Chon Dong Chiu Han Va Phan Lap Gen Cystatin Lien Quan Den Tinh Chiu Han o Cay Lac

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vn
B GIO DC V O TO I HC THI NGUYN
V TH THU THY
TO DNG CHU HN V PHN LP GEN CYSTATIN LIN QUAN N TNH CHU HN CY LC (Arachis hypogaea L.)
LUN N TIN S SINH HC
Thi Nguyn - 2011
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B GIO DC V O TO I HC THI NGUYN
V TH THU THY
TO DNG CHU HN V PHN LP GEN CYSTATIN LIN QUAN N TNH CHU HN CY LC (Arachis hypogaea L.)
LUN N TIN S SINH HC
Chuyn ngnh: Di truyn hc M s: 62 42 70 01
Hng dn khoa hc: 1. PGS.TS Chu Hong Mu 2. PGS. TS Nguyn Th Tm
Thi Nguyn - 2011
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LI CAM OAN
Ti xin cam oan bn lun n l cng trnh nghin cu ca ti di s hng dn ca PGS.TS. Chu Hong Mu v PGS.TS. Nguyn Th Tm, s gip ca cc cn b Khoa Sinh Trng i hc S phm, Khoa Khoa hc S sng- Trng i hc Khoa hc, i hc Thi Nguyn. Cc s liu nu trong lun n l trung thc. Mt s kt qu c cng b ring hoc ng tc gi, phn cn li cha c ai cng b trong bt k cc cng trnh no khc. Ti xin hon ton chu trch nhim v nhng s liu trong lun n ny.
Tc gi
V Th Thu Thy
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MC LC
Trang Li cam oan Li cm n Mc lc Danh mc cc k hiu, ch vit tt Danh mc bng Danh mc hnh M U . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chng 1. TNG QUAN TI LIU . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Cy lc v c tnh chu hn ca cy lc . . . . . . . . . . . . . . . . . . . . . 1.1.1. Ngun gc v c im nng sinh hc ca cy lc . . . . . . . . . . . . . 1.1.2. c tnh chu hn ca thc vt v ca cy lc . . . . . . . . . . . . . . . . . 1.2. Nghin cu nng cao kh nng chu hn ca cy trng bng cng ngh t bo thc vt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Chn dng t bo soma trong chn ging cy trng . . . . . . . . . . . .
15 16 i ii iii vi viii x 1 4 4 4 8
1.2.2. Pht sinh bin d trong qu trnh nui cy. . . . . . . . . . . . . . . . . . . . . 19 1.2.3. Cc phng php chn dng t bo . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.4. Mt s thnh tu trong chn dng chng chu yu t bt li ca ngoi cnh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 1.3. Phn tch, nh gi cc dng chn lc c ngun gc t nui cy m so . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.3.1. nh gi kh nng chu mt nc ca m, t bo thc vt . . . . . . . 23 1.3.2. nh gi kh nng chu hn giai on ht ny mm v giai on cy non. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 1.3.3. K thut RAPD trong nh gi h gen ca cc dng chn lc . . . . 1.4. Gen lin quan n tnh chu hn cy lc . . . . . . . . . . . . . . . . . . . .
26 28 20
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1.4.1. Cc gen lin quan n kh nng chu hn ca cy lc . . . . . . . . . . . 1.4.2. Nhm gen m ha protein iu khin hot ng phin m ca cc gen chu hn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 5. 1.5.1 Cystatin v vai tr ca cystatin thc vt . . . . . . . . . . . . . . . . . . . . c im cu to v phn loi cystatin . . . . . . . . . . . . . . . . . . . . . .
29 30 30 33 28
1.5.2. Cu trc khng gian v c ch c ch ca cystatin . . . . . . . . . . . . .
1.5.3. Chc nng ca cystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 1.5.4. Gen m ha cystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38
Chng 2. VT LIU V PHNG PHP NGHIN CU . . . . . . . . 43 2.1. 2.2. 2.3. Vt liu thc vt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ho cht v thit b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43 45
Phng php nghin cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

46
2.3.1. Phng php nui cy in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Phng php nghin cu ng rung . . . . . . . . . . . . . . . . . . . . . . . . 49 2.3.3. Phng php sinh l, ho sinh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. Phng php sinh hc phn t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5. Phng php phn tch s liu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.
50 52 55
a im nghin cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
57
Chng 3. KT QU NGHIN CU V THO LUN . . . . . . . . . . . 31. Kt qu to dng chu hn bng k thut x l m so trong h thng nui cy in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1 3.1.2 Sng lc dng m so chu tc ng ca thi kh . . . . . . . . . . . . . . nh hng ca tia gamma kt hp vi thi kh n t l sng st v ti sinh cy ca ging lc L18 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3 3.2. c im nng sinh hc ca cc qun th lc th h R0 v RM0 . Kt qu phn tch nhng dng lc chn lc qua cc th h . . . . . . .
57 57
66 69 74
3.2.1. c im nng sinh hc cc dng lc chn lc th h th Nht v th Ba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
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3.2.2. nh gi cc dng chn lc th h th Nm . . . . . . . . . . . . . . . . . 78 3.2.3. c im ca mt s dng lc u vit . . . . . . . . . . . . . . . . . . . . . . . 94 3.3. Phn lp v xc nh trnh t gen cystatin t cy lc . . . . . . . . . . . . 97
97
3.3.1. Khuych i gen cystatin t DNA h gen ca cy lc . . . . . . . . . . .
3.3.2. Kt qu tch dng v xc nh trnh t gen cystatin . . . . . . . . . . . . . 98 3.3.3. Kt qu so snh trnh t gen v protein cystatin . . . . . . . . . . . . . . . . 102 KT LUN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111
CC CNG TRNH CNG B LIN QUAN N LUN N . . . . . . 113 TI LIU THAM KHO .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 PH LC
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DANH MC CC K HIU, CH VIT TT
ABA AhNCED AhSP BAP bp CC CKH CTAB CYS DNA tg EDTA CHP HSP HvCPI IPTG kb kDa KLK krad LB LEA LTPs MS mRNA : absicis acid : Arachis hypogaea 9-cis-epoxycarotenoid dioxygenase : Arachis hypogaea serine proteinase : 6 Benzyl amino purin : base pair : Corn Cystatin : Cy khng ho : Cetyltrimethylammonium bromide : cystatin : Deoxyribose nucleic acid : ng tc gi : Ethylene diamine tetraacetic acid : Cy hi phc : heat shock protein : Hodeum vulgare Cysteine Proteinase Inhibitor : Isopropyl -D-1 thiogalactopyranoside : kilo base : kilo dalton : Khi lng kh : kilorad : Luria- Bertani : late embryogenesis abudant : lipid transfer proteins : Murashige- Skoog : messenger Ribonucleic acid
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OC OD PCR PEG PLD RAPD SDS TAE TTC VuC X-gal WC 2,4D - NAA : Oryza Cystatin : Optical Density : Polymerase chain reaction : Polyethylene glycol : Phospholipase D : Random amplified polymorphic DNA : Sodium Dodesyl Sulphate : Tris Acetate EDTA : 2,3,5 Trichlo tetrazolium chlorit : Vigna unguiculata cystatin : 5- Bromo- 4 Cloro- 3 indolyl D galactopyranoside : Wheat cystatin : 2.4 Dichlorphenoxyacetic acid : - Naphthaleneacetic acid
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DANH MC BNG
Trang Bng 1.1. Din tch, nng sut v sn lng lc ca Vit Nam giai on 2005-2009 . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 1.2. Gen cystatin lin quan vi kh nng chng chu hn mt s loi thc vt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 2.1. Bng 3.1. Bng 3.2. Bng 3.3. Mt s c im ca 10 ging lc nghin cu . . . . . . . . T l to m so v khi lng m so ca 10 ging lc mt nc ca m so sau x l bng k thut thi kh Kt qu kim tra kh nng chu mt nc ca m so 10 ging lc bng phng php nhum TTC . . . . . . . . . . . . Bng 3.4. T l sng st ca m so b x l bng thi kh sau 4 tun nui phc hi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 3.5. Bng 3.6. T l ti sinh cy ca m so sng st sau 6 tun . . . . . . nh hng ca chiu x kt hp vi thi kh 9 gi n t l sng st v ti sinh cy ca m so ging lc L18 Bng 3.7. c im nng hc ca qun th R0, RM0 ti sinh t m so mt nc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 3.8. c im nng sinh hc cc dng lc chn lc th h th Nht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 3.9. Bng 3.10. c im nng hc cc dng lc chn lc th h th Ba c im nng sinh hc v cht lng ht cc dng chn lc th h th Nm . . . . . . . . . . . . . . . . . . . . . . . Bng 3.11. Tng quan gia hot ca -amylase v hm lng ng giai on ht ny mm . . . . . . . . . . . . . . . . . . . Bng 3.12. T l cy sng, cy phc hi v ch s chu hn tng i ca cc dng chn lc th h th Nm . . . . . . . . . . 86 84 79 75 77 70 66 63 65 61 41 43 58 59 5
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Bng 3.13. S phn on DNA nhn bn ngu nhin v t l phn on a hnh ca 25 mi trong phn ng RAPD . . . . . . . Bng 3.14. T l sai khc v h gen ca cc dng chn lc v ging gc L18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 3.15. Bng 3.16. Bng 3.17. Ch th RAPD c trng ca cc dng lc chn lc . . . . Mt s c im ca 3 dng chn lc v ging L18 . . . . S sai khc v trnh t nucleotide trn gen cystatin ca ging v dng lc nghin cu . . . . . . . . . . . . . . . . . . . . . Bng 3.18. tng ng v sai khc on m ho mt s trnh t gen cystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bng 3.19. Thnh phn amino acid ca protein cystatin cy lc . . 104 109 103 91 93 95 90
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DANH MC HNH Trang
Hnh 1.1. Hnh 1.2. Hnh 1.3. Hnh 2.1. Hnh 3.1. Hnh 3.2.
Trnh t 102 amino acid ca OCI . . . . . . . . . . . . . . . . . . . . Cu trc khng gian ba chiu ca cystatin thc vt . . . . . . M hnh cu trc c ch hot ng ca oryzacystatin . . . . . M hnh th nghim tng qut . . . . . . . . . . . . . . . . . . . . . . . mt nc ca m so cc ging lc . . . . . . . . . . . . . . . Dung dch nhum TTC ca m so ging lc L18 cc ngng x l thi kh . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32 34 36 46 60
62
Hnh 3.3.
Mt s hnh nh cy ti sinh ca ging lc L18 giai on 6 tun tui . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 70 72 Cc dng lc chn lc trng v xun 2008 . . . . . . . . . . . . Hnh nh trong chn dng chu hn lc bng k thut nui cy in vitro S bin ng v hot ca -amylase trong iu kin hn sinh l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 83 S bin ng hm lng ng trong iu kin hn sinh l th hnh rada biu th s khc nhau v kh nng chu hn ca cc dng lc chn lc, ging L18 v ging L23 . . 87 89 Hnh nh in di sn phm RAPD vi mt s mi . . . . . . .. S m t mi quan h ca 7 dng chn lc vi ging gc L18 mc phn t . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Hnh nh qu v ht ca cc dng chn lc th h th Nm . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . 96 97 Hnh nh in di sn phm nhn gen cystatin t cy lc . . .
Hnh 3.4. Hnh 3.5.
Hnh 3.6.
Hnh 3.7 Hnh 3.8
Hnh 3.9. Hnh 3.10.
Hnh 3.11.
Hnh 3.12.
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Hnh 3.13. Hnh 3.14. Hnh 3.15. Hnh nh in di sn phm colony-PCR . . . . . . . . . . . . . . . Kt qu so snh trnh t nucleotide gen cystatin ca cy lc Mi quan h di truyn ca mt s on m ho gen cystatin thc vt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hnh 3.16. So snh trnh t amino acid ca 4 mu nghin cu . . . . . . . 105 107 99 102
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M U
1. L do chn ti Lc (Arachis hypogaea L.) l cy thc phm, cy cng nghip ly du trong nhm cy trng cn. Mc d cy lc c t lu i nhng vai tr ca cy lc mi c cng b t khong 100 nm tr li y. Trng lc mang li ngun li c gi tr v kinh t, v dinh dng, ng thi cn ci to t rt tt nh kh nng cng sinh vi vi khun Rhizobium.. [3]. Trn th gii, nhu cu s dng v tiu th lc ngy cng tng, cng l l do khuyn khch nhiu nc u t pht trin sn xut lc vi quy m lin tc m rng [145]. Vit Nam, sn xut lc c phn b trn tt c cc vng sinh thi nng nghip, vi khong 40% tng din tch gieo trng cc cy cng nghip ngn ngy. Trong , 2/3 din tch t trng lc ph thuc vo nc tri. So vi nhiu cy trng khc, cy lc c nhu cu c bit v nc bi r khng c lng ht v qu lc c hnh thnh di t. S bin i kh hu lm cho lng ma hng nm phn b khng u gia cc vng min v cc thi im trong nm. Trong khi , qu trnh sinh trng v pht trin ca cy lc c nhng giai on rt mn cm vi m, nu nhng giai on ny thiu nc s lm gim nng sut, cht lng ca lc. Chnh v vy, vic nh gi c bin php ci to ging lc theo hng nng cao kh nng chu hn l rt cn thit. Cc phng php v ang c nghin cu ng dng ci to, pht huy tim nng ca nhiu loi cy trng nh: lai ging, t bin thc nghim, cng ngh t bo thc vt v chuyn gen [30], [76], [131]. Hiu qu vic ng dng cng ngh t bo thc vt nng cao tnh chng chu bt li mi trng lin tc c khng nh. Trong qu trnh nui cy in vitro do nh hng ca mi trng, c bit l cht kch thch sinh trng, cc t bo c th b bin i vt cht di truyn. Larkin v Scowcroft (1981) gi nhng bin d l bin d soma hay bin d sinh dng. Nu kt hp nui cy m t bo vi x l t bin bng cc tc nhn vt l hay ho hc th tn s t bin
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c th tng thm [85]. iu ny c ngha c bit quan trng trong vic to ra ngun vt liu khi u cho chn ging [87]. Vit Nam, s ra i ca 2 ging la DR1 v DR2 c kh nng chu hn v chu lnh tt l minh chng u tin cho nhng kh nng [1]. n nay, nhiu dng cy c ngun gc in vitro mang tnh trng mong mun c to ra t qu trnh x l v nui cy m so c cng b [6], [34], [39], [130]. Kh nng chu hn ca thc vt l tnh trng do nhiu gen quy nh. Do vy, vic tm kim v phn tch gen lin quan n c tnh chu hn c nhiu nh khoa hc quan tm nghin cu. Mt s gen lin quan n tnh chu hn ca cy trng c phn lp v cng b nh: gen LEA [32]; gen DREB [113]; gen P5CS [10]; gen NCED, AhPLD [104], [128]... Gen cystatin (CYS) ca thc vt c cng b u tin trn cy la bi Abe v tg (1987) [40]. n nay, gen CYS c phn lp nhiu loi thc vt bc cao, cy mt l mm v hai l mm [17], [92], [95]... Cc nghin cu v gen cystatin bn lun nhiu v mi lin quan ca n vi kh nng chng chu hn, lnh, mn [52], [57], [96], [125]... Xut pht t nhu cu thc tin ca vic chn to ging lc theo hng nng cao kh nng chu hn, chng ti tin hnh ti ca lun n: To dng chu hn v phn lp gen cystatin lin quan n tnh chu hn cy lc (Arachis hypogaea L.). 2. Mc tiu nghin cu To c dng lc c kh nng chu hn cao hn ging gc bng cng ngh nui cy m t bo thc vt. Xc nh c s khc bit trong trnh t ca gen cystatin gia dng lc chn lc c ngun gc t m so v cc ging lc c kh nng chu hn khc nhau. 3. Ni dung nghin cu Sng lc dng m so chu mt nc di tc ng ca thi kh v tc ng ca chiu x tia gamma kt hp vi thi kh.

Phn tch s bin ng mt s tnh trng s lng, cht lng ht v kh nng chu hn ca cc dng chn lc. So snh v xc nh s sai khc trong h gen ca cc dng chn lc bng k thut RAPD. Khuch i, tch dng v xc nh trnh t gen cystatin ca dng lc chn lc v ging gc.
4. Nhng ng gp mi ca lun n i) Lun n xy dng c quy trnh chn dng lc chu hn theo cch s dng kt hp cc phng php nui cy m t bo, gy t bin bng tia gamma v s dng ch th phn t RAPD; tch dng v so snh s khc bit trnh t gen cystatin lin quan n tnh chu hn. Quy trnh ny c th rt ngn thi gian cn thit v nng cao hiu qu cho cng tc chn ging lc. ii) X l m so bi tia gamma lm gim chiu cao v t l ti sinh cy, bin i mu sc v hnh dng l. Pht hin c 5 ch th RAPD c trng cho hai dng lc chn lc RM47 v RM48: RM48/OPA07-750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348-200bp; RM47/OPH08-250bp. Pht hin gen cystatin ca cy lc thuc nhm I ca phytocystatin, c c s tng ng cao nht vi cystatin ca u xanh (81,3%), thp nht vi cystatin ca qu kiwi (42,9%). Gen cystatin c 1 intron v 2 exon, m ha cho protein c 98 amino acid. Cystatin ca dng RM48 c ngun gc t m so c x l bi tia gamma kt hp vi thi kh c 7 v tr amino acid sai khc so vi ging gc L18. S thay th amino acid ca dng RM48 bi amino acid ca ging gc cc v tr 29 (Glu Asp), 30 (HisThr), 31 (AsnThr), 32 (Lys Arg), 33 (Lys Asn), 34 (Glu Arg), 36 (Gly Ala). iii) Xc nh c s khc bit so vi ging gc L18 v kh nng chu hn ca ba dng lc RM48, RM47, R46 c ngun gc t m so chu mt nc c x l bi tia gamma (2krad) kt hp vi thi kh lin tc 9 gi trong h thng nui cy in vitro.

Chng 1
TNG QUAN TI LIU
1.1. CY LC V C TNH CHU HN CA CY LC 1.1.1. Ngun gc v c im nng sinh hc ca cy lc Cng vi thi gian khm ph ra chu M, nhiu dn liu chng minh cy lc c ngun gc t nhin t cc nc thuc vng Nam M. Theo cc nh nghin cu lch s t nhin, ngi Inca trng lc nh mt loi rau c tn l ynchis dc vng duyn hi ca Peru. Sau xut hin ph bin chu u, chu Phi, chu , qun o Thi Bnh Dng v cui cng ti vng ng Nam Hoa K. Tuy nhin, sn xut lc rng ri thuc cc nc vng nhit i v cc vng trn th gii t 400 v Bc n 400 v Nam [4]. u tin, trong nghin cu ca Linnaeu (1753) cng b Arachis hypogaea L. l tn mt loi. T cho n nm 1939 ch c chi lc trng Arachis hypogaea L. c m t. Hin ti, Arachis l mt chi trong tng Hedysareae ca b u (Leguminosae). Chi Arachis c 22 loi, u l loi cy hng nm sng trn mi trng cn; c l km v 3 n 4 l cht; hoa dng cnh bm, mu vng; c m phn sinh t; qu hnh thnh di t. Cu trc thng nht trong chi l tia qu phnh to t non, mi qu tht khong 1 n 5 t, mi t cha mt ht vi 2 l mm v mt phi thng [8]. Cy lc c ngun gc t Nam M v c mt nc ta vo khong th k XVII-XVIII. Theo Tng cc thng k nm 2009, vi chiu di hn 3000 km, tri dc t v tr 80 27 n 230 23 v Bc, cy lc c trng tt c cc vng sinh thi nng nghip ca nc ta [145].

Mc d cn kh khn, nhng nhiu a phng cy lc vn c coi l cy trng ch o. Nm 2008, FAO nh gi Vit Nam l mt trong 10 nc ng u th gii v sn xut lc, vi tng sn lng t 490.000 tn [144], tuy nhin s pht trin cy lc nc ta vn mc cha tng xng vi tim nng ca n [3]. Kt qu nghin cu trn bng 1.1 cho thy s khng n nh v cc ch s pht trin ca cy lc (cc ch s c tnh so vi nm trc bng 100%). C nhiu nguyn nhn hn ch s pht trin ca cy lc, trong c nguyn nhn do hn hn [145].
Bng 1.1. Din tch, nng sut v sn lng lc ca Vit Nam giai on 2005- 2009 Din tch Nm x 1000 ha Ch s pht trin (%) 2005 2006 2007 2008 2009 169,6 246,7 254,5 255,3 249,2 102,2 91,5 103,2 100,0 97,6 18,1 18,7 20,0 20,8 21,1 Nng sut t/ ha Ch s Sn lng x Ch s
pht trin 1000 tn pht trin (%) 102,0 103,0 107,0 104,0 101,0 489,3 462,5 510,0 530,2 525,1 (%) 104,3 94,5 110,3 104,0 99,0
Ngun: Tng cc thng k (2009)[145] Cc ging lc ca Vit Nam c tuyn chn t nhiu ngun khc nhau, v chng u thuc loi hnh thc vt spanish, c tp tnh sinh trng thnh cm [3].

Sn xut lc min Bc thng tp trung vo hai v chnh: v Xun H (gieo ht khong t trung tun thng 1 n trung tun thng 2 dng lch) v v Thu ng (gieo ht khong t trung tun thng 8 n trung tun thng 9 dng lch). thi im u v v cui v thng xy ra hn hn, do tuyn chn nhng ging lc c thi gian sinh trng ngn khc phc nhng tc ng xu ca hn c nhiu nh khoa hc quan tm nghin cu [4], [8]. Cy lc mc t ht, s pht trin ca cy lc chu nh hng ca nhiu yu t sinh thi nh nhit , m iu kin nhit t 250C n 300C, m 50-60% l mi trng rt thch hp cho s ny mm v pht trin cy lc [4]. Cy lc trng thnh gm c mt thn chnh (n) mc t t cui tr trn l mm, c cao t 12cm n 65cm, hai l mm i xng (n+1). H r ca cy lc gm 1 r cc v cc r con. B r cy lc pht trin mnh su t 5cm n 35cm, lan rng khong 12- 14cm. Trn t ct pha, b r c th di 90-130cm. Trn r cy lc c th c cc nt sn cha cc vi sinh vt thuc chng Rhizobium lm nhim v c nh m. V vy, vic trng lc va em li ngun kinh t c gi tr va c tc dng ci to t [8]. Cy lc bt u c hoa khong t 20 ngy n 30 ngy sau khi ny mm. Hoa lc t th phn (th phn cho ch khong 1,0% - 3,9%). Hoa thng n vo thi im c nh sng, khi thi tit lnh, m t hoa c th n mun hn. Trc khi hoa n 7- 8 gi, bao phn nt ra, nhy ch tip nhn ht phn trc 24 gi v sau 12 gi khi hoa n. Khi th phn c khong 6 gi th s th tinh din ra hon ton v sau khi th tinh, non pht trin xuyn qua hoa l ra tia c di, tia c pht trin m xung t 2cm mi pht trin thnh qu. Nu s pht trin ca tia c di ti 15cm m khng tip xc c vi t

s ho ngay. T khi hoa lc th tinh n khi qu chn khong 60 ngy [4], [8]... Theo Ng Th Dn v tg (2000), thi gian sinh trng ca lc c th xc nh theo hai cch: (i) cch th nht, tnh thi gian sinh trng l khong thi gian t khi gieo ht n khi thu hoch, (ii) hoc cch tnh th 2 l khong thi gian t khi ht ny mm n khi thu hoch. Trong , cch th hai c dng nhiu hn, v khi gieo ht nu gp iu kin khng thun li nh hn u v hay lnh u v (iu kin thng gp min Bc), ht lc c th nm trong t t 10 ngy n 20 ngy mi ny mm. i vi cy lc, sinh trng l mt c im ca kiu gen mc d chu nh hng rt ln ca ma v v mi trng [4]. Thc t min Bc, khng c ging lc no c thi gian sinh trng t hn 120 ngy. Do , da vo thi gian sinh trng ngi ta chia cc ging lc thnh 3 nhm: cc ging sinh trng ngn ngy (thi gian sinh trng t hn 120 ngy), ging sinh trng trung bnh (120 ngy- 140 ngy) v cc ging sinh trng di ngy (c thi gian sinh trng trn 140 ngy). C ba giai on pht trin ca cy lc c xc nh rt mn cm vi m, l: (i) thi k u ca sinh trng dinh dng; (ii) thi k ra hoa v m tia xung t; (iii) thi k qu chn. Nhng nh hng r rt ca s thiu nc n cy lc c th d dng quan st nh: thiu nc lm gim din tch l, thiu nc lm cho cy c ngn nh hn, cnh v hoa t hn... do nh hng n nng sut v cht lng lc [4], [111]... cng l nguyn nhn khin cho cc vng trng lc nh nc tri cha c nhng u t thch ng vo cy lc [4]. Kt qu nghin cu nh gi kh nng chu hn ca mt s ging lc trong iu kin c mi che vi trng lc c s ti nc b sung gi m n nh trn ng rung, cho thy cc yu t cu thnh nng sut (s qu chc, t l nhn, nng sut qu kh) ca nhm b hn gim so vi nhm c cung

cp m y . Tuy nhin, cc c im hnh thi (h sc t thn, mu sc l, dng hnh sinh trng) khng lin quan hoc t lin quan n kh nng chu hn ca cy lc [7], [14]. 1.1.2. c tnh chu hn ca thc vt v ca cy lc 1.1.2.1. C s sinh l, ha sinh v sinh hc phn t ca tnh chu hn Kh nng ca thc vt ngn nga tn thng khi b nh hng cc yu t bt li l tnh chng chu (stress resistance) [1]. Trong t nhin, hn hn l hin tng thng xuyn xy ra v lin quan trc tip n vn nc trong cy. Kramer (1983) cho rng, nhng tc ng ca mi trng xung quanh gy nn mt nc thc vt l hn [82]. Khi hn l yu t bt li vi thc vt s gy ra cc mc thit hi khc nhau. chng li s mt nc do nguyn nhn t cc tc ng bn ngoi, thc vt c ba c ch sinh l ch yu c tho lun, l c ch trn hn, c ch trnh hn v c ch chu hn. Thc vt c th hon thnh chu k sng sm hn trc khi s thiu nc nghim trng trong t v trong cy xy ra. Kiu chu mt nc ny gi l trn hn (drought escape). Mun trn c hn, cy phi c sc sinh trng v pht trin mnh, c th ra hoa v to qu sm thi im thiu nc khng nh hng n chu k sng ca cy. Nhm cy trn hn thng l cy sng vng sa mc, cy c thi gian sinh trng ngn. Chu trnh sng ca chng thng hon thnh trc khi ma kh hn ti [63]. trnh hn, thc vt c th gi nc trong t bo hoc gim s thot nc ra ngoi c th. Trong , phng thc trnh hn (drought avoidance) ch yu ca thc vt l hn ch s mt nc. V d, thay i m kh khng, gim din tch l hoc nhanh chng b li s thiu ht nc thng qua nhng bin i v hnh thi. nh hng s pht trin ca l theo hng gim s hp th bc x, tng cng s hp thu nc theo hng pht trin su v rng ca b r [49], [60].

