men bnh m, Bng 1.1). Hn na nhiugene l chung cho cc loi v c bit l khng thay i trong s tin ho qua. Chng hn, ch c 94 trong s1278 h protein trong b gene ngidng nh l c trng cho cc ng vt c xng sng. Chc nng csnht trong s cc chc nng ca t bo - chuyn ho cs, s phin mca DNA thnh RNA, s dch m ca RNA thnh protein, s ti bn DNA... - ch tin ho mt lc v hu nh gi nguyn khng i k t s tin

ho ca nm men n bo v vi khun.Bng 1.1 Kch thc b gene ca nhiu loiem so snh

Phm vi phnloi

Tn Latin Tn chung nS bp(x 10

6)

Genes (x10

3)

Prokaryote

Vi khun c 12 1 VSV c - 1,6-3,0 1,5-2,7

Vi khun 40 1 VSV vk - 0,6-7.0 0,5-6,6

Vi khun Escherichia coli2

khng c - 4,6 4,3

Eukaryote

Nm men bia S. cerevisiae2 men b/m 16 12 6

Giun C.elegans2 giun trn 5/6 97 19

Cn trng D.melanogaster rui gim 4 180 13,6

TV ht kn A. thaliana2 arabidopsis 5 125 25,5

TV ht kn Oryza sativa2 la go 12 400 ?

TV ht kn Zea mays2 ng 10 2400-3200 ?



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TV ht kn L.esculentum khoai ty 12 900 ?

TV ht kn Eucalyptus3 bch n 11 340-580 ?

Cy rng/TV httrn Thng3

thng 12 20.000-30.000 ?

Gm nhm Mus musculus2 chut 20 3.500 21-30

Linh trng Homo sapiens 2 ngi 23 3.400 26-311 S loi c cc trnh t b gene c gii y .2

Cc loi c s liu trnh t b gene y hoc hu nhy .3

S liu da trn nhiu loi.

(Ngun: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm)

Hnh 1.13 So snh s lng v mt gene ba loi E. coli, nm men bia nychi S. cerevisiae v giun trn C. elegans.

Genomics so snh khm ph c tnh bo tn ca gene nh th, gip cho vic hiu bit v suy ra chc nng ca mt gene c th t cc sliu thu c i vi cc gene tng ng ging nhau (similarhomologous genes) c nghin cu cc sinh vt khc. Kh nhiuchc nng ca cc gene cy rng c th hc c t cc s iu thu c cc sinh vt khc, chng hn nhArabidopsis. Trong s cc thch thckhc nhau l tnh phc t p v kch thc ln ca cc b gene cy ci(Bng 1.1). Kch thc ca b gene cy thng (20,000-30,000 triu cp base), chng hn, l ln gp 6 ssen 8 ln so vi b gene ngi (3,400triu cp base), v 150 n 200 ln ln hn b gen ca Arabidopsis (125triu cp base). Ngay c kch thc vt l tng i nh ca b gene cydng Populus (500 triu cp base), vn 40 ln b hn loi thng Pinustaeda ( c nghin cu rt k) s ging nh b gene cy rng u tin c gii ton b trnh t, s bng khong 4 ln b gen Arabidopsis(mc d ging vi la v 6 ln b hn ng, c hai hu nhu cgii trnh ty ).

Cng vi s pht trin nhanh chng v v cng hp dn nh vy ca



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lnh vc genomics, hng lot cc cng ty c phn doanh nghip nh nc(doanh nghip cng) v t nhn ua nhau ra i, c bit l cc quc giai u trong lnh vc ny nh: M , c, Php, Anh, Canada, B , Nht,c, Thuin, v.v.

Hnh 1.14 v 15 di y cho thy s pht trin tng tin v s lngca cc cng ty c phn doanh nghip cng M t nm 1994 n 2000,cng nh t trng u th ca cc hng genomics M so vi cc cngquc khc v cng ngh sinh hc.

Hnh 1.14 S pht trin tng tin v s lng ca cc cng ty c phn doanhnghip cng v genomics M t nm 1994 n nm 2000.

Hnh 1.15 T trng u th ca cc hng genomics cng v t nhn M sovi cc cng quc khc v cng ngh sinh hc.

IV. Cc nguyn tc nghin cu v phng php hc tp b mn

1. Cc nguyn tc nghin cuTrong nghin cu di truyn hc vi sinh vt v sinh hc ni chung c

cc nguyn tc chung cn tun th nh l phng php lun, sau y: (1)



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Ly t bo lm n v nghin cu; (2) Thng tin di truyn cha trong bgene t bo chi phi mi biu hin sng ca n m cc gene l n v ditruyn cs; (3) S hot ng ca cc gene trong qu trnh pht trin cth l c trng cho tng gene tng giai on c th; (4) Cc qu trnhtrong cc h thng sng phi c iu ha v kim sot m bo chos tn ti ca n l lin tc, trong ph bin l s tiu chnh bng cccchphn hi thng tin (feed-back mechanism); (5) S thng nht giacu trc v chc nng biu hin tt c cc mc t chc khc nhau cas sng; (6) Tt c cc t chc v qu trnh sng u tun theo cc quylut vt l v ha hc; (7) S sng trn tri t tri qua qu trnh tin hakhong 3,5 t nm qua, v vy khi so snh, nhng nt tng ng giachng cho thy tnh thng nht v mt ngun gc v nhng nt d bit cho

thy tnh pht trin, s phn ha a dng tt yu ca chng.2. Phng php hc tp b mn

Cng nh bt k mn hc no khc, vic hc tp di truyn hc vi sinhvt i hi phi nm vng lch s mn hc, i tng, nhim v, phng php nghin cu v h thng kin thc cn bn ca n. Bn cnh ccnguyn tc ni trn vn rt cn cho t duy trong hc tp, y xin nu vint chnh lin quan n phng php hc tp c th ca b mn.

(1) Nm vng cc kin thc lin mn (nh t bo hc, ha sinh hc,m trn ht l di truyn hc v vi sinh vt hc) v lin ngnh (nh tonthng k-xc sut, vt l v ha hu c).

(2) Nm vng h thng khi nim cbn cng nh cc thut ng mikhng ngng ny sinh. Chng hn, gene l khi nim cn bn c ni hmkhng ngng c pht trin su rng. c bit, trong thi i ngy nay,vi s mra mt k nguyn mi - K nguyn sau b gene (Post-genomicEra), hng lot thut ng v lnh vc nghin cu mi lin quan vi nhngcnh ca -ome v -omicsng lot ra i v gn lin vi s pht trin caTin-sinh hc (Bioinformatics) nh: genome vi genomics, proteome viproteomics, transcriptome vi transcriptomics,...

(3) Hiu r bn cht ca cc nguyn l di truyn trong tng chcng nh mi lin quan gia chng c th gii thch v vn dng tronggii quyt cc bi ton hoc tnh hung ca i sng v thc tin sn xut.

(4) nm kin thc v pht trin cc k nng t duy mt cch vngchc i hi phi bit vn dng kin thc vo gii bi tp cng nh cc knng thc hnh th nghim.

(5) Di truyn hc vi sinh vt l mt khoa hc thc nghim, nn thngtin thu c l nhcc quan st t th gii vi sinh vt, v phng php



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khoa hc chnh l cng c hiu bit cc quan st . Ni n phngphp nghin cu khoa hc l ni n cc bc tin hnh theo mt trnh tt chc cng vic cht ch sau y: Quan st Gi thuyt Don Thc nghim ( kim tra gi thuyt t ra) xut gi thuyt mi.

(6) Trong khi hc gio trnh, bn nn lm t nht mt tiu lun v mtvn cp nht m mnh yu thch. Cng vic ny i hi s say m tmti cc thng tin mi, c bit l thng qua mng internet, vit bi tnglun khoa hc v trnh by trong mt seminar. iu quan trng l phi tocho mnh mt hoi bo hc tp, mt kh nng v phng php t hc.

(7) Bi di truyn hc vi sinh vt v cc ng dng ca n l mt lnhvc khoa hc non tr nhng pht trin vi tc ht sc nhanh chng, chonn khi lng kin thc mi tch ly c l v cng phong ph v adng. c th cp nht thng tin v mn hc i hi phi tng cngkh nng s dng ting Anh v internet. ng k l cc trang web cgii thiu sau mi chng, hoc c th s dng ngay cc t kha (keywords) c cho tng ch tm kim vi cng c mnh nht hinnay l Google (http://www.google.com/), hoc bng cc cng c khcnh: MSN (http://www.msn.com/), Yahoo (http://www.yahoo.com/) hocWikipedia (http://www.wikipedia.com/).

