kỹ thuật gateway

I HC THI NGUYN TRNG I HC S PHM -------
LU TH C
PHN LP V THIT K VECTOR C CH BIU HIN GEN M HA ENZYME INVERTASE (-FRUCTOFURANOSIDASE) NHM TNG TR LNG SUCROSE CY MA
LUN VN THC S SINH HC
Thi Nguyn 2009
S ha bi Trung tm Hc liu i hc Thi Nguyn
http://www.Lrc-tnu.edu.vn
I HC THI NGUYN TRNG I HC S PHM -------
LU TH C
PHN LP V THIT K VECTOR C CH BIU HIN GEN M HA ENZYME INVERTASE (-FRUCTOFURANOSIDASE) NHM TNG TR LNG SUCROSE CY MA
LUN VN THC S SINH HC

Chuyn ngnh: Di truyn hc M s: 60.42.70
NGI HNG DN KHOA HC: TS. L QUNH LIN
Thi Nguyn 2009
LI CAM OAN Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s liu, kt qu nghin cu trong lun vn l trung thc v cha tng c ai cng b trong bt k mt cng trnh no khc. Tc gi
Lu Th C
LI CM N
Li u tin ti xin c by t lng bit n su sc ti TS. L Qunh Lin, Phng Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc, Vin Khoa hc v Cng ngh Vit Nam, l ngi thy tn tnh hng dn, ch bo, du dt v gip ti trong sut thi gian ti thc hin v hon thnh lun vn ny. Ti xin c by t lng bit n su sc ti GS.TS. L Trn Bnh, TS. Chu Hong H, KS. Tin Pht Phng Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc, l nhng ngi tn tnh ch bo, truyn t nhiu kinh nghim qu bu v gip ti trong sut thi gian thc tp v hon thnh lun vn. Trong thi gian thc tp nghin cu ti cng nhn c s h tr nhit tnh v nhng kin ng gp b ch ca cc c ch, cc anh ch, cc bn trong Phng Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc. Ti xin chn thnh cm n s gip qu bu . Ti xin gi li cm n chn thnh ti cc thy c gio trong khoa SinhKTNN v khoa Sau i hc, trng i hc S phm Thi Nguyn hng dn, truyn t kin thc cho ti trong sut qu trnh hc tp v nghin cu. Ti cng v cng cm n nhng tnh cm tt p ca nhng ngi thn trong gia nh, ng nghip v bn b lun dnh cho ti, ng vin v to mi iu kin tt nht cho ti trong sut thi gian hc tp, nghin cu. Thi Nguyn, ngy 25 thng 09 nm 2009 Hc vin
Lu Th C
MC LC
Trang M U 1 Chng 1. TNG QUAN TI LIU ..................................................................... 3 1.1. VAI TR V TM QUAN TRNG CA CY MA ................................ 3 1.1.1. S lc v cy ma ............................................................................................. 3 1.1.2. Tnh hnh sn xut ma Vit Nam ................................................................ 4 1.2. SINH TNG HP SUCROSE ........................................................................... 5 1.3. VN CHUYN SUCROSE TRONG T BO .............................................. 8 1.5. C CH BIU HIN GEN BNG PHNG PHP RNAi (RNA INTERFERENCE) .................................................................................... 10 1.5.1. Ngun gc RNAi .............................................................................................. 10 1.5.2. C ch gy bt hot gen .................................................................................. 10 1.6. K THUT GATEWAY ............................................................................... 12 1.7. NGHIN CU V TI SINH V CHUYN GEN CY MA ........... 14 Chng 2. NGUYN LIU V PHNG PHP NGHIN CU ........... 16 2.1. NGUYN LIU ................................................................................................... 16 2.1.1. Nguyn liu thc vt ........................................................................................ 16 2.1.2. Cc chng plasmid v enzyme ....................................................................... 16 2.1.3. Ha cht khc .................................................................................................... 16 2.1.3. Cc thit b my mc ....................................................................................... 17 2.2. PHNG PHP ................................................................................................. 17 2.2.1. Thit k mi....................................................................................................... 17 2.2.2. Tch RNA tng s ............................................................................................ 18 2.2.3. RT-PCR .............................................................................................................. 18 2.2.4. Tch dng v xc nh trnh t gen ............................................................... 19 2.2.5. Thit k vector ti t hp INV-RNAi ........................................................... 20
2.2.6. Ti sinh ma thng qua m so ...................................................................... 21 2.2.7. Th nghim chuyn gen gus-intron vo cy ma ....................................... 22 NI DUNG NGHIN CU: ...................................................................................... 2 Chng 3. KT QU V THO LUN ........................................................... 24 3.1. THIT K MI ................................................................................................... 24 3.2. TCH RNA TNG S ...................................................................................... 25 3.3. NHN DNG ON GEN M HA ENZYME INVERTASE ............ 27 3.4. TCH DNG GEN V XC NH TRNH T GEN ............................. 28 3.4.1. To plasmid ti t hp INV-pENTR ............................................................ 28 3.4.2. Bin np plasmid ti t hp INV_pENTR vo t bo kh bin E.coli TOP 10 ............................................................................................ 28 3.4.3. Chn lc plasmide ti t hp INV_pENTR bng PCR ............................. 29 3.4.4. Kt qu xc nh trnh t nucleotit ................................................................ 31 3.5. THIT K VECTOR TI T HP INV-RNAi........................................... 31 3.5.1. To vector ti t hp INV_RNAi bng k thut Gateway ....................... 31 3.5.2. Bin np vector INV_RNAi vo t bo kh bin E.coli ........................... 32 3.6. BIN NP VECTOR CHUYN GEN INV_RNAi VO CHNG VI KHUN A.TUMEFACIENS CV58C1. .......................................... 34 3.7. TI SINH V BC U BIU HIN GEN GUS MA ................... 35 3.7.1. Quy trnh ti sinh ma thng qua m so ..................................................... 35 3.7.3. Chn lc m so v ti sinh cy chuyn gen ............................................... 37 KT LUN V NGH ..................................................................................... 40 TI LIU THAM KHO ....................................................................................... 42
NHNG CH VIT TT
AS A.tumefaciens BAP bp cDNA Acetosyringone Agrobacterium tumefaciens 6-Benzyl Amino Purine (Benzyladeninpurin) Cp base Complementary DNA = DNA b sung c tng hp bng khun mRNA cs DEPC DNA dNTP EDTA EtBr E.coli gus IBA kb LB MS NAA OD PCR RNA RNase RT-PCR SDS TAE Taq 2,4D Cng s Diethyl pyrocarbonat Deoxyribonucleic Acid deoxynucleosit triphotphat (deoxynucleoside triphosphate) Ethylene diamine tetraacetic acid EtBrEthiium bromide Escherichia coli -glucuronidase Indole-3-Butyric Acid kilo base Luria and Bertani Mi trng nui cy theo Murashige v Skoog Naphthalene Acetic Acid Gi tr mt quang (optical density) Polymerase Chaine Reaction = Phn ng chui Polymerase Ribonucleic Acid Ribonuclease Reverse Transcriptase-PCR Sodium dodecylsulfat Tris-acetate-EDTA Thermus aquaticus DNA (polymerase) 2,4-Dichlorophenoxyacetic Acid
DANH MC CC BNG
Tn bng Trang
Bng 2.1. Cc plasmid s dng trong th nghim ................................................. 16 Bng 2.2. Chu k nhit cho phn ng RT-PCR mt bc ................................... 18 Bng 2.3. Chu k nhit cho phn ng PCR ............................................................ 19 Bng 2.4. Cc mi trng ti sinh cy ma ............................................................. 21 Bng 3.1. Trnh t v cc thng s cn thit ca cp mi 3INV v 5INV .......................................................................................................... 25 Bng 3.2. M s cc trnh t on gen Invertase ma trn ngn hng gen NCBI ................................................................................................... 25 Bng 3.3. Kh nng to m so v ti sinh ging ma ROC10 in vitro trn cc mi trng th nghim M1 - M4. .......................................... 36
DANH MC CC HNH
Tn hnh Hnh 1.1: Chu trnh sinh tng hp sucrose vi s tham gia ca cc enzyme chnh ............................................................................................... 6 Hnh 1.2. C ch gy bt hot gen RNAi................................................................ 11 Hnh 1.3. S m t k thut Gateway ................................................................ 13 Hnh 3.1. Kt qu in di RNA tng s tch t l v b thn non ca 2 ging ma ROC1 v ROC10 trn gel agarose 1% ............................... 26 Hnh 3.2. Kt qu in di sn phm RT-PCR trn gel agarose 0,8% ................ 27 Hnh 3.3. Kt qu in di sn phm PCR plasmid vi cp mi M13 (For/Rev) nhm kim tra s c mt ca on Invertase trong vector pENTR/D ....................................................................................... 30 Hnh 3.4. Kt qu so snh trnh t on gen Invertase phn lp c vi trnh t Invertase trong ngn hng gen c m s AY302083 .................................................................................................. 31 Hnh 3.5. M hnh cu trc chuyn gen INV_RNAi ........................................... 32 Hnh 3.6. Kt qu in di sn phm ct plasmid INV_RNAi t hp vi HindIII v XbaI ......................................................................................... 34 Hnh 3.7. in di sn phm PCR plasmid INV-RNAi trong A.tumefaciens vi cp mi c hiu 5INV v 3INV ....................... 35 Hnh 3.8. Quy trnh ti sinh ma ROC10 in vitro t m so ................................ 37 Hnh 3.9. Bin np gen gus-intron vo cy ma ROC10 in vitro thng qua trung gian A.tumefaciens ................................................................. 39 Trang
M U
ng l mt nhu cu cn thit trong i sng con ngi. Theo thng k, nhu cu tiu th ng trn th gii trung bnh tnh theo u ngi l 35 kg/1 ngi/1 nm. Ti Vit Nam, nm 1994 l 8 kg/1 ngi/1 nm, hin nay l 15 kg/1 ngi/1 nm v d kin nhu cu v ng cn tip tc tng na. Ti cc nc nhit i v cn nhit i nh Vit Nam, 75% sn lng ng c sn xut t cy ma. Ma l mt trong s t loi thc vt tch tr ch yu ng sucrose (-D-glucopyranosyl-1, 2-D-fructofuranose), ngun nguyn liu ban u sn xut ng. Do , Vit Nam ma tr thnh mt cy cng nghip trng yu v l cy xa i gim ngho ca chnh ph. Tuy nhin, cc ging ma ca Vit Nam c nng sut ng ch t mc trung bnh ca th gii. Vic nhp cc ging ma cao sn ca th gii kt hp vi phng php lai to truyn thng cha thc s c hiu qu trong vic to ging ma c hm lng ng cao li ph hp vi iu kin th nhng kh hu ca nc ta. Chn to ging ma c hm lng ng cao bng cng ngh sinh hc c tim nng gim gi thnh ng m khng cn tng din tch trng ma v thc y s pht trin nn cng nghip ma ng ti Vit Nam. Sinh tng hp sucrose l mt qu trnh phc hp, trong enzyme Invertase c xem nh l mt chic cha kha iu chnh s tch ly lng sucrose trong cy ma. N c vai tr phn hy sucrose trong t bo. V vy, mun tng tr lng sucrose trong cy ma th phi c ch c s biu hin ca gen m ha Invertase. C ch gy bt hot gen RNAi (RNA-interference) hin nay tr thnh mt bin php cng ngh hu hiu c th c ch hon ton biu hin ca gen ng vt, thc vt v c vi sinh vt [31]. thc vt,
2 RNAi c th c thc hin bng cch chuyn gen c cu trc biu hin s phin m cao RNA sense, anti-sense hoc RNA kp tc b sung chnh n m cha trnh t tng ng vi gen ch. Vi mc tiu nghin cu chn to ging ma c hm lng ng cao, chng ti chn ti Phn lp v thit k vector c ch biu hin gen m ha enzyme Invertase (-fructofuranosidase) nhm tng tr lng sucrose cy ma. MC TIU NGHIN CU: c ch biu hin ca Invertase dng ha tan nhm tng tr lng sucrose cy ma. NI DUNG NGHIN CU: 1. Phn lp on gen m ha cho enzyme Invertase cy ma in vitro ROC1 2. Thit k c vector c ch biu hin gen m ha Invertase (fructofuranosidase) cy ma. 3. Nghin cu h thng ti sinh cy ma phc v cho mc ch chuyn gen tip theo.
Chng 1 TNG QUAN TI LIU

