Ti liu chia HC THI NGUYN I s ti: wWw.SinhHoc.edu.

vn
TRNG I HC S PHM
----------- -------------

NCS. NG MNH DNG

BO CO SEMINA 2

PHN LP V XC NH TRNH T GEN M HA PROTEIN V (CP) CA VIRUS GY BNH XANH LN

Ngi hng dn khoa hc : PGS.TS. CHU HONG H

TS. L VN SN Ti liu chiaThi Nguyn - 2012 s ti: wWw.SinhHoc.edu.vn

Bnh xanh ln trn cy bng

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Theo thng k c khong 20 loi virus khc nhau gy bnh trn cy bng th bnh xanh ln hay cn gi l bnh xanh l (Cotton blue disease) l mt trong s nhng bnh virus gy thit hi nng n nht (Brown, 1992). Bnh xut hin ph bin ti cc nc c

chu Phi, chu v Nam M (Bonacic et al., 1997).

Vit Nam, trong cc loi bnh hi trn cy bng hin nay th xanh ln c xem nh l mt tc nhn gy thit hi nhiu nht.

Qua cc nghin cu ca vin Nghin cu cy bng v PTNN Nha

H cho thy tt c cc ging bng lui (Gossypium hirsutum L.) ang trng ph bin Vit Nam u b nhim bnh xanh ln, cc ging khng ca chu Phi, Nam M v Thi Lan sau khi c nhp ni v trng th nghin ti Nha H cng u nhim bnh. Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn Bnh xanh ln thng xut hin trong giai on cy non nn gy ra thit hi rt ln c th n 100% sn lng. Biu hin u tin trn nhng l non lm cho l phng ln, ra l cong

nh v pha di, nhng vng tht l gia cc gn chnh c mu

xanh sng, khi l gi th ton b l s b cong xung, cu trc gin, c mu xanh sm ng nht. Cy nhim bnh c t thn v cnh ngn li dng zic-zc, khng ha g, ln hoc nm b ra, s lng n hoa v qu, kch thc qu gim nhiu dn n gim c nng sut ln cht lng bng si (Bonacic et al., 1997). Bnh xanh ln c truyn trong t nhin nh mi gii duy nht l rp bng (Aphis gossypii) (Cauquil & Follin, 1983). Ngoi ra, bnh xanh ln c th truyn nhn to qua ghp cy, nhng khng truyn qua ht bng cng nh cc phng thc c hc khc. Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn Nguyn nhn gy bnh xanh ln c chn on l do virus gy ra. Da trn cc triu chng ca bnh v phng thc ly truyn thng qua rp bng, cc nh khoa d on virus gy bnh xanh ln c kh nng thuc h virus Luteoviridae. Gn y vo nm 2005, Corra v cng s thng qua phn lp, xc nh v so snh trnh t ca gen v v mt phn ca gen RdRp khng nh c virus gy bnh xanh ln l thuc h Luteoviridae,

c kh nng xp vo chi Polerovirus v t tn virus ny l Cotton

leafroll dwarf virus (CLRDV) (Corra et al., 2005).

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

CLRDV l virus RNA si n dng, h Luteorividae, chi Polerovirus.

Genome Polerovirus bao gm 6 ORF, c nh s t 0 n 6 (Mayo MA & Ziegler-Graff V,1996). Protein v (CP) chnh c m ha bi

ORF3 u 3 ca genome. Protein v virus cn c m ha bi

ORF4, ORF4 nm trong trnh t gen CP nhng khung c m khc (Tacke E et al., 1993).

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

VT LIU NGHIN CU

-Ngun bnh virus: L bng nghi nhim bnh xanh ln do Vin Nghin cu v Pht trin cy bng Nha H cung cp

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PHNG PHP NGHIN CU

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Phng php RT-PCR s dng cho nhn bn gen virus CLRDV Tch RNA: Khong 250 mg l nghi nhim bnh (CLRDV) c nghin trong nit lng. Bt nghin thu c cho vo ng 2 ml v b sung 1 ml Trizol (Invitrogen). Cc bc tch chit RNA tng s c thc hin theo phng php ca Napoli v cng s (1990).