Ti liu Tham khoPhm Thnh H. 2005.Nhp mn cng ngh sinh hc. NXB Gio Dc.Atlas, RM. 1995.Principles of Microbiology. St. Louis, Missouri: Mosby.

FAO: http://www.fao.org/DOCREP/003/X6884E/x6884e03.htm

Kimball J. 2004: http://users.rcn..com/jkimball.ma.ultranet/BiologyPages/

Madigan, MT and JM Martinko. 2006. Brock Biololy of Microorganisms.11th ed. Pearson Prentice Hall, Inc. Upper Saddle River, New Jersey.

Maloy, S. 2006. Microbial Genetics.

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/genetics/Nature -OMICS Gateway:

http://www.nature.com/omics/index.html

http://www.genomicglossaries.com/content/gloss_cat.asp

TIGR Microbial Genome Database.2005.

http://www.tigr.org/tdb/mdb/mdbcomplete.html

DOE Microbial Genome Program Report. 2005.

http://www.www.ornl.gov/hgmis/publicat/microbial/13doeproj.html



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Chng 1

Cc c im ca Di truyn hcVi sinh vt

I. Slc lch svi sinh vt hcVi sinh vt(microorganisms, microbials) l tn gi chung dng ch

tt c cc sinh vt c hnh th b nh m ch c th nhn thy di knhhin vi quang hc hoc knh hin vi in t. Lnh vc nghin cu ny gil Vi sinh hc (microbiology), ra i cch y hn 300 nm bi Antonivan Leeuwenhoek (1676; hnh 1.1) vi khm ph u tin cc vi sinh vtbng knh hin vi n gin.

V lch s pht trin ca vi sinh hc, cc tc gi khc nhau phn chiakhng ging nhau (bn c c th tham kho cc ti liu c gii thiudi y). Chng hn:

Theo Nguyn Thnh t (2005, tr.19-28), c th chia lm 4 giai on:giai on s khai (vi i din l A. van Leeuwenhoek), giai onPasteur, giai on sauPasteurv giai on hin ti.

Madigan v Martinko (2006, tr.9-20) xt qua 4 thi k: (i) Nhng gcr lch s ca vi sinh hc - Robert Hook, van Leeuwenhoek v Cohn; (ii)Pasteur, Koch v cc k thut nui cy thanh trng; (iii) Tnh a dng visinh vt v s ra i ca vi sinh vt hc i cng; v (iv) K nguyn hini ca vi sinh vt hc.

y, tm xt theo quan nim ca McKane v Kandel (1996). C thni rng t na sau th k XIX, s pht trin ca vi sinh hc gn lin vi

bn hng nghin cu chnh c tm lc nh sau:Tranh lun v thuyt tsinh (spontaneous generation controversy):

Hng lot cc bng chng v l l vt thng thuyt t sinh, tiu biu lcc cng trnh ca Spallanzani (1765), Schroeder v von Dusch (1854),Pasteur (1861) v Tyndall (1877).




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Hnh 1.1 A. van Leeuwenhoek, L. Pasteur v W. Flemming (t tri sang).

Nghin cu s ln men (fermentation): Nm 1837 Schwann xcnh nm men Saccharomyces cerevisiae chu trch nhim cho s ln mencn; n nm 1864 Pasteur (hnh 1.1) cu vng nn k ngh sn xut ru

vang Php nhpht trin k thut kim sot s ln men, v y l cscho phng php kh trng Pasteur hin i; nm 1897 Buchner khmph ra s ln men cn v bo.

Nghin cu di truyn hc phn t (molecular genetics) khi u tcng trnh ca Beadle v Tatum nm 1941 vi gi thuyt mt gene-mtenzyme v ko di cho n nay.

Nghin cu bnh ly nhim (infectous disease): Nm 1798 Jennergii thiu vaccine u tin, khi s dng mm bnh u b gy mindch chng li bnh u ma. Nm 1876 Koch chng minh bnh nguynhc ca bnh than; ch ra bn bc cho vic xc nh nguyn nhn ca ccbnh nhim trng. Nm 1881, Pasteur iu ch vaccine chng li bnhthan v n nm 1886 chnh ng li iu ch vaccine chng bnh di.Nm 1883 Metchnicoff chng minh vai tr ca cc t bo bch cu. Nm1929 Flemming (hnh 1.1) khm ph ra penicillin...

Cn lu rng, chnh t nghin cu u tin ca Jenner v vaccine hnh thnh mt nhnh min dch hc (immunology) m s pht trin can gn lin vi tn tui ca Pasteur nh cp. n nm 1954 Salk v1955 Sabin sn xut thnh cng cc vaccine chng virus polio gy bnhbi lit. Nm 1980 T chc Y t Th gii (WHO) tuyn b gii quytxong bnh u ma, v bnh AIDS cng bt u xut hin. Nm 1984Milstein, Koeller v Jeme sn xut cc khng th n dng; nm 1990Murry v Johnson s dng cc tc nhn c ch min dch thc hinthnh cng cc ca ghp m. n 1993 ca liu php gene u tin duy trkh nng ca bnh nhn suy gim min dch chng c bnh ly nhim...

II. Cc loi t bo vi sinh vt prokaryote v eukaryoteCc vi sinh vt khng phi l mt nhm ring bit hoc mt n v

phn loi, m thng bao gm nhiu nhm gii khc nhau rt a dng, tcc virus (xem chng 5), vi khun cho n cc vi sinh vt eukaryote.

Da vo phn loi hc phn t, nm 1977 Carl Woese chia sinh vtnhn s thnh 2 nhm da trn trnh t 16S rRNA, gi l nhm hay vc(domain) vi khun thc (Eubacteria) v vi khun c(Archaebacteria). ngl lun rng hai nhm ny, cng vi sinh vt nhn chun, tin ha c lp

vi nhau v vo nm 1990 nhn mnh thm quan im ny bng cch ara h thng phn loi 3 vc, bao gm vi khun (Bacteria), vi khun c




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(Archaea) v sinh vt nhn chun (Eukarya). Quan im ny ni chungc chp nhn rng ri gia cc nh sinh hc phn t.

Trc tin, ta hy hnh dung mi quan h v mt pht sinh chng loica cc prokaryote (gm bacteria v archaea) v cc eukaryote Hnh 1.2.

Cn lu rng, v mt tin ha, vi khun c coi l cc vi sinh vt khc, xut hin cch y chng 3,7 triu nm. Hai bo quan ty th(mitochondrion) v lc lp (chloroplast) c mt trong cc t bo eukaryotec xem l bt ngun t vi khun bng con ng ni cng sinh(endosymbiotic).

Hnh 1.2 Cy pht sinh chng loi ca s sng da trn so snh trnh t RNAribosome si n (ssrRNA). T gc cy s sng cho thy cc prokaryote phnthnh nhm (Domains) l Archaea v Bacteria. mc phn loi, cc sinh vt

phn nh ca cc nhnh Archaea i din cho cc B (Order); phn nh cacc nhnh Bacteria l cc ngnh (Phyla). Cc nhm vi khun a dng v quantrng nht, c nghin cu k nht l Cyanobacteria, Proteobacteria v cc vikhun Gram dng. (Ngun: Kenneth Todar, 2004).

1. Tbo v cc c tnh cbn ca n

T bo (t ting Latin cella, ngha l khoang nh; thut ng ny doRobert Hooke a ra) l n v cu trc v chc nng ca a s sinh vt(tr nhng dng sng tin t bo chng hn nh virus). Nhng sinh vtn bo nh vi khun, cth ch gm mt t bo. Cc sinh vt a bo cuto t nhiu t bo.

Hc thuyt t bo c xy dng t th k 19 v theo quan nim hinnay c th tm tt ni dung ca n nh sau:

1. Mi sinh vt c cu to t mt hoc nhiu t bo.




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2. Cc t bo chc sinh ra t nhng t bo trc .

3. Mi chc nng sng ca sinh vt c din ra trong t bo.

4. Cc t bo cha cc thng tin di truyn cn thit iu khin ccchc nng ca mnh, v

5. C th truyn vt liu di truyn ny cho cc th h t bo tip theo.Mi t bo l mt h thng m, t duy tr v t sn xut. Mi t bo

u c mt s kh nng nh: (i) Sinh sn thng qua phn bo; (ii) Trao icht v nng lng; (iii) Tng hp cc protein; (iv) p ng vi cc kchthch, hoc thay i ca mi trng bn trong v bn ngoi nh cc thayi v nhit , pH hoc ngun dinh dng; (v) Di chuyn cc ti tit.

