i Bộ giáo dục đào tạo Tr-ờng đại học s- phạm hà nội ================================= Hoàng thị thủy Nghiên cứu xây dựng hệ thống tái sinh b-ớc đầu chuyển gen thị vào khoai tây thông qua vi khuẩn Agrobacterium tumefaciens luận văn thạc sĩ sinh học Hà nội, 2010 ii LI CM N hon thnh khúa lun ny, tụi xin by t lũng cm n sõu sc ti PGS TS Lờ Huy Hm, Vin trng Vin Di truyn Nụng nghip ó to mi iu kin thun li cho tụi c hc v nghiờn cu ti Phũng thớ nghim trng im Cụng ngh T bo thc vt, Vin Di truyn Nụng nghip Tụi xin chõn thnh cm n TS Phm Th Lý Thu l ngi thy ó tn tỡnh hng dn giỳp tụi sut thi gian thc hin v hon thnh lun Tụi xin chõn thnh cm n s o to v giỳp nhit tỡnh ca cỏc giỏo viờn B mụn sinh Trng i hc s phm H Ni 2, nhng ngi thy ó dy tụi sut quỏ trỡnh hc tụi hon thnh chng trỡnh cỏc mụn hc cao hc ng thi tụi cng ó nhn c s giỳp nhit tỡnh ca th cỏn b khoa hc ca Phũng Thớ nghim trng im Cụng ngh T bo thc vt, Vin Di truyn Nụng nghip Tụi rt bit n nhng ngi thõn gia ỡnh tụi ó luụn luụn bờn tụi, quan tõm v to iu kin tt cho tụi hc v nghiờn cu Tụi cng vụ cựng cm n s ng viờn, khớch l ca cỏc ng nghip v bn bố ó dnh cho tụi Nhõn dp ny tụi xin by t lũng bit n chõn thnh i vi nhng s giỳp quý bỏu ú H Ni, ngy 05 thỏng 10 nm 2010 Hc viờn Hong Th Thy iii LI CAM OAN Tụi xin cam oan õy l cụng trỡnh nghiờn cu ca riờng tụi Cỏc s liu v kt qu nờu lun ỏn l trung thc v cha tng c cụng b bt k mt cụng trỡnh no khỏc H Ni, ngy 05 thỏng 10 nm 2010 Tỏc gi Hong Th Thy iv MC LC LI CM N i LI CAM OAN iii MC LC iv DANH MC CC Kí HIU, CC CH VIT TT vii DANH MC CC Kí HIU, CC CH VIT TT vii DANH MC CC BNG viii DANH MC CC HèNH .ix DANH MC CC HèNH .ix M U Chng TNG QUAN TI LIU 1.1 Mt s c im sinh hc ca cõy khoai tõy 1.1.1 H thng phõn loi cõy khoai tõy 1.1.2 c im hỡnh thỏi 1.2 Nhõn ging bng nuụi cy mụ t bo 1.2.1 C s khoa hc ca k thut nuụi cy mụ, t bo thc vt 1.2.2 Cỏc hng tỏi sinh cõy t mụ thc vt 1.2.3 Tỏi sinh cõy khoai tõy 1.2.4 Cỏc yu t nh hng n nuụi cy in vitro 1.3 C s khoa hc ca cụng ngh chuyn gen vo thc vt 11 1.3.1 Cỏc phng phỏp chuyn gen vo thc vt 11 1.3.2 H thng vector chuyn gen 21 1.4 Tỡnh hỡnh nghiờn cu v ngoi nc 23 1.4.1 Cỏc nghiờn cu sn xut, nhõn ging khoai tõy 23 1.4.2 Mt s thnh tu v cõy trng bin i gen 25 Chng 29 VT LIU, NI DUNG V PHNG PHP NGHIấN CU 29 2.1 Vt liu nghiờn cu 29 v 2.1.1 Vt liu thc vt 29 2.2.2 Vt liu di truyn 29 2.2 Ni dung nghiờn cu 30 2.2.1 Cỏc nghiờn cu to callus v tỏi sinh chi 30 2.2.2 Nghiờn cu tỏi sinh chi 30 2.3 Phng phỏp nghiờn cu 31 2.3.1 Phng phỏp thit k thớ nghim 31 2.3.2 Phng phỏp chun b mu thc vt 32 2.3.3 Phng phỏp chun b dch vi khun 32 2.3.4 Phng phỏp chuyn gen 33 2.3.5 Mụi trng nuụi cy 34 2.3.6 Phng phỏp ỏnh giỏ biu hin gen gus tm thi 35 2.4 B trớ thớ nghim 35 2.4.1 Nghiờn cu xõy dng h thng tỏi sinh 35 2.4.2 Nghiờn cu chuyn gen vo khoai tõy thụng qua