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PHNG PHP NGHIN CU

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Tng hp cDNA: cDNA c tng hp theo kt RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas). Phn ng cDNA c thc hin trong th tch 20 l gm c cc thnh phn sau: 10 ng - 5 g RNA tng s; 250 ng mi ngu nhin. B sung nc kh ion c x l DEPC ti th tch 12 l. 70C/5 pht, sau t vo . Tip tc b sung cc thnh phn vo ng phn ng trn: 4l m RT 5X tng hp cDNA; 1l riboLock Ribonuclease inhibitor (20u/l); 2 l dNTP (10 mM). Hn hp sau o v ly tm nh; 25C/5 pht. Tip tc b sung 1 l enzyme RevertAid (200/l) Thc hin chu trnh nhit nh sau: 25C/10 pht, 42C/60 pht, 72C/10 pht, 40C/10 pht. Ti liu chia s ti: wWw.SinhHoc.edu.vn

PHNG PHP NGHIN CU

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RT-PCR:

Cc on gen quan tm c nhn ln bng cc cp mi c hiu.

Si cDNA c dung lam khun, chu k nhit nh sau: 94C /3 pht, 25 chu k gm 94C/40 giy, t 50-56C /40 giy, 72C/1 pht 30 giy, 72C/10 pht v kt thc 4C. Sn phm c kim tra trn gel agarose 0,8 % .

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

Tch dng sn phm PCR Cc sn phm PCR c tch dng da trn nguyn tc b sung A-T s dng vector pBT. Phn ng gn gen vao vector tch dng pBT c thc hin theo thanh phn va chu k sau: Thanh phn phn ng gn gen vao vector pBT
STT 1 2 3 4 5 6 Thnh phn m gn gen vo vector 2X DNA mu Vector tch dng pBT rATP PEG 4000 T4 ligase Tng Th tch (l) 10 7,5 1,0 0,5 2,0 1,0 20l

Hn hp c nhit 22oC trong 2 gi v sau c bin np vo t bo kh bin E.coli DH5 v c cy tri trn mi trng LB c c cha ch th chn lc. Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn * Bin np plasmid ti t hp vo t bo kh bin E. coli bng sc nhit B sung 20 l vector gn gen vo ng ng t bo kh bin v

trn nh. Hn hp c t trong 30 pht, ri 420C 1 pht.

Sau t li vo 5 pht. B sung 300 l mi trng LB lng, hn hp c nui 370C, lc 200 v/p trong 1 gi. S dng que cy tri, tri 150- 250 l dch khun ln a mi trng LB c c cha 100 mg/l Ampicillin, X-gal 30 mg/l, IPTG 100 M. a 370C trong 16 gi. Tch chit plasmid t t bo vi khun Tch chit plasmid theo ch dn ca kit Plasmid Extraction Kit (Bioneer). Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Phng php xc nh v x l trnh t gen thu c Trinh t gen c xc nh trn my c t ng ABI PRIM 3100 Avant Genetic Analyzer bng cch s dng b ho cht sinh chun bigDye Terminator v3.1 Cycle Sequencing. Kt qu c x l bng phn mm DNAstar.

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KT QU

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Nhn bn gen ca CLRDV bng RT-PCR RNA tng s t mu bng nghi nhim bnh sau khi tch chit c s dng lm khun tng hp cDNA nh enzyme phin m ngc (Reverse transcriptase) vi cp mi ngu nhin di 6 nucleotide (random primer) (theo b Kit ca Fermentas). cDNA sau c lm mu cho phn ng PCR khuch i cc on DNA mang gen m ho cho protein v (CP) ca CLRDV vi cp mi c thit k c hiu bng 1 . Bng 1. Trnh t v cc thng s cn thit ca cc mi phn lp gen
Mi CP-CLRDV-F CP-CLRDV-R Trnh t (5-3) GAATTCATGAATACGGTCGTGGGTA CTCGAGTTTTGGATTGTGGAATTGG c im 14CG+10AT CP 12CG+12AT 56 615 Gen phn lp Tgm (oC) KT (bp)