2. Cc dng tbo

Ngi ta c th phn loi t bo da vo kh nng c th tn ti clp hay l khng. Cc sinh vt c th bao gm ch mt t bo (gi l sinhvt n bo) thng c kh nng sng c lp mc d c th hnh thnh

cc khun lc. Ngoi ra, sinh vt cng c th bao gm nhiu t bo (sinhvt a bo), trong mi t bo c bit ha v thng khng th sngst khi b tch ri. Nu xt v cu trc ni bo, cc t bo c th ch lm 2dng chnh (Hnh 1.3) sau y:

Tbo prokaryote thng c cu trc n gin, ch thy sinh vtn bo hoc tp on n bo. Trong h thng phn loi 3 gii, cc sinhvt prokaryote l thuc gii Archaea v Eubacteria.

Tbo eukaryote thng cha cc bo quan c mng ring. Sinhvt n bo eukaryote cng rt a dng nhng ch yu l sinh vt a bo.T bo eukaryote bo gm cc sinh vt l ng vt, thc vt v nm.

Hnh 1.3 Cc t bo prokaryote (vi khun) v eukaryote (ng vt).




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2.1. Cc t bo prokaryote

Prokaryote l nhm t bo khng c mng nhn. y l c imchnh phn bit vi cc t bo eukaryote. Prokaryote cng khng c ccbo quan v cu trc ni bo in hnh ca t bo eukaryote. Hu ht cc

chc nng ca cc bo quan nh ty th, lc lp, b my Golgi c tinhnh trn mng sinh cht. T bo prokaryote c 3 vng cu trc chnh l:

(i) tin mao (flagella), tim mao, hay lng nhung (pili) - cc proteinbm trn b mt t bo;

(ii) v t bo bao gm capsule, thnh t bo v mng sinh cht;

(iii) vng t bo cht c cha DNA genome, cc ribosome v cc thvn (inclusion body).

Cc c trng ca t bo prokaryote :

T bo cht l phn dch lng chim hu ht th tch t bo, khuchtn vt cht v cha cc ht ribosome nm t do trong t bo.

Mng sinh cht l lp phospholipid kp phn tch phn t bo chtvi mi trng xung quanh. Mng sinh hc ny c tnh bn thm, hay cngi l thm c chn lc.

Hu ht cc t bo prokaryote u c thnh t bo (trMycoplasma, Thermoplasma (archae) v Planctomycetales. Chng ccu to t peptidoglycan v hot ng nh mt ro cn ph chn lcnhng cht vo ra t bo. Thnh t bo cng gip vi khun gi nguynhnh dng v khng b tc ng ca p sut thm thu trong mi trngnhc trng.

Nhim sc th ca t bo prokaryote thng l mt phn t DNAdng vng (tr vi khunBorrelia burgdorferi v mt s khc; xem chng2). Mc d khng phi c cu trc nhn hon chnh, DNA c c ctrong vng nhn. T bo prokaryote cn cha nhng cu trc DNA ngoinhim sc th gi lplasmid, n cng c dng vng nhng nh hn DNAnhim sc th. Trn cc plasmid thng cha cc gene c chc nng bsung, v d khng khng sinh.

T bo prokaryote mang cc tin mao gip t bo di chuyn chng trong mi trng.

Cu trc t bo ca vi khun c m tHnh 1.4.




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Hnh 1.4 Cc thnh phn cu trc ca t bo E. coli.

2.2. Cc t bo eukaryote (Hnh 1.5)T bo eukaryote (ting Latin c ngha l c nhn tht s) thng lngp 10 ln v kch thc so vi t bo prokaryote do gp khong 1.000ln v th tch. im khc bit quan trng gia prokyryote v eukaryote lt bo eukaryote c cc xoang t bo c chia nh do cc lp mng tbo thc hin cc hot ng trao i cht ring bit. Trong , iu tinb nht l vic hnh thnh nhn t bo c h thng mng ring bo vcc phn t DNA ca t bo. T bo eukaryote thng c nhng cu trcchuyn bit tin hnh cc chc nng nht nh, gi l cc bo quan.

Cc c trng ca t bo eukaryote:

T bo cht thng khng nhn thy nhng th ht nh

prokaryote v rng phn ln ribosome ca chng c bm trn mng lini cht.

Mng t bo cng c cu trc tng t nhprokaryote tuy nhinthnh phn cu to chi tit li khc nhau mt vi im nh. Ch mt s tbo eukaryote c thnh t bo.

Vt cht di truyn trong t bo eukaryote thng gm mt s phnt DNA mch kp thng, c c c ch yu bi cc protein histone tonn cu trc nhim sc th. Mi phn t DNA c lu gi trong nhn tbo vi mt lp mng nhn bao bc. Mt s bo quan (ty th v lp th)ca eukaryote c cha DNA mch kp vng ring.

Mt s t bo eukaryote c th di chuyn nh tim mao hoc tinmao. Nhng tin mao thng c cu trc phc tp hn so vi prokaryote.

Roi

T bo cht

Vcht bo

Mng t bo

DNA

Nucleoid

Si lng

Plasmid

Mesosome

Ribosome

V bc




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Hnh 1.5 M hnh mt t bo ng vt in hnh. Cc bo quan: (1)-hchnhn; (2)- nhn; (3)- ribosome; (4)- ti tit; (5)- li ni cht ht, (6)- b myGolgi, (7)- khung xng t bo, (8)- li ni cht trn, (9)- ty th, (10)- khng

bo, (11)- t bo cht, (12)- lysosome, (13)- trung th.

3. So snh cc tbo eukaryote, eubacteria v archaea

Cc c im phn bit cc t bo eukaryote, eubacteria v archaeac tm tt Bng 1.1.

Bng 1.1 So snh cc t bo prokaryote v eukaryote

Prokaryotec im Eukaryote Eubacteria Archaeobacteria* Vng nhnMng nhn C Khng KhngHch nhn C Khng Khng

Vng nhn Khng C CS lng nhim sc th 2 1 1Cc NST cha histone C Khng KhngPhn chia t bo Nguyn phn Thg ct i Phn ct i* T bo chtDng t bo cht C Khng KhngCc ty th C Khng KhngCc lp th C thc vt Khng KhngCc ti mng C Khng KhngPhc hp Golgi C Khng KhngLi ni cht C Khng Khng

Kch thc ribosome 80 S 70 S 70 S* Cc lp b mtMng sinh cht C C C




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Cc lin kt lipid mng Ester Ester EtherCc sterol mng C Him khi KhngPeptidoglycan vch tbo Khng C KhngCc si lng, nu c Cc si thoi Cc lng t ???

V tr vn chuyn in tMng boquan Mng t bo Mng t bo

*ng knhT bo in hnh 2-25 m 0,3-2 m 0,5-2 m

(Ngun: dn theo Watson et al 1987; McKane v Kandel 1996)

III. c im ca vi sinh vt1. Vi nti cng vc im ca cc vi sinh vt

Kch thc b nh:

Cc vi sinh vt c kch thc rt b, o bng n v micromet (1m =10-6m) nh cc vi nm, vi khun hoc nanomet (1nm = 10-9nm) nh cc

virus. V d: Cc t bo nm men c ng knh 5 -10 m. Cc vi khunc ng knh chiu di cth thay i trong khong (0,2 - 2,0) (2,0 -8,0) m; hay nhE. coli chng hn rt b: 0,5 2,0 m v.v.

Hp th nhiu, chuyn ho nhanh:

Cc vi sinh vt tuy nh b nht trong sinh gii, nhng nng lc hp thuv chuyn ho ca chng c th vt xa cc sinh vt bc cao. Chng hn,vi khun lactic (Lactobacillus) trong 1 gi c th phn gii mt lngng lactose nng hn 1.000-10.000 ln khi lng cth chng...

Kh nng sinh sn nhanh:

So vi cc sinh vt khc th cc vi sinh vt c tc sinh trng v

sinh si ny ncc k nhanh. Chng hn, E. coli, trong iu kin thchhp, thi gian mt th h ko di khong 20 pht. Nu khng b cc iukin t nhin khng ch, ch sau mt ngy m t mt t bo ban u ssinh sn c 272 t bo, nng 4.722 tn!

