Plant biotechnology Lab 1

Strategies of gene 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

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

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’

Introduction Background Safety Start GUS staining assay Setup PCR reaction Gel electrophoresis of PCR