Biotechnology/Phytohormones Lab 1 Sept. 13 th 2010.
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Transcript of Biotechnology/Phytohormones Lab 1 Sept. 13 th 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
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
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