Kh nng thch ng rt cao v pht sinh bin d mnh:

Ni chung, cc vi sinh vt vn c cc cchiu ho chuyn ho thch ng c vi cc iu kin sng bt li. Trong mt t bo vi sinhvt, s lng cc enzyme thch ng chim ti 10% hm lng protein.Nu c mt thay i cht dinh dng th ch sau 1/1.000 giy, chng cth thay i thch ng ri. Mt s vi khun c th tin hnh quang hp

di tc dng ca nh sng, sng khng cn oxy; nhng nu chuyn votrong ti lp tc chng c th s dng oxy sng. Mt s vi sinh vt khig p cc iu kin khc nghit th chuyn sang trng thi bo t, ngng




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hot ng. Mt s c th sinh trng ngay cnhit rt cao 250oC,hoc sng y su i dng vi p sut khong 1.100 atm, v.v.

Lin quan ti kh nng thch ng cng nh s phong ph v chngloi, cc vi sinh vt cn c c tnh quan trng na l d pht sinh bin

d, vi tn s trung bnh 10-5

-10-10

. Nguyn do bi v cth chng thngl n bo vi b gene n bi, sinh sn nhanh, s lng nhiu, tip xctrc tip vi mi trng sng. Hnh thc bin d thng g p l cc tbin gene v ko theo cc bin i v hnh thi, cu to, kiu trao i cht,sn phm trao i cht, tnh khng nguyn, tnh khng ...

Phn b rng, chng loi nhiu:

Cc vi sinh vt phn b khp mi ni v pht trin nhanh chng nhng ni c thc n, m, v nhit ti u cho s phn chia v lnln ca chng. Chng c thc mang i bi gi t ni ny sang nikhc. C th ngi l ni c tr ca hng t vi sinh vt; chng trn da,ng rut, trong mi, ming v nhng ch hkhc ca cth. Chng c

trong khng kh, nc ung v thc n.V chng loi, c tnh c trn 100 nghn loi, trong nm chim

khong 69 nghn loi, vi to - 23 nghn, vi khun lam - 2,5 nghn, vi khun- 1,5 nghn, virus v ricketsi - 1,2 nghn...

2.c im ca vi khun

2.1. c im sinh sn

Vi khun sinh sn bng cch chia i (binary fission) hay trc phn(amitosis). Mc d khng c hnh thc sinh sn hu tnh (ch l sinh sncn hu tnh, parasexual reproduction), cc bin i di truyn vn xy ratrong tng t bo vi khun thng qua cc hot ng ti t hp di truyn.

C ba kiu ti t hp di truyn c pht hin vi khun:+Bin np (transformation): chuyn DNA trn t mt t bo vi khun

sang t bo khc thng qua mi trng lng bn ngoi, hin tng nygm c vi khun cht.

+ Ti np (transduction): chuyn DNA vi khun t t bo sang t bokhc thng qua ththc khun (bacteriophage).

+ Giao np hay tip hp (conjugation): chuyn DNA t vi khun nysang vi khun khc thng qua ng tip hp hay lng gii tnh (pilus).

Sau khi nhn c DNA t mt trong nhng kiu trao i thng tin ditruyn ni trn, vi khun s tin hnh phn chia v truyn b gene ti t

hp cho th h sau.2.2. Cc qu trnh trao i cht




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C rt nhiu kiu trao i cht khc nhau vi khun. Vi khun ddng(heterotroph) phi da vo ngun carbon hu cbn ngoi, trongkhi cc vi khun tdng(autotroph) c kh nng tng hp cht hu ct CO2 v nc. Cc vi khun t dng thu nhn nng lng t phn ng

oxy-ha cc hp cht ha hc gi l vi khun ha dng(chemotroph), vnhng nhm thu nng lng t nh sng thng qua qu trnh quang hpc gi l vi khun quang dng (phototroph). Ngoi ra, cc vi khuncn c phn bit nh vo ngun cht kh m chng s dng. Nhngnhm s dng hp cht v c(nh nc, kh hir, sulfua v ammoniac)lm cht kh c gi l vi khun v c dng (lithotroph) v nhngnhm cn hp cht hu c(nhng, acid hu c) gi l vi khun huc dng (organotroph). Nhng kiu trao i cht da vo ngun nnglng (quang dng hay ha dng), ngun cht kh (v cdng hayhu cdng) v ngun carbon (t dng hay d dng) c thc kth p khc nhau trong tng t bo, v nhiu loi c th thng xuynchuyn t kiu trao i cht ny sang kiu trao i cht khc.

Nhng cht dinh dng cn thit cho s pht trin bnh thng gmnit, lu hunh, phospho, vitamin v cc nguyn t kim loi nh natri,kali, canxi, ma-nh, mangan, st, km, cban, ng, nikel... Mt s loicn thm mt s nguyn t vt khc nh tungsten, vanai hay bo.

Vi khun quang v c t dng bao gm vi khun lam(cyanobacteria) l mt trong nhng loi c nht c bit n t ha thchv c l ng mt vai tr quang trng trong vic to ra ngun oxy chokh quyn. Chng l nhng tin phong trong vic s dng nc nh lngun electron v c (lithotrophic) v l sinh vt u tin dng b myquang hp phn r nc. Cc vi khun quang hp khc dng cc ngun

electron khc nn khng to ra oxy.Da vo phn ng vi oxy, hu ht cc vi khun c thc xp vo

3 nhm: mt s ch c th mc khi c oxy c gi l vi khun hiu kh(aerobe); mt s khc ch c th mc khi khng c oxy c - vi khun kkh(anaerobe); v mt s c th mc c khi c hay khng c oxy th thucnhm vi khun k kh ty (facultative anaerobe). Cc vi khun khng sdng oxy nhng vn c th mc khi c xy - vi khun chu oxy(aerotolerant). Nhng vi khun c th mc tt trong mi trng khcnghit i vi con ngi c gi l extremophile. Mt s vi khun sngtrong sui nc nng - vi khun chu nhit (thermophile); mt s khcsng trong h nc rt mn - vi khun chu mn (halophile); trong khi

c loi li sng trong mi trng acid hay kim - vi khun chu axit(acidophile) hay vi khun chu kim (alkaliphile) v cn mt s sng di




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lp bng h trong dy ni Alpes - vi khun chu hn (psychrophile).

2.3. Di ng

Vi khun di ng nh vo tin mao (flagellum), trt (bacterialgliding) hay thay i sc ni (buoyancy). Nhm xon khun (spirochaete)

c cc cu trc tng t tin mao gi lsi trc (axial filament). Chng cmt th xon c c bit quay trn khi di chuyn.

Tin mao ca vi khun c sp xp theo nhiu cch. Vi khun c thc mt tin mao mi cc ca t bo, hay c th c mt nhm nhiu tinmao mt u. Nhiu vi khun (nh E. coli) c hai kiu di ng khcnhau: di ng tin ti (bi) v quay vng.

Vi khun di ng khi b thu ht hay y ra bi mt s tc nhn kchthch, hot ng ny c gi l tnh hngng(taxes), chng hn nh:ha hngng(chemotaxis), quang hngng(phototaxis), chngng(mechanotaxis) v thngng(magnetotaxis).

2.4. Cc nhm phn loi v c im nhn bitVi khun c nhiu hnh dng khc nhau (Hnh 1.6 v 1.7). a s chnh que, hnh cu, hay hnh xon; cc vi khun c hnh dng nh vyc gi theo th t l trc khun (bacillus), cu khun (coccus), vxonkhun (spirillum). Mt nhm khc na lphy khun (vibrio) c hnh duphy. Hnh dng khng cn c coi l mt tiu chun nh danh vikhun, tuy nhin c rt nhiu chi c t tn theo hnh dng (v d nhBacillus, Streptococcus, Staphylococcus) v n l mt im quan trng nhn dng cc chi ny.

Mt cng c quan trng nhn dng khc l nhum Gram (mang tnca Hans Christian Gram, ngi pht trin k thut ny). Nhum Gram

gip phn bit cc vi khun thnh 2 nhm, da vo thnh phn cu to cavch t bo.

(a) (b) (c) (d)Hnh 1.6 (a) Cc t bo E. colitht i; (b) Streptococcus; (c) Bacillus anthracis

trong mt mao mch phi; (d) Staphylococcus aureus.




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Hnh 1.7 Hnh dng khc nhau ca cc vi khun.A. Hnh que - trc khun (Bacillus)B. Hnh cu (coccus) to thnh chui (strepto-) - lin cu khun (Streptococcus).C. Hnh cu to m (staphylo-) - t cu khun (Staphylococcus).D. Hnh trn sng i (diplo-) - song cu khun (Diplococcus).E. Hnh xon - xon khun (Spirillum, Spirochete).F. Hnh du phy - phy khun (Vibrio).