1.1. VAI TR V TM QUAN TRNG CA CY MA 1.1.1. S lc v cy ma Ma (Saccharum L.) thuc chi ma (Saccharum), h ha tho (Poaceae), b la (Poales), lp mt l mm (Monocotyledoneae). Chng l nhng cy c thn to, mp, chia t cao t 2 - 6 m. Cc loi thc vt trong chi ny a s l cc loi c sng lu nm bao gm khong 6 - 37 loi ty theo h thng phn loi, sng ch yu khu vc nhit i v n i trn th gii [2]. Cy ma cha hm lng ng rt cao chim khong 46% khi lng kh, trong sucrose chim ti 80%. Chnh v th, ma tr thnh mt trong nhng cy cng nghip quan trng ca ngnh cng nghip sn xut ng. Ngoi ra, cy ma cn cha cc cht m (protein), cht bt (carbohydrate), cht bo (lipid), cc cht khong v cc vitamin v th ma cn c tc dng thanh nhit, gii kht, tr gip tiu ha, cung cp nng lng v cc cht dinh dng cn thit cho c th. Theo ng y, ma l "v thuc" dng cha mt s bnh nh ho khan, i tin to, tiu tin bt li, au d dy, an thai Ma cn l loi cy c tc dng bo v t rt tt, c bit l chng xi mn t cho cc vng i trung du. Hn na, ma l cy r chm v pht trin mnh trong tng t t 0 - 60 cm (1 ha ma tt c th cho 13 - 15 tn r sau thu hoch), y l ngun cht hu c qu lm tng ph ca t. Phn b ma cha nhiu cellulose c th dng lm nguyn liu t l, hoc lm bt giy, ba cc tng, p thnh vn dng trong kin trc... Sn phm cn b cn li sau khi ch bin ng (bn lc) c th s dng sn xut nha, xrin, lm sn, xi nh giy... ph phm cn li dng lm phn bn rt tt. Trong tng lai b ma cn c th ngun nguyn liu lm bt giy, lm si thay th
S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.Lrc-tnu.edu.vn
4 cc loi cy rng b gim i. Khi m ngun nhin liu lng ngy cng cn kit nh hin nay, mt s nc pht trin trn th gii nh M, Brazil, n bt u s dng nhin liu sinh hc t cy ma b sung v thay th. Nh vy, cy ma c vai tr rt quan trng trong i sng kinh t ca con ngi. 1.1.2. Tnh hnh sn xut ma Vit Nam Hin nay c khong 200 quc gia v vng lnh th trn th gii trng v sn xut ma ng, sn lng trung bnh t khong 13.246 triu tn (gp 6 ln so vi c ci ng). Vit Nam, ma l cy trng ch o trong ngnh cng nghip ng ca c nc. D kin nin v 2009-2010 din tch ma nguyn liu c nc s vo khong 290.000 ha, tng 19.400 ha so vi v trc, trong din tch vng ma nguyn liu tp trung ca cc nh my l 221.816 ha vi nng sut ma bnh qun t 55 tn/ha v sn lng t 16 triu tn. Cy ma gp phn xa i gim ngho vng trung du, min ni nhiu tnh nc ta nh: Ha Bnh, Thanh Ha, Ngh An, Ph Yn, Bnh nh, Qung Ngi [3]... Nh nc h tr mt phn u t pht trin c s h tng giao thng, thy li cho vng trng ma tp trung, nghin cu chuyn giao khoa hc k thut v cng ngh nhm nng cao nng sut, cht lng, hiu qu sn xut ma ng [1]. Qu trnh th ha, cng nghip ha ngy mt gia tng cng vi s bin i mi trng kh hu nn din tch t trng trt c xu hng ngy mt thu hp. Hn na, nc ta hin nay c ti trn 60% cc ging ma l nhng ging c nh: ROC1, ROC10, F156, F127 hoc cc dng lai ghp ni chi phc tp. Cc ging ny c c im d canh tc, thch nghi rng vi nhiu vng sinh thi ca Vit Nam, nhng tr lng ng rt thp. Cn li cc ging ma nhp ni tuy c tr lng ng cao song khng ph hp vi kh hu Vit Nam nn nng sut thp. Chnh v th, Quyt nh s 26/2007/Q-
5 TTg ca Ph th tng Nguyn Sinh Hng Quy hoch pht trin ma ng n nm 2010 v nh hng n nm 2020 c ph duyt a quan im r rng l: ng thi vi vic nhp khu ging ma c nng sut, tr ng cao c nh gi tt ph hp vi Vit Nam th phi xy dng h thng vin nghin cu v cc trung tm ging ma iu kin trang thit b v nng lc cn b ch ng sn xut ging tt, c nng sut, tr lng ng cao ca Vit Nam, p ng yu cu sn xut [1]. 1.2. SINH TNG HP SUCROSE Trong lc lp ca ma c enzyme photphoenolpyruvat-cacboxilase hot ng rt mnh. Sn phm u tin ca quang hp ma l cc axit oxaloaxetic, malic, aspartic u gm c bn nguyn t cacbon trong phn t, do ma c gi l thc vt C4 [5]. Chu trnh C4 (hay c ch Hatch-Slack) l c ch c s chuyn ho trong vic thc hin chc nng quang hp ca cy C4: mt loi lc lp chuyn trch c nh CO 2, cn mt loi lc lp chuyn kh CO2 thnh cc cht hu c cho cy. V vy m hot ng quang hp ca cy C4 c hiu qu hn cc nhm thc vt khc v thng cho nng sut sinh hc rt cao. Sucrose l mt disaccharide ca glucose (-D-glucopyranoside) v fructose (-D-fructofuranosyl), c cng thc phn t C12H22O11. y l sn phm chnh ca qu trnh quang hp, c vai tr b sung nng lng cho qu trnh sinh trng pht trin ca thc vt cng nh cc sinh vt sng khc. Trong c th ng vt, sucrose l nguyn liu tng hp glucogen, khi tha s chuyn sang dng m d tr. Sucrose tch ly phn ln cc m ca thc vt, gip cho thc vt c kh nng thch nghi tt hn vi cc iu kin bt li ca mi trng nh: hn, lnh, mn v cng nh sng mnh... [7, 12, 17, 23, 26, 30]. N c tch tr ch yu cy ma, c ci ng v c
6 trong nhiu loi cy khc nh da, chui, m, mn, da hu, to, c rt y l ngun nguyn liu t nhin, rt d trng vi s lng ln v gi r. Sucrose rt d ha tan trong nc, khi b thy phn to thnh glucose v fructose. Sinh tng hp sucrose l mt chu trnh phc tp din ra cytosol (t bo cht) trong l ca cy trng vi s tham gia ca nhiu enzyme khc nhau, trong mt s enzyme chnh (key enzyme) c nh hng ln ti lng sucrose c tng hp. Cc enzyme ny xc tc cho cc dng phn ng: (1) Tng hp sucrose nh sucrose 6-phosphatephosphatase (SPS) hoc sucrosesynthase (SS) (2) Thy phn sucrose nh -fructofuranosidase (Invertase) (3) Vn chuyn cc hexose ti t bo cht v chuyn ha li thnh sucrose nh pyrophosphate fructose 6-phosphat 1 phosphostransferase (PFP) [6].
SUCROSE 6-P SPS Pi UDP SS UDP-GLUCOSE PPi
SUCROSE Invertase
UDP
FRUCTOSE
GLUCOSE
ATP
UTP PFP Pi ADP FRUCTOSE 1.6-P2 Pi ATP GLUCOSE 1-P ADP GLUCOSE 6-P
FRUCTOSE 6-P
Hnh 1.1: Chu trnh sinh tng hp sucrose vi s tham gia ca cc enzyme chnh
7 Thay i hot tnh cc enzyme ny thng dn ti nhng bin i ln trong lng sucrose tch ly trong t bo thc vt [6, 10, 11, 15, 31, 35]. Cc enzyme lin quan ti chu trnh sinh tng hp sucrose thc vt c nghin cu trn nhiu i tng gm c thc vt hai l mm v mt l mm [10, 15, 31]. Trong , SPS (sucrose 6-phosphatephosphatase) c coi l enzyme chnh ca chu trnh sinh tng hp sucrose thc vt, n xc tc qu trnh hnh thnh sucrose 6-phosphate, c cht cho phn ng tng hp cc phn t sucrose [7, 16]. SPS l enzyme c hot tnh t l thun vi sucrose trong cc m d tr ca khoai ty, ng, ci b xi [30]. ng, hot tnh SPS t l thun vi tc tng hp v vn chuyn sucrose [16]. Cy c chua chuyn gen tng cng biu hin SPS th lng sucrose tng cn lng tinh bt gim trong qu trnh quang hp [33]. Tng t, cy bng mang gen SPS ca rau bi-na (spinacia oleracea) cng c tc tng hp sucrose cao hn so vi i chng [13]. Ngc li, khi lm bt hot SPS, cc dng cy khoai ty chuyn gen s gim tr lng sucrose [10]. Gen m ha cho SPS c phn lp t nhiu loi thc vt khc nhau v cho ti nay ngn hng gen NCBI c thng tin v vi trm trnh t SPS ca cc loi thc vt hai l mm nh khoai ty, thuc l, rau bi-na, c ci ng; cy mt l mm nh la, ng, ma v c to lam [9, 10, 13, 14, 21, 27, 31, 33]. Lin quan cht ch vi SPS trong chu trnh sinh tng hp sucrose l enzyme thy phn sucrose - Invertase (fructofuranosidase). Khi hot tnh ca Invertase cao th lng ng tch ly trong cc m t bo gim v ngc li. Hot tnh ca enzyme pyrophosphate fructose 6-phosphotransferase (PFP) cng t l nghch vi lng sucrose trong t bo thc vt [11]. PFP xc tc chuyn ha fructose 6-phosphate thnh fructose 1,6-biphosphate. Do vy, nu hot tnh ca PFP gim, lng c cht cho phn ng tng hp sucrose s tng, dn ti lng sucrose cng tng tng ng. nh hng ny c chng minh trn cc dng ma c mang
8 cu trc antisense lm bt hot PFP. Lng sucrose trn cc cy ma chuyn gen cn non tng khong 50%. Tuy nhin, khi phn tch trn cc cy trng thnh, tng lng sucrose tng, nhng cha ng k so vi i chng [11]. Nhng nghin cu trn chng t c vai tr quan trng ca cc enzyme SPS, PFP, Invetase trong vic tng hp v tch ly sucrose thc vt. 1.3. VN CHUYN SUCROSE TRONG T BO Theo thuyt vn chuyn ng (hay thuyt Turgeon), ng sucrose c tng hp t bo cht ca cc t bo tht l trong quang hp s c vn chuyn ra khng bo, sau nh h thng cu trc lin kt gia cc t bo (si lin bo v cu sinh cht hay plasmodesmata) n s c chuyn t t bo ny sang t bo khc v nh ng dn phloem m sucrose i ti khp cc c quan ca thc vt bng con ng khuych tn. Cc phn t nh sucrose trong ng dn phloem s c polyme ha thnh nhng phn t ng ln v phc tp hn, lc ny cc phn t ng c y ra xa khi l n nhng phn khc ca cy, ni m n c s dng hay tch tr li, v do kch thc ca n qu ln nn n khng th chuyn ngc tr v l. Chnh c ch khuch tn ny to nn s vn chuyn lin tc sucrose t cc c quan quang hp (source tissue) ti cc c quan d tr (sink tissue). Bn cnh , sucrose cn c vn chuyn v tch tr ti khng bo lm nguyn liu cho chu trnh sinh tng hp tinh bt. Ngoi lng sucrose c vn chuyn lin tc, mt phn sucrose s c phn hy nhm cung cp nng lng cho qu trnh sinh trng v pht trin, ng thi ti to cc c cht khc. 1.4. ENZYME INVERTASE (-FRUCTOFURANOSIDASE) Invertase (-fructofuranosidase) c m ha bi 5 - 10 ng phn ty thuc tng loi thc vt khc nhau. Cy m hnh Arabidopsis thaliana c 5 ng phn Invertase, trong khi c chua hin nay phn lp c 8 ng phn. Hin nay, trn ngn hng gen c trnh t EST (Expressed Sequenced
