B GIO DC V O TO TRNG I HC NNG LM TP.

HCM B MN CNG NGH SINH HC ***000***

KHA LUN TT NGHIP

THIT LP QUY TRNH IN DI PROTEIN SDS PAGE V NG DNG NH GI PHN NG CA CY LA I VI THUC SINH HC KCH KHNG

Ngnh hc: CNG NGH SINH HC Nin kha: 2003 2007 Sinh vin thc hin: L NGUYN PHC SN

Thnh Ph H Ch Minh 9/2007

B GIO DC V O TO TRNG I HC NNG LM TP.HCM B MN CNG NGH SINH HC ***000***

THIT LP QUY TRNH IN DI PROTEIN SDS PAGE V NG DNG NH GI PHN NG CA CY LA I VI THUC SINH HC KCH KHNG

Gio vin hng dn: TS. L NH N

Sinh vin thc hin: L NGUYN PHC SN

Thnh Ph H Ch Minh 9/2007

LI CM T

c c thnh qu ngy hm nay, trc tin, con xin cm n b m v gia nh to mi iu kin thun li con c th yn tm hc tp, nghin cu v hon thnh tt lun vn ny. TI CHN THNH CM N Ban Gim Hiu trng i hc Nng Lm Thnh ph H Ch Minh B mn Cng ngh Sinh hc, i hc Nng Lm Thnh ph H Ch Minh B mn Bo v Thc vt, i hc Nng Lm Thnh ph H Ch Minh Vin nghin cu Cng ngh Sinh hc v Cng ngh Mi trng, i hc Nng Lm Thnh ph H Ch Minh to mi iu kin thun li cho ti trong sut thi gian thc tp hon thnh kha lun tt nghip ny. TI TRN TRNG BIT N Thy L nh n tn tnh hng dn, to mi iu kin gip chng ti hon thnh kha lun ny. Thy Bi Cch Tuyn, thy Bi Minh Tr to iu kin cho chng ti thc tp ti Vin nghin cu Cng ngh Sinh hc v Cng ngh Mi trng. Anh Nguyn Vn Lm nhit tnh ch dn ti trong sut qu trnh thc hin ti. Cc anh ch , Vin nghin cu Cng ngh Sinh hc v Cng ngh Mi trng. Cc anh ch B mn Bo v Thc vt, trng i hc Nng Lm Thnh ph H Ch Minh. Cc thnh vin lp Cng ngh Sinh hc 29 ng vin, gip chng ti trong thi gian thc tp.

iii

TM TT
ti Thit lp quy trnh in di protein SDS- PAGE v ng dng nh gi phn ng ca cy la i vi thuc sinh hc kch khng do L Nguyn Phc Sn thc hin t 15/03/2007 n 31/08/2007 ti b mn Bo v Thc vt, khoa Nng hc, trng i hc Nng Lm Thnh ph H Ch Minh v phng Cng ngh Sinh hc Thc vt, vin Nghin cu Cng ngh Sinh hc v Cng ngh Mi trng, trng i hc Nng Lm Thnh ph H Ch Minh. ti l mt hng nghin cu pht trin ca phng php sinh hc phn t trong iu kin phng th nghim, m bc u l hon thin phng php SDS PAGE p dng trn protein ca l la. Quy trnh tri qua cc giai on: 1. Chun b mu l la 2. Ly trch protein ca l la 3. in di SDS PAGE mu protein ly trch c t l la. Tm li, ti ny thit lp c quy trnh SDS PAGE trn i tng l protein ca l la nhng vn cha hon thin c quy trnh mc protein c tinh sch cao. Kt qu trong ti ny l c s ban u trong vic hon thin phng php phn tch proteomics phc v cho nhng nghin cu ng dng trong cng tc bo v thc vt.

iv

MC LC
LI CM T .......................................................................................................... iii TM TT ............................................................................................................... iv MC LC ............................................................................................................... v DANH SCH CC CH VIT TT .................................................................. viii DANH SCH CC HNH V CC BNG ......................................................... ix Chng 1 M U ................................................................................................. 1 1.1 t vn .......................................................................................................... 1 1.2 Mc ch............................................................................................................. 2 1.3 Yu cu ............................................................................................................... 2 Chng 2 TNG QUAN TI LIU ....................................................................... 3 2.1 Vi iu s lc v cy la ................................................................................ 3 2.1.1 Ngun gc v phn b................................................................................... 3 2.1.2 c im hnh thi ca la ............................................................................ 3 2.1.3 c im ht la............................................................................................ 5 2.1.4 iu kin ht la ny mm ....................................................................... 5 2.1.4.1 Nc ......................................................................................................... 5 2.1.4.2 Nhit .................................................................................................... 5 2.1.4.3 Khng kh ................................................................................................. 6 2.2 Mt s phng php tch chit protein tng s t thc vt ............................... 6 2.2.1 Quy trnh c s dng SDS ............................................................................ 7 2.2.2 Quy trnh c s dng phenol ......................................................................... 7 2.2.3 Quy trnh c s dng PMSF.......................................................................... 8 2.3 Phng php in di trn gel polyacrylamide ................................................... 8 2.3.1 S lc v lch s gel polyacrylamide v s pht trin ca phng php in di trn gel polyacrylamide ........................................................................................ 8 2.3.2 Gel polyacrylamide ....................................................................................... 9 2.3.3 Phng php SDS- PAGE .......................................................................... 10

2.3.4 Nhum gel sau khi in di .......................................................................... 11 2.3.5 Mt s yu t cn quan tm trong in di trn gel polyacrylamide ............ 13 2.3.6 Phng php in di hai chiu .................................................................... 14 2.4 Mt s nghin cu lin quan n in di protein SDS- PAGE ....................... 16 2.4.1 Nghin cu trong nc ............................................................................... 16 2.4.2 Nghin cu ngoi nc ............................................................................... 17 Chng 3 VT LIU V PHNG PHP TIN HNH .................................. 18 3.1 Thi gian v a im thc hin ti ............................................................. 18 3.2 Ha cht v vt liu dng trong th nghim ..................................................... 18 3.2.1 Thuc sinh hc ............................................................................................ 18 3.2.2 Ha cht dng trong ly trch........................................................................ 18 3.2.3 Ha cht in di .......................................................................................... 19 3.2.4 Trang thit b th nghim ............................................................................. 19 3.3 Phng php tin hnh nghin cu .................................................................. 19 3.3.1 Chun b mu v ly mu ............................................................................ 19 3.3.1.1 Chun b mu la ................................................................................... 19 3.3.1.2 X l thuc v ly mu .......................................................................... 20 3.3.2 Ly trch protein tng s t l la ................................................................. 21 3.3.2.1 Cc bc ly trch protein theo quy trnh c s dng SDS ..................... 21 3.3.2.2 Cc bc ly trch protein theo quy trnh c s dng phenol .................. 21 3.3.2.3 Cc bc ly trch protein theo quy trnh ci tin c dng SDS ............. 22 3.3.3 in di kim tra mu protein ly trch ..................................................... 23 3.3.3.1 Chun b ha cht ................................................................................... 23 3.3.3.2 Chun b mu v chy in di ................................................................ 24 3.3.3.3 Tin hnh in di v xem kt qu .......................................................... 24 3.3.4 Th nghim kho st chn iu kin in di ............................................... 25 3.3.5 Kho st nng gel ................................................................................... 25 3.3.6 in di cc mu protein kim tra phn ng ca cy la i vi thuc sinh hc kch khng ................................................................................................ 26

vi

Chng 4 KT QU V THO LUN .............................................................. 27 4.1 Kt qu ly trch protein tng s ........................................................................ 27 4.2 Kt qu kho st chn iu kin in di .......................................................... 29 4.3 nh hng ca nng gel n kt qu in di .............................................. 30 4.4 nh gi phn ng ca la i vi thuc sinh hc .......................................... 32 4.4.1 Kt qu in di ca tt c cc mu protein trong 12 nghim thc .............. 32 4.4.2 Kt qu in di mu protein ca la l II................................................. 33 4.4.3 Kt qu in di mu protein ca la l III ............................................... 34 Chng 5 KT LUN V NGH ................................................................... 35 5.1 Kt lun ............................................................................................................ 35 5.2 ngh ............................................................................................................. 35 TI LIU THAM KHO ...................................................................................... 36

vii

DANH SCH CC CH VIT TT

2- DE AFLP APS CBB DTT EDTA HPLC IEF KDa PCR PVDF RADP RFLP SDS PAGE Two- dimensional electrophoresis Amplified Fragment Length Polymorphism ammonium persulfate Coomassie Brilliant Blue Dithiotheitol Ethylene Diamine Tetra acetic Acid High performance liquid chromatography Isoelectric focusing kilo Dalton Polymerase Chain Reaction Polyvinylidene difluoride Randomly Amplified Polymorphic DNA Restriction Fragment Length Polymorphism Sodium dodecyl sulfate- polyacrylamide gel electrophoresis SNP SSR STS TEMED SDS TE w/v Single Nucleotide Polymorphism Simple Sequence Repeats Sequence Tagged Site N,N,N,N-tetramethylenediamide Sodium dodecyl sulfate
Tris EDTA