IV. Cc phng php nghin cu c th ca di truyn hc vi

sinh vt v mt s phng php sinh hc phn tthng dngi vi cc vi sinh vt, phn tch di truyn hc cng l phng php

duy nht nghin cu cc c tnh di truyn v bin d ca chng. Docc vi sinh vt thng c b gene n bi, c bit cc vi khun ch cmt nhm lin kt gene nn s phn tch di truyn hc chng l ngin hn cc eukaryote bc cao, gm cc giai on sau: (i) Xc nh ccgene; (ii) Xc nh trt t ca cc locus trn nhim sc th; v (iii) Xcnh cu trc tinh vi ca gene.

Tng qut, c cc phng php cbn c p dng cho phn tch ditruyn vi sinh vt nh sau: phn tch t bin, phn tch ti t hp, phntch sao chp, phn tch on khuyt v phn tch b sung.

1. Phn tch t bin

Phn tch t bin c p dng xc nh cc gene v c tinhnh bng cch o m cc kt qu cui cng ca s biu hin gene thnhra s bin i kiu hnh (c im hnh thi, ho sinh, khng nguyn hoctnh mn cm i vi cc tc nhn ho hc, vt l v sinh hc khc nhau)ca cc t bo vi khun. Vic pht hin mt t bin ngu nhin hay gyto ch ra s tn ti ca mt gene c th.

Sbin i hnh thivi sinh vt bao gm cc bin i v kch thc,hnh dng v s hnh thnh sc t ca cc khun lc do cc t bo btbin to nn trn cc mi trng dinh dng c cng nh s bin i ca

bn thn cc phn t ca t bo (v d s tng kch thc hoc mt lng ttrn b mt mng t bo). Sbin i ho sinh bao gm cc bin i lin




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quan ti vic t bo mt kh nng tng h p cc amino acid v vitaminhoc mt kh nng chuyn ho cc hp cht hydrat carbon. Cc bin ivkhng nguyn th hin ch vi khun b mt i nhng khng nguynnht nh. Cc bin i trong tnh bn vng ca vi khuni vi cc tc

nhn khc nhau lin quan ti s xut hin trong chng cc kh nng khng i vi s chiu x, vi cc ho cht khc nhau (k cc cc loithuc khng sinh) hoc vi phage v.v.

Do tn st bin vi khun l rt thp nn vic phn lp cc t bobt bin ch c th thc hin c trong cc th nghim vi cc qun tht bo. Nh th, v nguyn tc, trong trng hp ny c th s dng btk phng php no cho php tch c cc tht bin t cc qun th.Vic xc nh s lng cc t bin da trn cc phng php xc nhtn st bin. Thng thng, phn tch di truyn cn c cc ni tbin mang cc t bin v tr cho trc. Chng hn, i viB. subtilis, cth x l sb DNA gy bin np bng cc tc nhn gy t bin; E.

coli, c th gy cc t bin c v tr xc nh bng cch a vo t bo vikhun cc gene t bin nhcc phage ti np.

2. Phn tch ti thp

Phn tch ti t hp l phng php c trng c dng xc nhv tr v trt t ca cc gene trn nhim sc th. i vi vi khun, vicphn tch di truyn da vo cc qu trnh trao i vt liu di truyn nhbin np, ti np v tip hp hay cn gi l giao np (chng 5 v 6). cc vi nm, vic phn tch di truyn c tin hnh bng php phn tchb bn v da trn chu trnh cn hu tnh (chng 7).

Ni chung, s trao i di truyn cc vi khun v qu trnh hu tnh

cc c th bc cao l kh ging nhau. Vic truyn vt liu di truyn t vikhun thcho (donor) sang vi khun thnhn (recipient) c th coi nhnh s kt hp nhn ca cc t bo sinh dc (y l s to thnh cc thlng bi tng phn), cn s st nhp ca vt liu di truyn vo b geneca vi khun th nhn, v s hnh thnh nhim sc th ti t hp sau ,c th so snh vi cc kt qu ca gim phn. Chnh cc h thng ti thp ny l c scho phng php phn tch ti t hp v lp bn ditruyn vi khun. V d, trt t ca hu ht cc gene trn nhim sc thE.colic xc nh l nhs dng tip hp v ti np; B. subtilis nhtin p v bin n p; cn Salmonella typhimurium ch yu nh ti np.Ngoi ra, php phn tch ti t hp ny cn c s dng nghin cu

cu trc tinh vi ca gene.3. Phn tch sao chp




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Phng php ny cho php xc nh trt t cc gene trn nhim sc thda trn s tnh ton cc s liu v s bt u sao chp (ti bn) ca nhimsc th t mt im xc nh. Do thi gian sao chp ca mt phn nhimsc th nht nh ph thuc vo khong cch t phn n khi im

sao chp nn th t sao chp phn nh trnh t sp xp ca cc gene. Nhvy, bn nhim sc th ch c thc xy dng da trn cc dn liuv trt t sao chp ca cc phn ring bit ca nhim sc th.

4. Phn tch on khuyt

Php phn tch on khuyt c s dng xc nh v tr ca ccgene trn nhim sc th cung nh nghin cu cu trc tinh vi ca gene. N da trn vic tnh ton cc on khuyt trn nhim sc th. Nh sphn tch ny ngi ta pht hin c v tr ca hng lot gene E. coliv S. typhimurium, hiu bit c cu trc tinh vi ca cc gene trnoperon lactose E. coli. Phng php ny cng c s dng rng ri nghin cu cu trc tinh vi ca gene phage.

5. Phn tch bsung

Phng php ny c s dng pht hin chc nng ca cc genenht nh tham gia vo vic xc nh mt c tnh no ca vi khun,da trn hin tng b sung ca cc gene (ngha l s tng tc gia ccsn phm gene). Phng php ny do Lewis tm ra nm 1951 trong khinghin cu tnh allele rui gim. Di y ta hy xem xt php thcis-trans (u-lch) ny qua cng trnh ca Benzer.

Cc cng trnh nghin cu ca Seymour Benzer (t 1957 n 1961) vti t hp phage T4 cho thy rng, gene theo quan nim ca Morganc th chia nh thnh cc n v nh hn. ng a ra cc thut ngmuton, recon v cistronnh ngha cc n v khng chia nh tngng l t bin, ti t hp v chc nng. Bng cch lai cc tht binca cng mt gene c ngun gc c l p nhau trong khi cho ly nhimphage, lm xut hin phage kiu di. iu ny ch c th xy ra bi sti thp bn trong gene, nu nh cc phn nh ring bit ca gene u bt bin. iu ny chng t rng gene b phn chia thnh cc n v nhhn thng qua ti t hp v dt bin. Tuy nhin, v kch thc ca mutonv recon c coi l tng ng vi mt cp nucleotide, cho nn ngynay t thn hai n v ny khng cn gi tr s dng na.

Thut ngcistron ca Benzer c ngha l n v chc nng di truyn

khng chia nh. iu ny c th xc nh bng s phn tch b sung(complementation analysis), trong gene m c th l sn phm ca nc trc nghim v kh nng b p cho mt t bin ti mt gene tng




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ng trong cng t bo. S b sung lin tip lm phc hi kiu hnh di.cistron 1 cistron 2

S I P Kiu di(a)

cistron 1 cistron 2

S I P Kiu di(b) X X

cistron 1 cistron 2 X

S I P Kiu di

(c) X

cistron 1 cistron 2 X

S Tht bin

(d) X

Hnh 1.8 S minh ha trc nghim cis-trans: (a) con ng chuyn habnh thng; (b) trc nghim cis; (c) v (d) trc nghim trans. Ch thch: S-ccht (subtrate); I- sn phm trung gian (intermediate); P- sn phm cui cng(product), y l sc tc trng cho kiu hnh di; cc mi tn () ch ccenzyme sn phm sinh ra t cc cistron 1 v cistron 2.

C sca phn tch b sung l trc nghim cis-trans (cis-trans test),m ty ny sinh ra thut ng cistron, trong cc cp t bin btngun c lp c xt cc cu hnh cis (u) v trans (lch). Trcnghim cis c dng lm i chng, v nu nh c hai t bin u cmt trong mt b gene th b gene kia phi l kiu di c hai locus vsinh ra cc sn phm gene bnh thng, do cho ra kiu hnh di (hnh1.8b). Trc nghim trans l php th b sung v xc nh gi hn ca nv chc nng. Nu nh cc t bin nm trong cc gene khc nhau, khichng c mt cu hnh trans, mi mt b gene c th b sung sn phmm gene kia khng to ra c. Khi c tt c cc sn phm gene cnthit th t bo l kiu di (hnh 1.8c), ngha l c sbsung dng tnh(positive complementation). Nu nh c hai t bin thuc cng mt gene,khi chng c mt cu hnh trans, th mi mt b gene c th mang mtbn sao t bin ca gene v khng c sn phm hot ng chc nngc to ra trong t bo, ngha l khng c s b sung (hnh 1.8d).