Duy tr p sut thm thu ni bo c tc dng bo v hoc duy tr sc sng, n hi ca t bo ngay c khi b mt nc cc oan, l kh nng chu hn (drought tolerance). Trong iu kin hn hn, p sut thm thu ca t bo c iu chnh tng ln gip cho t bo c th duy tr lng nc. Tuy nhin, cng mt thi im b hn, thc vt s dng nhiu hn mt c ch chng hn [60], [98]. Tm hiu bn cht ho sinh v sinh hc phn t ca tnh chu hn l mi quan tm ca nhiu nghin cu [12], [47], [48], [49], [60]. Nghin cu tnh chng chu hn v cu trc t bo ngi ta nhn thy, hng lot nhng bin i su sc xy ra nhiu cp trong cc giai on khc nhau v lin quan n hot ng ca cc gen. Nghin cu ca Bray (1993) v nhng sn phm ca gen tham gia bo v t bo khi t bo b thiu nc c tip cn theo hng: (1) hot ng ca nhm gen to sn phm bo v t bo (protection of cellular) v (2) hot ng ca nhm gen tham gia iu chnh p sut thm thu trong t bo (osmotic adjustment) [51]. Theo hng th nht, (i) Sn phm ca gen l cc cht mi gii phn t (chaperones). Cht mi gii phn t tham gia vo qu trnh to cu trc khng gian ng cho cc phn t protein c to thnh trong t bo, hay to cu trc ng cho nhng phn t protein b bin tnh trong iu kin cc oan. Mt protein l c xc nh l cn thit sp xp cu trc ng ca mt phc hp enzyme ln trong lp lc, nhng protein l ny khng lin quan n vic lp rp cui cng ca phc enzyme. iu ny nhanh chng c xc nh rng, cng vi nhng protein ch o lp rp chnh, loi protein l ging ht vi mt trong nhiu protein c tng hp mnh khi cc t bo gp nhit cao. V th, nhm cht mi gii phn t th nht gi l "protein sc nhit", hoc HSP (HSP- heat shock protein). Trong iu kin bnh thng cc HSP vn c tng hp, nhng khi gp cc iu kin bt li nh hn hn, nhit cao, mui cao... chng c

tng hp gia tng. HSP thc vt gm nhiu loi khc nhau v kch thc, khi lng v s khu tr trong t bo. HSP70 l cht mi gii phn t c kch thc khong 70kDa, nhm ny tm thy trong cht nguyn sinh ca cc t bo. Bnh thng, HSP70 gn vi polypeptide ang tng hp trn ribosome, do chui polypeptide c n nh cho n khi tng hp xong. Trong iu kin cc oan, HSP70 ngn chn s co cm kt ta protein [71]. HSP60 (chaperones c kch thc khong 60kDa) l cht mi gii phn t tm thy trong mt s bo quan ca sinh vt nhn thc nh ty th, lc lp. HSP60 c cu trc hnh tr, c vai tr to cu trc ng cho protein. (ii) Cc sn phm ca gen tham gia o thi nhng cht b bin tnh, nhng cht c kh nng gy c cho t bo khi t bo gp tc ng ca iu kin bt li. Cc cht ny bao gm ubiquitin, protease, catalase, supeoxid dismutase... Do vy, hot ng ca nhm cht ny s gp phn gii c v bo v t bo. (iii) Cc cht bo v enzyme, c ch hot ng ca cc enzyme thy phn khc...do lm hn ch s phn gii nhng cht c hot tnh enzyme v bo v cc cu trc trong t bo, nhm ny gi chung l protease inhibitor. Nhm cht c ch enzyme gm nhiu loi, tuy nhin s hiu bit v chng cn hn ch [70]. Theo hng th 2, vic iu chnh p sut thm thu ca t bo khi b mt nc l rt quan trng. Cc cht iu ha p sut thm thu c vai tr khc nhau trong vic gi v ly nc vo t bo. l, vic iu chnh cc knh ion trn mng t bo. Sn phm ca gen thay th v tr nc ni xy ra cc phn ng ho sinh, hoc tng tc vi lipid, protein mng t bo, ngn chn s ph hu mng t bo v ngn chn s ph hy cc protein khc. LEA (late embryogenesis abudant) l protein giu amino acid a nc, khng cha cysteine v tryptophan, do rt thch hp vi vai tr mang nc v gip duy tr nhu cu nc ca t bo [110]. LTPs (lipid transfer proteins) l mt nhm protein h tr hnh thnh lp cutin, lp sp do bo v m trnh mt nc.

LTP tng vn chuyn phospholipid ti mng, do tng bn ca mng v kh nng gi nc ca t bo [78]. Sn phm ca gen iu chnh p sut thm thu bng cch iu khin s ra vo nc qua mng khng bo. Bn cnh , cc cht nh: ion K+, proline, estoine, cc cht ng sucrose, manitol...l nhng cht ng vai tr quan trng trong vic tng cng p sut thm thu ca t bo. 1.1.2.2. c tnh chu hn ca cy lc c tnh chu hn ca cy trng ni chung v ca cy lc ni ring c cu thnh bi mt s yu t nh din tch l, hm lng dip lc trong l, hiu sut quang hp, thot hi nc v hiu qu s dng nc, b r cy lc. Din tch l v hm lng dip lc trong l L lc thuc loi l kp, vi 3 hoc 4 l cht. Tng s l trn cy tng khong t ngy 21 n ngy th 90-100 sau khi trng. nh hng ca thiu nc r nht l gim tng trng ca t bo trn ton b cy, nh vy din tch quang hp s gim dn n gim sc pht trin ca cy. Ch s din tch l c th c ci thin bi t hp cc cht kch thch sinh trng v nguyn t vi lng [33]. Nghin cu xc nh ch s din tch l cho thy, y l ch tiu c mi lin quan vi hm lng dip lc, mt cht dip lc, hiu qu s dng nc... [119], [120]. Dip lc l sc t quang hp ch yu ca cy trng. V vy, mt cht dip lc rt quan trng trong vic nh gi kh nng quang hp ca cy. Mt cht dip lc c tnh bng hm lng cht dip lc trn mt n v din tch l. Mt cht dip lc n nh trong thi gian hn hn c th l mt ch tiu y ha hn trong vic la chn dng, ging lc c kh nng chu hn [44]. cng l l do nhm tc gi Nguyn Tn L v V nh Ngn (2010) tin hnh thm d nh hng ca CaCl2 v t hp mt s nguyn t vi lng ci thin hm lng dip lc trong l [11].

SCMR (SPAD chlophyll meter reading) phn nh c tnh hot ng hp th nh sng ca l. Ch s SCMR ph thuc vo hm lng dip lc trn mi n v din tch l. Arunyanark v tg (2009) cho rng, c th s dng SCMR nh mt cng c nh gi nhanh v tnh trng cht dip lc tng i ca lc cng nh gin tip la chn kh nng chu hn ca cy lc [43]. Hiu sut quang hp Hiu sut quang hp l ch tiu nh gi trn c s hm lng vt cht kh ca cy trng. Hiu sut quang hp phn nh nng sut cy trng. Hiu sut quang hp thay i theo cc giai on sinh trng ca cy. Giai on no c hot ng quang hp mnh nht th c hiu sut quang hp cao nht. C nhiu cch xc nh hiu sut quang hp ca cy nh: xc nh cng quang hp; xc nh khi lng vt cht kh.... Trong , cng quang hp ca cy c tnh thng qua lng CO2 cy hp thu, lng O2 cy thi ra hay lng cht hu c c tch lu trn mt n v din tch l trong mt n v thi gian xc nh [11], [33], [44]... Cng quang hp nh gi kh nng quang hp ca cc qun th cy trng khc nhau. Cng quang hp l mt ch tiu thay i, tu thuc vo ging cy trng, cc c quan, giai on sinh trng khc nhau ca cy, iu kin ngoi cnh Theo Arunyanark v tg (2008) hn hn lm gim ng k khi lng cht kh ca cy [44]. thc vt, khi mt mt lng nc khong 5-10% s thun li cho qu trnh quang hp. Nu bo ho ca nc trong cy thiu hn 10% th hiu sut quang hp b gim st. Quang hp ngng khi hm lng nc trong cy thiu khong t 30% tr ln. c tnh ny l biu hin thch nghi ca b my quang hp trn mi trng cn. Tuy nhin, tu theo kh nng chng chu hn ca cy m mc gim st quang hp s khc nhau. Thc vt c kh nng chng chu hn tt, khi thiu nc hiu sut quang hp s gim t hn. Ribulose 1,5 diphosphat cacboxylase xc tc s chuyn ha ribulose 1,5 diphosphat (RiDP) trong giai on u tin ca qu trnh c nh CO2.

Ribulose 1,5 diphosphat cacboxylase c nhiu trong l. Khi thiu nc, kh khng ng li, hot tnh ca ribulose 1,5 diphosphat cacboxylase b gim st, kh nng c nh CO2 gim dn n hiu sut quang hp ca l gim. V vy, hot tnh ca ribulose 1,5 diphosphat cacboxylase n nh c cho l yu t quan trng gp phn khng hn hn trong cy lc [108]. Thot hi nc v hiu qu s dng nc Thot hi nc l s bay hi ca nc t c th thc vt. Qu trnh thot hi nc xy ra ch yu l, khi kh khng m rng cho vic ly CO2 vo qu trnh quang hp. Qu trnh thot hi nc ca thc vt thc y s vn chuyn nc v cc cht khong t r ln l, ng thi lm mt l cy khi b t nng. Nghin cu s phn b kh khng cy lc nhn thy, s lng kh khng nh nhau c 2 mt l. Do , trong thi im sinh trng mnh, so vi cc cy trng khc cy lc cn cung cp lng nc gp i. chng mt nc, cy lc c c tnh khp l li v hng l song song theo hng tia nng chiu ti [4]. Hiu qu s dng nc ca cy lc c tnh bng t l gia sinh khi thu c trn t vi s thot hi. Theo Songsri (2009), hn hn lm gim hiu qu s dng nc, gim khi lng r kh v ch s thu hoch. Kt qu nghin cu ch ra rng, cy lc c ch s din tch l thp, SCMR cao s cho hiu qu s dng nc ln [119]. B r cy lc Hot ng ca b r cy lc vi c tnh chu hn, c xc nh thng qua ch s RLD (root length density). RLD l ch s lin quan n chiu di v mt phn b ca r, ch s ny c tnh bng t l ca tng chiu di r (cm) trn mt n v khi lng t (cm3). Songsri v tg (2008) nhn thy, ging lc c chiu di r ln t 40-100cm (RLD40 to 100cm) th c ch s chu hn cao v do n nh nng sut qu v ch s thu hoch trong iu kin hn hn [120].

Serine proteinase l enzyme thuc nhm subtilisin, hot ng c trong iu kin thiu nc (cc enzyme hot ng ti thch trong mi trng kim). Serine proteinase c tnh c hiu nhm tng i, hot ng ca serine proteinase thy phn nhiu loi lin kt peptide cng nh cc lin kt ester, nht l nhng lin kt c to thnh t cc amino acid thm. Di tc dng ca enzyme ny, mt s triglyceride cng c th b thy phn. Nghin cu ca Drame v tg (2007) cho thy, s biu hin ca gen AhSP (Arachis hypogaea serine proteinase) m ha serine proteinase trn cy lc gim mnh vi s thiu ht nc, c bit i vi ging chu hn tt. Khi thy ha tr li, ging chu hn tt cng l ging phc hi biu hin ca gen AhSP nhanh hn [59]. Phn tch ca Akcay v tg (2010) cho rng, mc tch t proline, kh nng hot ng ca ascorbate peroxidase v catalase tng l nhng c ch quan trng duy tr kh nng chu hn cy lc [42]. Phospholipase l enzyme thy phn lipid, c vai tr i vi hot ng bo v mng t bo. Hot ng ca phospholipase to ra nhiu lipid ng vai tr quan trng trong cu trc mng t bo nh phosphatidic acid, diacylglycerol hay inositol-triphosphat. Phospholipase gm nhiu loi, khc nhau v v tr phn ct trn phosphatidic acid, pH ti u, kch thc phn t... [104], [128]. Jacob v tg (1999) pht hin, cht ng phn PLD lin kt vi absicis acid c tc dng iu khin s ng kn l kh trong sut qu trnh hn hn [77]. Do , s gia tng tch ly bn sao ca PLD gp phn gim bt s mt nc trong t bo v bo v mng t bo ca cy lc [59]. Hot ng tch ly protein nh LEA cng c chng minh ng thi xy ra trong phn ng vi s thiu ht nc cy lc. Tch ly protein LEA bo v mng t bo khi s ph hy v chng chu vi s mt nc. Trong l lc hm lng protein LEA c tch ly cao khi c s thiu ht nc, s tch ly hm lng protein LEA khng r rt l kt qu nhn c trong cy lc nc [59].

iu chnh p sut thm thu ca cy lc, hot ng to cc cht tham gia vo qu trnh iu chnh p sut thm thu ca thc vt ni chung v cy lc ni ring l qu trnh phc tp vi s tham gia ca nhiu gen, enzyme v protein. Nghin cu s bin ng hm lng protein v proline giai on cy non vo cc thi im trc khi gy hn vi thi im sau 5 v 9 ngy gy hn, Akcay v tg (2010), Dinakar v tg (2008) nhn thy, hm lng proline c s gia tng khi thi gian gy hn ko di v c mi tng quan thun vi kh nng chu hn ca cy lc [42], [56]. Abscicis acid (ABA) l hoocmon thc vt lin quan n vic iu khin qu trnh ng m kh khng. S sinh tng hp ABA trong cy c iu khin bi 9-cis-epoxycarotenoid dioxygenase (NCED) [69]. cy lc, gen AhNCED1 (Arachis hypogaea 9-cis-epoxycarotenoid dioxygenase) m ha tng hp NCED c nghin cu s biu hin cho thy, hot tnh ca NCED tng ln ng k l v thn cy lc p ng vi tnh trng mt nc ca cy [127]. Nh vy, tt c cc loi thc vt u c c tnh sng thch hp trong tnh trng thiu ht nc. Khi s thiu nc do hn hn, thc vt s phn ng bng nhiu c ch khc nhau. Cy lc c kh nng chu hn km. Khi gp hn, cy lc pht huy c tnh chu hn thng qua nhng thay i v hnh thi, sinh l, ha sinh. Cho nn, vic tm kim cc yu t cu thnh c tnh chu hn ca cy trng ni chung v cy lc ni ring nng cao kh nng chng chu ca cy l cn thit. 1.2. NGHIN CU NNG CAO KH NNG CHU HN CA CY TRNG BNG CNG NGH T BO THC VT Hin nay, nui cy m t bo thc vt ang c ng dng trong cc lnh vc nhn ging in vitro, phc trng v to cy sch bnh, nui cy bao

phn v to dng thun, dung hp t bo trn v lai v tnh, ci tin v nng cao kh nng chng chu ca cy trng. ng dng cng ngh nui cy m t bo thc vt nhm nng cao kh nng chu hn ca cy trng c tip cn theo hng nghin cu h thng ti sinh phc v chuyn gen v chn dng t bo soma. Chuyn gen thc vt c th thc hin trc tip hoc gin tip, tuy nhin thc vt bc cao c thnh t bo dy, bn vng nn k thut chuyn gen gin tip t ra u th hn. to c cy chuyn gen bng phng php chuyn gin tip phi hon thin h thng ti sinh cy. n nay, vic chuyn mt s gen iu khin v gen hot ha s biu hin ca nhm gen lin quan n tnh chu hn thc vt c cng b bi Kosmas v tg (2006), Ying v tg (2009) [81], [131]. Nghin cu ca Wan v tg (2006) v chuyn gen AhNCED1 iu khin sinh tng hp ABA ca cy lc vo cy Arabidopsis cho thy, hm lng ABA ca cy Arabidopsis tng ln trong iu kin thiu nc [127]. Cc dng t bo c to ra t nhiu phn khc nhau ca cy. Qu trnh nui cy cc dng t bo lm xut hin nhiu bin d. Vic chn dng bin d soma, ti sinh cy v to cy hon chnh s l ngun vt liu khi u rt tt cho qu trnh chn to ging theo nh hng tng cng kh nng chng chu [1], [31], [80]. 1.2.1. Chn dng t bo soma trong chn ging cy trng Tt c cc t bo ca mt c th u mang b my thng tin di truyn ging nhau, mi t bo bt k ly t c th thc vt u c kh nng tim tng pht trin thnh mt c th hon chnh, l tnh ton nng ca t bo thc vt. iu ny c cc nh khoa hc chng minh bng cc th nghim nui cy m v t bo thc vt.

M hoc qun th t bo nui cy bao gm mt s lng ln cc t bo khng ng nht. V th qun th t bo nui cy c th xem nh qun th thc vt m cng din ra s thay i v kiu gen, kiu hnh v tui. Khi nhng t bo c ti sinh thnh cy s th hin thay i mc c th. Thm ch c nhng qun th t bo pht trin t mt t bo ban u nhng trong sut qu trnh sinh trng v pht trin n khi hnh thnh mt c th hon chnh din ra nhiu thay i di truyn do nh hng ca cc yu t mi trng nui cy, c bit l cc cht iu ho sinh trng. K thut chn dng t bo i vi thc vt bc cao nhm mc ch: (i) Chn dng chng chu vi iu kin bt li ca ngoi cnh phi sinh hc nh: nng, lnh, phn, mn, hn, ng... (ii) Chn dng t bo khng cc c t do tc nhn sinh hc gy ra nh: c t do nm v vi khun gy bnh tit ra, cc loi khng sinh do vi sinh vt sn sinh ra... (iii) Chn dng sn xut d tha cc loi sn phm ch yu l amino acid nhm mc tiu xy dng qui trnh sn xut theo quy m cng nghip cc sn phm . (iiii) Chn cc dng mang nhng c im nh du nghin cu di truyn (genetic marker). T bo nui cy in vitro c t l bin d di truyn ln, v th c th chn c cc c th t bin nhanh hn v c hiu qu hn so vi cc phng php chn ging thng thng khc p dng trn cy nguyn vn [22]. Nu kt hp vi x l t bin th tn s t bin cng cao hn [35], [101]. K thut nui cy m v t bo cn cho php lm gim ng k thi gian cn thit chn c nhng tnh trng mong mun [1], [36]. Theo L Trn Bnh v tg (1998), k thut chn dng t bo soma chng chu vi cc yu t bt li ca mi trng bao gm cc bc: (1) To m so trn mi trng MS c bn, b sung 2,4-D. X l m so vi cc iu kin cc oan ca mi trng (nng, lnh, hn, nhm, mui...) ngng di mc gy cht v chn dng t bo m so chng chu c cc iu kin bt

li; (2) Chn lc cc dng m so sng st a ln mi trng ti sinh cy v to cy hon chnh; (3) Trng, theo di v phn tch di truyn cc dng chn lc c; (4) nh gi kh nng chng chu mc cy hon chnh, phn tch cc c im ha sinh v sinh hc phn t [1]. Nguyn liu dng cho chn dng t bo thc vt c th l m so, t bo nui cy dch lng hoc t bo trn. Trong , thng dng hn c trong chn dng t bo thc vt l m so. M so (callus) l khi m thc vt gm nhng t bo cha phn ho, c kh nng phn chia lin tc. Trong cng ngh t bo thc vt, nui cy m so l mt k thut c bn cho php bin i cc b phn khc nhau ca cy thnh m so v t ti sinh c cy hon chnh. Trong nui cy in vitro, m so c to ra bng cch nui cy cc c quan ca thc vt (l, hoa, qu, thn...) mi trng v iu kin nui cy thch hp. S dng m so lm nguyn liu chn dng t bo thc vt c u im l n gin v thng c tnh bin ng di truyn cao. M so c s dng chn cc dng chng chu vi tc nhn ngoi cnh phi sinh hc, chu bnh v cc dng cho nng sut th cp cao. Nghin cu to m so v ti sinh cy t m l ca lc, McKently v tg (2000) nhn thy cy hon chnh trng trong nh li c kh nng sinh trng, pht trin bnh thng, khng c s bin i v kiu hnh [95]. T nhng mnh l mm trn mi trng thch hp, Nguyn Xun Ti v tg (2005) to mm a chi v mt s dng cy xanh c ngun gc t ging lc L14 sinh trng c khi nui cy nh li. Qun th R0 trng trong nh li c s xut hin bin d v hnh thi t dng ng sang dng trung gian (khong 35%). Thi gian xut hin hoa u tin ca cy ti sinh mun hn ging gc khong 20-30 ngy. Phn tch a hnh DNA ca qun th cy ti sinh th h R0 bng ch th SSR cc tc gi cng nhn c s khc bit

mc DNA t 8% n 35% so vi ging gc [22]. M so lc c ngun gc t thn mm chu nh hng ca tia gamma c kh nng ti sinh cao hn. Nghin cu cng thu c mt s cy ti sinh khng bnh thng, c bit l hnh dng l [101]. 1.2.2. Pht sinh bin d trong qu trnh nui cy iu kin nui cy m, t bo lm pht sinh bin d vi tn s xut hin khong 10-5- 10-10, v trong mi trng nui cy c cc cht kch thch sinh trng vi nng cao. C th kt hp vi x l t bin hoc x l stress tng tn s t bin t 10-100 ln [87]. Do vy, vic phi hp gy t bin cng vi qu trnh nui cy m t bo s gp phn chn c th t bin nhanh hn v hiu qu hn so vi nhng phng php chn ging thng thng khc p dng trn cy nguyn vn [58]. Tc nhn t bin c th s dng trong qu trnh nui cy m gm nhiu loi, l cc tc nhn ho hc gy t bin nh ethylen imine, nitroso methyl urea Cc tc nhn vt l gy t bin nh: nhit , tia bc x khng gy ion ho (tia UV), mc nng lng ca tia UV ch c th dn n nhng t bin im hay t bin cu trc nhim sc th. Tia bc x gy ion ho (tia X, tia gamma), trong sinh hc thng dng tia gamma t ngun phng x Co60 gy t bin v c bc sng ngn (1,25 MeV) nn xuyn su cao hn, y cng l loi tia c dng nhiu gy t bin v ci to ging cy trng [6], [29], [36], [112]. Tc nhn lm pht sinh t bin tc ng khng ging nhau vo cc loi sinh vt khc nhau, khng ging nhau cc b phn c quan trong c th, trong thng thy s mn cm ca ht ny mm hn so vi ht kh; cc c quan sinh dng nhy cm hn ht ng nghV vy, thu c tn s t bin cao, vi mi loi cy trng cn phi chiu x liu lng ln gy

ra nhng bin d di truyn c li m khng gy cht cng nh tng bt th, lm nh hng n cng tc chn to ging sau ny. xc nh hiu qu tc ng ca chiu x, ngi ta thng xc nh liu lng ti hn, tc l liu lng cn 30-40 % s cy sng st sau khi chiu x. Tuy nhin, ngi ta thng dng liu chiu x nh hn liu ti hn t 1,5 2,0 ln, tt nht l dng nhng liu lm gim t l ny mm v t km hm sinh trng [21]. Xc nh liu chiu x gy nh hng n sinh vt, cc n v l rad v rnghen thng c dng tng ng nhau v c tnh l lng phng x lm cho 1g m sinh vt sng b chiu x hp th nng lng; ngoi ra c n v gray v 1kilorad. Nui cy m pht sinh bin d, kt hp nui cy m vi cc tc nhn t bin gia tng th t bin. Tuy nhin, khi nim t bin c li l nhng du hiu do ngi nghin cu t quy c, bi vy tt c nhng du hiu t bin c chn nghin cu nh: thp cy, nhiu nhnh, chn sm, nng sut cao, khi lng qu, ht lnu xp vo cc t bin c li [6]. t bin lm xut hin nhng tnh trng mi, cc tnh trng mi di truyn cho th h sau thng qua sinh sn hu tnh s chu s phn ly di truyn theo quy lut t nhin [80], [88] 1.2.3. Cc phng php chn dng t bo Chn dng t bo c th thc hin theo nhiu cch: trc tip, gin tip v chn tng th. Chn dng trc tip thng qua u th v sinh trng hay s khc bit v mu sc c th chn c dng t bo mong mun t qun th t bo. iu kin chn lc y l cc cht chn lc nhng nng khc nhau gy tc ng trc tip ln sinh trng ca t bo. Phng php chn trc tip thng c ng dng chn dng chng chu v nhng dng cho sn phm th cp