Weight for volume

viii

DANH SCH CC HNH V CC BNG

Hnh 2.1 Hnh thi cy la (Oryza sativa L.) ........................................................... 4 Hnh 2.2 Cu trc protein trc v sau khi lm bin tnh bi SDS ....................... 11 Hnh 3.1 La mm trong hp nha 2 ngy ............................................................ 20 Hnh 4.1 Kt qu in di SDS PAGE mu protein ly trch ca cc quy trnh ..... 28 Hnh 4.2 Kt qu kho st iu kin in di ph hp vi protein l la................ 30 Hnh 4.3 Kt qu in di SDS PAGE cc mu protein kho st nng gel...... 31 Hnh 4.4 Kt qu in di SDS PAGE cc mu protein ca la trong 6 nghim thc (nghim thc 1, 2, 3,4, 5 v 6)................................................................. 32 Hnh 4.5 Kt qu in di SDS PAGE cc mu protein ca la trong 6 nghim thc (nghim thc 4, 5, 6, 7, 10 v 11). ........................................................... 32 Hnh 4.6 Kt qu in di SDS PAGE cc mu protein ca la trong 5 nghim thc (nghim thc 5, 8, 9, 10 v 12). ............................................................... 32 Hnh 4.7 Kt qu in di SDS PAGE mu protein ly trch t l II...................... 33 Hnh 4.8 Kt qu in di SDS PAGE mu protein ly trch t l III .................... 34 Bng 3.1 Bng b tr phn l th nghim theo nghim thc .................................. 20 Bng 3.2 Thi gian x l thuc trc khi ly mu theo tng nghim thc .......... 21 Bng 3.3 Th nghim kho st iu kin in di .................................................... 25

ix

Chng 1 M U
1.1 t vn La l cy lng thc quan trng, l nhu cu khng th thiu i vi con ngi, c bit cc nc Chu . Vit Nam vi c im kh hu nhit i gi ma, ma nhiu, m cao, rt thch hp cho vic trng la. Mt khc ngi Vit Nam cn c truyn thng canh tc cy la t rt lu i. Trc y, vi iu kin vt cht cn thiu thn, lng thc khng n ngi ta ch c nhu cu c n no. Hin nay vi mc sng ngi dn ngy cng c nng cao th ngoi nhu cu n no, vic n ngon, c dinh dng cao dn tr nn l nhu cu quan trng i vi mi ngi. Nhng hin nay cc bnh trn cy trng ngy cng cng honh hnh d di, gy cn tr trong sn xut v pht trin ngnh nng nghip. Ngy nay, vi nhng thnh cng ln trong cng ngh sinh hc trn th gii cng nh trong nc, nh hng nghin cu ng dng cng ngh sinh hc trong cng tc bo v thc vt c s chuyn hng r rt. Trc ht l nhng u t rt ln khai thc v ng dng cng ngh sinh hc trong vic phng tr nm bnh, su ry hi, virus, c di v cc loi cn trng gy hi trong sn xut nng nghip. Theo hng ng dng cng ngh sinh hc trong nghin cu cc ch phm sinh hc c bn cht l cc polyamin ang ngy cng pht trin. y l mt hng nghin cu mi c th tr thnh mt cng ngh sch trong phng tr su bnh hi nc ta nu c u t pht trin. Cng vi nhng thnh cng ln trong cng ngh sinh hc th cc k thut v sinh hc phn t ra i nh phng php Southern blot, RFLP (Restriction Fragment Length Polymorphism), RADP (Randomly Amplified Polymorphic DNA), SSR (Simple Sequence Repeats), SNP (Single Nucleotide Polymorphism), STS (Sequence Tagged Site), PCR (Polymerase Chain Reaction), AFLP (Amplified Fragment Length Polymorphism) to nn mt s chuyn bin r rt trong cc hng nghin cu v acid nucleic. Tuy nhin cc phng php sinh hc phn t

trn ch c th tp trung nghin cu v gen v cu trc gen m khng th cho ta bit c s biu hin ca gen. l nguyn nhn dn n s ra i ca ca cc phng php nh sc k lng cao p (HPLC), SDS PAGE, Western blot, 2 D PAGE. V nguyn tc th chng c mi lin h vi nhau v l cu ni cho nhau. Trong , phng php SDS PAGE c xem l mt k thut n gin m em li hiu qu cao v c nhiu ng dng trong sinh hc phn t. Trong phm vi cho php chng ti thc hin ti: Thit lp quy trnh in di protein SDS- PAGE v ng dng nh gi phn ng ca cy la i vi thuc sinh hc kch khng. 1.2 Mc ch Thit lp quy trnh ly trch protein tng s t l la. S dng phng php in di protein SDS PAGE xc nh phn ng ca cy la i vi thuc sinh hc kch khng. 1.3 Yu cu Ly trch c protein tng s t l la. Hon thin k thut in di SDS PAGE trn protein ca la. So snh xem s khc bit v protein tng s gia mu la khng x l thuc polyamin vi mu la x l thuc polyamin nng khc nhau. So snh s khc bit v protein ca cc mu la c x l thuc polyamin vi nng v thi gian tc ng khc nhau.

Chng 2 TNG QUAN TI LIU

2.1 Vi iu s lc v cy la 2.1.1 Ngun gc v phn b La l loi thc vt c canh tc t rt lu i. V ngun gc ca la trng cha c hiu r rng v c nhiu kin khc nhau. Hin nay trn th gii c h ai cy la trng. La trng Oryza sativa c thun ha Chu , nn c gi l la trng Chu . La trng Oryza glaberrima c thun ha Chu Phi nn c gi l la trng Chu Phi (Bi Huy p, 1999). Cc nh kho c hc M cho rng la trng xut hin rt sm cch y khong hn 9 10 nghn nm. Nhiu nh kho c hc khc cho l la trng xut hin cch y 6.000 nm, la trng chu Phi (Oryza glaberrima) xut hin cch y 3.500 nm. Cn mt s tc gi khc cho l la trng chu Phi xut hin rt mun, ch sau cng nguyn, cch y khong 1.800 1.900 nm. (Bi Huy p, 1999) V mt phn b th cy la l loi thc vt c din tch phn b kh rng, loi O. sativa phn b ko di t v 35 Nam n 50 Bc trn 110 quc gia. Din tch gieo trng chim khong 10% din tch t nng nghip trn th gii (144 triu ha). La go c trng t cao bng mc nc bin n cao 3.000 m so vi mc nc bin. La c trng t vng n i n vng nhit i. La go c th c trng trn nhiu loi t khc nhau t chua, t kim, t nhim phn. Nhng dng la c th c phn chia thnh 3 nhm sinh thi, Indica ( vng nhit i v cn nhit i), Javanica ( c trng Indonesia) v Japonica (vng n i). C hai dng c canh tc nhiu nht l Indica v Japonica. (Bi Huy p, 1999). 2.1.2 c im hnh thi ca la La trng l cy thn tho, h thng r chm, sng hng nm, c thi gian sinh trng thay i ty theo ging la, v trng, ni trng v cc iu kin sinh thi thi gian trng ko di trong khong t 75 250 ngy.

Thn cao t 70 150 cm, mt s ging la ni c thn cao 2 3 m, hay 5 6 m (la ni Bangladesh). t thn nhn v cch nhau bi nhng lng di, ngn khc nhau. Phin l thng hnh u, u l nhn, b mt phin l v mp l u rp. B l c tha la, l da hnh mi mc hay ch i, cc u ch u nhn. La thng to ra thnh nhiu nhnh (dnh la: tillers), bao gm cng v l c hoc khng c bng (panical). Nhnh bc 1 xut hin t nhng t gn thn chnh v nhnh bc hai, bc ba xut hin t nhng nhnh bc mt ny. L mc lin tip trn thn bao gm b l bao ly thn v phin l. C l ni gia phin l v b l c mt li b v 2 tha la t c l. Bng mc trn t trn cng ca thn t bn trong l c v mang nhiu hoa trong mt bng con. Cm hoa l mt chm tha, thng, hp, u hi cong xung, di 15 30 cm hoc di hn. Hoa nh hnh thun di, my hoa thun di hnh mi mc, hoa mu hng vng hay mu tm, c hoa lng tnh, t th phn. Hoa c 6 nh c mnh, bao phn di, bu hoa c vi, nhy ngn v hai u nhy c lng t [11].

Hnh 2.1: Hnh thi cy la (Oryza sativa L.) V c bn, la l cy thch nghi vi iu kin c nc. Ba giai on chnh trong qu trnh sinh trng v pht trin ca cy la theo vin nghin cu quc t IRRI l: (i) Giai on tng trng: t khi gieo ht cho n khi cy la lm ng. (ii)

Giai on sinh sn: t khi la lm ng n khi la tr bng. (iii) Giai on la chn: t khi la tr bng n khi la chn. (V Tng Xun, 1986) 2.1.3 c im ht la Ht la l mt loi qu, thuc loi qu dnh. Hnh thi v mu sc ca ht la ty ging la. La tin (Indica) c ht di cn la cnh (Japonica) th c ht trn. a s ht la c mu vng sng, mt s c mu vng sm hoc nu en nh np cm. (L Minh Trit, 2003) V c bn th cu trc ht la gm: V tru gm tru trn v tru di. Cm gm biu b, qu b v chng b (nucellus). Phi nh gm c lp aleuron v phi nh tch t tinh bt. Mm cy gm c phi (mm) l, phi r v tr phi gia phn di ca ht. Ht la l non so th tinh chn, c hai my tru nh trn v di, hai v tru trn v di, cung tru phn di ca ht v ui cht ht (ngn hoc di). Mt ht la c trng lng t 12 44 mg 0% m . 2.1.4 iu kin ht la ny mm 2.1.4.1 Nc Ht la sau khi thu hoch v phi kh tn tr, c cha mt lng nc nht nh, di 14% trng lng kh. Ht mun ny mm c th lng nc trong ht phi t khong 22 25%. Do cn phi ngm ht trc khi ging ht ht lng nc cn thit. Thi gian ngm ht lu hay mau ty thuc vo nhit ca nc ngm v khng kh, ty ht ging mi hay c. Nhit khng kh cao, nc m v v ht mng th ht ht nc nhanh, khng cn ngm qu lu, ht ht nhiu nc, ha tan, lm tiu hao cht d tr trong ht, ng thi lm ht b chua, nm bnh d pht trin, ht d b thi v ny mm yu. (V Tng Xun v ctv, 1986) 2.1.4.2 Nhit Ht la ny mm thch hp nhit 30 350C. Nhit cao trn 400C hoc thp hn 170C ht kh ny mm, nu ko di c th lm h mm. V vy sau khi ngm, cn ht nhit thch hp. Ma dm hoc ma lnh ht kh ny mm nn cn k. C th ti thm nc m lm tng nhit nu cn thit. Trong