T cc kt qu nghin cu ca Benzer cho thy: Cistron (hay gene cutrc) l mt on xc nh ca DNA mang thng tin cu trc ca mt

polypeptide c th m gii hn ca n c xc nh bng trc nghimcis-trans. Theo , kch thc trung bnh ca mt cistron ~1.200 cp base.




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6. Nng sut phn gii v mt sthut ngca di truyn hc vi sinh vt

Nng sut phn gii ca di tuyn hc c xc nh bi khong cchgia cc cu trc di truyn (gene) cn phn tch trn nhim sc th. ilng ny ph thuc vo s lng c thi con nghin cu thu c t

mt php lai c th; s con chu thu c cng ln th kh nng pht hincc th ti t hp him cng ln, tc nng sut phn gii ca phn tch ditruyn hc cng cao. Theo lut s ln ny, cc vi khun t ra rt thun litrong phn tch di truyn hc, v trong mt thi gian ngn c th thu cmt s lng cc k ln con chu t mt t bo vi khun, cng nh c ths dng cc mi trng nui cy khc nhau chn lc cc th ti t hp.

Cc thut ng v k hiu thng dng ca di truyn hc vi khun datrn ngh ca Demerec v cng sa ra nm 1966, vi t nhiu chnhl b sung cho n nay (xem chng 6).

7. Slc vmt sphng php thng dng ca sinh hc phn t

S tin b nhanh chng gn y ca sinh hc ni chung v cng nghsinh hc (biotechnology) ni ring l nhs pht trin mnh m ca ccphng php v k thut mi nh: Knh hin vi in t; tch chit v phntch nh tnh v nh lng th nucleic acid; xc nh trnh t nucleicacid, lai phn t nucleic acid, nh du ng v phng x v s dng ccmu d; khuych i gene hay phng php trng h p chui nh polymerase (Polymerase Chain Reaction = PCR); xy dng cc phn tDNA ti t h p vto dng DNA ti t h p; thu nhn gene bng cchthnh lp cc th vin gene, tng hp gene bng con ng ho hc vngn hng cDNA; gy bin i vt liu di truyn.

Trong khun kh ca chng ny chng ti ch gii thiu ba phng

php chnh: lai phn t, xc nh trnh t nucleic acid v PCR (c s dngmt s k thut lin quan nh mu d v nh du).

7.1. Lai phn t (molecular hybridization)

Ngi ta li dng s bin tnh v hi tnh ca DNA to ra cc phnt DNA lai bng cch lm lnh t t hn hp cc DNA bin tnh t hailoi khc nhau (hnh 1.9). K thut lai phn t (molecular hybridization)ny c ng dng rng ri xc nh mc tng ng DNA cacc nhm phn loi khc nhau. Chng hn, cc thc nghim cho thy ckhong 25% tng s DNA ngi v chut c th lai vi nhau. K thutny cn c ng dng rng ri nh v gene bng cch s dng cc vt

d c nh du ng v phng x (radioactive probe) hoc lai hunhquang ti ch(fluorescense in situ hybridization = FISH) v.v.




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Hnh 1.9 Bin tnh v hi tnh ca DNA v ng dng trong lai phn t nucleicacid (tri), v trong k thut s dng mu d DNA tm on ch.

7.2. Xc nh trnh t (nucleic acid)

Trong di truyn hc v ho sinh, xc nh trnh t (sequencing) cngha l xc nh cu trc chnh (hay trnh t chnh) ca mtpolymersinhhc cha c phn loi. Xc nh trnh t cho kt qu l s m t tuyntnh mt cch hnh nh hay cn gi l "chui".

Trong thut ng di truyn hc, xc nh

trnh t DNA l qu trnh xc nh trt tnucleotide ca mt on DNA. Hin nay, huht mi xc nh trnh t DNA u ctinhnh bng cch s dng phng php phn tch trnh t c pht trin biFrederick Sanger. Kthut ny dng phn tchtrnh t c th (sequence-specifictermination) ca mt phn ng tng hpDNA trong ng nghim (in vitro) dng chtnn nucleotide c chnh sa.

Ti sao phi xc nh trnh tDNA?Trnh t ca DNA m ha cc thng tin cn thit cho cc c th

Mu d

Mu d DNA

Mng lcnylon

on ch

Bn Gel

DNA bin tnhbi nhit DNA hi tnh

bi lm ngui

Lai DNA/RNA

Si DNASi RNA

Lai ho

Hnh 1.10 Mt phn cabn gel phn tch trnh tc nh du phng x.




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sng c th tn ti v ti sn sinh. Vic xc nh trnh t v th rt hu chvi cc nghin cu 'thun ty' l gii ti sao v bng cch no m cc cth tn ti, cng nh cc ch mang tnh ng dng. V bn cht quantrng ca DNA i vi cc sinh vt sng, hiu bit v trnh t DNA c th

trnn hu ch vi cc nghin cu sinh hc v ng dng. V d, trong ykhoa n c thc dng xc nh, chn on v pht trin cc phngphp iu tr cho cc bnh v di truyn hc. Tng t, cc nghin cu vopathogens c th gip iu tr cc bnh ly nhim (contagious diseases).Cng ngh sinh hc (biotechnology) l mt ngnh ang pht trin, vitim nng p dng cho cc sn phm v dch v hu ch.

7.3. Phng php PCR (Polymerase Chain Reaction)

V mi kiu sinh vt c cha DNA c trng ring, nn c th dngDNA xc nh ging nh mt "du vn tay". Cc th nghim di truynnh th s dng cc on nh du ca DNA duy nht t cc vi sinh vt bit d tm nhim sc th ca sinh vt cha bit. Mu d (probe) nys ch t hp vi DNA ca sinh vt cha bit nu nh nhim sc th can c cha mt on tng ng. Du (label) ch th ny c thc phthin sau . Tuy nhin, nu mu d DNA ny l c trng cho mt sinhvt khc th n s khng phn ng, v s khng pht hin c du. Ccmu d DNA c tnh c th v phn ng dng tnh l bng chng vtnh ng nht ca vi sinh vt. Nhng tin b ca cng ngh sinh hc ngynay c th cho mt DNA ca vi sinh vt "sinh trng" thm ch ngay ckhi sinh vt kh nui cy. Nh c cc mu DNA cho vic xcnh hu nh bt k vi sinh vt no c th thu c t mt mu tiu bnthm ch khng phi qua nui cy sinh vt . chnh l nh s phtminh ra phng php khuych i gene hay PCR (Gene amplification -Polymerase Chain Reaction; Hnh 1. 11) bi Kary Mullis nm 1985.7.3.1. PCR l g?

PCR l ch vit tt ca cm t Polymerase Chain Reaction (tm dchl phn ng chui trng h p nhpolymerase. PCR l mt k thut phbin trong sinh hc phn t nhm khuych i (to ra nhiu bn sao) mton DNA m khng cn s dng cc sinh vt sng nhE. coli hay nmmen. PCRc s dng trong cc nghin cu sinh hc v y hc phc vnhiu mc ch khc nhau nh: pht hin cc bnh di truyn, nhn dngvn tay DNA, chn on bnh, tch dng gene, xc nh huyt thng v.v.

7.3.2. Nguyn tc v quy trnh

PCR l mt k thut cho php khuych i nhanh mt mu DNA cth trong ng nghim (hn l trong cc t bo sng nh l E. coli). Vi




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quy trnh ny ngi ta c th to ra v s bn sao ca mt phn t DNAn. Quy trnh "to dng in vitro" ny c tm tt nh sau:

Hnh 1.11 S minh ha quy trnh k thut PCR.

- thc hin mt PCR, cn phi bit t nht mt on trnh t caphn t DNA quan tm (v d mt mu mu).

- Sau phi tng hp cc on mi (primer), tc cc oligonucleotidengn (cha khong hai chc nucleotide) m n b sung chnh xc vi trnhtu 3' ca mi mt si ca DNA cn khuych i.

- Mu DNA c un nng tch cc si n (bin tnh) v trn lnvi cc on mi.

- Nu nh cc on mi tm thy cc trnh t b sung trong DNA,chng s kt hp vo cc si .

- S tng hp bt u (bao gicng theo chiu 5' 3') bng cch sdng si gc lm khun.