cao. Tc nhn chn lc thng c s dng phi k n polyethylene glycol (PEG). Cc cng trnh nghin cu s dng PEG ca Matheka v tg (2008) v chn dng m so ng chu hn [94], chn dng la chu hn ca L Th Bch Thy (2008) [34] c cng b. Trong trng hp chn dng gin tip, c im ca dng c chn l kt qu biu hin khuyt tt ca t bo. Trn mi trng cha ClO3 nhng t bo c nitrat reductase s dng ClO3 nh NO3 v kh thnh clorit. Clorit tc dng nh mt c t, cho nn ch nhng t bo khng c nitrat reductase mi sng st [100]. Chn lc tng th cc t bo d dng thc vt thng c chn bng phng thc x l t bin v nui trn mi trng c cha yu t dinh dng cn thit, m yu t ny c khi li chnh l nhn t gy t bin. 1.2.4. Mt s thnh tu trong chn dng chng chu yu t bt li ca ngoi cnh i vi cc nh trng trt cng nh vi chin lc pht trin cy trng, vic chn v to ra ging cy c kh nng chng chu nhng tc ng ngoi cnh phi sinh hc v tc ng ca cc cht ho hc l mc tiu ca nhiu chng trnh nghin cu. Nhng ging c chn lc ngoi cc ch tiu v nng sut v phm cht cn cn phi c kh nng thch nghi vi nhng iu kin ht sc a dng pht trin tt nhiu vng sinh thi khc nhau. Yu t bt li ca mi trng gm nhiu loi nh phn, chua, mn, hn, nng, lnh... Nui cy m v t bo c th ng gp mt phn quan trng trong vic chn to dng chng chu cc yu t bt li . Bng cng ngh t bo thc vt, cc nh khoa hc nhn c nhng dng chu mui NaCl t m so la [37], thuc l [13], la m [117]. Kt qu nghin cu ca Singh v tg (2004) cho thy, ABA l cht c lin quan n

vic tng hp cc protein mi (c bit cc protein c khi lng phn t 26 kDa) xut hin cc dng chu mui [117]. Mi tng quan gia ABA, tnh chu mui v s tng hp cc protein c bit l vn ang cn tip tc nghin cu. Vn chn dng chu hn, chu nhit thp c thng bo l rt thnh cng trn cy la [1]. Vic tin hnh chn lc cc dng la chu nng, chu phn cng bc u c nghin cu bi Nguyn Th Tm v tg (1999) [28], Nguyn Th Vinh v tg (1995) [38]. lm tng tn s t bin ca cc cy trng, nghin cu phi hp tc nhn t bin vo qu trnh nui cy in vitro c cng b. L Xun c v tg (2006) to c dng la TB06 thp cy, ngn ngy t ging la Tm xoan. Dng TB06 c c kho nghim trong h thng kho nghim Quc gia v c nh gi l dng c trin vng [6]. Theo hng ci thin tnh chu hn, L Th Bch Thu v tg (2008) x l m so la bng tia gamma trc khi ti sinh cy, sau thu ht ca cc dng cy ti sinh v sng lc bng PEG cng vi ch th phn t SSR v STS lin quan n hn. Nhm tc gi thu c ba dng trin vng ngh tip tc nh gi v gii thiu kho nghim ging chu hn [34]. Nh vy, vic nghin cu xy dng v hon thin cc h thng nui cy m t bo thc vt nhm nng cao kh nng chng chu vi cc iu kin bt li ca mi trng c nhiu nh khoa hc quan tm ch . Trong , nhiu dng cy xanh to ra c kh nng sinh trng pht trin bnh thng nh cy la, cy lc... Chn dng theo hng chng chu bt li mi trng rt thnh cng trn mt s i tng nh la, thuc l...Nhng thnh tu thu

c l gi chng ti la chn nghin cu nng cao kh nng chng chu hn trn cy lc. 1.3. PHN TCH, NH GI CC DNG CHN LC C NGUN GC T NUI CY M SO 1.3.1. nh gi kh nng chu mt nc ca m, t bo thc vt X l gy mt nc ca m hay t bo thc vt thng s dng lung kh v trng ca box cy hoc dng cc cht lm thay i p sut thm thu ca t bo. nh gi kh nng chu mt nc ca m so thng qua vic xc nh mt nc v kh nng chu mt nc. Kt qu nh gi cho php xc nh ngng chn lc dng t bo, y l hng kh hiu qu trong nghin cu ng dng cng ngh nui cy m t bo thc vt nng cao kh nng chu hn ca cy trng. Matheka v tg (2008) dng PEG 6000 nng 20% x l m so ng, sau chn dng t bo sng st trn mi trng cha manitol [96]. Vi mc tiu to vt liu khi u phc v chn dng chu hn cy u xanh, Chu Hong Mu v tg (2010) to c m so t phi mm ca ht u xanh trn mi trng MS c bn c b sung 2,4-D 10mg/l. Sau 10 ngy nui cy, cc m so c thi kh v nui phc hi. nh gi t l m sng st, m sng st ti sinh cy, nhm tc gi xc nh c ngng x l thi kh chn dng chu hn ca hai ging u xanh khc nhau l 5 gi (VN6) v 7 gi (VN5) [15]. Nghin cu kh nng chu mt nc mc m so ca lc ch yu mi dng li vic nh gi mt nc, t l sng st v kh nng ti sinh cy cc ngng x l khc nhau. Vi mt nc t 80,40% n 86,49% m so ca nhiu ging lc c nh gi l c kh nng ti sinh. Tuy nhin, kh nng ti sinh khc nhau ty thuc vo ging v thi gian chu thi kh

[25]. Do vy, cc cy ti sinh c to thnh dng hon chnh s l ngun vt liu khi u c gi tr trong chn dng. 1.3.2. nh gi kh nng chu hn giai on ht ny mm v giai on cy non Trn c s ngun vt liu khi u c, nghin cu nh gi cc dng chn lc chnh l qu trnh phn tch, tm ti nhm chn ra cc dng mang tnh trng mong mun bi dng to ging mi. Qu trnh nh gi cc dng chn lc ging nh qu trnh chn ging trn cy nguyn vn. C nhiu cch tip cn gii quyt vn ny, tuy nhin m hnh chung l t kt qu th nghim, nhn xt hin tng v rt ra kt lun mang tnh quy lut. Tnh a dng di truyn ca sinh vt biu hin s a dng cc tnh trng v hnh thi, v cc c im sinh l, ho sinh; v kh nng chng chu cc iu kin bt li ca ngoi cnh, v s bin ng hm lng cc cht trong t bo Khi nghin cu chn ging cy trng ngi ta thng quan tm n cc tnh trng c th cn, ong, o, m c (cc tnh trng s lng). Nghin cu cc tnh trng s lng cho php xc nh mi lin quan n nng sut sinh vt, n cht lng sn phm. S di truyn cc tnh trng s lng tun th cc nguyn tc c bn ca quy lut di truyn [16]. Hai giai on sinh trng ca thc vt c cho l mn cm vi hn l giai on ht ny mm v giai on cy non. Nhng giai on ny thng c s dng nh gi kh nng chng chu vi yu t bt li ca mi trng. Kh nng chu hn giai on ht ny mm ca cy c nh gi ch yu trn c s s bin ng ca cc ch tiu ha sinh. Trong giai on ht ny mm, nhiu enzyme thy phn c tng hp mnh phn gii cht d tr cung cp dinh dng cho cy. Hot ca mt s enzyme tng l minh chng cho tnh chng chu ca cy trng c ngun gc t m so b x l c ci thin. Nghin cu ca Nguyn Th Tm v L Trn Bnh (2003) nhn thy, c

mi tng quan thun cht ch v s bin ng ca hm lng ng tan v hot ca -amylase cc dng la chn lc t m so chu nng [27]. Trong , tng p sut thm thu ca t bo thng qua cc phn t ng tan s lm tng kh nng chng chu ca cy. giai on cy non, kh nng chu hn c nh gi thng qua xc nh cc ch tiu sinh l, ha sinh. n giai on cy non c im hnh thi ca cy c th quan st c. S bin ng hm lng cc cht vn din ra bnh thng, theo mt quy lut nht nh. Quan st s pht trin cc c im quan trng cy khoai ty nh: chiu di v trng lng r, chiu di v trng lng chi, tc pht trin ca cy con iu kin gy hn bng PEG 6000 trong ng nghim v cy con chn lc trng ngoi ng rung, kt qu nghin cu khng nh, c th la chn kh nng khng hn ca cc dng khoai ty chn lc cc mc thiu ht nc khc nhau thng qua quan st kiu hnh [83]. Nghin cu ca Nguyn Th Tm v tg (2009) v vic nh gi c im ha sinh ht ca cc dng la c ngun gc t m so chu mt nc th h R4 so vi ging gc, cc ch tiu phn tch gm protein, lipid, ng u c s gia tng trong ht, kt qu nghin cu khng nh cc dng chn lc c ci thin v thnh phn ha sinh ht [23]. Trng Th Bch Phng v tg (2005) tin hnh phn tch thnh phn protein trong ht ca 3 dng la chn lc c ngun gc t m so b x l ABA trn mi trng b sung 15% manitol, nhm tc gi nhn thy tt c cc dng chn lc u tng v s lng hoc hm lng tiu phn protein so vi ging gc [19]. Phn tch a hnh protein bng k thut in di hai chiu ca Hong Tn Qung v tg (2008) cng nhn c kt qu tng t [20]. S a hnh protein trong ht l do biu hin ca gen lin quan n hn trong cc dng chn lc in vitro c hot ha khi gp iu kin bt li mi trng lm xut hin protein mi. Rt c th

l kt qu ca gen tng hp protein d tr c bin i nh qu trnh nui cy v chn lc ng bin d soma. Nhng thay i lm xut hin nhng tnh trng mi l c s chn to dng mang cc tnh trng mong mun. Phn tch s bin ng hm lng cc cht trong iu kin stress khc nhau chn lc dng mang tnh trng mong mun. Cc dng chn lc to ra bng k thut nui cy m t bo thc vt c s tng hm lng proline c xc nh l ch th lin quan n kh nng chu hn ca la. V vy, c th cn c vo hm lng proline trong cy la chn dng c kh nng chu hn [34]. 1.3.3. K thut RAPD trong nh gi h gen ca cc dng chn lc K thut RAPD c William v tg (1990) s dng pht hin hin tng a hnh DNA nh s nhn bn ngu nhin ca cc mi c trnh t ngn, di khong 9-12 nucleotide, c nhit gn mi thp (350C -370C). Sn phm ca phn ng c phn tch bng phng php in di trn gel agarose, nhum gel bng ethydium bromide, sau quan st v phn tch kt qu nh nh sng ca tia cc tm (UV-ultraviolet) [129]. Phn tch tnh a hnh ca DNA da trn s xut hin hay khng xut hin cc phn on c cng kch thc c khuch i t h gen nh s thay i cc im gn ca mi... S dng k thut RAPD phn tch h gen c c im l n gin, nhanh chng, khng cn bit nhng thng tin v trnh t. V vy, nhiu nghin cu cho thy s dng RAPD kh hiu qu trong phn tch qun th, xc lp ngun gc loi, lp bn di truyn v c bit so snh h gen ca cc dng chn lc thu c lm c s la chn dng trin vng [54], [55], [72], [107]. Nguyn c Hong v tg (2008) s dng k thut RAPD vi 10 mi ngu nhin nhn thy h gen ca cc dng la chn lc th h R3 c ngun

gc t m so chu mt nc c s thay i so vi ging gc, mc khc bit di truyn l 10% [9]. Vi cc dng lc chn lc, kt qu phn tch RAPD cng thu c s khc bit ca dng chn lc vi ging gc trong khong 3,18- 20,55% [24]. Cc kt qu nghin cu u chng t c s bin i vt cht di truyn trong qu trnh nui cy in vitro ca cc dng chn lc so vi ging gc . Ch th RAPD c nh gi l c gi tr trong vic pht hin t bin khi nui cy chi cy u tng, mc d kt qu nghin cu thu c s mi c t l a hnh khng cao song tc gi cng khng nh y l cng c hu ch trong vic pht hin cc t bin khi nui cy phi bng in vitro trn cy u tng [73]. Mc xut hin khng ging nhau v t l cc bng a hnh, v mc bin d trong h gen bng k thut RAPD, cc kt qu nghin cu trong chn dng u khng nh s sai khc trong phn tch l cn c to ging mi. Tuy nhin, nghin cu ca L Th Bch Thy v tg (2008) khng nh, khng phi tt c cc dng chu chiu x u to ra s khc bit trong h gen, t kt qu phn tch RAPD tc gi vn nhn thy nhiu dng la ging nhau hon ton, ging vi i chng v ging vi m khng chiu x. S phn ly do t bin la khng theo mt quy lut nht nh [34]. Bn cnh vic xc nh s thay i trong h gen, mt s nghin cu cng ch ra vic la chn k thut RAPD cng rt hiu qu vi vic lin kt vi cc gen c gi tr ca qu trnh chn ging. Ch th RAPD c cng b l c lin quan n gen khng bnh ph nm mc trng u, cy mang gen khng bnh c chn lc th h F2 cho kt qu a hnh gia cy b v cy m [62]. Kt qu phn tch v khng nh, ch th RAPD lin kt vi gen kim sot tnh khng c cy ngh [121]. Trn cy lc, nghin cu ca Mondal v tg (2007) cho bit ch th RAPD c mi lin kt vi gen khng bnh g st

[99] Tuy nhin, vic tm ch th lin quan n tnh chu hn ca thc vt ni chung v cy lc ni ring cn l vn cn c tho lun. 1.4. GEN LIN QUAN N TNH CHU HN CY LC Tnh chu hn ca thc vt l tnh trng do nhiu gen quyt nh. Xu hng tm kim gen chu hn l quan tm nghin cu ca nhiu tc gi. Tuy nhin, n nay vn cha tm thy gen c hiu no quyt nh tnh chu hn ca thc vt m ch c cc gen lin quan n kh nng chu hn. C th chia cc gen lin quan n tnh chu hn lm hai nhm: Cc gen lin quan n kh nng chu hn ca thc vt v nhm gen m ha protein iu khin hot ng phin m ca cc gen lin quan n tnh chu hn. 1.4.1. Cc gen lin quan n kh nng chu hn ca cy lc Cc gen lin quan n kh nng chu hn ca thc vt c th chia thnh cc nhm: (i) Cc gen tham gia vo qu trnh bo v thnh t bo; (ii) Cc gen tham gia vo qu trnh tng hp cc cht iu ha p sut thm thu v (iii) Cc gen lin quan n s pht trin b r. Gen LEA l mt trong nhng nhm gen lin quan ti s mt nc ca thc vt. Bnh thng, sn phm ca gen LEA c to ra vi lng ln trong giai on mun ca qu trnh hnh thnh phi. Khi t bo b mt nc protein LEA c tng hp nhiu hn, mc phin m ca LEA c iu khin bi ABA. Biu hin mnh ca gen LEA s lm gim mt nc v tng p sut thm thu trong t bo [51], [67]. Nghin cu ca Su v tg (2010) trn ging lc Luhua14 pht hin, c t nht 8 loi gen LEA. Cc gen LEA ca lc k hiu AhLEA bao gm AhLEA8-1, AhLEA7-1, AhLEA6-2, AhLEA6-1, AhLEA4-2, AhLEA4-1, AhLEA1-3, AhLEA1-1. Tm gen AhLEA c kch thc dao ng trong khong 299-575bp [122].

Nhm gen P5CS m ha tng hp pyrroline 5 carboxylate synthetase. Enzyme P5CS tham gia vo qu trnh tng hp proline, biu hin ca gen P5CS mnh s lm tng hm lng proline, c vai tr tng p sut thm thu ca t bo v cy trong iu kin hn hn.... Gen NCED m ha tng hp 9-cis epoxycarotenoid dioxygenase. Theo nghin cu ca Guerrini v tg (2005), s bt li ca iu kin v hn, mui lm cho gen iu khin tng hp 9-cis epoxycarotenoid dioxygenase biu hin mnh hn. Hot ng ca 9-cis epoxycarotenoid dioxygenase s bin i neoxathin thnh xathoxin, y chnh l giai on trc khi hnh thnh ABA. Do , hot ng ca NCED c xc nhn c mi lin quan vi kh nng chng chu ca thc vt [69]. Gen AhNCED ca lc c phn lp bi Wan v tg (2005) c kch thc 2486bp, m ha mt protein gm 610 amino acid, vi trng lng phn t l 66,86 kDa [128]. Gen PLD m ha phospholipase D, hot ng ca phospholipase D mnh s to mt phn t phospholipid v mt gc cha nhm hydroxyl t do, hot ng ca cc PLD c khng nh c lin quan n hn [59], [74], [126]. Tch dng v xc nh trnh t gen PLD ht lc, nhm nghin cu ca Nakazawa (2006) pht hin trong ht lc c hai gen PLD, k hiu l AhPLD1 v AhPLD2. Trong , AhPLD1 m ha phospholipase D1 gm 807 amino acid, v AhPLD2 m ha phospholipase D2 gm 794 amino acid [104]. C cht ca cc phospholipase l phosphatidylcholine, hot ng ti thch vi mi trng c pH=5. Trong iu kin b hn, cDNA m ha phospholipase D ca lc c phn lp c kch thc 556bp [59]. 1.4.2. Nhm gen m ha protein iu khin hot ng phin m ca cc gen chu hn

Cc gen m ha protein iu khin phin m c kh nng hot ha hoc c ch biu hin ca cc gen lin quan n tnh chu hn thng qua vic bm vo trt t DNA iu khin (cis acting element) trn vng khi ng gen (promoter) v tng tc vi RNA polymerase to thnh phc hp khi u qu trnh phin m cc gen lin quan n tnh chu hn. Theo cc nghin cu v nhn t phin m ca mt s tc gi c cng b trn Ngn hng gen quc t, c nhiu yu t khi u phin m iu khin tnh chu hn ca thc vt nh: DREB, bZIP, MYB.[79], [113], [116]. Gen DREB (dehydration responsive elemen binding) c tch ra t l, hoa, thn v r ca lc u c kch thc 687bp m ha phn t protein gm 228 amino acid [86]. Gen cystatin thc vt m ha protein cystatin c ch hot ng ca cysteine proteinase. Trong nhng nm gn y, cystatin c bn lun nhiu bi mi lin quan ca n vi tnh chng chu yu t bt li ca ngoi cnh, bao gm cc yu t v hn, lnh, mn ... [93], [125], [132]. 1.5. CYSTATIN V VAI TR CA CYSTATIN THC VT Cystatin (CYS) l mt dng protein c ch hot ng ca cysteine proteinase (cysteine proteinase inhibitor-CPI). Cystatin phn b rng ri ng vt, thc vt v vi sinh vt cho thy y l mt nhm cht ng vai tr quan trng vi s tn ti, sinh trng v pht trin ca sinh vt [106]. Trong nhng nm gn y, nhiu cng trnh nghin cu cng b v gen cystatin ch ra rng, cystatin l sn phm ca mt h gen, tuy nhin, nhng hiu bit v gen cystatin thc vt cn nhiu hn ch [46], [89], [92]. 1.5.1. c im cu to v phn loi cystatin Phn tch cystatin cc gii sinh vt, kt qu nghin cu cho thy cc loi protein cystatin khc nhau c mi quan h vi nhau v cu trc bc 1, c

bit l nhng trnh t amino acid c trng trong vic c ch hot ng ca cysteine proteinase. S sai khc v kch thc, khi lng ca phn t protein, v s lng v v tr cc lin kt disulphide l cn c phn loi cystatin thnh bn h: stefin, cystatin, kininogen v phytocystatin. Stefin Stefin l h CYS th nht, gm nhng protein c khi lng phn t khong 11 kDa. Chui polypeptid cha khong 100 amino acid, khng c lin kt disulphide v cc nhm cacbonhydrat lin kt trong thnh phn cu to. Vng hot ng ca stefin l v tr G gn u NH2 v 5 amino acid trong trung tm phn ng [115]. Cystatin Cystatin l h th hai, c khong 120-126 amino acid, khi lng khong 13,4-14,4 kDa. H cystatin th 2 c 2-3 lin kt disulphide v a s l khng glycosyl ho. Tt c cystatin h 2 c trnh t tripeptide bo th l phenylalanine- alanine-valine (FAV) gn u NH2 v 1 dipeptide bo th gn u COOH l phenylalanine- tyrosine (PW). Cc trnh t bo th c ghi nhn l rt quan trng trong vic kt hp vi enzyme ch [124]. Kininogen Kininogen l h CYS th ba, bao gm nhng glycoprotein c kch thc ln, khi lng ln. Khi lng ca kininogen khong 60-120 kDa. Cc kininogen ng mt vai tr quan trng trong qu trnh ng mu [103]. a s cystatin ca h th nht v h th ba ging nhau trnh t pentapeptide trong trung tm phn ng, vng bo th l glutamine-valinevaline-alanine-glycine (QVVAG). Cc thnh vin ca h th 2 c s thay th amino acid v tr th 2 v th 4 ca chui, v vy c trnh t pentapeptide

tng ng l QxVxG (trong x, c thay th bng mt amino acid bt k) [45]. Phytocystatin Phytocystatin (phycys) l h CYS th t. Nhm phytocystatin l cc cystatin thc vt, c to thnh mt phn h c lp trn cy pht sinh loi ca siu h CYS [90]. im chung ca cc phycys l tt c u cha thm so vi cystatin ca cc sinh vt khc mt trnh t vi nhng amino acid bo th [LVI]- [AGT]- [RKE]- [FY]- [AS] [VI] vng xon anpha u NH2 ca chui polypeptide. Trong , cc v tr trong ngoc ([ ]) c th thay th cho nhau [125]. Cht gy c ch cysteine proteinase ca thc vt c phn lp t nhiu loi, kt qu thu c cho thy chng c kch thc khc nhau, thng dao ng t 12 kDa n 85 kDa. Cystatin thc vt c th chia thnh hai nhm da theo cu trc phn t protein v trng lng ca chng. Nhm th nht c to thnh bi cc phytocystatin cha mt min CYS, c kch thc 12-16 kDa, khng c cu disulphide v vng glycosilation gi nh, gen m ho chng khng c intron hoc c 1 intron on gia LARFAV v QxVxG. Phytocystatin nhm I c nghin cu k v sm nht l cystatin la, k hiu OCI [40].
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Hnh 1.1. Trnh t 102 amino acid ca OCI (m s trn GenBank: AAA33903) [143]

Oryzacystatin I tng ng vi cystatin lng trng trng, kt qu phn tch cho thy OCI thiu cu ni disulphide, khi lng vo khong 11,5 kDa vi 102 amino acid. Vng CYS t v tr 20 n 95, trong vng bo th ca OCI (m t trn hnh 1.1 l cc v tr in m) gm glycine v tr s 5 (G5), nm peptide (pentapeptide) vng trung tm hot ng ko di t v tr 53 n 57 l glutamine53-valine54-valine55-alanine56-glycine57 v proline83tryptophan84 (PW). Trnh t c trng ca h (LARFAV) ko di t v tr 26 n 31. Nhm phytocystatin I cng l nhm ch yu ca thc vt [40]. Nhm phytocystatin th hai s hu nhiu min cystatin (multicystatin), vi nhng vng bo th lp i lp li. Khi lng phn t ca phytocystatin nhm 2 ln, dao ng trong khong 80-120 kDa. Gen m ho chng c 3 intron, chui polipeptide c mt phn m rng u COOH [89], [92]. Phn tch PMC (Potato MultiCystatin) l cystatin a min c phn lp t cung c khoai ty cho thy, khi lng ca PMC khong 85 kDa [64]. PMC c 8 vng ca cystatin, cc vng gn ng thi vi nhiu cysteine proteinase [105]. Kt qu phn tch v cu trc ca cystatin thc vt cho thy, y thc s l nhm cht phc tp, cn thit c nhng nghin cu su v khuyn co c ci nhn su sc v vai tr ca cystatin trong c th sng. 1.5.2. Cu trc khng gian v c ch c ch ca cystatin 1.5.2.1. Cu trc khng gian ca cystatin Cu trc khng gian c bn ca cystatin thc vt bao gm 4 n 5 phin gp np nm song song v ngc chiu nhau. Cc phin lin kt vi xon vng u NH2. Cystatin thc vt m t trn hnh 1.2 cho thy, v tr c bn ca cc vng bo th v hot ng gm: (i) Mt on th ra gn u NH2, cha

trnh t G (ii) vng xon cha trnh t c trng cho cystatin thc vt l LARFAV (iii) hai vng kp tc gn vo trung tm hot ng ca cysteine proteinase, trong : vng gn u NH2 cha trnh t bo th QxVxG, vng th 2 gn u COOH cha PW.
Vng xon cha LARFAV u COOH
u NH2 Vng kp u tin (QxVxG)
Vng kp th hai (PW)
Hnh 1.2. Cu trc khng gian ba chiu ca cystatin thc vt (theo Girard v tg, 2007) [66]
1.5.2.2. Cysteine proteinase i tng c ch ca cystatin l cysteine proteinase. y l nhm enzyme thuc phn lp th 3, theo cch phn loi ca hip hi Ho sinh quc t (thng k hiu: EC 3.4.22-). Cysteine proteinase xc tc phn ct chui peptide v tr bn trong mch polypeptide (endopeptidyl hydrolase). Trung tm hot ng ca enzyme ny c cha nhm sunfuhydryl (hay cn gi l

thiol, nhm SH), y l nhm c kh nng phn ng cao. Nhm sunfuhydryl tham gia vo nhiu hot ng bin i ha hc. V d nh, nhm SH tham gia vo s ion ha, acyl ha, phosphoryl ha, oxy ha, ankyl ha... v ngi ta cng thy rng, hng lot cc enzyme b c ch khi nhm SH chc nng ca chng b kha. Qu trnh nghin cu khng r bn cht ca c cht [45]. Qu trnh tng hp cysteine proteinase xy ra polysome trn li ni cht (RER- rough endoplasmic reticulum), sau peptide tn hiu b loi b, nhng enzyme cha c hot tnh. Cysteine proteinase tip tc c vn chuyn n khng bo (vacuole) d tr, sau loi b trnh t tn hiu u NH2, mi tr thnh enzyme c hot tnh [102]. V vy, qu trnh tng hp cysteine proteinase thnh dng hot ng l mt qu trnh phc tp. Trong cc loi cysteine proteinase th h papain c nghin cu k nht. Theo Fischer v tg (2000), papain c sinh ra dng cha hot ng hoc t hot ng (zymogen), c kch thc t 40-50 kDa. Qu trnh bin i phn t sau dch m lm cho cc phn t cysteine proteinase mi sinh b ct hn ch khong 3 n 4 ln mi tr thnh cysteine proteinase hot ng. Kch thc ca cysteine proteinase trng thnh khong 22-35kDa. a s cc cysteine proteinase c pH cc thun vng axit [61]. Trong c th sng, cysteine proteinase thc hin nhiu chc nng quan trng nh: trng thnh protein, lm mi protein ph hp vi nhng thay i bn ngoi v loi b protein bt thng, protein nh gp cun [68]. Trong cc cy h u, cysteine proteinase dng papain hin din nh proteinase ch yu ca qu trnh thy phn protein d tr. a s cystatin thc vt c ch hai loi cysteine proteinase thuc nhm papain v legumain. Nhm papain (nhm C1) b c ch bi cc phn t cystatin c khi lng phn t 12-16kDa, nhm legumain (nhm C13) b c

ch bi cystatin 23 kDa [92]. Gy hn giai on cy la m non vi mc thm ht nc khong 60%, kt qu phn tch trong iu kin hn hn, hot ng ca cysteine proteinase gim biu hin cc ging c kh nng chu hn, tuy nhin n li khng thay i hoc l tng cc ging chu hn km. Kt qu nghin cu ch ra rng hot ng phn gii protein thp hn v gim s biu hin ca cysteine proteinase trong giai on u thiu ht nc c th coi l mt ch s nh gi kh nng chu hn ca la m [118]. 1.5.2.3. C ch c ch ca cystatin Kt qu nghin cu trong bo co ca Merops (2010), Rawlings v tg (2008) ch ra rng, cystatin ng vai tr nh c cht xm nhp vo trung tm hot ng ca enzyme v ngn chn vic i vo ca cc c cht protein khc [97], [109]. Cc thnh phn trong cu trc khng gian ba chiu hnh thnh nn dng hnh chc ba c cm vo trong trung tm hot ng ca enzyme ch gm 2 vng lp (k hiu l L1, L2) v 1 vng u NH2.
Hnh 1.3. M hnh cu trc c ch hot ng ca oryzacystatin

