Biotechnology/Phytohormones Lab 1 Sept. 13 th 2010

Strategies of gene expression analysis Promotor analysis Function of gene/protein Expression pattern of genes 1. GUS staining 2. RT-PCR

GTL1 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 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 → indigo 3.Dark blue color

RT-PCR PCR cDNA Synthesis L.V. Kendall et al, ’ 3’ 5’ Can be RT (HIV-RT 3’-5’ endonuclease activity, J.J. DeStefano et al, 1991) 3’ 5’ 3’ 5’

Start GUS staining assay Setup PCR reaction 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 P uL = 1ml Use for: uL 200uL = 0.2ml Use for: uL 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 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. 20 th ) Treat dwarf plants with GA or Control solution Treat tall plants with GA inhibitor or Control solution

Experiment C+D Effect of BR/BR inhibitor on Arabidopsis growth BR and control treatmentBR 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. 20 th ) 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!

PCR 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 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 : GA 20 Traces of GA 1 Tall: Contains: GA 1