Agrobacterium tumefaciens 37 2.5 Cỏc ch tiờu ỏnh giỏ 39 2.6 X lý s liu 39 2.7 Thit b nghiờn cu 39 2.8 a im v thi gian nghiờn cu 39 KT QU NGHIấN CU V THO LUN 40 3.1 Xõy dng quy trỡnh to callus v tỏi sinh cõy khoai tõy 40 3.1.1 nh hng ca cỏc nn khoỏng ti kh nng to callus t cỏc dng mụ khỏc cõy khoai tõy 40 3.1.2 nh hng ca auxin ti kh nng to callus khoai tõy 42 3.1.3 nh hng ca cỏc loi xytokinin ti kh nng to callus khoai tõy 46 3.1.4 nh hng ca xaccaroz ti kh nng to callus khoai tõy 49 3.1.5 nh hng ca Casein hydrolysate (CH) ti kh nng to callus 52 3.1.6 Nghiờn cu tỏi sinh chi 55 vi 3.2 Nghiờn cu chuyn gen vo khoai tõy thụng qua Agrobacterium tumefaciens 56 3.2.1 La chn chng vi khun A tumefaciens thớch hp cho chuyn gen ging khoai tõy Atlantic 56 3.2.2 La chn vector thớch hp cho chuyn gen vo cõy khoai tõy thụng qua vi khun A tumefaciens 59 3.2.3 Nghiờn cu nh hng ca mt vi khun (OD600nm) lờn t l biu hin tm thi ca gen gus 61 3.2.4 Nghiờn cu nh hng ca hm lng Acetosyringone (AS) lờn t l biu hin tm thi ca gen gus 62 63 KT LUN V NGH 66 TI LIU THAM KHO 67 Ph lc1: Thnh phn mụi trng MS (Murashige and Skoogs, 1972) 73 Ph lc 2: Thnh phn mụi trng N6 (Chu v cng s, 1975) 74 Ph lc 3: Mụi trng nuụi cy s dng nghiờn cu 76 vii DANH MC CC Kí HIU, CC CH VIT TT & cs v cng s 2,4D Dichlorophenoxy acetic acid AS Acetosyringone BAP 6- Benzyl amino purin bar Gen mó húa tng hp phosphinothricin acetyl transferase HST Cht iu hũa sinh trng thc vt GA Gibberellic Acid gus Gen mó húa tng hp -glucuronidase IAA - Indole- Acetic Acid Kinetin 6- furfuryl amino purin LB Luria Bertani Medium MS Murahige and Skoogs, (1962) NAA Napthalene acetic acid N6 Chu & cs, 1975 viii DANH MC CC BNG Bng 3.1 nh hng ca cỏc nn khoỏng ti kh nng to callus t mụ lỏ cõy khoai tõy .40 Bng 3.2 nh hng ca cỏc nn khoỏng ti kh nng to callus t on thõn 41 cõy khoai tõy .41 Bng 3.3 nh hng ca auxin ti kh nng to callus t mụ lỏ cõy khoai tõy 43 Bng 3.4 nh hng ca auxin ti kh nng to callus t on thõn cõy khoai tõy 45 Bng 3.6 nh hng ca cỏc loi xytokinin ti kh nng to callus t on thõn cõy khoai tõy 48 Bng 3.8 nh hng ca xaccaroz ti kh nng to callus t on thõn cõy khoai tõy 51 Bng 3.10 nh hng ca casein hydrolysate ti kh nng to callus t on thõn cõy khoai tõy .54 Bng 3.12 La chn chng vi khun thớch hp cho chuyn gen ging khoai tõy Atlantic 56 Bng 3.13 .59 cõy khoai tõy Atlantic thụng qua vi khun A tumefaciens .59 Bng 3.14 La chn vector thớch hp cho chuyn gen vo on thõn 60 cõy khoai tõy Atlantic thụng qua vi khun A tumefaciens .60 Bng 3.15 nh hng ca mt vi khun ti t l biu hin tm thi ca gen gus cõy khoai tõy chuyn gen .61 64 ix DANH MC CC HèNH Hỡnh 1.1.Cu trỳc Ti-plasmid 18 Hỡnh 1.2 Cu trỳc on T-DNA .18 Hỡnh 1.3 Quỏ trỡnh chuyn T-DNA vo t bo thc vt 19 Hỡnh 1.4 S cu trỳc vector nh th 21 Hỡnh 1.5 S cu trỳc vector liờn