Eργαστηριακές Ασκήσεις Βιοτεχνολογίας Φυτών_2013

1

&

2013

2

A.

(

)

.

T-DNA

Agrobacterium .

Agrobacterium

DNA ( -DNA)

. T-DNA

vir ,

T-DNA

( -RB LB). DNA

.

.

vir . T-DNA

DNA.

, T-DNA ,

(Koncz et al, 1989, Mathur et al,

1998)

,

,

DNA Agrobacterium ,

.

in vitro .

- Arabidopsis thaliana,

in planta

. ,

.

3

(Clough and Bent, 1998; Bechtold

et al. 1993) floral-dip,

,

Agrobacterium.

,

, .

.

()

,

.

(~ 1%)

.

4

A1.

Arabidopsis thaliana Columbia-0 (Col-0)

(>

250 Einsteins m-2 sec-1), 8h / 16h

18 h / 6 h

, 220 C

20o C.

1: A rabidopsis thaliana Columbia

1. (1510cm)

, .

~ 9cm.

.

2. ,

.

4-6 . ,

.

5

A2. Agrobacterium tumefaciens

Agrobacterium tumefaciens

GV3101 Ti pM90(pTiC58T-DNA)

. o

pBIN GUS 35S .

1. T LB

(

50 mg/L 28 C, 250 rpm 18-24 h (O.D.600 1.8-2).

2. 10 min

5500 g.

3. MS, 5% / 0.05% Silwet L77 (

) O.D.600= 0.1-0.5. A 200-300 ml

2-5 ( ).

GUS

6

A3.

1. Agrobacterium

2-3 min.

.

2. ,

, 16-24 .

3. 3-5

(siliques) .

4 C.

2: rabidopsis thaliana Agrobacterium

tumefaciens

A4.

,

Arabidopsis thaliana

(1 ).

1. 70% 30-60

sec 30% (2.625% )/

7

0.05% Tween20 5 min. .

, ,

(100 ml 3 ml Cl)

4 15h.

2. E 1/2 MS , 0.8% 50

g/ ml . vancomycin 500mg/l

. ,

20-30 min.

3.

100-150 (~15 mg)

2.5 ml 0,05% . 10-15 min,

.

. .

4. Parafilm. 4 C 1

(50-100 Einsteins m-2

sec-1) 7-10 .

.

5. 1.5% Phytagel,

.

6. .

3: rabidopsis thaliana

8

1


Agrobacterium tumefaciens.

1. Agrobacterium O.D.600~ 2. 10 ml .

2. ( ) 10 4-5rpm.

3. E 10 ml IM .

4. .

5. 90 ml 100 ml OD~ 0,2.

6. SILWET L-77 1 min.

7. .

8. A .

9. 2 min.

10. , 5 min .

11. 2 min.

12. .

\

13. ( 18 /6 210C).

9

2

1

.

1. .

2. 1 0 (eppendorf no. 1).

3. eppendorf 1 ml ETOH 70% () (eppendorf no. 2).

4. eppendorf 3 , .

5. eppendorf 1 ml 20% / 0.05% Tween 20 (eppendorf no. 3) 5 min.

6. 5-6 1 ml ( no. 1).

7. 1ml , eppendorf ( + ) (MS medium + Km50). .

8. .

9. parafilm.

10. ( 18/6 210C).

10

B. gus


E

(

).

- (uidA / gus), GFP,

(lux & luc)

(cat).

4: gus gfp

rabidopsis thaliana

- (GUS)

. -

(. - )

( ). ,

.

11

B1.

in situ X-gluc

(5-bromo-4 chloro-3-indolyl -D-glucuronide). GUS

,

. .


GV3101::35S::GUS ( 1).

1. 1-2 Arabidopsis thaliana

GV3101::35S::GUS ( 1) 500l

. eppendorf 1-2 RT 15

.

2. 1-2 dH2O X-

Gluc.

3.