9 Tags) ca enzyme Invertase phn lp t mt s ging ma [35]. Invertase trung tnh (c trong t bo cht) c tinh sch t thn ma trng thnh. Tuy nhin trnh t gen m ha cho dng enzyme ny vn cha c cng b trn GenBank. Invertase c mt hu ht cc m thc vt tch tr ng v tn ti nhiu dng khc nhau: dng ho tan (soluble acid Invertase) c nhiu trong khng bo (dch t bo); dng lin kt vi mng (cell wall Invertase) c trong thnh t bo; dng c lp (neutral Invertase) c ch yu trong ht. N l mt enzyme xc tc qu trnh thy phn sucrose trong khng bo thnh hai ng n l glucose (Aldohexose) v fructose (Ketohexose) [6]. V th, mc biu hin ca n c nhiu nh hng ln s sinh trng pht trin ca thc vt [6]. C th, khi Invertase c hot tnh cao n s lm gim mt lng sucrose ng k trong thn cy ma. Nghin cu cho thy nhng dng ma c sn lng ng cao thng l cc dng c hot tnh Invertase thp v ngc li [35]. Tng t, mt s dng c chua ngt c tch ly nhiu ng l do hot tnh ca Invertase thp. Invertase c hot tnh cao trong khng bo ca t bo thuc l v u tng cng lm gim lng sucrose trong cc c quan ny [6]. Bt hot Invertase lm tng t l tch tr sucrose trong cy c chua chuyn gen l v qu, ng thi cng lm thay i t l ng n (hexose) trong cc dng cy ny [18, 24]. L ca cc dng khoai ty biu hin gen m ha Invertase phn lp t nm men tch tr ch yu glucose v fructose hn l sucrose. Hn na, nhng dng ny hnh thnh t c v nh hn cc dng i chng, nhng cha nhiu ng hn l tinh bt. iu ny chng t rng Invertase c lin quan cht ch ti hm lng v vn chuyn ca sucrose thc vt.
10 Nh vy, Invertase c xem nh l mt chic cha kho iu chnh s tch ly sucrose d tr trong thc vt. Do , c ch biu hin ca Invertase c th tng tch ly sucrose trong cy ma. 1.5. C CH BIU HIN GEN BNG PHNG PHP RNAi (RNA INTERFERENCE) 1.5.1. Ngun gc RNAi RNAi l mt c ch cn bn kim sot chui thng tin di truyn hay cch v hiu ho hot ng ca cc gen xc nh do hai nh khoa hc Andrew Z. Fire v Craig C. Mello khm ph ra v cng b trn tp ch Nature vo ngy 19/12/1998 [4]. Andrew Fire v Craig Mello nghin cu c ch iu khin biu hin gen giun trn (Caenorhabditis elegans) v cho rng khi mRNA chiu dch m v chiu i m gp nhau th chng s kt hp li thnh nhng mRNA si kp. Hai ng kim chng li gi thuyt ca mnh bng cch tim cc phn t mRNA si kp cha cc mt m di truyn quy nh nhiu protein khc ca giun trn. Kt qu u thu c protein c m ha bi cc gen khng c tng hp. Qua Fire v Mello rt ra c kt lun rng c th RNA dng chui kp lm cc gen b bt hot. Cng trnh c cng b v c trao gii Nobel Y hc nm 2006. 1.5.2. C ch gy bt hot gen RNAi (RNA interference) c coi nh mt phng thc min dch t nhin gip sinh vt chng li s xm nhp ca virus RNA bng cch phn hu cc trnh t nucleotide tng ng ca chng [8]. N lm trung gian khng li c acid nucleic ngoi bo v ni bo, cng nh iu khin s biu hin gen m ha protein. N c thc hin khi c s xut hin ca phn t RNA mch kp trong c th sinh vt gy nn c ch s biu hin gen ca mt loi trnh t c hiu.
11
Hnh 1.2. C ch gy bt hot gen RNAi Hnh 1.2 cho thy, C ch RNAi c bt u bng vic phn ct phn t RNA chui kp (dsRNA) bi enzyme Dicer - mt trong nhng enzyme thuc h RNase III, to thnh cc phn t RNA c ch nh (siRNA) c kch thc khong 21 - 26 nucleotide [4]. Cc siRNA ny c gii xon v mt mch gn kt vi mt phc hp protein mt cch chn lc gi l phc hp cm ng s bt hot RNA (RISC RNA Induced Silencing Complex). Argonaute (protein Argonaute) trong RISC c cha RNase-H hot ng nh mt endonuclease s tch siRNA thnh nhng chui RNA n, trong ch c mt chui n RNA c u 5 c lc bt cp base (base pairing) nh nht c chn tip tc i vo phc h RISC. Sau , RISC s thu nhn cc phn t phin m mRNA ni sinh ca t bo c trnh t tng ng vi trnh t ca chui siRNA ang c mt trong phc h bng cch bt cp vi cc base theo
12 nguyn tc b sung. Khi c nhn din cc mRNA nhanh chng b ct t khong gia ca chui xon kp siRNA-mRNA v b tiu hy bi cc RNA nuclease (Helicase) c trong RISC. Si RNA b phn ct, tip tc hnh thnh cc siRNA. Qu trnh tip din lin tc nh vy s phn hy cc bn m sao hnh thnh, kt qu l c ch biu hin ca gen mong mun [4]. C ch can thip RNAi em li nhng ng dng v cng to ln v ang l cng c nghin cu hu ch trong nhiu ngnh sinh hc, nng nghip v y dc hc. N c bit n nh mt k thut sinh hc hin i c hiu qu trong vic chuyn gen phng chng bnh do virus, vi khun, hay lm tng cng, c ch mt tnh trng mong mun no sinh vt. Phng php ny c ng dng thnh cng thay i thnh phn cht bo trong du, loi caffein trong c ph, tng hm lng lysine trong ng hoc loi cc cht gy d ng to v c chua [19, 20, 25]. RNAi l mt hng mi cho php cc nh khoa hc nghin cu nhng ng dng trong cc liu php tr bnh cho con ngi trong tng lai cng nh phn tch chc nng h gen cy trng v.v... 1.6. K THUT GATEWAY K thut Gateway (Invitrogen) l mt k thut dng ha ph bin, n mang li hiu qu cao v nhanh chng khi phn tch chc nng, biu hin protein v dng ha on DNA. K thut ny cho php chuyn on DNA gia cc vector tch dng khc nhau m vn duy tr nh hng chnh v cu trc c, thay th vic s dng cc enzyme gii hn v cc enzyme ni hiu qu trong thi gian ngn. K thut ny c hiu qu cao (90%) i vi vic tch dng c nh hng ca cc sn phm PCR. Hn na cc phn ng n gin, d thc hin, nhanh, mnh v t ng. y l k thut hu ch cho nhng c tnh ti t hp c hiu v tr ca vi khun lambda, gip cho phn
13 ng ti t hp lambda (Lambda reconstruction: LR) xy ra mt cch hiu qu v gn cc on trnh t DNA vo nhiu h thng vector.
Hnh 1.3. S m t k thut Gateway - K thut Gateway c thc hin bi hai bc chnh: + To dng tip nhn entry clone bng cch chn gen biu hin (expression gene) vo vector tip nhn (pENTR/D). + To dng biu hin (expression clone) bng cch ti t hp gia entry clone vi mt vector ch (destination vector) m c cha cc trnh t attR1 v attR2 v marker c kh nng khng chn lc ccdB. Trn hnh 1.3 cho thy dng vector nhn c cc im ti t hp attL1 v attL2 s phn ng vi cc im ti t hp trn vector ch attR1 v attR2 to ra mt cu trc mi attB1 v attB2. Mt khc, on gen quan tm c
14 gn vo trn vector nhn s trao i cho vi on ccdB trn vector ch v th thu c trn dng biu hin c cu trc ca on gen mong mun. 1.7. NGHIN CU V TI SINH V CHUYN GEN CY MA Ti sinh cy c xem l mu cht quan trng, quyt nh s thnh cng ca cc th nghim chuyn gen. Hin nay, h thng chuyn gen thc vt thng qua A.tumefaciens mang li nhng thnh tu ln trong thi gian ngn c th to ra cc ging cy trng c nhng c tnh tt mong mun. ma chuyn gen thnh cng vi hai phng php l sng bn gen v thng qua vi khun A.tumefaciens, trong chuyn gen thng qua A.tumefaciens l hiu qu hn hn. Snyman v cs (2006) ti sinh v chuyn gen thnh cng cy ma bng phng php sng bn gen t m so [29]. M so to ra bng cch t nhng lt ct nh dy 1 - 2 mm phn nh ngn ca cy ma ln mi trng cm ng to m so l (MS + 30 g/l sucrose + 0,5 g/l casein + 0,6 mg/l 2,4D +5 g/l agar), pH = 5,8 trong iu kin ti, 28oC. Trc 4 gi chuyn gen bng k thut sng bn gen, cc m so c t ln mi trng c b sung thm 0,2 M Sorbitol; 0,2 M manitol. Sau chuyn gen cc m so c ng nui cy trong ti, 3 ngy. Tip theo m so c chuyn sang mi trng chn lc v thu c cy chuyn gen hon chnh vi cc mi trng c b sung 45 mg/l geneticin. Manickavasagam v cs (2004) ti sinh v chuyn gen thnh cng ma thng qua A.tumefaciens t cc m phn sinh ca chi bng con ng to a chi vi hiu qu thu c l 49,6% cy chuyn gen [22]. Ly nhim A.tumefaciens vo cc m non b lm thng ca ma trong 10 pht. Sau thm kh trn giy thm v t ln mi trng MS trong 3 ngy. Cc m c dit khun bng nc ct kh trng c b sung 500 mg/l cefotaxime.
15 ng to c cc cy chuyn gen trn cc mi trng chn lc ti sinh c b sung 5 mg/l ppt. Santosa v cs (2004) chuyn gen thnh cng vo m so ca ma thng qua A.tumefaciens GV2260. Kt qu kim tra cc cy sau chn lc thy rng c khong 85 - 100% cy sng st c mang gen chuyn [34]. M so c to t on thn non trn mi trng [4,3 g/l MS + 30 g/l sucrose + 0,5 g/l NZamine + 0,3 ml vitamin (0,1 g / 75 ml hydrochloride thiamine; 0,05 g / 75 ml biotin; 1 g / 75 ml pyrodoxine hydrochlorid; 0,25 g / 75 ml myo-inositol) + 3 mg/l 2,4D + 100 ml nc da + 8 g/l agar, pH = 5,8] trong iu kin ti, 1 thng. Trc 7 gi bin np vi A.tumefaciens b sung thm 75 l cht chng xyha vo dch khun. A.tumefaciens c ha tan vi mi trng cm ng to chi c b sung 5 ml cht chng xyha vi OD578 = 0,2 v c ly nhim vi cc mnh m so nh (2 - 3 mm) trong 5 - 10 pht. Dit khun v chuyn m so ln mi trng c b sung 500 mg/l cefotaxime 28oC, lc 60 vng/2 ngy. Cy chuyn gen c to thnh vi cc mi trng chn lc c b sung 500 mg/l cefotaxime v 100 mg/l kanamycin. Zhangsun v cs (2007) ti sinh v chuyn gen ma t m so thng qua A.tumefaciens (OD600=0,2; 0,4; 0,6) trong thi gian 10 - 20 pht vi khng sinh chn lc 500 mg/l carbenicillin v 1 mg/l ppt [28]. Nh vy, cc tc gi ti sinh v chuyn gen thnh cng ch yu t m so v thng qua vi khun A.tumefaciens. thnh cng vic chuyn gen vo thc vt ni chung cng nh ma th nht thit phi c mt h thng ti sinh hon chnh v mt quy trnh chuyn gen hot ng hiu qu. Trong th nghim ny chng ti th nghim v ci bin h thng ti sinh v quy trnh chuyn gen ca cc nghin cu thnh cng v ma trn.
16
Chng 2 NGUYN LIU V PHNG PHP NGHIN CU