hp 23 Hỡnh 2.1 S cu trỳc vector pCAMBIA1301 .29 Hỡnh 2.2 Cu trỳc vector p6d35S-GUS 30 Hỡnh 3.1 nh hng ca cỏc nn khoỏng ti kh nng to callus t mụ lỏ 41 cõy khoai tõy (1.Mụi trng: N6; Mụi trng: MS) .41 Hỡnh 3.2 nh hng ca cỏc nn khoỏng ti kh nng to callus t on thõn khoai tõy (3 Mụi trng: N6; Mụi trng: MS) 42 Hỡnh 3.3 nh hng ca auxin ti kh nng to callus t mụ lỏ cõy khoai tõy 44 CT1(1); CT9(2); CT(7) .44 Hỡnh 3.4 nh hng ca auxin ti kh nng to callus t on thõn cõy khoai tõy CT1(1); CT9(2); CT(6) .46 Hỡnh 3.5 nh hng ca xytokinin ti kh nng to callus t mụ lỏ cõy khoai tõy 48 Hỡnh 3.6 nh hng ca cỏc loi xytokinin ti kh nng to callus t on thõn cõy khoai tõy .49 Hỡnh 3.7 nh hng ca xaccaroz ti kh nng to callus t mu lỏ cõy khoai tõy 51 Hỡnh 3.8 nh hng ca xaccaroz ti kh nng to callus t on thõn cõy khoai tõy 52 Hỡnh 3.9 nh hng ca casein hydrolysate ti kh nng to callus t mu lỏ cõy khoai tõy 53 Hỡnh 3.10 nh hng ca casein hydrolysate ti kh nng to callus t on thõn cõy khoai tõy .54 x Hỡnh3.11 Tỏi sinh chi t mu callus to thnh t on thõn cõy khoai tõy Attlantic .56 Hỡnh 3.12 Biu hin tm thi ca gen gus trờn mu lỏ khoai tõy Atlantic sau lõy nhim vi cỏc chng vi khun A tumefaciens khỏc 58 Hỡnh 3.13 Biu hin tm thi ca gen gus trờn on thõn khoai tõy Atlantic sau lõy nhim vi cỏc chng vi khun A tumefaciens khỏc 58 Hỡnh 3.14 La chn vector chuyn gen thớch hp vo mu lỏ khoai tõy Atlantic(AA39: p6d35S-GUS; AA43: pCAMBIA1301) .60 Hỡnh 3.15 La chn vector chuyn gen thớch hp vo on thõn khoai tõy Atlantic (AA39: pCAMBIA1301; AA43: p6d35S-GUS) 60 Hỡnh 3.16 nh hng ca hm lng AS ti t l biu hin tm thi ca gen gus lờn mu lỏ khoai tõy 63 Hỡnh 3.17 nh hng ca hm lng AS lờn t l biu hin tm thi ca gen gus thõn khoai tõy 63 Hỡnh 3.18 nh hng ca thi gian lõy nhim lờn t l biu hin tm thi ca gen gus on thõn khoai tõy 65 64 Thi gian lõy nhim nh hng rt ln n kt qu ca quỏ trỡnh bin np Nu thi gian lõy nhim quỏ ngn thỡ s khụng m bo hiu qu xõm nhim ca vi khun vo mụ thc vt Vi mi i tng thc vt s cú thi gian lõy nhim nht nh Do ú thớ nghim ny chỳng tụi nghiờn cu thi gian lõy nhim cỏc thi gian khỏc ln lt l 20 phỳt, 40 phỳt v 60 phỳt Kt qu thớ nghim c trỡnh by bng 3.17 Bng 3.17 CT (%) (phỳt) (%) /c 0 20 52,78 81,67 40 78,89 96,11 60 55,55 73,89 Kt qu bng 3.17 cho thy: T l biu hin tm thi ca gen gus i vi mu lỏ bin np thp nht thi gian lõy nhim l 20 phỳt ( 52,78%) Tng thi gian lõy nhim lm tng hiu qu bin np T l biu hin tm thi ca gen gus t cao nht thi gian lõy nhim 40 phỳt chim 78,98%, mc biu hin mu xanh chm m Tuy nhiờn tng thi gian lõy nhim lờn 60 phỳt thỡ li gim hiu qu bin np xung cũn 55,55% i vi mu bin np l on thõn: T l biu hin tm thi ca gen gus thu c cao hn so vi mu lỏ bin np c th cỏc cụng thc CT1, CT3 ln lt cú t l l 81,67%, 73,89% so vi CT2 l 96,11% chng t rng t l biu hin tm thi ca gen gus cao nht