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

Kt qu in di sn phm RT-PCR trn hnh

1 cho thy, sn phm RT-PCR rt c hiu, ch cho mt phn on DNA duy nht vi kch thc tng ng tnh ton l thuyt. c th kt lun chnh xc cc phn on ny c phi l ca virus CLRDV hay khng, cc phn on ny tip tc c dng ha bng cch gn vo vector pBT v bin np vo t bo kh bin E.coli DH5 vi mc ch la chn nhng dng khun lc dng tnh, tch chit plasmid xc nh trnh t.
750 500

M 1

Hnh 1. Kt qu in di sn phm RT-PCR nhn gen CP ca CLRDV M: Thang DNA chun 1 kb; 1: CLRDV-Nha H; 2: i chng m

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Ti liu chia s ti: wWw.SinhHoc.edu.vn Dng ha gen vo vector pBT Sn phm RT-PCR sau khi thi gel v gn vo vector pBT c bin np vo t bo kh bin E. coli DH5. Da vo phng php chn lc khun lc xanh trng v colony-PCR, nhng dng khun lc dng tnh c la chn tinh sch phc v cho vic c trnh t. pUC18F/pUC18R l cp mi nm trn vector pBT, nu on gen c chn vo ng v tr ca plasmid th theo l thuyt sn phm colony-PCR chn dng pBT/CP-CLRDV l 779 bp (615+164) v so vi i chng m pBT l 164 bp.

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Ti liu chia s ti: wWw.SinhHoc.edu.vn Sn phm colony-PCR c in di trn hnh 2 cho thy, cc phn on thu c c kch thc tng ng vi tnh ton l thuyt chng t vic
bp M 1 2 3 4 5

dng ha gen vo vector pBT

thnh cng. Chng ti tip tc la chn mt dng
1000 750

dng tnh nui lng tch chit

plasmid phc v c trnh t.
Hnh 2. Kt qu in di sn phm colony-PCR chn dng pBT/CP-CLRDV M. marker 1 Kb. 1-4: Dng khun lc 1-4; 5: i chng m (khun lc xanh-mang plasmid khng gn gen quan tm)

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

Xc nh v so snh trnh t gen ca CLRDV

Gen c c trnh t trn my c t ng ABI PRIMS 3100 Avant Genetic Analyzer bng cch s dng b ho cht sinh chun BigDye Terminator v3.1 Cycle Sequencing. Sau khi c trnh t, trnh t ny c so snh trong BLAST ca NCBI. Kt qu cho thy, on gen phn lp c l on gen CP ca virus CLRDV.

Bng cc phn mm my tnh chuyn dng nh BioEdit, DNAstar,

Chromas; trnh t CP thu c c x l v phn tch. Di y l hnh nh v kt qu c trnh t gen CP ca dng virus CLRDV-Nha H (hnh 3).