Theo Nakazawa v tg (2000), m hnh cu trc c ch hot ng ca oryzacystatin c m t (hnh 1.3) gm: pha trc (hnh A) v pha sau (hnh B) oryzacystatin tng tc vi enzyme papain (mu xm trng). Cc amino acid ca cystatin tng tc vi papain l: vng u NH2 t glycine v tr s 5 ti glycine v tr s 11, glutamine v tr s 53 ti tyrosine v tr s 60 cho vng c ch u tin v tryptophan v tr s 80 n glutamine v tr s 91 cho vng c ch th hai (mu vng) [104]. V tr cc vng bo th ca cc cystatin khc nhau l khc nhau, tuy nhin thng gp trong cc cystatin l: v tr glycine- vng u NH2, vng lp th nht l on bo th QxVxG, nm trong vng trung tm ca cystatin v vng lp th 2 l v tr W (tryptophan) vng u COOH. Phn protein cn li khng xm nhp vo khe hot ha, nhng li ng gp rt ng k ti qu trnh lin kt v nh hng mnh ti tnh c hiu ca cystatin vi cysteine proteinase [92]. 1.5.3. Chc nng ca cystatin Kt qu d on v phn ng ca t bo khi b thiu ht nc, Bray (1993) cp sn phm ca cc gen tham gia duy tr chc nng t bo trong sut giai on mt nc, l nhm cht c ch hot ng ca protease (protease inhibitor) [51]. Khi gen ny hot ng, sn phm to thnh s tham gia vo bo v cc enzyme, hn ch s phn gii cc cht do bo v cc cu trc trong t bo. Nghin cu ca chng ti v cystatin gi rng, phi chng enzyme b c ch l cysteine proteinase v cht c ch ca n chnh l cystatin. Diop v tg (2004) phn lp trnh t gen VuC1 t mRNA (cDNA) m ho cystatin t l cy u c ve (Vigna unguiculata (L.) Walp.). Cc protein VuC1 ti t hp (rVuC1) c biu hin t cDNA l mt cht c ch

hiu qu hot ng ca papain. S biu hin ca gen VuC1 trong t bo phn ng vi tc ng ca cc yu t bt li phi sinh hc (hn hn, ABA ngoi sinh) c phn tch bi phng php Northern blot v Western blot. Kt qu cho thy, trong cc m l ca cy u c ve thuc hai ging khc nhau v kh nng chu hn, cc yu t bt li phi sinh hc to ra s tng tch t cc polypeptide VuC1. S khc nhau v mt tch t cystatin tng ng vi mc chu ng ca cy u c ve [57]. Vic nghin cu gen cystatin khng nhng gii quyt mi lin quan ca gen vi tnh chng chu hn m cn lin quan n kh nng chng vi sinh vt, chng cn trng gy bnh i vi cy trng [57], [70], [125], [132]. 1.5.4. Gen m ha cystatin 1.5.4.1. Gen m ha cystatin thc vt Gen m ho cystatin thc vt c phn lp u tin trn cy la, gi l oryzacystatin I, k hiu l OCI. Kt qu tch chit OCI t mRNA ca la c on m ha di 309 bp, m ha phn t protein di 102 amino acid [40]. Gen cystatin th 2 phn lp t h gen la l oryzacystatin II do Kondo v tg (1991). OCII l mt phn t c kch thc khong gn 2,5 kb, cha 1534 bp. OCII c 3 exon v 2 intron, on m ha di 321 nucleotide m ha phn t cystatin gm 106 amino acid (M s: X57658) [140]. Kt qu nghin cu ng cho thy, c 10 gen cystatin ca ng, cc gen k hiu l CC, k hiu cc gen theo th t t 1 n 10, tng ng l CC1, CC2, CC3, CC4, CC5, CC6, CC7, CC8, CC9 v CC10 [93]. Trong 10 gen ng, c 9 gen c phn lp t mRNA, ch c 1 gen CCI phn lp t DNA. Kch thc gen CCI cha 2024bp, trong c 3 exon v 2 intron, 3 exon m ha phn t protein di 134 amino acid (M s: D63342) [141].

Trn cy u xanh, tc gi Chu Hong Mu v tg (2008) phn lp gen cystatin t DNA ca cc ging khc nhau, u nhn c gen c kch thc 1115 nucleotide, cha 2 exon v 1 intron. on m ha cystatin u xanh di 267 nucleotide m ha phn t protein di 88 amino acid. So snh trnh t gen ny vi cc trnh t gen cystatin c kh nng chu hn khc nhau trn ngn hng gen quc t, nhm tc gi khng nh khng c s sai khc v trnh t gen cystatin [17]. Cng thuc nhm cy u , gen cystatin cy u tng kh ln, kch thc m nhm tc gi Misaka (2000) phn lp cha ti 5863 nucleotide, trn c 4 exon v 3 intron [142]. So vi gen cystatin phn lp t DNA h gen, chng ti nhn thy gen cystatin phn lp t mRNA chim s lng ln. Cystatin trong h Hodeum vulgare do by gen m ho, cc gen c phn lp t mRNA k hiu t HvCPI1 n HvCPI7 [91]. Tuy nhin cn c vo trnh t protein suy din, Abraham v tg (2006) cho bit ch c 1 trong 7 trnh t cystatin l lin quan n cht c ch cysteine proteinase [41]. Trong ht la m c t nht 4 gen cystatin, gen cystatin ca la m k hiu l WC1, WC2, WC4, WC5. Cc gen phn lp t cDNA c tng ng vi nhau t 63% n 93%, trong WC1 v WC3 ging nhau 100% v trnh t amino acid [84]. 1.5.4.2. S biu hin ca gen cystatin S biu hin ca gen c ch protease thng b gii hn bi cc t chc c bit hoc khng ging nhau trong nhng giai on pht trin khc nhau ca cy. Minh chng trn mt s loi thc vt cho thy, s c mt ca cystatin cc thi k pht trin khc nhau ca cy. Biu hin ng trong giai

on ht ny mm [93], trong qu trnh rng l sm ca cy rau dn [125], ht kh, ht ny mm v trong hoa ca cy Arabidopsis [75]. Nghin cu ca Massonneau v tg (2005) nhn thy, trong 10 gen ng th, CC1 u tin biu hin trong c non, hai gen CC8, CC10 biu hin trong vic pht trin ht, 7 gen CC cn li biu hin trong 2 hoc nhiu m khc nhau. Ring trong iu kin chu nh hng ca s thiu ht nc c ti 5 gen cng biu hin, l cc gen CC2, CC3, CC4, CC v CC9 [93]. Vi bn gen CYS ca la m c ba gen WC1, WC2, WC4 biu hin mnh trong giai on u ca qu trnh trng thnh ht ging v qu trnh ht ny mm. Gen WC1 biu hin nhiu trong lp alleuron v ni nh. Gen WC4 biu hin lp alleuron v phi. Cn gen WC5 biu hin trong cc m mm giai on u cy ti sinh v trong lp alleuron ca ht trng thnh [53]. Trong iu kin bt li (nguyn nhn do nng, hn, mui...) s biu hin ca gen cystatin tng cao hn trong nhiu loi cy trng. Zhang v tg (2008) phn lp gen cystatin t l cy Arabidopsis thaliana nhn c hai loi, k hiu l AtCYSa v AtCYSb, trong iu kin mi trng bt li c hai gen AtCYSa v AtCYSb u biu hin nhiu hn, l l do nhm tc gi khuyn co c th s dng cystatin nng cao kh nng chng chu ca thc vt vi mi trng bt li [132]. c tnh tng s biu hin ca gen cystatin trong iu kin bt li v mi trng cn c pht hin trn nhiu cy trng khc v t nhiu phn khc nhau ca cy, nhng kt qu thu c u chng t c mi lin quan ca cystatin vi kh nng chng chu hn ca thc vt (bng 1.2). Kt qu tm kim gen cystatin lin quan n kh nng chng chu hn thc vt trn bng 1.2 cho thy, ngun tch chit gen cystatin c th t nhiu

phn khc nhau ca cy. Cc phn thng s dng phn lp gen cystatin l l, r, thn. Trong , hai b phn l v r ca cy thng chu nh hng mnh bi cc yu t bt li ca mi trng [3], [4], [44], [119]. Bng 1.2. Gen cystatin lin quan vi kh nng chng chu hn mt s loi thc vt Tn loi thc vt Tn gen cystatin u cove (Vigna unguiculata L. Walp) Rau dn (Amaranthus hypohondiracus) Cy ci bin (Cakile maritima) La m ng (Triticum aestivum) TaMDC1 l, r CmC l AhCPI thn, r VuC1 Ngun tch chit l Tn tc gi, nm cng b Diop v tg, 2004 [57] Valdes v tg, 2007 [125] Megdiche v tg, 2009 [96] Christova v tg, 2006 [52]
So vi cc thc vt khc, nghin cu v cystatin v gen cystatin cy lc cn t c cng b. Kt qu nghin cu bi Yan v tg (2004) cho thy mRNA tch trong ht ca loi Arachis hypogeae l mt polynucleotide di 297 nucleotide, m ho chui polypeptide di 98 amino acid (AY722693), vng nghin cu c cho l c ch cysteine proteinase c trnh t QVVAG ko di t v tr 49 n 53 [134]. c im cu trc ny cng l kt qu nghin cu ca nhm tc gi Srinivansan v tg (2008) khi tch chit cystatin t loi Arachis diogoi (M s: EU723567) [133].

Nh vy, cystatin l protein c ch hot ng ca cysteine proteinase c tm thy trong nhiu loi sinh vt. thc vt, cystatin c gi l phytocystatin bi s c mt mt on trnh t bo th v c trng ca gii l LARFAV. Cc vng CYS c ch trung tm hot ng ca cysteine proteinase rt bo th v c xc nh trn nhiu loi thc vt. Biu hin ca cystatin nhiu giai on sinh trng- pht trin khc nhau. Chc nng ca cystatin c bn lun bi mi lin quan ca n vi tnh chng chu cc yu t bt li ca ngoi cnh, c bit l tnh chng chu hn... l cn c chng ti tip tc nghin cu, phn tch cystatin trn cy lc.

Chng 2
VT LIU V PHNG PHP NGHIN CU

2.1. VT LIU THC VT Vt liu nghin cu ca lun n l 10 ging lc c ngun gc khc nhau. Trong , 8 ging (L05, L16, L18, L23, L24, V79, MD7, MD9) c cung cp bi Trung tm Nghin cu v pht trin u , Vin Cy Lng thc v Cy Thc phm Vit Nam. Ging SD30 do Trung tm Khuyn Nng tnh Nam nh cung cp. Ging lc ( BG) do Trung tm pht trin u huyn Vit Yn, Bc Giang cung cp. Mt s c im ca cc ging nghin cu c trnh by bng 2.1. Bng 2.1. Mt s c im ca 10 ging lc nghin cu
Ging lc L05 Nhp ni t ICRISAT, nm 1999 L16 Ging lai to ca Trm Xuyn/ V79/ICGV871 57, nm 1992 L18 Nhp ni t Trung Quc, nm 2004 120-130 Sc sinh trng tt, l xanh m, khng bnh l v bnh ho xanh vi khun kh. T l nhn 6971%. Nng sut 55-70 t/ha, qu to, eo qu trung bnh, gn r, v la mu hng. Khi lng 100 ht 60-65g. Kh nng chu hn km. L23 Nhp ni t 120-135 Thn ng, tn gn, l mu xanh m, khng Trung Quc, bnh l v bnh ho xanh vi khun kh. T l Ngun gc TGST (ngy) 110-120 L to, mu xanh nht, nhim bnh ho xanh vi khun. T l nhn 73-75%. Nng sut 24-27 t/ha, ht lc mu hng sng, khi lng 100 ht 43-52g. Kh nng chu hn kh 120-125 Sinh trng kho, l mu xanh vng, nhim bnh l trung bnh. V qu mng nhn, ht lc mu hng cnh sen. Khi lng 100 ht 55-60g. Kh nng chu hn kh. c im sinh hc, kh nng chng chu

nm 2001 nhn 70-72%. Nng sut 45-55 t/ha, qu to, eo v gn qu nng, v la mu hng. Khi lng 100 ht 60-65g. Kh nng chu hn tt L24 Nhp ni t Trung Quc, nm 2001 120-135 Thn ng, tn gn, l mu xanh m, khng bnh l v bnh ho xanh vi khun kh. T l nhn 70-72%. Nng sut 45-55 t/ha, qu to, eo v gn qu nng, v la mu hng. Khi lng 100 ht 60-65g. Kh nng chu hn kh. V79 Ging t bin to ra t ging lc Bch sa. (Trung Quc), nm 1995 MD7 Nhp t Trung Quc, nm 2000 120-130 Thn ng, tn gn, l mu xanh m, khng bnh l v bnh ho xanh vi khun kh. T l nhn 73-75%. Nng sut 27,9 t/ha, qu trung bnh, gn qu nng, v la mu hng. Khi lng 100 ht 48-50g. Kh nng chu hn kh. 100-120 Thn ng, tn gn, l nh mu xanh m, khng bnh l v bnh ho xanh vi khun cao. T l nhn 71-73%. Nng sut 35-40 t/ha, qu to, eo v gn qu nng, v la mu hng. Khi lng 100 ht 55-60g. Kh nng chu hn kh. MD9 Nhp t Trung Quc, nm 2006 125-130 Thn ng, tn gn, l mu xanh nht, khng bnh l v bnh ho xanh vi khun tt. T l nhn 70-72%. Nng sut 38-48 t/ha, qu trung bnh, v la mu hng. Khi lng 100 ht 5560g. Kh nng chu hn kh. SD30 Nhp t Trung Quc 120-135 Thn ng, tn gn, l nh, dy mu xanh m, khng bnh l v bnh ho xanh vi khun kh. T l nhn 70-72%. Nng sut 35-40 t/ha, qu to, eo v gn qu nng, v la mu hng. Khi lng 100 ht 55-60g. Kh nng chu hn kh. 120-130 Thn ng, tn gn, l mu xanh m, khng bnh l v bnh ho xanh vi khun tt. T l nhn 70-75%. Nng sut 13,5 t/ha, qu trung bnh, v la mu . Khi lng 100 ht 5560g. Kh nng chu hn tt.
BG
Ging a phng

2.2. Ho cht v thit b S dng cc loi ho cht tinh khit v chuyn dng c ngun gc t cc hng ni ting trn th gii, bao gm: - Cc cht kch thch sinh trng: 2,4-D; BAP; - NAA ca hng Sigma. - Cc ho cht dng trong phn lp gen nh Taq- polymerase, m PCR, EDTA, SDS, Tris HCL, agarose... ca hng Invitrogen. - Vector tch dng pBT, mi RAPD... do phng Cng ngh t bo thc vt- Vin Cng ngh Sinh hc cung cp. - Cp mi nhn gen cystatin c thit k da trn trnh t nucleotide ca gen cystatin cng b (2004) ti ngn hng gen quc t vi m s AY722693. Sau c tng hp ti hng DNA- alpha. - Cc ho cht thi gel, tch plasmid l sn phm do hng Fermentas cung cp. Thit b Cn in t Sartorius (c), my li tm lnh Hettich (c) my quang ph UV- Cintra 40 (c), t sy Carbolite (Anh), box cy Nuaire (Ty Ban Nha), ni kh trng Tomy (Nht Bn); my PCR Amplied Biosystems (USA/Singapore), my soi v chp nh gel Logic 1500 (Kodak- M), cng vi mt s thit b khc. 2.3. PHNG PHP NGHIN CU Th nghim c nghin cu theo m hnh tng qut c trnh by hnh 2.1.

HT LC
TO M SO
GY MT NC
TO T BIN KT HP VI GY MT NC
SNG LC CC DNG CY TI SINH THNG QUA S N NH CH TIU NNG SINH HC CC TH H
PHN TCH NNG SUT, CHT LNG HT, KH NNG CHU HN CA CC DNG CHN LC
PHN TCH S THAY I TRONG H GEN V TRNH T GEN CYSTATIN CA CC DNG CHN LC
TUYN CHN V GII THIU DNG U T
Hnh 2.1. M hnh th nghim tng qut 2.3.1. Phng php nui cy in vitro Cc k thut c bn ca qu trnh nui cy in vitro thc hin theo Nguyn Th Tm v tg (2006) [26], c ci tin. Cc k thut gm:

- To m so: Ht lc nguyn vn c kh trng trong dung dch javen 60% (30 pht), cn 70% (1 pht). Tip tc lm sch ht bng nc ct v trng v to m so trn mi trng MS c b sung sucrose (3%), agar (0,9%), 2,4D (12mg/l), pH= 5,8. Nui cy hon ton trong ti, nhit 250C 10C. nh gi t l to m so t phi lc sau 10 ngy nui cy theo cng thc: Ci ( % ) = Trong : Ncp: S phi to m so Nt : Tng s phi nui cy Ci : T l to m so Tnh tc sinh trng ca m so bng cch cn khi lng cc m c to thnh sau 10 ngy. - X l to t bin: M so c to thnh sau 10 ngy em chiu x tia gamma (ngun Co60) ti trung tm chiu x Quc Gia (T Lim, H Ni) vi 5 liu chiu 0,5krad; 1krad; 2krad; 3krad; 4krad. - X l mt nc: Cc m so bao gm nhng m chu chiu x v m khng chu chiu x c ct gn theo chiu ngang t pha u thn phi thnh cc n v c khi lng khong t 100mg n 150mg. Sau , thi kh m so bng lung kh v trng ca box cy (hng sn xut Nuaire, 2003, Ty Ban Nha) vi cc ngng thi gian xc nh 3 gi, 6 gi, 9 gi, 11 gi. Nhit phng n nh 250C. Xc nh mt nc ca m so cc ngng thi gian theo cng thc: N cp Nt 100%

Wl ( % ) =
Trong : Wl: mt nc (%) Wf: Trng lng m ti (mg) Wd: Trng lng m kh (mg) - Ti sinh cy: Cc khi m x l mt nc c chuyn ln mi trng ti sinh (MS, sucrose 3%, agar 0,9%, BAP 2mg/l, pH=5,8) nui di nh sng n ca phng cy vi cng 2000lux, thi gian chiu sng 17/24 gi, nhit 250C 10C. Xc nh kh nng chu mt nc ca m so sau 4 tun nui phc hi theo cng thc:
Wf Wd 100% Wf
Sv ( % ) =
Trong :
N sv 100% Nt
Sv : T l m so sng st (%) Nsv: Tng s m so sng st Nt : Tng s m so x l Xc nh kh nng chu mt nc thng qua t l sng st ca m so sau x l bi thi kh v kh nng ti sinh cy sau 6 tun nui cy. T l cy ti sinh c tnh theo cng thc:
Rc ( % ) =
Trong :
Nr 100% N SV
Rc: Kh nng ti sinh cy Nr: Tng s m ti sinh cy Nsv: Tng s m sng st

- To cy hon chnh: Mi chi cy sng st v t kch thc khong 2,0 - 2,5cm c chuyn vo mi trng ra r (MS, sucrose 3%, agar 0,9%, NAA 0,5mg/l, 0,2% than hot tnh pH=5,8, b sung 20% nc da, nui cy trong nh sng n ca phng cy cng 2000 lux, nhit 250C 10C to cy hon chnh. - Ra cy v ch chm sc: Sau 8 n 10 tun to cy hon chnh, ton b s bnh ra r c chuyn ra khi phng nui cy, trong phng c nh sng t nhin, nhit bnh thng khong 2 ngy; nhc cy c b r to kho ra khi bnh nui, ra sch lp thch bm xung quanh r cy, trng trn nn t ti xp, ti lin tc m bng dung dch MS pha long 10 ln. Nu cng nh sng mnh cn hn ch bng cch s dng bt che chn, tt nht l dng tm li cch nhit. Sau khong 3 tun ra cy, s cy con sng st c r bm t, xut hin r mi, thn v l c mu xanh l bt u sinh trng pht trin. 2.3.2. Phng php nghin cu ng rung -Trng v theo di ngoi ng rung: Cy ti sinh t m so (Qun th R0, RM0) c chuyn ra trng ngoi ng rung. Trong : Cy R k hiu l cy ti sinh t m so chu mt nc; cy RM l k hiu cy ti sinh t m so chu x l chiu x tia gamma kt hp vi gy mt nc. Mi cy ca R0, RM0 c gi l mt dng. Cc dng c nh du bng cc s, theo di v thu hoch ring trng v tip theo. Ht ca cy R0, RM0 c gi l th h R1, RM1. Tng t, cy ca ht R1, RM1 c gi l R2, RM2... Ch chm sc ca cc dng v ging nh nhau (ph lc 3). - Xc nh cc ch tiu nng hc v nng sut vo thi k chn ca cy vi cc ch s: Chiu cao thn chnh (cm); s nhnh/cy; s qu chc/cy; khi lng 100 qu; khi lng 100 ht theo V Cng Hu v tg (1995) [8].

2.3.3. Phng php sinh l, ho sinh 2.3.3.1. Phng php phn tch ha sinh - nh lng protein ho tan theo phng php Lowry; Chit protein trong m phosphat citrat (pH=10) qua m nhit 40C, to phn ng mu ca dch chit vi thuc th foling v i chiu vi th xy dng bng protein chun bc sng 750nm xc nh lng protein trong nguyn liu. Protein tan c tnh bng % khi lng kh (Nguyn Vn Mi, 2001) [18]. - nh lng lipid bng my Soxhlet. Lipid c chit rt ra khi mu bng CHCl3 trn my Soxhlet. Hm lng lipid c trong nguyn liu c tnh theo n v % kh tuyt i bng hiu s ca khi lng mu trc v sau khi chit lipid (Nguyn Vn Mi, 2001) [18]. - Xc nh hot ca -amylase theo phng php Heinkel trn nguyn tc so snh da vo s gim cng mu ca hn hp phn ng vi thuc th I2 [5]. + Chit enzyme trong m phosphat pH=6,8 + Trn phn ng gm 0,5ml tinh bt 1% trong m phosphat 0,5ml NaCl 0,1% 1,0ml m phosphat 0,5ml dch chit enzyme Lc u 300C trong 30pht. B sung 2,5ml dung dch sunfosalisilic acid 20%. Ly 0,5ml dch trn, b sung 5ml I2 1% long 150 ln. Lc u 5 pht v so mu bc sng 560nm. ng kim tra tin hnh song song vi ng th nghim nhng thay dung dch axit vo trc sau mi cho dch chit enzyme. ng chun xc nh hot -amylase xy dng bng tinh bt chun.