thi gian lõy nhim l 40 phỳt v on thõn kh nng lõy nhim t cao hn 65 Hỡnh 3.18 nh hng ca thi gian lõy nhim lờn t l biu hin tm thi ca gen gus on thõn khoai tõy Nguyờn nhõn ca hin tng ny cú th l thi gian lõy nhim cao s tng kh nng cnh tranh gia cỏc t bo vi khun lm nh hng ti hiu qu ca quỏ trỡnh bin np Do ú chỳng tụi s dng thi gian lõy nhim l 40 phỳt cho cỏc thớ nghim chuyn gen tip theo trờn cõy khoai tõy Atlantic 66 KT LUN V NGH Kt lun Da trờn cỏc kt qu nghiờn cu thu c, chỳng tụi rỳt mt s kt lun nh sau: a/ Nghiờn cu xõy dng h thng tỏi sinh: - Mụi trng MS l mụi trng khoỏng c bn cho kh nng to callus t mu lỏ v on thõn tt hn, tc hỡnh thnh callus nhanh hn, callus cú mu trng, sỏng cú xp hn so vi callus hỡnh thnh trờn mụi trng N6 - Mụi trng MS cú b sung 0,5-1 mg/l 2,4-D v 0,1 mg/l IAA cho t l to callus t mu lỏ, on thõn khoai tõy tt nht - BAP v kinetin cú nh hng tớch cc ti kh nng to callus t mu lỏ v on thõn khoai tõy Mụi trng cú b sung 0,5 mg/l BAP v mg/l kinetin cho t l to callus cao nht - Kh nng to callus t mu lỏ hoc on thõn khoai tõy tụt nht b sung 60 g/l xaccaroz vo mụi trng nuụi cy - Mụi trng cú b sung 400 mg/l casein hydrolysate ó cú tỏc dng lm tng kh nng to callus t mu lỏ v on thõn khoai tõy b/ Nghiờn cu chuyn gen: mt s yu t chớnh nh hng n hiu qu quỏ trỡnh chuyn gen vo cõy khoai tõy ó c ti u húa nh sau: Chng vi khun A tumefaciens: AGL-1 Vector chuyn gen: p6d-35SGUS Mt vi khun lõy nhim: OD600 = 0,8 Nng acetosyringone b sung vo dch vi khun lõy nhim v mụi trng ng nuụi cy: 200 àM Thi gian lõy nhim: 40 phỳt ngh Cn tip tc cỏc nghiờn cu hon thin h thng tỏi sinh cõy khoai tõy nhm to ngun vt liu ban u cho nghiờn cu chuyn gen Tip tc cỏc nghiờn cu ci thin hiu qu chuyn gen vo cõy 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Chinese Sci Bulletin, 45(17), pp 1537-1546.Zupan J., Muth T R., Draper O., Zambryski P (2000), The transfer of DNA from A tumefaciens into plants: a feast of fundamental insights, Plant J., 23, pp 11-28 50 Zupan J., Muth T R., Draper O., Zambryski P (2000), The transfer of DNA from A tumefaciens into plants: a feast of fundamental insights, Plant J., 23, pp 11-28 72 Ti liu khỏc 51 http://www.biochem.uiowa.edu/donelson/Database%20items/LB%20media.htm 52 http://thuviensinhhoc.com/FlashPaper/Cong_nghe_sinh_hoc/chuong7.swf 53 http://www.cuctrongtrot.gov.vn/Tech_Science.aspx?index=detail&type=a&idtin=208 54 http://www.hua.edu.vn/tc_khktnn/Upload%5Claiducluu_nh4%2652006.pdf 73 Ph lc1: Thnh phn mụi trng MS (Murashige and Skoogs, 1972) Thnh phn mụi Tờn hoỏ cht Nng (mg/l) trng a lng Vi lng St Cht hu c v Vitamin NH4NO3 1650 KNO3 1900 MgSO4 7H2O 370 KH2PO4 170 CaCl2 2H2O 440 H3BO3 6,2 MnSO4 4H2O 17,0 ZnSO4 7H2O 10,5 KI 0,83 CuSO4 5H2O 0,025 MoO4Na2 2H2O 0,25 CoCl2 6H2O 0,025 FeSO4 7H2O 27,8 Na2EDTA 37,3 Axit Nicotinic (B5) 