2.1. NGUYN LIU 2.1.1. Nguyn liu thc vt Chng ti s dng ging ma ROC1 v ROC10 in vitro ang c gi ti Phng Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc, Vin Khoa hc v Cng ngh Vit Nam. 2.1.2. Cc chng plasmid v enzyme Bng 2.1. Cc plasmid s dng trong th nghim Plasmid pENTRTM/DTOPO pK7GWIWG2(II) Kch thc (bp) 2580 Khng sinh chn lc Ngun cung cp Invitrogen (M) Trng i hc Ghent (B)
Kanamycin Spectinomycine, streptomycine
12904
- Cc chng vi khun: E.coli One Shot TOP 10 v A.tumefaciens chng CV58C1 mang plasmid pGV2260 - Cc enzyme gii hn: HindIII , XbaI ca hng New England Biolabs - Cc enzyme T4 ligase, T4 kinase do hng Fermentas cung cp 2.1.3. Ha cht khc - Cp mi 3INV/5INV nhn on gen m ha enzyme Invertase v cp mi M13for/rev kim tra s c mt ca gen Invertase trong vector tch
17 dng pENTRTM/D-TOPO do chng ti thit k v t tng hp ti hng Bioneer (Hn Quc). - Gateway LR ClonaseTMII Enzyme Mix Kit (Invitrogen, M) - B kit kit Trizol Regents (Invitrogen, M) - B kit Qiagen (QIAquick Gel Extraction) - B kit QIAprep Spin Miniprep (QIAGEN). - Cc ha cht thng dng trong sinh hc phn t (thang marker chun, agarose, phenol, chloroform, isoamylalcholhol... ) v cc ha cht cho nui cy m u thuc phng Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc mua t cc hng ni ting nh Invitrogen, Merk, Amersham Parmacia Biotech, Fermentas, New England Biolabs... 2.1.3. Cc thit b my mc Pipetman, my soi gel (Bio-Rad), my chp nh (Amersham Pharmacia Biotech), my li tm (Ependorf), my o pH (Mettler), b in di (Bio-Rad), my ht chn khng (Savant), my PCR (MJ Research), b n nhit, ni kh trng, my bin np bng xung in, my o OD, my vortex, my xc nh trnh t nucleotid t ng... ca Phng th nghim Cng ngh T bo Thc vt, Vin Cng ngh Sinh hc. 2.2. PHNG PHP 2.2.1. Thit k mi Khai thc d liu trong GenBank tm ra tt c cc trnh t gen m ho Invertase ca cy ma (Bng 3.2). S dng phn mm chuyn dng DNAstar so snh tng ng gia cc trnh t Invertase thu c v thit k cp mi c hiu ti cc vng c bo th cao nht.
18 2.2.2. Tch RNA tng s S dng ha cht Trizol Regents (Invitrogen, M) tch chit RNA tng s t cc mu l v b thn non ca 2 ging ma in vitro ROC1 v ROC10 theo hng dn km theo. 2.2.3. RT-PCR Phn lp gen m ha enzyme Invertase ma bng k thut RT-PCR mt bc (RT-PCR one step) theo chu k sau: Bng 2.2. Chu k nhit cho phn ng RT-PCR mt bc Bc 1 2 3 4 5 6 7 Phn ng Tng hp cDNA Bin tnh Bin tnh Gn mi Ko di chui Hon tt chui Kt thc phn ng Nhit (oC) 50 95 94 52 72 72 8 Thi gian 30 pht 5 pht 20 giy 1 pht 1 pht 10 pht 24 gi Chu k 1 1 35 35 35 1 1
Thnh phn ca phn ng vi tng th tch 25 l c b sung vo ng effendorf 0,5 ml c x l DEPC theo hng dn ca nh sn xut (Bioneer) gm c: 2X dung dch m RT-PCR; 1 g RNA tng s; 0,4 - 0,6 mM 3INV; 0,4 - 0,6 mM 5INV; 0,5 g enzyme RT. Mix mu nh nhng v thc hin phn ng RT-PCR theo chu k nhit nh bng 2.2. Sn phm c kim tra bng phng php in di trn gel agarose 0,8 - 1,5% trong dung dch m TAE 1X v nhum bn gel trong ethidium
19 bromide (EtBr). Sn phm PCR c tinh sch bng QIAquick Gel Extraction Kit. 2.2.4. Tch dng v xc nh trnh t gen Sn phm PCR tinh sch s c gn vo vector tch dng pENTR/D ca hng Invitrogen (M) to vector t hp c mang on gen m ha Invertase mong mun (INV_pENTR). Phn ng c thc hin theo hng dn ca nh sn xut. Sau , vector ti t hp INV_pENTR c bin np vo t bo kh bin E.coli Top 10 v nhn nui lng ln trong mi trng LB c b sung khng sinh chn lc 50 mg/l kanamycin. Plasmid ti t hp c tch chit theo phng php Sambroock v c ct gi -20oC. INV_pENTR c kim tra bng phng php PCR vi cp mi c hiu M13for/rev. Thnh phn phn ng PCR vi cp mi c hiu M13for/rev vi tng th tch 25 l bao gm: 1X dung dch m PCR, 50 ng plasmid, 50 mM MgCl2, 2 mM dNTPs, 10 ng M13for, 10 ng M13rev, 0,5 g Taq polymerase. Chu k ca phn ng nh sau: Bng 2.3. Chu k nhit cho phn ng PCR Bc 1 2 3 4 5 6 Phn ng Bin tnh Bin tnh Gn mi Ko di chui Hon tt chui Kt thc phn ng Nhit (oC) 95 94 52 72 72 4 Thi gian 5 pht 20 giy 1 pht 1 pht 10 pht Chu k 1 35 35 35 1 1
20 Mu plasmid ti t hp INV_pENTR sau khi kim tra s c loi RNA v thc hin xc nh trnh t nucleotide trn my ABI PRIMS 3100 Avant Genetic Analyzer ti Phng th nghim Trng im Cng ngh gen. 2.2.5. Thit k vector ti t hp INV-RNAi INV_pENTR s c gn vo vector pK7GWIWG2(II) theo k thut Gateway to plasmid ti t hp mang on gen m ha Invertase (INV_RNAi). Trong k thut ny, vector pENTR/D mang v tr ti t hp attL trong khi vector nhn l attR. Cc v tr ny gip phn ng ti t hp lambda (Lambda reconstruction: LR) xy ra khi c mt ng thi plasmid INV_pENTR vi vector nhn pK7GWIWG(II) gn on gen Invertase theo c 2 chiu xui-ngc vo vector nhn, to nn cu trc INV_RNAi mong mun. Cc dng plasmid ti t hp sau c nhn ln trong t bo E.coli trn a mi trng thch LB c b sung khng sinh chn lc 100 mg/l spectinomycin, 40 mg/l streptomycin v 50 mg/l chloramphenicol 37oC qua m. Sau tin hnh tch plasmid theo kit QIAprep Spin Miniprep (QIAGEN). Plasmid ti t hp INV_RNAi thu c s c phn lp v kim tra s c mt ca on gen Invertase bng phng php ct gii hn vi hai enzyme HindIII v XbaI sau c kim tra bng phng php in di trn gel agarose 0,8%. Dng t bo mang vector INV_RNAi s c bin np vo A.tumefaciens CV58C1 bng xung in 400 / 2,5 KV / 25 F. Plasmid ti t hp c nhn nui trong mi trng c b sung 100 mg/l streptomycin, 100 mg/l spectinomycin v 50 mg/l rifamycin 28oC qua m. Tch plasmid theo Sambroock v kim tra bng phng php PCR vi cp mi c hiu 5INV v 3INV.
21 2.2.6. Ti sinh ma thng qua m so Chng ti tin hnh ti sinh ma theo phng php ca hai nhm nghin cu Santosa v cs (2004); Zhangsun v cs (2007) c ci bin nh sau: ct nhng on thn ma non c cha nh sinh trng ca cy ma in vitro di khong 0,5 cm sau t ln mi trng mi trng to m so (M1 M4). Cc mu cy s c nhn nui trong bung ti 25 oC trong vng 15 20 ngy th thu c cc m so ln tt. Cc m so nhn ln trong mi trng to m so (M1) lng c b sung 3 mg/l 2,4D trong ti, 27oC, lc 90 vng/pht, 1 tun t kch thc ln phc v cho vic bin np. Sau , cc m so c loi b phn thn xanh v t ln mi trng cm ng to chi Mc. Khi chi c kch thc khong 1- 3 cm (25 - 30 ngy) th chuyn sang mi trng to r Mr. Bng 2.4. Cc mi trng ti sinh cy ma Mi trng Nhn ma Thnh phn M0: MS + 0,8 mg/l BAP + 0,4 mg/l IBA + 30 g/l sucrose + 9 g/l agar M1: MS + 3 mg/l 2,4D + 30 g/l sucrose + 9 g/l agar To m so M2: MS + 4 mg/l 2,4D + 30 g/l sucrose + 9 g/l agar M3: MS + 5 mg/l 2,4D + 30 g/l sucrose + 9 g/l agar M4: MS + 6 mg/l 2,4D + 30 g/l sucrose + 9 g/l agar Nhn m bng nui lng lc To chi To r Mc: MS +1,5 mg/l kinetin + 2 mg/l BAP + 30 g/l sucrose +9 g/l agar Mr: MS + 1 mg/l NAA + 30 g/l sucrose +9 g/l agar 5,8 M1 lng: MS + 3 mg/l 2,4D + 30 g/l sucrose 5,8 5,8 pH 5,8
22
2.2.7. Th nghim chuyn gen gus-intron vo cy ma 2.2.7.1. To huyn ph vi khun Ly mt khun lc t a nui c nui trong mi trng LB c b sung 100 mg/l spectinomycin + 50 mg/l chloramphenicol + 50 mg/l kanamycine (hoc 100 mg/l spectinomycin + 50 mg/l rifamycin) 28oC, lc 200 vng/pht qua m. Ly ra 5 ml khun nui phc hi trong 30 ml mi trng LB lng mi, lc tip trong khong 2 - 3 gi trong cng iu kin. Sau ly tm khun 5000 vng/pht trong 10 pht thu sinh khi t bo ri ha tan khun vo MS khng ng, (pH = 5,8) c b sung AS 100 mM. 2.2.7.2. ng nui cy vi huyn ph A.tumefaciens v ti sinh M so sau khi c nhn sinh khi t mi trng lng s c ly ra tch thnh tng mnh nh. Qu trnh ly nhim khun c tin hnh theo hai hng l thi kh m so v chuyn ln mi trng cm ng to chi vi cc ngng thi gian khc nhau. Sau m so c ly ra v ngm trong huyn ph vi khun thi gian t 10 - 30 pht. Sau thm kh v cy ln mi trng ng nui cy M1 (thi kh) hoc Mc (cm ng to chi). Sau 3 ngy, m so c cy chuyn sang mi trng dit khun M1 c b sung 500 mg/l cefotaxim trong 1 tun. Cc m so sng st s c chuyn ln mi trng ti sinh Mc + 500 mg/l cefotaxim v Mc + 500 mg/l cefotaxim + 1 mg/l ppt. Cc chi ti sinh c chuyn sang mi trng chn lc Mr c b sung 500 mg/l cefotaxim v 1 mg/l ppt to cy hon chnh. Chng ti kim tra biu hin gen gus bng cch nhum m so sau ng nui cy vi dung dch X-gluc, 37oC trong ti trong khong t 12 16 gi, sau ra vi cn 70% v soi trn knh hin vi.
23 KHI QUT S TH NGHIM
Cy ma in vitro
Ct on thn st gc (5 cm) Thit k mi M so Tch RNA tng s
ng nui cy RT_PCR Dit khun
Tch dng v xc nh trnh t gen Chn lc m so
Ti sinh cy
Thit k vector ti t hp INV_RNAi
24
Chng 3 KT QU V THO LUN