qu trnh cn trn u ng hoc xc tr cho nhit phn phi u th ht mi mc u. (V Tng Xun v ctv, 1986) 2.1.4.3 Khng kh So vi nhiu loi ht ging khc th ht la khi ny mm cn t oxi hn, nhng khng th thiu. Nu ngp trong nc su 15 cm, ht la cng c th ny mm c. Nhng trong iu kin thiu oxi th mm la nh ra trc v vn di hn, r s t v mc chm. V s pht trin ca r la cn nhiu oxi hn, do , mun iu khin khng cho r ra di, ta c em ht ngm trong nc, ngc li, ta tip tc v thng xuyn trn cho ht oxi, r s ra u v khe. Ngm ging khng tt th t l mc mm thp, mc mm khng u, nh hng xu n s pht trin cy la ngay t u, la pht trin khng tt. (V Tng Xun v ctv, 1986) 2.2 Mt s phng php tch chit protein tng s t thc vt Trong cc nghin cu sinh hc phn t v protein bc u u phi tch chit cho c protein tng s tin hnh nhng th nghim tip theo. Vn quan tm y l lm sao thu nhn c lng protein v cc protein thu c gi c trng thi nguyn vn, khng b nh hng bi cc tc nhn l ha. Thng th vic tch chit protein tri qua mt s bc c bn sau. 1. Ph v vch t bo v gii phng protein. Protein nm trong t bo cht ca t bo nn phi ph v mng t bo gii phng protein. Bc ny thng thng c thc hin bng cch nghin mu ng thi thm mt s ha cht xc tc ph v mng t bo nh 2 mecaptoethanol. 2. Loi b thnh phn khng mong mun trong mu. bc ny thng l thm mt s mui lm dung mi ha tan protein nh sodium chlorid, sodium phosphate. 3. Ta protein v thu nhn protein dng kh. Bc ny thng c tin hnh khi thc hin xong cc bc c bn trong ly trch. Lc ny protein ang c ha tan trong dung mi, khi thm cc cht ta protein nh acetone, amonium sulfate sau em ly tm s thu c protein di dng c c. Phi kh v thm cht bo qun gi protein trnh khi s phn hy ca cc enzyme.

V c bn th vic tch chit protein tng s phi tri qua nhng bc trn. Nhng vi nhng i tng khc nhau th s c nhng quy trnh tch chit c th khc nhau. V vy, trong nghin cu c c quy trnh tch chit protein tng s n nh, cn tin hnh nhiu th nghim khc nhau t rt ra quy trnh thch hp vi i tng cn nghin cu. Di y l mt s quy trnh tch chit protein. 2.2.1 Quy trnh c s dng SDS (Samuel S.M.Sun, 1994) 1. Nghin 0,2 g mu l vi 1 ml dung dch ly trch (0,25 M NaCl, 1% SDS, 1% 2 mercaptoethanol, 0,05M sodium phosphate pH 7,5) thnh dch lng. 2. Chuyn dch nghin vo mt eppendorf 1,5 ml, v ly tm vi tc 14.000 vng/pht trong 10 pht nhit phng. 3. S dng pipette ht dch ni vo mt eppendorf 1,5 ml mi; trnh ht lp lipid trn cng. 4. Ly tm dch ni vi tc 14.000 vng/pht trong 10 pht nhit phng. 5. Tip tc ht dch ni vo mt eppendorf khc. 6. Thm vo 500 l acetone 80%, ly tm 5.000 vng/pht trong 5 pht 40C. 7. b dch thu nhn kt ta, tr mu protein 40C cho ti khi s dng. 2.2.2 Quy trnh s dng phenol (Hurkman v Tanaka, 1986) 1. Nghin 1g mu l ti trong nit lng thnh bt mn. 2. Thm 2,5 ml Tris- phenol pH 8,8 v 2,5 ml dch ly trch (0,1 M Tris-HCl pH8,8, 10 mM EDTA, 0,4% 2 mercaptoethanol, 0,9 M sucrose). Tip tc nghin mu trong 30 giy, sao chuyn dch nghin vo trong ng Falcon 15 ml. Pha long dch mu vi t l 1:5 trong dch ly trch v phenol pH 8,8. 3. Vortex hn hp dung dch trong ng Falcon n khi ng nht. 4. Ly tm 5.000 vng/pht trong 10 pht 40C. 5. Loi b phenol v thm vo 2,5 ml dch ly trch v 2,5 ml phenol. Vortex cho hn hp ng nht. Ly tm 5.000 vng/pht trong 10 pht 40C. 6. Loi b phenol, thm 5 ml 0,1 M amonium acetate trong methanol 100% ( tr mu -200C).

7. Vortex v mu -200C trong 1 gi hoc qua m. Thu kt ta bng cch ly tm 20.000 vng/pht trong 20 pht 40C. 8. Ra kt ta vi 0,1 M amonium acetate trong methanol, tip tc ra vi acetone 80%, v cui cng l ra vi ethanol 70%. Mi ln ra nh vy th mu -200C trong 15 pht. Sau b dch v thu kt ta. 9. Thm acetone 80% cha 10 mM DTT. Tr -800C cho ti khi s dng. 2.2.3 Quy trnh c s dng PMSF (Fan shuguo, 2002) 1. 1 g mu c nghin trong ci, lc v giy lc. 2. a vo dung dch nh sau (100 mM/l Tris HCl , 4 M/l EDTA, 0,5 M/l DTT v 1 M/l PMSF). 3. Mu c chuyn sang ng Falcon 15 ml v ly tm vi vn tc 5.000 vng/pht 40C trong 15 pht. 4. Ht ly dch ni chuyn sang ng Falcon 50 ml v thm vo 40 ml acetone. 5. Ly tm loi b EDTA, lipid, ng saccharide. 6. Ly tm mu vi vn tc 5.000 vng/pht 40C trong 5 pht. Loi b acetone, thm vo 40 ml acetone khc. (Ch c th thm (NH4)2SO4 cho kt ta). 7. Lp li 3 ln loi b acetone nh trn sau ly phn ta. 8. Thm acetone vo trn u. 9. Ht dch v chuyn vo eppendorf 1,5 ml sau ly tm vn tc 16.000 vng/pht nhit 40C trong thi gian 20 pht. 10. Loi b acetone, gi kt ta trong t m 370C trong 5 pht phi kh. 11. Pha kt ta li vi 1x TE v s dng. 2.3 Phng php in di trn gel polyacrylamide 2.3.1 S lc v lch s gel polyacrylamide v s pht trin ca phng php in di trn gel polyacrylamide in di l mt phng php c s dng ch yu trong nghin cu sinh hc phn t. N cho php phn tch trng lng ca cc phn t sinh hc, t ta c th xc nh c trng lng phn t ca chng.

Vo khong thp nin 1930 phng php in di trn gel u tin c bit n. Raymond v Winstraub (1959) ln u tin gii thiu v gel polyacrylamide. Ornstein v Davis (1964) thc hin in di trn gel polyacrylamide khng lin tc. Beber v Osborn (1969) gii thiu v tc nhn gy bin tnh protein v sodium dodecyl sulfate. Laemmli (1970) pht trin phng php SDS PAGE trong phn tch 28 thnh phn ca T4 phage. 2.3.2 Gel polyacrylamide Acrylamide l mt n phn t c cu trc: CH2=CHCONH2 v bisacrylamide c cu trc: CH2=CHCONHCH2NHCOCH=CH2. Acrylamide l mt c t thn kinh mnh, khi ht phi s lm m mt. N c kh nng thm qua da khi tip xc trc tip. Hiu qu c t s tch t dn. V vy khi thc hin lm th nghim vi acrylamide nn eo khu trang v gng tay. Tuy nhin dng c polymer ha thnh polyacrylamide th li khng c. Nhng khi tip xc trc tip vi n vn nn mang gng tay bo v v c th s cn mt lng nh acrylamide vn cha c polymer ha. (Sambrook v ctv, 2001) Gel polyacrylamide l gel c to thnh do s polymer ha cc n phn t acrylamide v N,N-methylene-bis-acrylamide. Kch thc l gel ph thuc vo chiu di ca chui polymer v mc polymer ha. Gel polyacrylamide c m t qua 2 c im: %C v %T. 1. Tng lng n phn t acrylamide cha trong gel (%T) %T= (g acrylamide + g bis- acrylamide) x 100% Tng th tch (ml) 2. Lng bis- acrylamide cha trong gel (%C) %C= g bis- acrylamide x 100% g acrylamide + g bis- acrylamide Qu trnh polymer ha c xc tc bi ammoniumpersulfate (APS), N,N,N,N-tetramethylethylenediamine (TEMED). S dng gel polyacrylamide chy in di th vic chun b gp nhiu kh khn hn so vi chy in di trn gel agarose.

10

S di chuyn ca cc phn t sinh hc trn gel ph thuc vo kch thc ca l gel, in tch ca phn t cn phn tch v in trng. Gii hn trng lng phn t ca cc phn t sinh hc m gel c th phn tch ty thuc vo nng acrylamide v bis-acrylamide: nu nng gel thp s to ra l gel ln, cho php phn tch cc phn t sinh hc c kch thc ln v ngc li nu nng gel cao s to ra l gel nh, cho php phn tch cc phn t sinh hc c kch thc nh (xem ph lc 2). 2.3.3 Phng php SDS-PAGE (Laemmli, 1970) Phng php SDS- PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) cn c gi l phng php in di trn gel acrylamide khng lin tc v mu protein c gy bin tnh (discontinuous and denaturing). SDS l mt tc nhn lm bit tnh v m tnh ha cc phn t protein. Trong k thut ny protein c x l vi cht ty SDS v tc nhn kh cu ni disulfite l 2mercaptoethanol hoc dithiotheitol (DTT). Khi x l protein vi 2- mercaptoethanol hay dithiotheitol th cc tc nhn ny s ct t cc cu ni disulfite (S S) ca protein, v vy protein t cu trc bc 2, 3, 4 c bin i thnh chui polypeptide bc mt v nh s c mt ca SDS tt c cc protein u c tch in m. Nh , di tc ng ca in trng s di chuyn trong gel ca cc phn t protein ch ph thuc vo kch thc, nhng phn t c kch thc ln s di chuyn chm hn nhng phn t c kch thc nh khi i qua mt l gel c kch thc nht nh. Di tc dng ca in trng cc phn t tch in m s di chuyn v cc dng. a cc protein trong mu v cng mt vch xut pht ng thi to ra s phn tch tt hn, ngi ta thng s dng phng php in di trn gel khng lin tc (discontinuous gel). Trong phng php ny gel gm 2 lp: Lp gel gom (stacking gel): Thng thng lp gel ny c nng gel thp vo khong 4 5%, v nm pha trn ni to ging bm mu. Khi c tc ng ca in trng cc protein c trong mu s bt u di chuyn t nhng ging mu qua lp gel ny. Nh , cc protein trong mu c dn li v to mt lp bng mng

11

nm ngay pha trn lp gel phn tch, to iu kin thun li hn trong vic phn tch protein [5]. Lp gel phn tch (separating gel) (lp di): Lp gel ny c nng gel cao hn so vi lp gel gom, v nm pha di. C tc dng chnh trong vic to ra cc bng protein c trng lng phn t, kch thc khc nhau t mt hn hp protein xut pht ban u [5]. K thut SDS- PAGE c th xc nh c nhng phn t protein c trng lng phn t t 10.000 200.000 Daltons. Nhng phn t protein c trng lng ln hn 200.000 Daltons thng c xc nh trn gel c nng acrylamide t hn 2,5% [5].