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

M13-R

Bam HI

CP-CLRDV-F

.
CP-CLRDV-R

Ti liu chia s ti: wWw.SinhHoc.edu.vn

1 1 1

31 31 31

61 61 61

91 91 91

121 121 121

151 151 151

181 181 181

10 20 30 ------------------+-------------------+-------------------+A T G A A T A C G G T C G T G G G T A G A A G A A C G A T C CP-CLRDV-Agh A T G A A T A C G G T C G T G G G T A G A A G A A C G A T T CP-CLRDV Bra A T G A A T A C G G T C G T G G G T A G A A G A A C G A T T CP-CLRDV VN 40 50 60 ------------------+-------------------+-------------------+A A T G G A A G A A G A C G A C C A C G T A G G C G C A A C CP-CLRDV-Agh A A T G G A A G A A G A C G A C C A C G T A G G C G C A A C CP-CLRDV Bra A A T G G A A G A A G A C G A C C A C G T A G G C G C A A C CP-CLRDV VN 70 80 90 ------------------+-------------------+-------------------+A G G C G T C G G C A A A A T C A G C C A G T G G T T G T G CP-CLRDV-Agh A G G C G T C G G C A A A A T C A G C C A G T G G T T G T G CP-CLRDV Bra A G G C G T C G G C A A A A T C A G C C A G T G G T T G T G CP-CLRDV VN 100 110 120 ------------------+-------------------+-------------------+G T C C A A G C C C C T C G G A A C A C A C A A C G C A G A CP-CLRDV-Agh G T C C A A G C C C C T C G G A A C A C A C A A C G C A G A CP-CLRDV Bra G T C C A A G C C C C T C G G A A C A C A C A A C G C A G A CP-CLRDV VN 130 140 150 ------------------+-------------------+-------------------+A G A C G A C G A A G A C G A G G A G G T C G T A A T A G G CP-CLRDV-Agh A G A C G A C G A A G A C G A G G A G G T C G T A A T A G G CP-CLRDV Bra A G A C G A C G A A G A C G A G G A G G T C G T A A T A G G CP-CLRDV VN 160 170 180 ------------------+-------------------+-------------------+A C A G G A G G A C G C A T T C C T G G A G G A C C A G G A CP-CLRDV-Agh A C A G G A G G A C G C A T T C C T G G A G G A C C A G G A CP-CLRDV Bra A C A G G A G G A C G C A T T C C T G G A G G A C C A G G A CP-CLRDV VN 190 200 210 ------------------+-------------------+-------------------+G C T T C A A G C G A G A C A T T T G T T T T C T C A A A A CP-CLRDV-Agh G C T T C A A G C G A G A C A T T T G T T T T C T C A A A A CP-CLRDV Bra G C T T C A A G C G A G A C A T T T G T T T T C T C A C A A CP-CLRDV VN

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

211 211 211

241 241 241

271 271 271

301 301 301

331 331 331

361 361 361

391 391 391

220 230 240 ------------------+-------------------+-------------------+G A C A G T C T C T C G G G A A G T T C C T C A G G A T C A CP-CLRDV-Agh G A C A G T C T C T C G G G A A G T T C C T C A G G A T C A CP-CLRDV Bra G A C A G T C T C T C G G G A A G T T C C T C A G G A T C A CP-CLRDV VN 250 260 270 ------------------+-------------------+-------------------+A T C A C G T T C G G G C C G T C T T T A T C A G A T T G C CP-CLRDV-Agh A T C A C G T T C G G G C C G T C T T T A T C A G A T T G C CP-CLRDV Bra A T C A C G T T C G G G C C G T C T T T A T C A G A T T G C CP-CLRDV VN 280 290 300 ------------------+-------------------+-------------------+C C G G C A T T C A G C A A T G G A A T A C T C A A G G C C CP-CLRDV-Agh C C G G C A T T C A G C A A T G G A A T G C T C A A G G C C CP-CLRDV Bra C C G G C A T T C A G C A A T G G A A T G C T C A A G G C C CP-CLRDV VN 310 320 330 ------------------+-------------------+-------------------+T A C C A T G A A T A T A A G A T C T C A A T G G T C T T A CP-CLRDV-Agh T A C C A T G A A T A T A A G A T C T C A A T G G T C T T A CP-CLRDV Bra T A C C A T G A A T A T T A G A T C T C A A T G G T C T T A CP-CLRDV VN 340 350 360 ------------------+-------------------+-------------------+T T G G A G T T C A T C T C C G A G G C C T C T T C A A C A CP-CLRDV-Agh T T G G A G T T C A T C T C C G A G G C C T C T T C A A C A CP-CLRDV Bra T T G G A G T T C A T C T C C G A G G C C T C T T C A A C A CP-CLRDV VN 370 380 390 ------------------+-------------------+-------------------+T C C T C C G G T T C C A T C T C T T A C G A A G T G G A T CP-CLRDV-Agh T C C T C C G G T T C C A T C T C T T A C G A A G T G G A T CP-CLRDV Bra T C C T C C G G T T C C A T C T C T T A C G A A G T G G A T CP-CLRDV VN 400 410 420 ------------------+-------------------+-------------------+C C A C A C T G T A A A T T G T C A A C C C T A T C C T C C CP-CLRDV-Agh C C A C A C T G T A A A T T G T C A A C C C T A T C C T C C CP-CLRDV Bra C C A C A C T G T A A A T T G T C A A C C C T A T C C T C C CP-CLRDV VN