3.1. THIT K MI nhn c on gen m ha cho enzyme Invertase bng k thut RT-PCR iu quan trng nht l phi c c cp mi c hiu cho trnh t ca on gen m ha cho enzyme Invertase. Chng ti s dng t kha Invertase Sugarcane tm kim trong ngn hng d liu gen NCBI cc trnh t gen Invertase c cng b (www.ncbi.nlm.nih.gov). Kt qu thu c su trnh t ca gen m ha cho enzyme Invertase cy ma, trong c 5 trnh t m ha cho enzyme Invertase ha tan (soluble acid Invertase) vi m s AF062734, AF 062735, AF083855, AF083856, AY302083; v mt trnh t m ha cho enzyme Invertase lin kt mng (cell wall Invertase) c m s AY302084 (Bng 3.2). S dng chng trnh MegAlign (DNAstar) so snh tng ng ca cc trnh t trn vi nhau cho thy nm trnh t ca Invertase ho tan tng ng cao mc d c phn lp t cc ging ma khc nhau v c tng ng 46% vi on trnh t m ho cho Invertase lin kt mng. Trnh t y ca gen ny vi m s AY302083 c di 1923 bp. Trong nghin cu ca chng ti on gen di 435 nucleotide (t nucleotide v tr 384 ti v tr 818 bp - vng c trnh t bo th cao nht) nm trn gen m ho enzyme Invertase ha tan (AY302083) c la chn tch dng. Sau vi mc ch gip sn phm PCR gn c vo vector tch dng pENTR/D chng ti gn thm ng thi vo mi u 3 mt on trnh t CACC to v tr bm cho enzyme TOPO-Isomerasse (GTGG) gn trn vector nhn dng PENTR/D-TOPO. Cp mi c t tng hp bi hng BIONEER, Hn Quc. Trnh t cp mi c hiu thu c bng 3.1 nh sau:
25 Bng 3.1. Trnh t v cc thng s cn thit ca cp mi 3INV v 5INV Mi (5INV) (3INV) Trnh t mi 5-CATCTGGGGCAACAAGATC-3 5-CACCAAGTGCACGAAGTCCTTG-3 Tm(oC) 52,3 59,1 %GC 52,6 54,5
Bng 3.2. M s cc trnh t on gen Invertase ma trn ngn hng gen NCBI STT 1 2 3 4 5 M s AY302083 AF062734 AF083856 AF083855 AF062735 Chiu di 2274 bp 1808 bp 1402 bp 494 bp 1808 bp Dng mRNA mRNA Tn Invertase ha tan Tc gi Peters,K.F., Grof,C.P.L. and Botella,J.R. Albert,H.H., Zhu,Y.J. and Moore,P.H. Albert,H.H., Zhu,Y.J. and Moore,P.H. Albert,H.H., Zhu,Y.J. and Moore,P.H. Albert,H.H., Zhu,Y.J. and Moore,P.H. Peters,K.F., Grof,C.P.L., Albertson,P.L. and Botella,J.R.
Invertase ha tan Invertase ha mRNA tan Invertase ha mRNA tan Invertase ha mRNA tan mRNA Invertase lin kt mng
AY302084
1889 bp
3.2. TCH RNA TNG S Do c tnh ca RNA l mt loi phn t khng bn, d b phn hy bi cc enzyme ribonuclease (RNase). RNase c mt khp ni trong t bo v c hot tnh rt mnh v vn c hot tnh mnh nhit cao (100oC trong khong 1 gi). Do , vic tch chit RNA i hi phi ht sc cn thn trnh cc tp nhim cha RNase t mi trng v tt c cc dng c th nghim tch RNA u phi c x l trong dung dch DEPC 0,1% loi tr RNase.
26 tch RNA tng s t l v thn non ca ma chng ti tin hnh thu mu l v b non ca hai ging ROC1 v ROC10. Cc mu c nghin nhanh trong N2 lng thnh bt mn vi ci chy s c kh trng ph v t bo. Sau phi b sung ngay dung dch Trizol vo v nu khng cc RNase ni bo s c gii phng v phn ct RNA. Cc thnh phn trong dung dch Trizol nh: phenol, guanidine isothiocyanate s nhanh chng lm kt ta protein v bt hot cc RNase ni bo. B sung chloroform:isoamyl (24:1) lm sch cc protein cn st li. Tip theo vic b sung isopropanol vo lm kt ta RNA. Cui cng sn phm RNA tng s c ha tan trong 20 l nc ct c DEPC 0,01%. RNA tng s s c kim tra cht lng bng phng php in di trn gel agarose 1%. Hnh 3.1 cho thy cc mu RNA t thn v l c hm lng ln v c 2 bng RNA riboxom r nt nn khng nh rng RNA tng s cha b phn hy v ti u tin hnh cc th nghim tip theo. Nh vy, chng ti tch chit thnh cng RNA tng s t l v thn non ca ma ROC1 v ROC10 in vitro. L 10 Thn 1 10
ROC 1
Hnh 3.1. Kt qu in di RNA tng s tch t l v b thn non ca 2 ging ma ROC1 v ROC10 trn gel agarose 1% RNA tng s s c loi DNA bng DNase s dng cho phn ng RT-PCR.
27 3.3. NHN DNG ON GEN M HA ENZYME INVERTASE RNA tng s sau khi c tinh sch s s dng lm khun cho phn ng RT-PCR nhn on gen m ha cho enzyme Invertase vi cp mi 5INV v 3INV thit k. Nu phn ng ny xy ra c hiu theo l thuyt th s c mt bng duy nht c kch thc di tng ng vi tnh ton l thuyt l 435 bp ca on Invertase cn phn lp c nhn ln. INV M
450 bp
Hnh 3.2. Kt qu in di sn phm RT-PCR trn gel agarose 0,8% (M): Marker 1kb; (INV): sn phm thi gel Phn ng RT-PCR c th b gim hiu qu do mt s nguyn nhn nh cht lng v hm lng RNA tng s ca gen m ha enzyme Invertase, nhit bt cp vi mi cha ph hp... V vy khi tin hnh phn ng chng ti iu chnh lng mu, nng mi, Mg2+, nhit bt cp vi mi... m bo cho phn ng RT-PCR xy ra c hiu nht. Thnh phn hn hp ca phn ng v chu k nhit ca phn ng c ti u ha trnh by mc 2.2.1.3. Sn phm ca phn ng RT-PCR mt bc vi khun l RNA tng s thu c t b ma non (ging ROC1) c kim tra bng phng php in di trn trn gel agarose 0,8% (Hnh 3.2). Kt qu cho thy chng ti
28 nhn c 1 on DNA c kch thc khong 450 bp (khi so snh vi thang DNA chun 1 kb), kch thc ny ph hp vi kch thc on DNA d on khi thit k cp mi c hiu. 3.4. TCH DNG GEN V XC NH TRNH T GEN 3.4.1. To plasmid ti t hp INV-pENTR khng nh chc chn rng on DNA thu c t phn ng RT-PCR chnh xc l on gen m ha enzyme Invertase, chng ti tin hnh vic tip theo l tch dng v xc nh trnh t gen. vic tch dng t c hiu qu nht chng ti tin hnh tinh sch sn phm RT-PCR theo b kit Qiagen ca hng QIAquick Gel Extraction. Qu trnh tch dng c thc hin bng cch gn sn phm RT-PCR vo vector tch dng pENTR/D ca hng Invitrogen (M) to entry clone INV_pENTR. Vector pENTR/D c kch thc 2580 bp, trn vector ny c hai v tr gn attL1 v attL2, on gen cn chuyn Invertase s c gn vo gia hai v tr ny. Vector ny cn c gen khng khng sinh kanamycin v c v tr gn mi M13 phc v cho vic chn dng cc khun lc mang vector ti t hp INV_pENTR. Ngoi ra, enzyme Topoisomerase s gip cho vic gn gen vo vector din ra trong mt khong thi gian rt ngn (khong 5 pht) v c th t hiu qu cao ti 90%. Thnh phn v chu trnh ca phn ng c trnh by mc 2.2.4.1. Kt qu ca phn ng s thu c vector ti t hp INV_pENTR. 3.4.2. Bin np plasmid ti t hp INV_pENTR vo t bo kh bin E.coli TOP 10 Vector ti t hp INV_pENTR thu c sau phn ng lai s c bin np vo t bo kh bin E.coli TOP 10 bng phng php sc nhit nhm tch dng v nhn nhanh plasmid ti t hp vi s lng ln. Hiu qu ca phn ng ny ph thuc vo cht lng sn phm vector ti t hp v t bo
29 kh bin. y, ton b vector ti t hp INV_pENTR s c bin np vi t bo kh bin v c cy tri khun trn mi trng c b sung khng sinh chn lc kanamycine 50 mg/l v 37oC, qua m. Kt qu thu c cc khun lc mu trng trn a mi trng. Song khng phi tt c cc khun lc u mang plasmid ti t hp INV_pENTR v bn thn plasmid ny c gen khng khng sinh cn t bo kh bin E.coli li khng khng bt k khng sinh no. V vy, c th c mt lng nh no cc t bo cha plasmid pENTR/D nhng li khng gn gen Invertase, do enzyme TOPO hai u vector b mt v plasmid t ng vng. Vy trong s nhng khun lc thu c, khun lc no mang vector cha on gen m ha Invertase v khun lc no khng c? xc nh r cc plasmid ti t hp chnh l dng INV_pENTR mong mun, chng ti tin hnh tch chit plasmid theo Sambrock t nhng khun lc sng st trn mi trng c b sung khng sinh chn lc v thc hin phn ng PCR plasmid thu c vi cp mi c hiu M13for/rev. 3.4.3. Chn lc plasmide ti t hp INV_pENTR bng PCR vi cp mi M13 (F/R) Qu trnh bin np plasmid ti t hp vo t bo kh bin E.coli bng sc nhit c hiu qu c th ti 95%. Song rt c th cn c nhng gen khng c gn vo v chng s tn ti bn trong hoc xung quanh nhng khun lc mc trn a mi trng chn lc. Do , PCR plasmid vi cp gen c hiu 3INV, 5INV vn c th thu c nhng bng c kch thc khong 435 bp nhng khng c on gen Invertase c gn vo vector. V vy, chng ti tin hnh phn ng PCR vi cp mi M13for/rev c hiu ca vector pENTR/D vi cc plasmid ca cc dng khun s 1, 2, 3 c kim tra chnh xc xem on gen Invertase gn c vo vector tch dng hay cha. Theo
30 l thuyt th v tr gn mi M13for/rev trn vector pENTR/D c thit k c hiu v cho php nhn mt on DNA t nucleotide v tr 537 ti v tr 861 trn vector pENTR/D. Nh vy, PCR vi cp mi ny s thu c cc sn phm c chiu di khong 750 bp hoc 324 bp t cc dng plasmid INV_pENTR dng hoc m tnh tng ng khi kim tra bng phng php in di trn gel agarose 0,8%.
750 bp
Hnh 3.3. Kt qu in di sn phm PCR plasmid vi cp mi M13(For/Rev) nhm kim tra s c mt ca on Invertase trong vector pENTR/D (M): Marker 1kb; (1), (2),( 3): sn phm PCR t plasmid INV_ pENTR/D; (-): i chng m Vi kt qu thu c hnh 3.3 cho thy rng 2 dng plasmid ti t hp tch t dng khun s 1 v 2 cho cc bng c kch thc khong 750 bp ng vi kch thc ca tnh ton l thuyt ca cc dng plasmid INV_pENTR dng tnh. Nh vy, c th kt lun rng on gen Invertase c gn v to entry clone INV_pENTR dng khun s 1 v 2. Dng plasmid INV_pENTR ti t hp c gi i xc nh trnh t on gen Invertase.
31 3.4.4. Kt qu xc nh trnh t nucleotit on gen m ha Invertase c gi i xc nh trnh t nuclotide trn my xc nh trnh t t ng ABI PRIMS 3100 Avant Genetic Analyzer. Sau , chng ti s dng phn mm DNAstar v BioEdit tin hnh so snh trnh t nucleotide ca on gen m ha enzyme Invertase ny vi trnh t Invertase c m s AY302083 c s dng thit k cp mi. Kt qu nh sau:
AY302083 amplified 310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AGGAACTGGATGAACGACCCCAATGGCCCGGTGTACTACAAGGGCTGGTACCACCTGTTCTACCAATACAACCCGGACGGCGCCATCTGGGGCAACAAGA -----------------------------------------------------------------------------------CATCTGGGGCAACAAGA 410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TCGCGTGGGGCCACGCCGTCTCCCGCGACCTCATCCACTGGCGCCACCTCCCGCTGGCCATGCTGCCCGACCAGTGGTACGACACCAACGGCGTCTGGAC TCGCGTGGGGCCACGCCGTCTCCCGCGACCTCATCCACTGGCGCCACCTCCCGCTGGCCATGGTGCCCGACCAGTGGTACGACACCAACGGCGTGTGGAC 510 520 530 540 550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| GGGCTCCGCCACCACGCTCCCCGACGGCCGCCTCGCCATGCTCTACACCGGCTCCACCAACGCCTCCGTGCAGGTGCAGTGCCTCGCCGTGCCCGCCGAC GGGCTCCGCCACCACGCTCCCCGACGGCCGCCTCGCCATGCTCTACACGGGCTCCACCAACGCCTCCGTGCAGGTGCAGTGCCTCGCCGTGCCCGCCGAC 610 620 630 640 650 660 670 680 690 700 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| GACGCCGACCCGCTGCTCACCAACTGGACCAAGTACGAGGGCAACCCGGTGCTGTACCCGCCGCCGGGCATCGGGCCCAAGGACTTCCGCGACCCCACCA GACGCCGACCCGCTGCTCACCAACTGGACCAAGTACGAGGGCAACCCGGTGCTGTACCCGCCGCCGGGGATCGGGCCCAAGGACTTCCGCGACCCCACCA 710 720 730 740 750 760 770 780 790 800 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| CGGCGTGGTTCGACCCGTCGGACAACACCTGGCGCATCGTCATCGGCTCCAAGGACGACGCCGAGGGCGACCACGCCGGCATCGCCGTGGTGTACCGCAC CGGCGTGGTTCGACCA------------------------------------------------------------------------------------