- Xc nh hm lng ng bng phng php vi phn tch [2]. + Chit ng bng nc ct + Trn phn ng gm 2ml dch ng 2ml kaliferixianua. Lc u cch thy si 15 pht 4ml hn hp Fe2(SO4)3 0,1% v gelatin 10% (t l 20:1) nh mc thnh 20ml, lc u. o bc sng 585nm vi ng chun lm bng glucose. 2.3.3.2. Phng php nh gi nhanh sc sng ca t bo v m trong qu trnh nui cy in vitro bng cch nhum mu vi TTC. M so ca 10 ging lc sau khi tin hnh x l thi kh mt nc c xc nh nhanh bng phng php nhum TTC theo Towill v tg (1975) [123]. Cc bc tin hnh nh sau: (1) Cn 0,6g TTC pha long trong 25ml nc ct. (2) Cn 0,3g m so cho vo ng nghim, thm 0,5ml MS pha long 10 ln v 0,25ml dung dch TTC. ti 12 gi nhit 250C. (3) Gn b phn nc trong ng nghim. Ra m so nhum bng nc ct. Sau thm vo 5ml cn 90%, thy phn nhit 600C trong 1gi. (4) o dung dch thu c bc sng 485nm. i chng l cn 90%. 2.3.3.3. Phng php nh gi kh nng chu hn giai on ht ny mm Ht lc ngm trong nc sch khong 4 gi, sau trong dung dch sorbitol 7%. Sau 3 ngy, 5 ngy, 7 ngy, 9 ngy mu ly ra xc nh ng thi hot ca -amylase v hm lng ng theo m t mc 2.3.3.1. 2.3.3.4. Phng php nh gi kh nng chu hn giai on cy non - nh gi kh nng chu hn giai on cy non bng phng php gy hn nhn to theo L Trn Bnh v tg (1998) [1]: Ht lc ny mm em gieo vo cc chu (kch thc 22cm x 10cm) cha ct vng ra sch, mi

chu trng 10 cy. Thi gian u ti nc cho m, khi cy c 3 l tht, ngng ti nc v theo di cc ch tiu lin quan n kh nng chu hn. Mc ho ca cy c xc nh sau 1 ngy, 3 ngy, 5 ngy, k t khi l th nghim bt u c cy ho. Sau 5 ngy hn, tin hnh ti nc phc hi. Cc ch tiu theo di: T l cy khng ho; T l cy phc hi; Ch s chu hn tng i. Kh nng chu hn ca cy c xc nh bng ch s chu hn tng i v biu din bng th hnh rada, gm cc trc a, b, c, d, e, f, tng ng vi cc ch tiu theo di. Sn= 1/2 * sin * (anbn+ bncn + cndn +dnen+enfn+fnan) Trong : =3600/6= 600 n: k hiu ca ging v cc dng nghin cu a: % cy khng ho sau 1 ngy hn b: % cy phc hi sau 1 ngy hn c: % cy khng ho sau 3 ngy hn d: % cy phc hi sau 3 ngy hn e: % cy khng ho sau 5 ngy hn f: % cy phc hi sau 5 ngy hn Cy c ch s chu hn tng i cng ln th kh nng chu hn cng cao v ngc li. 2.3.4. Phng php sinh hc phn t 2.3.4.1. Tch chit DNA tng s Tch chit DNA tng s theo phng php ca Gawel v tg (1991) [65]. : l gc c to bi hai trc mang tr s gn nhau v

- Thnh phn m ra: Tris HCl 1M: 2ml; EDTA 0,5M, pH=8: 0,2ml; Sorbitol 2M: 3,5ml; NaH2PO4 0,4%; Nc ct va : 20ml. - Thnh phn m chit: Tris HCl 1M, pH=8: 1ml; NaCl 5M: 2,8ml; EDTA 0,5M, pH 4,8: 0,4ml; CTAB 4%; Nc ct va 10ml. Cc bc c bn gm ph v mng t bo bng nghin l ti vi nit lng, chit DNA bng dung dch CTAB, loi protein v tp cht bng hn hp chlorofom/isoamyl vi t l 24:1. Xc nh hm lng v tinh sch DNA bng cch o quang ph hp th trn my quang ph UVvis Cintra 40 (Australia) v in di trn gel agarose 0,8% trong TAE. 2.3.4.2. K thut RAPD - Phn tch a hnh DNA bng k thut RAPD, vi 25 mi ngu nhin theo William v tg (1990) [129]. Cc mi do phng Cng ngh t bo thc vt, Vin Cng ngh Sinh hc cung cp (ph lc 2). Mi ng phn ng PCR c 25l dung dch cha: 2,0l m PCR 1X, 2,0l MgCl2 (25mM), 1,2 l dNTP (2,5mM), 1,6l mi (10mM), 0,4l Taq polymerase (5U/l), 0,8l DNA h gen (10ng/l), nc ct va . Phn ng PCR-RAPD thc hin theo chu trnh nhit gm 6 bc: 940C trong 3 pht, 920C trong 1 pht, 350C trong 1 pht, 720C trong 1 pht, t bc 2 n bc 4 lp li 45 chu k, 720C trong 10 pht, gi 40C. Phn tch sn phm PCR bng cch in di trn gel agarose 1% trong TAE 1X, sn phm in di nhum trong dung dch ethidium bromide v chp nh di nh sng n cc tm. 2.3.4.3. K thut phn lp gen cystatin Phn lp gen cystatin c thc hin theo m t ca Chu Hong Mu (2008) [16], cc bc c bn gm:

- Nhn bn gen: gen cystatin c khuch i bng k thut PCR vi cp mi c thit k da trn trnh t nucleotide ca gen cystatin cng b ti Ngn hng gen quc t vi m s AY722693 [134]. Cp mi Cys-AraF/CysAraR c thit k c trnh t nucleotide nh sau: Cys-AraF: 5ATGGCAGCAGTGGGTGCA 3 Cys-AraR: 5 TTAAGCATTGGAGCCATCAC 3 Phn ng c tin hnh trong hn hp gm: DNA h gen (100ng/l) 1l; mi (10pmol/l): 2,0l; dNTPs (2,5mM): 2,0l; Mg+2(25nM): 2,5l; Taq polymerase (5U/l): 0,5l; m PCR (10X): 2,5l; Nc kh ion: 25 l. Hn hp c thc hin vi 30 chu k phn ng, cc giai on gm: 940C/3 pht, 940C/30 giy, 560C/1 pht, 720C/1 pht, 720C/10 pht v lu gi sn phm 40C. Sn phm PCR c lm sch bng b kit ca hng Fermentas. To plasmid ti t hp Ghp ni on gen lm sch vo vector tch dng pBT bng b kit pGEM-T System. Thnh phn phn ng ghp ni c: Vector pBT: 1l; m ligation 10X: 2l; T4-ligase (2U/ml): 0,5l; Sn phm thi gel: 7,0l; Nc kh ion va 20l. Hn hp c 220C, trong 1 gi 30 pht, sau bo qun 40C. Bin np plasmid ti t hp vo khun E.coli chng DH5 bng phng php sc nhit: Ly 7-9l plasmid ti t hp vo 200l t bo kh bin. t hn hp vo 30 pht, sau chuyn vo b n nhit 420C trong 1 pht, ri li chuyn vo 5 pht.

B sung 400l LB (1% pepton, 1% NaCl, 0,5% cao nm men, nc ct va ), nui lc 200 vng/pht trong 60 pht 370C. Sn phm nui lc cy tri trn mi trng LB (c cha agar 1,6%), b sung ampicilin 100mg/l, IPTG 0,1mM, X-gal 40mg/l. Nui a khun 370C khong 16 gi. Tch v chn dng gen c tin hnh theo Sambrook v tg (2001) [114]. Chn khun lc trng nui trong mi trng LB lng c b sung ampicilin, kim tra sn phm chn dng bng phn ng colony-PCR. Thnh phn phn ng tng t nh thc hin phn ng khuch i gen nhng thay DNA khun c tinh sch bng khun lc mang DNA ti t hp. Sn phm chn dng c in di kim tra trn gel agarose 1%. Tinh sch plasmid Thu nhn khun bng cch ly tm dch nui trn 3000 vng/ pht trong 7 pht. Sau lm sch v thu nhn plasmid bng b kit Gene JETTM Plasmid Miniprep (Fermentas) Xc nh trnh t DNA c thc hin trn my xc nh trnh t DNA t ng ABI PRISM 3100 Avant Genetic Analizer. 2.3.5. Phng php phn tch s liu - Phn tch cc tr s thng k bng phn mm Excel theo hng dn ca Chu Hong Mu (2008) [16]. Cc gi tr bao gm: gi tr trung bnh ( X ), phng sai (2), lch (), sai s (S X ), v h s bin ng (Cv%). - Phn tch s liu RAPD bng phn mm NTSYSpc version 2.0 (Applied Biostatisc Inc., USA, 1998) theo hng dn ca Chu Hong Mu (2008) [16]. Da trn s xut hin hay khng xut hin ca cc phn on DNA khi in di sn phm RAPD bng DNA h gen cc dng chn lc vi cc mi

ngu nhin lm c s cho phn tch s liu. Kt qu phn tch s sai khc di truyn v s hnh cy th hin mi quan h mc phn t ca cc dng chn lc so vi ging gc. Tnh h s di truyn theo cng thc:
H RAPD = f i 2
i n
HRAPD l h s a dng di truyn; fi l tn sut ca alen th i - Trnh t gen c x l trn my vi tnh bng phn mm BioEdit v DNAstar. So snh trnh t gen, trnh t amino acid bng phn mm MegAlign v BioEdit. 2.4. A IM NGHIN CU Th nghim c tin hnh ti cc phng th nghim Di truyn hc, Cng ngh t bo thc vt, Ho sinh hc ca Trng i hc S phm; Phng th nghim Sinh hc ca Trng i hc Khoa hc; Phng th nghim Sinh hc phn t v Cng ngh gen ca Vin Khoa hc S Sng, thuc i hc Thi Nguyn. X l chiu x bi tia gamma, ngun Co60 ti Trung tm chiu x Quc gia, T Lim- H Ni. Trnh t DNA c xc nh trn thit b gii trnh t DNA t ng ti Vin Cng ngh Sinh hc. Th nghim ng rung thc hin ti T Rng Vu, Phng Quang Vinh- Thnh ph Thi Nguyn.

Chng 3
KT QU NGHIN CU V THO LUN
3.1. KT QU TO DNG CHU HN BNG K THUT X L M SO TRONG H THNG NUI CY IN VITRO
3.1.1. Sng lc dng m so chu tc ng ca thi kh 3.1.1.1. Kh nng to m so v sinh trng ca m so 10 ging lc nghin cu Vi mc ch nh gi kh nng to m so ca cc ging lc trong h thng nui cy in vitro lm c s cho cc nghin cu to dng lc chu hn bng cng ngh t bo thc vt, chng ti tin hnh thm d kh nng to m so t cc t bo phi mm ca ht v tc sinh trng ca m so 10 ging lc nghin cu, kt qu c trnh by bng 3.1. Bng 3.1 cho thy, tt c cc ging lc nghin cu u c kh nng to m so. cng mt iu kin nui cy, cc ging c s sai khc v t l to m so v kch thc khi m so to thnh. T l to m so t phi mm ca 10 ging lc dao ng t 82,71% (L18) n 98,55% (V79). Cc ging lc c t l to m so t trn 97% l cc ging MD7, MD9, V79. Ging c t l to m so thp hn 90% gm cc ging L18, L23, BG. Trong , t l to m so thp nht l ging L18 (82,71%). Khi lng m so ca cc ging lc c to thnh dao ng t 115,00mg (L18) n 198,00mg (V79). Cc ging L05, L23, MD7, MD9, SD30, V79 c khi lng m so t trn 180mg. Bn ging L16, L18, L24 v BG c khi lng m so to thnh nh hn 160mg.

Bng 3.1. T l to m so v khi lng m so ca 10 ging lc
STT 1 2 3 4 5 6 7 8 9 10 Ging lc L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG T l to m so sau 10 ngy (%) 95,16 5,83 95,48 2,14 82,71 5,44 89,93 5,42 90,74 3,53 98,40 4,52 97,01 3,36 91,05 5,17 98,55 5,19 86,03 4,12 Khi lng m so (mg/m) 193,33 5,42 142,00 5,83 115,00 6,45 181,87 5,63 151,67 9,46 185,71 8,75 194,26 5,27 196,18 4,53 198,00 6,63 158,33 5,87
Trong cng mi trng nui cy in vitro, t l to m so v tc sinh trng ca m so phn nh s biu hin khc nhau ca kiu gen cc ging lc. Kt qu thu c cho thy, kh nng to m so v s sinh trng ca m so 10 ging lc p ng cho cc nghin cu tip theo trong chn dng t bo. 3.1.1.2. Kh nng chu mt nc ca m so cc ging lc nghin cu xy dng quy trnh chn lc dng t bo chu mt nc lc, chng ti ct cc khi m thu c thnh cc phn c khi lng tng ng nhau, khong t 100mg n 150mg. Sau , xc nh ngng chu mt nc ca t bo m so cc ging lc bng k thut thi kh.

Vi cc mc thi gian 3 gi, 6 gi, 9 gi, 11 gi thi kh lin tc, lng nc trong m so gim dn ti mc rt t c th bay hi c. nh gi kh nng chu mt nc ca m so c tin hnh thng qua xc nh mt nc, kh nng chu mt nc v kh nng ti sinh cy. mt nc ca m so mt nc ca m so c tnh thng qua kt qu xc nh khi lng m trc v sau khi thi kh. Kt qu xc nh mc mt nc ca m so sau x l bng k thut thi kh c trnh by bng 3.2 v hnh 3.1. Bng 3.2. mt nc ca m so sau x l bng k thut thi kh (% so vi khi lng m ban u)
Thi gian thi kh (gi) 3 67,37 0,88 69,11 0,58 70,04 0,77 66,21 0,90 58,66 0,64 71,66 0,86 61,45 0,81 60,73 0,67 73,01 0,58 6 78,90 0,64 83,68 0,65 84,71 0,62 84,18 1,09 84,67 0,81 84,99 0,48 77,19 0,30 78,47 0,57 81,85 0,51 83,03 0,52 9 80,59 1,28 89,89 0,43 85,81 0,32 86,00 0,60 86,03 0,82 86,64 0,73 82,61 0,31 86,19 0,79 84,05 0,28 85,03 0,61 11 84,34 0,69 90,08 0,47 86,60 0,10 87,05 0,88 87,09 0,84 86,82 0,58 85,18 0,50 87,14 0,09 87,97 0,48 88,11 0,48
STT
1 2 3 4 5 6 7 8 9 10
Ging
L05 L16 L18 L23 L24 MD7 MD9 SD30 V79
BG 72,33 0,33

Kt qu trnh by bng 3.2 cho thy, sau 3 gi thi kh lin tc, m so ca cc ging lc b mt nc t 58,66% (L24) n 73,01% (V79) so vi khi lng m ban u. M so ca cc ging u tng mt nc khi thi gian thi kh cng di. mt nc 6 gi dao ng trong khong 77,19% (MD9) n 84,99% (MD7). Mc nc mt sau 9 gi t 80,59% (L05) n 89,89% (L16). Sau 11 gi thi kh lng nc mt dao ng t 84,34% (L05) n 90,08% (L16).
mt nc (%)
100 80 60 40 20 0 3h 6h 9h 11h Thi gian thi kh (gi)
L05 V79
L16 MD7
L18 MD9
L23 SD30
L24 BG
Hnh 3. 1. mt nc ca m so cc ging lc
T l mt nc ca m so tng nhanh trong nhng gi thi kh u, gim dn nhng khong thi gian sau. So vi thi im 3 gi, sau 6 gi mt nc tng thm t 8,84% (V79) n 26,01% (L24). n 9 gi, mt nc tip tc tng t 1,10% (L18) n 7,72% (SD30) v sau 11gi, lng nc ch mt thm 0,79% (L18) n 3,92% (V79). Nhn chung, v tc mt nc ca m so gia cc ging lc khng c s sai khc ng k .

Kh nng chu mt nc ca m so Kh nng chu mt nc ca m so cc ging lc c xc nh thng qua kt qu nh gi nhanh sc sng ca m so bng phng php nhum TTC v xc nh t l sng st ca m so sau x l bi thi kh. nh gi nhanh sc sng ca m so bng phng php nhum TTC
Bng 3.3. Kt qu kim tra kh nng chu mt nc ca m so 10 ging lc bng phng php nhum TTC
STT Ging lc 3 1 2 3 4 5 6 7 8 9 10 L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG 6 9 11 Gi tr OD485 sau cc ngng x l thi kh (gi)
0,232 0,004 0,163 0,003 0,133 0,001 0,093 0,011 0,112 0,001 0,082 0,001 0,173 0,003 0,148 0,003 0,162 0,002 0,103 0,001 0,195 0,002 0,140 0,002 0,134 0,012 0,180 0,004 0,193 0,011 0,175 0,002 0,182 0,036 0,130 0,001 0,133 0,003 0,102 0,003
0,127 0,003 0,093 0,002 0,073 0,001 0,053 0,001 0,055 0,001 0,042 0,001 0,128 0,003 0,065 0,020 0,075 0,001 0,053 0,001 0,123 0,001 0,082 0,001 0,106 0,035 0,062 0,002 0,154 0,020 0,058 0,002 0,108 0,001 0,077 0,001 0,092 0,002 0,055 0,001

Cc t bo cn sng bt mu vi thuc nhum, t bo cht khng bt mu. Khi ngm vi cn 90% trong 60 pht 600C, mu s phai ra dung dch. o mt quang ca dung dch bc sng 485nm phn nh sc sng ca t bo [123]. Xc nh gi tr OD bc sng 485nm ca dung dch thuc nhum TTC vi m so cc ging lc thu c kt qu khc nhau gia cc ging v cc thi im m so chu x l. Gi tr OD485 ngng x l thi kh ca cc ging u cho kt qu cao nht ngng thi kh 3 gi, sau gim dn. Trong cc ngng thi kh, cng mu ca ng nhum m so ging lc L18 lun nht nht v c gi tr OD485 thp nht (bng 3.3).
3 gi
6 gi
9 gi
11 gi
Hnh 3.2. Dung dch nhum TTC ca m so ging lc L18 cc ngng x l thi kh (C: ng i chng bng cn 90% )

Xc nh t l sng st ca m so sau 4 tun Cy chuyn ton b cc t bo sau khi x l bng thi kh gy mt nc sang mi trng ti sinh cy. Sau 4 tun nui cy, xc nh t l m sng st so vi tng s m c cy chuyn, kt qu nghin cu c trnh by bng 3.4. Bng 3.4. T l sng st ca m so b x l bng thi kh sau 4 tun nui phc hi (%) STT
1 2 3 4 5 6 7 8 9 10
Ging lc
L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG
Thi gian thi kh (gi)
3
100,00 0,00 72,41 0,63 72,00 0,72 76,47 0,64 89,58 0,54 90,97 1,03 79,55 0,73 82,05 0,55 88,24 0,52 84,62 0,54
6
75,61 0,64 61,76 0,54 40,74 0,43 53,13 0,81 56,52 0,46 82,05 0,42 56,67 0,50 81,06 0,47 63,33 0,54 63,89 0,44
9
48,39 1,08 50,00 0,45 10,81 0,52 25,00 0,30 50,00 0,44 41,67 0,64 33,33 0,41 29,27 0,59 42,42 0,64 34,21 0,34
11
19,05 0,49 19,05 0,44 9,26 0,60 13,33 0,52 12,50 0,54 15,00 0,66 14,29 0,60 15,91 0,39 19,01 0,54 14,29 0,61
Quan st kh nng phc hi ca cc m so b thi kh trn mi trng ti sinh cy chng ti nhn thy, nhng m so sng st trn mi trng ti sinh chuyn mu xanh v c s gia tng dn v kch thc m so so vi kch thc khi m ban u. Ngc li, cc m khng c kh nng sng st c mu trng xm v sau cht dn (nh C, hnh 3.3). Kh nng chu mt nc ca m so c nh gi thng qua t l sng st ca m so sau 4 tun nui phc hi. T l sng st sau 4 tun ca

cc m so mt nc t l nghch vi thi gian thi kh. Sc sng ca m so c s khc nhau gia cc ging lc cng mt ngng thi gian thi kh. Sau 3 gi thi kh lin tc, t l sng st ca m so cc ging lc dao ng t 72,00% (L18) n 100% (L05; MD7). Sau 6 gi thi kh t l sng ca m so t t 40,74% (L18) n 82,05% (MD7). Ging L18 lun c t l sng st thp nht. Kt qu xc nh t l sng st ca m so sau x l bng thi kh th hin sc chu ng ca t bo v y l c s sng lc dng t bo [1], [34]. Cc m so chu mt nc thu c l ngun nguyn liu thc hin ti sinh cy phc v chn dng lc chu hn. Kh nng ti sinh cy ca m so chu mt nc Kh nng ti sinh cy l mt ch tiu quan trng quyt nh thnh cng trong chn dng t bo v chu nh hng ca nhiu yu t [1]. Kt qu nghin cu cho thy, tt c cc ging lc s dng trong th nghim u c kh nng ti sinh cy t cc m so sng st. Kt qu bng 3.5 cho thy, s lng m sng st ca cc ging lc c kh nng ti sinh cy cc ngng thi gian thi kh khc nhau. Sau 3 gi thi kh, m so sng st ca cc ging lc u c kh nng ti sinh cy vi t l t 66,13% n 90,27%. Thi gian thi kh ko di lm gim t l sng st. Tuy nhin, cc t bo sng st li c kh nng ti sinh cy cao. M so ca 10 ging lc sau 9 gi n 11 gi thi kh (m mt t 84,05% n 89,89% nc so vi khi lng m ti) c kh nng sng st cn 10,81% n 19,05%. Cc m sng st cho t l ti sinh cy t 83,33% n 100,00%. Cn c v kt qu nghin cu, chng ti chn thi im m mt trn 84% nc so vi khi lng ti ban u v t l m sng st trong khong 10% n 20% l ngng sng lc dng.

Bng 3.5. T l ti sinh cy ca m so sng st sau 6 tun (%)
STT 1 2 3 4 5 6 7 8 9 10
Ging lc L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG 3 66,63 2,37 74,97 2,17 66,13 0,57 87,70 1,01 81,00 0,23 83,23 1,24 85,10 3,55 71,13 1,19 90,27 1,68 75,60 1,79
Thi gian thi kh (gi) 6 67,10 1,87 94,23 1,28 68,47 2,14 93,43 0,35 93,27 3,64 83,70 3,77 83,43 3,67 83,87 2,00 91,97 0,98 94,13 1,23 9 93,00 4,13 100,00 0,00 83,33 3,61 96,30 3,70 95,57 2,63 90,67 2,16 93,33 3,70 95,23 4,77 100,00 0,00 100,00 0,00 11 100,00 0,00 97,23 2,77 53,33 3,33 94,43 3,94 100,00 0,00 91,67 1,93 96,30 3,70 100,00 0,00 100,00 0,00 100,00 0,00
X l m so lc bng k thut thi kh m so, chng ti xc nh ging lc L18 c kh nng chu mt nc km nht v la chn mc thi kh 9 gi lm ngng sng lc. Theo nghin cu ca mt s tc gi, cc t bo c kh nng chu mt nc trong iu kin cc oan l nhng t bo c kh nng chng chu tt [1], [51]. nh hng ca thi kh gy mt nc cho t l sng st ca m so 10 ging lc khc nhau, ph thuc vo kh nng chu mt nc ca cc ging. Kt qu nh gi ph hp vi cng b do Trung tm u , Vin Cy Lng thc v Cy Thc phm Vit Nam cung cp (bng 2.1), ph hp vi kh nng chu hn ca 10 ging lc giai on ht ny mm khi b hn sinh l v giai

on cy non khi b hn nhn to. Trong 10 ging lc nghin cu, ging L18 c kh nng chu mt nc km nht, c hot - amylase v hm lng ng nhm thp nht trong cc ngy ht ny mm khi b hn, c ch s chu hn thp nht (ph lc 4, ph lc 5). 3.1.2. nh hng ca tia gamma kt hp vi thi kh n t l sng st v ti sinh cy ca ging lc L18 Ging lc L18 c xc nh l ging c kh nng chu hn, chu mt nc km nht trong s 10 ging lc nghin cu. Bn cnh , ging L18 c nng sut cao (bng 2.1). V vy, vi mc tiu ci thin kh nng chu hn ca ging lc nng sut cao ny, chng ti tin hnh thm d nh hng kt hp ca chiu x tia gamma vi sng lc dng chu mt nc bng k thut thi kh i vi m so ging lc L18. Bng 3.6. nh hng ca chiu x kt hp vi thi kh 9 gi n t l sng st v ti sinh cy ca m so ging lc L18
Liu chiu x kt hp thi kh 9 gi 0,5krad 1,0krad 2,0krad 3,0krad 4,0krad
T l m sng st (% ) 34,23 0,37 33,65 0,52 28,42 0,43 18,87 0,59 12,51 0,42
T l ti sinh (% ) 47,63 2,37 88,87 5,57 38,87 5,54 26,17 4,97 21,30 6,03 Hnh thi cy ti sinh
Bnh thng Bnh thng Bnh thng L nh, mp l xon, vng v rng L nh, mp l xon, vng v rng

Bng 3.6 trnh by kt qu thm d nh hng ca 5 liu lng chiu x mc: 0,5krad; 1,0krad; 2,0krad; 3,0krad; 4,0krad kt hp vi thi kh m so trong 9 gi. Hiu qu tc ng kt hp cc liu lng chiu x khc nhau vi thi kh lin tc c xc nh thng qua t l m sng st v t l m sng st ti sinh cy. Kt qu xc nh t l m sng st ti sinh cy cho thy, kh nng ti sinh cy ca m so chu nh hng kt hp ca chiu x vi thi kh dao ng t 21,30% n 88,87%. So vi kh nng ti sinh cy ca m sng st ch x l thi kh (bng 3.5), th vic x l m so bng chiu x kt hp vi thi kh lm gim t l ti sinh cy. Kt qu nghin cu nh hng ca tia gamma n t l ti sinh ca cy lc cng tm thy s tng t cy la [35]. S khc bit quan trng ca cy ti sinh chu tc ng ca chiu x kt hp vi thi kh biu hin r nt c im hnh thi, c bit v mu sc v hnh dng l. Vi liu chiu x thp (0,5krad; 1krad; 2krad), l cy vn c mu xanh nh nhng cy i chng. Nhng vi liu chiu x cao nh 3krad v 4krad th mu xanh ca l xut hin trong khong 1-2 tun u sau l bt u chuyn mu vng a. Hnh thi l nh v xut hin hin tng xon l. Tc sinh trng ca cy t l nghch vi cng ca liu chiu x. liu chiu x cao cy c hin tng li dn, thn cn ci. Chiu cao cy ti sinh thp hn rt nhiu so vi cy i chng (hnh 3.3). Kt qu nghin cu nh hng ca cc liu chiu x khc nhau n kh nng ti sinh cy v hnh thi l ca ging lc L18 c im tng t nh kt qu nghin cu ca Muthusamy v tg (2007) cng b [101].
Hnh 3.3. Mt s hnh nh cy ti sinh ca ging lc L18 giai on 6 tun tui

A: Cy ti sinh khng chu thi kh B: Cy ti sinh chu thi kh 9 gi C: Liu chiu x 0,5krad + thi kh 9 gi D: Liu chiu x 2,0krad + thi kh 9 gi E: Liu chiu x 3,0krad + thi kh 9 gi G: Liu chiu x 4,0krad + thi kh 9 gi

T nhng nh gi kh nng sng st v ti sinh ca m so sau khi x l bng tia gamma, chng ti xc nh c liu ti hn ca m so lc l liu chiu x 4krad. Nu vt qu gii hn 4krad a s m so cht, khng th to s dng m ln phc v nhng nghin cu tip theo, c bit l nghin cu chn dng. Tuy nhin, thu c nhiu t bin cn xc nh liu lng chiu x thch hp, va to ra nhng bin d di truyn c li, va c t l cc dng m sng st v ti sinh cao. Do vy, ngi ta thng dng liu chiu x nh hn liu ti hn t 1,5 n 2,0 ln to t bin [21]. Nhn xt v kt qu sng lc dng m so chu chiu x v thi kh Liu chiu x thch hp cho ging lc L18 l t 0,5krad n 2krad v liu lng 2krad kt hp vi thi kh 9 gi c la chn sng lc dng m v to cy hon chnh. Kt qu x l m so trong h thng nui cy in vitro ca ging lc L18 bng thi kh lin tc 9 gi v chiu x liu lng 2krad kt hp thi kh lin tc 9 gi thu c 167 dng m v t c 198 dng lc c to ra. 3.1.3. c im nng sinh hc ca cc qun th lc th h R0 v RM0 Phn tch mc bin d v c im nng sinh hc ca cy nh chiu cao thn chnh, s nhnh/cy, s qu/cy v s qu chc/cy ca cc dng thuc 2 qun th R0 v RM0. Kt qu nghin cu c trnh by bng 3.7 v hnh 3.4. Kt qu nghin cu mt s c im ca cc qun th lc th h R0, RM0 bng 3.7 cho thy, cc dng lc chuyn t ng nghim ra trng ngoi ng rung (v xun, 2008) c s bin ng di truyn ln hn nhiu so vi ging gc tt c cc ch tiu nghin cu.