- Hn h p phn ng phi cha tt c bn loi deoxynucleotidetriphosphate (dATP, dCTP, dGTP v dTTP) v mt DNA polymerase(loi chu nhit, v d Taq polymerase c chit xut t vi khunThermus aquaticus sng sui nc nng).

- S trng hp c tip din chng no mi si n c tng hp micn cha v tr c nhn bit bi on mi khc.

- Lc ny ta c hai phn t DNA ging ht phn t ban u.- By gita ly hai phn t ny cho bin tnh v lp li qu trnh .

Phn t DNA cnkhuych i

Cc si DNA mi c to thnh,v n lt li t nhn i

Cc si DNA tch ra bi nhit. Cc on mibm vo mi si u 5' v bt u tng hpDNA mi nh enzyme DNA polymerase

Cc on mi(oligonucleotide)c tng hpbng ho hc

Thu c hng triu bn saoca DNA c ti bn

Quy trnh ny c lp li 20-60 ln




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- Sau mi chu k s phn t DNA li tng gp i.

Nhs dng cc thit b tng, mi chu k ti bn c th hon thnhcha y 5 pht. Sau 30 chu k, t mt phn t DNA ban u ckhuych i ln hn mt t bn sao (230 = 1,02 x 109). Nh vy, v

nguyn tc, vi phng php PCR ta c th khuych i s DNA tmt chn tc hay mt git mu xc nh trnh t DNA.

7.3.3. S pht trin v mrng cc ng dng gn y ca PCR

T khi ra i n nay, phng php PCRng vai tr cch mng hotrong nhiu lnh vc nghin cu v ng dng khc nhau nh: chn onnhanh, gii trnh t DNA b gene, gy t bin im nh hng, v.v. Cth thc hin PCRin situ (ngay trong t bo) vi c DNA v RNA.

Do phng php PCRn gin, d thc hin v c nhiu ng dngrng ri nn n c hon thin khng ngng. Tht vy, tuy ch trong mtthi gian ngn k t lc ra i, nhiu bin dng ca PCR mi ln lt ra

i. Chng hn:(i) RT- PCR (reverse transcriptase PCR): k thut m RNA c th

c s dng lm khun cho s khuych i PCR sau khi chuyn ithnh cDNA, cn gi l RNA-PCR hat RT-PCR. K thut ny t ra nhyhn cc phng php khc c dng cho s phn tch RNA.

(ii)RT-PCR cnh tranh (competitive RT-PCR): k thut thng cs dng trong vic nh lng cc loi RNA chuyn bit.

(iii)Real-Time PCR l mt k thut PCRnh lng, n c th gippht hin cc sn phm PCR tch luc ti thi im thc t trong qutrnh khuych i gene. Nhvy c thnh gi s tch lu sn phm vnh lng qPCR (quantitative PCR).

(iv) PCR-ELISA: s kt hp PCR vi th nghim min dch lin ktenzyme (ELISA = enzyme linked immunoassay) trong chn on.

Hnh 1.12 di y cho thy mt my phn tch DNA k hiu iCyclerThermal Cycler, vi cc tin ch sau:

Cho chnh xc cao i vi PCRnh lng thi gian thc (real-time quantitative PCR).

C kh nng quay vng chu k nhit nhanh chng, un nng tc ln ti 3,3 C mi giy v lm ngui tc ln n 2,0 C mi giy.

m bo chnh xc cao v nhit n nh ng b ...




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(a) (b)

Hnh 1.12 (a) My PCR v (b) my phn tch DNA (DNA analyzer)Ngun: (a) http://vi.wikipedia.org/; (b) http://www.bio-rad.com/

* Lch sca phng php PCR

Phng php cn bn chy PCR c Kary Mullis pht minh, ng otgii Nobel v Ha hc vo thng 10 nm 1993 cho thnh tu ny, chsau 7 nmkhi ng a ra tng. kin ca Mullis l pht trin mt quy trnh m DNA cth nhn ln nhiu ln mt cch nhn to qua nhiu chu k sao chp bienzyme DNA polymerase.

DNA polymerase c t nhin trong sinh vt sng, ni m n thc hin chcnng nhn DNA khi t bo phn chia. N lm vic bng cch ni vi si DNA vto si b sung. Theo quy trnh PCR gc ca Mullis, enzyme c dng trongphn ng in vitro (iu khin mi trng bn ngoi c th sinh vt). Si DNA ib tch thnh 2 si n khi un nng 96C. Tuy nhin, nhit ny DNApolymerase b ph hy v vy cn b sung enzyme sau mi giai on nung nngca mi chu k. Quy trnh PCR gc ca Mullis khng c hiu qu cao v n mtnhiu thi gian, cn mt lng ln DNA polymerase, v phi lin tc lu sut

trong qu trnh PCR.Sau , quy trnh gc ny c pht trin bng cch dng DNA-Polymerasely t vi khun a nhit(thermophilic) sng trong mch nc phun nhit trn 110C. DNA polymerase t sinh vt ny l n nh nhit cao(thermostable) v khi dng trong PCR n khng b ph v khi hn hp cnung nng tch si DNA. T, khng cn phi them DNA-polymerase vomi chu k, qu trnh sao chp DNA c thn gin v tng hn.

Mt trong nhng DNA-polymerase chu nhit u tin c phn lp ctThermus aquaticus v c gi l Taq. Taq polymerasec dng rng ritrong thc nghim PCR (5/2004). Nhc im ca Taq l thnh thong n nhmln trong qu trnh sao chp DNA, dn n kt cp sai trong chui DNA, v nthiu tnh sa sai exonuclease 3-5. Cc polymerase nh Pwo hay Pfu, cphn lp t Archaea c c ch sa sai v c th lm gim mt cch ng k s

t bin xy ra trong chui DNA c sao chp. Ngy nay, s kt hp gia Taqv Pfu c th cung cp c tin cy cao ln s khuch i chnh xc ca DNA.




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7.3.4. Cc ng dng ca PCR

Cc ng dng cbn ca PCR c th k l: nhn dng du vn tay ditruyn (genetic fingerprinting), chn on bnh di truyn, kim tra huytthng, tch dng gene (cloning), gy t bin im nh hng (site-

directed mutagenesis), phn tch mu DNA c, xc nh allele ca t binhoc a hnh c mt c th thng qua s dng PCR c th cho allele(allele-specific PCR), so snh mc biu hin ca gene nhRT-PCR vReal-Time PCR.

Sn phm PCR c th c xc nh thng qua kch thc ca nbng phng php in di trn bn gel agarose (agarose gelelectrophoresis). Kiu in di ny l mt quy trnh bao gm vic bmDNA ln trn bn gel agarose v sau cho mt dng in chy qua bngel. Kt qu l cc si DNA b hn s di chuyn nhanh hn cc si lnhn dc theo bn gel hng v dng in dng. Kch thc ca snphm PCR c th xc nh bng cch so snh vi mt thang DNA (DNAladder), vn c cha cc on DNA c kch thc bit cng nm trongbn gel (Hnh 1.13).

(A) (B)

Hnh 1.13 (A) Sn phm PCR c i chiu vi ging DNA trn bn gelagarose. Thang DNA (ging 1), sn phm PCR nng thp (ging 2), v nng cao (ging 3). Ngun: Helmut W. Klein, Institute of Biochemistry,University of Cologne, Germany.

(B)in di cc on DNA c khuych i bng PCR: (1)- Ngi cha, (2)-Ngi con, (3)-Ngi m. a con c di truyn mt s ch khng phi tt c

du vn tay ca mi mt b m; y cho thy mt du vn tay mi, c nht.V. Vai tr ca vi sinh vt trong i sng v sn xut




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1. Vi khun c ch v vi khun gy hi

Vi khun c th c ch hoc c hi cho mi trng v ng vt, k ccon ngi. Vai tr ca vi khun trong gy bnh v truyn bnh rt quantrng. Mt s l tc nhn gy bnh (pathogen) gy ra cc bnh nh: un

vn, st thng hn, giang mai, t, bnh ly qua thc phm v lao. Nhimkhun huyt, l hi chng nhim khun ton cth gy sc v gin mch,hay b phn gy ra bi cc vi khun nh streptococcus, staphylococcushay nhiu loi Gram m khc. Mt s nhim khun c th lan rng ra khpcth v trthnh ton thn. thc vt, vi khun gy m l, chy l vho cy. Cc hnh thc ly nhim gm qua ti p xc, khng kh, thcphm, nc v cn trng. Vt ch (host) b nhim khun c th tr bngthuc khng sinh, c chia lm hai nhm l dit khun (bacteriocide) vkm khun (bacteriostasis), vi liu lng m khi phn tn vo dch cthc th tiu dit hoc km hm s pht trin ca vi khun.