Hnh 2.3 Cu trc protein trc v sau khi bin tnh bi SDS 2.3.4 Nhum gel sau khi in di Nu protein khng c nh du phng x, th sau khi in di thy c s phn tch ca cc bng protein trn gel chng ta cn phi tin hnh nhum gel. S la chn phng php nhum c xc nh da trn nhiu yu t bao gm nhy, gii nhum, tin dng, tnh kinh t, thit b nhum, thit b c sn c. Coomassie Brilliant Blue R- 250 thch hp dng nhum cho hu ht cc protein v thng c s dng. i vi Coomassie Brilliant Blue G- 250 nn s dng nhum i vi nhng protein c trng lng phn t polypeptide thp. Nhum bc

12

(Rabilloud, 1990) l phng php rt nhy i vi protein v nn s dng nhum gel khi in di nh gi s tinh khit ca mu protein. Nhum bc cho php pht hin mt cch nhanh chng v nhy cc bng protein trn gel ngoi tr nhng protein khng c phn tch trn gel. a) Nhum Coomassie Brilliant Blue Nhum bng Coomassie Brilliant Blue R- 250 l phng php nhum ph bin nht thng c s dng pht hin protein trn gel polyacrylamide (Wilson, 1983). Rt d pht hin khi c khong 0,1 1 g protein trn bng. Dung dch nhum bao gm cc thnh phn nh: 0,1% Coomassie Brilliant Blue R- 250 (w/v), 40% methanol (v/v), 10% acid acetic (v/v). Ty theo hm lng protein m ta c th thay i t l thnh phn cc cht cng nh cht nhum. Nu trn gel cha polypeptide c trng lng phn t thp th ta c th nhum trong hn hp dung dch 0,1% Coomassie Brilliant Blue R- 250 (w/v), 50% methanol (v/v), 10% acid acetic (v/v) khong mt gi. Ngoi ra, chng cng c th c nhum vi 0,025% Coomassie Brilliant Blue G- 250 (w/v) trong methanol v acid acetic t 1 2 gi. b) Nhum bc Phng php ny c th pht hin protein nhy gp trm ln so vi phng php nhum bng Coomassie Brilliant Blue (Merril, 1990). Nhng bng cha khong 10 100 ng protein hay nucleic acid c th d dng c nhn thy. Thng thng ngi ta s dng mui bc nitrate. Cc bc tin hnh nhum bc phc tp hn rt nhiu so vi nhum bng Coomassie Brilliant Blue [9]. Cc bc nhum (Kyle Coachman, Jeff Ranish v Steve Hahn, 2002) 1. Ngm gel trong acid acetic 7% khong 10 pht. 2. Ngm gel trong methanol 50% khong 20 pht. 3. Ra gel trong nc siu sch khong 10 pht. 4. Chun b dung dch nhum: Chun b dung dch A gm: 0,8 g bc nitrate, 4 ml nc siu sch.Chun b dung dch B gm: 21 ml nc siu sch, 250 l NaOH 30%, 1,4 ml NH4OH 14,8 M.

13

5. Nh t t tng git dung dch A vo dung dch B. Ngay lp tc thm vo hn hp dung dch 76 ml nc siu sch. 6. Ngm gel trong dch nhum khong 15 pht. 7. Ra gel tht k li vi nc siu sch. 8. Ngm gel trong dch gii nhum (200 ml nc siu sch, 1 ml citric acid 1%, 100 l formaldehyde 37%) cho ti khi c th thy r cc bng protein. Thng thng t 2 15 pht. 2.3.5 Mt s yu t cn quan tm trong in di trn gel polyacrylamide a) H m in di v cc tc nhn gy bin tnh protein H m quyt nh nhu cu v nng lng v nh hng n s phn tch protein. H m bao gm m dng pha gel v m dng chy in di. H m ca gel khng lin tc u tin c pht trin bi Ornstein (1964) v Davis (1964). H ng dng phn tch protein nguyn th, tc l nhng protein cha b bin tnh. H m h s dng gm bn yu t: (i) gel gom dng m Tris HCl pH 6,8; (ii) gel phn tch dng m Tris HCl pH 8,8, (iii) m in di Tris glycine pH 8,3; (iv) mu cn phn tch dng m Tris HCl pH 6,8. Nhng vi h m c s dng nh vy th trong m hnh ny h khng th tin hnh phn tch c mt khong rng v trng lng protein. Da trn m hnh v h m ca Ornstein v Davis, Laemmli pht trin trong vic s dng thm tc nhn kh cu ni disulfite v SDS. Nh , vic phn tch protein trn gel ch ph thuc vo trng lng phn t ca protein. Do vic phn tch protein tr nn d thc hin hn v khng b ph thuc vo in tch ca cc phn t sinh hc. Tuy nhin m hnh ca ng cng gp mt s kh khn. Nhiu protein khng c phn tch theo mong mun khi s dng SDS PAGE (Andrews, 1986; Hames, 1990; See v Jackowski, 1989). Hin tng ny xy ra l do: (i) cu trc disulfite ca protein khng b tc ng, v protein khng b m tnh ha bo ha bi SDS. (ii) Nhng glycoprotein v lipoprotein trong mu khng b m tnh ha bo ha bi SDS, bi v thnh phn khng phi protein ca chng khng tng tc vi SDS.

14

b) iu kin nng lng Nng lng cung cp trong in di l in nng. C rt nhiu thit b ngun in khc nhau c sn xut phc v trong in di. Mt s thit b cho php chy t ng sau khi ta chnh cc thng s mong mun (hiu in th, cng dng in, thi gian in di) v cng c mt s thit b i hi phi c s gim st v thi gian (Allen v ctv, 1984). Gii hn chnh ca cc m hnh in di l kh nng lm tht thot nng lng khi in nng chuyn ha thnh nng lng in trng. S tht thot l do mt phn nng lng in c chuyn ha thnh nhit nng (Wooolley, 1987). Nhit nng ny c th gy ra mt s nh hng xu n kt qu in di nh: bng protein b mo, cong; tng s khuch tn; s hot ng tr li ca enzyme trong mu; s bin tnh protein. Do mt thit b in di tt phi m bo c s chuyn nhit t gel ra mi trng ngoi. Thng thng, in di nn chnh hiu in th (volt) v cng dng in (ampe) sao cho qu trnh in di xy ra nhanh chng m vn m bo c s phn tch protein mu. 2.3.6 Phng php in di hai chiu K thut in di hai chiu trn gel polyacryla mide (2 DE) l mt k thut c kh nng phn tch hn 1.800 protein trn cng mt bn gel, n l mt cng c quan trng c bn trong nhng th nghim m cc protein phc tp c tch ra phn tch ng thi.Cc bc c bn trong k thut 2 DE: Chun b mu v lm ha tan mu: Phng php chun b mu ty thuc vo mc ch nghin cu. Cc yu t nh tan, in tch, kch thc v im ng in ca protein u c lu trong qu trnh chun b mu. Vic chun b mu cng gip lm gim bt s phc tp ca mt hn hp protein. Cc on protein c dng trong chy in di hai chiu phi c duy tr trong dung dch m bin tnh c lc ion thp duy tr tnh nguyn vn ca protein v gi chng trng thi ha tan. in di theo chiu th nht: Protein c tch ra da trn im ng in pI ca chng, pH m protein khng tch in v khng di chuyn trong in trng.

15

K thut ny c gi l isoelectric focusing (IEF). i vi in di hai chiu, IEF l cng c tt nht thc hin phn tch protein theo nng pH. Protein l mt phn t ion lng tnh. Khi m protein c t trong mi trng vi gradient pH v b ph thuc vo mt trng in t, ban u n s di chuyn n in cc tri du vi n. Trong sut qu trnh tn ti ca chng qua gradient pH, protein s c th nhn hay mt proton. Cn bng trng thi ca protein: trng thi bnh thng protein c th mang in tch m, in tch dng hay cn bng v in tch. iu ph thuc vo s lng nhm ( NH2) v (COO) ca cc phn t amino acid cu to nn protein. Do , trc khi thc hin in di theo chiu th hai, cc phn t protein cn phi c gy bin tnh v ng nht v in tch. bc ny ngi ta x l protein vi 2-mercaptoethanol hay dithiotheitol, cc tc nhn ny s ct t cc cu ni disulfite (SS) ca protein, v vy protein t cu trc bc 2, 3, 4 tr thnh chui polypeptide v nh s c mt ca SDS tt c cc protein u c tch in m. Phn tch protein theo chiu th hai: Di tc dng ca in trng do protein mang in tch m sau khi cn bng trng thi nn protein s di chuyn t cc m sang cc dng. Khi protein di chuyn qua gel polyacrylamide, s c st ca cc ht acrylamide vi phn t protein to ra lc khng lm ngn cn s di chuyn ca protein. Protein c trng lng phn t cng ln th lc cn cng mnh. Nhum protein: c th nhn thy c protein sau khi in di, gel phi c nhum, tr khi chng ta nh du protein. S la chn phng php nhum c xc nh da trn nhiu yu t bao gm nhy, di nhum, tin dng, tnh kinh t, v thit b nhum, thit b c sn c. Thu nhn v phn tch hnh nh protein: Vic thu nhn hnh nh theo k thut s l mt trong nhng yu t quan trng lm cho phng php 2-DE l phng tin thc t trong vic thu nhn d liu trong nghin cu. Phn mm phn tch protein PDQuest (Bio Rad) cho php chng ta phn tch c cc hnh nh gel, ch gii cc im protein v thu nhn d liu. Phn mm PDQuest cng tham