Bng 3.7. c im nng hc ca qun th R0, RM0 ti sinh t m so mt nc
( X : Gi tr trung bnh;
SX :
Sai s trung bnh mu; Cv%: H s bin ng )
Ch tiu theo di Cao thn chnh (cm) S nhnh/cy (nhnh) S qu/cy (qu) S qu chc/cy T l qu chc (%)
Ging gc 37,00 0,58 2,70 8,33 0,33 6,93 20,67 0,67 5,59 16,33 0,33 79,00
Qun th R0 57,00 4,61 25,58 3,00 0,39 41,57 22,80 1,81 25,05 14,80 1,50 64,91
Qun th RM0 18,50 1,91 10,32 6,75 0,57 8,38 14,00 1,96 13,98 8,17 1,36 54,65
X SX
Cv %
X SX
Cv %
X SX
Cv %
X SX
R0 A. Khu vc th nghim qun th R0, RM0
Ging gc
RM0
B. Cy v qu giai on thu hoch
Hnh 3.4. Cc dng lc chn lc trng v xun nm 2008

Chiu cao cy l c im sinh trng ph thuc vo ging, mi trng v iu kin gieo trng. Chiu cao thn chnh ca cy lc thng dao ng trong khong t 12cm n 65cm [8]. Nghin cu c im sinh trng ca hai qun th chu mt nc v chu chiu x kt hp vi gy mt nc, chng ti nhn thy s xut hin rt r cc t bin v chiu cao cy. Trong , qun th R0 xut hin nhiu t bin tng chiu cao ca cy, trung bnh chiu cao cy ca qun th ny l 57,00cm. Khc vi qun th R0, qun th RM0 xut hin nhiu dng t bin thp cy, vi chiu cao trung bnh t 18,50cm. Chiu cao cy ca qun th R0 bin ng mc 25,58% v qun th RM0 l 10,32%. Nghin cu nh hng ca liu chiu x 7krad trn ging la Tm Xoan v c im chiu cao cy, tc gi L Xun c v tg (2006) cng thu c nhiu t bin thp cy, mc thp cy ca mt s dng th h RM1 ch bng 50% so vi i chng [6]. Cy t qun th trong ng nghim nhn chung cho t l ra nhnh thp. T l nhnh ca qun th R0 thp hn nhiu so vi ging gc, mc bin ng di truyn l 41,57% so vi i chng l 6,93%; S nhnh ca qun th RM0 t 6,75 nhnh/cy v l ch tiu c mc bin ng nh (Cv=8,38%). Kt qu nghin cu cho thy, s qu/cy qun th R0 ca cc dng lc chu mt nc cao hn so vi ging gc (ging gc trng t ht), s bin ng trong qun th R0 mc 25,05%. Lng qu ca qun th RM0 t 13,33 qu/cy, mc bin ng l 13,98%. T l qu chc ca cy c to ra t ng nghim u thp hn so vi ging gc. Nh vy, qu trnh chn dng lc chu hn bao gm: (1) To m so v x l m so; (2) Ti sinh cy v to cy hon chnh (3) Trng, theo di v phn tch di truyn cc dng chn lc (hnh 3.5).
Hnh 3.5. Hnh nh trong chn dng chu hn lc bng k thut nui cy in vitro
A. To m so; B. Ti sinh cy t m so sau x l thi kh; C. To cy hon chnh; D. Cy R0 ngoi ng rung; E. C ca mt dng chn lc th h Ba G. Cc dng trng th h th Nm

X l m so lc trong nui cy in vitro, chn lc m so sng st, ti sinh cy v theo di qun th R0 v RM0 c th rt ra mt s nhn xt sau: (1) Cc dng c ngun gc t m so chu thi kh c chiu cao cy tng hn so vi ging gc; cn cc dng c ngun gc t m so chu chiu x kt hp vi thi kh c chiu cao cy gim so vi ging gc. (2) Cc qun th cy c to ra bng k thut nui cy m t bo thc vt c s nhnh/cy t hn so vi ging gc. Qun th R0 t 3 nhnh/cy, qun th RM0 t 6,75 nhnh/cy, ging gc c 8,33 nhnh/cy. (3) Qun th R0 c 22,80 qu/cy, nhiu hn ging gc (t 110,30% so ging gc), qun th RM0 c s qu/cy t hn ging gc, ch t 14,00 qu/cy (67,73% so ging gc). (4) H s bin ng di truyn cc ch tiu nghin cu ca 2 qun th cy c to ra bng k thut nui cy m t bo thc vt ln hn so vi ging gc. C rt nhiu yu t tham gia vo vic cu thnh nng sut cng nh cht lng ht lc, cn thit phi c s theo di s n nh ca cc tnh trng trong mi th h v qua cc th h, c bit l kh nng chu hn ca cc ging lc. T kt qu nh gi th h R0, RM0, chng ti chn c 7 dng c ngun gc t m so chu x l ca ging lc L18, trong 3 dng c ngun gc t m so chu nh hng ca thi kh l R44, R46, R48 v 4 dng c ngun gc t m so chu nh hng ca chiu x kt hp vi thi kh l RM46, RM47, RM48, RM49 lm vt liu khi u nh gi, chn lc cc th h tip theo.

3.2. KT QU PHN TCH NHNG DNG LC CHN LC QUA CC TH H 3.2.1. c im nng sinh hc cc dng lc chn lc th h th Nht v th Ba Ht lc rt kh bo qun, t l ny mm thp khi thi gian bo qun ko di. V vy, ton b ht ca 7 dng lc chn lc qun th R0, RM0 chng ti trng k tip vo cc v trng trong nm. Th h th Nht (R1, RM1) c trng v thu ng nm 2008. T kt qu thu c trnh by bng 3.8 c th rt ra mt s nhn xt nh sau: Cy th h th Nht vn duy tr s sai khc v chiu cao thn chnh, trong cy ca cc dng c ngun gc t m so chu nh hng ca chiu x thp hn (khong t 17,67cm n 22,33cm) so vi dng chu thi kh (t 49,60cm n 61,70cm). Tt c cc dng chn lc u c mc bin ng v ch tiu ny thp hn ging gc. S nhnh/cy ca cc dng chn lc dao ng t 4,67 nhnh n 8,00 nhnh. Mc bin ng s nhnh trong khong 8,25% n 19,99%; ba dng RM47, RM48, RM49 c s nhnh/cy cao hn ging gc. S qu/cy ca cc dng nghin cu dao ng t 14,67 qu n 22,17 qu/cy. Hai dng R46 v R48 c s qu/cy cao hn so vi ging gc. S qu chc/cy ca cc dng chn lc trong khong 10,00- 13,67 qu/cy, tt c cc dng chn lc u c t l qu chc/cy thp hn so vi ging gc. So vi cc cy trng khc nh la, la m, u xanh, u tng... s qu/cy ca lc t hn. V vy, s qu/cy ca cc dng chn lc trong nhng th h u rt kh khn cho vic thc hin phn tch tnh chu hn ca cc dng ny. Chnh v vy, chng ti la chn ht v trng lin tc cc v xun h v thu ng hng nm.

Bng 3.8. c im nng sinh hc cc dng lc chn lc th h th Nht (n<30; =0,05) ( X : Gi tr trung bnh;
SX :
Sai s trung bnh mu; Cv %: H s bin ng)
Cao thn chnh (cm) Ch tiu theo di X SX R44 R46 R48 RM46 RM47 RM48 RM49 L18 53,40 3,22 49,60 2,34 61,70 1,19 19,33 0,67 17,67 1,45 22,33 1,33 21,25 0,83 29,90 1,84 Cv % 19,08 14,91 6,11 3,45 8,22 5,97 3,91 19,47
S nhnh/cy X SX 5,14 0,53 5,32 0,33 5,03 0,26 4,67 0,64 7,00 0,58 7,33 1,45 8,00 1,00 6,11 0,55 Cv % 12,61 19,99 16,33 14,29 8,25 19,81 12,50 18,34
S qu/cy
S qu chc/cy Cv % 16,42 14,38 16,25 6,93 9,09 16,08 6,78 6,70
X SX
18,33 1,23 22,17 1,30 20,50 1,36 17,33 1,20 14,67 1,32 19,00 3,06 19,67 1,33 19,83 0,54
X SX
12,40 0,45 10,00 0,56 11,25 0,49 13,67 0,88 10,67 1,06 11,63 2,40 11,33 0,63 14,00 0,60
Cv % 12,23 17,64 12,34 24,05 9,94 20,67 5,56 12,07
Th h th Nht trng v Thu ng, nm 2008, ti T Rng Vu, Phng Quang Vinh, Thnh ph Thi Nguyn

Vi mc tiu to ra cc dng lc c kh nng chu hn cao, chng ti la chn kt qu nh gi cc dng chn lc th h Ba v th Nm bo co. y l nhng th h trng lin tc cc v Thu ng nm 2009, 2010. V Thu ng thng trng trong khong t 25 thng 8 hng nm n u thng 1 ca nm k tip. Thi im ny min Bc thng c c im lnh v kh, y cng l hng nghin cu ca lun n. Th h th Ba cha c lng qu ln, v vy kt qu nh gi cc dng chn lc th h ny thng qua phn tch c im nng hc (bng 3.9). Nhng ch tiu nghin cu ca cc dng chn lc th h th Ba c duy tr n nh tng ng so vi ging gc. dao ng ca cc dng chn lc mi ch tiu nghin cu l khng nhiu. So vi th h th Nht, chiu cao cy ca dng chu chiu x kt hp vi thi kh c s ci thin mnh, kt qu th hin trn bng 3.9 cho thy, trong 7 dng nghin cu qua 3 th h ch c dng RM49 th h th Ba c ngun gc t m so chu chiu x kt hp vi thi kh c chiu cao cy thp hn ging gc. th h th Ba c 2 dng R46, R48 c t l nhnh cao hn ging gc; s bin ng ca tnh trng s nhnh/cy trong khong t 3,17 n 10,34%. Phn tch s lng qu/cy, s qu chc/cy ca cc dng chn lc th h th Ba chng ti nhn thy, dng RM47 c s n nh v s biu hin ca cc tnh trng nghin cu u mc cao hn ging gc. S biu hin cc tnh trng s lng phn nh c im phn ng ca kiu gen cc ging lc trc tc ng ca mi trng v ma v. S n nh kh nhanh ca cc tnh trng nghin cu ngay t th h th Nht ca cy lc c ngun gc t m so gp phn khng nh gi tr ca k thut nui cy m t bo thc vt c th to ging mi v rt ngn thi gian to ging, tng hiu qu ca qu trnh chn ging.

Bng 3.9. c im nng sinh hc cc dng lc chn lc th h th Ba (n<30; =0,05) ( X : Gi tr trung bnh; S X : Sai s trung bnh mu; Cv %: H s bin ng )
Cao thn chnh (cm) Ch tiu theo di X SX 42,75 1,52 41,64 0,81 48,60 2,84 37,25 1,23 37,08 1,05 45,20 1,38 32,20 2,67 34,79 1,73 Cv % 4,65 1,95 5,85 3,31 2,84 3,05 8,30 4,66
S nhnh/cy X SX 4,83 0,40 8,60 0,27 8,25 0,48 6,00 0,33 5,80 0,25 4,85 0,15 5,60 0,40 8,00 0,52 Cv % 10,34 9,81 11,61 5,03 4,30 3,17 7,14 5,81
S qu/cy
S qu chc/cy Cv % 5,46 5,34 5,83 4,89 9,18 5,22 17,54 8,04
X SX
20,00 1,09 20,11 1,07 17,88 1,04 23,50 1,15 32,83 3,02 18,30 0,96 19,00 3,33 20,33 2,04
X SX
18,14 0,91 17,90 0,91 16,22 0,98 18,63 0,84 25,83 1,60 13,60 0,65 14,20 2,01 18,78 1,10
Cv % 13,29 16,12 18,18 4,53 6,19 4,80 14,15 17,62
R44 R46 R48 RM46 RM47 RM48 RM49 L18
Th h th Ba trng v Thu ng, nm 2009, ti T Rng Vu, Phng Quang Vinh, Thnh ph Thi Nguyn

3.2.2. nh gi cc dng lc chn lc th h th Nm Cc dng chn lc th h th Nm trng v Thu ng nm 2010. th h th Nm lng qu v ht ln thc hin cc th nghim lin quan n vic nh gi kh nng chu hn ca cc dng chn lc. Chng ti tip tc nh gi cc dng chn lc th h th Nm thng qua s phn tch mt s c im nng sinh hc; xc nh cc yu t cu thnh nng sut nh khi lng 100 qu, khi lng 100 ht; nh gi cht lng ht trn phng din ha sinh thng qua xc nh hm lng protein, hm lng lipid; nh gi kh nng chu hn giai on ht ny mm, giai on cy non v s sai khc trong h gen bng k thut RAPD. 3.2.2.1. c im nng sinh hc v cht lng ht Kt qu nh gi s n nh cc tnh trng nng hc ca mt s dng chn lc th h th Nm c trnh by bng 3.10. Bng 3.10 cho thy, cc ch tiu nng sinh hc v cht lng ht c s bin ng thp, trong khong 1,14% n 7,88%. S n nh biu hin ca cc tnh trng ny mt ln na khng nh s dng k thut nui cy m t bo thc vt gp phn rt ngn thi gian chn dng. th h th Nm, khi lng 100 qu lc ca cc dng chn lc dao ng t 83,11g n 145,12g. C 5/7 dng c khi lng 100 qu mc qu to (107,05g 145,12g), chim 71,43%. Ba dng gm R44, R46, RM46 c khi lng 100 qu cao hn so vi ging gc (cao hn 116,59g). Khi lng 100 ht ca cc dng chn lc th h th Nm dao ng trong khong 41,11g n 56,27g, cc dng chn lc c khi lng 100 qu ln cng l dng c khi lng 100 ht cao.

Bng 3.10. c im nng sinh hc v cht lng ht cc dng chn lc th h th Nm ( X : Gi tr trung bnh; S X : Sai s trung bnh mu; Cv%: H s bin ng )
Ch tiu theo di Cao thn chnh (cm) S nhnh/cy S qu/cy S qu chc/cy Khi lng 100 qu (g) X SX Cv% 2,10 3,48 3,72 7,79 4,31 6,45 1,68 2,43 Khi lng 100 ht (g) X SX 49,23 0,89 56,27 1,32 44,07 3,47 52,40 0,78 41,11 2,50 46,53 2,89 45,00 3,52 47,81 0,55 Cv% 1,80 2,34 7,88 1,48 6,08 6,21 7,82 1,14 Hm lng protein (% KLK) X SX 32,33 3,28 32,08 2,54 26,58 2,04 29,09 2,29 38,97 4,25 27,72 1,96 25,72 2,89 34,47 2,85 Hm lng lipid (% KLK) X SX 38,11 0,89 34,67 0,77 36,44 0,89 38,22 2,70 36,89 0,89 38,67 0,88 38,22 0,44 36,00 2,67
X SX R44 R46 R48 RM46 RM47 RM48 RM49 L18 45,62 0,76 45,77 0,79 48,08 0,85 43,27 0,77 41,63 0,54 35,46 1,02 31,47 1,13 30,62 0,84
Cv% 1,66 1,72 2,20 1,78 1,30 2,88 3,59 2,73
X SX 6,50 0,18 5,77 0,20 5,20 0,20 5,80 0,20 4,73 0,19 5,67 0,22 6,53 0,57 7,69 0,29
Cv% 2,88 3,49 3,85 3,45 4,12 3,97 4,19 3,72
X SX 23,36 1,19 19,15 0,69 15,64 0,96 20,22 1,26 27,72 1,31 27,08 1,84 22,33 1,18 26,80 1,22
Cv% 5,07 3,59 6,16 5,06 4,70 6,78 5,29 4,55
X SX 17,25 1,25 13,08 0,63 11,75 0,59 13,26 2,76 19,44 1,38 14,55 0,99 12,69 0,40 14,45 0,78
Cv%
7,23 121,33 2,54 4,84 145,12 5,05 5,04 83,11 3,10
5,74 120,95 9,42 7,07 103,43 4,46 6,84 108,27 6,98 3,14 107,05 1,80 5,39 116,59 2,84
Th h th Nm trng v Thu ng, nm 2010, ti T Rng Vu, Phng Quang Vinh, Thnh ph Thi Nguyn

S bin ng v ch tiu qu v ht ca cy trong qun th th nghim mc thp (mc bin ng l Cv =1,14% - 7,88%). Hm lng lipid trong ht l mt c trng ca ging. c bit, cy lc l cy cng nghip ly du, s tch lu du t ti a nu thu hoch ng chn [8]. Cc dng chn lc th h th Nm c hm lng lipid t 34,67% khi lng kh (KLK) n 38,67% KLK. Dng c hm lng lipid cao nht l RM48, thp nht l dng R46. Hm lng protein trong ht khng ch phn nh cht lng ht m cn lin quan n kh nng chng chu ca cy trng. Cc dng chn lc th h th Nm c hm lng protein khong t 25,72% KLK n 38,97% KLK. Dng c hm lng protein cao nht l RM47, thp nht l dng RM49. Cn c vo kt qu nghin cu v c im nng sinh hc, cht lng ht ca cc dng lc chn lc c ngun gc t m so chu mt nc v m so chu nh hng chiu x kt hp vi x l gy mt nc, chng ti chn c mt s dng c c im ni bt, bao gm: (1) Dng R46 c khi lng 100 qu v ht cao; (2) Dng RM47 c hm lng protein cao; (3) Dng RM48 l dng c hm lng lipid cao. Trong cng mt iu kin trng trt nh nhau, cc dng chn lc c s thay i v kiu hnh, l l do chng ti tip tc nghin cu s thay i mc phn t. 3.2.2.2 Kt qu nh gi kh nng chu hn Kh nng chu hn ca thc vt ni chung v ca cy lc ni ring l mt tnh trng kh kim sot ngoi ng rung. Do vy, chng ti tin hnh nh gi kh nng chu hn ca cc dng lc chn lc th h th Nm giai on ht ny mm v giai on cy non. Kt qu nh gi c thc hin

ng thi vi ging gc v ging L23 c kh nng chu hn tt nht trong 10 ging lc nghin cu. Kh nng chu hn giai on ht ny mm nh gi kh nng chu hn ca ging lc L18, L23 v cc dng lc chn lc, chng ti la chn phng php gy hn sinh l giai on ht ny mm bng cch x l ht bng dung dch sorbitol 7% nghin cu. giai on ht ny mm, cc cht trong ht chuyn trng thi t t hot ng sang giai on hot ng mnh m, nhiu enzyme mi c tng hp, trong c -amylase. S tng hot ca -amylase lm tng hm lng ng do tinh bt b thy phn. Kh nng chu hn ca ging lc L18, L23 v cc dng chn lc c chng ti nh gi thng qua xc nh hot -amylase v hm lng ng. Kt qu nghin cu trnh by ph lc 6, hnh 3.6 v hnh 3.7.
0,7 0,6 VH/mg ht ny mm 0,5 0,4 0,3 0,2 0,1 0 3 5 7 Ngy tui ny m m 9
R44 RM48
R46 RM49
R48 L18
RM46 L23
RM47
Hnh 3.6. S bin ng v hot ca -amylase trong iu kin hn sinh l

Kt qu nghin cu cho thy, di tc ng ca hn sinh l s bin ng hot -amylase ca ging lc L18, L23 v cc dng chn lc c s sai khc nhau ng k. giai on 3 ngy tui, hot ca -amylase dao ng trong khong t 0,15 VH/mg ht ny mm n 0,23 VH/mg ht ny mm. X l ht bng sorbitol 7% gy hn sinh l, hot ca amylase c xu hng tng t 3 ngy tui n 7 ngy tui sau gim dn giai on 9 ngy tui. Ging c hot ca -amylase cao nht ngy ny mm th 7 l RM48 (0,66 VH/mg ht ny mm). Cng ngy ny mm th 7, hot ca -amylase cao th 2 l dng R46 (0,63 VH/mg ht ny mm), sau l ca ging L23 (0,59 VH/mg ht ny mm). Dng c hot ca amylase thp ngy ny mm th 7 l R44 (0,48 VH/mg ht ny mm). Kh nng hot ng ca -amylase trong iu kin b hn ph thuc vo kh nng chu hn ca cy lc. Cn c vo hot ca -amylase giai on 7 ngy tui, chng ti xp ging lc L18, L23 v cc dng chn lc theo mc chu hn thnh 4 nhm: (1) Nhm 1: c 3 dng (RM48, R46, RM46), hot ca -amylase trong khong 0,60-0,66 VH/mg ht ny mm. (2) Nhm 2: hot ca -amylase trong khong 0,58-0,59 VH/mg ht ny mm, gm c ging L23 v dng RM47. (3) Nhm 3: c 2 dng (RM49, R48) v ging L18, c hot ca -amylase trong khong 0,50-0,55 VH/mg ht ny mm. (4) Nhm 4: c 1 dng (R44), hot ca -amylase t 0,48 VH/mg ht ny mm.

ng trong c th thc vt l nhm cht lin quan n s iu ha p sut thm thu ca t bo khi gp iu kin cc oan [50]. Xc nh hm lng ng trong ht ny mm ca ging lc L18, L23 v cc dng chn lc bng phng php vi phn tch, chng ti nhn thy tt c cc dng v ging lc nghin cu u c quy lut bin ng hm lng ng tng t khi ht ny mm n giai on ht ny mm 7 ngy tui, sau gim dn (ph lc 6 v hnh 3.7)
2 1,8 1,6
% ht ny mm
1,4 1,2 1 0,8 0,6 0,4 0,2 0 R44 R46 R48 RM46 RM47 RM48 RM49 L18 L23
3 ngy
5 ngy
7 ngy
9 ngy
Hnh 3.7. S bin ng hm lng ng trong iu kin hn sinh l
Theo quan im hin nay, vic tch ly cc cht ha tan trong t bo ng nht vi khi nim iu chnh p sut thm thu. y l phng thc thch nghi ca c th thc vt i vi cc yu t bt li ca mi trng. Khi t bo mt nc cc cht ha tan s c tch ly dn chng li s mt nc ng thi tng kh nng gi nc ca cht nguyn sinh. Qu trnh thy phn

polysaccharid d tr l ngun cung cp cht tan cho qu trnh iu chnh p sut thm thu ca t bo khi b mt nc ng thi gp phn thc y qu trnh phc hi cy [50], [51]. Hm lng ng c to thnh nhiu nht 7 ngy tui theo th t gim dn ca cc ging v dng chn lc l: RM48>R46>L23>RM47>R48>L18>RM46>RM49>R44. Trong iu kin bt li (hn, lnh, nng...), kt qu nghin cu trn cy thuc l ca Nguyn Hong Lc (1992) v cy la ca Nguyn Th Tm v tg (2003) cho thy c s gia tng hm lng ng [13], [27]. Tuy nhin, trong iu kin bt li th hot ca -amylase, hm lng ng cng ch tng mt t l nht nh v ph thuc vo kh nng chu hn ca cy. Bng 3.11. Tng quan gia hot ca -amylase v hm lng ng giai on ht ny mm STT 1 2 3 4 5 6 7 8 9 Ging v dng chn lc R44 R46 R48 RM46 RM47 RM48 RM49 L18 L23 Phng trnh hi quy 1,13x + 0,72 1,61x + 0,57 1,04x + 0,77 1,83x + 0,34 0,54x + 1,04 2,66x + 0,66 1,04x + 0,68 1,07x +0,70 2,26x + 0,19 H s tng quan 0,9881 0,8972 0,8379 0,9811 0,9945 0,9999 0,9862 0,8466 0,9983

Phn tch mi tng quan gia s bin ng hot ca -amylase v hm lng ng ca ging lc L18, L23 v cc dng chn lc giai on ht ny mm khi b hn sinh l cho thy, hm lng ng ph thuc tuyn tnh vo hot -amylase, vi h s tng quan (R) dao ng t 83,79% n 99,99%. Hot ca -amylase cao s lm qu trnh phn gii tinh bt thnh ng xy ra mnh m, m bo cung cp cht dinh dng cho qu trnh ny mm ca ht, c bit l iu chnh p sut thm thu ca t bo trong iu kin cc oan. S sai khc v hot ca -amylase v ng cc dng, ging lc trong cc thi im ht ny mm khc nhau lin quan n kh nng chu hn ca chng. Dng chn lc c hot ca -amylase v hm lng ng cao hn ging gc minh chng cho hiu qu ci thin kh nng chu hn ca cy bng k thut nui cy m t bo thc vt. Kh nng chu hn giai on cy non Kt qu nh gi kh nng chu hn giai on cy non ca cc dng chn lc th h th Nm c trnh by bng 3.12 v hnh 3.7 . Theo di t l cy sng st, cy phc hi thi im 1 n 5 ngy hn, bng 3.12 cho thy, cc dng lc c ch s chu hn tng i khc nhau, dao ng t 3672,48 n 10089,20. Dng RM48 c ch s chu hn cao nht (10089,20%), sau n dng R46 v ging lc L23, thp nht l cc dng R44, RM49 v ging gc L18. Trong 7 dng lc chn lc v ging gc L18 c 5 dng chn lc c ch s chu hn tng i cao, trong 2 dng c ngun gc t m so chu thi kh (R46; R48) v 3 dng c ngun gc t m so chu nh hng ca chiu x kt hp vi thi kh (RM46; RM47; RM48).