AY302083 amplified
AY302083 amplified
AY302083 amplified
AY302083 amplified
Hnh 3.4. Kt qu so snh trnh t on gen Invertase phn lp c vi trnh t Invertase trong ngn hng gen c m s AY302083 Trn hnh 3.4 cho thy, trnh t gen Invertase phn lp c cy ma ROC1 in vitro c tng ng kh cao vi trnh t gen AY302083 c s dng thit k cp mi c hiu (90,1%). iu ny chng t chng ti phn lp chnh xc on gen m ha Invertase mong mun cy ma ROC1 in vitro. 3.5. THIT K VECTOR TI T HP INV-RNAi 3.5.1. To vector ti t hp INV_RNAi bng k thut Gateway K thut Gateway Cloning l mt k thut nhn dng ph bin v hiu qu cho vic gn trnh t DNA vo h thng mt hoc nhiu vector. Trong th
32 nghim ny chng ti thc hin phn ng LR vi cc thnh phn trnh by mc 2.2.2.1. gn vector INV_pENTR vi vector pK7GWIWG2(II) nhm thu c vector chuyn gen INV_RNAi ca gen m ha Invertase. Phn ng ny c tin hnh vi s xc tc ca enzyme LR-clonase v Proteinase K nhit phng. pK7GWIWG2(II) l mt vector chuyn gen c cu trc bi hai vng gn c bit l: attR1-ccdB-attR2 c chiu ngc nhau v c ni vi nhau nh mt on intron. Do , khi thc hin phn ng lai LR th s to ra sn phm l mt plasmid INV_RNAi c hai v tr gn mang on gen Invertase c chiu ngc nhau: sense-intron-antisense (Invertase-intron-antiInvertase). on intron ca vector pK7 c vai tr rt quan trng trong vic to on tht nt (vng) RNA si i d c hnh thnh (Invertase-intronantiInvertase) v hot ng mt cch n nh trong genome ca vt ch khi n c chuyn vo. y chnh l cu trc RNAi cn thit k chuyn gen vo cy ma nhm tng tr lng ng ca ma cao hn mt cch n nh.
Hnh 3.5. M hnh cu trc chuyn gen INV_RNAi 3.5.2. Bin np vector INV_RNAi vo t bo kh bin E.coli Vector ti t hp INV_RNAi c bin np vo t bo kh bin E.coli TOP 10 bng phng php sc nhit 42 oC vi khong thi gian l 1 pht 30 giy. Sau , khun s c nui phc hi vi 250 l LB lng 37oC t lc khong 60 pht. Tip theo khun s c nui cy trn mi trng chn lc
33
c khng sinh 100 mg/l spectinomycin, 40 mg/l streptomycin v 50 mg/l chloramphenicol. Kt qu thu c mt lng ln cc khun lc mu trng mc trn mi trng chn lc. Vector pK7GWIWG2(II) l vector c cu trc mang gen khng cc khng sinh spectinomycin, streptomycin, chloramphenicol v gen ccdB m ha cho plasmid F gy c ch sinh trng ca t bo E.coli, cn vector INV_RNAi khng c gen ccdB do gen Invertase chn vo thay th. V vy, cc khun lc mc trn a mi trng chn lc ch c th l nhng khun lc c mang plasmid pK7GWIWG2(II) nhng v tr cha on gen ccdB b t bin v cc khun lc c mang plasmid INV_RNAi. Chn ngu nhin 3 khun lc c nh s t 1 n 3 nui trong mi trng lng c cc khng sinh chn lc tng t v tin hnh tch plasmid t cc dng khun . kim tra cc plasmid thu c c chnh xc l INV_RNAi hay l pK7GWIWG2(II) nhng v tr cha on gen ccdB b t bin. Chng ti thc hin phn ng ct enzyme gii hn c hiu XbaI v HindIII i vi 3 plasmid t khun lc s 1, 2 v 3. Theo tnh ton trn l thuyt th sn phm ca phn ng ct enzyme s cho 3 on c kch thc l: on 1 di 978 bp mang on Invertase chiu xui, on 2 di 2825 bp mang on Invertase chiu ngc v cui cng l phn cn li ca vector nhn pK7GWIWG(II) di 8114 bp. Hnh 3.6 cho thy sn phm ca phn ng ct enzyme thu c cc phn on c cc kch thc tng ng vi d tnh c 3 plasmid 1, 2 v 3 v c s khc bit r rt so vi i chng l vector khng bin np pK7GWIWG(II), chng t cu trc INV_RNAi c thit k.
34
Hnh 3.6. Kt qu in di sn phm ct plasmid INV_RNAi t hp vi HindIII v XbaI (M): Marker 1kb; (1), (2), (3): cc dng plasmid ti t hp; (-): i chng vector pK7GWIWG(II) 3.6. BIN NP VECTOR CHUYN GEN INV_RNAi VO CHNG VI KHUN A.TUMEFACIENS CV58C1. kim tra hot ng ca cu trc INV_RNAi trn vector ti t hp trong cy trng cng nh to nguyn liu cho qu trnh chuyn gen c ch biu hin gen m ha Invertase cy ma, th cu trc INV_RNAi phi c bin np vo vi khun A.tumefaciens CV58C1 bng phng php xung in. y l mt phng php bin np c hiu qu cao trong thi gian ngn. Plasmid ti t hp INV_RNAi s 2 c bin np vo A.tumefaciens bng xung in vi thnh phn v cc bc tin hnh bin np c trnh by mc 2.2.2.3. Kt qu cho thy trn a mi trng c b sung khng sinh chn lc 100 mg/l streptomycin, 100 mg/l spectinomycin v 50 mg/l rifamycin thu c nhng khun lc mu trng. Sau chn ngu nhin 2 dng khun lc nhn nui trong mi trng lng c b sung cc khng sinh chn lc 100 mg/l streptomycin, 100 mg/l spectinomycin v 50 mg/l rifamycin tch plasmid.
35
Hnh 3.7. in di sn phm PCR plasmid INV-RNAi trong A.tumefaciens vi cp mi c hiu 5INV v 3INV (M): Marker 1kb; (1), (2): cc dng plasmid ti t hp; (-): i chng m Tch plasmid theo Sambroock v kim tra s c mt ca INV_RNAi trong A.tumefaciens CV58C1 bng phng php PCR vi cp mi c hiu 5INV v 3INV. Kt qu kim tra hnh 3.7 cho thy cc dng khun lc u cho kt qu dng tnh kch thc khong 450 bp mong mun. Nh vy, chng t chng ti to c vector chuyn gen INV_RNAi trong chng vi khun A.tumefaciens CV58C1. y l ngun nguyn liu phc v cho th nghim chuyn gen tip theo nhm to dng ma bt hot gen m ho Invertase, tng lng sucrose tch tr. 3.7. TI SINH V BC U BIU HIN GEN GUS MA 3.7.1. Quy trnh ti sinh ma thng qua m so T cc nghin cu ti sinh v chuyn gen thnh cng cy ma cho thy rng t l ti sinh thu c cao v hiu qu thng qua nguyn liu ban u l m so. Trong th nghim ny, chng ti tin hnh th nghim to m so vi ging ma ROC10 in vitro trn mi trng MS i chng khng c 2,4D v cc mi trng cm ng to m so M1-M4 (Bng 2.4). Kt qu thu c cho thy mi trng i chng MS (khng c 2,4D) hon ton khng
36 to c m so. Cn cc mu trn mi trng cm ng c b sung hm lng cht kch thch sinh trng 2,4D u to c m so v cho hiu qu ti sinh ma c th hin thng qua bng sau: Bng 3.3. Kh nng to m so v ti sinh ging ma ROC10 in vitro trn cc mi trng th nghim M1 - M4. Mi 2,4-D Tng mu trng (mg/l) cy M1 M2 M3 M4 3 4 5 6 3x130 3x130 3x130 3x130 T l to m so (%) T l mu to S chi / 1 chi khi m so (%)
83,07 0,44 67,95 1,36 42,66 1,45 86,15 3,11 46,41 3,33 20,66 2,96 71,79 0,68 63,08 1,33 32,33 1,45 83,33 2,89 37,95 1,12 17,66 4,33
Bng 3.3 cho thy, cc mu mi trng M1 c b sung 3 mg/l 2,4D cho t l to m so v hiu sut ti sinh cao hn hn so vi cc mi trng M2 - M4 c b sung 4, 5, 6 mg/l 2,4D; trong t l to chi t 67,95% v s chi trn mt khi m so t khong 42,66. Nh vy, mi trng M1 c b sung 3 mg/l 2,4D cho kt qu to m so v ti sinh cy ma ROC10 in vitro tt nht trong th nghim ny. Do , chng ti chn mi trng M1 c b sung 3 mg/l 2,4D lm mi trng cm ng to m so cho vic ti sinh v chuyn gen ma trong cc th nghim tip theo. Qu trnh ti sinh cy t m so n cy hon chnh c minh ha trong hnh 3.8. Mt khc, chng ti tip tc nhn nui m so trong mi trng lng lc M1 c b sung 3 mg/l 2,4D trong vng 1 tun, 27oC, lc 90 vng/pht, trong iu kin ti phc v cho vic bin np th nghim gen gus-intron.
37
Cy ma ROC10 in vitro
on thn st gc di 0,5 cm trn mi trng cm ng to m so
M so
To cy hon chnh
M so ln mm xanh
M so ang nh mm
Hnh 3.8. Quy trnh ti sinh ma ROC10 in vitro t m so 3.7.3. Chn lc m so v ti sinh cy chuyn gen Quy trnh chuyn gen c hiu qu l yu t rt quan trng trong vic chuyn gen vo thc vt. Sau khi th nghim c mi trng ti sinh cy ma thng qua m so, chng ti tin hnh th nghim vic chuyn gen vo cy ma thng qua vic s dng gen ch th gus-intron. Trong nghin cu ny, chng ti th nghim hai chng vi khun A.tumefaciens gian nhim khun l 10 v 30 pht. Chng ti th nghim ly nhim khun vi m so theo phng php thi kh sau nhn nui trong mi trng lng lc M1. Song chng ti khng thu c kt qu chuyn gen mong mun khi kim tra biu hin gen ch th gus-intron vi dung dch X-gluc. Do , chng ti tin hnh hng nghin cu th hai l chuyn m so sau nhn nui sang mi trng cm ng to chi Mc sau mi tin hnh ly nhim khun. thi gian bin np 10
khc nhau l
EHA1300 v CV58C1 cng mang vector chuyn gen pPTN289, ngng thi
38 pht cc m so c hai chng vi khun u khng thu c mu c biu hin gen gus. Trong khi thi gian nhim khun 30 pht, mu c biu hin gen gus xut hin c hai cng thc vi hai chng khun nghin cu. T l biu hin gen gus trn m so t 31,67% vi chng CV58C1 v 11,67% khi s dng chng EHA1300. Khi s dng hai chng vi khun ny vi ngng thi gian nhim khun l 30 pht, chng ti u thu c cc chi ma sng st trn mi trng chn lc (vi CV58C1 l 18,57% v EHA1300 l 7,14%). Bng 3.4. Kt qu bin np gen gus-intron vo cy ma Thi gian T l biu Chng vi khun bin np hin gen gus A.tumefaciens (pht) (%) CV58C1 EHA1300 30 30 T l mu ti sinh (%) T l mu sng st trn mi trng c b sung 2 mg/l ppt (%) 18,57 1,28 7,14 0,69
31,67 0,65 81,43 1,56 11,67 0,89 92,85 0,31
Nh vy, bc u chng ti thu c kt qu khi tin hnh chuyn gen gus vo cy ma thng qua m so theo phng php cm ng to chi. Tuy nhin do thi gian c hn, cc yu t nh hng n kt qu chuyn gen vn cha hon ton c ti u. Mc d vy, vic thu c cc m so ti sinh c biu hin ca gen ch th gus cho thy vic s dng cng ngh RNAi to cy ma chuyn gen lm tng kh nng tch tr ng trong cy ma l mt hng i trin vng trong tng lai.
39
M so bin np trn mi trng ng nui cy Mc
M so biu hin gus khi nhum vi dung dch X-gluc
Chi cy trn mi trng chn lc Mr c b sung 500 mg/l cefotaxime v 2 mg/l ppt
M so to chi trn Mc c b sung 500 mg/l cefotaxime
Hnh 3.9. Bin np gen gus-intron vo cy ma ROC10 in vitro thng qua trung gian A.tumefaciens
40
KT LUN V NGH
KT LUN: 1. Phn lp c on gen m ho Invertase c kch thc khong 435 bp t ging ma ROC1 in vitro xc tc qu trnh phn hu sucrose. 2. Thit k c vector chuyn gen INV-RNAi c ch s biu hin ca Invertase v bin np thnh cng chng vi khun A.tumefaciens CV58C1. 3. Chn lc mt s mi trng thch hp ti sinh cy ma thng qua m so (mi trng to m so M1, mi trng to chi Mc, mi trng to r Mr) v bc u chuyn gen ch th gus-intron vo cy ma. NGH: Tin hnh chuyn vector c ch biu hin m ha Invertase (INVRNAi) cy ma nhm tng tr lng ng ca cc ging ma Vit Nam.
BI BO CA TC GI C LIN QUAN N LUN VN C CNG B