16

gia iu khin cc thit b ghi nhn hnh nh v h thng thu thp protein im ca h thng thit b phn tch protein Spot Cutter. 2.4 Mt s nghin cu ng dng in di protein SDS- PAGE 2.4.1 Nghin cu trong nc ng dng in di protein SDS- PAGE Nm 2005, Trn Linh Thc (i hc Khoa hc t nhin Thnh ph H Ch Minh) s dng phng php SDS- PAGE trong ti Nghin cu cc h thng biu hin gen trong E.Coli sn xut protein ti t hp chng minh s biu hin ca gen v s hin din ca protein ti t hp. Nm 2005, nhm nghin cu (Khoa Nng nghip v Sinh hc ng dng, trng i hc Cn Th nghin cu ng dng phng php in di protein SDSPAGE nh gi thun ca mt s b ht ging rau cc loi. Ht ging rau a vo nh gi l loi ht lai F1 c sn xut trong nc v nhp khu. Nm 2005, V Th Hong Mi (i hc M Bn cng Thnh ph H Ch Minh) s dng phng php SDS- PAGE kt hp vi phng php Western blot trong ti Cc phng php chun on thng hn thng dng vi mc ch phn tch cc thnh phn protein khng nguyn S. typhi. Nm 2007, V Cng Thnh v ctv (Khoa Nng nghip v Sinh hc ng dng trng i hc Cn Th) ng dng k thut in di protein SDS-PAGE ci thin thnh cng phm cht nhiu ging la c sn ang b thoi ha ca ng bng sng Cu Long. Trong chn to ging la, k thut ny gip pht hin nhanh nhng tnh cht ni bt nh mi thm, protein, amylose cc nh khoa hc chn lc c nhng dng, ging c phm cht tt. Mt s ging la c sn c ci thin phm cht thnh cng v nhiu ging la trin vng ra i bng k thut ny. Nm 2007, Phm Vn Phng (i hc Cn Th) ng dng k thut in di SDS-PAGE protein nghin cu c im di truyn v chn ging la. K thut in di SDS-PAGE protein gip nh chn ging sm tuyn chn chnh xc nhng c th mong mun ngay t th h u ca cc t hp lai nn rt ngn thi gian tuyn chn so vi phng php truyn thng. K thut in di SDS-PAGE protein gip nh gi a dng di truyn v phn bit hai loi la hoang ng bng

17

sng Cu Long da vo mc n mu Coomassie ca bng protein ch th dng basic glutelin. 2.4.2 Nghin cu ngoi nc ng dng in di protein Fan Shuguo (1999) ng dng in di protein nh gi tnh chu mn khc nhau ca mt s loi cy trng. Nazrul Islam, M. Lonsdale, N. M. Upadhyaya, T. J. Higgins, H. Hirano v R. Akhurst (2004) thc hin nghin cu ti ly trch protein t l la cho in di hai chiu v ng dng n trong phn tch h protein ca la. Scott A. Young v ctv (1995) thc hin nghin cu s tch ly peroxidase trong mch g ca la trong sut qu trnh phn ng khng tng thch vi Xanthomonas oryzae pv oryzae. H bt u chng X. oryzae pv oryzae cho la khi la c 2 3 l. Sau ct ly nhng mu l khi chng c 48 gi, tin hnh ly trch protein ca l sau tin hnh in di trn gel polyacrylamide trong iu kin protein mu khng b bin tnh (Discontinuous and nondenaturing). Chn ly bng protein c kch thc tng ng vi peroxidase (42 kD), tip tc tin hnh in di SDS- PAGE v lai trn mng PVDF. Vch protein s c pht hin trn mng nh nhum CBB. Sau ra v thu mu gii trnh t.

18

Chng 3 VT LIU V PHNG PHP TIN HNH

3.1 Thi gian v a im thc hin ti Thi gian: ti c thc hin t ngy 15 thng 03 nm 2007 n ngy 31 thng 08 nm 2007. B mn Bo v Thc vt, Khoa Nng hc, i hc Nng Lm Tp. HCM. Phng th nghim Cng ngh Sinh hc Thc vt Vin Cng ngh Sinh hc v Cng ngh Mi trng Trng i hc Nng Lm Tp. HCM. 3.2 Ha cht v vt liu dng trong th nghim 3.2.1 Thuc sinh hc Ch phm sinh hc s dng x l cho la l thuc sinh hc tr su ry v virus c tn thng hiu l AMINO 15SL, c sn xut ti cng ty Hp Danh Sinh hc Nng nghip Sinh Thnh. Thnh phn gm c polyamid + polyamin 15%, humic acid 5%. Liu lng s dng theo hng dn: pha 50 ml ch phm vi 16 lt nc phun cho 500 m2 cy trng. Phun ln 2 sau ln th nht 1 tun. Cng dng theo nh sn xut a ra: (i) c ch men tiu ha ca su ry gy cht trit k c thnh trng ca bm, ry cnh di; (ii) kim hm s nhn bn ca virus trong t bo cy trng; (iii) cung cp cht dinh dng c hiu cho cy trng mau phc hi, duy tr nng sut. 3.2.2 Ha cht dng trong ly trch protein Nit lng Acetone (Merck) NaCl (Merck) Sodium phosphate (Merck) Sucrose (Merck) TE 1x Dithiothreitol (Bio- Rad) Sodium dodecyl sulfate (Merck) 2- mercaptoethanol (Merck) Ethanol (Merck) EDTA Tris- HCl (Merck)

19

Amonium acetate (Merck) 3.2.3 Ha cht in di Ammonium persulfate (Merck) Methanol (Merck) Axit acetic (Merck) 2- mercaptoethanol (Merck) Glycerol (Merck) CBB R- 250 (Bio- Rad) 3.2.4 Trang thit b th nghim Hp nha ng ong 100 ml My ht v t cy v trng (Anh Quc) Eppendorf 0,2 ml; 1,5 ml Pipet cc loi (Bio Rad) My ly tm lnh (Hettich c) Ni hp (Autoclave- Tomy) B ngun in di (Bio Rad )

Methanol (Merck)

Sodium Dodecyl Sulfate (Merck) Tris- HCl (Merck) Bromophenol blue (Merck) Dithiothreitol (Bio- Rad) Glycine (Merck) TEMED (Bio- Rad)

Acrylamide v N,N-methylene-bis-acrylamide (Bio- Rad)

Becher 50 ml Gng tay Chn s v chy (c) Cn phn tch 4 s (Ohaus - M) u tp cc loi (c) My vortex My o pH

Bn in di ng Mini-PROTEAN 3 Cell (Bio Rad) T lnh 40C, t lnh -200C v t lnh -700C (Sanyo Nht) 3.3 Phng php tin hnh nghin cu 3.3.1 Chun b mu v ly mu la 3.3.1.1 Chun b mu la Ht la c ngm 36 gi trong nc. ht nhit 30 350C, 9 gi, trong ti. Khi ht nt nanh, chn nhng ht nt nanh u nhau, gieo trong cc hp nha (hnh 3.1). Th nghim c b tr 3 l, gm 12 nghim thc, mi nghim thc gm 3 hp v c nh du nh bng 3.1. Sau khi gieo, tip tc ti trong 6 gi hoc cho ti khi ht ln mm trng di 1 2 mm. Bt u cung cp nh sng, nc, phn bn y cy m pht trin mnh, khe n giai on 4 l.

20

La mm

Hnh 3.1 La mm sau khi gieo trong hp nha 2 ngy 3.3.1.2 X l thuc v ly mu la Khi cy m pht trin n giai on 4 l, bt u tin hnh x l thuc sinh hc kch khng theo b tr bng 3.2. L I: L i chng, khng x l thuc sinh hc kch khng. L II: X l nng thuc theo hng dn ca nh sn xut. L III: X l nng thuc gp 2 ln so vi hng dn ca nh sn xut. Bng 3.1 Bng b tr phn l th nghim theo nghim thc Nghim thc L 1 I C1 2 C2 3 C3 4 C4 5 C5 6 C6 7 C7 8 C8 9 C9 10 11 12 C12

C10 C11

II

X1

X2

X3

X4

X5

X6

X7

X8

X9

X10 X11 X12

III

Y1

Y2

Y3

Y4

Y5

Y6

Y7

Y8

Y9

Y10 Y11 Y12

Ch thch: C: Mu i chng, X: Mu x l nng thuc theo hng dn ca nh sn xut, Y: Mu x l nng thuc gp 2 ln so vi hng dn ca nh sn xut

21

Bng 3.2 Thi gian x l thuc trc khi ly mu theo tng nghim thc
Nghim thc Thi gian x l thuc trc khi ly mu 1 6 gi 2 12 gi 3 18 gi 4 24 gi 5 30 gi 6 36 gi 7 42 gi 8 48 gi 9 54 gi 10 60 gi 11 66 gi 12 72 gi

3.3.2 Ly trch protein tng s t l la Trong nghin cu ny chng ti thc hin kho st 3 quy trnh ly trch protein tng s: quy trnh c s dng SDS (Samuel S.M. Sun, 1994), quy trnh c s dng phenol (Hurkman v Tanaka, 1986) v quy trnh ci tin. Sau khi ly trch tin hnh in di cc mu protein ly trch c chn ra quy trnh tt nht ly trch tt c mu la cn li cho cc th nghim sau. 3.3.2.1 Cc bc ly trch protein theo quy trnh c s dng SDS 1. Nghin 0,2 g mu l vi 1 ml dung dch ly trch (0,25 M NaCl, 1% SDS, 1% 2 mercaptoethanol, 0,05 M sodium phosphate pH 7,5) trong ci thnh dch. 2. Chuyn dch nghin vo mt eppendorf 1,5 ml, v ly tm vi tc 14 .000 vng/pht trong thi gian 10 pht nhit 200C. 3. S dng pipette ht dch ni vo mt eppendorf 1,5 ml mi; trnh ht lp lipid trn cng. 4. Ly tm dch ni va ht vi tc 14.000 vng/pht trong thi gian 10 pht nhit 200C. 5. Tip tc ht dch ni vo mt eppendorf khc. 6. Thm acetone 80%, ly tm 5000 vng/pht trong 5 pht 40C. Thu kt ta, phi kh v thm vo eppendorf 100 l 1x TE, tr nhit 40C ti khi dng. 3.3.2.2 Cc bc ly trch protein theo quy trnh c s dng phenol 1. Nghin 0,5 g mu l ti trong nit lng bng ci v chy thnh bt mn, cho vo eppendorf 1,5 ml.