Bng 3.12. T l cy sng, cy phc hi v ch s chu hn tng i ca cc dng chn lc th h th Nm
Dng, ging gc
% CKH sau 1 ngy hn 78,6 70,8 73,3 54,6 95,8 90,0 62,5 66,7 72,8
% CKH sau 3 ngy hn 57,3 50,0 53,3 36,4 75,0 80,0 30,8 50,0 51,5
% CKH sau 5 ngy hn 36,7 20,8 26,7 18,2 20,8 20,0 7,7 30,3 25,8
% CHP sau 1 ngy hn 14,3 53,8 20,0 41,7 16,0 50,0 21,7 16,7 50,8
% CHP sau 3 ngy hn 14,3 57,7 46,7 50,0 20,0 60,0 39,1 33,3 57,0
% CHP sau 5 ngy hn 28,6 69,2 53,3 58,3 24,0 70,0 52,2 50,0 69,0
Ch s chu hn tng i
Xp th t
R44 R46 R48 RM46 RM47 RM48 RM49 L18 L23
4327,07 7662,38 5713,17 5019,50 5272,80 10089,20 3672,48 4725,09 7898,30
4 2 3 3 3 1 4 4 2
Cn c vo ch s chu hn tng i ca cc dng lc chn lc v ging gc L18 (bng 3.12 v hnh 3.8) c th xp cc dng chn lc v ging gc 4 mc chu hn khc nhau. Mc 1: c ch s chu hn tng i trn 10000 (c mt dng RM48); Mc 2: ch s chu hn tng i trn 7000; c dng R46, ging L23; Mc 3: ch s chu hn tng i t 5000 n 6000 c cc dng: R48, RM46, RM47; Mc 4: ch s chu hn tng i nh hn 5000 gm ging gc L18, dng R44 v dng RM49.

% CKH sau 1 ngy hn 100,0 80,0 % CHP sau 5 ngy hn 60,0 40,0 20,0 0,0 % CKH sau 3 ngy hn
% CHP sau 3 ngy hn
% CKH sau 5 ngy hn
% CHP sau 1 ngy hn
R44 RM48
R48 RM49
R46 L18 gc
RM46 L23
RM47
Hnh 3.8. th hnh rada biu th s khc nhau v kh nng chu hn ca cc dng lc chn lc, ging L18 v ging L23
Cc dng lc chn lc c ngun gc t m so chu mt nc v m so chu chiu x kt hp vi thi kh gy mt nc c s khc nhau v mt s c im nng sinh hc, c im ha sinh ht v kh nng chu hn. Bn cht ca cc thay i ny lin quan n h gen nh th no l l do chng ti tip tc phn tch s sai khc mc phn t ca cc dng chn lc v ging gc. 3.2.2.3. Kt qu so snh s sai khc v h gen ca cc dng chn lc v ging gc Vi mc tiu nghin cu s thay i h gen gia cc dng lc chn lc th h th 5 so vi ging gc, chng ti tin hnh tch chit DNA t h gen cy lc v phn tch a hnh DNA bng k thut RAPD.

Kt qu tch chit DNA tng s L non ca cc dng lc c s dng tch chit DNA. Sau khi tch chit v tinh sch, hm lng v sch DNA c xc nh bng phng php o quang ph hp th bc sng 260nm v 280nm. ng thi, DNA tng s c in di trn gel agarose 1% v chp nh trn my soi gel nh gi cht lng dung dch DNA. Kt qu thu nhn hm lng DNA tch c t 0,5g l lc non trong khong t 462g/ml n 1693g/ml. T s OD260nm / OD280nm ln hn 1,8. Hnh nh DNA trn gel sc nt v gn, m bo cht lng thc hin cc k thut sinh hc phn t tip theo (hnh nh khng trnh by). Kt qu phn tch a hnh DNA bng k thut RAPD Phn tch tnh a hnh ca DNA da trn s xut hin hay khng xut hin cc phn on c cng kch thc c khuch i t h gen nh s thay i cc im gn ca cc mi trong phn ng RAPD [16], [129]. Phn ng RAPD c thc hin vi 25 mi ngu nhin c chiu di 10 nucleotide (ph lc 2). Kt qu phn tch thu c tng s 1254 phn on, vi kch thc c on ca cc phn on trong khong t 0,2kb n 4,2kb. Tnh ton kt qu ca th nghim vi 25 mi nghin cu (bng 3.13), chng ti nhn thy, phn ng RAPD vi 25 mi lm xut hin trong 7 dng v ging gc t 16 phn on n 110 phn on DNA. C 7 mi (OPA05, OPA10, OPA13, OPH04, OPF09, OPN05, OPQ02) cho kt qu n hnh, hai mi (OPA13 v OPF09) ch xut hin 2 bng vch n hnh trn c 7 dng v ging gc. Mi c s phn on nhiu nht l OPA04, vi 110 phn on DNA. Hnh 3.9 trnh by hnh nh in di sn phm RAPD vi mt s mi, trong cc bng xut hin v din mt c so snh vi ging gc.

1 2 3 4 5 6 7 8 M M 1 2 3 4 5 6 7 8
OPA08
M 1 2 3 4 5 6 7 8 M 1 2 3
OPB05
4 5 6 7 8
OPH08
1 2 3 4 5 6 7 8 M M 1 2
OPQ02
3 4 5 6 7 8
OPQ05
UPC348
Hnh 3.9. Hnh nh in di sn phm RAPD vi mt s mi

Bng bin mt Bng mi xut hin
M: Marker chun; 1.L18 2.RM46 3.RM47 4.RM48 5.RM49 6.R44 7.R46 8.R48 89 Tai lieu chia se tai: wWw.SinhHoc.edu.vn

Bng 3.13. S phn on DNA nhn bn ngu nhin v t l phn on a hnh ca 25 mi trong phn ng RAPD
TT K hiu mi S phn on DNA Tng n s a T l on a hnh (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 OPA01 OPA02 OPA03 OPA04 OPA05 OPA06 OPA07 OPA08 OPA09 OPA10 OPA11 OPA12 OPA13 82 63 63 110 64 62 29 48 77 40 31 41 16 64 56 56 104 64 48 28 28 64 40 24 8 16 18 21,95 7 11,11 7 11,11 6 0 5,45 0,00 14 15 16 17 18 19 20 21 22 23 24 OPA14 OPA15 OPB05 OPB10 OPH04 OPH08 OPF09 OPF10 OPN05 OPQ02 OPQ05 36 44 59 21 24 31 16 46 64 64 68 55 1254 24 31 32 16 24 16 16 32 64 64 40 40 999 TT K hiu mi S phn on DNA Tng n s a T l on a hnh (%) 12 33,33 13 29,55 27 45,76 5 23,81 0 0,00
hnh hnh
hnh hnh
14 22,58 1 3,45
15 48,39 0 0,00
20 41,67 13 16,88 0 0,00
14 30,43 0 0 0,00 0,00
7 22,58 33 80,49 0 0,00
28 41,18 15 27,27 255 20,33
25 UPC348 Tng cng

Tng s phn on a hnh l 255 phn on, chim 20,33% s phn on c nhn bn. T l phn on a hnh cao nht mi OPA12 vi 33/41 phn on c nhn ln, chim 80,49%. Da trn s xut hin hay khng xut hin cc phn on khi thng k kt qu in di sn phm RAPD trn gel agarose 1% thit lp mi quan h di truyn ca cc dng chn lc so vi ging gc mc phn t. Kt qu nghin cu c trnh by bng 3.14 v hnh 3.10. Bng 3.14. T l sai khc v h gen ca cc dng chn lc v ging gc L18 (%) R44 R44 R46 R48 RM46 RM47 RM48 RM49 L18 0,000 6,061 3,752 1,521 1,522 6,043 3,420 5,069 0,000 3,146 1,540 1,531 5,437 2,814 4,762 0,000 4,807 4,757 9,143 4,898 3,875 0,000 1,844 6,464 3,147 3,805 0,000 6,405 3,757 5,066 0,000 6,573 8,002 0,000 6,536 0,000 R46 R48 RM46 RM47 RM48 RM49 L18
Bng 3.14 l kt qu so snh v t l sai khc mc phn t v h gen ca cc dng chn lc so vi nhau v so vi ging gc. Kt qu cho thy, 8 h gen c t l sai khc t 1,521% n 9,143%. Mc sai khc ln nht gia

h gen ca dng RM48 vi h gen ca dng R48 (t l sai khc l 9,143%). Hai dng chn lc c h gen ging nhau nht l RM46 v R44 (t l sai khc nh nht vi 1,521%) Thit lp mi quan h ca 7 dng chn lc v ging gc mc phn t da trn s liu a hnh RAPD vi 25 mi ngu nhin c th hin s hnh 3.10.
Hnh 3.10. S m t mi quan h ca 7 dng chn lc vi ging gc L18 mc phn t
Hnh 3.10 cho thy, 7 dng lc chn lc u c s sai khc v h gen so vi ging gc L18. S m t mi quan h ca cc dng chn lc v ging gc c phn b thnh 2 nhm vi khong cch di truyn l 22% (1- 0,88). Nhm I: Ch c mt dng RM48, y l dng c ngun gc t m so chu tc ng kt hp gia chiu x v thi kh, c khong cch di truyn so vi cc dng cn li v ging gc nhnh 2 l 22%. Nhm II: gm cc dng R48, RM49, RM47, R46, R44, RM46 v ging gc L18. Khong cch di truyn ca cc dng chn lc v ging gc L18 l 9,5% (1 - 0,905 = 0,095). Nhm II chia lm hai nhnh, nhnh th nht l

ging gc (L18); nhnh th hai, chia thnh nhiu nhnh nh bao gm cc dng chn lc cn li v hai dng R44, R46 cng c ngun gc t m so chu mt nc. Hai dng ny c sai khc so vi ging gc mc thp 1,521% v 1,522%. H s a dng di truyn v cc phn on DNA c trng ca cc dng chn lc Cn c vo tn sut xut hin cc alen trong tng locus, s dng cng thc tnh h s a dng di truyn (Genetic Diversity Index) da trn cc d liu phn tch RAPD, chng ti xc nh c h s a dng di truyn (HRAPD) ca 7 dng chn lc trong phm vi 25 mi ngu nhin. Kt qu tnh ton cho thy cc dng chn lc biu hin s a dng di truyn mc phn t vi h s HRAPD = 11,09%. y chnh l ngun gen phong ph cung cp cho chn dng lc. Bng 3.15. Ch th RAPD c trng ca cc dng lc chn lc Dng lc chn lc R44 R46 R48 RM46 RM47 RM48 RM49
OPA07 750bp -
OPA08 500bp -
OPB05 900bp -
UPC348 200bp -
OPH08 250bp -

Kt qu nghin cu bng 3.15 cho thy, dng RM48 xut hin 4 kch thc c trng vi 4 mi. Trong , mi OPA07 kch thc c trng xut hin 750bp (RM48/OPA07-750bp); mi OPA08 kch thc c trng xut hin 500bp (RM48/OPA08-500bp); mi OPB05 c kch thc c trng 900bp (RM48/OPB05-900bp); mi UPC348 kch thc c trng xut hin 200bp (RM48/UPC348-200bp). Mi OPH08 xut hin mt c trng kch thc 250bp vi dng RM47 (RM47/OPH08-250bp). Nh vy, s dng 25 mi trong phn ng RAPD nhn bn cc phn on DNA t h gen ca 7 dng lc c ngun gc t m so sau khi x l bi thi kh v chiu x kt hp vi thi kh xc nh c 5 ch th phn t RAPD c trng 2 dng lc chn lc vi 5 mi ngu nhin (OPA07, OPA08, OPB05, UPC348, OPH08). nh gi cc dng chn lc thng qua phn tch mt s c im nng hc, ch tiu nng sut, cht lng ht v kh nng chu hn th h th Nm, chng ti tuyn chn c 3 dng u vit tip tc nh gi, theo di, bi dng gii thiu kho nghim ging. l cc dng: (1) Dng RM48 c kh nng chu hn cao nht, hm lng lipid cao nht (t 38,67% KLK); Dng R46 c kh nng chu hn cao th hai v c khi lng 100 qu, 100 ht cao (145,12g/100 qu; 56,27g/100 ht ); Dng RM47 c c kh nng chu hn cao, hm lng protein ln nht (t 38,97% KLK). 3.2.3. c im ca mt s dng lc u vit chn lc Tp hp cc kt qu phn tch v c im nng sinh hc, cht lng ht, kh nng chu hn v c im sinh hc phn t ca cc dng chn lc c ngun gc t m so chu thi kh v m so chu chiu x kt hp vi thi kh ca ging L18 c trnh by bng 3.16 v hnh 3.11.

Bng 3.16. Mt s c im ca 3 dng chn lc v ging L18 c im
Ngun gc
RM48
RM47
R46
L18
Nhp t Trung Quc, nm 2004
Cy ti sinh t Cy ti sinh t Cy ti sinh t m so chu m so chu m so chu chu chiu x + chiu x + thi kh thi kh thi kh 35,46 1,02 41,63 0,54 45,77 0,79
Chiu cao cy (cm) Khi lng 100 qu (g) Khi lng 100 ht (g) Hm lng protein (% KLK) Hm lng lipid (% KLK) Ch s chu hn Ch th RAPD c trng
30,62 0,84
108,27 6,98
103,43 4,46
145,12 5,05
116,59 2,84
46,53 2,89
41,11 2,50
56,27 1,32
47,81 0,55
27,72 1,96
38,97 4,25
32,08 2,54
34,47 2,85
38,67 0,88
36,89 0,89
34,67 0,77
36,00 2,67
10089,20
5272,80
7662,38
4725,09
RM48/OPA07-750bp; RM48/OPB05-900bp;
RM48/OPA08-500bp; RM48/UPC348-200bp;
RM47/OPH08-250bp;
RM48
RM48
RM47
RM47
R46
R46
L18
L18
Hnh 3.11. Hnh nh qu v ht ca cc dng chn lc th h th Nm

3.3. PHN LP V XC NH TRNH T GEN CYSTATIN T CY LC 3.3.1. Khuch i gen cystatin t DNA h gen ca cy lc Sau khi tch chit DNA tng s, tinh sch v pha v nng thch hp, chng ti tin hnh nhn bn gen cystatin bng phng php PCR vi DNA ca 7 dng chn lc, ca ging L18, v ging L23. Kt qu nhn gen c kim tra trn gel agarose 1% cng vi marker (hnh 3.12).
Hnh 3.12. Hnh nh in di sn phm nhn gen cystatin t cy lc
Sn phm ca phn ng PCR thu c hnh 3.12 cho thy, ch c 1 bng DNA, kch thc khong gn 500 bp, ph hp vi tnh ton l thuyt v kch thc gen cystatin c phn lp t mRNA do nhm tc gi Yan cng b (2004) [134]. Do , chng ti hy vng rng, kt qu ca phn ng PCR vi cp mi Cys-AraF/Cys-AraR c nhn ln t DNA h gen ca ging lc L18, L23 v 7 dng lc chn lc l s c mt ca gen cystatin trong h gen cy lc. Tuy nhin, khng nh chnh xc sn phm c khuych i t h gen l gen cystatin phi thc hin tch dng, xc nh trnh t v so snh vi trnh t gen cystatin cng b.

Chng ti tin hnh tch dng v xc nh trnh t gen cystatin ca 2 dng R46, RM48 (2 dng c kh nng chu hn cao nht), ging gc L18 (kh nng chu hn km) v ging L23 (c kh nng chu hn tt) nhm mc ch so snh trnh t gen ca cc dng v ging ny. 3.3.2. Kt qu tch dng v xc nh trnh t gen cystatin Sn phm PCR gen cystatin c lm sch bng b kit ca Fermentas, sau c gn vo vector pBT to vector ti t hp. Vector ti t hp c bin np vo t bo E. coli DH5 bng phng php sc nhit. Nui khun E.coli bin np trn mi trng LB c b sung thch agar, ampicilin, X-gal v IPTG 100mM nh m t mc 2.3.3.3. Sau 16 gi nui n nh 370C, trn a thch xut hin cc khun lc mu xanh v trng. Chn cc khun lc mu trng, hnh trn u trn a thch thc hin ng thi phn ng colony- PCR vi chnh cp mi nhn gen v nui khun trong 3 ml mi trng LB lng c ampicilin. Kt qu kim tra (hnh 3.13) chng t vic ghp ni sn phm PCR vo vector tch dng, chn dng v phn ng clony-PCR hy vng t c kt qu nh mong mun. Tinh sch plasmid bng b kit plasmid miniprep ca Fermentas, sau xc nh trnh t nucleotide ca gen cystatin trn my t ng ABI PRISM 3100 Avant Genetic Analizer ca hng Ampplied Biosystem ti Vin Cng ngh Sinh hc.
L18
R46
RM48 L23
Hnh 3.12. Hnh nh in di sn phm colony-PCR Hnh 3.13. Hnh nh in di sn phm colony-PCR Kt qu xc nh trnh t nucleotide thu c t plasmid ca cc mu nghin cu c x l bng phn mm DNAstar cho thy, gen cystatin ca ging lc L18, L23, dng R46 v RM48 u c kch thc 461 nucleotide. So snh cc trnh t ny vi trnh t nucleotide phn lp t cDNA ca 2 gen cystatin cy lc trn ngn hng gen quc t c m s AY722693 l trnh t dng thit k mi v trnh t mang m s EU723567, chng ti nhn thy gen m ho protein cystatin m chng ti phn lp gm 2 exon v 1 intron. on exon th nht ca gen gm 102 nucleotide bt u t v tr 1 n 102 v on exon th hai c 195 nucleotide, t v tr 267 n 461; vng intron gia c 164 nucleotide, t v tr 103 n 266 (hnh 3.14).
10 20 30 ------------------+-------------------+-------------------+L18 R46 RM48 L23 EU723567 AY722693 A A A A A A T T T T T T G G G G G G G G G G G G C C C C C C A A A A A A G G G G G G C C C C C C A A A A A A G G G G G G T T T T T T G G G G G G G G G G G G G G G G G G T T T T T T G G G G G G C C C C C C A A A A A A C C C C C C C C C C C C T T T T T T C C C C C C G G G G G G C C C C C C G G G G G G A A A A A A A A A A A A G G G G G G T T T T T T A A A A A A

40 50 60 ------------------+-------------------+-------------------+L18 R46 RM48 L23 EU723567 AY722693 G G G G G G C C C C C C C C C T C C G G G G G G G G G G G G A A A A A A A A A A A A A A A A A A C C C C C C G G G G G G A A A A A A G G G G G G A A A A A A A A A A A A C C C C C C A A A A A A G G G G G G C C C C C C C C C C C C T T T T T T C C C C C C G G G G G G A A A A A A G G G G G G A A A A A A T T T T T T C C C C C C G G G G G G A A A A A A T T T T T T
L18 R46 RM48 L23 EU723567 AY722693
70 80 90 ------------------+-------------------+-------------------+A G T C T T G C T C G C T T T G C T G T C G A T G A A C A C A G T C T T G C T C G C T T T G C T G T C G A T G A A C A C A G T C T T G C T C G C T T T G C T G T T G A T G A C A C A A G T C T T G C T C G C T T T G C T G T T G A T G A A C A C A G T C T T G C T C G C T T T G C T G T C G A T G A A C A C A G T C T T G C T C G C T T T G C T G T C G A T G A A C A C 100 110 120 ------------------+-------------------+-------------------+A A C A A G A A A C A G G T T T C T T T C T C T C T C T A C A A C A A G A A A C A G G T T T C T T T C T C T C T C T A C A C A A G A A A C A G G G T T T C T T T C T C T C T C T A G A A C A A G A A A C A G G T T T C T T T C T C T C T C T T G A A C A A G A A A C A G - - - - - - - - - - - - - - - - - A A C A A G A A A C A G - - - - - - - - - - - - - - - - - 130 140 150 ------------------+-------------------+-------------------+C G A T C A T C G C T T T G G C T C T G G T T T T G G T T C C G A T C A T C G C T T T G G C T C T G G T T T T G G T T C C G A A G A T C G C T T T G G C T C T G G T T T T G G T T C C G A A G A T C G C T T T C G C T T T G G T T T T G G T T C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 160 170 180 ------------------+-------------------+-------------------+G T T T T G T A T G A T T G A T C A T G A T T A G T G T T C G T T T T G T A T G A T T G A T C A T G A T T A G T G T T C G T T T T G T A T G A T T G A T C A T G A T T A G T G T T C G T T T T G T G T G A T T G A T C A T G A T T A G T G T T C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 190 200 210 ------------------+-------------------+-------------------+-
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
G G G G -
A A A A -
T T T T -
T T T T -
T T T T -
T T T T -
G G G G -
A A A A -
T T T T -
T T T T -
T T T T -
A A A A -
A A A A -
T T T T -
T T T T -
T T T T -
T T T T -
A A A A -
A A A A -
A A A A -
A A A A -
T T T T -
A A A A -
A A A A -
T T T T -
T T T T -
T T T T -
G G G G -
G G G G -
G G G G -
L18 R46 RM48 L23 EU723567 AY722693
220 230 240 ------------------+-------------------+-------------------+T G G T T G T T T T G G A T T T T T A T C T A A T G T G A C T G G T T G T T T T G G A T T T T T A T C T A A T G T G A C T T G T T G T C T T G G A T T T T T A T C T A A T G T A A C T G G T T G T T T T G G A T T T T T A T C T A A T G T G A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

L18 R46 RM48 L23 EU723567 AY722693 250 260 270 ------------------+-------------------+-------------------+G A A T T A A A T T G G G A A T G A T G A T G C A G A A T G G A A T T A A A T T G G G A A T G A T G A T G C A G A A T G G A A T T A A A T T G G G A A T G A T C A T G C A G A A T G G A A T T A A A T T G G G A A T G A T C A T G C A G A A T G - - - - - - - - - - - - - - - - - - - - - - - - - - A A T G - - - - - - - - - - - - - - - - - - - - - - - - - - A A T G
L18 R46 RM48 L23 EU723567 AY722693
280 290 300 ------------------+-------------------+-------------------+G C C T T C T C G A G T T T A A G A G G G T T A T A A G T G G C C T T C T C G A G T T T A A G A G G G T T A T A A G T G C C C T T C T C G A G T T T A A G A G G G T T A T A A G T G C C C T T C T T G A G T T T A A G A G G G T T A T A A G T G G C C T T C T C G A G T T T A A G A G G G T T A T A A G T G G C C T T C T C G A G T T T A A G A G G G T T A T A A G T G 310 320 330 ------------------+-------------------+-------------------+C T A A G C A G C A A G T T G T T G C T G G G A C T T T G C C T A A G C A G C A A G T T G T T G C T G G G A C T T T G C C T A A G C A G C A A G T T G T T G C T G G G A C T T T G C C T A A G C A G C A A G T T G T T G C T G G G A C C T T G C C T A A G C A G C A A G T T G T T G C T G G G A C T T T G C C T A A G C A G C A A G T T G T T G C T G G G A C T T T G C 340 350 360 ------------------+-------------------+-------------------+A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G A C C A C A T C A C T T T G G A G G C A G C A A G T G G T G 370 380 390 ------------------+-------------------+-------------------+A T A G T A A G A A T G T T T A T G A A G C C A A G G T G T A T A G T A A G A A T G T T T A T G A A G C C A A G G T G T A T A G T A A G A A T G T T T A T G A A G C C A A G G T G T A T A G T A A G A A T G T T T A T G A A G C C A A G G T T T A T A G T A A G A A T G T T T A T G A A G C C A A G G T G T A T A G T A A G A A T G T T T A T G A A G C C A A G G T G T 400 410 420 ------------------+-------------------+-------------------+G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G G G G A A A A G C C A T G G A T G A A C T T C A A G G A G G 430 440 450 ------------------+-------------------+-------------------+T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T T T C A G G A G T T C A A G C T T G C T G G T G A T G G C T
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693
L18 R46 RM48 L23 EU723567 AY722693

L18 R46 RM48 L23 EU723567 AY722693 460 --------------------+C C A A T G C T T A A C C A A T G C T T A A C C A A T G C T T A A C C A A T G C T T A A C C A A T G C T T A A C C A A T G C T T A A
Hnh 3.14. Kt qu so snh trnh t nucleotide gen cystatin ca cy lc 3.3.3. Kt qu so snh trnh t gen v protein cystatin 3.3.3.1. So snh trnh t nucleotide ca gen cystatin Kt qu so snh trnh t nucleotide ca gen cystatin ca 2 ging lc L18, L23 vi hai dng lc R46, RM48 c to ra bng cng ngh nui cy m t bo thc vt c s sai khc. Trong , trnh t nucleotide ca ging L18 v dng R46 c ngun gc t m so chu mt nc ging nhau hon ton. Dng RM48 c 19 v tr nucleotide sai khc so vi ging gc (L18). Nucleotide ca gen cystatin ging lc L18 c 14 v tr sai khc so vi ging L23. Gen cystatin ca cy u xanh tng i bo th, khng c s sai khc v gen cystatin ca ging chu hn tt v ging chu hn km [17]. Kt qu xc nh s sai khc nucleotide trn gen cystatin cy lc (bng 3.17) cho thy s sai khc v gen cystatin lc xut hin c vng exon v vng intron. Trong vng exon 1, ging L18 c 2 v tr sai khc vi L23 v 12 v tr sai khc vi dng RM48. vng exon 2, ging L18 c 4 v tr sai khc vi ging L23 v c 1 v tr sai khc vi dng RM48. Ngoi ra, gen cystatin ca ging L23 v RM 48 cn c 11 v tr sai khc nm trong on intron, s sai khc c th c trnh by bng 3.17 v cc v tr ng khung trn hnh 3.14. Nh vy, cng c ngun gc t ging L18, hai dng lc chn lc c s sai khc nhau v trnh t nucleotide ca gen cystatin, ng thi cng biu hin sai khc gia ging c kh nng chu hn km (L18) vi ging c kh nng chu hn tt hn (L23).