. 370C 1-2 .

4. .

2.

gus

4-MUG (4-methylumbelliferryl--D-glucuronide),

4-U (4-methylumbelliferone). O

MU

pM/min/mg .

MU

19,82g MU (4-Methylumbelliferone),(Sodium salt, Sigma M1508)

1 ml 100 mM.

4 0C .

.

12

1. 10 l MU (4-Methylumbelliferone) (100 mM) 990 l

(0,2 M a2CO3).

1 mM (106 nM).

2. 1 nM, 10 nM, 100 nM,

103 nM, 104 nM 105 nM :

100l(106nM) 900l (0,2M a2CO3)10

5nM.

100l(105nM) 900l (0,2M a2CO3)10

4nM.

100l(104nM) 900l (0,2M a2CO3)10

3nM.

100l(103nM) 900l (0,2M a2CO3)10

2nM.

100l(102nM) 900l (0,2M a2CO3)10nM. 100l(10nM) 900l (0,2M a2CO3)1nM.

.

4. :

Excitation= 365 nm, Emission= 445 nm, Excitation Slit Width= 2.5, Emission Slit

Width= 10, Emission filter= open or 1% attenuator. Excitation filer= clear (auto

cutoff on).

5. ( 0,2M

a2CO3). .

2X103nM.

,

U, .

Bradford,

.

1. - Arabidopsis thaliana,

,

. 100 l .

2. 13.000 / .

13

3. eppendorf. M

80 0C.

4. E eppendorf

900 l (0,2M a2CO3).

5. eppendorf 50 l 500 l

GUS (-) (GAB).

6. 37 0C.

7. 1 100 l,

1, 900 l

.

8. .

9. 6

37 0C 100 l

No 2. .

10. 11

( 3) 16 ( 4).

. (

(1-10 ) (1-10

).

( BRADFORD)

A. BSA

1. eppendorf BSA 1, 2, 5, 10, 20

mg/ml.

2. 3-5 l 900 l

Bradford.

3. 1000 l ddH2O.

4. 595 nm.

.

5.

BSA.

.

BRADFORD

1. T eppendorf 100 l

.

14

2. 13000 / .

3. eppendorf.

80 0C.

4. 3-5 l 95 l ddH2O 900 l

Bradford.

5. H 595 nm.

.

()

50 mM Na2PO4 (pH:7), 10 mM Na2EDTA (pH:8) , 0,1% (w/v) N-lauroyl-sarcosyl,

1% (v/v) Triton X-100, 0,07% -.

GUS (X-GLUC)

1-2 mM 5-bromo-4-chloro-3-indoly;--D-glucuronide

(X-GLUC)

GUS (MUG)

1-2 mM 4-methyl-umbelliferyl-glucuronide (MUG)

Bradford stock

100 mg Coomasie blue G250, 100 ml 3P4 (85%), 50 ml ,

50 ml ddH2O.

15

3

1. - Arabidopsis thaliana,

,

. 100 l

.

2. 13.000 / .

3. E eppendorf

900 l (0,2M a2CO3).

4. 50 l eppendorf 500 l

GUS (-) (MUG).

5. 100 l,

to, 900 l

. t .

6. MUG

37 0C.

7. 5

37 0C 100 l

t1. t1 .

8. 20 .

16

.

. ,

( ) .

,

(. ). ,

.


( ) (agroinfection). o

( ).

Agrobacterium Nicotiana

benthamiana ().

1.

Nicotiana benthamiana

(> 250 Einsteins m-2 sec-1), 18 h / 6 h

, 24 0 C 20 o C.

(4-8 )

.

2. Agrobacterium tumefaciens


GV3101, pBIN m-gfp5-ER (Haseloff et al ,1997),

GFP 35S.

17

1. T LB

() 28 C,

250 rpm 18-24 h ( ).

2. 20 min

1000 g

3. O.D.600= 0.4-0.5.

4. 30 min.

3.

1.