Lu Th C, Tin Pht, Chu Hong H, L Trn Bnh, L Qunh Lin (2009) Phn lp v thit k vector c ch biu hin gen m ho Invertase (-fructofuranosidase) cy ma. Hi ngh Cng ngh Sinh hc ton quc_Thi Nguyn, thng 11 nm 2009.
TI LIU THAM KHO

TI LIU TING VIT 1. Quyt nh s 26/2007/Q-TTg, v vic ph duyt Quy hoch pht trin ma ng n nm 2010 v nh hng n nm 2020. 2. Hong Th Sn (2003) Phn loi hc thc vt. Nxb Gio dc 3. Trn Vn Si (2003) Cy ma. Nh xut bn Ngh An. 4. Nng Vnh (2007) Cng ngh can thip RNA (RNAi) gy bt hot gen v tim nng ng dng to ln. Tp ch Cng ngh Sinh hc 5(3): 265-275. 5. V Vn V (1999) Sinh l thc vt ng dng. Nxb Gio dc. TI LIU TING ANH 6. Avigad G and Dey PM (1997) Carbohydrate metabolism: storage carbohydrates. Plant biochemistry Acedemic Press Ltd, pp: 143-204. 7. Balibrea ME, Rus-Alvarez AM, Bolarin MC, Perez-Alfocea F (1997) Fast changes in soluble carbohydrates and proline content in tomato seedlings in response to ionic and nonionic iso-osmostic stress, J. Plant physiol. (151): 222-226. 8. Baulcomble D (2004) RNA silencing in plants. Nature 431 (7006): 356-63. 9. Chen S, Hajirezaei M, Bornke F (2008) Differential expression of sucrose phosphate synthase isoenzymes in tobaco reflects their functional specialization during dark governed starch mobilization in source leaves. Plant Cell Environ. 31(1): 165-76 elegans. Nature. 391: 806-811. 10. Geigenberger P, Reimholz R, Deiting U, Sonnewald U and Stitt M (1999) Decreased expression of sucrose phosphate Synthase Strongly inhibits the water stress-induced synthesis of sucrose in growing potato tubers. The plant Journal. 19: 119-129.
11. Groenewald J, Botha FC (2007) Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in Sugarcane enhances sucrose accumulation in immature internoods. Transgenic Res. 12. Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu. Rev. Plant physiol. Plant Mol. Biol. 41: 187223. 13. Haigler CH, Singh B, Zhang D, Hwang S, Wu C, Cai WX, Hozain M, Kang W, Kiedaisch B, Strauss RE, Hequet EF, Wyatt BG, Jwiden GM, Holaday AS (2007) Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions. Plant Mol Biol. 63 (6): 815-32. 14. Hesse H, Sonnewald U, Willmitzer L (1995) Cloning and expression analysis of sucrose phosphate synthase from sugar beet (Beta vulgaris L.). Mol Gen Genet. 247 (4): 515-20. 15. Huber SC. and Huber JL. (1992) Role of sucrose-phosphate Synthase in Sucrose Metabolism in leaves. Plant physiol. 99: 1275-1278. 16. Huber SC, and Huber JL(1996) Role and regulation of Sucrose phosphate synthase in higher plants. Annu. Rew. Plant physiol. Plant Mol.Biol. 47: 431-444. 17. Ingram J, Chadler JW, Gallagher L, Salamini F, Bartels D (1997) Analysis of cDNA clones encoding sucrose-phosphate synthase in relation to sugar interconversions associated with dehydration in the resurrection plant, Craterostigma plantagineum Hochst. Plant Physiol. 115:113-121. 18. Klann EM, Hall B, Bennett AB (1996) Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit. Plant Physiol. 112 (3): 1321-30.
19. Le QL, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S, Sonnewald U (2006b) Reduced allergenicity of tomato fruits harvested from Lyc e 1 silenced transgenic tomato plants. J. Aller. Clin. Immunol. 118(5):1176-83. 20. Liu Q, Singh SP, Green AG (2002) High-stearic and High-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol. 129(4): 1732-43. 21. Lunn JE, Gillespie VJ, Furbank RT (2003) Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants. J Exp Bot. 54 (381): 223-37 22. Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B, Selvaraj N, Vasudevan A, Kasthurirengan S (2004) Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species bybrids) using axillary buds. Plant Cell Rep 23: 134-143. 23. Morgan JM (1984) Osmoregulation and water stress in higher plant, Ann Rev. Plant physiol. 35: 299-319 24. Nguyen-Quoc B, Foyer CH (2001) A role for futile cycles involving invertase and sucrose synthase in sucrose metabolism of tomato fruit. J Exp Bot. 52 (358): 881-9. 25. Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H (2003) Producing decaffeinated coffee plants. Nature 423(6942): 823. 26. Quick P, Siegl G, Neuhaus E, Feil R, Stitt M (1989) Sort term water stress leads to stimulation of sucrose synthesis by activating sucrose-phosphate synthase. Planta. 177: 535546. 27. Salerno GL, Pagnussat GC, Pontis HG (1998) Studies on sucrose phosphate synthase from rice leaves. Cell Mol Biol 44(3): 407-16 28. Santosa DA, Hendroko R, Farouk A v Greiner R (2004) A rapid and highly efficient method for transformation of sugarcane callus. Molecular Biotechnology 28: 113-119.
29. Snyman SJ, Meyer GM., Richards JM, Haricharan N, Ramgareeb S, Huckett BI (2006) Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency. Plant Cell Rep. 25: 10161023. 30. Stitt M (1989) Product inhibition of potato tuber pyrophosphate, Fructose 6-phosphate phosphotransferase by phosphate and pyrophosphate. Plant physiol. 89: 628-633. 31. Sugihartor B, Sakakibara H, Sumadi and Sugiyama T (1997) Differential Expression of Two Genes for Sucrose-phosphate-Synthase in Sugarcane: Molecular Cloning of the cDNAs and Comparative Analysis of Gene Expression. Plant cell physiol. 38: 961-965. 32. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, eddective and high-throughput gene silencing in plants. Plant J. 27(6): 581-90. 33. Worrell AC, Bruneau JM, Summerfelt K, Boersig M, Voelker TA (1991) Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning. Plant Cell. 3(10): 1121-30. 34. Zhangsun D, Luo S, Chen R, Tang K (2007) Improved Agrobacteriummediated genetic transformation of GNA transgenic sugarcane. Section Cellular and Molecular Biology 62 (4): 386-393. 35. Zhu YJ, Komor E, and Moore PH. (1997) Sucrose Accumulation in the Sugarcane Stem 1s Regulated by the Difference between the Activities of Soluble Acid Invertase and Sucrose Phosphate Synthase. Plant Physiol. 115(2): 609-616.
PH LC
Mi trng MS c bn: - MS I: KNO3:1900 mg/l; NH4NO3:1650 mg/l; MgSO4.7H2O:370 mg/l; KH2PO4:170 mg/l - MS II: CaCl2.2H2O mg/l - MS III: H3BO3:6,2 mg/l; MnSO4: 22,3 mg/l; ZnSO4: 8,6 mg/l; KI: 0,83 mg/l; Na2MoO4: 0,25 mg/l; CuSO4: 0,025 mg/l; CoCl2: 0,025 mg/l - MS IV:Na2EDTA: 37,3 mg/l; FeSO4.7H2O: 27,8 mg/l - MS V: Glycin: 2 mg/l; Nicotinic: 0,5 mg/l; B1: 0,5 mg/l; B6: 0,5 mg/l Mi trng LB: - LB lng: Mi trng LB lng: 0,5% (5 g/l) dch chit nm men (Yeast extract), 1% (10 g/l) bacto-Tryptone, 1% (10 g/l) NaCl. Hoc dng 25g LB bt LB pha sn cho 1l LB lng. - LB c: LB lng b sung 1,5% (15g/l) Bactor-aga. Dung dch m tch plasmid: - Sol I: 50 mM glucose, 25 mM Tris HCl pH 8, 10 mM ETDA pH8 - Sol II: 0.2 NaOH, SDS 1% - Sol III: 60 ml CH3COOK 5M; 11,5 ml CH3COOH; 28,5 ml H20 Cc mi trng c kh p sut 4 atmotphe v 117oC trong 15 pht. m in di gel agarose: - TAE 50 X (100 ml) : Tris -base 24.2 g, axit axetic 5.71g , EDTA 0.1M pH8 50ml thm nc n 100 ml. - m tra mu (loading buffer): 0,1 ml bromophenol blue 10%; 0,3 ml glycerol 100%, thm nc ct cho 1 ml