Bng 3.17. S sai khc v trnh t nucleotide trn gen cystatin ca ging v dng lc nghin cu STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 V tr 33 81 87 88 89 90 92 93 95 96 99 100 101 119 120 124 125 134 138 158 212 218 238 260 271 278 326 389 L23 T T A C A C A C A G A C A T G A G C T G G T G C C T C T L18 C C A C A C A C A G A C A A C T C G C A G T G G G C T G R46 C C A C A C A C A G A C A A C T C G C A G T G G G C T G RM48 C T C A C A C A G A C A G A C A G G C A T C A C C C T G Vng sai khc EXON EXON EXON EXON EXON EXON EXON EXON EXON EXON EXON EXON EXON INTRON INTRON INTRON INTRON INTRON INTRON INTRON INTRON INTRON INTRON INTRON EXON EXON EXON EXON

Kt qu nghin cu chng t, hiu qu vic x l tia gamma kt hp vi thi kh lm tng tn s t bin v gen cystatin so vi m so ch chu thi kh. 3.3.2.2. So snh trnh t nucleotide vng m ha V gen cystatin phn lp t DNA h gen ca lc c cha vng intron khng tn ti trong qu trnh dch m, nn chng ti tin hnh loi b intron v ghp ni cc on exon, kt qu thu c on m ha cystatin di 297 nucleotide. So snh vi 6 trnh t on m ha protein ca gen cystatin thc vt c kch thc tng ng cng b trn ngn hng gen quc t (ph lc 1). Kt qu nh gi thng qua phn tch sai khc, tng ng v xc nh mi quan h v gen cystatin thc vt trnh by bng 3.18 v hnh 3.15. Bng 3.18. tng ng v sai khc on m ho mt s trnh t gen cystatin
tng ng (Percent Identity)
sai khc(Divergence)
Trong : 1. Ging lc L18 (Arachis hypogaea L.)

2. Dng chn lc R46 3. Dng chn lc RM48 4. Ging lc L23 (Arachis hypogaea L.) 5. Lc (Arachis hypogaea- m s gen trn NCBI: AY722693) [134];

6. La (Oryza. Sativar -m s gen trn NCBI: S49967) [135]; 7. Kiwi (Actinidia deliciosa -m s gen trn NCBI: AY390352) [136]; 8. La (Oryza sativar japonica -m s gen trn NCBI: AB125973) [137]; 9. u xanh (Vigna radiata -m s gen trn NCBI: AM712476) [138]; 10. i mch (Hordeum ulgare subsp-m s gen trn NCBI: Y12068) [139].
Kt qu nghin cu trn bng 3.16 cho thy 4 trnh t on m ha gen cystatin lc c mc tng ng vi 6 trnh t cystatin ca cc cy trng khc cng b trn ngn hng gen quc t t 45,9% n 100%. Cc trnh t nucleotide ca lc c tng ng t 98,0% n 100%. c bit, khng c bt k s sai khc no v nucleotide ca ging lc L18 vi dng chn lc R46 v trnh t nucleotide ca ging Arachis hypogaea L. trn ngn hng gen quc t (m s AY722693) [134]. on m ha ca gen cystatin lc c tng ng gn nht vi cystatin u xanh (81,3%) v xa nht vi cystatin qu kiwi (45,9%).
Hnh 3.15. Mi quan h di truyn ca mt s on m ho gen cystatin thc vt S hnh 3.15 th hin mi quan h di truyn ca on m ha gen cystatin ca lc vi 6 cy trng khc. Trn c s phn tch trnh t on m

ho gen cystatin, kt qu trn s cho thy 10 trnh t c phn thnh 2 nhm chnh: Nhm I phn thnh 3 nhm ph ln v nhiu nhm ph nh: Nhm ph ln 1 c nhm ph nh 1 gm on m ha cystatin ca cc dng v ging lc c quan h gn gi vi nhau t l phn tn ch trong khong 0,0 n 2,1. Nhm lc ny c quan h gn nht vi cystatin u xanh (ging MN93 ngun u nhp ni t Thi Lan, m s AM712476) [138]. Nhm ph nh 2 l s phn b ca cystatin la (m s S49967) [135]. Nhm ph ln 3 gm i mch (m s Y12068) [139] v la (m s AB125973) [137]. Nhm II c khong cch xa nht vi cc nhm cn li v ch c mt trnh t on m ho gen cystatin phn lp t qu kiwi (m s AY390352) [136], c t l sai khc vi cystatin cy lc l 41,5%. S thay i trnh t nucleotide c th c hoc khng lm thay i trnh t cc amino acid ca protein. Kt qu nghin cu cystatin trn u xanh ca Chu Hong Mu v tg (2008) khng nhn c s sai khc no trn chui polypeptid [17]. c bit vi phn t cystatin, kh nng hot ng ca chng ph thuc rt ln vo cc on bo th. Vng bo th ca a s cystatin l glycine (G) gn u NH2, vng trung tm cha 5 amino acid l glutaminamino acid bt k-valine- amino acid bt k-glycine (QxVxG) v u COOH cha tryptophan (W)... Cystatin ca cy lc nh th no l l do chng ti tip tc nghin cu v trnh t amino acid trn chui polypeptide. 3.3.2.3. So snh trnh t amino acid ca protein cystatin Trnh t nucleotide on m ho gen cystatin ca ging lc L23, L18 v 2 dng lc nghin cu c suy din bng phn mm DNAstar to phn t protein gm 98 amino acid, vi m m u AUG quy nh tng hp methionine (M) v m kt thc l UAA. Kt qu so snh 4 trnh t nghin cu bng phn mm chuyn dng BioEdit biu din hnh 3.16.

So snh trnh t amino acid ca cc dng lc nghin cu, chng ti d dng pht hin c 4 trnh t amino acid nghin cu u cha 2 vng bo th ca nhm l LARFAV v QVVAG. V tr c th ca hai on bo th ny l: L22A23R24F25A26V27 v Q49V50V51A52G53. i chiu vi cch phn loi gen cystatin ca Margis v tg (2008), Martinez v tg (2009), chng ti pht hin gen cystatin ca cc dng v ging lc nghin cu thuc nhm phn loi I ca phytocystatin [89], [90].
Hnh 3.16. So snh trnh t amino acid ca 4 mu nghin cu
Kt hp vi kt qu phn tch trn gen, nghin cu ca chng ti ch ra rng: v tr ca glutamine 34 (Q34) v asparagine 35 (N35) l cp amino acid c m ho bi nhng b ba pha u on ni 2 exon ca cystatin. V tr tng ng vi n l v tr arginine 34 (R34) v asparagine 35 (N35) ca cystatin dng RM48. Hai amino acid v tr 34 khc nhau l glutamine v arginine, l 2 amino acid khc nhau v c im cu to gc R, trong glutamine l amit ca glutamic acid; khc vi arginine l amino acid thuc nhm amino acid kim. S sai khc ny c lin quan g n cu trc phn t v hot ng ca cystatin trong c ch hot ng ca cysteine proteinase, l gi cho nhng nghin cu tip theo v cystatin lc.

Bn cnh s sai khc amino acid v tr 34, kt qu so snh trnh t amino acid ca ging lc L23, L18 v 2 dng R46, RM48 chng ti pht hin thm 6 v tr sai khc trn chui amino acid, nng tng s amino acid thay i trn cystatin lc l 7 v tr. S sai khc c th th hin v tr s 29, 30, 31, 32, 33, 34 v 36 ca cc dng v ging nghin cu (hnh 3.16). Nu ly cystatin ca ging L18 l cn c so snh, chng ti nhn thy, ging lc L23 c mt v tr sai khc l v tr amino acid 36, alanine thay th glycine. S sai khc ln nht ca cystatin ging lc L18 l cystatin ca dng RM48 (7 v tr sai khc). Phn tch cc mi lin quan khc trnh t amino acid ca cc dng, ging phn tch vi mc chu hn khc bit cho thy, ging L23 c kh nng chu hn tt, sai khc vi cc trnh t ca nhm chu hn km v tr 36, cystatin dng RM48 cng c s sai khc ny. cc v tr 29, 30, 31, 32, 33, 34 l nhng v tr ln cn trong vng CYS ca dng RM48 c s khc bit vi cc trnh t nghin cu, rt c th s khc bit ny gp phn lm tng i lc ca cht c ch vi trung tm hot ng ca enzyme, do lm tng cng kh nng chng chu hn cng nh chng chu vi cc yu t bt li khc ca ngoi cnh.

Bng 3.19. Thnh phn amino acid ca protein cystatin cy lc Amino acid k nc * + + * + + + * * * + + + + + + * + * + 45,92% so vi tng amino acid 54,08% so vi tng amino acid 28,57% so vi tng amino acid Amino acid a nc Amino acid khng thay th
Loi amino acid A (Ala) D (Asp) E (Glu) I (Ile) M (Met) R (Arg) C (Cys) G (Gly) L (Leu) F (Phe) N (Asn) Q (Gln) H (His) K (Lys) P (Pro) S (Ser) V (Val) T (Thr) W (Trp) Y (Tyr) (*) Amino acid k nc (+) Amino acid a nc (-) Amino acid khng thay th
RM48 14 5 9 3 2 5 0 6 7 4 8 3 2 7 2 6 9 4 2 1 45 53 28
L18 13 4 10 3 2 3 0 7 7 4 7 4 3 9 2 6 9 2 2 1 45 53 28
L23 14 4 10 3 2 3 0 6 7 4 7 4 3 9 2 6 9 2 2 1 45 53 28

Kt qu phn tch thnh phn amino acid ca cystatin ging lc L23, L18 v 2 dng chn lc R46, RM48 trn bng 3.19 cho thy c s khc bit kh ln v amino acid ca cc dng v ging lc. Tuy c s khc bit v mt s loi amino acid trong thnh phn protein cystatin cy lc, nhng kt qu thng k tng s cc amino acid a nc, k nc hay nhm cc amino acid thit yu cho ngi khng nhn c s khc bit gia cc dng v ging chn lc. Lng amino acid k nc chim t l tng i ln trong thnh phn cystatin lc (45,92%) cho php d on s c tr ca cystatin lin quan n tnh chu hn ca cy lc. R rng nhng protein k nc s phn b trn mng t bo hoc h mng, cn protein a nc tan trong dch nguyn sinh. Do , vi chc nng c ch hot ng cysteine proteinase rt c th cystatin cy lc tham gia bo v mng t bo s c hiu qu trong iu kin thiu nc cc oan. Phn tch gen cystatin lc vi cystatin ca nhiu loi thc vt khc, chng ti xc nh c v tr ca cystatin lc thuc nhm I ca h phytocystatin. nh hng ca chiu x kt hp vi thi kh lm thay i trnh t nucleotide trn gen v thay i amino acid trn protein cystatin ca cc dng lc chn lc so vi ging gc. Tuy nhin, kh nng chu hn ca thc vt l tnh trng a gen v bn thn gen cystatin l gen lin quan n nhiu kh nng chng chu khc ca thc vt. Kt qu nghin cu thu c s l cn c nghin cu su hn v gen cystatin lc.

KT LUN
1. X l m so ca 10 ging lc bng k thut thi kh xc nh c ging lc L18 c kh nng chu mt nc thp nht. ngng chn lc thi kh 9 gi kt hp vi chiu x tia gamma 2krad lm gim t l ti sinh cy v lm xut hin cc kiu hnh thp cy lc. 2. ti sinh c 198 dng lc c ngun gc t m so chu mt nc v m so chu chiu x tia gamma kt hp vi thi kh gy mt nc ca ging lc L18. tuyn chn c 3 dng lc c kh nng chu hn cao l RM48, R46 v RM47. Dng RM48 c khong cch di truyn so vi ging gc l 22%, dng RM47 v dng R46 c khong cch di truyn vi ging gc L18 l 9,5%. 3. H s a dng di truyn ca cc dng lc chn lc th h th Nm l 11,09%. C 5 ch th RAPD c trng c pht hin 2 dng lc RM48 v RM47: RM48/OPA07-750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348-200bp; RM47/OPH08-250bp. 4. Tch dng thnh cng gen cystatin t DNA h gen ca mt s dng lc chn lc v ging gc. Gen cystatin ca cy lc c 461 nucleotide, c 2 exon v 1 intron, thuc nhm I ca phytocystatin. on m ha gen cystain ca cc dng, ging lc nghin cu c s tng ng cao nht vi cystatin ca u xanh (81,3%), thp nht vi cystatin ca qu kiwi (42,9%). Protein do gen m ha c 98 amino acid, trong 45,92% amino acid k nc, 54,08% amino acid a nc v 28,57% amino acid thit yu so vi tng amino acid ca protein cystatin. 5. Trnh t amino acid ca cystatin dng lc RM48 c s sai khc ln nht so vi ging gc L18 7 amino acid. S thay th amino acid ca dng RM48 bi amino acid ca ging gc cc v tr 29 ((Glu Asp), 30 (HisThr),
31 (AsnThr), 32 (Lys Arg), 33 (Lys Asn), 34 (Glu Arg), 36 (Gly


Ala). Nhng thay i amino acid l gi tip tc tm kim mi lin
quan ca cystatin vi kh nng chng chu hn. NGH 1. Tip tc theo di, phn tch v bi dng 3 dng lc u vit l R46, RM47, RM48 gii thiu kho nghim ging. 2. Thit k vector mang gen cystatin v chuyn vo cy lc cng nh cc loi cy trng khc nghin cu nh hng ca cystatin n kh nng chu hn v mt s c tnh khc thc vt.

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Ph lc 1. on m ho ca gen cystatin mt s ging cy trng
STT 1 2 3 4 5 6 7 8 9 10 11
Tn khoa hc Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis diogoi Arachis hypogaea Oryza sativar(OCI) Actinidia deliciosa Oryza sativa japonica group (gKOCIII) Vigna radiata Horrdeum ulgare subsp
Tn thng dng Ging lc L18 Dng chn lc R46 Dng chn lc RM48 Ging lc L23 Lc Lc La kiwi La u xanh (MN93) i mch
M s FN811133 HE578279 FR745399 FR691053 EU723567 AY722693 S49967 AY390352 AB125973 AM712476 Y12068
s nucleotide 297 297 297 297 297 297 309 351 327 267 327
Nm cng b 2010 2011 2010 2010 2008 2004 1993 2004 2008 2007 2005
Ti liu trch dn
133 134 135 136 137 138 139
Tai lieu chia se tai: wWw.SinhHoc.edu.vn Ph lc 2. Trnh t 25 mi ca phn ng RAPD

TT 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Tn mi OPA1 OPA2 OPA3 OPA4 OPA5 OPA6 OPA7 OPA8 OPA9 OPA10 OPA11 OPA12 OPA13 OPA14 OPA15 OPB05 OPB10 OPH04 OPH08 OPF09 OPF10 OPN05 0PQ02 OPQ05 UPC348 Trnh t (5->3) TCCACGGGCA CACCATCGTG CCTGGGTCCA ACCCAGGTTG GTGACCAGAG GACGACCGCA CTTCGCTGTC CCGCCGGTAA CCGCGTTGAG ACGCGAACCT AGCCAAGGAC TTCCCGCACT ACCCTGAGGA CAAGCCGTGA GTGGATCGTC CAGAGGTTCC GACCAGAGGT GGCTCTGGGT GGGATAAGGG ACGGCAATGG CCCGAGAAAC GGTTGGGCCA GACCAGCCCA CCCGAAGCAA CACGGCTTCC
Tai lieu chiatrng lc thuwWw.SinhHoc.edu.vn nilon Ph lc 3. Cch se tai: ng khng dng cng ngh che ph
- Thi v gieo trng: Lc thu ng c th gieo trng t 15/8 30/9, tt nht t 15/8 10/9. - Chn t : Chn t ct pha tht nh ch ng ti tiu v d thot nc. Lm t nh, sch c di, t l ht t c ng knh nh hn 1cm chim trn 70%, ln lung rng 90cm, cao 15cm, rnh rng 25cm. Nu t t c th p dng phng php lm t ti thiu. - Phn bn v phng php bn phn Liu lng phn bn (tnh cho 1 so Bc b) Phn chung hoai: 300 350kg Phn ln super: 15 20kg m ur: 2,5 3kg. Kali clorua: 4 5kg Vi bt: 20kg Phng php bn phn: + Bn lt : 100% PC + 100% ln + 50% m vo cc hng rch. + Bn thc ln 1: Lc cy lc c 2 3 l tht bn 50% lng m kt hp vi xi ph vng to iu kin cho vi sinh vt nt sn hot ng. + Bn thc ln 2: Khi cy lc c 6 7 l tht, bn ton b lng kali. + Bn thc ln 3: Khi cy tt hoa, bn 50% lng vi cn li, kt hp vi vun cao lung chng v to t ti xp, thun li cho cy lc m tia, lm c. Mt , khong cch v phng php gieo ht Mt trung bnh t 34 36 cy/m2. Khong cch thch hp t 18 20cm x 30cm. Tin hnh rch 3 hng dc theo lung su 3 4cm ri gieo ht, gieo 2 ht/hc theo khong cch nh trn. Nu p dng phng php lm t ti thiu phi s dng t mn bng cch trn phn chung ( c vi ln) vi tru v t bt hoc t hun ph ln trn ht sau khi gieo (gieo hc vi khong cch nh trn). Mt s bin php k thut khc Lc cn c phi li trn nong, nia, di nng nh 2 ngy trc khi gieo (phi c c). Chn nhng ht tt gieo, ht cn c ngm ny mm trc khi gieo... Phun Boocdo 1%, Zinep 0,3%, Danconil 0,2% khi thy c biu hin ca bnh g st, m l. Phun Padan 95SP, Opatox, Beettox khi thy lc b su xanh, su khoang, b tr, b phn hay rp mui gy hi. Thu hoch: Thu hoch lc khi c t 80 90% s c gi. Sau khi thu hoch cn gom nilon li mt ch v t, trnh nhim mi trng . http://agriviet.com/nd/724-cach-trong-lac-dau-phong-thu-dong-dat-nang-suatcao/[17 - Aug - 2007]

Ph lc 4. Hot ca -amylase v hm lng ng ca 10 ging lc trong iu kin hn sinh l
Hot ca - amylase thi im gy hn (VH/ mg ht ny mm/30/300C) Tn ging lc

3 ngy 5 ngy 7 ngy 9 ngy
Hm lng ng kh thi im gy hn (% mg ht ny mm)

X SX
L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG 0,17 0,01 0,41 0,05 0,29 0,05 0,42 0,01 0,11 0,01 0,19 0,01 0,11 0,01 0,37 0,02 0,48 0,04 0,31 0,04
X SX
0,43 0,08 0,44 0,03 0,35 0,01 0,44 0,03 0,38 0,04 0,36 0,03 0,48 0,02 0,42 0,04 0,51 0,02 0,42 0,04
X SX
0,60 0,06 0,48 0,01 0,48 0,01 0,52 0,02 0,48 0,04 0,44 0,02 0,56 0,11 0,55 0,10 0,70 0,05 0,44 0,03
X SX
0,56 0,03 0,27 0,02 0,22 0,01 0,35 0,03 0,45 0,06 0,40 0,05 0,46 0,07 0,31 0,07 0,59 0,03 0,17 0,01
X SX
2,79 0,21 1,29 0,02 2,47 0,52 2,78 0,03 1,46 0,25 1,73 0,02 2,09 0,33 1,90 0,03 1,50 0,22 1,54 0,33
X SX
6,16 0,86 3,24 0,57 2,50 1,33 5,60 1,05 4,05 0,48 3,25 0,66 5,18 0,90 6,46 0,75 4,09 0,40 2,83 0,61
X SX
6,52 0,64 4,90 0,22 4,96 0,45 6,53 0,56 4,24 0,21 4,10 0,35 7,79 0,36 6,76 0,34 3,86 0,54 3,10 0,29
X SX
4,68 0,71 3,31 0,27 4,56 0,56 5,18 1,05 2,78 0,24 3,51 0,66 5,29 0,42 0,44 0,38 2,93 0,45 2,85 0,35

Ph lc 5. T l cy sng, cy phc hi v ch s chu hn tng i ca 10 ging lc trong iu kin gy hn nhn to
% Tn ging lc CKH sau 1 ngy hn L05 L16 L18 L23 L24 MD7 MD9 SD30 V79 BG 70,5 69,5 56,7 80,5 68,5 66,7 66,0 68,5 70,5 76,7
% CKH sau 3 ngy hn 58,4 49,1 33,5 61,5 52,0 52,3 55,4 56,0 60,1 56,6
% % CKH sau 5 CHP sau 1 ngy hn ngy hn 31,2 33,5 25,3 45,3 33,5 34,4 32,7 28,5 28,0 49,5 31,0 32,5 26,7 30,5 25,0 30,1 31,5 27,6 27,5 28,5
% CHP sau 3 ngy hn 31,2 33,5 43,3 45,3 35,0 32,5 32,0 37,6 28,0 39,0
% CHP sau 5 ngy hn 52,0 50,5 40,5 50,5 52,5 52,7 51,8 49,5 48,8 51,4
Ch s chu hn tng i
Xp th t
5699,35 5385,08 3736,30 7297,52 5391,12 5425,24 5448,35 5416,26 5311,62 6760,26
3 3 4 1 3 3 3 3 3 2

Ph lc 6. Hot ca - amylase v hm lng ng trong iu kin hn sinh l ca cc ging v dng chn lc th h th Nm
Hot amylase thi im gy hn Tn dng v ging lc

R44 R46 R48 RM46 RM47 RM48 RM49 L18 L23 VT 3 ngy 5 ngy 7 ngy 9 ngy
Hm lng ng kh thi im gy hn
X SX
0,18 0,01 0,19 0,01 0,19 0,02 0,20 0,01 0,20 0,04 0,23 0,01 0,15 0,04 0,20 0,03 0,22 0,01
X SX
0,42 0,06 0,52 0,05 0,50 0,03 0,45 0,04 0,53 0,05 0,54 0,02 0,47 0,02 0,52 0,06 0,47 0,03
X SX
0,48 0,04 0,63 0,04 0,54 0,04 0,60 0,02 0,58 0,04 0,66 0,07 0,55 0,03 0,53 0,02 0,59 0,01
X SX
0,14 0,02 0,19 0,03 0,19 0,05 0,25 0,03 0,21 0,05 0,20 0,04 0,23 0,02 0,23 0,06 0,26 0,02
X SX
0,94 0,04 1,07 0,04 1,05 0,13 1,30 0,03 1,17 0,13 1,42 0,05 0,93 0,18 1,07 0,03 1,11 0,02
X SX
1,22 0,01 1,21 0,03 1,15 0,02 1,24 0,04 1,32 0,06 1,51 0,08 1,14 0,02 1,14 0,02 1,28 0,03
X SX
1,23 0,05 1,74 0,12 1,40 0,14 1,37 0,03 1,45 0,09 1,81 0,10 1,28 0,02 1,38 0,07 1,51 0,03
X SX
0,85 0,07 0,93 0,03 0,98 0,09 0,77 0,10 1,16 0,09 0,59 0,03 0,93 0,04 0,95 0,02 0,77 0,04
VH/ mg ht ny mm/30/300C
% mg ht ny mm

Chon Dong Chiu Han Va Phan Lap Gen Cystatin Lien Quan Den Tinh Chiu Han o Cay Lac

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LUN N TIN S SINH HC

Thi Nguyn - 2011

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LUN N TIN S SINH HC

Chuyn ngnh: Di truyn hc M s: 62 42 70 01

Hng dn khoa hc: 1. PGS.TS Chu Hong Mu 2. PGS. TS Nguyn Th Tm

Thi Nguyn - 2011

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

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1.5.2. Cu trc khng gian v c ch c ch ca cystatin . . . . . . . . . . . . .

1.5.3. Chc nng ca cystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 1.5.4. Gen m ha cystatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Phng php nghin cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

2.3.1. Phng php nui cy in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.1. c im nng sinh hc cc dng lc chn lc th h th Nht v th Ba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

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3.3.1. Khuych i gen cystatin t DNA h gen ca cy lc . . . . . . . . . . .

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

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Hnh 3.4. Hnh 3.5.

Hnh 3.7 Hnh 3.8

Hnh 3.9. Hnh 3.10.

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TNG QUAN TI LIU

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