.

1-2 ml.

2. To 48 h.

4.

18

4

1) Agrobacterium tumefaciens (GV3101::35S::GFP).

10 ml 4000 rpm 10 .

2) 3ml MMA

().

3) MMA OD600: 0.1-0.5

4) AS .

5) RT >30 (60-90) 19-23 C.

6) 2ml

MMA .

1-2 ml GV3101::35S::GFP.

7) 48 GFP

UV .

MMA .

19

LB 1 : 10 g peptone, 10 g NaCl, 5 g Yeast Extract

S (Murasige & Schoog) -

1 : 2,2 g Murashige and Skoog 0,5 g MES pH 5,7

KOH 7 g r

50 mg Kan/l, ~40 C.

Murashige and Skoog (MS)a.

(mg/l)

Macroelementsb

NH4NO3 1650

KNO3 1900

CaCI2.2H2O 440

MgSO4.H2O 370

KH2PO4 170

Microelementsc

KI 0,83

H3BO3 6,2

MnSO4.H2O 22,3

ZnSO4.7H2O 8,6

Na2MoO4.H2O 0,25

CuSO4.5H2O 0,025

CoCI.6H2O 0,025

Iron sourcec

FeSO4 7H2O 27,8

Na2EDTA.2H2O 37,3

Organic supplementc

Myoinositol 100

Nicotinic acid 0,5

Pyroxide- HCI 0,5

Thiamine- HCI 0,5

Glycine 2

20

10 S, pH 6.5

10 mM MgCl2

150 M acetosyringone

()


1% (v/v) Triton X-100, 0,07% -.

GUS (GAB)

1-2 mM 4-methyl-umbelliferyl-glucuronide (GUS)

Bradford stock

100 mg Coomasie blue G250, 100 ml 3P4 (85%), 50 ml ,

50 ml ddH2O.

()


1% (v/v) Triton X-100, 0,07% -.

21

. In silico

1.

,

cDNA ,

.

cDNA .

) )

) .

.

,

, ,

80 90.

NCBI

(www.ncbi.nlm.nih.gov),

.

in silico

. ,

,

cDNA ,

.

(Arabidopsis

thaliana, Medicago truncatula, Zea mays, Lycopersicon esculentum, Oryza sativa

.) . ,

.

Arabidopsis thaliana The Arabidopsis Information Resource (TAIR,

(www.arabidopsis.org).

,

.

,

PCR.

/ ,

22

.

,

.

( ). ,

(.

http://tgrc.ucdavis.edu/Data/Acc/dataframe.aspx?start=CoreTaxon.aspx&navstar

t=nav.html).

Arabidopsis

thaliana. T-DNA

TAIR

Blast.

TILLING

http://www.gabi-

till.de/project/up/arabidopsis.html.

Arabidopsis thaliana RNAi

, Argikola.

http://www.agrikola.org/index.php?o=/agrikola/main.

.

Real time-PCR.

Arabidopsis

thaliana /.

,

,

.

,

.

NCBI TAIR.

23

,

.

.2 5

1.

Arabodopsis thaliana (..

).

2. TAIR Atg (tg number)

. www.arabidopsis.org

3.

www.ncbi.nlm.nih.gov .

4. .

5.

. - -

.

6.

.

7.

.

8. .

References:

Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated

gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci.

Paris, Life Sciences 316:1194-1199.

Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-

mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.

Haseloff, J., Siemering, K.R., Prasher, D.C. and Hodge, S. (1997) Removal of a

cryptic intron and subcellular localization of green fluorescent protein are required

to mark transgenic Arabidopsis plants brightly. Proc. Natl Acad. Sci. USA 94:

21222127

Jefferson RA (1987) Assaying chimaeric genes in plants: the GUS gene fusion

system. Plant Molecular Biology Reporter 5: 387-405.

Eργαστηριακές Ασκήσεις Βιοτεχνολογίας Φυτών_2013

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