Biotechnology/Phytohormones Lab 1 Sept. 13 th 2010.

Biotechnology/PhytohormonesBiotechnology/PhytohormonesLab 1Lab 1

Sept. 13th 2010

Strategies of gene expression analysisStrategies of gene expression analysis

Promotoranalysis

Function of gene/protein

Expressionpattern of

genes

1.GUS

staining

2.RT-PCR

GTL1 geneGTL1 gene

• Research project in Prof. Hasegawa’s lab

• GTL1 is member of GT-2 family

• Act as a transcription factor that binds to GT-elements (GGTTAA, GGTAAT, or GGTAAA)

• Data suggests dark/light regulation of GTL1

GUS reporter systemGUS reporter system Reporter gene: GUS (beta-glucuronidase) Substrate: X-gluc (5-bromo-4-

chloro- 3-indolyl glucuronide)

• Plants have very low X-GLUC cleavage activity

• GUS is fused to native promotor of gene of interest (GOI)

• Enables analysis of temporal and spatial expression pattern of GOI

1. GUS cleaves the substrate X-gluc

2. One product → indigo3. Dark blue color

RT-PCRRT-PCR

PCR

cDNA Synthesis

L.V. Kendall et al, 2000

5’ 3’

5’

Can be RT (HIV-RT 3’-5’ endonuclease activity, J.J. DeStefano et al, 1991)

3’ 5’

5’

5’ 3’

5’

Start GUS staining assay

Setup PCR reaction

1.

2.

- label 4 eppendorf tubes- seedlings grown under diff. conditions- add 90% aceton – 30min room temp.- discard- add ddH2O – discard- add X-Gluc staining solution – keep at 37C overnight

- label 4 PCR tubes-Take 10 µl of cDNA, pos./neg. control- add 40 µl of Mast Mix to each tube- the tubes go into the PCR machine

in between the GUS staining (30min incubation)

Pipettes

P1000

P200

P20

1000uL = 1ml

Use for: 200-1000uL

200uL = 0.2ml

Use for: 20-200uL

20uL = 0.02ml

Use for: 2-20uL

Phytohormones

Effects of GA/GA inhibitor on

peas

Effects of BR/BR inhibitor on Arabidopsis

Gibberellic acid (GA) Brassinosteroids (BR)

Stem length

Seed germination

Transition to flowering

Pollen development

Cell division

Cell elongation

Photomorphogenesis

Reproductive development

Leaf senescence

Stress responses

H

H

H

O

H

O

HO

HO

OH

OHH

Brassinolide

Experiment A+B: Effect of GA/GA inhibitor on pea growth

GA or Control solution GA inhibitor or Control solution

•Label pots (group and lab number, treatment)•Measure internode length of 2x 5 dwarf and 2x 5 tall pea plants

• next measurement next week (MONDAY Sept. 20th)

Treat dwarf plants with GA or Control solution

Treat tall plants with GA inhibitor or Control solution

Experiment C+DEffect of BR/BR inhibitor on Arabidopsis growth

BR and control treatment BR inhibitor and control solution

Choe et al., 1999

•Label pots (group and lab number, treatment)•Measure rosette diameter of 2x 10 dwarf and 2x 10 tall Arabidopsis plants

• next measurement next week (MONDAY Sept. 20th)

Treat dwarf plants with BR or Control solution

Treat tall plants with BR inhibitor or Control solution

Flow Chart

1. Start GUS staining assay

2. Set up PCR reactions

3. GA/GA inhibitor treatment on peas

4. BR/BR inhibitor treatment on Arabidopsis

Enjoy!

PCRPCR• Polymerase Chain Reaction (PCR)

• in vitro DNA polymerization with DNA dependent DNA polymerase

• First described 1971 by Kepple et al. but fresh enzyme added each cycle

• Kary Mullis, 1984, introduced the idea of using thermostable DNA -polymerase from Thermus aquaticus (Taq)

Kary Mullis

Reverse transcriptaseReverse transcriptase• Reverse Transcriptase (RT) is a RNA-

dependent DNA polymerase

• RNA (retro)viruses and transposons in eukaryotes use RT

• Catalyzes RNA-directed extension of 3'-end of DNA strand by 1 deoxynucleotide at a time

• Cannot initiate a chain de novo → requires a RNA or DNA primer

• DNA can also serve as template Human immunodeficiency virus (HIV) uses RT without proof reading function

Source: PDB entry 1HMV

GA biosynthesis pathway

Sumagic (Uniconazole-P)

Mutation in dwarf pea

Ultradwarf:No GAs

Dwarf:Contains :GA20

Traces of GA1

Tall:Contains:GA1