Trong t, cc vi sinh vt sng trong nt r (rhizosphere) bin nit

thnh ammoniac bng cc enzyme ca chnh mnh. Mt s khc li dngphn t kh nitlm ngun m cho mnh, chuyn nitthnh cc hp chtca nit; qu trnh ny gi l qu trnh cnh m. Nhiu vi khun ctm thy sng cng sinh trong c th ngi hay cc sinh vt khc. V dnh s hin din ca cc vi khun cng sinh trong rut gi gip ngn cns pht trin ca cc vi sinh vt c hi.

Vi khun c kh nng phn gii cc hp cht hu cmt cch ngkinh ngc. Mt s nhm vi sinh "chuyn ha" ng mt vai tr rt quantrng trong vic hnh thnh cc khong cht t mt s nhm hp cht huc. V d, s phn gii cellulose, mt trong nhng thnh phn chim a strong m thc vt, c thc hin ch yu bi cc vi khun hiu kh thucchi Cytophaga. Kh nng ny cng c con ngi ng dng trong cngnghip v trong ci thin sinh hc (bioremediation). Cc vi khun c khnng phn hy hydrocarbon trong du m thng c dng lm schcc vt du loang v.v.

Vi khun cng vi nm men v nm mc c dng ch bin ccthc phm ln men nh ph-mai, da chua, nc tng, da ci bp(sauerkraut), gim, ru, v yoghurt. S dng cng ngh sinh hc, cc vikhun c thc "thit k" (bioengineer) sn xut thuc tr bnh nhinsulin, hay ci thin sinh hc i vi cc cht thi c hi.

2. Nhng ch li bt ngun tcc vi sinh vt v cc hotng ca chng

Ni chung, vi nng lc chuyn ho mnh m v kh nng sinh snnhanh chng ca cc vi sinh vt cho thy tm quan trng to ln ca chng




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trong thin nhin cng nh trong cc hot ng ci thin cht lng sngca con ngi nhhiu bit v cc hot ng sng ca chng (Bng 1.2).

Ngoi ra, cc vi sinh vt cn l i tng cho cc nghin cu cbnca di truyn hc. T dn ti s hnh thnh cc lnh vc di truyn hc

sinh-ho v di truyn hc vi sinh vt trong thp nin 1940, hai nn tngchnh cho s ra i ca di truyn hc phn t v cng ngh DNA ti thp sau ny (nh cp Bi mu).

Bng 1.2 Nhng ch li bt ngun tcc vi sinh vt v cc hotng ca chng(Theo McKane v Kandel 1996)

*Trong cc mi trng t nhin

Hot ng ch li

Phn hu xc hu c Quay vng cc cht dinh dng trong sinh quyn.

Sn xut oxy Cc vi sinh vt (VSV) quang hp thu sinh to ra

khong mt na oxy ca kh quyn.

Ngn nga dch bnh Cc bnh cn trng c th gip phng tr cc dch

bnh ph hoi ma mng.

Cnh nit Mt vi vi khun bin i nitbu kh quyn thnh

ra mt dng m thc vt c th d dng s dng.

S sng st ca ccloi nhai li

Cc vi sinh vt tiu ho cellulose trong rut tru b,cu ...cho php ng vt s dng thc n m n

khng th tiu ho bng cch khc.

Cc chui thc nthu sinh

Cc vi sinh vt quang hp nc cung cp nnglng v dinh dng t chng duy tr v nui

sng cc tt c cc sinh vt tiu th thu sinh.Cc chui thc n

trong tS phn hu ca VSV cung cp cc cht dinh dng

cho cc sinh vt quang hp m n h trcc chui

thc n thuc t kh. Mt sng vt t sngbng cc sinh vt thu sinh, qua kt ni ccchui thc n nc v t.

Ph hu cc c t Cc sn phm gy c ca mt s sinh vt c kh

c mt cch t nhin nhhot ng ca VSV.

*i vi ng dng ca con ngi

Hot ng ch li

Ln men cn Sn xut bia, ru vang v cnSn xut khng sinh Nhiu dc phm c dng chng li cc bnh




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ngi v cc ng vt khc.

Cc thuc dit bnhbng sinh hc

Cc VSV c kh nng c bit git cn trng cdng thay th cc ho cht chng li cc dch

bnh gy hi ma mng m khng phi git cc

ng vt c ch hoc lm nhim mi trng.X l rc thi sinh hc Cc VSV c dng lm sch cc cn b du v

phn hu cc c t v cc ph liu cng nghip.

Cng ngh sinh hc Cho php cc nh khoa hc to ra cc ni VSV mi

c cc c tnh c o c th dng trong sn xutinsulin hoc cc ch phm y-sinh hc khc...

Sn xut thc phm Yaourt, phomat... v nhiu thc n khc c 'rippen'

bng s ln men vi sinh vt.

SX ho cht c/nghip Cn, cc amino acid, vitamin, cc enzyme hu ch

Protein n bo B sung thc phm ha hn cu i khp ton cu.

Cc VSVsinh trng trn cc hp cht hu cngin (thm ch cc cht thi) c th sn xut nhanhthc phm cht lng cao dng trong chn nui...

Sn xut cc vaccine Cc vt gy bnh sinh trng qua nui cy nh l

ngun vt liu ngoi lai c s dng dng bini (khng gy bnh) tim chng cho ngi vkch thch min dch chng li bnh tng ng.

Test Ames i vi ccho cht gy ung th

Cung cp test xc nh nhanh hng ngn ho cht,nhs dng kh nng ca chng gy cc bin

i di truyn vi khun nh l mt cht ch th v

tim nng gy ung th ca chng.Khai thc mng v

uraniumCc vi khun phn hu cho php cc hot ng

khai thc kim loi t qung m bng cch khc

hiu qu kinh t rt thp. Cc vi khun ny cungcp khong 10% lng ng c khai thc.

X l nc thi Hot ng VSV gip lm sch nc thi v git cc

sinh vt gy bnh trc khi a tr li mi trng.

Cc ngun nng lng Kh methane t nhin v ethanol l hai sn phm cht

t ca cc VSV sinh trng bng cch bin isinh hc bin cc ph thi thnh nhin liu.

*Cc m hnh cho nghin cu c bnKhm ph Cc ng gp ca vi sinh vt




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DNA l vt cht ditruyn

Cc vi khun v virus cung cp cng c cho ccth nghim chng minh vt cht di truyn l DNA.

Cch biu hin gene Cc vi khun v virus c dng tm hiu cch

thc thng tin m ho trong cc gene to ra cc

protein c th m t hnh thnh nn tnh trng.M di truyn Cc vi khun cung cp cc enzyme cho cc nghin

cu dch m di truyn bng cch thit k cc trnht RNA c th v qua gii tt c m di truyn.

Cc con ngchuyn ho cbn

Nhiu con ng sinh ho (chu trnh Krebs chnghn) m c khm ph v tin hnh cc vi

khun l trung tm ca s chuyn ho hu ht ttc cc sinh vt (k c con ngi).

Enzyme phin mngc

Mt enzyme cc virus gy bnh AIDS v mt svirus gy ung th cho php cc virus RNA hp

nht cc bn sao vt cht di truyn ca chng voDNA ca cc nhim sc thng vt.

Cc enzyme gii hnv splicing gene

Cc enzyme vi khun cung cp cch m cc nhkhoa hc li dng chuyn cc gene t sinh vt

ny sang sinh vt khc, qua mra cnh ca chok thut di truyn v cc i l mi cho nghin cudi truyn cbn.

Cu hi v Bi tp

1. Hy cho bit cc c im chung trong cu to v hot ng sng ca

cc vi sinh vt v ngha ca chng.2. S khc nhau gia cc t bo prokaryote (eubacteria v archaeobac-teria) v eukaryote l g?

3. Hy cho bit cc ch li ca vi sinh vt i vi mi trng t nhin, ivi cc ng dng ca con ngi?

4. Chng minh rng cc vi sinh vt l i tng quan trng trong ccnghin cu ca di truyn hc v sinh hc phn t.

5. Cc vi sinh vt c tm quan trng nh th no trong s pht trin ca kthut di truyn v cng ngh DNA ti t hp?

6. C nhng phng php no c s dng trong phn tch di truyn hc

vi sinh vt? Th no l phng php phn tch b sung? Cho v d v nucc kh nng ng dng ca chng trong phn tch di truyn vi sinh vt.


giao trinh di truyen hoc vsv va ung dung _ hoang trong phan, truong thi bich phuong

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