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Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn 430 440 450

421 421 421 ------------------+-------------------+-------------------+A C G A T T A A C A A A T T C G G A A T C A C C A A G A A T CP-CLRDV-Agh A C G A T T A A C A A A T T C G G A A T C A C C A A G A A C CP-CLRDV Bra A C G A T T A A C A A A T T C G G A A T C A C C A A G A A C CP-CLRDV VN 460 470 480 ------------------+-------------------+-------------------+G G G A G G A A G C A A T T T G C G G C G T C T T T C A T C CP-CLRDV-Agh G G G A G G A A G C A A T T T G C G G C G T C T T T C A T C CP-CLRDV Bra G G C A G G A A G C A A T G T G C G G C G T C T T T C A T C CP-CLRDV VN 490 500 510 ------------------+-------------------+-------------------+A A T G G A C A G G A A T G G C A T G A C A C C T C C G A G CP-CLRDV-Agh A A T G G A C A G G A A T G G C A T G A C A C C T C C G A G CP-CLRDV Bra A A T G G A C A G G A A T G G G A T G A C A C C T C C G A G CP-CLRDV VN 520 530 540 ------------------+-------------------+-------------------+G A C C A A T T C A G A A T C T T A T A C A A A G G C A A T CP-CLRDV-Agh G A C C A G T T C A G A A T C T T A T A T A A A G G C A A T CP-CLRDV Bra G A C C A G T T C A G A A T C T T A T A T A A A G G C A A T CP-CLRDV VN 550 560 570 ------------------+-------------------+-------------------+G G T T C C T C G T C G A T A G C T G G C T C T T T T A A G CP-CLRDV-Agh G G T T C C T C G T C G A T A G C T G G C T C T T T T A G G CP-CLRDV Bra G G T T C C T C G T C G A T A G C T G G C T C T T T T A G G CP-CLRDV VN 580 590 600 ------------------+-------------------+-------------------+G T C A C A A T T C G G T G C C A A T T C C A C A A T C C A CP-CLRDV-Agh G T C A C A A T T C G G T G C C A A T T C C A C A A T C C A CP-CLRDV Bra G T C A C A A T T C G G T G C C A A T T C C A C A A T C C A CP-CLRDV VN -----------A A A T A G A A A T A G A A A T A G

451 451 451

481 481 481

511 511 511

541 541 541

571 571 571

601 601 601

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CP-CLRDV-Agh CP-CLRDV Bra CP-CLRDV VN

Ti liu chia s ti: wWw.SinhHoc.edu.vn

Hnh 4. Kt qu so snh gen CP-CLRDV phn lp Vit Nam (CP-CLRDV VN) vi cc trinh t c cng b ca Braxin (CP-CLRDV Bra) v Aghentina (CP-CLRDV Agh).

Cc trnh t thu c c so snh vi cc trnh t gen ca virus CLRDV cng b trn ngn hng gen th gii cho thy: Trnh t gen CP phn lp t CLRDV Vit Nam c tng ng vi trnh t gen

cng b t 98,2-99,2%. Kt qu ny chng t y ng l cc trnh t

ca gen CP-CLRDV. Trnh t gen CP ny ging vi trnh t gen CPCLRDV Braxin hn trnh t CP-CLRDV Aghentina (hnh 4). Ti liu chia s ti: wWw.SinhHoc.edu.vn

Ti liu chia s ti: wWw.SinhHoc.edu.vn KT LUN phn lp c gen m ha protein v (CP) ca virus gy bnh xanh ln trn cy bng Vit Nam. on gen ny s l ngun nguyn liu tin hnh cho cc nghin cu tip theo ca ti.

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