REPORTER GENESSubmitted by Swati sohni (230) Manish (221) Sec C,Msc Biotech (2 sem)

MARKER GENESMonitoring and detection of plant transformation systems in order to know whether the DNA has been successfully transferred into recipient cells is done with the help of a set of genes. Marker genes are categorized into two types: Reporter genes Selectable marker genes

REPORTER GENES

A Reporter gene is a test gene whose expression results in quantifiable phenotype. A synthetic modification is introduced (or the protein coding sequence is deleted and replaced by another gene) in order to either simplify the detection of the gene product or to distinguish it from identical genes in the genome.

MECHANISM

FEATURES OF REPORTER GENES

Detection with high sensitivity Assay should be non destructive Product of reporter gene should be resistant to the chemicals used in the assay Should not be toxic to the cell Assay should require a minimal amount of effort and expense

COMMONLY USED REPORTER GENES

Opine synthase Chloramphenicol acetyl transferase (CAT) GUS Bacterial luciferase (Lux F2) Firefly luciferase (Luc) Green fluorescent protein (GFP) Anthocyanins

OPINE SYNTHASE

Octopine or nopaline synthase genes (ocs, nos) are present in the T DNA of Ti plasmid of Agrobacterium The presence of opine in any plant material clearly indicates the transformed cells

TECHNIQUE

Extraction of protein Incubation of extraction with opine precursor Arginine ,pyruvate and NADH for ocs Arginine,KGA and NADH for nos Separation of reaction product by paper chromatography

CHLORAMPHENICOL ACETYL TRANSFERASE (CAT)

The cat gene was the first bacterial gene to be introduced in the plant cells The most commonly used reporter gene was found in the transposable element Tn9 The structural gene codes for the enzyme CAT ,which carries out acetylation of cholamphenicol

ACETYLATION OF CHLORAMPHENICOL

TECHNIQUEThe substrate required for enzymatic action are acetyl CoA and labelled chloramphenicol (14 C) Incubation of labelled chloramphenicol with crude extract of plant tissue in buffer Autoradiography to detect presence of acetyl chloramphenicol

THE FIREFLY LUCIFERASE (LUC)

Luciferase catalyzes the oxidative decarboxylation of luciferin (6-hydroxybenzothiazole to oxyluciferin in the presence of ATP, oxygen and magnesium ions The activity of luciferase is measured as initial maximum light intensity upon mixing the enzymes with substrate. Luminescence in terms of light production can be recorded in luminometer

GREEN FLUORESCENT PROTEIN

GFP is a small protein of 238 amino acids This owes its visible absorbance and fluorescence to beta hydroxy benzylidine imidazolinone chromophore Detection of green fluorescence can be detected under fluorescent microscope

ANTHOCYANINS

Anthocyanins are prominent red and purple pigments of flowering plants The regulatory genes C1 and B/R coordinately regulate the activity of structural genes

TECHNIQUE

Chimeric gene constructs have been prepared with C1 and B/R regulatory genes Introduction of above gene into plant cells Production of red coloration Identification of red spots visually or microscopically

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