22

2. Thm vo eppendorf cha mu 0,5 ml Tris- phenol pH 8,8 v 0,5 ml dch ly trch (0,1 M Tris-HCl pH 8,8, 10 mM EDTA, 0,4% 2 mercaptoethanol, 0,9 M sucrose).
3. Vortex u hn hp dung dch trong eppendorf. (Lu bc ny nn thc hin nhanh v phenol c th gy bin tnh protein)

4. Ly tm 5000 vng/pht trong thi gian 10 pht nhit 40C. 5. Thu dch ni vo eppeendorf 1,5 ml khc, thm vo 1 ml dch ly trch. Vortex cho hn hp ng nht. mu nhit -200C trong mt gi. Ly tm 12.000 vng/pht trong thi gian 10 pht nht 40C. 6. Ht dch ni vo eppendorf 1,5 ml khc. Thm 1 ml 0,1 M amonium acetate trong methanol 100%. 7. Vortex v mu nhit -200C trong 1 gi hoc qua m. Thu kt ta bng cch ly tm 12.000 vng/pht trong thi gian 20 pht nhit 40C. 8. Ra kt ta vi 0,1 M amonium acetate trong methanol, tip tc ra vi acetone 80%, v cui cng l ra vi ethanol 70%. Mi ln ra nh vy th mu -200C trong 15 pht. Sau b dch v thu kt ta. 9. Thm acetone 80% cha 10 mM DTT. Tr -800C cho ti khi s dng. 3.3.2.3 Cc bc ly trch protein theo quy trnh c s dng SDS ci tin 1. Nghin 0,5 g mu l la trong nit lng thnh bt mn, cho vo eppendorf 1,5 ml. 2. Thm 1ml dch ly trch (0,5 M EDTA, 0,05 M sodium phosphate 1% SDS, 0,25 M NaCl , 1% 2- mercaptoethanol, 1 M Tris- HCl pH 8,0) vo eppendorf, vortex cho ng nht. mu nhit -200C trong mt gi. 3. Ly tm 13.000 vng/pht trong 15 pht 40C. 4. Ht dch ni vo eppendorf khc. Thm 400 l ( acetone 80%, 0,2 M DTT), vortex hoc lc u. Ta protein nhit -200C trong mt gi hoc qua m. 5. Ly tm 13.000 vng/pht trong thi gian 15 pht nhit 40C. 6. dch trong. Thm 100 l 1x TE, 370C trong 1 gi. 7. Thm 400 l ethanol 70%. Ly tm 13.000 vng/pht trong 5 pht 40C.

23

8. dch trong, lp li tng t bc 7. 9. dch trong, phi kh mu, sau pha mu vi 100 l 1x TE tr mu nhit -700C n khi s dng. 3.3.3 in di kim tra mu protein ly trch 3.3.3.1 Chun b ha cht a) Ha cht gel Pha gel phn tch (separating gel) c nng 12%T Cho cc thnh phn sau theo th t vo ng Falcon 15 ml sch : 4 ml 30% Acrylamide/Bis (29:1), 2,5 ml Tris HCl 1,5 M pH 8,8, 3,4 ml nc ct 2 ln kh ion, 0,1 ml 10% SDS. Sau khi cho y cc thnh phn trn theo ng th tch vo ng Falcon. Tip tc thm vo ng Falcon 50 l ammonium persulfate 10%, 10 l TEMED v lc nh ng Falcon vi ln (trnh to bt kh), dng pipettte loi 100 1000 l bm t t dung dch gel vo khun, gel sao cho mc dung dch gel cao hn 7 cm (trnh to bt kh trong khun gel). Sau , nh nhng t mt lp nc ct ln trn lp gel mt gel c phng. Ch gel ng (khong 20 30 pht). Vi 10 ml dung dch gel c th c 2 gel 100 x 100 x 0,75 mm. Pha gel gom (Stacking gel) c nng 4%T Cho cc thnh phn sau theo th t vo ng Falcon 15 ml khc: 1,3 ml Acrylamide/Bis (29:1) 30%, 2,5 ml Tris HCl 0,5 M pH 6,8, 6,1 ml nc ct 2 ln kh ion, 0,1 ml SDS 10%. Sau khi gel phn tch trng ngng hon ton, ht nc bn trn. Tin hnh lp gel gom bng cch dng pipette loi 100 1000 l ht 2 ml dung dch gel gom vo becher 50 ml, thm vo dung dch 10 l ammonium persulfate 10%, 2 l TEMED. Dng pipette trn u v bm vo khun gel. ng thi a lc vo gel to cc ging mu (Lu : trnh to bt kh trong khun gel). Sau khi gel gom trng ngng hon ton, ta tin hnh tho lc ra khi khun gel cn thn trnh to bt kh trong cc ging (Lu : nu c bt kh trong ging ta dng
pipette loi 100 1000 l bm kh mnh vo ging lm v bt kh). Tip theo ta lp

da gel vo b phn in di, cho dung dch in di vo bn.

24

b) Chun b dung dch np mu (sample buffer) v dung dch m in di (electrode buffer) Pha dch np mu gm: 3,55 ml nc ct 2 ln kh ion; 1,25 ml Tris-HCl 0,5 M pH 6,8; 2,5 ml glycerol; 2 ml SDS 10% (w/v); 0,2 ml bromophenol blue 0,5%(w/v). Khi dng thm vo 50 l 2- Mercaptoethanol hoc 0,5 ml dithiothreitol 2 M (DTT). Dung dch m in di l Tris glycine, pH 8,3. Pha 1l Tris glycine (196mM glycine, 0,1% SDS, 50 mM Tris HCl pH 8,3). c) Chun b dung dch nhum v dung dch gii nhum Dung dch nhum Coomassie Brilliant Blue R- 250 Pha hn hp theo t l nh sau vo mt hp nha c np: 0,2% Coomassie Brilliant Blue R- 250 (w/v), 45% methanol (v/v), 45% nc ct hai ln kh ion (v/v), 10% acid acetic (v/v). y kn li v gi trong ti n khi s dng. Dung dch gii nhum Pha hn hp sau theo ng t l vo mt hp nha khc: 25% methanol (v/v), 65% nc ct hai ln kh ion (v/v), 10% acid acetic (v/v). y kn, gi trong ti n khi s dng. 3.3.3.2 Chun b mu v chy in di Dng pipette loi 0,5 10 l ht 3 l dung dch np mu v 7 l mu protein vo mt eppendorf 0,2 ml sch, trn u, y np v gia nhit 950C t 3 5 pht gy bin tnh protein. Sau khi gia nhit c th ly tm 10.000 vng/pht trong 3 pht. 3.3.3.3 Tin hnh in di v xem kt qu 1. Dng pipette loi 0,5 10 l np mu vo cc ging (10 l/ging). 2. Tin hnh chy in di hiu in th 100 V, cng dng in 15 mA, trong thi gian 120 pht hoc in di cho ti khi vch mu ca dch np mu cch y gel khong 0,5 1cm. 3. Sau khi in di, tho gel ra khi khun, ra sch gel bng nc 5 pht (cn
thn trnh lm t gel). Nhum gel trong thi gian 30 45 pht.

4. Sau khi nhum xong, ra gel vi nc 5 pht. Tin hnh gii nhum bng cch ngm gel trong dung dch gii nhum cho n khi nn gel tr nn trong sut

25

khng mu. Sau khi gii nhum xong, protein c pht hin nh cc vch mu xanh lam trn nn gel trong sut. 3.3.4 Th nghim kho st chn iu kin in di iu kin in di nh hng ln n kt qu in di. Nu iu kin in di khng ph hp vi protein th rt c th s lm cc bng protein b cong, hoc cc protein b bin tnh do s hot ng tr li ca cc enzyme. ng thi n cng c th lm cho cc bng protein khng phn tch r rng. Trong th nghim ny chng ti tin hnh kho st iu kin in di cho ph hp vi protein ca l la nhm chn ra iu kin in di thch hp. B tr 3 th nghim khc nhau (bng 3.3) Bng 3.3 Th nghim kho st iu kin in di Th nghim 1 2 3 iu kin in di Gel gom
200 V, 13 mA, 20 pht 100 V, 20 mA, 10 pht 100 V, 15 mA, 20 pht

Gel phn tch

200 V, 18 mA, 100 pht 100 V, 20 mA, 80 pht 100 V, 15 mA, 100 pht

Mu s dng trong th nghim ny l mu protein ly trch theo quy trnh tt nht trong 3 quy tnh kho st. Ch tiu theo di: hnh dng cc bng protein v s phn tch ca cc bng trn gel. 3.3.5 Kho st nng gel Nng gel nh hng rt ln n phn tch ca cc bng protein trn gel. V nu nng gel cng cao th kch thc l gel cng nh li v s cn tr s di chuyn ca ca cc protein c kch thc cng ln. Ni cch khc l vi mi mt nng gel th cho php phn tch protein mt khong trng lng phn t xc nh. thy c mt cch r rng cc bng protein ca la trn gel, cn kho st nng ca gel phn tch cho ph hp vi protein tng s c ly trch t l la. Phi la chn nng gel sao cho cc bng protein trn gel c phn tch r rng.