kỹ thuật gateway

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

kỹ thuật gateway

Uploaded by

Copyright:

Available Formats

I HC THI NGUYN TRNG I HC S PHM -------

LUN VN THC S SINH HC

Thi Nguyn 2009

S ha bi Trung tm Hc liu i hc Thi Nguyn

I HC THI NGUYN TRNG I HC S PHM -------

LUN VN THC S SINH HC

NGI HNG DN KHOA HC: TS. L QUNH LIN

Thi Nguyn 2009

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 1 TNG QUAN TI LIU

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 2 NGUYN LIU V PHNG PHP NGHIN CU

Kanamycin Spectinomycine, streptomycine

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

23 KHI QUT S TH NGHIM

Ct on thn st gc (5 cm) Thit k mi M so Tch RNA tng s

ng nui cy RT_PCR Dit khun

Tch dng v xc nh trnh t gen Chn lc m so

Thit k vector ti t hp INV_RNAi

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 3 KT QU V THO LUN

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

on thn st gc di 0,5 cm trn mi trng cm ng to m so

31,67 0,65 81,43 1,56 11,67 0,89 92,85 0,31

S ha bi Trung tm Hc liu i hc Thi Nguyn

M so bin np trn mi trng ng nui cy Mc

M so biu hin gus khi nhum vi dung dch X-gluc

M so to chi trn Mc c b sung 500 mg/l cefotaxime

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

BI BO CA TC GI C LIN QUAN N LUN VN C CNG B

S ha bi Trung tm Hc liu i hc Thi Nguyn

TI LIU THAM KHO

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn