1 I HC NNG LM TP.

H CH MINH VIN CNG NGH SINH HC & MI TRNG

THC HNH

CNG NGH SINH HC CY TRNG

THNG 12/2007 Lu hnh ni b

NI DUNG THC HNH

Chuyn gen bar khng thuc dit c glufosinate trn cy u xanh (Vigna radiata (L.) Wilczek) bng vi khun Agrobacterium tumefaciens
- Cc ging u xanh c s dng trong th nghim l ging TN 182 ca Cng ty TNHH-TM Trang Nng, ging HL 89-E3 ca Cng ty c phn ging cy trng Nha H. - Vi khun Agrobacterium tumefaciens chng EHA101pIB-GUS do Hans-Jrg Jacobsen thit k, mang plasmid pGII0229 Trgus cp148 luc, plasmid ny mang gen gusA, gen nptII v gen bar vi promoter l CaMV 35S v terminator l nos polyA.

Hnh 1. Plasmid pGII0229 Trgus cp148 luc

PHN 1. KH TRNG HT, CHUN B MI TRNG

I. Vt liu a. Kh trng ht Mu ht. Dng c cy m: pince, dao cy, n cn. Ha cht kh trng: Cn 70o, cn 96 o, Javel, khng sinh Tetracilin. Nc ct tit trng * Mi trng tin nui cy (pre-culture medium): to iu kin cho m thch nghi vi mi trng nui cy. * Mi trng (inoculation medium): mi trng to iu kin cho vi khun xm nhp vo m thc vt. * Mi trng ng nui cy (co-culture medium): mi trng thun li cho s pht trin ca m v vi khun. II. Cch tin hnh a. Kh trng ht theo quy trnh sau y b. Ra ht bng nc tit trng 3 ln (mi ln 5 pht). Ra ht bng cn 960 3 ln, (mi ln 5 pht). Ra bng nc tit trng 1 ln. Ra bng nc javel m c 3 ln, (mi ln 10 pht). Ra bng nc ct tit trng 1 ln. Ra ht bng khng sinh 500mg/l Tetraciclin trong 5 pht. Ra bng nc tit trng 1 ln. Ngm ht bng nc tit trng trong 1 gi. Ngm ht qua m.

b.

Chun b mi trng

Chun b mi trng

Mi trng tin nui cy (pre-culture medium) Mi trng B5 (lng) + 1 mg/l BA Thnh phn mi trng B5 Thnh phn Nng (g/l)

KNO3 (NH4)2SO4 CaCl2.2H2O MgSO4.7H2O NaH2PO4.H2O MnSO4*H2O H3BO3 ZnSO4*7H2O NaMoO4*2H2O CuSO4*5H2O CoCl2*6H2O

25 1.34 1.5 2.5 1.5 1 0.3 0.2 0.025 0.0025 0.0025

* Mi trng (inoculation medium): B5, 20g/l glucose, 2 g/l MES, pH 5.5 -5.6. * Mi trng ng nui cy (co-culture medium) - B5 - 2 g/l - 0.5 g/l - 0.87 - 0.5 g/l - 0.8 g/l MES (Morpholino ethan sulfonic acid) KNO3 g/l CaCl2 MgCl2 * 7H2O glutamine

- 10 mg/l gluthatione - 2.2 mg/l TDZ (Thidiazuron) - 10 g/l - 10 g/l - 4 g/l - pH 5.2 - Acetosyringone (0; 50; 100 M) (b sung sau khi hp kh trng) sucrose glucose agar

PHN 2. GIEO HT
I. Vt liu Ht kh trng v ngm qua m.

II.

Dng c nui cy m: pince, dao cy, n cn, a petri (hp nha) cha mi trng agar. Loi b lp v ngoi (seed coat) ca ht u xanh v t ht vo mi trng

Cch tin hnh agar (10 g/l).

Hnh 2. Hnh thi ca thc vt 1 l mm v 2 l mm

PHN 3. TCH TR DI L MM (hypocotyl) v TNG SINH VI KHUN

I. Vt liu Ht ging ny mn. Dng c nui cy m: pince, dao cy, n cn, a petri (hp nha) cha mi trng agar. a petri cha mi trng tin nui cy. Fancol 15ml. Mi trng LB. Ct tr di l mm ti im mt l mm (cotyledonary node) bng cch loi b tr trn l mm (epicotyl) v r mm (radicle). Sau t cc tr di l mm (kch thc trung bnh 0,5 0,6 cm) trong a petri cha mi trng tin nui cy (preculture medium) trong iu kin chiu sng. Nhn sinh khi vi khun Agrobacterium tumefaciens trong mi trng LB trn my lc vi vn tc 150 vng/pht, 280C, qua m.

II. Cch tin hnh

PHN 4. NG NUI CY TR DI L MM V VI KHUN

I. Vt liu

Tr di l mm. Vi khun. Dng c nui cy m: pince, dao cy, n cn, a petri (hp nha) cha mi trng agar. a petri cha mi trng ng nui cy. Fancol 15ml. a. Thu sinh khi vi khun

II. Cch tin hnh

o OD (optical density) vi khun bc sng 600nm (blank bng mi

trng LB). Khi gi tr OD 0,6 1 th tin hnh chuyn gen (1 OD tng ng khong 3 x 109 bacteria/ml).

Ly tm trong ng fancol 15ml 3000rpm trong 10 pht, 240C. Thu ta, ha tan trong mi trng . b. ng nui cy Cho tr di l mm (to vt thng/khng to vt thng) vo mi trng Sau thi gian , cy mu vo mi trng ng nui cy cha cc nng

trong thi gian t 10 30 (ty theo nghim thc). Acetosyringone khc nhau, trong thi gian 3 ngy (Xem bng di y) B tr th nghim Thi gian Khng to vt thng (mu) To vt thng (mu)
Acetosyringone (M)
10 30 30 30 30 20 30 30 30 30 30 30

30

30

30

30

30

30

30

30

30

50

100

50

100

50

100

PHN 5. NHUM V QUAN ST MU

I. Vt liu Dng c cy m Nc ct Phosphate buffer Equilibration buffer

II. Cch tin hnh

- Sau 3 ngy ng nui cy, cc tr mm c ra bng nc ct tit trng cho

sch vi khun, ct b nhng phn dp nt, ch tr mm ra lm i v vi phosphate - buffer trong 5 pht. m. Ly tr mm ra soi di knh hin vi quan st mu xanh chm (sn phm phn ng ca enzyme glucuronidase vi X-gluc). Loi b phosphate buffer, tr mm bng Equilibration buffer 370C qua

Hnh 3. Phn ng to ra sn phm 5,5-dibromo-4,4-dicloro-indigo c mu xanh.

PH LC
1. Thnh phn mi trng LB (Lauria Broth) (Sambrook et al. , 1989) - 10 g/l tryptone - 5 g/l yeast extract - 8 g/l NaCl - pH 7.2 2. Phosphate buffer 0,1 M NaH2PO4 10ml stock/100ml 0,1 M Na2HPO4 10ml stock/100ml 10 mM EDTA 2ml stock/100ml 0.5mM K-ferrocyanide (K4[Fe(CN)6] 10ml stock/100ml 0.5mM K-ferricyanide (K3[Fe(CN)6] 10ml stock/100ml 2% formaldehyde 0,1 % Trition X-100 iu chnh pH 7.0 bng KOH; bo qun trong ti 4oC 3. Equilibration Buffer (bo qun trong ti, 4oC) Phosphate Buffer 100ml 1 mg / ml x-Gluc (ha tan trong DMSO)