26

Tin hnh th nghim bng cch cc ming gel c nng gel phn tch khc nhau v th nghim trn cc mu protein. Nng ca gel gom c gi nguyn l 4% T, nng ca gel phn tch c thay i 12% T, 13% T, 14% T. Ta tin hnh chy in di cng iu kin vi hiu in th n nh 100 V, cng dng in 15 mA, thi gian in di l 120 pht, nhum gel trong 0,2% CBB (w/v) nhit phng 250C. 3.3.6 in di cc mu protein kim tra phn ng ca cy la i vi thuc sinh hc kch khng Th nghim 1: Tin hnh in di tt c cc mu protein ly trch c t tt c cc mu la v so snh qua li gia 12 nghim thc mu protein ca la. Cch tin hnh 1. Chun b 6 ming gel c nng gel phn tch l 12%, nng gel gom l 4%. Chun b sn tt c cc mu protein ly trch ca 12 nghim thc. 2. Dng micropipette np mu vo cc ging vi t l 3 l dch np mu v 7 l mu. Trc khi np mu vo ging, hn hp mu v dch np mu c gia nhit 950C trong thi gian 5 pht. 3. Tin hnh in di tt c cc ming gel hiu in th 100 V, cng dng in 15 mA, thi gian 120 pht. 4. Sau khi in di xong, cn thn ly cc gel ra khi khun. Nhum gel trong dung dch nhum CBB 0,2% (w/v) trong thi gian 30 pht. Sau khi nhum xong, cn thn ly gel ra, ra sch gel bng nc v ngm gel trong dung dch gii nhum cho n khi xut hin cc bng protein trn gel hay cho ti khi nn gel tr nn trong sut. Nu c protein trong mu th s xut hin cc bng mu lam trn gel. Th nghim 2: in di kim tra kt qu ca mu protein c ly trch t l II. Thc hin in di 2 ming gel theo iu kin ging th nghim 1. Gel 1: np cc mu t X1 X9 hnh 4.11. Gel 2: np cc mu t X4 X12 hnh 4.12. Th nghim 3: in di kim tra kt qu ca mu protein c ly trch t l III. Tin hnh nh th nghim 2 i vi cc mu Y1 Y12.

27

Chng 4 KT QU V THO LUN

4.1 Kt qu th nghim cc quy trnh ly trch protein Giai on tch chit protein tng s l mt giai on tuy d thc hin nhng khng phi l n gin. Giai on ny rt quan trng v n l yu t hng u quyt nh s thnh cng ca th nghim. C nhiu phng php ly trch protein khc nhau nhng mc ch cui cng ca cc phng php l lm sao thu c lng protein c tinh khit cao phc v cho nghin cu. Mun vy, ngoi vic c c mt phng php ly trch th kinh nghim trong thao tc cng ng vai tr quan trng. Vic tch chit phi m bo sao cho protein khng b bin tnh, t gy hoc tht thot. Tin hnh kho st cc quy trnh ly trch chn ra quy trnh ti u trong vic ly trch protein tng s t l la. i vi quy trnh c s dng SDS tin hnh ly trch th nghim 6 mu la. Kt qu (hnh 4.1a) cho thy, mu protein ly trch c sau khi tin hnh in di SDS- PAGE cho t bng protein, v cc bng protein rt m. iu ny c th do cc mu protein tng s ly trch c theo quy trnh ny cn ln nhiu tp lipid, polysaccharide nn khi in di cc bng khng phn tch c r rng. Hoc do protein b bin tnh trong qu trnh ly trch, c bit l khi bo qun mu 40C cc protein l enzyme c th s hot ng c trong nhit ny v phn hy cc protein trong mu. Qu trnh nghin mu la vi dch ly trch nn thc hin nhanh, v trong dch ly trch c 2- mercaptoethanol c kh nng ct t cu ni (S S) trong protein v lm protein bin tnh. Khi protein bin tnh nu tip tc nghin c kh nng s lm t gy protein. Bc ta protein nn thc hin nhit m su s gip ta ti a protein v lm gim ti thiu lng protein b bin tnh do chnh enzyme ni bo gy ra. Cc mui gip ha tan protein trong dung dch cn c gi pH 8.0, v pH ny protein n nh.

28

(a)

(b)

(c)

Hnh 4.1 Kt qu in di SDS PAGE mu protein ly trch ca cc quy trnh (a) Mu protein c ly trch theo quy trnh 1 (b) Mu protein c ly trch theo quy trnh 2 (c) Mu protein c ly trch theo quy trnh 3

kho st quy trnh ly trch c s dng phenol, tin hnh ly trch th nghim 6 mu la. Tt c 6 mu c ly trch trong quy trnh ny u cho kt qu khng tt (hnh 4.1b). Mi mu ch cho t 2 4 bng protein trn gel, cc bng khng phn tch r. gii thch cho kt qu ny chng ti cho rng l do phenol c kh nng ph hy hp cht polysaccharide v n cng c kh nng ph hy c protein. Khi thc hin bc ny nn tin hnh nhanh, trnh n mc ti a s phn hy protein do phenol gy ra. Trong quy trnh ny c mt s u im: (i) EDTA c s dng trong dch ly trch vi mc ch kh quan trng l ngn cn s hot ng ca cc emzyme ni bo bng cch tham gia gn cc ion Mg++, Ca++. V cc enzyme ni bo mun hot ng mnh phi gn vi cc cation ha tr II c bit l vi Mg++. (ii) Amonium acetate c s dng trong bc ta protein vi mc ch l m lm tng kh nng ta protein. Sau khi tin hnh th nghim 2 quy trnh ly trch trn, mu protein thu c trong 2 quy trnh khng tt v khng iu kin tin hnh in di. Do , sau khi

29

rt ra mt s kinh nghim chng ti th nghim ci tin quy trnh c s dng SDS ly trch 6 mu la. Kt qu ly trch 6 mu t c l kh tt (hnh 4.1c). Mu protein sau khi in di SDS- PAGE c s phn tch cc bng kh r rng, t b tp (smear rt m). Kt qu tt t c trong quy trnh ny so vi 2 quy trnh trc l tng hp ca nhiu yu t: (i) khi nghin mu vi nit lng cc m s mm hn v cc t bo d dng tch ri nhau hn; khi vi tc ng ca 2 mercaptoethanol mng t bo s b ph v v phng thch protein; (ii) vic s dng EDTA trong quy trnh ngn cn cc enzyme ni bo hot ng trong dch ly trch. (iii) cc mui trong dch ly trch kt hp vi Tris- HCl pH 8,0 gi n nh protein ha tan trong dung dch; (iv) khi ta protein bng acetone -200C s ngn cn nhng enzyme ni bo hot ng. T kt qu ly trch ca quy trnh ci tin, nhn thy mu protein c ly trch theo quy trnh ny iu kin tin hnh in di. Thc hin ly trch tt c mu la cn li theo quy trnh ny lm th nghim. Sau y l mt s im cn lu khi tin hnh ly trch protein tng s l la chng ti rt ra t thc nghim: 1. Dng c cn kh trng cn thn phng nga tp nhim. 2. Mang bao tay bo v v kh trng bng cn 70% cn thn trnh tp nhim. 3. Mu sau khi ly ra khi t -200C phi c ct v ngm ngay trong nit lng. 4. Thao tc nh nhng khi nghin mu l la tht nhuyn thnh bt mn. 5. Bc ta protein trong acetone nn thc hin nhit m su. 6. Nu c s dng phenol trong ly trch th nn thao tc nhanh, trnh s phn hy protein 7. Khi tr mu nn tr nhit di -700C. Nhng tt nht l s dng ngay sau khi ly trch. 4.2 Kt qu th nghim kho st chn iu kin in di Kt qu in di iu kin th nghim 1 (hnh 4.2a) cho thy cc bng protein b cong, mo. Hin tng ny c th l do thi gian in di lu 120 pht trong hiu in th 200 V lm nhit mi trng in di tng. Do chng ti tin hnh th nghim 2 tng Ampe ln 20 mA v gim hiu in th xung cn 100 V. Kt qu

30

t c trong th nghim ny l s phn tch cc bng protein khng r rng, iu ny l do qu trnh in di xy ra nhanh cc bng protein cha th phn tch. Rt kinh nghim t 2 th nghim trn chng ti tin hnh th nghim 3. Trong th nghim ny chng ti tin hnh gim Ampe xung cn 15 mA v vn gi hiu in th l 100 V. Kt qu (hnh 4.2c) cho thy cc bng protein phn tch r rng v khng b cong, mo. T kt qu chng la chn iu kin in di theo th nghim 3 tin hnh cc th nghim sau.

