plant biotechnology lab 1. strategies of gene analysis promotor analysis function of gene/protein...

Plant biotechnology Lab 1Plant biotechnology Lab 1

Strategies of gene analysisStrategies of gene 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

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

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’

Introduction

Background

Safety

Start GUS staining assay

Setup PCR reaction

1. 2.3.

Gel electrophoresis

of PCR