0,5 Thiamin HCl Myo-Inositol 100 Pyrodoxin 0,5 ng 60 Thch 74 Ph lc 2: Thnh phn mụi trng N6 (Chu v cng s, 1975) Thnh phn mụi Tờn hoỏ cht Nng (mg/l) trng a lng Vi lng St Cht hu c v Vitamin (NH4 )2SO4 463,00 KNO3 2830,00 MgSO4 90,27 KH2PO4 400,00 CaCl2 125,33 H3BO3 0,80 MnSO4 H2O 3,33 ZnSO4 7H2O 1,50 KI 0,80 Fe NaEDTA 36,70 Axit Nicotinic (B5) 0,50 Thiamin HCl 1,00 Glyxin 2,00 Pyrodoxin HCL 0,50 ng 60 Thch Thnh phn mụi trng LB Yeast extract Typtone( or peptone) NaCl H2 pH 5g 10g 8g 900ml 7.2 75 Ph lc 3: Mụi trng nuụi cy s dng nghiờn cu NC = MS + 60-80 g/l xaccaroz + 100 mg/l myoinositol + g/l agar CI = MS + 30g/l xaccaroz + 100 mg/l myoinositol + g/l agar LCM = MS + 30g/l xaccaroz + 100 mg/l myoinositol + g/l agar + mg/l 2,4-D + 500 mg/l Casein Hydrolysate(CH) AM1 = CI + mg/l 2,4-D + 0,1 mg/l IAA AM2= CI + 0,5 mg/l 2,4-D + 0,1 mg/l IAA BKM1 = CI + mg/l 2,4-D + 0,1 mg/l IAA+ 0,5 mg/l BAP + mg/l kinetin BKM2 = CI + 0,5 mg/l 2,4-D + 0,1 mg/l IAA+ 0,5 mg/l BAP + mg/l kinetin XM1= CI(khụng cú xaccaroz) + 60 g/l xaccaroz + mg/l 2,4-D + 0,1 mg/l IAA + 0,5 mg/l BAP + mg/l Kinetin XM2 = CI (khụng cú xaccaroz) + 60 g/l xaccaroz + 0,5 mg/l 2,4-D + 0,1 mg/l IAA + 0,5 mg/l BAP + mg/l Kinetin CHM1= CI (khụng cú xaccaroz) + 60 g/l xaccaroz + mg/l 2,4-D + 0,1 mg/l IAA + 0,5 mg/l BAP + mg/l Kinetin+ 400 mg/l Casein hydrolysate CHM2 = CI (khụng cú xaccaroz) + 60 g/l xaccaroz + 0,5 mg/l 2,4-D + 0,1 mg/l IAA + 0,5 mg/l BAP + mg/l Kinetin + 400 mg/l Casein hydrolysate SM = MS + mg/l BAP + mg/l kinetin + 400 mg/l (CH) + 30 g/l xaccaroz + 100 mg/l myoinositol + g/l agar 76 S CC TH NGHIM TI SINH Cõy khoai tõy in vitro Lỏ on thõn Cy mu trờn mụi trýng to callus (5 mulỏ/a ;10 mu on thõn/a) (TN1-TN5) Nuụi 26 C, t n nhit, thi gian 25-30 ngy Cy chuyn mu callus (5 mulỏ/a ;10 mu on chi 26 C, chiu sỏng 8thõn/a) sang mụi trýng Nuụi tỏi sinh (SM) 10h/ ngy, thi gian 8-10 Nuụi 26 C, chiu sỏng 8-10h/ ngy, thi gian 8-10 ngy ngy Chi t chiu cao 1-2 cm c cy chuyn sang bỡnh nuụi cú cha mụi trng NC Nuụi 26 26 chiu 8- ngy, thi gian Nuụi C,C, chiu sỏngsỏng 8-10h/ 10h/ ngy ngy, thi gian 10-15 10-15 ngy To cõy tỏi sinh hon chnh 77 S TH NGHIM CHUYN GEN Chun b mu thc vt (t thõn, Mu lỏ) Nuụi vi khun Agrobacterium Cy gt Agrobacterium trờn mt LB + KS thớch hp, nuụi 28C, 2-3 ngy Chun b dch vi khun lõy nhim Lõy nhim: Thm khụ mu ct trờn giy lc ó kh trựng, Lõy nhim VK-mu 30 phỳt ng nuụi cy: Cy chuyn mu (20 mu/a) sang mt LCM-AS Loi b Agrobacterium: Cy chuyn mu (20 mu/a) sang mt LCM + 500 mg/l cefotaxime Nu cũn Agrobacterium mu, mu x bng mt MS + 500 mg/l cefotaxime Chn lc v tỏi sinh chi Cy chuyn mu (20 mu/a) sang mt LCM + 500 mg/l cefotaxime + KS chn lc Chuyn mu callus to thnh sang mt CSM1 + KS chn lc Nuụi iu kin chiu sỏng cao (3000lux), 10-15 ngy Tỏi sinh chi trờn mụi trng chn lc 78