(a) (c) (b) Hnh 4.2 Kt qu kho st iu kin in di ph hp vi protein l la (a) Kt qu in di vi iu kin th nghim 1 (b) Kt qu in di vi iu kin th nghim 2 (c) Kt qu in di vi iu kin th nghim 3 4.3 Kt qu th nghim kho st chn nng gel Cc ming gel c chy in di vi hiu in th n nh 100V, 15mA, trong thi gian 120 pht. Kt qu in di (hnh 4.3) cho thy mc phn tch cc mu protein trn gel 12% T, 13% T, 14% T l kh ging nhau . Th hin r mu Y6, Y7 v Y8 trn gel 13% T v trn gel 12% T c phn tch r . Mu protein Y5 gel 13% T ch thy rt t bng c th khng phi do nh hng ca nng gel. Hin tng c th l do protein trong mu t (t hn 0,1 g) khng n mu Coomassie Brilliant Blue. Hm lng protein t c th l do qu trnh bo qun mu khng tt, ly trch mu khng t hoc do khi gia nhit bin tnh trc khi np

31

mu protein bi t gy. Nhng mu Y6, Y8 gel 12% T v X4, X5 gel 14% T n mu CBB m c th do hm lng protein trong mu nhiu hn 1 g. Ngoi ra ta cn thy nng gel 14% T, khong cch phn tch gia cc bng hp hn so vi khong cch phn tch gia cc bng ca gel 13% T, v khong cch i vi gel 12% T l rng nht. Nh vy, iu kin hiu in th n nh v cng dng in khng i, nu nng gel cng cao th khong thi gian chy in di phi cng nhiu, s phn tch cc bng protein tt hn v r hn. T kt qu trn chng t nng gel t 12% T 14% T khng nh hng nhiu n kt qu in di. Do chng ti quyt nh s dng gel 12% T tin hnh in di cho cc th nghim sau nhm tit kim kinh ph.

a)

b)

c)

Hnh 4.3 Kt qu in di SDS PAGE cc mu protein kho st nng gel a) L hnh in di protein l la trn gel c nng 12% T b) L hnh in di protein l la trn gel c nng 13% T
c) L hnh in di protein l la trn gel c nng 14% T

32

4.4 nh gi phn ng ca la i vi thuc sinh hc 4.4.1 Kt qu in di ca tt c cc mu protein trong 12 nghim thc

Hnh 4.4 Kt qu in di so snh mu protein ca 6 nghim thc (nghim thc 1, 2, 3, 4, 5 v 6)

Hnh 4.5 Kt qu in di so snh mu protein ca 6 nghim thc (nghim thc 4, 5, 6, 7, 10 v 11)

Hnh 4.6 Kt qu in di so snh mu protein ca 5 nghim thc (nghim thc 5, 8, 9, 10 v 12)

33

T kt qu in di SDS- PAGE tt c cc mu protein trong 12 nghim thc (hnh 4.4, hnh 4.5, hnh 4.6) so snh cc hnh chng ti nhn thy rng tt c cc mu protein u cho mt di bng ng hnh. Khng c s khc bit no v cc di bng ng hnh . C th l do thuc cha kp tc ng ln s sinh trng v pht trin ca cy la do thi gian tc ng cn t (sau 72 gi), hoc do thi im x l thuc cn sm (giai on la 4 l) nn h protein ca la c th cha c s bin i. Ngoi ra c th vi iu kin trng la di nh sng n neon, v trong phng s gy ra mt s hn ch v sinh trng v pht trin dn n la khng th hp thu c thuc. Cng khng loi tr kh nng phng php SDS- PAGE cha nhy pht hin s khc bit. 4.4.2 Kt qu in di mu protein ca la l II Tin hnh in di 12 mu protein ca l 2, kt qu (hnh 4.7) khng tm thy s khc bit v bng protein gia cc mu protein ca la c x l thuc theo hng dn ca nh sn xut. Cc mu protein ly trch c trong l la ny cho kt qu l mt di bng protein ng hnh. iu ny xy ra c th l do thi gian tc dng thuc khng , la cha kp phn ng vi thuc nn h protein ca la cha c thay i ng k. Tuy nhin nu c xy ra s thay i nh trong h protein ca la th cng rt kh pht hin: c th lng protein khc bit quan tm trong qu trnh ly trch khng nhn c, hoc lng protein qu t khng th pht hin bng nhum CBB. Ngoi ra khng loi tr kh nng vi phng php SDS- PAGE th cha nhy pht hin s khc bit.

Hnh 4.7 Kt qu in di SDS PAGE mu protein ly trch t l II Thc hin in di nng gel 12% T, hiu in th n nh 100V cng dng in 15 mA, thi gian in di 120 pht.

34

4.4.3 Kt qu in di mu protein ca la l III

Hnh 3. 8 Kt qu in di SDS PAGE mu protein 4.8 protein ly trch t l III

Thc hin in di di nng gel 12% T, 12% hiu in th n nh 100V, cng Thc hin in nng gel T, hiu in th n nh 100V in 15 mA, thi gian in di 120 pht. cng dng dng in 15 mA, thi gian in di 120 pht.

Kt qu thu c trong hnh 4.8 cng khng cho s khc bit no d trong th nghim ny ta tc dng thuc vi liu lng gp i so vi hng dn ca nh sn xut. T kt qu ny ta c th gii thch rng thi gian tc dng thuc cha nh hng ln sinh l ca cy m. Do thi gian ta tc dng thuc v ly mu cn ngn, ch trong vng 72 gi ng h. Ngoi ra vi iu kin trng la trong phng nh vy th kh nng hp th thuc cng kh xy ra nhanh chng do sinh l ca cy khng bnh thng.

35

Chng 5 KT LUN V NGH

5.1 Kt lun Trong ti ny chng ti hon thnh c mt s ni dung: 1. Thit lp c quy trnh ly trch protein trn l la. 2. Thit lp c phng php in di SDS- PAGE trn mu protein l la. 3. iu kin trng la trong phng vi nh sng n neon, khi x l thuc sinh hc AMINO 15SL ln la trong thi gian t 6 72 gi th cha tm thy s khc bit no v protein tng s khi phn tch bng k thut SDS- PAGE. 5.2 ngh 1. Tip tc th nghim ch phm sinh hc nhng tng thi gian tc dng thuc v tng liu lng thuc 2. M rng th nghim thuc khi la c trng trong nh li v trn iu kin th nghim ngoi ng rung khng nh r hiu qu ca thuc. 3. Tip tc nghin cu h protein ca cy la bng cc phng php phn tch proteomics khc nh in di 2 chiu, sc k.

36

TI LIU THAM KHO

TING VIT 1. Bin Tun An, 2006. Kho st h vi khun methylobacterium sp. trn la (oryza sativa L.) Ty Ninh. Kha lun tt nghip K s Cng ngh Sinh hc, i hc Nng Lm, TP. H Ch Minh, Vit Nam. 2. Bi Huy p, 1999. Mt s vn v cy la, NXB Nng Nghip. 3. Chu L Hi Anh, 2006. Kho st nh hng ca vi khun methylobacterium sp. ln s pht sinh c quan cy la (ozyra sativa L.) nui cy in vitro. Kha lun tt nghip K s Cng ngh Sinh hc, i hc Nng Lm, TP. H Ch Minh, Vit Nam. 4. L Minh Trit, 2003. Bi Ging Mn Hc Cy La. Khoa Nng Hc, i Hc Nng Lm, TP. H Ch Minh, Vit Nam. 5. Nguyn Th Lang, 2002. Phng Php C Bn Trong Nghin Cu Cng Ngh Sinh Hc. Nh xut bn Nng Nghip TP. H Ch Minh. 6. Nguyn Tin Thng, 2004. Ti liu hng dn thc tp: Cng Ngh Enzyme v Protein. Vin Sinh Hc Nhit i, TP. H Ch Minh. 7. V Tng Xun, Trn Thanh B, Nguyn Ngc , Dng Ngc Thnh v Vn X, 1986. Trng la nng sut cao. Nh xut bn Thnh ph H Ch Minh, H Ch Minh. 83 trang. TING NC NGOI 8. Allen R. C., Saravis C. A. and Maurer H. R., 1984. Gel electrophoresis and Isoelectrics Focusing of protein: Selected Techniques. De Gruyter, Berlin. 9. Davis B. J., 1964. Disc electrophoresis. II. Method and appication to human serum proteins. Ann. N. Y. Acad. Sci. 121, 404 427. 10. David E. Garfin, Jay A. Glasel and Murray P. Deutscher, 1995. Introduction to Biophysical Methods for Protein and Nucleic Acid Research. Electrophoretic Method. pp. 53 109.

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11. Department of health and ageing office of the gene technology regulator, 2005. The biology and ecology of rice (O. sativa) in Australia. 12. Hurkman and Tanaka, 1986. Phenol extraction followed by methanolic ammonium acetate precipitation an effective protocol for sample preparation from protein-poor, recalcitrant tissues such as plants. Plant Physiology, Vol. 81, pp. 802-806. 13. IRRI, 2002. IRRI rice almanac, Third edition, Manila, Philippine, IRRI. p. 253. 14. Laemmli U. K., 1970. Cleavage of structural proteins during the asembly of the head of bacterio- phage T4. Nature. London. 227, 680 685. 15. Nazrul Islam, Lonsdale M., Upadhyaya N.M., Higgins T. J., Hirano H. and Akhurst R., 2004. Protein extraction from mature rice leaves for twodimensional gel electrophoresis and its application in proteome analysis. Proteomics, Vol. 4, pp. 1903-1908. 16. Ornstein L., 1964. Disc electrophoresis. I . Background and theory. Ann. N. Y. Acad. Sci. 121, 321 349. 17. Sambrook and Russell. Molecular cloning-A laboratory manual. Volume 2, 3rd edition, 2001. Cold spring harbor laboratory press, New York. 18. Samuel S.M.Sun, 1994. Methods in plant molecular biology and agricultural biotechnology: a laboratory training manual. Asian Vegetable Research and Development Center. Shanhua, Tainan, Taiwan (ROC). 94 p. 19. Scott A. Young, Ailan Cuo, James A. Cuikema, Frank F. White, v Jan E. Leach, 1995. Department of Plant Pathology, Throckmorton Hall (S.A.Y., F.F.W., J.E.L.), and Division of Biological Sciences (J.A.G.), Kansas State University, Manhattan, Kansas 66506-5502; and Waksman Institute, Rutgers University, Piscataway, New Jersey 08855 (A.G.) 20. Woolley P., 1987. Thermal instability of electrophoresis gel. Electrophoresis 8, 339 345.

PH LC
Ph lc 1: Bng pha gel theo cc nng khc nhau

Phn trm gel 4% 5% 6% 7% 8% 9% 10% 11% 12% 13% 14% 15% 16% 17%

Nc kh ion (ml) 6.1 5.7 5.4 5.1 4.7 4.4 4.1 3.7 3.4 3.1 2.7 2.4 2.1 1.7

30% acrylamide/ bis (29:1) (ml) 1.3 1.7 2.0 2.3 2.7 3.0 3.3 3.7 4.0 4.3 4.7 5.0 5.3 5.7

*m in di (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

10% w/v SDS (ml) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

* m gel phn tch: 1,5 M Tris-HCl, pH 8,8 m gel gom: 0,5 M Tris-HCL, pH 6,8

Ph lc 2: Khong phn tch protein theo nng gel polyacrylamide Acrylamide (%) 15 10 7,5 5,0 Khong phn tch protein (kDa) 12 43 16 68 36 94 57 212